Immunotoxicology strategies for pharmaceutical safety assessment

IMMUNOTOXICOLOGY STRATEGIES FOR PHARMACEUTICAL SAFETY ASSESSMENT IMMUNOTOXICOLOGY STRATEGIES FOR PHARMACEUTICAL SAFET...

Author: Danuta J. Herzyk | Jeanine L. Bussiere

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IMMUNOTOXICOLOGY STRATEGIES FOR PHARMACEUTICAL SAFETY ASSESSMENT

IMMUNOTOXICOLOGY STRATEGIES FOR PHARMACEUTICAL SAFETY ASSESSMENT

Edited by DANUTA J. HERZYK AND JEANINE L. BUSSIERE

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2008 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 527-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data Immunotoxicology strategies for pharmaceutical safety assessment / [edited by] Danuta J. Herzyk, Jeanine L. Bussiere. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-12238-9 (cloth) 1. Immunotoxicology. 2. Immune system–Effect of drugs on. 3. Toxicity testing. 4. Drugs–Toxicology. I. Herzyk, Danuta J. II. Bussiere, Jeanine L. [DNLM: 1. Drug Toxicity–immunology. 2. Drug Design. 3. Drug Evaluation–methods. 4. Immune System–drug effects. 5. Immunotoxins–adverse effects. 6. Risk Assessment. QV 600 I337 2008] RC582.17.I47 2008 616.97′071–dc22 2008015104 Printed in the United States of America. 10

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CONTENTS

Preface

ix

Contributors

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Introduction to Immunotoxicology

xvii

Jack H. Dean

PART I CURRENT REGULATORY EXPECTATIONS FOR IMMUNOTOXICITY EVALUATION OF PHARMACEUTICALS 1

Current Regulatory Expectations for Immunotoxicity Evaluation of Pharmaceuticals

1

3

Kenneth L. Hastings

PART II WEIGHT OF EVIDENCE REVIEW: A NEW STRATEGY IN IMMUNOTOXICOLOGY 2.1

Clinical Pathology as Crucial Insight into Immunotoxicity Testing

11

13

Ellen Evans

2.2

Histomorphology of the Immune System: A Basic Step in Assessing Immunotoxicity

27

Patrick Haley

2.3

Need for Specialized Immunotoxicity Tests

45

Kazuichi Nakamura v

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2.4

CONTENTS

Specific Drug-Induced Immunotoxicity: Immune-Mediated Hemolytic Anemia

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Raj Krishnaraj

PART III NONCLINICAL CORE IMMUNOTOXICITY TESTING IN DRUG DEVELOPMENT

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3.1.1

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T Cell-Dependent Antibody Response Tests Joseph R. Piccotti

3.1.2

Natural Killer Cell Assay and Other Innate Immunity Tests

77

Lisa Plitnick

3.1.3

Cellular Immune Response in Delayed-Type Hypersensitivity Test

87

Karen Price

3.2

Evaluation of Drug Effects on Immune Cell Phenotypes

103

Laurie Iciek

PART IV EXTENDED IMMUNOTOXICOLOGY ASSESSMENT: EX VIVO MODELS

125

4.1

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Functional Cellular Responses and Cytokine Profiles Elizabeth R. Gore

4.2

Application of Flow Cytometry in Drug Development

141

Padma Narayanan, Renold J. Capocasale, Nianyu Li, and Peter J. Bugelski

PART V EXTENDED IMMUNOTOXICOLOGY ASSESSMENT: IN VIVO MODELS

161

5.1

163

Animal Models of Host Resistance Gary R. Burleson and Florence G. Burleson

5.2

Approaches to Evaluation of Autoimmunity

179

Danuta J. Herzyk

PART VI IMMUNOTOXICITY TESTING IN BIOPHARMACEUTICAL DEVELOPMENT 6.1

Differentiation between Desired Immunomodulation and Potential Immunotoxicity Jeanine L. Bussiere and Barbara Mounho

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CONTENTS

6.2

Relevant Immune Tests across Different Species and Surrogate Models

199

Jeanine L. Bussiere

6.3

Antidrug Antibody Responses in Nonclinical Studies and Their Implications

209

Barbara Mounho

PART VII 7.1

DEVELOPMENT OF VACCINES

Pharmacological Immunogenicity and Adverse Responses to Vaccines

217

219

Mary Kate Hart, Mark Bolanowski, and Robert V. House

7.2

Immunotoxicological Concerns for Vaccines and Adjuvants

229

Catherine Kaplanski, Jose Lebron, Jayanthi Wolf, and Brian Ledwith

PART VIII TESTING FOR DRUG HYPERSENSITIVITY

239

8.1

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Systemic Hypersensitivity Raymond Pieters

8.2

Nonclinical Models to Assess Respiratory Hypersensitivity Potential

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Curtis C. Maier

PART IX TESTING FOR DEVELOPMENTAL IMMUNOTOXICITY

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9.1

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Developmental Immunotoxicity in Rodents Rodney R. Dietert and Leigh Ann Burns-Naas

9.2

Developmental Immunotoxicity in Nonhuman Primates

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Pauline L. Martin and Eberhard Buse

PART X NEW METHODS IN ASSESSING IMMUNOMODULATION, IMMUNOTOXICITY, AND IMMUNOGENICITY 10.1

Alternative Animal Models for Immunomodulation and Immunotoxicity Peter J. Bugelski

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10.2

CONTENTS

Animal Models for Preclinical Comparative Immunogenicity Testing

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Daniel Wierda

10.3

T Cell Epitopes and Minimization of Immunogenicity

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Harald Kropshoffer and Thomas Singer

PART XI BRIDGING IMMUNOTOXICOLOGY TO CLINICAL DRUG DEVELOPMENT

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11

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Bridging Immunotoxicology to Clinical Drug Development Ian Gourley and Jacques Descotes

Index

385

PREFACE

This book is the first edition entirely dedicated to immunotoxicology testing during pharmaceutical drug development. Immunotoxicology is a highly specialized discipline that addresses potential adverse effects on the immune system, including immunosuppression, immunogenicity, hypersensitivity and other immune system functional disorders. A broad spectrum of xenobiotics, including agents present in our environment as well as pharmaceutical molecules may adversely affect the immune system. In pharmaceutical drug development, testing of drug candidates involves not only potential immuntoxic hazard identification but also risk assessment in the context of therapeutic use of a given drug. Thus, testing for potential immunotoxicity of drug candidates should be an integral part of overall safety evaluation in both preclinical and clinical phases of drug development. This approach is different from immunotoxicity testing of environmental agents where any immunotoxicity is hazardous and unacceptable in light of potential uncontrolled exposure of healthy population. In the development of novel therapeutic entities (chemicals, proteins and vaccines), strong immunotoxicity signals can be detected by hematology and/or lymphoid tissue histopathology evaluation as part of standard toxicity studies. However, potential immunotoxicity related to immune dysregulation by drugs, may only manifest at the functional level during an immune response to a challenge with an antigen (e.g., foreign protein, pathogen, toxin). Thus, evaluation of the functional immune system requires studies involving ‘activated’ immune cells, organs or entire hosts in response to an outside challenge. To detect and characterize such hazards, immunotoxicology assessment involves not only conventional toxicology endpoints (i.e., hematology, clinical chemistry and histology) but also a broad spectrum of specialized testing to evaluate potential immune dysregulation, including specific immune response tests (cellular or humoral), immunophenotyping, cytokine expression, immunoassays to address immunogenicity, and in vivo models of immune disorders to characterize potential impairment of host defense to infections, tumors and autoimmune diseases. ix

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PREFACE

This book is focused on discussions of strategies for application of immunotoxicology testing during drug development, in other words it addresses ‘what’ and ‘how’ can be performed in such evaluation. We hope this new book will be found valuable to both the experienced and novice immunotoxicologists who work, teach and study immune-related aspects of safety assessment of drug candidates in pharmaceutical development. Danuta J. Herzyk Jeanine L. Bussiere

CONTRIBUTORS

Mark Bolanowski, Senior Scientist, DynPort Vaccine Company LLC, 64 Thomas Johnson Drive, Frederick, MD 21702, E-mail: mbolanowski@csc. com Chapter 7.1: Pharmacological Immunogenicity and Adverse Responses to Vaccines Peter J. Bugelski, PhD, Senior Research Fellow and Head of Experimental Pathology,Toxicology and Investigational Pharmacology, Centocor Research and Development, Inc., 145 King of Prussia Road, Radnor, PA 19087, E-mail: [email protected] Chapter 4.2: Application of Flow Cytometry in Drug Development Chapter 10.1: Alternative Animal Models for Immunomodulation and Immunotoxicity Florence G. Burleson, PhD, BRT-Burleson Research Technologies, Inc., 120 First Flight Lane, Morrisville, NC 27560, E-mail: fburleson@BRT-LABS. com Chapter 5.1: Animal Models of Host Resistance Gary R. Burleson, PhD, BRT-Burleson Research Technologies, Inc., 120 First Flight Lane, Morrisville, NC 27560, E-mail: gburleson@BRT-LABS. com Chapter 5.1: Animal Models of Host Resistance Leigh Ann Burns-Naas, PhD DABT, Drug Safety Research and Development, Pfizer Global Research and Development, San Diego, CA 92064, E-mail: [email protected] Chapter 9.1: Developmental Immunotoxicity in Rodents xi

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CONTRIBUTORS

Eberhard Buse, Prof. Dr., Director of Pathology, Covance Laboratories GmbH, Kesselfeld 29, 48163 Muenster, Germany, E-mail: eberhard.buse@ covance.com Chapter 9.2: Developmental Immunotoxicity in Non-Human Primates Jeanine L. Bussiere, PhD, Executive Director of Toxicology, Department of Toxicolog,y MS/29-2-A, Amgen Incorporation, One Amgen Center Drive, Thousand Oaks, CA 91320-1799, E-mail: [email protected] Chapter 6.1: Differentiation Between Desired Immunomodulation and Potential Immunotoxicity Chapter 6.2: Relevant Immune Tests across Different Species and Surrogate Models Renold J. Capocasale, Research Scientist, Biologics Discovery and Development Sciences, Centocor, Inc., 145 King of Prussia Road, Radnor, PA 19087, E-mail: [email protected] Chapter 4.2: Application of Flow Cytometry in Drug Development Jack H. Dean, PhD, ScD, Diplomat ABT, Fellow ATS, Research Professor, Departments of Pharmacology & Toxicology, University of Arizona, 10331 N. Wild Creek Drive, Tucson, AZ 85742, E-mail: [email protected] Chapter: Introduction to Immunotoxicology Jacques Descotes, MD, PharmD, PhD, Fellow ATS, Poison Center and Pharmacovigilance Unit, Lyon University Hospitals, 162 Avenue Lacassagne, 69424 Lyon cedex 03, France, E-mail: [email protected] Chapter 11: Bridging Immunotoxicology to Clinical Drug Development Rodney R. Dietert, PhD, Professor of Immunotoxicology, Department of Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, E-mail: [email protected] Chapter 9.1: Developmental Immunotoxicity in Rodents Ellen Evans, DVM, PhD, Diplomate ACVP, Senior Director, Clinical Pathology, Immunotoxicology and Comparative Medicine, Schering Plough Research Institute, P.O. Box 32, Lafayette, NJ 07848, E-mail: ellen.evans@ spcorp.com Chapter 2.1: Clinical Pathology as Crucial Insight into Immunotoxicity Testing Elizabeth R. Gore, MA, 1136 Ashbridge Road, West Chester, PA 19380, E-mail: [email protected] Chapter 4.1: Functional Cellular Responses and Cytokine Profiles Ian Gourley, MD, MRCP, Senior Director, Early Development and Clinical Pharmacology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, E-mail: [email protected] Chapter 11: Bridging Immunotoxicology to Clinical Drug Development

CONTRIBUTORS

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Patrick J. Haley, DVM, PhD, Executive Director, Toxicology, Incyte Corporation, Experimental Sation, Rt 141 & Henry Clay Road, Building 400, Wilmington. DE 19880, E-mail: [email protected] Chapter 2.2: Histomorphology of the Immune System: A Basic Step in Assessing Immunotoxicity Mary Kate Hart, Director of Nonclinical Research, DynPort Vaccine Company LLC, 64 Thomas Johnson Drive, Frederick, MD 21702, E-mail: [email protected] Chapter 7.1: Pharmacological Immunogenicity and Adverse Responses to Vaccines Kenneth L. Hastings, DrPH, DABT, Associate Vice-President for Regulatory Policy, Sanofi-Aventis, 4520 East West Highway, Suite 210, Bethesda, MD 20814, E-mail: [email protected] Chapter 1: Current Regulatory Expectations for Immunotoxicology Evaluation of Pharmaceuticals Danuta J. Herzyk, PhD, Senior Scientific Director of Compound Management, Department of Safety Assessment, Merck Research Laboratories, 770 Sumneytown Pike, Mail stop: WP 45-233, West Point, PA 19486-0004, E-mail: [email protected] Chapter 5.2: Approaches to Evaluation of Autoimmunity Robert V. House, PhD, President and CSO, DynPort Vaccine Company LLC, 64 Thomas Johnson Drive, Frederick, MD 21702, E-mail: rhouse2@csc. com Chapter 7.1: Pharmacological Immunogenicity and Adverse Responses to Vaccines Laurie Iciek, PhD, Principal Toxicologist, Translational Sciences, MedImmune, 1 MedImmune Way, Gaithersburg, MD 20878, Email: [email protected] Chapter 3.2: Evaluation of Drug Effects on Immune Cell Phenotypes Catherine V. Kaplanski, DVM, PhD, Associate Director, Clinical Pathology and Immunology, Department of Safety Assessment, Merck Research Laboratories, 770 Sumneytown Pike, WP 45-321, West Point, PA 19486-0004, E-mail: [email protected] Chapter 7.2: Immunotoxicological Concerns for Vaccines and Adjuvants Raj Krishnaraj, DVM, MSc, PhD, Study Director, Toxicology/Pathology Scientific Services—Madison, Covance Laboratories Inc., 3301 Kinsman Blvd., Madison, WI 53704-2523, E-mail: [email protected] Chapter 2.4: Case Study in Immunotoxicology Practice: Immune-Mediated Hemolytic Anemia in Dogs

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CONTRIBUTORS

PD Harald Kropshoffer, PhD, Head of Immunosafety of Biotherapeutics, F. Hoffmann La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland, E-mail: [email protected] Chapter 10.3: T-Cell Epitopes and Minimization of Immunogenicity Jose Lebron, PhD, Associate Director, Biologics Safety Assessment, Merck Research Laboratories, WP45-325, West Point, PA 19486-0004, E-mail: jose_ [email protected] Chapter 7.2: Immunotoxicological Concerns for Vaccines and Adjuvants Brian Ledwith, PhD, MBA, Senior Director, Safety Assessment, Merck Research Laboratories, WP 45-215, West Point PA 19486-0004, E-mail: [email protected] Chapter 7.2: Immunotoxicological Concerns for Vaccines and Adjuvants Nianyu Li, PhD, Scientist, Comparative Biology and Safety Sciences, Amgen. Inc., 1201 Amgen Court West, E-mail: [email protected], Seattle, WA 98119 Chapter 4.2: Application of Flow Cytometry in Drug Development Curtis C. Maier, PhD, Head of Immunologic Toxicology, Department of Safety Assessment, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, UE0360, King of Prussia, PA 19406, E-mail: [email protected] Chapter 8.2: Nonclinical Models to Assess Respiratory Hypersensitivity Potential Pauline L. Martin, PhD, Associate Director, Toxicology, Centocor Research and Development, Inc, 145 King of Prussia Road, Radnor, PA19087, E-mail: [email protected] Chapter 9.2: Developmental Immunotoxicity in Non-Human Primates Barbara Mounho, PhD, DABT, Principal Scientist, Toxicology, Comparative Biology and Safety Sciences, Amgen, Inc., One Amgen Center Drive, Mail Stop MS-29-2-A, Thousand Oaks, CA 91320, E-mail: bmounho@amgen. com Chapter 6.1: Differentiation Between Desired Immunomodulation and Potential Immunotoxicity Chapter 6.3: Anti-Drug Antibody Responses in Preclinical Studies and Their Implications Kazuichi Nakamura, Shionogi & Co., Ltd., Developmental Research Laboratories, 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan, E-mail: kazuichi. [email protected] Chapter 2.3: Need for Specialized Immunotoxicity Tests Padma Narayanan, Padma Narayanan, DVM, PhD, Director, Comparative Biology and Safety Sciences, Amgen, Inc, 1201 Amgen Court West, Seattle, WA 98119, E-mail: [email protected] Chapter 4.2: Application of Flow Cytometry in Drug Development

CONTRIBUTORS

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Joseph R. Piccotti, PhD, Fellow, Immunotoxicology, Drug Safety and Metabolism, Schering-Plough Research Institute, 556 Morris Avenue, Summit, NJ 07901, Email: [email protected] Chaper 3.1.1: T-Cell Dependent Antibody Response Tests Raymond Pieters, PhD, Associate Professor, Group Leader Immunotoxicology, Institute for Risk Assessment Sciences (IRAS), Utrecht University, PO Box 80.177, 3508 TD Utrecht, Netherlands, E-mail: [email protected] Chapter 8.1: Testing for Drug Hypersensitivity Lisa Plitnick, PhD, Research Fellow, Head, Biologics Release Testing and Immunotoxicology, Department of Safety Assessment, Merck Research Laboratories, 770 Sumneytown Pike, WP 45-318, West Point, PA 19486-0004, E-mail: lisa_plitnick2merck.com Chapter 3.1.2: Natural Killer Cell Assay and Other Innate Immunity Tests Karen Price, MSc, Manager, Department of Immunotoxicology, Drug Safety Evaluation, Bristol-Myers Squibb Company, 6000 Thompson Road, East Syracuse, New York 13057-5050, E-mail: [email protected] Chapter 3.1.3: Cellular Immune Response in Delayed Type Hypersensitivity Test Thomas Singer, DVM, Global Head of Non Clinical Safety, F. Hoffmann La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland, E-mail: [email protected] Chapter 10.3: T-Cell Epitopes and Minimization of Immunogenicity Daniel Wierda, PhD, Research Fellow, Division of Toxicology, Eli Lilly and Company, 2001 W Main Street, Greenfield, Indiana 46140, E-mail: dwierda@ lilly.com Chapter 10.2: Animal Models for Preclinical Comparative Immunogenicity Testing Jayanthi Wolf, PhD Research Fellow, Department of Safety Assessment, Merck Research Laboratories, 770 Sumneytown Pike, WP45-338, West Point, PA 19486-0004, E-mail: [email protected] Chapter 7.2: Immunotoxicological Concerns for Vaccines and Adjuvants

INTRODUCTION TO IMMUNOTOXICOLOGY Jack H. Dean University of Arizona

It is important to briefly describe the organization and importance of the immune system to set the stage for the more focused chapters that follow. It is equally important to explain how the interest in immunotoxicology developed and flourished among toxicologists within regulatory bodies and the pharmaceutical industry. The earliest interest in immunology among toxicologist started with the observation that certain environmental chemicals (e.g., dioxins, PCBs, some pesticides, etc.) appeared to target the immune system and alter its function (Vos, 1977). In parallel, the discipline of immunopharmacology caught the interest of many in the 1970s, including this author, as the search for new chemical entities (NCEs) possessing immunological activity of therapeutic potential flourished. In many pharmaceutical companies, immunological active NCEs and cytokines became a development target (see reviews of Talmadge and Dean, 1994) for the treatment of cancer, viral diseases, and immune deficiencies. During the 1980s, toxicologists in industry were confronted for the first time with the safety assessment of protein therapeutics (e.g., monoclonal antibodies, IFNs, lymphokins, cytokines, etc.) and NCEs with novel immune activity. Consequently, most of the early scientists working in the developing discipline of immunotoxicology got their training in either basic immunology or immunopharmacology. Recognizing the importance of this topic, the Gordon Research Conference on Drug Safety in the summer of 1978 devoted 2 days to the topic of Immunotoxicology which represented one of the first symposia in this area. Shortly thereafter, I organized the first Workshop on Methods and Approaches for Assessing Immunotoxicology in Williamsburg, VA (Dean, 1979), which was attended by 50 scientists and physicians. The Williamsburg meeting was immexvii

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diately followed by an NIH Consensus Meeting at Research Triangle Park, NC, with the objective of defining specific research needs for this newly emerging field. The discipline of immunotoxicology soon captured the imagination of the International Program on Chemical Safety (IPCS) of the World Health Organization (WHO) and the first international meeting was held in 1984 during which a formal definition was developed (Berlin et al., 1987). By the mid- to late 1980s, most major pharmaceutical companies began recruiting toxicologists or immunologists trained in immunotoxicology and established small groups to monitor for inadvertent immune side effects of new drug candidates. In 1998 we reported (Dean et al., 1998) that there were 12 pharmaceutical companies with groups involved in the safety assessment of NCEs for immunotoxic potential. During this period there were also efforts at standardizing and validating these methods and the tiered approaches in rodents (Luster et al., 1988), as well as multiple ring studies. The field of immunotoxicology has matured considerably since this early period when most of the focus was on methods development and validation in rodents. This maturation has been successfully natured by the Immunotoxicology Specialty Section, charted by the Society of Toxicology (United States) in 1985, and the Immunotoxicology Technical Committee of the Health and Environmental Scientists Institute (HESI) of ILSI established in 1992.

THE IMMUNE SYSTEM: ORGANIZATION AND FUNCTION It is now well established that the immune system is a complex multicellular organ system consisting of granulocytes, macrophages, lymphocytes, and dendritic cells with various functions and unique phenotypic characteristics, which can produce various soluble mediators (for detail, see Dean et al., 2007). The cells that constitute the immune system are of hemopoietic origin and in adults, are found in the peripheral blood, lymphatic fluid, and organized lymphoid tissues, including bone marrow, spleen, thymus, lymph nodes, tonsils, and mucosa-associated lymphoid tissue. Since the immune system is in a constant state of self-renewal involving cell proliferation, differentiation, activation, and maturation, it is vulnerable to agents that disrupt any of these cellular processes. The immune system appears to exist principally to defend the host against invasion by infectious and opportunistic microorganisms and spontaneously arising neoplasia. This network of cells and soluble mediators that contribute to host defense are highly regulated and interdependent, and must not only discriminate self from nonself, but also be able to react to nonself with a variety of defensive responses (Paul, 1999). In addition, the immune system can occasionally develop a response to a chemical or drug or their reactive metabolite that might bind to or alter a host protein, resulting in an allergic or autoimmune response. It is now well established that the immune system of experimental animals, although exhibiting some obvious differences


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from that of humans, is still sufficiently similar that data obtained from lower species are instructive of a potential human response (Haley, 2003) and these data can be used for risk assessment. Nonspecific and Acquired Host Defense The host defense functions of the immune system are provided by two major mechanisms: a nonspecific or innate mechanism that does not require prior sensitization with the inducing agent to elicit a response, and a specific or acquired mechanism directed against the eliciting agent to which the individual has been previously sensitized (immunological memory). Penetration of the skin or mucosal defense barriers by an invading microorganism results in nonspecific reactions by phagocytic cells (granulocytes and macrophages [MØ]). If the microorganism is not controlled by these cells and persists, specific responses involving antibody production and the induction of effector lymphocytes can follow. Effector lymphocytes respond through cytokine mediators to seek out and destroy the invading microorganism. Both antibodyproducing lymphocyte responses (B cell mediated) and thymus-dependent lymphocyte responses (T cell mediated) are triggered by the presentation of foreign antigen to appropriate lymphocytes by dendritic cells, macrophages, or other antigen-presenting cells (APCs). Following antigen-induced activation, B cells proliferate and differentiate into plasma cells (PCs), with the support of T-helper 2 (Th2) cells, which produce large quantities of antigenspecific immunoglobulins (antibodies). Antibodies enter the plasma, where they bind the foreign material and neutralize, lyse, or facilitate phagocytosis of the agent. Antibody–antigen interactions are expanded by actions of the complement (C′) system and other inflammatory mediators (e.g., prostaglandins and leukotrienes). With the support of Th1 cells, another population of T cells, referred to as cytotoxic T cells, proliferate and recognize viral infected cells that they can destroy before viral replication is complete. The immune responses that characterize acquired host defenses represent a series of complex events that occur following the introduction of foreign antigenic material into the body. The two major types of specific immune response are (1) cell-mediated immunity (CMI), which is initiated by specifically sensitized T cells and is generally associated with delayed type hypersensitivity (DTH), rejection of tumors or foreign grafts, and resistance to viral agents; and (2) humoral immunity (HI), which involves the production of antibodies by PCs following sensitization to a specific antigen and is important in resistance to extracellular pathogens. Origin and Development of the Cellular Constituents The cellular elements of the immune system arise from pluripotent stem cells, a unique group of unspecialized cells that have self-renewal capacity. These cells are found in the blood islands of the embryonic yolk sac and in the liver of the

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fetus during fetal development, and later in the bone marrow. The pluripotent stem cell differentiates along several pathways, giving rise to erythrocytes, myeloid series cells (i.e., MØ and PMNs), megakaryocytes (platelets), or lymphocytes. Maturation generally occurs within the bone marrow, although lymphoid progenitor cells are disseminated through the blood and lymphatic vessels to the primary lymphoid organs where they undergo further differentiation under the influence of the humoral microenvironment of these organs. The primary lymphoid organs include the thymus in all vertebrates and the bursa of Fabricius (in birds) or bursa-equivalent tissue in other vertebrates; the latter are believed to be bone marrow and gut-associated lymphoid tissue in mammals. The primary lymphoid organs are lymphoepithelial in origin and are derived from ectoendodermal junctional tissue in association with gut epithelium. During the beginning of the second half of embryogenesis (days 12 to 13 in the mouse), stem cells migrate into the epithelia of the thymus and bursa-equivalent areas, where they differentiate independently of antigenic stimulation into immunocompetent T and B cells, respectively. The thymus is an organization of lymphoid tissue located in the chest, above the heart. Thymus development occurs during the sixth week of embryological development in humans and day 9 of gestation in the mouse. The thymus reaches its maximum size at birth or shortly thereafter in most mammals and then begins a slow involution that is complete between the ages of 5 and 15 years in humans. Histologically, the thymus consists of multiple lobules, each lobule containing a cortex (outer) and a medulla (inner). Lymphocyte precursors from bone marrow proliferate in the cortex of the lobules and then migrate to the medulla. In the medulla, they further differentiate, under the influence of thymic epithelium and hormonal factors, into mature T lymphocytes before emigrating to secondary lymphoid tissues. The neonatal/postnatal thymus has a significant endocrine function supported by nonlymphoid thymic epithelium cells. These cells produce a family of thymic hormones essential for T lymphocyte maturation and differentiation. The mammalian bursa-equivalent tissue, where B cells are formed, is believed to be the fetal liver, neonatal spleen, gut-associated lymphoid tissue, and adult bone marrow, depending on age. Mature B lymphocytes migrate from the bursa-equivalent tissue to populate the B-dependent areas of the secondary lymphoid tissues. Neonatal removal or chemical destruction of primary lymphoid organs prior to the maturation of lymphocytes into T or B cells or prior to their population of secondary peripheral lymphoid tissue dramatically depresses the immunological capacity of the host. However, removal of these same organs in adults has little influence on immunological capacity. In addition, neonatal thymectomy in mammals dramatically impairs the development of CMI but does not generally influence the generation of immunoglobulin-producing cells involved in antibody-mediated immunity. In contrast to the removal of primary lymphoid organs, removal of secondary lymphoid organs does not inhibit the development of immune competence, although it may suppress the magnitude or alter the tissue location of the responsive cells.


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CELLS AND CYTOKINES: FUNCTION AND RESPONSES Humoral Immunity The principal function of B lymphocytes is production of specific antibody in response to antigenic stimulation. B cells recognize antigen via a specific receptor, comprised of membrane immunoglobulins associated with accessory proteins either directly or in the presence of an APC. Binding of the receptor with its cognate antigen triggers transmembrane signaling, leading to activation of the B cell. The antigen is subsequently internalized, where it is processed and associated with class II major histocompatibility complex (MHC) molecules. Antigen-derived peptides, along with MHC proteins, are then transferred to the cell surface, where they are free to interact with helper T cells. Within 3 to 5 days following antigen exposure, this T/B cell interaction results in the B lymphocytes differentiating into blast cells, then into immature PC, and finally into antibody-secreting PC. The establishment of humoral immunity is characterized by an early rise in IgM antibody titer in the serum, followed several days later by the appearance of IgG antibodies. During this differentiation process, some of the lymphocytes develop into long-lived or memory cells (sensitized but non-blast cells), so subsequent antigen encounters result in an enhanced (secondary) response. This secondary response is characterized by a shorter latency for antibody appearance, as well as an increased affinity and synthesis of IgG antibodies. Antibody molecules react with specific antigenic determinants (epitopes) on their target, facilitating its removal (e.g., lysis or enhanced phagocytosis). Based on chemical structure and biological function, the five classes of antibody molecules in mammals are IgM, IgG, IgA, IgD, and IgE. Antibodies operate via several mechanisms to protect the host from infectious agents. Some of these mechanisms include virus neutralization, in which antibodies bind and prevent virus particles from infecting target cells; opsonization, the process by which antibody molecules react with infectious agents and thus enhance their phagocytosis; and antibody-dependent cellular cytotoxicity, the process whereby antibody-coated target cells are killed by Fc receptor-bearing lymphocytes. Of increasing interest is the concept that naturally occurring IgM antibodies (that is, antibodies that are secreted in the absence of antigen stimulation) may play an important role in immune surveillance against neoplasia (Vollmers and Brandlein, 2005).

Cell-Mediated Immunity Cell-mediated immunity (CMI), often referred to as T cell-mediated immunity, refers broadly to any host resistance mechanism in which cellular elements play a direct role and which is part of the acquired arm of immunity. This is in comparison to humoral immunity, in which there are certainly cellular interactions but the final host resistance products are soluble factors such as

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antibody. A number of host defenses are mediated directly by cells including MØ-mediated cytolysis, antibody-dependent cellular cytotoxicity, and natural killer (NK) cell cytotoxicity although cytotoxic T lymphocytes (CTLs) usually predominate, particularly in the destruction of virus-infected cells. Functions associated with CMI are commonly considered the province of T lymphocytes, although immune cells (e.g., B cells and MØ), as well as nonimmune cells (e. g., fibroblasts and dendritic cells) contribute to the development of CMI. As the primary effector cell in CMI, the T cell represents one of the most complex and multifunctional immune cells. Antigens that generally elicit CMI include tissue-associated antigens, chemicals and drugs that covalently bind to autologous proteins, and antigenic determinants on persistent intracellular microorganisms. The route of exposure also plays a major role in the type of response generated; for example, sheep erythrocytes elicit antibody production (but not CMI) when injected intravenously in humans, but elicit both when injected intracutaneously. The induction of CMI proceeds when small lymphocytes differentiate into large pyroninophilic cells and ultimately divide, giving rise to cells responsible for effector function, as well as immunological memory. In contrast to humoral immunity, which is more effective against extracellular pathogens, CMI helps protect against intracellular bacteria, viruses and neoplasia, and is responsible for graft rejection. T cells can differentiate into populations responsible for either regulatory or effector function. For example, regulatory and inducer T cell functions are provided by CD3/CD4+ T-helper cells. The T-helper function facilitates antibody responses by B cells and assists in other T cell responses. For most antigens, B cells require assistance from T cells for differentiation into PCs. T-helper cells are integral in the B cell response by participating in two distinct mechanisms: (1) major-histocompatibility locus-restricted B and T cell collaborations, and (2) cytokine-mediated differentiation. Helper function is a result of interactions between surface molecules on T-helper cells and B cells, as well as the production and secretion of immunoregulatory cytokines. Effector functions take the form of cytotoxic activity (CD3/CD8 phenotype), manifested by cytotoxic T lymphocytes (CTLs). These cells are able to specifically lyse target cells via the release of various bioactive molecules. Another effector function is the ability of T cells to mediate suppressor activity for both T and B cell responses. Suppressor activity is also mediated by cells bearing the CD3/CD8 phenotype, although recent studies suggest that this activity may be the result, at least in part, of differential cytokine production by this population. This responsibility for both helper and suppressor activities indicates the crucial roles of T cells in normal immune function. T-Helper 1 and T-Helper 2 Cells An important conceptual breakthrough in immunology was the finding that two major populations of T-helper cells exist that have different, sometimes opposing functions. Mosmann et al. (1986) first established the concept by


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demonstrating that cloned murine T cells exhibited differential patterns of cytokine production. One population designated T-helper 1 cells (Th1), was found to produce interleukin-2 (IL-2), IFN-gamma, and lymphotoxin. The second major population (designated Th2 cells) produces IL-4, IL-5, IL-10, and IL-13. Both populations of T cells produce IL-3, granulocyte–macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF). Later, a third population, Th0, was described and was found to exhibit an intermediate pattern of cytokine production. These cells are less well defined but may be an early precursor of Th1 and Th2, or, alternatively, they may represent an intermediate stage in development of the other two populations. Although there were initial doubts that human T cells followed this paradigm, it is now known that a similar paradigm exists for human T cells (Romagnani, 1995). The major differences appear to be in the profile of cytokine production, cytokine response (e.g., human Th1 and Th2 proliferate in response to IL-4 while only Th2 cells proliferate in the presence of IL-4 in rodents), and cytolytic potential. Despite these differences, the human and rodent systems are similar enough to make experimental rodent models meaningful for understanding the human immune response. Recent studies suggest that Th1 and Th2 cells may not necessarily represent distinct lineages descending from a common precursor but rather may be seen as points in a continuum; for example, development of each population is influenced by type, location, and concentration of eliciting antigen. More important may be the cytokine milieu. For example, the cytokines IL-12 and IFN-gamma-inducing factor (from MØ) and IFN-gamma (from NK cells) drive the development of Th1 cells, whereas IL-4 (from the ill-defined “T-accessory” cell, mast cells, or other sources) drives the development of Th2 cells (O’Garra, 1998). The Th1/Th2 paradigm is important for immunotoxicology in that certain immunopathologies have been associated with the predominance of one helper cell type over another, particularly in human disease states; for example, Th1 polarization has been associated with organ-specific autoimmune diseases such as multiple sclerosis and Hashimoto’s thyroiditis, whereas systemic autoimmune conditions, such as rheumatoid arthritis and Sjögren’s syndrome, lack a clear T cell polarization (Del Prete, 1998). On the other hand, strong Th2-type responses appear to result in many hypersensitivity disorders, including asthma. It is possible that assignation of Th1/Th2 patterns may eventually become much more important when designing and performing mechanistic immunotoxicology studies (Selgrade et al., 1997). Bone Marrow The bone marrow functions as a primary lymphoid organ and serves as the principal source of uncommitted stem cells, including both myeloid and erythroid precursor cells. The bone marrow architecture is highly organized and complex, consisting of a matrix or cellular stroma derived from local mesenchymal cells, as well as cells of hemopoietic parenchyma that are descendants

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of circulating stem cells. The bone marrow matrix consists of reticular– dendritic cells, fibroblast-like cells, and immune cells within the bone marrow microenvironment. Bone marrow stem and stromal cells have been shown to possess a significant capacity for metabolic activation because they contain cytochromes of the P450 and P448 families, as well as peroxidases, and can generate reactive oxygen species, which could also activate xenobiotics via oxidant-dependent mechanisms (Twerdok and Trush, 1988). This metabolic activity is thought to contribute to the sensitivity of bone marrow cells to toxicants such as benzene, which is extensively metabolized within the bone marrow. In light of the cell proliferation and differentiation occurring within the marrow, this tissue is also one of the most sensitive tissues to drugs or chemicals affecting cell division. Dose-limiting bone marrow toxicities are a significant end point with the use of anti-proliferative cancer drugs, including cytotoxic agents, antifolates, AIDS therapeutics, as well as certain cytokines (Greenberger, 1991; Rosenthal and Kowolenko, 1994). Mononuclear Phagocytic System Whether an antigen induces CMI, antibody production, or both depends on the physical and chemical characteristics of the antigen, the mode of presentation of the antigen to lymphocytes, the pattern of antigen distribution within lymphoid tissue, and the molecular configuration of the antigen. In many instances, antigen is initially phagocytized and processed by APCs. Antigenic peptides are transported to the cell surface, where they are presented to lymphocytes through cell surface interactions via specific surface proteins (e.g., class II MHC antigens). Cells of the MØ/monocyte lineage are found in many tissues, including liver (Kupffer cells), lung (alveolar and interstitial MØ), skin (Langerhans cells), and brain (astrocytes and microglia). These cells, because of their proximity to portals of entry, are often the first cells to interact with drugs, chemicals, and physical agents entering the organism via air, food, or blood. The capacity of cells of the mononuclear phagocytic system to carry out these functions is associated with their state of activation, which in turn is a function of both endogenous (e.g., IFN-gamma) and exogenous (e.g., bacterial lipopolysaccharide [LPS]) stimuli. Natural Killer Cells Natural Killer (NK) cells are a population of non-B, non-T lymphocytes that exhibit cytotoxicity toward a variety of target cells, including tumor cells and virally infected cells. NK cells express a unique panel of cell surface markers (e.g., asialo GM1) and are morphologically distinct, being larger than other lymphocytes. In addition, they contain numerous granules, leading to their designation as large granular lymphocytes (LGLs) (Smyth et al., 2005). Unlike CMI, NK cell-mediated cellular cytotoxicity is MHC unrestricted and does not


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require prior exposure to the target. NK cells have been seen principally as mediators of so-called “immune surveillance,” resulting in the concept of a constant removal of spontaneously arising neoplastic cells (Pross and Lotzová, 1993). In fact, the standard methodology for assessing NK cell function relies on the in vitro lyses of tumor target cells; however, NK cells are more likely to play a role in resisting the progression and metastatic spread of tumors once they develop, rather than preventing initiation (Herberman, 2001). NK cells respond to, and produce, key immunoregulatory cytokines and thus play an important role in the normal immune response. In fact, studies of individuals with NK cell deficiency states, most of which are associated with single gene mutations, have helped identify a role for NK cells in defense against human infectious disease. A resounding theme of NK cell deficiencies is susceptibility to herpesviruses (Orange, 2002). Cytokines and Chemokines Cytokines are glycoproteins that are generally produced in response to cellular activation. Most cytokines studied to date have multiple and overlapping actions and they frequently function via cascading mechanisms referred to as the cytokine network, interacting with each other both synergistically and antagonistically. Two important features of cytokines are that they usually act at a local level and they are rapidly cleared from the circulation. This combination of features helps ensure that cytokines remain compartmentalized, undoubtedly an important consideration given the potent bioactivity of these molecules (Dinarello, 1997). Cytokines serve as immune system mediators and regulators. They are produced proportionally by T-helper lymphocytes but are not exclusive to the immune system; in fact, some cytokines are phylogenetically ancient and highly conserved. Furthermore, both IL-1 and TNF are intrinsically involved in apoptosis and cellular proliferation, both fundamental biological processes. Thus, cytokines should be recognized for their role as conveyers of bio-information, rather than as simple effector molecules involved in a single physiological process such as immunity and host resistance. For convenience, cytokines may be grouped into several classes (see Dean et al., 2007). These classifications are necessarily arbitrary due to the overlapping activity of these molecules. Another related group of molecules are the chemokines. Chemokines are small peptide molecules that, like cytokines, were originally associated with the immune system, but which now are recognized as being produced by almost all cells of the body and involved in a multitude of biological functions. Chemokines play many roles, including modulation of the Th1/Th2 balance associated with autoimmunity and hypersensitivity (Montovani et al., 1998), as mediators of allergic inflammation (Bacon and Schall, 1996), and modulation of the function of leukocytes in disease states such as rheumatoid arthritis and asthma.

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CONCLUSIONS AND FUTURE DIRECTION When you finish reading this book, you should appreciate how the discipline of immunotoxicology has evolved and grown in importance in multiple aspects of pharmaceutical development and toxicology since its inception in the mid-1970s. The field has progressed from the identification of chemicals that may cause immunosuppression, modulation or allergic contact dermatitis to the validation of sensitive and quantitative assays that serve as biomarkers of immune system alterations in both animals and humans. In recent years, academic, industrial, and government scientists have taken a more mechanistic approach to define how environmental chemical and therapeutic agents might alter immune function at a cellular and molecular level. This understanding has contributed to our understanding. Basic mechanism involved in the induction of allergy and contact dermatitis were defined by such studies. Immunotoxicity data derived from experimental and human immunosuppression and hypersensitivity studies now play an increasing role in establishing health standards and defining permissible levels of toxic chemical or NCE exposure in humans. I believe we still need better correlation between findings in animals with known immune modifying drugs and clinical studies with these agents to better define the predictive value of our method when applied to human populations that might have been occupationally or environmentally exposed to an immunotoxicant. We also need to be diligent in evaluating any NCE or protein therapeutic that interacts with receptors on cells of the immune system as was recently demonstrated by (Suntharalingam et al., 2007). In this report, six healthy young male volunteers at a contract research organization were enrolled in the first Phase 1 clinical trial of TGN1412, a novel super agonist anti-CD28 monoclonal antibody that directly stimulates T cells. Within 90 minutes after receiving a single intravenous dose of the drug, all six volunteers had a systemic inflammatory response characterized by a rapid induction of proinflammatory cytokines and accompanied by headache, myalgias, nausea, diarrhea, erythema, vasodilatation, and hypotension. Within 12 to 16 hours after infusion, they became critically ill, with pulmonary infiltrates and lung injury, renal failure, and disseminated intravascular coagulation. Severe and unexpected depletion of lymphocytes and monocytes occurred within 24 hours after infusion. All six patients were transferred to an intensive care unit, where they received intensive cardiopulmonary support (including dialysis), highdose methylprednisolone, and an anti-interleukin-2 receptor antagonist antibody. Prolonged cardiovascular shock and acute respiratory distress syndrome developed in two patients, who required intensive organ support for 8 and 16 days. Despite evidence of the multiple cytokine-release syndrome (“cytokine storm”), all six patients survived. This Phase 1 study demonstrates the kind of difficulty that can occur when the immune system is significantly modulated by novel therapeutics and further supports the need for continued vigilance for immunotoxic potential of such agents. The recent loosening of the ICH regulatory guidelines (Weaver et al., 2005) in favor of a case-by-case approach


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to immunotoxicity assessment should not allow us to become complacent about the importance or potential danger of modulating the immune system by novel drug candidates, recombinant protein therapeutic, or gene therapy agents.

REFERENCES Bacon KB, Schall TJ. Chemokines as mediators of allergic inflammation. Int Arch Allergy Immunol 1996;109:97–109. Berlin A, Dean J, Draper M, Smith EMB, Spreafico F. Synopsis, conclusions, and recommendations. In: Immunotoxicology, edited by Berlin A, Dean J, Draper M, Smith EMB, Spreafico F, pp. xi–xxvii. Dordrecht: Martinus Nijhoff, 1987. Dean JH. Drug & Chemical Toxicology, Special Issue on Immunotoxicology, edited by Dean JH, 1979 (nos. 1&2); 2:1–179. Dean JH, Hincks JR, Remandet B. Immunotoxicology assessment in the pharmaceutical industry. Toxicol Lett 1998;102/103:247–255. Dean JH, House RV, Luster MI. Immunotoxicology: effects of and response to drugs and chemicals. In: Principles and Methods of Toxicology, 5th eds., edited by A. Wallace Hayes, pp. 1761–1793. Philadelphia, PA: Taylor and Francis, 2007. Del Prete G. The concept of type-1 and type-2 helper T cells and their cytokines in humans. Int Rev Immunol 1998;16:427–455. Dinarello CA. Role of pro- and anti-inflammatory cytokines during inflammation: experimental and clinical findings. J Biol Reg Homeostat Agents 1997;11:91–103. Greenberger JS. Toxic effects on the hematopoietic microenvironment. Exp Hematol 1991;19:1101–1109. Haley PJ. Species differences in the structure and function of the immune system. Toxicology 2003;188:49–71. Herberman RB. Immunotherapy. In: Clinical Oncology, edited by Lenhard RE, Jr, Osteen RT, Gansler T, pp. 215–223. Atlanta, GA: American Cancer Society, 2001. Luster MI, Munson AE, Thomas PT, Holsapple MP, Fenters JD, White KL, Lauer LD, Germolec DR, Rosenthal GJ, Dean JH. Development of a testing battery to assess chemical-induced immunotoxicity. National Toxicology Programs Criteria for Immunotoxicity evaluation in mice. Fundam Appl Toxicol 1988;10:2–19. Montovani A, Allavena P, Vecchi A, Sozzani S. Chemokines and chemokine receptors during activation and deactivation of monocytic and dendritic cells and in amplifications of Th1 versus Th2 responses. Int J Clin Lab Res 1998;28:77–82. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348–2357. O’Garra A. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 1998;8:275–283. Orange JS. Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 2002;4:1545. Paul WE. Fundamental Immunology, 4th ed. Philadelphia, PA: Lippincott, 1999.

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Pross HF, Lotzová E. Role of natural killer cells in cancer. Nat Immunol 1993;12: 279–292. Romagnani S. Biology of human Th1 and Th2 cells. J Clin Immunol 1995;15:121–129. Rosenthal GJ, Kowolenko M. Immunotoxicological manifestations of AIDS therapeutics. In: Immunotoxicology and Immunopharmacology, 2nd eds., edited by Dean JH, Luster MI, Munson AE, Kimber I, pp. 249–265. New York, NY: Raven Press, 1994. Selgrade MK, Lawrence DA, Ullrich SE, Gilmour MI, Schuyler MR, Kimber I. Modulation of T-helper cell populations: potential mechanisms of respiratory hypersensitivity and immune suppression. Toxicol Appl Pharmacol 1997;145:218–229. Smyth MJ, Cretney E, Kelly JM, Westwood JA, Street SE, Yagita H, Takeda K, van Dommelen SL, Degli-Esposti MA, Hayakawa Y. Activation of NK cell cytotoxicity. Mol Immunol 2005;42(4):501–510. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, Panoskaltsis N. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. New Engl J Med 2007;355:1018–1028. Talmadge JE, Dean JH. Immunopharmacology of recombinant cytokines. In: Immunotoxicology and Immunopharmacology, 2nd eds., edited by Dean JH, Luster MI, Munson AE, Kimber I, pp. 221–247. New York, NY: Raven Press, 1994. Twerdok LE, Trush MA. Neutrophil derived oxidants as mediators of chemical activation in bone marrow. Chem Biol Int 1988;65:261–273. Vollmers HP, Brandlein S. The ‘early birds’: natural IgM antibodies and immune surveillance. Histol Histopathol 2005;20(3):927–937. Vos JG. Immune suppression as related to toxicology. CRC Crit Rev Toxicol 1977; 5:67–101. Weaver JL, Tsutsui N, Hisada S, Vidal J-M, Spanhaak S, Sawada J-I, Hastings KL, van der Laan JW, Van Loveren H, Kawabata T, Sims J, Durham SK, Fueki O, Matula TI, Kusunoki H, Ulrich P, Nakamura K. Meeting report: development of the ICH Guidelines for immunotoxicology evaluation of pharmaceuticals using a survey of industry practices. J Immunotoxicol 2005;2:171–180.

PART I CURRENT REGULATORY EXPECTATIONS FOR IMMUNOTOXICITY EVALUATION OF PHARMACEUTICALS

1 CURRENT REGULATORY EXPECTATIONS FOR IMMUNOTOXICOLOGY EVALUATION OF PHARMACEUTICALS Kenneth L. Hastings

Immunotoxicology is generally perceived to be a relatively new area of concern in the development of human pharmaceuticals, but in fact adverse effects on immune function have been a significant problem for decades. For example, penicillin, arguably the most important pharmaceutical ever developed from a public health perspective, is also one of the most allergenic drugs. Penicillin is the most common cause of fatal drug-related anaphylaxis, which is a Type I immunopathy (Joint Task Force, 1998; Neugut et al., 2001). The problem is that anaphylaxis, as well as drug allergy in general, has not traditionally been considered a form of immunotoxicity. However, when all forms of drug-induced immune reactions are combined, immunotoxicity probably accounts for around 10% of total adverse drug reactions (Bala et al., 2005). These may manifest as increased susceptibility to infections and tumors, hemolytic anemias, systemic inflammatory reactions, and organ-specific autoimmune disease. It is the spectrum of immunotoxic drug reactions that has led to a misunderstanding of how important the subject really is. A related issue is the actual interpretation of the term immunotoxicity; this has traditionally been applied to drugs that impair immune function primarily by bone marrow and/or lymphatic system toxicity. It has become increasingly clear that drug allergy is likely to be more Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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important than direct immunotoxicity. Thus, ICH S8 defines immunotoxicity as both unintended immunosuppression and immunoenhancement (ICH Harmonised Tripartite Guideline, 2006). However, the entire linear paradigm of immunotoxicity, in which a drug either suppresses or enhances immune function, appears to be misleading. A more accurate term is adverse immunomodulation, which captures the concept that drugs may disrupt immune function with often unpredictable results (House and Hastings, 2004). Nevertheless, the regulatory approach to evaluating the potential immunotoxicity of drugs still relies on this linear concept, and is reflected in current guidance documents.

IMMUNOSUPPRESSION There are many methods for assessing the potential of drugs to produce immunosuppression. Most of these are adaptations of standard immunology methods, many of which are quite old. As a practical matter, the most important tools are those used to evaluate general toxicity, and rely on the skill of pathologists examining tissues from drug-treated animals for signs of immunotoxicity (Kuper et al., 2000). There is a commonly cited list of effects observable in standard nonclinical acute and repeat-dose toxicity studies which have proven to be sufficiently reliable in order to screen for “unintended immunosuppression” (USFDA, 2002). These include: (i) hematological changes; (ii) changes in immune system organ weights and/or histology; (iii) changes in serum immunoglobulin levels; (iv) increased incidence of infections; and (v) increased incidence of tumors. Of course, these signs are subject to many interpretations and should be considered carefully in evaluating potential relationship to immunosuppression. Hematological Changes In most nonclinical toxicology studies, the following clinical hematology parameters are assessed: packed cell volume (hematocrit), red blood cell counts, total hemoglobin levels, various red cell parameters (mean and variability in cell volume, mean hemoglobin, and mean hemoglobin concentration), white cell counts, absolute and relative white cell types (granulocytes, lymphocytes, monocytes, eosinophils, and basophils), and platelet counts (see Chapter 2.1). Each of these parameters could provide a signal that the drug being tested has an adverse effect on immune function. What should be kept in mind is that observed changes may not always have a simple relationship to effects such as bone marrow toxicity. Anemia is an excellent example of how complex interpretation of findings can be. Often, the first assumption made when treatment-related anemia is observed is that the drug being tested is a bone marrow toxin. Evaluating this possibility can be relatively straightforward. Examination of the bone marrow, usually by simple microscopic examination of “smears,” can give a clue that

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the basis of anemia is destruction of red cell precursors. Flow cytometry can also be used to obtain more accurate cell counts, although morphologic signs that could be useful are not obtained by this method. Other considerations would be whether reticulocyte counts have changed: a decrease (“right shift”) would be consistent with bone marrow toxicity, whereas an increase (“left shift”) would indicate some other reason for the observed anemia (such as a hemolytic process). Other clues would be white cell and platelet counts: these tend to decrease before observing anemia in bone marrow toxicity, and neutrophil counts are especially sensitive indicators. Of course, as is stated in ICH S8, it is important to consider the intended use of the drug. For example, traditional cytotoxic chemotherapeutic agents being developed to treat cancer often produce bone marrow toxicity, and anemia is a likely finding in nonclinical toxicity studies. However, even in this situation, it could be useful to determine the relative sensitivities of bone marrow progenitor cells. Erythrocyte and granulocyte colony-forming cell assays have been used to estimate safe doses for clinical trials in cancer patients, for instance. When anemia is observed in the absence of bone marrow toxicity, determination of cause can be especially difficult. Direct drug-induced hemolysis appears to be a relatively rare phenomenon, but in vitro assessment can be informative. Also, red cell morphology can be a clue: poikilocytosis is a likely finding where intravascular hemolysis is occurring. However, a much more difficult problem is immune-mediated hemolysis. First, this represents the “other end” of the traditional immunotoxicology continuum: the immune system has been adversely stimulated in some way. Where anemia is observed in nonclinical toxicology studies and bone marrow toxicity has been excluded as the cause, it may be useful to perform a direct Coombs’ test for antibodies bound to red cells. A positive finding likely indicates drug (hapten)-bound red cells which have induced an immune reaction; that is, a form of drug hypersensitivity rather than unintended immunosuppression (USFDA, 2002). An even more complex situation has been observed with recombinant proteins (biotherapeutic drugs, biopharmaceuticals). ICH S8 does not apply to these drugs, but increasingly adverse immune effects are being observed with biopharmaceuticals. Probably the best-known example involved recombinant erythropoietin (EPO), indicated for patients with anemia associated with cancer chemotherapy. For reasons that are not been completely understood, reformulated recombinant EPO, when administered to patients, was associated with pure red cell aplastic anemia. These patients developed neutralizing antibodies to EPO, resulting in ablation of both endogenous and recombinant molecule activity (Schellekens and Jiskoot, 2006). With respect to hematology, examples similar to anemia have been observed with both leukocytes and thrombocytes: the range of causes can vary from bone marrow toxicity to drug-induced antibody formation. An important consideration is that bone marrow toxicity probably is more often associated with exaggerated pharmacodynamics, whereas antibody-mediated cytopenia often is not. Unfortunately, as is clear from ICH S8, test methods for the latter effect

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are not readily available, and require imaginative problem solving. Another consideration is that leukocytosis can be an indicator of infection, and thus paradoxically associated with unintended immunosuppression. Changes in Immune System Organ Weights and Histology Immune organ weights are relatively insensitive indicators of immunotoxicity, but should not be ignored. Potent immunosuppressants can produce significant decreases in thymus, spleen, and lymph node weights. One consideration is that necropsy technique is an important cause of variability, resulting in statistically insignificant findings. Thus, individual animal findings may yield important signals. Another consideration in evaluating immune organ weights is that both increases and decreases may be signs of immunotoxicity. There are situations in which increased lymph node weights can be associated with immunosuppression, as this effect may be related to increased susceptibility to infections, especially viral. Thus, lymphadenopathy could represent a counterintuitive effect observed in nonclinical toxicology studies. It is even conceivable that lymph node weights could exhibit a dose-related bell-shaped curve, with mean decreases in the high-dose groups and increases, compared to controls, in lower-dose groups. Inflammation due to drug-induced autoimmunity should also be considered in evaluating immune organ weight increases. Often these changes are not given the consideration needed for proper evaluation of toxicology study findings. Histologic examination of immune system tissues obtained from animals in nonclinical toxicology studies is the most important single method for detection of immunotoxicity (see Chapter 2.2). This issue has been the subject of much debate, but as a practical matter, it is an obvious conclusion. The immune system should not be considered so significantly different from other organ systems that histology could not be considered the benchmark determination. Obviously there are potential functional effects of drugs that may not be detected by morphological examination, but as a practical consideration, these are likely to represent a minority. ICH S8 contains a list of tissues that should be specifically evaluated by histological examination for signs of immunotoxicity: thymus, spleen, lymph nodes draining the anatomical site of maximum drug exposure (assumed to be the route of administration), at least one lymph node distal to the site of maximum drug exposure, bone marrow, Peyer’s patch for drugs administered by the oral route, and bronchus- and nasal-associated lymphoid tissues for drugs administered by the inhalation or nasal route. For intravenously administered drugs, the spleen is considered to be the draining lymph node equivalent. Much has been made of the “enhanced histopathology” concept. In reality, this is simply a reminder to pay close attention to the histology of immune system tissues. ICH S8 recommends that a semiquantitative description of observed changes be used. This reflects the fact that lymphoid tissues demonstrate cellular dynamics indicative of potential functional changes. Once again,

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hypocellularity is not the only effect that can signal immunotoxicity. For example, drug-related increases in lymphoid germinal centers (usually taken to indicate “immune activation”) can indicate increased susceptibility to infections and/or hypersensitivity/autoimmunity. It is in this context that immunohistochemistry can be especially valuable: demonstrating increased expression of markers associated with inflammation could indicate potential adverse drug reactions not generally thought of as immunotoxicity. Changes in Serum Immunoglobulin Levels Basal immunoglobulin changes are not considered to be sensitive indicators of toxicity, but this conclusion is arguably an artifact of standard immunotoxicity studies conducted in rodents. These studies are usually 28-day repeat-dose toxicity studies, and basal immunoglobulin levels are unlikely to change significantly in this context. Experience suggests that chronic repeat-dose toxicity studies (of at least 3-month duration) are needed to detect the potential of xenobiotics to produce alterations in basal immunoglobulin concentrations in the blood. This is likely due, at least in part, to the circulating half-life of immunoglobulins. As discussed in Chapter 3.1, antigen challenge assays involving specific antibody response are much more sensitive indicators of immunoglobulin levels. However, elevations in serum immunoglobulin levels may be observed occasionally in repeat-dose toxicology studies, but this appears to be rare. When observed, under most circumstances it is likely associated with infection, and thus could constitute a pattern indicative of immunosuppression. This is especially important when accompanied by leukocytosis and signs of inflammation. Another potential consideration is unintended immune enhancement, such as induction of autoimmunity. Although not likely to be associated with drugs covered under ICH S8, this could be an important signal when evaluating the safety of biotherapeutics (Chapters see 6 and 7). Increased Incidence of Infections Increased infections in repeat-dose toxicology are an important, and all too often overlooked, indicator of unintended immunosuppression. There are several issues that should be considered. With respect to rodents used in GLP studies, these are assumed to be free of specific pathogens, so if treatmentrelated infections are observed, this should be taken as a presumptive sign of unintended immunosuppression. For non-rodents, it is less likely that these can be assumed to be completely free of potential pathogens, especially in the case of nonhuman primates. Thus, non-rodents may be more sensitive to immunosuppressive drug effects. If signs of infection are observed in nonclinical toxicology studies, an attempt should be made to isolate and identify the causative organism(s). In rodents, an important finding would be infections due to opportunistic saprophytes (especially given that these are assumed to be free of known pathogens). In nonhuman primates, a more important pos-

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sibility is activation of silent infections due to potential pathogens. An especially interesting situation would be emergence of infections such as malaria or other parasitic diseases in wild-caught nonhuman primates. These findings could be considered “adventitious” host-resistance assays. Unfortunately, the most important examples of drug-associated infections have been related to biopharmaceuticals where arguably the adverse effects represented exaggerated pharmacodynamics. Two examples in particular are therapeutic monoclonal antibodies directed at TNFα and α4-integrin, which in clinical use induced active tuberculosis and progressive multifocal leukoencephalopathy, respectively. Neither was found to have infection-inducing potential in nonclinical toxicology studies, and the causative organisms, Mycobacterium tuberculosis and JC virus, have not been used in host-resistance assays. Increased Incidence of Tumors One of the most difficult issues in drug development is evaluating potential carcinogenicity. As a general rule, drugs that produce compelling signs of genotoxicity are not likely to be developed (with obvious exceptions such as cancer chemotherapeutics). Therefore, when significant drug-related carcinogenicity is demonstrated in standard lifetime rodent bioassays, it is often important to determine the probable mechanism. Chronic immunosuppression is a known cause of tumors, especially types that appear to have viral etiology. When assessing the cause of positive carcinogenicity findings, especially where other non-genotoxicity mechanisms have been excluded (e.g., hormonal effects, liver enzyme induction), unintended immunosuppression should be investigated. Since carcinogenicity due to immunosuppression appears to be both dose- and duration-related, information useful in risk management could be obtained from proper evaluation of immune impairment parameters.

IMMUNE ENHANCEMENT ICH S8 includes unintended immune enhancement as an adverse effect, which should be considered in evaluating the potential immunotoxicity of drugs. Specifically excluded are drug-specific allergenicity and autoimmunity. This is perhaps a confusing exemption and should be discussed. First, it was recognized by the authors of ICH S8 that signs consistent with immune enhancement could, and often are, observed in nonclincial toxicology studies. These include, for example, leukocytosis, splenomegaly/lymphadenopathy, or other findings, characteristic of organ-specific and/or systemic inflammation. As is discussed above, these signs may in fact be associated with unintended immunosuppression. Also, changes in a hematologic parameter, which is red cell count, can be due to drug-induced immune stimulation. A classic example is penicillin-induced hemolytic anemia. Associated with long-term use of the drug, it is caused by hapten-red blood cell complexes

SUMMARY

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inducing primarily antidrug antibodies. This usually results in a Gell & Coombs Type II immunopathy (antibody-mediated cytolysis), but can also present as immune-complex disease (Type III), and even result in autoimmune hemolytic anemia (where autoantibodies to red cell antigens persist after withdrawal of penicillin therapy). It may be possible to model such reactions in animals, but more likely this would be observed clinically. Adverse immunostimulation is in fact a broad category of effects and can be associated with both chemical drugs and biopharmaceutical products. One example is anaphylactoid reaction, often referred to as a type of “pseudoallergy.” This reaction appears to be IgE mediated (Type I), but in fact involves only the effector mechanisms of anaphylaxis. There are three known primary causes: (i) direct drug interation with mast cells/basophils; (ii) activation of the alternate complement pathway; and (iii) dysregulation of arachidonic acid metabolism. The signs of anaphylactoid reaction resemble anaphylaxis (especially angioedema, urticaria, and cardiopulmonary crisis associated with release of histamine and other endogenous vasoactive compounds), but are not caused by IgE. To make the situation even more complex, some drugs known to cause anaphylactoid reactions (certain radioimaging agents, fluoroquinolone antibiotics) may also induce true IgE-mediated anaphylaxis. One interesting aspect of anaphylactoid reactions is that they can be detected in standard nonclinical toxicology studies and animals can be used to model reaction parameters, such as intravenous infusion rates, useful in risk management. Finally, in very rare instances elevations in particular immunoglobulin classes, especially IgE or IgA may be detected upon evaluation of serum immunoglobulins. IgE elevations may indicate allergenic potential, but it is highly unlikely to be observed outside of special assays such as adaptations of the murine local lymph node assay. Elevated IgA levels are associated with certain human diseases, such as a form of glomerulonephritis, but this phenomenon appears to be rarely observed in animal studies and even when seen, would be of uncertain predictive value. There are also examples of drugs that produce systemic effects consistent with adverse immunostimulation. The recent example of an anti-CD28 monoclonal antibody is an extreme case, and is in a class not covered by ICH S8.

SUMMARY ICH S8 contains a list of issues to consider in interpreting findings of unintended immunomodulation in drug development. An overall consideration is the intended use of the drug. Therapeutics intended to have effects on immune function should be evaluated in a context different from those that are not. Many biotherapetics are intended to alter immune function and safety evaluation is often integral to pharmacodynamic studies. This is the primary reason that ICH S8 does not apply to this class of pharmaceuticals.

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For drugs covered by ICH S8, where signs of immunotoxicity are observed, additional considerations should be given to statistical and biological significance of effects, as well as severity of effects. Some immunotoxic effects may be statistically significant, but are of dubious biological relevance. This is a very complex and contentious subject and is not amenable to simplification. However, consider statistically significant, dose- and duration-related increases in albumin/globulin ratios. Although this could reflect decreases in basal serum immunoglobulin levels, the basis of the effect could be unrelated to immune impairment, and could in fact be toxicologically insignificant based on the actual degree of the change. For drugs not covered by ICH S8—protein biotherapeutics—evaluation of potential immunotoxicity is likely to be conducted as an integral component of overall pharmacology as opposed to separate studies driven by observed “cause for concern.” There will be examples of protein therapeutics that may need to be assessed for unintended adverse effects on immune function independent of pharmacology studies, but these cases are unlikely to be common. The single general exception is likely to be immunostimulatory drugs, where such issues as “cytokine release syndrome” may need to be assessed. Even in this case, it is likely that adverse immune effects would be exaggerated pharmacodynamics. This is essentially the approach advocated by ICH S6, and even if the document is updated in the near future, it is unlikely that the basic approach would be changed.

REFERENCES Bala S, Weaver J, Hastings KL. Clinical relevance of preclinical testing for allergic side effects. Toxicology 2005;209:195. House RV, Hastings K. Multidimensional immunomodulation. J Immunotoxicol 2004; 1:123. ICH Harmonised Tripartite Guideline. Immunotoxicity Studies for Human Pharmaceuticals (S8). 2006. Available at http://www.ich.org/ Joint Task Force on Practice Parameters, American Academy of Allergy, Immunology, American College of Allergy, Asthma and Asthma and Immunology, and the Joint Council of Allergy, Asthma and Immunology. The diagnosis and management of anaphylaxis. J Allergy Clin Immunol 1998;101:S465. Kuper CF, Harleman JH, Richter-Reichhelm HB, Vos J. Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 2000;28:454–466. Neugut A, Ghatak A, Miller R. Anaphylaxis in the United States: an investigation into its epidemiology. Arch Intern Med 2001;161:15. Schellekens H, Jiskoot W. Eprex-associated pure red cell aplasia and leachates. Nat Biotechnol 2006;24:613–614. United States Food and Drug Administration (USFDA), Center for Drug Evaluation and Research. Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs; 2002, Washington, DC.

PART II WEIGHT OF EVIDENCE REVIEW: A NEW STRATEGY IN IMMUNOTOXICOLOGY

2.1 CLINICAL PATHOLOGY AS CRUCIAL INSIGHT INTO IMMUNOTOXICITY TESTING Ellen Evans

Thorough assessments of clinical pathology parameters, particularly hematology data, are crucial in weight of evidence review of data obtained from standard toxicity testing for immunotoxicity evaluation (ICH S8, 2006). Clinical pathology data often provide the earliest indication of effects of drugs or chemicals on immune status. It should be acknowledged that hematology and serum chemistry provide information about aspects of both “innate” and “acquired” immune systems, and that often effects on acquired immune functions may be manifested by changes in parameters generally associated with the innate immune system and vice versa. It is critical that data are not evaluated in a vacuum; that is, individual clinical pathology parameters have little meaning unless they are considered in the context of all other available clinical pathology data, clinical signs, and anatomic pathology data. It is important to distinguish between those findings which are present as a consequence of the perturbation (such as a neutrophilia with a left shift in response to an infection) and those findings which suggest a direct effect on one or more components of the immune system and/or a mechanism (such as a lymphopenia secondary to administration of a lymphotoxic agent). In the case of pancytopenia, for example, regardless of the mechanism for the pancytopenia, the consequence is likely to be diminished ability to respond to an infection. Conversely, pancytopenia can also be the consequence of an immune-mediated destruction of stem cells. For a new pharmaceutical entity, an attempt should Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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CLINICAL PATHOLOGY AND IMMUNOTOXICITY TESTING

be made to identify parameters which can be applied in clinical settings to identify efficacy, toxicity, and/or reversibility. This evaluation should identify those new molecular entities which should be tested further for immunotoxic potential and assist in selecting the most appropriate functional assays. Clinical pathology data should be evaluated by individuals with training in clinical pathology and knowledge of multiple species; ideally a board certified veterinary clinical pathologist with a solid understanding of immunology. The veterinary clinical pathologist also contributes knowledge of naturally occurring and systemic disease states that impact the immune system, which aids in determining whether findings indicate a direct effect on the immune system, or an immune response to other toxicities. The clinical pathology parameters most associated with assessment of immunotoxicity include hematology, which includes basic complete blood count (CBC) evaluation and bone marrow evaluation when warranted by hematology or histopathologic findings, lymph node cytology, and selected serum chemistry parameters: indicators of organ involvement in hypersensitivity (e.g., increased transaminases in immune-mediated hepatitis) globulins and, when warranted to characterize unexplained increases in globulins, serum protein electrophoresis and immunoelectrophoresis. The scope of this document does not permit a thorough discussion of hematopoiesis, pathogenesis, or normal homeostasis. It is also assumed that the reader has a basic understanding of the function of each of the hematopoietic cell types. Excellent references on these topics are available as background information (Feldman et al., 2000; Beutler et al., 2001; Greer et al., 2003; Latimer et al., 2003).

HEMATOLOGY In interpreting hematology data, certain generalizations apply. Normal findings in peripheral blood do not rule out effects on the immune system. For example, there are so many factors influencing the cells in circulation which are unrelated to immune status that profound changes can be occurring in lymphoid tissues without affecting circulating lymphocyte numbers. Conversely, abnormal findings in peripheral blood do not always reflect immunotoxicity. For example, excitement upon sample collection may cause epinephrine-mediated increases in circulating lymphocytes and other white blood cells; a stress response may result in corticosteroid-mediated redistribution of lymphocytes to lymph nodes and lymphopenia in peripheral blood. The increasing sophistication of automated cell counters has resulted in virtual replacement of many manual determinations, such as the white blood cell (WBC) differential (Hunt, 2004). Because the instrument is evaluating thousands of cells individually, as opposed to the traditional 100 used to perform a manual differential, the instrument differential is almost always more accurate than the manual differential. However, notable exceptions

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include samples with large numbers of immature cells or very small total nucleated cell counts. Generally the instrument will flag the results outside the normal range, prompting the operator to examine the smear. In terms of identifying bands in the neutrophil series, or changes in morphology of red cells or any of the leukocyte lineages, these instruments are often of little value and can actually be inaccurate. There is not always a flag in these cases that a manual evaluation should be done. Before accepting instrument flags on a given instrument or relying on the flags to prompt a manual review, it is important to perform a validation of that instrument against manual evaluation. In risk assessment, one cannot afford to potentially miss a subtle hemolysis, a left shift in the leukogram, or morphologic change in neutrophils or other cell types, and it is important to evaluate stained blood smears, at least for high dose and control animals, and at least once in a program per toxicology species. The ideal hematologic assessment combines the greater accuracy and efficiency of the automated differential with manual evaluation of morphology. It is important to note that reference intervals should be generated for the specific populations being evaluated, with separate ranges for males and females. Age ranges should include juvenile, young adult, and adult. If a geriatric study is conducted, the reference intervals should be appropriate for the geriatric population. For studies involving dogs, rabbits, and monkeys, pretest data should be obtained, because generally small numbers of animals are used, and there is significant inter-animal variability. This is less important for rodents, because typically larger numbers are used, and the data are more consistent. When pretest sampling is done, there should be at least two pretest intervals to allow for some acclimation and to assess intra-animal variability. The data should be interpreted in comparison to pretest (when available), concurrent controls, and reference intervals (historical controls), in that order. Extrapolation of findings in one species to other species should be done cautiously (Hall and Everds, 2003). There are significant species differences in bone marrow capacity, propensity to produce cells outside the bone marrow (extramedullary hematopoiesis), major circulating blood cell types, half-life in circulation, and dynamics in health and disease. In general, laboratory animal species are not as well studied as humans in terms of the normal dynamics of hematologic cells or clinical consequences of changes in hematologic parameters. In other words, while interpretation of alterations from “normal” may be well characterized across species, the extrapolation to risk assessment is not always straightforward. For example, while circulating neutrophil counts generally average 5.5 × 103/μL (the Schering-Plough Research Institute reference interval is 1.8 to 11.0 × 103/μL) in island-origin cynomolgus monkeys, it is not unusual for this species to demonstrate, sporadically, circulating neutrophil numbers of 0.5 × 103/μL with no apparent cause or consequences. Such a value in a human or dog generally suggests increased susceptibility to infection. There are differences in lymphocyte subtypes in circulation, as well. While the main lymphocyte in circulation is the T cell for both rats and monkeys, the

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TABLE 2.1-1 Lymphocyte Phenotypes in Peripheral Blood: Sprague-Dawley Rats and Cynomolgus Monkeys CD3 T Cells

Rat (SEM) N=9 Monkey (SEM) N = 12 1 2 3

CD3/CD4 T Cells

CD3/CD8a T Cells

CD45R1 or CD202 B Cells

×103/μL

%

×103/μL

%

×103/μL

%

×103/μL

%

6.36 (0.71)

58 (2.77)

4.33 (0.47)

40 (1.84)

2.13 (0.28)

19 (1.34)

4.18 (0.48)

39 (2.66)

3.88 (0.46)

84 (2.04)

2.28 (0.31)

49 (2.86)

1.183 (0.19)

253 (3.22)

0.30 (0.06)

6.5 (1.10)

B cell marker in rats. B cell marker in monkeys. N = 11.

percentage of total lymphocytes identified as B cells is much higher in rats than in monkeys (Table 2.1-1). Interpretation of Hematologic Findings Pancytopenia. Decreases in peripheral blood of all four major lines of hematopoietic cells, i.e., granulocytes, lymphocytes, red blood cells, and platelets, is termed pancytopenia. This finding requires a thorough review of the bone marrow, which should include cytology and histopathology. Bone marrow cultures can be employed to help determine mechanism and/or provide a means of extrapolating relative risk from animal data to human patients. In evaluating CBC data, it is critical to take into account the time course of the change, since the lineages have varying normal half-lives and cell turnover. While normal circulating half-lives for neutrophils (∼10 hours) and platelets (5–9 days) are similar across species, there is wide variation in erythrocyte turnover. For example, the half-lives of red blood cells in rats and dogs are 45–68 days and 110 days, respectively. As a rule of thumb, neutropenia precedes thrombocytopenia, and anemia is the last manifestation of diminished stem cell proliferation/differentiation. Evaluation of reticulocyte numbers may be useful to identify diminished red blood cell production prior to a decline in circulating erythrocytes. The consequences of pancytopenia include life-threatening anemia, spontaneous bleeding, and severely compromised host defense. Pancytopenia can be caused by direct toxicity to stem cells or bone marrow infrastructure; myelophthisis (occupation of marrow by fibroplasia, osteosclerosis, neoplastic cells or other cells not normally found in the marrow); effects on mitosis, differentiation, or maturation; or immune-mediated (antibody, cytotoxic T cell) destruction of stem cells. Any pancytopenia that is unexpected based on the mechanism of the test article warrants thorough investigation.

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White Blood Cells. In evaluating the leukogram (white blood cell data), there are several things to keep in mind. A sample of circulating peripheral blood is a “snapshot” of one compartment and one moment in time. This compartment is very dynamic and may or may not be reflective of the situation in the other compartments (bone marrow, lymphoid tissues, vessel margins, peripheral tissues). The peripheral blood leukocyte population varies by species; for example, rodents have predominantly lymphocytes in circulation, dogs have predominantly neutrophils, and cynomolgus monkeys are generally in between. White blood cell counts and differentials also vary by physiologic state (stress, excitement, circadian rhythm, bleeding order, relationship to feeding time, etc.). Maintaining constancy is important; at each interval of the pretest, dosing, and recovery phase of a study, blood should be drawn roughly at the same time of day and in the same temporal relationship to feeding and other activities in the room, and the order of handling and sampling should be randomized across dose groups. Neutrophils Neutrophils are produced and stored in the bone marrow, then released into circulation where a proportion (depending on species) are adhered to blood vessel walls (marginating pool) and the remainder circulate freely (circulating pool); peripheral blood samples are assessing the circulating pool. Neutrophils will either emigrate to tissues, be removed by spleen, bone marrow or liver, or transmigrate through mucous membranes. They do not recirculate. These basic concepts are useful in evaluating hematology findings. In peripheral blood, neutrophils are assessed in terms of numbers and morphology. Neutropenia (decreased numbers of neutrophils in circulation) occurs with direct myelotoxicity or effects on proliferation, severe infection in which demand exceeds the capacity of the bone marrow to respond, transient margination of neutrophils (e.g., due to endotoxin, adhesion molecule expression, or slowing of circulation), immune-mediated destruction of neutrophils, and suppression of release from marrow. Immune causes and/or consequences should always be considered in the face of neutropenia. In contrast, there are several causes of neutrophilia (increased numbers of circulating neutrophils) that are unrelated to immune status. Neutrophilia can be part of a corticosteroid-mediated stress response (increased release of mature neutrophils from marrow and decreased egress to tissues), or an epinephrine-mediated “physiologic” response (demargination) due to excitement or fear. Neutrophilia can also be seen with hemorrhage or hemolysis which accelerates red blood cell production. This is thought to be due to generalized bone marrow stimulation and, in the case of hemolysis, cell destruction may result in increased demand for neutrophils (Latimer et al., 2003). Inflammation is a common cause of neutrophilia, and is generally distinguishable from other causes by the presence of a left shift, i.e., a shift toward

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immaturity characterized by increased numbers of bands or even earlier neutrophil stages. Morphologic findings consisting of Döhle bodies, basophilia, toxic granulation or vacuolation (known collectively as “toxic change”) may be seen in neutrophils. Toxic change was once thought to be pathognomonic for infection, but can be seen in any particularly brisk inflammatory response. In the context of immune status evaluation, inflammation can be responding to infection or immune-mediated destruction of erythrocytes, platelets, and/or tissues. However, inflammation may also occur in response to tissue necrosis due to direct, i.e., nonimmune-mediated, toxicity. Therefore, it is important to investigate the cause of any neutrophilia with a left shift. Neutrophilia may occur in certain adhesion molecule or receptor deficiencies or blockade, or other defects in emigration to tissue. Morphologic evaluation of neutrophils (e.g., a blood smear or bone marrow evaluation) often provides valuable information, and is especially important when abnormalities are seen (e.g., increased or decreased neutrophil counts, clinical signs suggestive of infection). In addition to the toxic change previously discussed, morphologic findings important in a toxicology setting include asynchronous maturation, which suggests bone marrow toxicity, phagocytosed organisms in peripheral blood which indicate septicemia, and hypersegmentation, which suggests prolonged time in circulation due to diminished egress into tissues or diminished apoptosis. Lymphocytes In contrast to neutrophils, lymphocytes recirculate extensively. They “home” to specific lymphoid tissues based on receptor activation and binding. The distribution pattern may be altered in abnormal states. Although other white blood cells are typically affected, lymphopenia (decreased numbers of lymphocytes) is the most consistent peripheral blood finding across species under conditions of stress, which results in endogenous corticosteroid-mediated homing to lymphoid tissues and can result in decreased proliferation or lysis. Other drug-induced causes of lymphopenia include direct toxicity to lymphocytes or their ability to proliferate, perturbations of cytokines responsible for lymphocyte proliferation, or immunocompromise resulting in acute systemic infection (homing to lymphoid tissues in response to infection and/or stress response in the presence of infection). The most common cause of lymphocytosis (increased numbers of circulating lymphocytes) is termed “physiologic” and is mediated by epinephrine, which results in diminished homing to lymphoid tissues. Other causes of lymphocytosis include effects on cytokines or receptors responsible for lymphocyte production and/or trafficking, chronic antigenic stimulation by infectious agents, recent vaccination, hypoadrenocorticism and lymphoid neoplasia. Abnormal morphologic findings of lymphocytes include atypia, activation, or circulating blast cells. In summary, lymphocytosis does not exclusively indicate immune stimulation; lymphopenia does not necessarily represent lymphoid

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depletion, and the absence of changes in peripheral blood lymphocytes does not rule out test article effects on lymphocytes. These generalizations underscore the need for a weight of evidence of approach which takes into account clinical signs, gross necropsy and histopathology findings, and the entire hematology picture. Monocytes Monocyte decreases are rarely appreciable, as generally, the number of circulating monocytes is normally low. Among many primary and secondary roles in both acquired and innate immunity, monocytes and macrophages, which are derived from monocytes, play a major role in removing dead or abnormal cells and foreign material. Therefore, monocytosis may suggest necrosis or inflammation and is a common feature of immune-mediated hemolytic anemia and/or thrombocytopenia (Feldman et al., 2000; Beutler et al., 2001; Latimer et al., 2003). As with other leukocytes, administration of cytokines or therapeutic entities which affect cytokines involved in macrophage activation, production, and/or circulation may affect circulating monocyte numbers. Factors that stimulate neutrophilia may also stimulate monocytosis, since both lineages share a common pluripotent stem cell. During the recovery period from a bone marrow toxicant, peripheral blood monocytosis often occurs prior to the appearance of neutrophils, because monocytes lack a storage pool in bone marrow. Monocytosis may also be seen as part of the stress response, particularly in dogs. Morphologic findings, which suggest effects on the immune system and require further investigation, include an activated appearance or the presence of phagocytosed material (i.e., organisms in infection), erythrocytes (in the case of generalized stimulation of monocytes or immunemediated hemolysis), or drug substance. Eosinophils and Basophils In evaluating eosinophil numbers, it is important to consider the reference interval population, which may have a particular background influencing eosinophil numbers influencing baseline and control data; for example, environmental factors such as parasite load and exposure to antigens. Causes of eosinophilia include hypersensitivity reactions, fungal infection, some drug reactions (e.g., tetracycline), hypoadrenocorticism, some non-hematopoietic tumors, hematopoietic tumors such as mast cell tumors and eosinophilic and/or basophilic leukemias, and hypereosinophilic syndromes. Eosinopenia is difficult to document because reference intervals for healthy animals generally start at zero, but may be appreciable, particularly in rodents, with larger numbers of animals on study. The most common cause of eosinopenia is “stress,” i.e., increased production of corticosteroids; however, eosinopenia is a less reliable indicator of the stress response than lymphopenia. Other causes include epinephrine release and acute infection.

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Basophils are the least numerous and least well characterized of the circulating leukocytes. While they are not derived from the same progenitor cell as tissue mast cells, basophils and mast cells have several similar functions, including their participation in hypersensitivity reactions. Basophils have also been attributed roles in plasma lipolysis, parasite immunity, tumor cell cytotoxicity, and hemostasis. Increases have been associated with persistent lipemia, parasitism, allergic diseases, certain neoplastic diseases, and administration of certain drugs (e.g., heparin, penicillin). Typical Leukograms Four types of leukograms illustrate commonly encountered findings in animals and humans. They include three most commonly seen in toxicology studies: (i) “physiologic” leukocytosis, (ii) “stress leukogram,” and (iii) inflammatory leukogram; and rarely seen (iv) hematopoietic neoplasia. A brief discussion of the mechanisms contributing to these leukograms is included in the previous paragraphs, which discuss individual cell types. Physiologic leukocytosis is mediated by epinephrine release, and is characterized by an increase in mature neutrophils and lymphocytes. Other leukocytes may also increase, but less dramatically. Physiologic leukocytosis occurs at times of excitement or acute stress, can be related to circadian rhythm (e.g., feeding time), and generally has no direct relationship to the immune system. The so-called “stress leukogram” reflects corticosteroid-mediated effects. As classically presented, this is characterized by a mature neutrophilia, lymphopenia, and depending on the species, monocytosis. Eosinopenia typically occurs but may be difficult to appreciate, given the normally low numbers of eosinophils. Because the neutrophils remain in circulation longer, they may be hypersegmented. The stress leukogram can occur under many circumstances and can mimic direct effects on the immune system. Endogenous corticosteroid release occurs under situations of discomfort or pain, illness, fear, perturbations of a variety of organ systems, infection, exercise, change in environment, etc. Determining whether a stress response is due to endogenous corticosteroid release or secondary to direct effects on the immune system can be challenging, and is discussed in further detail later in the chapter. As mentioned in the neutrophil discussion, inflammation is identified by neutrophilia or neutropenia with left shifts, which help distinguish inflammation from stress neutrophilia. Chronic inflammation may not always present with left shifts, because the production of neutrophils will be adjusted to meet the demand. There may be other hematologic findings, i.e., lymphopenia (due to stress), monocytosis, and “anemia of chronic disease.” The latter is a minimal to mild non-regenerative anemia, which occurs as a result of IL-1 and TNFmediated iron sequestration by macrophages. Inflammation should be considered indicative of an effect on innate or acquired immunity unless proven otherwise (e.g., trauma, response to organ damage, pharmacologic, etc.).

CLINICAL CHEMISTRY

21

A final category of leukograms illustrating potential effects on immunity includes leukograms suggestive of hematopoietic neoplasia. In the subleukemic form, leukemia may present as cytopenias in that hematopoiesis in all but the neoplastic line is suppressed, and the affected line may be confined to the bone marrow. In these cases, bone marrow evaluation generally reveals the diagnosis. In the leukemic form, the affected cell line is increased in peripheral blood and may be characterized by mature or blast forms; the remaining leukocyte lines are generally reduced in number. Non-regenerative anemia is typical in both forms. Red Blood Cells and Platelets. While leukocyte changes are the primary focus of the clinical pathologist in identifying effects on the immune system, erythrocytes and platelets are often “innocent bystanders” in situations of adverse immune stimulation. Regenerative (determined by the presence of increased reticulocytes) anemias are suggestive of hemorrhage or hemolysis. Possible immune mechanisms of hemolysis include generalized stimulation of monocytes/macrophages or immune-mediated destruction. There are also nonimmune causes, such as injury to erythrocytes (altered lipid content and oxidative damage are two of the more common examples). Other findings, which are consistent with hemolysis but are not exclusively associated with immune-mediated hemolysis, include inflammatory leukograms, hyperbilirubinemia, and in the case of intravascular hemolysis, artifactually increased mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), hemoglobinuria and hemoglobinemia. Spherocytosis, while not pathognomonic for extravascular hemolysis, is highly suggestive of immune-mediated red blood cell destruction. Immune-mediated thrombocytopenia (ITP), with or without concurrent immune-mediated hemolysis, is often associated with administration of pharmaceutical entities. Typically, the thrombocytopenia in ITP is highly regenerative, with increased numbers of megakaryocytes in the bone marrow, unless the antibody is also found on megakaryocytes. As mentioned previously, anemia and/or thrombocytopenia may also be part of generalized bone marrow toxicity; and infection and inflammation can result in anemia of chronic disease and/or thrombocytopenia due to consumption of platelets in disseminated intravascular coagulation (DIC). CLINICAL CHEMISTRY Serum Chemistry The most relevant serum chemistry parameter for assessment of immune status is the measure of globulins, which includes predominantly immunoglobulins, lipoproteins, and acute phase proteins. Although measurement of globulins provides an insensitive and nonspecific means of assessing immune status, abnormalities may prompt further investigation. Large decreases may suggest lymphotoxicity; however, assessment of T cell-dependent antibody

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response is more sensitive and specific than changes in globulins, providing a better assessment of the ability of lymphocytes to respond to a challenge. Globulins can increase with advancing age, immune stimulation, certain lymphoid tumors, nephrotic syndrome, or in the presence of inflammation. In situations of abnormal globulin concentration, serum electrophoresis and/or immunoelectrophoresis may be helpful to delineate the various fractions. Electrophoresis separates globulins into α, β, and γ fractions. Lipoproteins and acute phase proteins of inflammation are seen in α and β fractions. Some immunoglobulins (IgM, IgA) may influence the β fraction, but most immunoglobulins migrate in the γ fraction. If the β, and/or γ fractions appear increased, immunochemical or radioimmunologic methods may be useful to identify and quantify individual globulins. Other serum chemistry parameters may also be altered in situations of hypersensitivity and autoimmune disorders when one or more tissues are involved in the reaction. For example, hypoalbuminemia and hypercholesterolemia are typical findings in immune-mediated glomerulonephritis. This may progress to renal failure and be seen as an increase in blood urea nitrogen (BUN) and creatinine. Increased transaminases are typically seen when the liver is involved. In these examples, the serum chemistry findings are reflective of the pathology of the tissues involved and not a good predictor of an immune-mediated effect, as nonimmune mechanisms of toxicity are much more commonly the cause of changes in these parameters. Furthermore, nonclinical safety studies are generally poor predictors of systemic and most organ-specific hypersensitivity reactions in the clinic. Urinalysis A few urinalysis parameters may provide clues to immunotoxic effects of pharmaceuticals, but are not useful in this regard unless other findings are taken into account. Briefly, the urinary tract may be a site of opportunistic infection; clues include hematuria, increased pH, pyuria, proteinuria, and/or bacteriuria (depending on collection method). Intravascular hemolytic anemia may result in hemoglobinuria, and extra- or intravascular hemolysis may result in hyperbilirubinuria. A high level of proteinuria is a hallmark, but not pathognomonic for glomerulonephritis, in which case it is generally accompanied by hypoalbuminemia (decreased albumin in peripheral blood). Glomerulonephritis is typically an immune-mediated syndrome and should be confirmed by histopathologic evaluation of the kidneys. CYTOLOGY The most common use of cytology in risk assessment is evaluation of bone marrow, which will be discussed in greater detail below. In drug safety evaluation, it is rarely used for other tissues; however, it may be useful in some situations, for example, assessment of enlarged lymph nodes, or sites of abscess,

BONE MARROW EVALUATION

23

extramedullary hematopoiesis or macrophage proliferation. Although histopathology provides an excellent assessment of structure and architecture, it has the potential to introduce fixation and slicing artifacts which limit evaluation of individual cells (see Chapter 2.2). In contrast, cytologic evaluation provides individual cellular detail. While cytologic evaluation is not practical or necessary as a routine practice in nonclinical safety studies, it may have a place in situations where characterization of changes in individual cells or identification of closely related cell types is useful. Because cells can be obtained by minimally invasive methods, i.e., fine needle aspirate, cytology provides a means of evaluating progression or recovery in the same individual, and may be adaptable to monitoring patients in the clinic.

FLOW CYTOMETRY Flow-assisted cell sorting technologies have become a standard of clinical pathology laboratories in a variety of settings: human and veterinary diagnostic laboratories, universities, and safety evaluation centers. In fact, hematology instruments providing automated differential cell counts utilize flow technology. The most common use of flow cytometry, other than routine hematology, in safety assessment is the delineation of specific subsets of lymphocytes. Flow cytometric methods of assessing bone marrow are being refined and have been incorporated into toxicologic evaluations in a variety of settings (Criswell et al., 1998; Saad et al., 2000). Additional detail regarding lymphocyte subset analysis and other applications of flow cytometry in assessing immunotoxicity are covered in Chapters 3.2 and 4.2.

BONE MARROW EVALUATION Changes in bone marrow are often reflective of immunotoxicity, since bone marrow may be either a direct target of pharmaceuticals, resulting in immunosuppression, or responsive to situations of altered immune status. The three aspects of bone marrow review are hematology, histopathology, and cytology. Hematology and bone marrow histopathology should be assessed in every standard toxicity study. Hematology provides information about circulating hematopoietic cells, and is generally reflective of the status of the bone marrow. Histopathology provides valuable information about overall cellularity (i.e., semiquantitative/qualitative estimation of total cell population relative to control or normal), architecture and morphologic infrastructure, and adequacy of megakaryocyte numbers. Routine processing, however, can obscure cellular detail, making it difficult to discern lineage, stage of maturation, or morphology of individual cells on hematoxylin and eosin (H&E)-stained sections; for these assessments, cytologic preparation and evaluation are necessary.

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Cytologic bone marrow evaluation is warranted when there are unexplained cytopenias, abnormal morphology, or inappropriate presence of immature cells in peripheral blood; abnormal findings on histopathology which require further characterization; or particular mechanisms which target hematopoietic cells and/or cell trafficking. Cytologic evaluation provides a qualitative or semiquantitative differential (erythrocytes, granulocytes, monocytes/ macrophages, lymphocytes/plasma cells), maturation progression of each hematopoietic lineage, and individual cell morphology. An important aspect of cytologic evaluation is the myeloid/erythroid (M/E) ratio, which is useful when there is a preferential effect on neutrophils compared with red blood cell production (or vice versa), or to determine if the bone marrow is mounting an appropriate response to a peripheral effect. Normal M/E ranges are wide and variable among and between species and at various ages, so comparison with concurrent controls is critical. Because bone marrow evaluation is a critical part of immunotoxicity assessment, it is important that appropriate techniques be employed. The site of bone marrow collection, consistency in the collection, processing and evaluation of marrow, and expertise of the individual(s) evaluating each component of the assessment are important.

STRESS AS EVALUATED BY CLINICAL PATHOLOGY PARAMETERS One of the most hotly debated topics within companies and among regulators and immunotoxicologists is the determination of “stress” and its consequences. Corticosteroids have well-documented effects on lymphoid cell populations, and it is commonly accepted that animals under stressful conditions produce high levels of endogenous corticosteroids. In addition, it is reasonable to expect exposures to pharmaceutical entities at levels deliberately intended to produce toxicity, which might also create a stressful condition. What follows from this logic is that findings in lymphoid tissues such as atrophy and depletion (even the terminology can be controversial) are often attributed to “stress” and not considered a direct effect of the compound on the immune system. This term should not be utilized lightly, as reflected in current regulatory guidances, which request corroborating evidence for a “stress” designation. It is extremely difficult to obtain meaningful results by measuring serum or plasma hormone levels of corticosteroids in the context of a routine safety study, particularly in the species commonly used for standard toxicity testing. Even assessing an abnormal adrenal cortex via ACTH challenge tests is difficult in rats and monkeys, and these tests were not designed to assess a normally functioning adrenal gland that is simply responding to systemic stress. Although not currently validated for this purpose or in widespread use, measurement of 24-hour urine corticosterone (for rats) or cortisol (dogs and monkeys) or urine cortisol/ACTH ratios may be useful in supporting a conclusion of stress

SUMMARY

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response instead of an immunotoxic response. Even in the context of data routinely measured, however, there are clues that a stress response is occurring. For example, there may be a stress leukogram, characterized primarily by lymphopenia and mature neutrophilia +/− eosinopenia and monocytosis. Adrenal cortical hyperplasia is often present, and the gluconeogenic effects of corticosteroids may result in hyperglycemia.

FINDINGS IN ROUTINE CLINICAL PATHOLOGY FOR IMMUNOTOXICITY TESTING Clinical pathology data should always be interpreted in its entirety; that is, a change in a single parameter is usually meaningless without consideration of changes or the lack of changes in other parameters. Even the constellation of findings in clinical pathology data should be interpreted in light of other data collected in a standard toxicity study, i.e., clinical signs, exposure to the pharmaceutical and its metabolites, and histopathology findings. If a cause for concern is raised, subsequent studies should be based on specific findings, mechanism, or patient population. For example, suspected immune-mediated hemolytic anemia, thrombocytopenia, or neutropenia could be assessed via flow-based immunofluorescence assay for cell-bound antibody or Coombs’ test in the case of anemia. Pancytopenia, neutropenia, abnormal hematopoietic cell morphology, or abnormal maturation of one or more hematopoietic lineages may suggest the need for bone marrow culture, which could be used to assess relative liability in human versus animal hematopoietic lineages. Lymphopenia may suggest a need to conduct immunophenotyping via immunohistochemistry in tissues or flow cytometry of peripheral blood or tissue cells. T cell-dependent antibody response (TDAR), NK activity, and/or B and T cell proliferation are often warranted in situations of lymphoid depletion in the absence of clear evidence of systemic toxicity/stress. In the face of infection, particularly fungal or bacterial infection, neutrophil and/or macrophage function should be assessed in addition to assessments of acquired immunity. Abnormal globulin values may prompt the need to conduct serum electrophoresis to characterize the decrease or increase or TDAR to determine whether the decrease in globulins represents a diminished ability to respond to challenge.

SUMMARY Clinical pathology plays a prominent role in the weight of evidence assessment of immunotoxic potential of pharmaceutical entities and identifying means of monitoring clinical patients. The routine parameters should be evaluated as a whole, since individual parameters are extremely dynamic, interrelated, and rarely interpretable out of context. Furthermore, individual clinical pathology

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parameters should be interpreted in the context of normal species responses, clinical signs, organ weights, necropsy, and histopathology data, as well as time course and mechanism of action.

REFERENCES Beutler E, Lichtman MA, Coller BS, Kipps TJ, editors. Williams Hematology, 6th ed. New York, NY: McGraw-Hill, 2001. Criswell KA, Bleavins MR, Zielinski D, Zandee JC. Comparison of flow cytometric and manual bone marrow differentials in Wistar rats. Cytometry 1998;32:9–17. Feldman BF, Zinkl JG, Jain NC, editors. Schalm’s Veterinary Hematology, 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2000. Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, editors. Wintrobe’s Clinical Hematology, 11th ed. Philadelphia, PA: Lippincott Williams & Wilkins, 2003. Hall RL, Everds NE. Factors affecting the interpretation of canine and nonhuman primate clinical pathology. Toxicol Pathol 2003;31(Suppl):6–10. Hunt W. Hematology automation: 50 years and counting. Advance/Laboratory 2004;43–46. ICH S8 Harmonised Tripartite Guideline: Immunotoxicity Studies for Human Pharmaceuticals. April 2006. Available at http://www.ich.org/cache/compo/502-272-1. html Latimer KS, Mahaffey EA, Prasse KW, editors. Duncan & Prasse’s Veterinary Laboratory Medicine: Clinical Pathology, 4th ed. Ames, IA: State Press, 2003. Saad A, Palm M, Widell S, Reiland S. Differential analysis of rat bone marrow by flow cytometry. Comp Haematol Int 2000;10:97–101.

2.2 HISTOMORPHOLOGY OF THE IMMUNE SYSTEM: A BASIC STEP IN ASSESSING IMMUNOTOXICITY Patrick Haley

Over the last several years, the histomorphologic assessment of the immune system has moved to the forefront of the tools for identifying immunotoxicity. Numerous scientific forums have attempted to address the sensitivity, specificity, and consistency of histopathology for identifying immunotoxicity risks of new chemical entities (NCE) developed as pharmaceuticals. Suggestions for advanced pathology training and harmonization of terminology have been proposed, and a “Best Practices” paper, along with an extensive monograph on the subject has been published (Haley et al., 2005; Maronpot, 2006), each with the intent of providing anatomic pathologists with the tools necessary to accurately and consistently characterize intended and unintended druginduced alterations of the immune system. The reader is strongly encouraged to review these, along with a number of other excellent references (Kuper et al., 1987, 2000, 2002; Jones et al., 1990). However, despite the availability of all of these aids, the morphologic characterization of lymphoid tissues remains difficult and elusive. This chapter will attempt to delineate approaches to assist anatomic pathologists as they go about identifying, categorizing, and putting into context changes to the immune system, both subtle and obvious. While regulations are concerned about “unintended immunosuppression” (the working definition of immunotoxicity), even in the case of intended immunosuppression via immunomodulatory drugs, histopathology is an extremely important tool in determining “how much Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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HISTOMORPHOLOGY OF THE IMMUNE SYSTEM

immunosuppression is too much.” Moreover, merely knowing basic histomorphology of lymphoid organs is not enough when dealing with the pharmacology of new drug candidates that target pivotal receptors, cytokines, and/or products of the immune response. Tampering with the biology of key molecular components of the immune system can have numerous and important downstream effects that express themselves morphologically, and while perhaps not deemed “toxicity” by some, such changes may be critical when identifying No Adverse Effect Levels (NOAEL) in toxicity studies. Therefore, the pathologist needs to have a sound understanding of immunobiology in order to put such changes in the appropriate perspective. A detailed review of the immune response is also beyond this chapter and the reader is encouraged to review any of the large number of available textbooks on the subject.

REVIEW OF THE BEST PRACTICE GUIDELINE FOR THE ROUTINE PATHOLOGY EVALUATION OF THE IMMUNE SYSTEM The methods and application of histological procedures for identifying immunotoxicity have been unclear to many, and the terminology used by pathologists has been ambiguous and confusing to some scientific disciplines. As a result, the Society of Toxicologic Pathology formed a Working Group (STP WG) to identify and draft a position paper on the Best Practices for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005). The STP Best Practice Guideline for the Routine Pathology Evaluation of the Immune System, as well as several other pertinent and recent references on the subject (see below), can now be referenced for discussions of lymphoid organ histopathology. This Best Practice Guideline covers several basic approaches that are designed to simplify and harmonize the morphologic assessment of the immune system. In addition, Maronpot (2006) has recently published a monograph entitled “Enhanced Histopathology of the Immune System.” This monograph replete with color photomicrographs, is a valuable tool for the appropriate characterization of lesions of the immune system. Before describing the overall approach, two basic principles need to be stated. First, it is of value to clarify that “enhanced histopathology” is not: (i) lymphoid tissue immunohistochemistry; (ii) blind scoring of lymphoid tissues; (iii) morphometry of lymphoid tissues; or (iv) flow cytometry of lymphoid tissue cell suspensions. These are specialized techniques that are done after the initial assessment of organ weight changes and/or hematoxylin and eosin (H&E) stained tissue sections show that a change has occurred. Such specialized techniques as listed above should be directed at answering a specific scientific question and are not expected be used as routine screening tools in large-scale safety assessment studies of NCEs conducted under Good Laboratory Practices (GLP). And secondly, like most tissues, lymphoid tissue has a limited repertoire of possible responses to damage or stimuli that include, but

REVIEW OF THE BEST PRACTICE GUIDELINE

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are not limited to: (1) hyperplasia, (2) hypertrophy, (3) atrophy, (4) necrosis, and (5) neoplasia. In fact, some lymphoid tissue changes may merely be a reflection of the normal function of the lymphoid tissue to filter lymph and need to be recognized as such. Examples of such changes may include, but are not limited to: (i) antigens leading to immune stimulation and follicular hyperplasia; (ii) particulates (foreign material captured within nodal macrophages), sinus erythrocytosis or the accumulation of red blood cells draining from another site [not nodal hemorrhage]) leading to nonspecific lymphoid hyperplasia; (iii) lymphocytes and other cells migrating through lymph nodes. With regard to the collection and weighing of lymphoid tissue, the Best Practice Guideline for the Routine Pathology Evaluation of the Immune System determined that: (i) recording and evaluating thymic and splenic weights should be done routinely; (ii) interpretation of these organ weights should only be done in the context of all other clinical, histopathology, and clinical pathology data from the study; (iii) alterations of spleen and thymus weights (along with histopathology) are reasonable indicators of systemic immunotoxicity; and (iv) spleen and thymus weights are likely to be more reliable indicators than are changes in the weight of peripheral lymph nodes. The Best Practice Guideline also reinforced the principle that histopathologic examination of lymphoid tissues as indicators of systemic immunotoxicity requires that: (i) each animal should receive a thorough macroscopic examination of the spleen, thymus, and lymph nodes; and (ii) thymus, spleen, draining lymph nodes, bone marrow in situ, and any gross lesions of a lymphoid organ represent the minimum of tissues for routine evaluation of the lymphoid system. The STP WG carefully considered the utility of routine histologic assessment of peripheral lymph nodes as indicators of systemic immunotoxicity and reached a consensus that the histology of normal peripheral lymph nodes can be highly variable, often overlaps with that of altered node morphology, and cannot be unequivocally used as an indicator of systemic immunotoxicity (Figures 2.2-1 and 2.2-2). Minor differences in collection, embedding, and sectioning combined with high intrinsic variability make consistent histologic characterization of lymph nodes problematic. Therefore the collection and examination of peripheral lymph nodes that do not drain the site of xenobiotic application are not recommended by the STP WG. This statement does not pertain to lymph nodes that actually drain the site of drug administration as such nodes represent a site of high, first-pass exposure of lymphoid tissue to drug and may therefore provide clues to potential effects on the immune system. Thus, if a drug is applied to the skin, then the closest regional draining lymph node should be examined histologically. In orally dosed drugs, the gutassociated lymphoid tissue (GALT), including the Peyer’s Patches and mesenteric lymph nodes, which should be collected in any standard necropsy, would be considered the most proximal nodes to drug administration. Likewise, intrapulmonary administered drugs would drain to the bronchus-associated lymphoid tissue (BALT) and tracheobronchial lymph nodes, making these

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Figure 2.2-1 Sections of different popliteal lymph nodes taken from normal SpragueDawley rats using standardized collection techniques. Note high degree of variability of node size and architecture.

Figure 2.2-2 Multiple sagittal sections of a normal popliteal node of a dog. Note the different morphologic presentation across the sections.

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tissues appropriate for examination. And if the nasal route is used, then examination of the nasal-associated lymphoid tissue (NALT) should be considered. For example, during the development of intranasally administered corticosteroids, examination of the NALT in two species resulted in a predictable decreased size and increased apoptosis of these lymphoid tissues (NDA 20-272, 1997). Enhanced histopathology involves the semiquantitative description of lymphoid tissue changes and is considered central to the Best Practice Guideline for lymphoid tissue microscopic examination. This approach is rooted in the concept that: (i) each lymphoid organ has separate compartments that support specific immune functions; (ii) these compartments can and should be evaluated individually for changes; and (iii) descriptive, rather than interpretative terminology, should be used to characterize changes within these compartments. The histopathologic examination of any tissue (lymphoid and nonlymphoid alike) requires that all compartments of each tissue be examined and notations made according to the compartmental architecture of the particular organ. The application of such an approach to lymphoid tissue is consistent with standard practice. Most, if not all, tissues have compartments or substructures that can be used to more clearly specify the location and the particular cell type involved in a given lesion. For example, the liver has centrilobular, midzonal and periportal hepatocytes, and changes in one zone versus another can give specific clues as to the pathogenesis of the lesion identified. The same is true for each of the lymphoid organs. A listing of the major lymphoid organ compartments is shown in Table 2.2-1. The Lymphoid Organs Thymus. Thymus organ weight, especially relative to brain, is an essential first step in the identification of immunotoxicity. The thymus is considered to be

TABLE 2.2-1 List of the Major Lymphoid Organ Compartments Thymus

Spleen

Lymph Node

Cortex

White Pulp PALS Lymphoid follicles Germinal center

Medulla Cortical-medullary ratio

Marginal zone Red pulp

Cortex Subcapsular sinus Lymphoid follicles Germinal centers High endothelial venules Paracortex Medulla Cords Sinuses

Bone Marrow Erythroid component Lymphoid component

Fat Stroma Megakaryocytes

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one of the more sensitive lymphoid organs to drug-induced changes, and detectable weight changes combined with histological changes are good indicators of systemic effects on the immune system (The ICICIS Group Investigators, 1998). The normal microanatomy of the thymus includes the cortex, medulla, stroma, Hassall’s corpuscles, and other epithelial elements. Hassall’s corpuscles are made up of concentric arrays of epithelial cells which contain keratohyalin and bundles of cytoplasmic filaments. Keratinization is limited in young rats but increases with age accompanied by cystic change as the thymus undergoes involution. On the other hand, keratinization is prominent in young dogs and nonhuman primates. Because the thymic epithelium is responsible for the production of thymic hormones (thymosin and thymopoietin that are pivotal in the differentiation and maturation of thymic lymphocytes), perturbation of this cell type by drugs can result in histomorphologic changes of the medulla (Cheville, 1983; personal observation). Histologic changes of the thymus that are frequently encountered include decreased cells of thymic cortex accompanied by increased lymphocyte apoptosis (Figure 2.2-3) and increased tingible-bodied macrophages (macrophages containing the stainable [tingible] pieces of apoptotic lymphocytes), and decreased delineation between cortex and medulla as cortical cells are lost resulting in a tattered appearance (Figure 2.2-4). Not all of these changes will necessarily be present at the time of tissue collection. Histomorphologic examination captures a tissue in a snapshot of time. Depending on the magnitude of the insult, apoptosis may occur suddenly throughout the organ and be just as rapidly cleared, or may occur slowly over a protracted period of time. Thus if the apoptosis is caused suddenly after the first dose, there may be little-to-no histologic evidence of the event, other than decreased numbers of cells, after 14 days or longer (Figure 2.2-5). Alternatively, if the apoptosis is associated

Figure 2.2-3 Normal rat thymus. Low magnification (left photo) shows robust cellularity of cortex. Right photo shows minimal presence of apoptotic cells often seen in control animals.


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Figure 2.2-4 Appearance of rat thymus with increased apoptosis.

Figure 2.2-5 Appearance of rat thymus with increased apoptosis after apoptotic bodies have been cleared.

with chronic low-level stress or toxicity, then the histologic evidence of ongoing apoptosis may still be present when the tissue is collected (Figure 2.2-6). The release of endogenous corticosteroids due to nonspecific stress has often been credited with such thymic effects. It is well documented that chemical, psychological, and physical stressors can activate the hypothalamicpituitary-adrenal axis thereby culminating in increased levels of endogenous

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Figure 2.2-6 Acute apoptosis of the thymus cortical lymphocytes with the notable tingible-bodied macrophages present.

glucocorticoids that cause apoptosis of thymocytes (Crabtree et al., 1979; Tecoma and Huey, 1985). The link between thymic changes induced by increased endogenous or applied corticosteroids appears to be scientifically sound and generally accepted (Sternberg et al., 1992; Cupps and Fauci, 1982; Greaves, 2000). For example, brief or prolonged psychological stress suppresses immune responses and increases infectious disease in man (Dorian and Garfinkel, 1987; Dantzer and Kelley, 1989). Importantly, before attributing tissue changes to endogenous corticosteroids or stress, lymphoid tissue changes should be accompanied by other organ toxicity and/or significant alterations in food consumption and/or body weight that would be indicative of systemic toxicity. In fact, decreased food consumption has been clearly identified as a significant cause of increased apoptosis of the thymic cortex (Levin et al., 1993). Clinical pathology alterations suggestive of stress consist of a “stress leukogram” of increased monocytes and neutrophils, accompanied by decreased lymphocytes (see Chapter 2.1). If the animal is showing definitive evidence of systemic toxicity, then alterations of the thymus histomorphology are likely to be secondary, rather than primary effects (Smialowicz et al., 1985). The goal in any immunotoxicity assessment is to determine if the lymphoid system is a primary target of the drug effects. There are examples of direct drug toxicity to the thymus, such as that of cyclophosphamide, which results in cell depletion in many lymphoid tissues, but especially of the thymic cortex. Also as noted above, the thymic cortex is not the only thymic compartment vulnerable to drug effects. Perturbation of thymic hormones and/or homeostasis may produce changes to the medulla such as increased presence of keratinized and cystic Hassall’s corpuscles (personal observation). The classic example of drug-induced alteration of the thymic medulla is that of cyclosporin A, which causes a decrease in the thymic medulla (Figure 2.2-7A; 2.2-7B) and decreased expression of MHC-II in medullary stroma with loss of dendritic cells (Beschorner et al., 1987).


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A

B

Figure 2.2-7 Rat thymus: (A) normal; (B) thymus of rat treated with cyclosporine A; note complete loss of medulla.

Spleen. The normal histology of the spleen in the rat and dog is shown in Figures 2.2-8 and 2.2-9, respectively. An important difference between these two species is that the function of spleen of the rat is primarily that of a lymphoid organ, demonstrated by the extensive periarteriolar lymphoid sheaths (PALS), numerous and often large lymphoid follicles, and thin capsule and trabeculae. In comparison, the dog spleen has relatively small PALS, few follicles and thick capsule and trabeculae; the dog spleen functions more as a blood storage and filtering organ, rather than a primary lymphoid organ. Comparatively, the monkey spleen has an appearance closer to that of the rat with moderate-to-large amounts of lymphoid tissue with well-developed PALS and frequently large germinal centers (Figure 2.2-10A; 2.2-10B). It is not unusual for these germinal centers to contain moderate-to-large amounts of amorphous eosinophilic material centrally. The robust germinal centers are likely a reflection that the cynomolgus monkeys used for safety studies are

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Figure 2.2-8

Normal rat spleen.

Figure 2.2-9 Normal dog spleen.

still obtained from regions in which malaria and nematode parasitism are endemic. The capsule and trabeculae of the monkey spleen are thin, as in the rat. Spleen weight, especially relative to brain, is an essential first step in the analysis and decreased spleen weight has been found to be a good indicator of systemic immunotoxicity, especially when combined with histomorphology. However, because the dog spleen can store large amounts of blood, incomplete exsanguination can result in erroneously high spleen weights making their utility problematic. Careful histologic examination can help determine if exsanguination has been complete (Figure 2.2-11).


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A

B

Figure 2.2-10 Normal monkey spleen: (A) spleen from a normal female cynomolgus monkey; general appearance is similar to the rat with a relatively thin capsule, small muscular trabeculae, and abundant white pulp; (B) high magnification of a normal monkey spleen; note large lymphoid follicle with centrally located homogenous eosinophilic material in germinal center.

Possible Lesions and What They Mean The characterization of the periarteriolar lymphoid sheaths (PALS) and red pulp is essential. It is very important to accurately identify the relative size and cellularity of the PALS of treated compared to control animals. This includes not only the large and obvious PALS but also the relative number of smaller lymphoid aggregates scattered throughout the spleen. Alteration of

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Figure 2.2-11 Photomicrograph of a dog spleen showing incomplete exsanguination (right side of image) that can result in increased weight.

PALS is suggestive of systemic immune organ effects, and changes in red pulp will likely reflect alterations of circulating lymphoid populations. Another splenic zone that appears to be increasingly affected by immunomodulatory drugs is the marginal zone. While it is known that cells in this zone support T cell independent humoral immune responses, the pathogenesis and biological implications behind the loss of this zone is yet to be delineated. Lymph Nodes. The reader is strongly encouraged to review the manuscripts by Sainte-Marie et al. (1982), and that of Tilney (1971), as they lay the foundation for the accurate characterization of lymph node architecture and drainage patterns. At the very least, characterization of lesions of the lymph node as to their location within the cortex, paracortex, or medulla is a must for providing insight into any putative drug effects on lymph node structure and function (Figures 2.2-12 and 2.2-13). If however, lymph node lesions involve all compartments to a similar degree, it is acceptable to record such lesions as occurring diffusely or generally within the node without specifying each compartment separately. The mesenteric lymph node and typically the mandibular lymph node are collected in most short-term and chronic toxicity studies. Both nodes tend to be highly reactive because they drain mucosal surfaces that frequently encounter microbes introduced via the oral cavity. Because the mesenteric node drains the gastrointestinal tract, this node is viewed as an indicator of direct, as well as systemic, toxicity in cases of oral dosing of compound. A practical issue with the mesenteric node is that of its extensive length and the normal variability encountered depending on the section of the node that is examined (Figure 2.2-14). Numerous secondary follicles with germinal centers


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Figure 2.2-12 Photomicrograph and diagram delineating the compartments of a rat lymph node.

Figure 2.2-13 Photomicrograph and diagram of a dog lymph node showing the lymph node compartments.

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Figure 2.2-14 Normal rat mesenteric node (montage). Note variability of number and size of follicles and cortical units.

accompanied by high numbers of plasma cells within the medulla are commonly encountered in the mandibular node. Likewise, the mesenteric node may also have a variably high number of germinal centers with high lymphocyte turnover. Possible Lesions and What They Mean: The “Reactive Lymph Node” The reactive lymph node may show complex changes involving one, several, or all of its anatomic subunits, and may involve resident cell populations (one or more), and/or migrating cell populations (one or more). The response can be immunologic or nonspecific, and can present with a follicular, sinus, diffuse, or mixed pattern (Ioachim, 1994). Peripheral lymph nodes, such as the mandibular or popliteal nodes that drain cutaneous or mucosal sites have a greater tendency to show nonspecific reactivity, as well as mixed specific reactivity as compared to deeper lymph nodes because of the numerous and varied materials encountered, some of which are antigenic. Nonspecific reactivity of nodes is more often seen in lymph nodes from larger outbred animals that are not kept under specific pathogen-free conditions, as most laboratory rodents are (Figure 2.2-15). It should be kept in mind that lymph node changes must be considered in the context of their own inherent marked variability and judgments as to compound-induced effects be rendered based on an adequate number of animals and a thorough comparison with age- and sex-matched controls.


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Figure 2.2-15 Nonspecific reactive lymphoid hyperplasia of a tracheobronchial lymph node in a cynomolgus monkey. Note the numerous reactive large germinal centers scattered deep in the paracortex.

Bone Marrow. It is very important to check multiple sites, such as the head of the femur, the shaft of the femur, and the vertebral bodies, as the amount of hematopoietic tissue in each of these sites is often different. Also, there are species differences in the relative proportion of fat to hematopoietic tissue that changes with age and location. Careful comparisons to age- and sexmatched control animals, and inherent species-specific degrees of normal variability are vital for making accurate statements concerning compound-induced changes. Also, since the bone marrow is a site of erythroid, as well as lymphoid progenitors, any alteration of cellularity needs identification of the cell type involved, if possible. Bone marrow biology is a science unto itself and much has been written pertaining to specific bone marrow toxins and their mechanisms that is beyond the scope of this chapter. However, much less has been reported concerning the nonspecific effects of stress on bone marrow as compared to the thymus and spleen. Levin et al. (1993), Ogawa et al. (1985), Oishi et al. (1979), Pickering and Pickering (1984), and Smith and Spivak (1985) provide important insight as to the exquisite sensitivity of the bone marrow to food restriction in rats, a phenomenon that is likely mediated by both reduced nutritional intake, as well as increased endogenous corticosteroids. They reported marrow necrosis, along with thymic atrophy in rats undergoing severe food restriction (25% of control intake). Likewise we have identified severe bone marrow depletion in a study that was restricted to high-dose animals that developed severe gastrointestinal lesions, decreased food intake, and body weight loss (personal observation). Mucosal-Associated Lymphoid Tissue (MALT). MALT is pivotally important in local immune responses at mucosal surfaces by providing protection at

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the main portal of entry for microorganisms. MALT presents histologically as dispersed aggregates of nonencapsulated lymphoid tissue and can be subtyped according to its anatomic location as nasal (NALT), bronchus (BALT), or gut (GALT). The GALT represents an important site of immune system exposure when considering oral dosing of animals with drug candidates, either by oral gavage or by diet. Lamina propria lymphocytes (LPLs) and intraepithelial lymphocytes (IELs), along with Peyer’s patches (PPs) and other intestinal lymphoid nodules, combine to form one of the largest and most unappreciated, lymphoid cell populations in the body (Cerf-Bensusan and Guy-Grand, 1991). Significant alterations to these populations, as well as the PPs, can open the door to potentially lethal bacterial and protozoal overgrowth followed by systemic invasion. The GALT is pivotal in maintaining the health of an individual because the extensive mucosal surface is constantly under assault by numerous microbes and pathogens. Conditions leading to alteration of these essential lymphoid populations may result in decreased antigen-specific and innate immunity of gut, thus allowing overgrowth and loss of immunotolerance against normal gut flora, followed by enteritis and septicemia. Once these GALT immune defenses are breached, resultant septicemia induces additional lymphocyte and gastrointestinal tract epithelial apoptosis, thereby enhancing enteropathy and resulting in severe morbidity. Accurate characterization of LPLs and IELs, while difficult and requiring a careful comparison with controls, can demonstrate significant compound-induced alterations by immunomodulatory drugs (personal observation). Possible Lesions and What They Mean Like lymph nodes, the PPs have specific functional zones populated by specific cell types, changes of which provide clues as to the cellular targets and pathogenesis of the lesions. Depletion of B cell or T cell areas mirrors similar changes in other lymphoid tissues, and cell depletion with concomitant increases in tingible-bodied macrophages can be an indicator of both specific drug effects, as well as secondary stress effects. Knowledge of potential molecular targets can be helpful in identifying subtle effects on the LPLs and IELs as well.

SUMMARY The information provided above is designed to simplify and demysticize the characterization of lymphoid system lesions. It is accurate to say that standard pathology practices, applied throughout the entire drug development process, have been very successful at identifying potential immunotoxicants. But as we

REFERENCES

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enter the realm of more sophisticated immunomodulatory agents, the line between intended pharmacodynamics and unintended excessive immunosuppression becomes more difficult to see, and characterization of more subtle effects is needed to determine No Observable Effect Levels (NOELs) and No Observable Adverse Effect Levels (NOAELs). The pivotal question that must be answered is “how much (immunosuppression) is too much?” Herein lays the real challenge in risk assessment as histopathology may be one of the more sensitive tests for early effects on the immune system. Not only must it be clear whether there is or is not a histologic change (lesion?), but based on the nature of the pathology and the biology of the particular cell population and/or organ effected, it should be determined if the change is reversible, and if it is of sufficient magnitude as to constitute a risk to the patient. Such risk–benefit ratios have not been part of the early debate concerning immunotoxicity (immunosuppression) associated with environmental or occupational exposure to chemicals. Indeed it is this concept of risk versus benefit that separates industrial chemical-induced immunotoxicity from drug-induced immunotoxicity from intentional therapeutic immunomodulation.

REFERENCES Beschorner WE, Namnoum JD, Hess AD, Shinn CA, Santos GW. Cyclosporin A and the thymus. Immunopathology. Am J Pathol 1987;126(3):487–496. Cerf-Bensusan N, Guy-Grand D. Intestinal intraepithelial lymphocytes. Mucosal immunology I: basic principles. Gastroentero Clin North Am 1991;20:549–576. Cheville NF, editor. Cell Pathology, 2nd ed. Ames, IA: The Iowa State University Press, 1983. Crabtree GR, Gillis S, Smith KA, Munck A. Glucocorticoids and immune responses. Arthritis and Rheum 1979;22:1246–1256. Cupps TR, Fauci AS. Corticosteroid-mediated immunoregulation in man. Immunol Rev 1982;65:133–153. Dantzer R, Kelley KW. Stress and immunity: an integrated view of relationships between the brain and the immune system. Life Sci 1989;44:1995–2008. Dorian B, Garfinkel PE. Stress, immunity and illness—a review. Pyschol Med 1987; 17:393–407. Greaves P. Histopathology of Preclinical Toxicity Studies. Interpretation and Relevance in Drug Safety Evaluation, pp. 107–108. New York, NY: Elsevier, 2000. Haley P, Perry R, Ennulat D, Frame S, Johnson C, Lapointe J-M, Nyska A, Snyder PW, Walker D, Walter G. STP position paper: best practices guideline for the routine pathology evaluation of the immune system. Toxicol Pathol 2005;33:404–407. Ioachim HL. Lymph Node Pathology, pp. 167–177. JB Philadelphia, PA: Lippincott, 1994. Jones TC, Ward JM, Moher U, Hunt RD, editors. Monographs on Pathology of Laboratory Animals. Hematopoietic System. New York, NY: Springer-Verlag, 1990.

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Kuper CF, Schuurman H-J, Vos JKG. Pathology in immunotoxicology. In: Methods in Immunotoxicology, Vol 1, edited by Burleson GR, Dean JH, Munson AE, pp. 397– 436. New York, NY: Wiley-Liss, 1987. Kuper CF, Herleman JH, Richter-Reichelm HB, Vos JG. Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 2000;28:454–466. Kuper CF, deHeer E, Van Loveren H, Vos JG. Chapter 39, immune system. In: Handbook of Toxicologic Pathology, 2nd eds., Vol 2, edited by Hascheck W, Rousseaux CG, Wallig MA, pp. 585–646. San Diego, CA: Academic Press, 2002. Levin S, Semler D, Ruben Z. Effects of two weeks of feed restriction on some common toxicologic parameters in Sprague-Dawley rats. Toxicol Pathol 1993;21:1–14. Maronpot RR. A monograph of histomorphologic evaluation of lymphoid organs. Toxicol Pathol 2006;34(5):409–696. NDA 20-272 Nasonex: SCH 320-88, mometosone furoate monohydrate, 1997. Ogawa Y, Matsumoto K, Kamata E, Ikeda Y, Kaneko T. Effect of feed restriction on peripheral blood and bone marrow cell counts of Wistar rats. Exp Anim 1985; 34:407–416. Oishi S, Oishi H, Hiraga K. The effect of food restriction for 4 weeks on common toxicity parameters in male rats. Toxicol Appl Pharmacol 1979;47:15–22. Pickering RG, Pickering CE. The effects of reduced dietary intake upon the body and organ weights, and some clinical chemistry and haematological variates of the young Wistar rat. Toxicol Lett 1984;21:271–277. Sainte-Marie G, Peng FS, Belisle C. Overall architecture and pattern of lymph flow in the rat lymph node. Am J Anat 1982;164:275–309. Smialowicz RJ, Luebke RW, Riddle MM, Rodgers RR, Rowe DG. Evaluation of the immunotoxic potential of chlordecone with comparison of cyclophosphamide. J Toxicol Environ Health 1985;15:561–574. Smith RRL, Spivak JL. Marrow cell necrosis in anorexia nervosa and involuntary starvation. Br J Haemotol 1985;60:525–530. Sternberg EM, Chrousous GP, Wilder RL, Gold PW. The stress response and the regulation of inflammatory disease. Ann Intern Med 1992;117:854–866. Tecoma ES, Huey LY. Minireview: psychic distress and the immune response. Life Sci 1985;36:1799–1812. Tilney N. Patterns of lymphatic drainage in the adult laboratory rat. J Anat 1971;109:369–383. The ICICIS Group Investigators. Report of validation study of assessment of direct immunotoxicity in the rat. Toxicology 1998;125(2–3):183–201.

2.3 NEED FOR SPECIALIZED IMMUNOTOXICITY TESTS Kazuichi Nakamura

Standard toxicity studies are routinely conducted during the course of pharmaceutical development. Available data obtained especially from repeat-dose toxicity studies can be utilized to signal a cause for concern if the data are adequately evaluated. In some cases, however, changes in the immune function may not be reflected in the morphology of lymphoid organs or hematology changes. Hence additional attentions should be paid to other factors such as pharmacological properties and tissue distribution of drugs in the nonclinical setting. Immune function tests are not ultimate methods to evaluate immunotoxicity of drugs. There are merits and demerits in each immune function test as discussed in other sections (see Chapter 3). Consideration for extrapolation of the data into the clinical situation is essential. Feedback from the clinical studies and retrospective views on the data from other nonclinical studies are also important. Once cause(s) for concerns are identified, additional immunotoxicity testing should be conducted to understand the mechanism of toxicity and the data should be carefully interpreted considering the risk and benefit of the drug. The weight of evidence review from standard toxicity studies relies on pathology to identify immunotoxicity since immune function tests are not generally conducted at this stage. Therefore, the sensitivity of histopathology should be improved and augmented by understanding the structure and the functions of lymphoid organs (see Chapter 2.2). A wealth of knowledge on the functional anatomy of the immune system is required to evaluate the Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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immunotoxicity as similar effects can be seen with immune-mediated and nonimmune-mediated toxicities. Connection between the morphology and immune function is an important subject of immunotoxicology evaluation of pharmaceuticals. Since immunotoxicology is an applied science and requires very broad expertise, conversation between pathologists and immunotoxicologists is essential to enhance the immunotoxicology evaluation. Another intricate factor to consider is pharmacological properties of drugs, which are indicated in earlier stages of the development. The pharmacological properties should be considered from the immunological aspects even though they may be seemingly unrelated to the immune system. Several receptors for endogenous humoral mediators (i.e., hormones, neuromediators, etc.) have been discovered on immunocompetent cells including T cells, B cells, dendritic cells, and NK cells. Such associations may also raise a cause for concern. In this section, the functional anatomy of lymphoid organs and the pharmacological properties are described to judge the need for further specialized immunotoxicology studies.

FUNCTIONAL ANATOMY OF LYMPHOID ORGANS Each lymphoid organ constitutes histological compartments, which have different and significant functions. Considerable expertise has been accumulated regarding the function of each compartment in the lymphoid organs and appreciation of the functional anatomy of the compartments is extremely important. This section will attempt to bridge the morphological changes of lymphoid organs with the changes in the function of the immune system. Function of the Thymus The thymus is a site for education of T cells. The diverse antigen receptors of T cells are generated by somatic gene recombination. The genes are randomly reconstituted and the receptors recognizing nonself- or self-antigens are expressed on thymocytes. In most cases, T cells that respond to nonselfantigens appear in the periphery (outside the thymus). Thymocytes with antigen receptors showing relatively weak affinity with nonself-peptides on class I major histocompatibility complex (MHC) antigens expressed on thymic epithelial cells are selected in the thymic cortex (positive selection), whereas those showing strong affinity with self-peptides are eliminated in the medulla (negative selection). Cell entry, exit, and migration in the thymus have been intensively studied in relation to T cell development. T Lymphocyte Selection and Maturation in the Thymus Bone marrow cells enter the thymus as prothymocytes through the large venules at the corticomedullary region (Ceredig and Schreyer, 1984). P-

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selectin expressed on the thymic epithelium binds to the prothymocytes (Rossi et al., 2005). Along the epithelial cells running from the perivascular region to the subcapsule (van de Wijngaert et al., 1984), early thymocyte precursors migrate to the subcapsular region and appear as CD4CD8 double negative (DN) cells. Several chemokines derived from thymic stroma cells regulate thymocyte trafficking. Expression of CXCR4 leads the progenitors to the subcapsular region (Plotkin et al., 2003), and that of CCR9 from the subcapsular region to the deeper cortex (Uehara et al., 2006). Positive selection takes place in the thymocytes descending back again from the subcapsular regions to the cortex. Prior to the positive selection, thymocytes acquire two molecules, i.e., CD4 and CD8. Such thymocytes are called CD4CD8 double positive (DP) cells and are located throughout the cortex (Figure 2.3-1). The DP cells lose either CD4 or CD8 and become CD4+CD8− single positive (SP) cells or CD4−CD8+ SP cells as the selection proceeds. The maturation of thymocytes induces migration of both SP cells from the cortex to the medulla with expression of chemokine receptor, CCR7 (Campbell et al., 1999). Self-reactive T cells are deleted or become in the state of anergy through interaction with thymic epithelial cells expressing Aire gene in the medulla (Zuklys et al., 2000). Finally, mature thymocytes egress from the thymus through venules at the corticomedullary region or lymphatics. Sphingosine-1-phosphate receptor is involved in the emigration of thymocytes from thymus (Matloubian et al., 2004).

Immature CD3low cells in cortex

Immature CD4CD8 DP cells in cortex

Mature CD3high cells in medulla

Cytokeratin (brown)/CD3 (blue)

Mature CD4 SP/CD8 SP cells in medulla

CD4 (brown)/CD8 (blue)

Figure 2.3-1 Immunohostochemistry of mouse thymus: double staining of cytokeratin/CD3 lymphocytes and CD4/CD8 lymphocytes. Peroxidase (PO) and alkaline phosphatase (AP) were used as the label for the secondary antibody. PO was visualized by H2O2 and diaminobenzidine, while AP by AS-MX phosphate and fast blue as the substrate and chromogen, respectively. DP: double positive; SP: single positive.

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The target cells of immunotoxicity can be identified by distinguishing between the compartments of the thymus. Histopathological findings can indicate disorders during the maturation process of T cells. For instance, DP cells are sensitive to glucocorticoid especially in synergy with endogenous ouabain, a steroid hormone produced by adrenal glands and hypothalamus under stress conditions (Rodrigues-Mascarenhas et al., 2006). Accumulation of mature SP T cells is observed in the thymus from animals treated with an immunosuppressive drug FTY720 (Yagi et al., 2000). Function of the Spleen The spleen plays pivotal roles in the immune responses to bloodborne pathogens and B cell maturation. Morphological changes of each compartment in the spleen (see Chapter 2.2) can indicate the functional impairment of lymphocytes residing there. Hence the histopathology of the spleen can guide additional immunotoxicity testing and influence study design of an immune function study. There are three compartments in the white pulp of the spleen, i.e., lymphoid follicles sometimes bearing a germinal center, periarteriolar lymphoid sheaths (PALS), and marginal zones. The splenic artery enters from the hilum and branches out into penicillar arterioles and central arterioles (Pillai et al., 2005). The penicillar arteriole flows into the red pulp, while the central arteriole runs through the white pulp and terminates at the marginal sinus which surrounds the lymphoid follicle and the PALS, and demarcate these two areas from the marginal zone. The marginal zone lies adjacent to the red pulp and outlines the lymphoid follicle and the PALS. These three compartments are easily distinguishable in H&E staining preparations (Figure 2.3-2). Identification of compartments gives us substantial information on potential effects on each lymphocyte subpopulation, i.e., T cells and B cells even without conducting immunohistochemistry or flow cytometry since T cells and B cells reside in well-defined areas. B cells are populated in the lymphoid follicle and marginal zone, whereas T cells accumulate in the PALS. B Lymphocyte Differentiation in the Spleen B cells in the bone marrow are still immature and differentiate into the follicular B cells and marginal zone B cells after arriving at the spleen (Pillai et al., 2005) (Figure 2.3-3). At least five types of B cells are identified in the murine spleen. Very immature cells are called transitional stage 1 newly formed B cells with the phenotype of IgMhighIgDlowCD23-CD21lowCD1dlow. These cells transform into transitional stage 2 follicular B cell precursors (IgMhighIgDhighCD23+ CD21intermediateCD1dlow) and subsequently into marginal zone B cell precursors (IgMhighIgDhighCD23+CD21highCD1dhigh). Finally, these B cell precursors maturate as follicular B cells (IgMlowIgDhighCD23+CD21intermediateCD1dlow) and marginal zone B cells (IgMhighIgDlowCD23−CD21highCD1dhigh), respectively. Mature

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FUNCTIONAL ANATOMY OF LYMPHOID ORGANS

PALS

LF

LF MZ

LF

PALS (T cell area) GC

MZ LF (B cell area) PALS GC LF

MZ (B cell area)

Low Magnification

High Magnification

Figure 2.3-2 Rat spleen in H&E preparation. PALS: periarteriolar lymphoid sheath; MZ: marginal zone; LF: lymphoid follicle; GC: germinal center.

Murine Spleen Transitional stage 1

Newly formed B cells IgMhighIgDlowCD23-CD21lowCD1dlow Lymphoid Follicle

Transitional stage 2

Mature

Follicular B cell precursors IgMhighIgDhigh CD23+CD21intermediate CD1dlow

Marginal zone B cell precursors IgMhighIgDhigh CD23+CD21high CD1dhigh

Lymphoid Follicle

Marginal Zone

Follicular mature B cells IgMlowIgDhigh CD23+CD21intermediate CD1dlow

Marginal zone mature B cells IgMhighIgDlow CD23-CD21high CD1dhigh

Figure 2.3-3 Schematic illustration of B cell differentiation in the murine spleen based on the literature (Pillai et al., 2005).

follicular and marginal zone B cells can be selectively affected by treatment with immunosuppresants. Production of Antibodies to Foreign Antigens The marginal zone is a frontline of the immune response in the spleen. Pathogens via the blood circulation arrive at the marginal zone and are ingested by

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dendritic cells. Foreign antigens are processed and expressed on their MHC class II antigens of dendritic cells and recognized by CD4 T cells in the PALS through T cell receptors with co-stimulatory molecules such as CD28 receiving the second signal from CD80/CD86 on the dendritic cell (Figures 2.3-4 and 2.3-5). Follicular B cells activated by T cells undergo blastogenesis and form germinal centers for antibody production and immunological memory. B cells, after recombination of immunoglobulin genes, differentiate into plasma cells producing and secreting specific antibodies. B cells populated in the marginal zone were identified as a distinct lineage quickly responding to T cellindependent type 2 antigens on the capsule of pathogens such as Streptococcus pneumoniae and Haemophilus influenzae. Current studies showed that marginal zone B cells are also involved in T cell-dependent antibody responses. In contrast to follicular B cells, marginal B cells act as the antigen presenting cells for T cell-dependent antigens and activate naive CD4 T cells (Attanavanich and Kearney, 2004). Furthermore, marginal zone B cells rapidly produce even IgG after class switching from IgM, but a large proportion of marginal zone B cells remain as IgM memory cells (Song and Cerny, 2003). Marginal zone B cells therefore consist of heterogeneous populations.

MHC class II ANTIGEN PEPTIDE

ANTIGEN-PRESENTING CELL

CD4

CD4 T CELL

TCR/CD3 COMPLEX Lck

PIP3

PI3K LAT

ZAP-70

SLP-76

Grb 2

Sos

CELL MEMBRANE

Gads

Vav

PLCγ

IP3

Ras

DAG

Raf

ITAM PTK

Rac

ER

PKC

Adapter molecule

ERK

GDP-GTP exchange factor

Ca

2+

CN

Kinase/Phosphatase Small GTP-binding protein

JNK/p38

NFAT

CYTOPLASM NUCLEUS

Transcription factor AP-1 NFAT

(Gene Expression) DNA

Figure 2.3-4 Illustration of signal transduction through T cell receptor (TCR) in CD4 T cell. Antigen recognition by T cell receptor triggers the signal transduction. AP-1 and NFAT bind each other in the nucleus to induce cytokine gene expression.

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PHARMACOLOGICAL PROPERTIES OF DRUGS

ANTIGEN-PRESENTING CELL

CD80/CD86 CD28

T CELL PI3K

CYTOPLASM

IKKα IKKβ

IKK

IKKγ

IKKγ

P IκB

IκB

NF-κB

NF-κB

Ub Ub P

Ub

Ub Ub Ub

P

P

IκB P

NF-κB

P [Degradation]

[Nuclear Translocation]

CYTOPLASM NF-κB

P

NUCLEUS

Figure 2.3-5 IKK/NF-κB cascade. Activation of the nuclear factor κB (NF-κB) pathway occurs after receiving a signal from the co-stimulatory molecule CD28 on T cells.

PHARMACOLOGICAL PROPERTIES OF DRUGS Lymphocytes have several receptors for hormones (Drazen and Nelson, 2001; Beagley and Gockel, 2003) and neurotransmitters (Franco et al., 2007; Nance and Sanders, 2007) affecting the immune function. Agonists and antagonists of these endogenous humoral mediators may affect the immune system even at the low and therapeutic dose. Which immune function test should be conducted is sometimes dependent on the pharmacological properties of the drug indicated by the pharmacology studies. Two well-known examples are given to understand the possible unintended immunotoxicological effects. It is recommended, however, to keep abreast of advances in understanding and knowledge on such effects of drugs. Prostaglandin E2 (PGE2) Prostaglandins are eicosanoids produced at the basin of the arachidonic acid cascade which starts with inflammation stimuli, and follows with the release of arachidonic acid from phospholipids in the cell membrane mediated by phospholipase A2. Arachidonic acid is converted into prostaglandin H2 by cyclooxygenases 1 and 2 (COX-1and COX-2) at the middle of the cascade. Prostaglandin H2 is transformed into various prostaglandins such as PGI2, PGF2α, PGD2, and PGE2.

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Several studies have shown that PGE2 affects immune function, as well as inflammation. CD4+CD25+ regulatory T cells expressing FOXP3 suppress autoreactive T cells to maintain self-tolerance and also have inhibitory effects on immune responses against viral infections and cancers. Adaptive regulatory T cells developing from naive T cells in the periphery express COX-2 and produce PGE2. The suppressive effect of adaptive regulatory T cells on effector T cells is reversed by COX inhibitors and PGE2 receptor-specific antagonists (Mahic et al., 2006). Activated B cells also express COX-2 and produce PGE2. PGE2, at least certain doses, up-regulates antibody production and class switching (Mongini, 2007). PGE2 has different and sometimes opposite effects on the maturation and cytokine secretion patterns of dendritic cells (Harizi and Gualde, 2006). Therefore, drugs that lower COX-2 activity may have effects on the immune functions.

Cannabinoids It is well known and established that cannabinoids affect the immune function in humans, as well as in in vitro and in vivo animal experiments (Klein et al., 1998). There are two types of the cannabinoid receptor called CB1 and CB2 receptors. CB1 receptors are mainly expressed in the brain, and CB2 receptors are almost exclusively found on immune cells including B cells, NK cells, dendritic cells, and T cells. The CB1 receptors are also expressed on immune cells, but the extent of expression is higher in the central nervous system. Immunological functions of CB2 receptors have been investigated by immunohistochemistry (Rayman et al., 2004). The N terminus (phosphorylated active form) of the CB2 receptor is highly expressed in the germinal center, whereas the C terminus (nonphosphorylated inactive form) of the CB2 receptor is observed in the mantle zone surrounding the germinal center in the lymphoid follicle and the marginal zone. CB2-expressing B cells in the germinal center co-express the co-stimulatory molecule CD40 and the proliferation marker Ki67. The study suggested that the activation of the CB2 receptor triggers migration of B cells from the marginal zone to proliferate and differentiate in the germinal center. Administration of Δ9-tetrahydrocannabinol, a component of marijuana, decreases the cellularity in the thymus and spleen. It has been reported that some CB2-selective agonists have an immunosuppressive property by inducing apoptosis in immune cells (Lombard et al., 2007). Possible immunosuppression causing bacterial infection as unintended immunotoxicity in patients should be avoided and managed by finding appropriate clinical use. The cannabinoid agonists, however, are thought to be promising immunomodulators to treat several inflammatory diseases or autoimmune diseases (Croxford and Yamamura, 2005; Lunn et al., 2006).

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SUMMARY The objective of the immunotoxicology evaluation is to identify the immunotoxicological risk and to reveal its extent. After the risk is assessed through the nonclinical studies, manageability of the risk for the patients and clinical volunteers is considered. Pharmaceuticals are administered in controlled manner to patients and clinical volunteers, who are informed of the risks of the therapeutic agent and can be monitored for potential immunotoxic effects during the clinical trials. If the benefit outweighs the risk, the drug should be developed for clinical use. A type of disease for which the drug is developed should be also considered. For example, immunosuppressive drugs are basically inappropriate for HIV-infected patients, but may be beneficial for patients suffering from atopy or autoimmune diseases. The pharmacological property in conjunction with the clinical use should be meticulously examined to distinguish the unintended immunotoxicity and intended immunomodulation in the risk–benefit decision process.

REFERENCES Attanavanich K, Kearney JF. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J Immunol 2004;172(2):803–811. Beagley KW, Gockel CM. Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone. FEMS Immunol Med Microbiol 2003; 38(1):13–22. Campbell JJ, Pan J, Butcher EC. Developmental switches in chemokine responses during T cell maturation. J Immunol 1999;163(5):2353–2357. Ceredig R, Schreyer M. Immunohistochemical location of host and donor derived cells in the regenerating thymus of radiation bone marrow chimeras. Thymus 1984; 6(1–2):15–26. Croxford JL, Yamamura T. Cannabinoids and the immune system: potential for the treatment of inflammatory diseases? J Neuroimmunol 2005;166(1–2):3–18. Drazen DL, Nelson RJ. Melatonin receptor subtype MT2 (Mel 1b) and not mt1 (Mel 1a) is associated with melatonin-induced enhancement of cell-mediated and humoral immunity. Neuroendocrinology 2001;74(3):178–184. Franco R, Pacheco R, Lluis C, Ahern GP, O’Connell PJ. The emergence of neurotransmitters as immune modulators. Trends Immunol 2007;28(9):400–407. Harizi H, Gualde N. Pivotal role of PGE2 and IL-10 in the cross-regulation of dendritic cell-derived inflammatory mediators. Cell Mol Immunol 2006;3(4):271–277. Klein TW, Newton C, Friedman H. Cannabinoid receptors and immunity. Immunol Today 1998;19(8):373–381. Lombard C, Nagarkatti M, Nagarkatti P. CB2 cannabinoid receptor agonist, JWH-015, triggers apoptosis in immune cells: potential role for CB2-selective ligands as immunosuppressive agents. Clin Immunol 2007:122(3);259–270.

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Lunn CA, Reich E-P, Bober L. Targeting the CB2 receptor for immune modulation. Expert Opin Ther Targets 2006;10(5):653–663. Mahic M, Yaqub S, Johansson CC, Tasken K, Aandahl EM. FOXP3+CD4+CD25+ adaptive regulatory T cells express cyclooxygenase-2 and suppress effector T cells by a prostaglandin E2-dependent mechanism. J Immunol 2006;177(1):246–254. Matloubian M, Lo CG, Cinamon G, Lensneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004;427(6972):355–360. Mongini PKA. COX-2 expression in B lymphocytes: links to vaccines, inflammation and malignancy. Clin Immunol 2007;125(2):117–119. Nance DM, Sanders VM. Autonomic innervation and regulation of the immune system (1987–2007). Brain Behav Immun 2007;21(6):736–745. Pillai S, Cariappa A, Moran ST. Marginal zone B cells. Annu Rev Immunol 2005; 23:161–196. Plotkin J, Prockop SE, Lepique A, Petrie HT. Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus. J Immunol 2003;171(9):4521–4527. Rayman N, Lam KH, Laman JD, Simons PJ, Louwenberg B, Sonneveld P, Delwel R. Distinct expression profiles of the peripheral cannabinoid receptor in lymphoid tissues depending on receptor activation status. J Immunol 2004;172(4):2111–2117. Rodrigues-Mascarenhas S, dos Santos NF, Rumjanek VM. Synergistic effect between ouabain and glucocorticoids for the induction of thymic atrophy. Biosci Rep 2006; 26(2):159–169. Rossi FMV, Corbel SY, Merzaban JS, Carlow DA, Gossens K, Duenas J, So L, Yi L, Ziltener HJ. Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1. Nature Immunol 2005;6(6):626–634. Song H, Cerny J. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J Exp Med 2003;198(12):1923–1935. Uehara S, Hayes SM, Li L, El-Khoury D, Canelles M, Fowlkes BJ, Love PE. Premature expression of chemokine receptor CCR9 impairs T cell development. J Immunol 2006;176(1):75–84. van de Wijngaert FP, Kendall MD, Schuurman H-J, Rademakers LHMP, Kater L. Heterogeneity of epithelial cells in the human thymus. An ultrastructural study. Cell Tissue Res 1984;237(2):227–237. Yagi H, Kamba R, Chiba K, Soga H, Yaguchi K, Nakamura M, Itoh T. Immunosuppressant FTY720 inhibits thymocyte emigration. Eur J Immunol 2000;30(5):1435–1444. Zuklys S, Balciunaite G, Agarwal A, Fasler-Kan E, Palmer E, Hollaender GA. Normal thymic architecture and negative selection are associated with Aire gene expression, the gene defective in the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). J Immunol 2000;165(4):1976–1983.

2.4 SPECIFIC DRUG-INDUCED IMMUNOTOXICITY: IMMUNEMEDIATED HEMOLYTIC ANEMIA Raj Krishnaraj

Drug-induced immune-mediated hemolytic anemia occurs when certain drugs initiate an immune reaction against red blood cells (RBCs). In some instances, the drugs interact with the RBC membrane, causing the cell to become antigenic. Antibodies elicited may be directed either against a combination of the drug and certain membrane components, or against epitopes of the drug molecule that are bound tightly to the RBC surface. Antibodies formed against the RBCs attach to RBCs and can cause their premature destruction resulting in anemia. In other instances, small doses of a precipitating drug can induce severe hemolytic attacks. This chapter is focused on nonprotein drug-induced immune activities that affect erythrocyte life span. Immunogenicity of protein biopharmaceuticals that can also be involved in induction of hemolytic anemia is discussed in another chapter (see Chapter 6). Some of the well-known drugs that can cause immune-mediated hemolytic anemia (IMHA) are penicillin and its derivatives, cephalosporins, levodopa, alphamethyldopa, quinidine, and some anti-inflammatory drugs. In particular, human cases of drug-induced IMHA associated with first-, second-, and thirdgeneration cephalosporins, including some with a fatal outcome have been reported. For example, the second-generation agents associated with fatal reactions include cefotetan, cefoxitin, and ceftriaxone (cited in Shammo et al., 1999). Antibodies to ceftizoxime have been demonstrated by both the drugadsorption (hapten) and immune-complex mechanisms (Calhoun et al., 2001). Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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Drug-dependent antibodies causing IMHA have been found in AIDS patients (Gonzalez et al., 2003). Other cases of IMHA after the administration of teicoplanin, a glycopeptide antibiotic (Coluccio et al., 2004) and by a metabolite of etodolac, a nonsteroidal anti-inflammatory drug (Cunha et al., 2000), have also been reported. Hemolytic anemia associated with antibodies to older drugs like tolbutamide and phenacetin have been known for decades (Bird et al., 1972). Patients may develop two types of immune response to drugs: (a) Drug-dependent antibodies: antibodies that react with RBCs only in the presence of the drug and/or its metabolites; these antibodies may be of the IgG and/or IgM type and activate complement in most cases, cause immediate intravascular hemolysis, and can be expected to occur suddenly and after a short-term drug dosing. The resulting IMHA generally resolves after the dug administration is stopped and is cleared from the circulation. The recovery suggests that such drugs do not create sustained immunotoxicity. (b) Drug-independent antibodies or autoantibodies: antibodies that react with RBCs in the absence and presence of the drug. Such antibodies, usually IgG autoantibodies, may not necessarily activate complement and can cause mild to severe extravascular immune hemolysis and therefore anemia. MECHANISMS OF IMMUNE-MEDIATED HEMOLYTIC ANEMIA The mechanisms involved in IMHA vary. Antibodies may be formed against the drug complexed with proteins, or antibodies are formed against a component of the blood cell, in this case, the RBC membrane protein(s), independent of the drug. Drugs may interact with RBCs by binding tightly or loosely. Drugs binding covalently to RBC membrane components may stimulate haptendependent antibodies, e.g., penicillin. Antibody can be demonstrated by testing against normal RBCs treated with the drug. The second type of drugdependent antibody binds to RBCs in the presence of drug in a soluble form (Johnson et al., 2007). Anti-RBC antibodies can easily be demonstrated by conventional techniques (Salama and Mueller-Eckhardt, 1996). However, the presence of anti-RBC antibodies may not always result in the destruction of target cells. If they do not activate complement, no lysis will occur. These are called incomplete antibodies. On the other hand, absence of specific anti-RBC antibodies in a test system cannot completely rule out the possibility of an immune-mediated hemolysis. Pruss et al. (2003) and Packman (2007) provide concise reviews of theoretical background for immune-mediated hemolysis. IMHA may involve a direct erythrolysis due to activation of complement, usually the terminal complement components, C5b-9. This results in intravascular immune hemolysis. An intravascular hemolytic episode is invariably

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associated with hyperhemoglobinemia. Alternatively, IMHA can be a result of antibody and/or C3b-mediated cell phagocytosis. This leads to extravascular immune hemolysis. Extravascular hemolysis could take place without obvious hemoglobinemia (Mollison et al., 1997). Mild hemoglobinemia or hemoglobinuria associated with massive extravascular hemolysis presumably occurs due to RBC damage resulting from the attachment of these antibody-coated cells to monocytes/macrophages (Jandl et al., 1957; Jandl and Kaplan, 1960; LoBuglio et al., 1967). Thus, the three basic mechanisms of IMHA appear to be (1) hapten or drug-adsorption mechanism; (2) ternary (immune) complex mechanism; and (3) true autoantibody mechanism. Drug-modified RBC may be partially or completely phagocytosed. Macrophages express surface receptors for the Fc region of IgG (especially IgG1 and IgG3) and for opsonic fragments of complement proteins C3 and C4. When present together on the RBC surface, IgG and complement fragments act synergistically to enhance phagocytosis. Typically, partial phagocytosis results in spherocyte formation. After internalization of a portion of attached RBC membrane by the macrophage, the remainder of the RBC escapes back into the circulation. More rigid and less deformable than normal RBCs, these spherocytes are prone to further damage and destruction in the spleen or liver.

EVALUATION OF IMMUNE-MEDIATED HEMOLYTIC ANEMIA IN PRECLINICAL STUDIES Cases of drug-induced anemia in animal studies have been described in several species especially in rats, dogs, and nonhuman primates, those commonly used during preclinical drug development. Phenylhydrazine causes direct lysis of RBCs by nonimmune mechanisms. In rats, its long-term administration can cause pronounced anemia, leukocytosis, notably in the lymphocyte population. Phenylhydrazine can cross-link red cell band 3 protein (senescent antigen), resulting in the binding of autologous IgG. Macrophages in the spleen and liver recognize this complex by Fc receptor mechanisms and trigger erythrophagocytosis. Thus, in addition to alterations in RBC membrane proteins, phenylhydrazine can stimulate the production of the autologous IgG that recognizes these sites and promotes lymphoid blastogenesis, most notably in the B cell lineage (Naughton et al., 1990). Hemolytic anemia was reported to be caused by DQ-2511, an antiulcer drug, when injected to beagle dogs for 4 weeks (Ohno et al., 1993). However, in these dogs, blood smear evaluation indicated an increase in the number of cells containing Heinz bodies that supported nonimmune hemolytic anemia. Therefore, before entertaining the idea of drug-induced anemia as an adverse immune-mediated reaction to a potential drug candidate during a toxicology study, an effort should be made to rule out nonimmunological causes of hemolytic anemia. For example, oxidative damage to RBCs can be a possible cause.

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Oxidative damage to hemoglobin may lead to its precipitation (e.g., by acetaminophen) and present as Heinz bodies that can be detected by morphological evaluation of blood smears. Heinz body is a rounded, often refractile, projection from the surface of the RBC that is due to oxidation and denaturation of hemoglobin. Typically, the detection of Heinz bodies associated with hemolytic anemia is believed to be indicative of nonimmune-mediated anemia. In contrast, supportive evidence of the involvement of immune system components in anemic animals could include a positive autoagglutination of blood cells. A novel 2-fluorenonyl carbapenem antibiotic, when injected intravenously to rhesus monkeys (Macaca mulata) for 2–4 weeks induced a hemolytic anemia (extravascular hemolysis, splenomegaly, Coombs’ test positive for IgG, and up to 25% reduction in the erythron). After 3 weeks of recovery, the erythron had returned to normal; after additional 2 months the animals were Coombs’ test negative. Of note, other species tested, i.e., rats, mice, and squirrel monkeys (Saimiri sciureus) did not show any sensitivity to the drug, suggesting an increased sensitivity in rhesus monkeys compared to other species (Lankas et al., 1996). When anemia, along with one or more of other findings such as hyperbilirubinemia, hemoglobinuria, and histopathologic changes, is present in animal studies, a Coombs’ test could indicate whether or not IMHA is the cause. The histopathologic findings associated with anemia could include enhanced destruction of RBCs in spleen and/or bone marrow and regenerative response (reticolositosis, erythroid hyperplasia, or extramedullary hematopoiesis) in the absence of hemorrhage. While the spleen may enlarge as it processes a huge number of damaged RBCs, splenomegaly can occur due to other causes also. In addition, the differential diagnosis of a declining hematocrit value should include drug-related hemolysis. Since differential diagnosis between potential nonimmune- and immune-mediated hemolytic anemia may be challenging in drug development, approaches to such evaluations in toxicology studies are described herein in more details.

LABORATORY DIAGNOSTIC METHODS Moderate to severe anemia, e.g., low hematocrit value, is typically accompanied by marked reticulocytosis (increased absolute reticulocyte count), polychromasia, leukocytosis, significant spherocytosis, and sometimes, thrombocytopenia. A CBC shows low RBC count and hemoglobin value. During IMHA, the stimulation of bone marrow may be strong enough to bring about leukocytosis. Quantitative details of diagnostic criteria for IMHA are well described (Weiss and Tvedten, 2003). They describe the following diagnostic features of canine IMHA: identification of large numbers of spherocytes (e.g., 2+ or more) and autoagglutination (persistent after saline dilution) or both; a CBC pattern consistent with moderate to severe anemia (e.g., PCV = 16%),

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marked reticulocytosis (e.g., 625,000/μL), polychromatia (e.g., 3+ or 4+), and marked leukocytosis (e.g., 54,000/μL). Morphology of Red Blood Cells Blood smears that are normally collected during the study may be re-evaluated for signs of Heinz bodies, spherocytosis, red cells in macrophages, presence of RBC fragments, autoagglutination, polychromasia, and poikilocytosis. Autoagglutination, seen in severe cases of IMHA, is caused by the crosslinking of RBC membrane-associated antibodies resulting in irregular threedimensional clusters of RBCs. This should be distinguished from rouleaux formation. Spherocytes are produced when an RBC (membrane) is incompletely destroyed by phagocytic cells. Such cells escape back to the circulation. A normal RBC is biconcave on both sides and slightly paler centrally. After a portion of its membrane is attacked and perhaps lost, it reshapes into a more spherical shape with a denser red color (a geometric shape with the lowest surface area: volume ratio). Spherocytes are most easily observed in dogs (Figure 2.4-1). A spherocyte count of above 10 spherocytes per 1000× magnification (100× oil objective) observed microscopically in the blood smear can be considered diagnostic for canine IMHA (Weiss and Tvedten, 2003). It should be remembered that spherocytosis is a subjective morphologic microscopic evaluation. One may attempt to determine the definitive underlying cause for the spherocytosis. The production of spherocytes is the body’s

Figure 2.4-1 Spherocytes. Blood smear from a dog with autoimmune hemolytic anemia, Wright-Giemsa, 100× oil immersion. Erythrocytes exhibit increased polychromasia and anisocytosis, with many spherocytes present. Spherocytes lack central pallor and are of smaller diameter than that of erythrocytes. Courtesy of Dr. John W. Harvey; © 2006; Merck & Co., Inc., Whitehouse Station, NJ. Published in educational partnership with Merial Ltd.

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physiological reaction to blood loss so that more RBCs can be generated. Some of the causes for spherocytosis in dogs other than IMHA include hypophosphotemia or zinc intoxication. Presence of a toxicologically significant amount of zinc in the test article formulation should be ruled out. Serum iron levels can also be measured. It would be also good to rule out blood parasites which can be missed during the quarantine procedures, especially when using nonhuman primates. Hemaglutination Tests An established method of diagnosis of IMHA is Coombs’ test, also known as direct agglutination test (DAT). First described by Coombs in 1945, it is performed to detect either erythrocyte-directed IgG in plasma or IgG, or complement coating the surface of circulating RBCs (Figure 2.4-2). In general, these molecules do not cause direct agglutination of erythrocytes. To demonstrate their presence, anti-species globulin (AG), preferably a monoclonal antibody, with specificity for IgG or various complement proteins, is added to a suspension of RBCs. Monospecific antisera to IgM or IgA may also be employed in this test. The direct Coombs’ test detects sensitization of RBCs to a drug in vivo by looking for the presence of polyclonal anti-RBC antibodies or complement on the surface of RBCs. A species-specific antibody reagent (AG) used on patient’s EDTA blood detects agglutination of RBCs in a majority of cases of IMHA. Therefore, in a suspected case of drug-induced IMHA, fresh blood must be collected as soon as possible to conduct the direct Coombs’ test. This test is still the state of the art for the diagnosis of IMHA. In this specific test,

Figure 2.4-2 Illustration of Coombs’ test: direct antiglobulin and indirect antiglobulin tests. Courtesy of Zarandona JM, et al. CMAJ 2006;174:305–307.

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RBC-bound immunoglobulins may be detected even after hematocrit values reach normalcy and clinical improvement, thus facilitating a slightly delayed diagnostic confirmation. However, sometimes, false-positive results are possible during inflammation or infectious diseases which might lead to ineffective attachment of antibodies to RBC surfaces. The indirect Coombs’ test measures free anti-RBC antibodies in serum. If fresh blood was not collected or could not be collected, an alternative is to evaluate serum if available. The indirect Coombs’ test is not commonly used to diagnose IMHA. However, when plasma or serum samples are saved from animals with suspected IMHA, they can be used for additional evaluation of potential reactivity with the drug or development of an assay for screening similar drugs in developmental process. More frequently, in humans, the indirect assay is used to determine whether an individual might have a reaction to a blood transfusion. In short, the three types of drug-dependent IMHA that can show positive Coombs’ test results include: (1) The drug combines with its antibody and the complex is adsorbed nonspecifically onto the RBCs and fixes complement, e.g., stibophen. (2) The drug is readily attached to RBCs, which are then either directly agglutinated by antidrug antibody or, if the antibody is weak, it is revealed by a positive indirect Coombs’ test, e.g., penicillin. (3) The drug either alters the RBC membrane or affects the antibodyforming cells so that an autoantibody with a specificity toward RBCs is generated without direact reaction with the drug, e.g., methyldopa. Newer methods are being tried to improve the sensitivity of laboratory detection to diagnose IMHA. In one such approach, flow cytometric evaluation of RBCs was reported to be more sensitive than the Coombs’ test (Kucinskiene et al., 2005). In this test, presence of IgG on RBCs (from anemic dogs) was detected with goat- or rabbit anti-dog IgG reagents. An ELISA-based test was also reported (Jones et al., 1987). The ELISA methodology identifies and quantitates RBC-bound immunoglobulins and complement in canine blood. The method identifies cases of IMHA which are negative by the Direct Coombs’ test and the amount of antibody present on RBCs showed a correlation to the hemoglobin level. However, these are more expensive, time-consuming, nonroutine experimental assays compared to the Coombs’ tests. Additional methods may be used to address other quantitative and qualitative changes related to IMHA. Examples of immunologic studies of drugdependent antibodies involve detection of drug-coated antibodies and immune complexes (Garratty, 2004). Analysis could also include determination of the antibody type, subclass, and titer of antibodies eluted from the RBCs. Diminished antioxidant reserve, i.e., a state of oxidative stress, may exist in dogs with IMHA as suggested by elevated plasma malonaldehyde and lower serum alpha, gamma, and delta tocopherol levels (Pesillo et al., 2004).

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SUMMARY Drug-induced immune-mediated hemolytic anemia caused by drug-related antibody formation is a serious adverse reaction. When signals for this type of immunotoxicity are detected in preclinical and/or early clinical studies, the mechanistic and diagnostic evaluation of such drug effects must be addressed during the preclinical drug development process. REFERENCES Bird GW, Eeles GH, Litchfield JA, Rahman M, Wingham J. Haemolytic anaemia associated with antibodies to tolbutamide and phenacetin. Br Med J 1972;1(5802):728–729. Calhoun HW, Junsanto T, Donoghue MDT, Naureckas E, Baron JM, Baron BW. Ceftizoxime-induced hemolysis secondary to combined drug adsorption and immune-complex mechanisms. Transfusion 2001;41(7):893–897. Coluccio E, Villa MA, Villa E, Morelati F, Revelli N, Paccapelo C, Garratty G, Rebulla P. Immune hemolytic anemia associated with teicoplanin. Transfusion 2004; 44(1):73–76. Cunha PD, Lord RS, Johnson ST, Wilker PR, Aster RH, Bougie DW. Immune hemolytic anemia caused by sensitivity to a metabolite of etodolac, a nonsteroidal anti-inflammatory drug. Transfusion 2000;40(6):663–668. Garratty G. Review: Drug induced immune hemolytic anemia—the last decade. Immunohematology 2004;20(3):138–146. Gonzalez CA, Guzman L, Nocetti G. Drug-dependent antibodies with immune hemolytic anemia in AIDS patients. Immunohematology 2003;19(1):10–15. Jandl JH, Richardson-Jones A, Castle WB. The destruction of red cells by antibodies in man. I. Observations on the sequestration and lysis of red cells altered by immune mechanisms. J Clin Invest 1957;36:1428. Jandl JH, Kaplan ME. The destruction of red cells by antibodies in man. III. Quantitative factors influencing the pattern of hemolysis in vivo. J Clin Invest 1960;39:1145–1156. Johnson ST, Fueger JT, Gottschall JL. One center’s experience: the serology and drugs associated with drug-induced immune hemolytic anemia—a new paradigm. Transfusion 2007;47(34):697–702. Jones DRE, Stokes TJ, Gruffydd-Jones TJ, Bourne FJ. An enzyme-linked antiglobulin test for the detection of erythrocyte-bound antibodies in canine autoimmune haemolytic anaemia. Vet Immunol Immunopathol 1987;6:11–21. Kucinskiene G, Schuberth HJ, Leibold W, Pieskus J. Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs. Vet J 2005;169:303–307. Lankas GR, Coleman JB, Klein HJ, Bailey Y. Species specificity of 2-aryl carbapeneminduced immune-mediated hemolytic anemia in primates. Toxicology 1996; 108(3):207–215. LoBuglio AF, Cotran RS, Jandl JH. Red cells coated with immunoglobulin G: binding and sphering by mononuclear cells in man. Science 1967;158:1582–1585.

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Mollison PL, Engelfriet CP, Contreras M, Jones J. Blood Transfusion in Clinical Medicine. Oxford: Blackwell, 1997. Naughton BA, Dornfest BS, Bush ME, Carlson, CA, Lapin DM. Immune activation is associated with phenylhydrazine-induced anemia in the rat. J Lab Clin Med 1990; 116(4):498–507. Ohno H, Tojo H, Kakihata K, Nomura M, Takayama S. Heinz body hemolytic anemia induced by DQ-2511, a new anti-ulcer drug, in dogs. Fundam Appl Toxicol 1993; 20(2):141–146. Packman CH. Hemolytic anemia due to warm autoantibodies. Blood Rev 2007 [Epub ahead of print]. Pesillo SA, Freeman LM, Rush JE. Assessment of lipid peroxidation and serum vitamin E concentration in dogs with immune-mediated hemolytic anemia. Am J Vet Res 2004;65(12):1621–1624. Pruss A, Salama A, Ahrens N, Hansen A, Kiesewetter H, Koscielny J, Dörner T. Immune hemolysis-serological and clinical aspects. Clin Exp Med 2003;3:55–64. Salama A, Mueller-Eckhardt C. Nachweis von erythrozytären Antigenen und Antikörpern. In: Transfusionsmedizin, edited by Mueller-Eckhardt C, pp. 587–596. Berlin, Heidelberg, New York: Springer, 1996. Shammo JM, Calhoun B, Mauer AM, Hoffman PC, Baron JM, Baron BW. First two cases of immune hemolytic anemia associated with ceftizoxime. Transfusion 1999;39(8):838–844. Weiss D, Tvedten H. Chapter 3, Erythrocyte disorders. In: Small Animal Clinical Diagnosis by Laboratory Methods, 4th eds., edited by Willard MD, Tvedten H. Saunders, Philadelphia, 2003.

PART III NONCLINICAL CORE IMMUNOTOXICITY TESTING IN DRUG DEVELOPMENT

3.1.1 T CELL-DEPENDENT ANTIBODY RESPONSE TESTS Joseph R. Piccotti

The use of a T cell-dependent antibody response (TDAR) assay is endorsed by regulatory agencies as a first-line immune function test for evaluating the immunotoxicity potential of new drug candidates (CPMP, 2000; FDA, 2002; ICH, 2006). The TDAR measures a humoral immune response to antigen, which requires the participation of antigen-presenting cells (e.g., macrophages and dendritic cells), T lymphocytes, and B lymphocytes. Although the end point measured in the TDAR is antigen-specific antibody production, alterations in antibody responses may reflect effects on any or all of these cell populations. Thus, if effects on the TDAR are seen, additional studies may be needed to identify the cell type(s) affected. This section provides an overview of the TDAR assay and gives guidance on the use of this test in immunotoxicity assessment of drug candidates. The plaque-forming cell (PFC) assay, the prototypic method used to evaluate a primary TDAR, is a modification of the hemolytic or Jerne plaque assay (Jerne and Nordin, 1963). In the PFC assay, mice or rats are immunized with sheep red blood cells (sRBC) typically by intravenous injection (Temple et al., 1993; Wilson et al., 1999). The spleens are harvested at the peak of IgM production (approximately 4–5 days after immunization), processed into singlecell suspensions, and then suspended in agar with sRBC and complement. In the presence of complement, the IgM-antigen complexes form a plaque, which is caused by a single, specific antibody-forming B cell. The number of antibodyproducing cells is determined by counting the number of plaques. The PFC Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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assay was shown by Luster et al. (1992) to be the most sensitive stand-alone functional assay for detecting immunosuppression in mice. By itself, this assay showed a 78% prediction rate, which was more sensitive than other traditional functional assays such as mitogen-induced proliferation, natural killer cell activity, mixed lymphocyte reaction, and delayed-type hypersensitivity and cytotoxic T lymphocyte responses. Only lymphocyte subset analysis by flow cytometry alone was shown to be more predictive (83%) than the PFC assay, although routine immunophenotyping does not provide a functional measurement of immunocompetence. The TDAR assay also was shown to be a more sensitive indicator of immunotoxicity compared to standard end points such as hematology, lymphoid organ weights, and histopathology of lymphoid tissues (Dean et al., 1983; Luster et al., 1992). As an alternative to the PFC assay, the primary antibody response to sRBC also can be measured in serum (Temple et al., 1993; Ladics et al., 1995, 1998; Wilson et al., 1999). In this approach, immunization with sRBC is conducted similarly to the PFC assay. Antigen-specific IgM responses in serum are measured by enzyme-linked immunosorbent assay (ELISA). The wells of a microtiter plate are coated with sRBC membranes, which provide the capture antigen for binding of specific antibody. The preparation of the capture antigen in this approach is considered critical. The peak antibody response in vivo appears to be about 1–2 days delayed, compared to the generation of plaqueforming cells. The PFC assay and measurement of serum anti-sRBC IgM were shown to be equally sensitive to the immunosuppressive effects of cyclophosphamide and benzo[a]pyrene in mice and rats (Temple et al., 1993). In contrast, serum anti-sRBC IgM in rats was shown to be more sensitive to immunosuppression by dioxin-like chemicals than the PFC assay (Johnson et al., 2000). Measuring serum IgM specific for sRBC in rats also was shown to be more sensitive to immunosuppression than standard immunotoxicity end points and lymphocyte subset analysis (Ladics et al., 1995, 1998). Advantages of measuring serum antibodies by ELISA over the PFC assay include the option to freeze serum samples for later analysis, the possibility of serial sampling, the potential for automation, and assessment of in vivo total antibody formation versus spleen-specific responses. In addition, measuring antibodies by ELISA is technically easier and less labor intensive than the PFC assay. Measurement of a TDAR in vitro is another variation of the PFC assay. This approach was originally developed by Mishell and Dutton (1967). Splenocytes are treated in vitro with test compound or vehicle and are sensitized with sRBC for approximately 4–5 days. Following the incubation period, the cultures are then resuspended in agar with sRBC and complement. As in the PFC assay, the number of antibody-producing cells is determined by counting the number of plaques. The Mishell–Dutton assay requires less test substance compared to in vivo approaches and thus lends to the possibility of testing multiple compounds concurrently. The use of sRBC as a T cell-dependent antigen has been shown to be useful in assessing drug- and chemical-induced immunosuppression (Luster et al.,

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1992; Temple et al., 1993; Ladics et al., 1995, 1998; Descotes et al., 1996; ICICIS Group Investigators, 1998). In recent years however, TDAR assay development and application has refocused on using keyhole limpet hemocyanin (KLH) as a T cell-dependent antigen (Smith et al., 2003; Gore et al., 2004; Ulrich et al., 2004; Finco-Kent and Kawabata, 2005; Piccotti et al., 2005). KLH is a soluble and highly immunogenic protein. Antibody responses to KLH are measured by ELISA, which provides the same advantages as mentioned above for assessing responses to sRBC by this method. While the kinetics of antiKLH IgM response in rats (Gore et al., 2004; Piccotti, unpublished data) is consistent with the kinetics of the primary IgM response to sRBC (Temple et al., 1993), KLH provides a better standardized and characterized reagent compared to sRBC. Importantly, the inter-assay variability often seen with measuring anti-sRBC antibodies by ELISA, due primarily to variations in sRBC membrane coating preparations, is not seen with KLH. Kim et al. (2007) recently published a meta-analysis of TDAR data from numerous pharmaceutical and chemical companies, which compared sRBCand KLH-specific antibody responses in Sprague-Dawley and Wistar Han rats using multiple study designs specific for each participating laboratory. The results of these analyses demonstrated that the key elements contributing to the differences between TDAR tests included antigen type, rat strain, and route of immunization. However, the pattern of responses (i.e., reduction in antibody production) seen with potent immunosuppressive agents (cyclophosphamide, cyclosporine, and dexamethasone) was shown to be similar for sRBC and KLH despite differences among the study protocols used.

TDAR TEST METHODS Well-characterized T cell-dependent antigens such as KLH and sRBC that induce a robust primary antibody response are recommended for TDAR tests (ICH, 2006). Utilizing adjuvants to enhance antigen immunization is not recommended, except for the use of alum in nonhuman primates (ICH, 2006). However, robust IgM and IgG responses to KLH in cynomolgus monkeys can be obtained without the use of an adjuvant (Piccotti et al., 2005). Validation of the TDAR assay should characterize antigen dose, administration route, and the timing of antibody production in the relevant toxicology species (rodents and non-rodents). The route of administration is an important variable since immunization route will influence the kinetics and robustness of the antibody responses (Gore et al., 2004). In addition to the immunization paradigm, validation of the analytical assay(s) used to measure antibody concentrations should be completed before applying the TDAR assay to immunotoxicity assessment. The methods commonly used to detect antibody levels in test samples include measuring antibody titers (derived from serial dilution of a test sample) or antibody concentrations (extrapolated from a standard curve generated with known concentration of purified control antibody).

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Expression of antibody levels as optical densities is not recommended (ICH, 2006). Published studies that evaluated the sensitivity of the primary antibody responses to KLH used known immunosuppressive drugs (e.g., cyclophosphamide, cyclosporine, azathioprine, FK506) at doses that do not cause overt systemic toxicity, but alter standard immunotoxicologic end points, including hematology and lymphoid tissue morphology (Smith et al., 2003; Gore et al., 2004; Ulrich et al., 2004). The significant inhibition of the anti-KLH antibody responses at these doses confirmed the immunosuppressive effects of the tested drugs. However, the sensitivity of the primary TDAR to less potent immunomodulatory compounds needs to be established, especially considering the inherently high animal-to-animal variability seen in antibody responses in outbred species. APPLICATION IN IMMUNOTOXICITY ASSESSMENT Methods such as the TDAR assay have been shown to predict immunotoxicity in humans (Vos and Van Loveren, 1998). The factors to consider for conducting a TDAR assay are defined in the ICH S8 guidance (2006). This assay should be used along with standard immunotoxicity end points, results of other specialized immunotoxicity tests, and clinical data, when available, in a weight-ofevidence approach (see Chapter 2). Species Selection It is recommended that the TDAR is evaluated in a relevant species used in toxicity testing (FDA, 2002; ICH, 2006). Species selection is often determined by signs of immunotoxicity observed in standard toxicity studies, in that the species where evidence of immunosuppression is seen in repeated-dose toxicity studies should be evaluated to assess immunotoxicity potential. Immunotoxicity testing in nonhuman primates may be needed when a test compound has pharmacological activity in nonhuman primates, but not in rodents (e.g., biologicals), or when evidence of immunotoxicity is observed in toxicity studies using nonhuman primates. An important consideration when measuring a TDAR is the potential for pre-existing IgM against KLH, which has been reported for cynomolgus monkeys, dogs, and humans (Korver et al., 1984; Finco-Kent and Kawabata, 2005; Piccotti et al., 2005). A possible reason for pre-existing IgM is exposure to KLH in diet or presence of natural antibodies cross-reacting in the ELISA. Natural antibodies generated against shared epitopes can be found in both human and monkey serum (Guilbert et al., 1982; Soos et al., 2003). TDAR Protocols and Dose Selection Figure 3.1.1-1 gives an example of a stand-alone 28-day TDAR protocol in rats. A 28-day study is recommended, although other study durations may be

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A Initiate dosing

Serum collection

Day 1

Day 29 KLH Day 24 Specific anti-KLH IgM

B Initiate dosing

Serum collection

Day 1

Day 20

Day 29

KLH Day 15 Specific anti-KLH IgM (Day 20) and IgG (Day 29)

Figure 3.1.1-1 Example of a stand-alone primary TDAR protocol in rats. Animals (8–10/sex/group) are treated with test compound (low, mid, and high doses) or vehicle by a clinically relevant route for 28 consecutive days. (A) Rats are given a single intravenous injection of KLH on Day 24, and serum collected on Day 29 for determination of IgM specific for KLH. (B) Alternatively, animals are immunized with KLH on Day 15, and serum collected on Days 20 and 29 for measurement of anti-KLH IgM and IgG, respectively.

used with justification (FDA, 2002; ICH, 2006). Dosing of the test article (and vehicle) is initiated on Day 1, and animals are treated daily for 28 consecutive days. To assess primary anti-KLH IgM responses (Figure 3.1.1-1A), rats are immunized in this example with a single intravenous dose of KLH on Day 24, which typically is 5 days prior to study termination (on Day 29). As an alternative, animals are immunized earlier, Day 15 in this example, and the effects of compound treatment on the production of IgM and IgG specific for KLH are assessed (Figure 3.1.1-1B). This illustration is based on peak anti-KLH IgG responses approximately 14 days post-immunization (Gore et al., 2004); however, robust IgG responses can be seen up to 21 days after immunization (Piccotti, unpublished data). As mentioned previously, characterizing the timing of the antibody responses is an important component of TDAR assay validation.

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Evaluation of primary anti-KLH IgG responses in rats treated with cyclosporine was shown by Gore et al. (2004) to be more sensitive to immunosuppression than IgM. The greater reduction seen in IgG production compared to IgM in this study could be due to either increased sensitivity of IgG to immunosuppression or longer exposure to the drug during antigen-specific IgG formation. Since the kinetics of the primary anti-KLH IgM and IgG responses in rat typically do not overlap (peak IgM and IgG occur at approximately 4–7 days and 14–21 days post-immunization, respectively), a direct comparison of the sensitivity of IgM and IgG antibodies to immunosuppression in the same study is not feasible in rats. However, in monkeys the kinetics of the anti-KLH IgM and IgG responses have been shown to overlap (Piccotti et al., 2005) and common time point(s) for measuring IgM and IgG in animals treated for the same duration with test compound could be studied. In general, measuring both anti-KLH IgM and IgG responses would provide a more comprehensive evaluation of potential compound effects on humoral immune function (Gore, 2006). Selection of appropriate compound dose in a TDAR assay is another important component of protocol design. The high dose administered in a TDAR study should be above the no observed adverse effect level (NOAEL); however, potential secondary effects on the immune system due to stress should be avoided. It is recommended that the high dose should not decrease body weight gain by more than 10% compared to vehicle controls (Descotes, 2006). Animals should be given a low dose in which no effects on the TDAR are anticipated. A mid-dose also should be included to assess a potential dose relationship. In addition, a positive control (e.g., cyclophosphamide, cyclosporine) is recommended to verify assay performance. The positive control should be included periodically at a minimum; however, adding a positive control to each study is recommended at least until experience is gained in assay conduct and historical data are generated. Another variable to take into consideration when conducting a TDAR assay is the appropriateness of immunizing the main study animals or adding satellite groups in standard toxicity studies. Ladics et al. (1995) reported that immunization of rats with sRBC does not affect hematology or clinical chemistry parameters. In addition, injection of sRBC did not alter lymphoid organ weight or histology, with the exception of anticipated changes in splenic morphology. However, less is known about the potential effects of KLH immunization on interpretation of standard toxicity end points. Until more experience is gained on the effects of KLH immunization, satellite groups are recommended. Alternatively, a stand-alone TDAR study may be conducted (Figure 3.1.1-1). Conducting a stand-alone study in nonhuman primates is often not practical due to animal cost and availability. In these instances, incorporation of antigen immunization in the standard toxicity studies is justified and generally believed not to affect the interpretation of compound effects on standard toxicity end points.

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EXAMPLES OF TDAR APPLICATION

EXAMPLES OF TDAR APPLICATION A weight-of-evidence approach is recommended when evaluating the immunotoxicity potential of test compounds (FDA, 2002; ICH, 2006). Figure 3.1.1-2 illustrates when the TDAR assay could be used during the drug development process. If no signs of immunotoxicity are seen in standard toxicity studies and other factors such as pharmacological class, intended patient population and clinical data are not causes for concern, then specialized immunotoxicity testing such as a TDAR assay typically should not be needed. Importantly, evaluation of standard immunotoxicity end points (i.e., hematology and lymphoid organ morphology) has been shown to predict immunotoxicity risk (Weaver et al., 2005). If unintended immunotoxicity signals are detected in

Discovery

Initial causes-forconcern?

Preclinical

No

Clinical

Std toxicity studies

Causes-forconcern?

Yes

Consider in vitro/in vivo TDAR

No

Yes

Rank order & select optimal candidate(s) No further testing needed

Neg

TDAR in appropriate species

Pos Factors to consider: Standard toxicity findings Pharmacology Off-target effects Patient population Class history Clinical data

Consider follow up mechanistic studies

Figure 3.3.1-2 Application of the TDAR assay in immunotoxicity assessment. When evaluating unintended immunosuppression, the TDAR assay may be conducted when evidence of immunotoxicity is seen in repeated-dose toxicity studies. In these instances, the assay should be conducted prior to Phase 3 or earlier, depending on factors such as the severity of the findings and the intended patient population. The TDAR assay also could be used early in the drug development process to screen for immunotoxicity potential. This approach may be useful particularly to help de-risk unintended offtarget immunomodulation or when a novel target/mechanisms may alter immune function.

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standard toxicity studies, then testing the drug candidate in a TDAR assay can be used to evaluate potential functional consequences of these findings, especially when a specific cell target is not identified. This assay would provide information on the effects of the test compound on immune function and its potential to be immunotoxic. In these instances, the TDAR assay should be conducted in the relevant species prior to clinical trials in large patient populations, typically before Phase 3. In a stand-alone study conducted later in the drug development process, existing nonclinical and clinical data could be used to aid in dose selection. However, testing may be needed earlier depending on the severity of the findings and whether the patient population is at risk (e.g., immunocompromised). As mentioned previously, if effects on the TDAR assay are seen, then follow-up mechanistic studies may be needed to dissect the mechanism of immunosuppression (see Chapters 4.1 and 5.1). The TDAR assay also could be used earlier in the drug development process prior to standard repeated-dose toxicity testing to assess the immunomodulatory potential of drug candidates, for example, to help de-risk unintended offtarget immunosuppressive effects in development of anti-inflammatory or immunomodulatory agents. In this scenario, a TDAR assay could be used to help rank order compounds and select the best candidate(s) to move forward. Because of limited amount of test compound available at this early stage of drug development, a mouse TDAR study or alternatively the in vitro Mishell– Dutton assay could be considered. Aligned with this strategy, the TDAR assay could be used to test a compound series with a novel target not intended to alter immune function but with potential effects on immune cells (e.g., receptor/target found on lymphocytes). In this scenario, it may be helpful to evaluate potential immunosuppression early to help select the best drug candidate(s). In addition, the TDAR assay could be used to screen backup candidates in cases when a selected lead compound tested in initial standard toxicity studies showed an immunotoxicity signal that could impede the development of the compound. In this example, identifying the amount of immunosuppression that is believed to be an acceptable risk, if any, is important.

SUMMARY The TDAR assay is believed to be one of the more predictive functional assays for assessing the immunotoxicity potential of drug candidates. This assay could be used to investigate the functional consequences of alterations seen in repeated-dose toxicity studies and/or clinical trials, and to provide an early read on the immunomodulatory potential of discovery candidates. The TDAR assay has been shown to predict immunotoxicity hazard. However, because of the inherent inter-animal variability seen in the TDAR particularly in outbred species, the assay should not be used as the definitive test but as an integral component of a weight-of-evidence approach for evaluating immunotoxicity risk.

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REFERENCES CPMP (Committee for Proprietary Medicinal Products). Note for Guidance on Repeated Dose Toxicity. CPMP/SWP/1042/99, 2000. Dean JH, Luster MI, Boorman GA, Lauer LAD, Leubke RW, Lawson L. Selective immunosuppression resulting from exposure to the carcinogenic congener of benzopyrene in B6C3F1 mice. Clin Exp Immunol 1983;52:199–206. Descotes J. Methods of evaluating immunotoxicity. Expert Opin Drug Metab Toxicol 2006;2(2):249–259. Descotes G, Pinard D, Gallas J-F, Penacchio E, Blot C, Moreau C. Extension of the 4week safety study for detecting immune system impairment appears not necessary: examples of cyclosporin A in rats. Toxicology 1996;112:245–256. FDA (Food and Drug Administration, Center for Drug Evaluation and Research). Guidance for Industry. Immunotoxicology Evaluation of Investigational New Drugs, 2002. Finco-Kent D, Kawabata TT. Development and validation of a canine T-cell-dependent antibody response model for immunotoxicity evaluation. J Immunotoxicol 2005;2: 197–201. Gore ER. Immune function tests for hazard identification: a paradigm shift in drug development. Basic Clin Pharmacol Toxicol 2006;98:331–335. Gore ER, Gower J, Kurali E, Sui J-L, Bynum J, Ennulat D, Herzyk DJ. Primary antibody response to keyhole limpet hemocyanin in rat as a model for immunotoxicity evaluation. Toxicology 2004;197:23–35. Guilbert B, Dighiero G, Avrameas S. Naturally-occurring antibodies against nine common antigens in human sera. I. Detection, isolation, and characterization. J Immunol 1982;128:2779–2787. ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006. ICICIS Group Investigators. Report of validation study of assessment of direct immunotoxicology in the rat. Toxicology 1998;125:183–201. Jerne NK, Nordin AA. Plaque formation in agar by single antibody-producing cells. Science 1963;140:405. Johnson CW, Williams WC, Copeland CB, DeVito MJ, Smialowicz RJ. Sensitivity of the SRBC PFC assay versus ELISA for detection of immunosuppression by TCDD and TCDD-like congeners. Toxicology 2000;156:1–11. Kim CJ, Berlin JA, Bugelski PJ, Haley P, Herzyk DJ. Comparison of immune functional tests using T-dependent antigens in immunotoxicology studies: a meta-analysis. Per Exp Clin Immunotox 2007;1:60–73. Korver K, Zeijlemaker WP, Schellekens PTA, Vossen JM. Measurement of primary in vivo IgM- and IgG-antibody response to KLH in humans: implications of pre-immune IgM binding in antigen-specific ELISA. J Immunol Methods 1984; 74:241–251. Ladics GS, Smith C, Heaps K, Elliott GS, Slone TW, Loveless SE. Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study. Toxicology 1995;96:225–238.

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Ladics GS, Smith C, Elliott GS, Slone TW, Loveless SE. Further evaluation of the incorporation of an immunotoxicological function assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study. Toxicology 1998;126:137–152. Luster MI, Portier C, Pait DG, White KL, Gennings C, Munson AE, Rosenthal GJ. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam Appl Toxicol 1992;18:200–210. Mishell RI, Dutton RW. Immunization of dissociated spleen cell cultures from normal mice. J Exp Med 1967;126:423–442. Piccotti JR, Alvey JD, Reindel JF, Guzman RE. T-cell-dependent antibody response: assay development in cynomolgus monkeys. J Immunotoxicol 2005;2:191–196. Smith HW, Winstead CJ, Stank KK, Halstead BW, Wierda D. A predictive F344 rat immunotoxicology model: cellular parameters combined with humoral responses to NP-CγG and KLH. Toxicology 2003;194:129–145. Soos JM, Rodd M, Polsky RM, Keegan SP, Bugelski P, Herzyk DJ. Identification of natural antibodies to interleukin-18 in the sera of normal humans and three nonhuman primate species. Clin Immunol 2003;109:188–196. Temple L, Kawabata TT, Munson AE, White KL. Comparison of ELISA and plaqueforming cell assays for measuring the humoral immune response to SRBC in rats and mice treated with benzo[a]pyrene or cyclophosphamide. Fundam Appl Toxicol 1993;21:412–419. Ulrich P, Paul G, Perentes E, Mahl A, Roman D. Validation of immune function testing during a 4-week oral toxicity study with FK506. Toxicol Lett 2004;149:123–131. Vos JG, Van Loveren H. Experimental studies of immunosuppression: how do they predict for man? Toxicology 1998;129:13–26. Weaver JL, Tsutsui N, Hisada S, Vidal J-M, Spanhaak S, Sawada J, Hasting KL, van der Laan JW, Van Loveren H, Kawabata TT, Sims J, Durham SK, Fueki O, Matula TI, Kusunoki H. Meeting report: development of the ICH guidelines for immunotoxicology evaluation of pharmaceuticals using a survey of industry practices. J Immunotoxicol 2005;2:171–180. Wilson SD, Munson AE, Meade BJ. Assessment of the functional integrity of the humoral immune response: the plaque-forming cell assay and the enzyme-linked immunosorbent assay. Methods 1999;19:3–7.

3.1.2 NATURAL KILLER CELL ASSAY AND OTHER INNATE IMMUNITY TESTS Lisa Plitnick

Innate immunity is considered the body’s first line of defense against pathogens and malignant transformation, and initiates within minutes to hours following exposure to pathogens. This phase of the immune response consists of both cellular and soluble mediators including phagocytic cells (macrophages and neutrophils) and natural killer cells (NK cells) and molecules such as complement proteins, and cytokines, respectively. During the safety evaluation of therapeutics, toxicities that are specific to innate immunity may be observed and there are specific assays available to determine which aspect of innate immunity is affected. These assays include NK cell, macrophage and neutrophil function assays, and the assessment of soluble mediators in serum. A selection of these assays will be described in this section. INNATE IMMUNITY AS IMMEDIATE DEFENSE OF THE IMMUNE SYSTEM Role of Natural Killer Cells Natural killer (NK) cells are non-B, non-T lymphocytes that are an integral part of the first line of defense in innate or “natural” immunity. These cells are considered large granular lymphocytes (LGLs) as they are larger than B and Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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T lymphocytes, and contain cytoplasmic granules with perforin and granzymes that facilitate spontaneous killing of target cells. NK cells recognize triggering receptors on target cells and in the absence of a second negative (inhibitory) signal, the cells are lysed. Conversely, upon interaction with normal cells, an inhibitory signal is delivered through the inhibitory receptor on the NK cell, thus preventing the lysis of normal cells (Bryceson et al., 2006; Lodoen and Lanier, 2006). Specifically, NK cells are involved in targeted lysis of tumor and virally infected cells by virtue of their aberrant expression of “self” peptides associated with MHC Class I molecules (known as the “missing self” hypothesis). In addition to these well-described functions, it is now thought that NK cell responses to some viruses, bacteria, and parasites are indirect and mediated through accessory cells such as monocytes, macrophages, or dendritic cells (Newman and Riley, 2007). These accessory cells provide soluble (IL-12, IFNα, IFNβ, IL-18, IL-2, IL-15, and TNF), as well as contact-dependent (GITR, CD28, CD40L, 2B4, NKG2D, and NKp80) signals that lead to cell activation (required for inactivated NK cells to become lymphokine-activated killer [LAK] cells and effectively kill target cells), as well as increases in effector functions (cytokine release, cytotoxicity) of NK cells. This interaction also aids in shaping the adaptive immune response. These accessory cells also secrete soluble factors that lead to either direct (TGFβ acting directly on NK cells) or indirect (IL-10 acting on accessory cells) down-regulation of NK cell function to prevent immunopathology due to over-activation. NK cells constitute only a small proportion of lymphoid cells (5 to 20%) (Andoniou et al., 2006). However, their ability to respond to multiple ligands, as opposed to T cells which respond to only one antigen, allows a substantial number of NK cells to respond to a specific insult during the innate immune response. In addition, NK cells constitutively express cytokine receptors allowing them to respond rapidly (Yokoyama et al., 2004). These two characteristics allow for a robust response despite their relatively low numbers. Evaluation of Natural Killer Cell Activity. The European Agency for the Evaluation of Medical Products (EMEA) guidance for Repeat Dose Toxicity Testing calls for assessing NK cell function as part of routine toxicity testing (EMEA, 2000). However, as the EMEA has adopted the ICH S8 guidance on immunotoxicity, this type of assessment is now recommended only as a followup to immunophenotyping studies that demonstrate a change in number of NK cells, or if the routine toxicity study demonstrates increased viral infection rates, or in response to other factors (ICH, 2006). Those other factors may include increased tumor formation or increased incidence of bacterial infections. These may be triggers to perform follow-up testing that may include the assessment of NK cell function. NK Cell Function Assays. The ICH S8 guidance specifically mentions the chromium-release assay as a means to assess NK cell function but suggests

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that new methods that involve nonradioactive labels can be used if adequately validated. Of the assays used to assess the function of NK cells, the one most widely used is the chromium-release assay (Brunner et al., 1968; Friberg et al., 1996). In general, tumor cells known to be sensitive to NK cell-mediated cytotoxicity are loaded with a radioisotope (typically 51Cr) and incubated with single-cell suspensions of lymphoid cells either from treated animals or naive cells incubated directly with drug. Following this incubation, the levels of radioactivity released into the supernatant are measured and used as an indication of the amount of target cell lysis. This technique may be used in rodents, nonhuman primates and canines. The main differences in these species-specific assays are the target cells used. YAC-1 cells are typically used in rodent assays (Friberg et al., 1996), K562 for primate assays (Condevaux et al., 2001), and canine adenocarcinoma cell line (CTAC) cells for canine assays (Gondolf et al., 1996). Despite being the “gold standard,” chromium-release assays have several inherent drawbacks: use of radioactivity, high spontaneous release of the detection reagent from target cells, not appropriate for integration into routine rodent toxicity studies since spleens are required as sufficient numbers of cells may not be obtained from peripheral blood in rodents. In addition to being inconvenient and potentially hazardous, laboratories in some countries are prohibited from using radioactivity. Therefore, several groups have developed nonradioactive flow cytometry-based assays to assess NK cell function (Prosperi et al., 1986; Radosevic et al., 1993; Karawajew et al., 1994; Johann et al., 1995; Lee-MacAry et al., 2001; and Chapter 4.2). Some of these assays still have some issues with target cell leakage, which leads to a high spontaneous background rate; however, Marcusson-Ståhl et al. (2003) described a novel flow cytometry-based method in rats that is comparable to the 51Cr-release method in terms of sensitivity. The assay involves staining target cells with the nonradioactive dye carboxy-fluoresceine succinimidyl ester (CFSE) followed by staining with propidium iodide (PI) and analysis by flow cytometry. The number of CFSE-stained cells represents total target cells and those that also stained with PI are identified as “killed” target cells (Marcusson-Ståhl et al., 2003). The assay itself is somewhat longer; however, no radioactivity is required in this method and other end points such as immunophenotyping may be included by incorporating into a rat toxicity study at multiple time points. This test also allows the assessment of numbers of viable cells and avoids the complication of spontaneous leakage of detection reagent from target cells. A nonradioactive alternative to the chromium-release assay has also been developed in canines (Gondolf et al., 1996). This assay is a colorimetric method that utilizes the dye Rose Bengal (RB). Leukocytes isolated from dog peripheral blood (PBL, effector cells) are incubated with CTAC cells (target cells). Effector and lysed target cells are removed by washing, and the surviving adherent target cells are stained with RB. The optical density (OD) of the remaining target cells corresponds to the number of surviving cells, and thus

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is inversely correlated to the cytotoxic activity. The RB assay is also as sensitive as the chromium-release assay, is quick and easy to perform, inexpensive and avoids radioactive materials and waste. The method is, however, restricted to adherent target cells. As this assay is performed on PBL as in the CFSE assay described above, the benefits are similar (may be included in routine toxicity studies, etc.). Given that the target cells for nonhuman primates and humans are the same, the alternative human assays should be useful when assessing NK cell function in nonhuman primates (Flieger et al., 1995; Johann et al., 1995; GodoyRamirez et al., 2000). Phagocytic Cells Phagocytic cells, or phagocytes, recognize, ingest, and destroy many pathogens without the aid of the adaptive immune response and thus are integral in innate immunity. There are two major families of phagocytes involved in innate immunity: mononuclear phagocytes (macrophages) and polymorphonuclear neutrophilic leukocytes (neutrophils). Macrophages mature from circulating monocytes and migrate into the tissues where they reside in major organs, including spleen, lung (alveolar macrophages), liver (Kupffer cells), and skin (Langerhans cells). Neutrophils, which are abundant in circulation but are not found in healthy tissues, act in concert with macrophages to eliminate pathogens. Once a pathogen has invaded the tissue, macrophages are the first to recognize them and initiate the recruitment of neutrophils. Despite both cell types being classified as phagocytes, there are aspects of each that are distinct. Macrophages are long-lived and have the ability to go through more than one round of phagocytosis, whereas neutrophils are short-lived and die soon after undergoing phagocytosis. In addition, macrophages have the ability to present antigen to T cells thereby activating the adaptive immune system, but neutrophils are thought to lack this function. Both of these phagocytes identify pathogens via cell surface receptors that recognize specific constituents of the pathogen. Once engulfed and internalized into a phagosome, pathogens are killed in one of two ways: the phagosome may become acidified itself or it may fuse with a lysosome which contains antimicrobial components, generating what is termed a phagolysosome. The latter is termed respiratory burst, a process during which oxygen derivatives that are directly toxic to bacteria are generated within the phagolysosome (Janeway et al., 2005; Parham, 2005). Evaluation of Phagocyte Activity. As mentioned above, the ICH S8 guidance on immunotoxicity testing calls for follow-up testing as necessary. If it is determined that follow-up testing is required and the cell types affected in the initial evaluation of a test article are not involved in the more general assay for immune function (the T-Dependent Antibody Response Assay), specific assays may be conducted to assess the function of a particular cell type such as

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phagocytes. It is possible to measure changes in many different aspects of phagocyte function, including phagocytosis and intracellular killing. Phagocytosis. Phagocytosis may be evaluated using various in vitro techniques. These techniques range from microscopic evaluation (Bravo-Cuellar et al., 1991; Hampton and Winterbourn, 1999; Hubbard, 1999; Esteban et al., 2004) to flow cytometry (Hasui et al., 1989; Hampton and Winterbourn, 1999; van Eeden et al., 1999; Bassøe et al., 2000; Lehmann et al., 2000; Webb et al., 2007) to measurement of phagocytized particles in supernatants (Betjes et al., 1994; Hampton and Winterbourn, 1999). In some of these techniques, several aspects of phagocyte function may be measured simultaneously using purified phagocytes or evaluated in whole blood, eliminating the need to generate purified populations of cells. Measurement of phagocytosis via microscopy involves the incubation of the phagocytes of interest with target particles (labeled or unlabeled beads [Hubbard, 1999; Esteban et al., 2004], bacteria [Hampton and Winterbourn, 1999], and fungi [Bravo-Cuellar et al., 1991]) and the quantitation of ingested particles using a microscope. This assessment provides a direct visual assessment of phagocytosis but when counting small particles such as bacteria, quantitation and the determination of ingestion is somewhat difficult. Flow cytometry is a rapid and simple way to assess phagocytosis. A variety of targets: bacteria (Hasui et al., 1989; van Eeden et al., 1999; Lehmann et al., 2000; Webb et al., 2007), fungi (Lehmann et al., 2000), beads (Bassøe et al., 2000; Lehmann et al., 2000), zymosan (Lehmann et al., 2000), yeast, or other particles (van Eeden et al., 1999) may be evaluated using this technique. In general, phagocytes and fluorescently labeled targets are incubated for a period of time prior to analysis by flow cytometry. Macrophages and neutrophils are separated from lymphocytes using forward and side scatter based on their size and granularity, respectively, and analyzed for associated fluorescence based on the label on each target (see also Chapter 4.2). In vivo methods to measure phagocytosis are also available and can be performed using radioactive (Hofhuis et al., 1981) and nonradioactive (Ou et al., 1989) techniques. Hofhuis et al. have described a method for assessing phagocytosis and bacterial killing by mouse peritoneal macrophages. Mice were injected intraperitoneally with [3H]-thymidine labeled Listeria monocytogenes. Following injection, peritoneal lavages were collected and radioactivity of the samples used to determine the amount of phagocytosis and killing. Ou et al. have also used an in vivo method to assess phagocytosis in mice. The benefit of this assay is that it does not involve the use of radioactivity but rather the cells are stained and viewed via microscopy. Respiratory/Oxidative Burst Respiratory burst of macrophages and neutrophils may be measured via chemiluminesence (Bravo-Cuellar et al., 1991) or more commonly via flow cytom-

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etry (Hasui et al., 1989; van Eeden et al., 1999; Bassøe et al., 2000; Lehmann et al., 2000; Webb et al., 2007; Chapter 4.2). The flow cytometric technique measures “oxidant product formation” and is based on the conversion of nonfluorescent substrates into fluorescent products when exposed to intracellular reactive oxygen intermediates produced during respiratory burst. The benefits of this assay are that it is straightforward to perform, semiquantitative, and may be performed on small sample volumes. It may also be performed on mixed cell samples such as whole blood. Measurement of Target Killing Microbiological Assays. The assessment of bacterial killing involves measuring the viability of bacteria by various methods, e.g., absorbance measurements using MTT dye (Hubbard, 1999), growth on agar plates (Hofhuis et al., 1981; Betjes et al., 1994; Hampton and Winterbourn, 1999), thymidine uptake, or staining with fluorescent dyes (Hampton and Winterbourn, 1999) following incubation with phagocytic cells. Hampton and Winterbourn (1999) offer advantages and disadvantages of these analyses. Tumor Killing. Tumor cell killing may be assessed both in vivo (Ou et al., 1989) and in vitro (Betjes et al., 1994; Hubbard, 1999). Ou et al. described an assessment of tumor cell killing in which leucite- and mineral oil-induced plasmacytomas induced in mice were evaluated for growth in the presence of a test compound thought to decrease the ability of phagocytes to kill tumor cells. When assessed in vitro, tumor cells are incubated with phagocytic cells and their survival assessed by measurement of radioactivity (if tumor cells are radioactively labeled) (Hubbard, 1999) or incubation with MTT dye (Betjes et al., 1994) which stains viable tumor cells. SOLUBLE MEDIATORS OF INNATE IMMUNITY Complement Complement proteins are found naturally in circulation and the classical and alternative pathways work together to clear pathogens. Although the classical pathway requires pathogen-specific antibodies, the alternative complement pathway does not require this specificity and is therefore useful during the innate immune response. When a pathogen is recognized, the rate of hydrolysis and activation of one of C3, the complement components, is increased. This kicks off a cascade of binding and cleavage of additional complement proteins ultimately leading to the binding of C3b to the surface of the pathogen. The C3b molecules are then amplified and eventually coat the surface of the pathogen making it a target for phagocytosis by macrophages. Alternatively, C3b can combine with other complement proteins forming pores which lyse the pathogen directly (Janeway et al., 2005).

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Measurement of Complement Proteins. Complement proteins may be measured in serum by ELISA or in tissues by immunohistochemical techniques (Morita et al., 2006) using commercially available labeled antibodies specific for individual complement components in several different species. These methods allow the incorporation of complement measurement into toxicity studies rather than needing to conduct stand-alone studies to evaluate complement-related changes.

SUMMARY In order to understand potential immunotoxicity observed during the safety evaluation of therapeutics, assays are available to assess whether specific components of innate immunity are affected. These assays include functional assays for the cell types involved in this phase of the immune response (NK cells, macrophages, and neutrophils) and measurement of complement proteins. The majority of these assays may be incorporated into toxicity studies thereby allowing for simultaneous measurement of specific immunotoxicological end points within the same study as toxicological end points.

REFERENCES Andoniou CE, Andrews DM, Degli-Esposti MA. Natural killer cells in viral infection: more than just killers. Immunol Rev 2006;214:239–250. Bassøe C-F, Smith I, Sørnes S, Halstensen A, Lehmann AK. Concurrent measurement of antigen- and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils. Methods 2000;21:203–220. Betjes MGH, Havenith CEG, van de Loosdrecht AA, Beelen RHJ. Methods for studying immuno-effector functions and antigen presenting activity of human macrophages. J Immunol Methods 1994;174:215–222. Bravo-Cuellar A, Homo-Delarche F, Ramos-Zepeda R, Dubouch P, Cabannes J, Orbach-Arbouys S. Increased phagocytic activity of peripheral blood monocytes after intravenous injection of phospholipase A2 to monkeys. Immunol Lett 1991; 28:5–10. Brunner K, Mauel J, Cerottini JC, Chapus B. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labelled allogenic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 1968;14:181–196. Bryceson YT, March ME, Ljunggren H-G, Long EO. Activation, coactivation, and costimulation of resting human natural killer cells. Immunol Rev 2006;214: 73–91. Condevaux F, Guichard J, Forichon A, Aujoulat M, Descotes J. Compared effects of morphine and nickel chloride on NK cell activity in vitro in rats and monkeys. J Appl Toxicol 2001;21:431–434. EMEA. Note for Guidance on Repeated Dose Toxicity. CPMP/SWP/1042/99, 2000.

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Esteban S, Nicolaus C, Garmundi A, Rial RV, Rodriguez AB, Ortega E, Ibars CB. Effect of orally administered L-tryptophan on serotonin, melatonin, and the innate immune response in the rat. Mol Cell Biochem 2004;267:39–46. Flieger D, Gruber R, Schlimok G, Reiter C, Pantel K, Rietmüller G. A novel non-radioactive cellular cytotoxicity test based on the differential assessment of living and killed target and effector cells. J Immunol Methods 1995;180:1–13. Friberg D, Bryant JL, Whiteside TL. Measurements of natural killer (NK) activity and NK-cell quantification. Methods Enzymol 1996;9:316–326. Godoy-Ramirez K, Franck K, Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow-cytometry. J Immunol Methods 2000;239:35–44. Gondolf C, Burkhardt E, Failing K, Stitz L. A new colorimetric method for measuring cell-mediated cytotoxicity in dogs. Vet Immunol Immunopathol 1996;55:11–22. Hofhuis FMA, van der Meer C, Kersten MCM, Rutten VPMG, Willers JMN. Effects of dimethyldioctadecyl ammonium bromide on phagocytosis and digestion of Listeria monocytogenes by mouse peritoneal macrophages. Immunology 1981;43:425–431. Hampton MB, Winterbourn CC. Methods for quantifying phagocytosis and bacterial killing by human neutrophils. J Immunol Methods 1999;232:15–22. Hasui M, Hirabayashi Y, Kobayashi Y. Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. J Immunol Methods 1989;117:53–58. Hubbard AK. Effects of xenobiotics on macrophage function: evaluation in vitro. Methods 1999;19:8–16. ICH. S8. Immunotoxicity Studies for Human Pharmaceuticals. 2006. Janeway CA, Travers P, Walport M, Shlomchik M. Immunobiology: The Immune System in Heath and Disease, 6th ed., pp. 37–100. New York, NY: Garland Science Publishing, 2005. Johann S, Blümel G, Lipp M, Förster R. A versatile flow-cytometry-based assay for the determination of short- and long-term natural killer cell activity. J Immunol Methods 1995;185:209–216. Karawajew L, Jung G, Wolf H, Micheel B, Ganzel K. A flow-cytometric long-term cytotoxicity assay. J Immunol Methods 1994;177:119–130. Lee-MacAry AE, Ross EL, Davies D, Laylor R, Honeychurch J, Glennie MJ, Snary D, Wilkinson RW. Development of a novel flow-cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J Immunol Methods 2001;252:83–92. Lehmann AK, Sørnes S, Halstensen A. Phagocytosis: measurement by flow cytometry. J Immunol Methods 2000;243:229–242. Lodoen MB, Lanier LL. Natural killer cells as an initial defense against pathogens. Curr Opin Immunol 2006;18:391–398. Marcusson-Ståhl M, Cederbrant K. A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies. Toxicology 2003;193:269–279. Morita H, Suzuki K, Mori N, Yasuhara O. Occurrence of complement protein C3 in dying pyramidal neurons in rat hippocampus after systemic administration of kainic acid. Neurosci Lett 2006;409:35–40.

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Newman KC, Riley EM. Whatever turns you on: accessory-cell dependent activation of NK cells by pathogens. Nat Rev Immunol 2007;7:279–291. Ou DW, Shem M-L, Luo Y-D. Effects of cocaine on the immune system of Balb/c mice. Clin Immunol Immunopathol 1989;52:305–312. Parham P. The Immune System, 2nd ed., pp. 227–260. New York, NY: Garland Science Publishing, 2005. Prosperi E, Croce AC, Bottiroli G, Supino R. Flow-cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein. Cytometry 1986;7(1):70–75. Radosevic K, Schut TC, van Graft M, De Grooth BG, Greve J. A flow-cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process. J Immunol Methods 1993;161(1):119–128. van Eeden SF, Klut ME, Walker BAM, Hogg JC. The use of flow cytometry to measure neutrophil function. J Immunol Methods 1999;232:23–43. Webb C, McCord K, Dow S. Neutrophil function in septic dogs. J Vet Intern Med 2007;21:982–989. Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu Rev Immunol 2004;22:405–429.

3.1.3 CELLULAR IMMUNE RESPONSE IN DELAYED-TYPE HYPERSENSITIVITY TESTS Karen Price

As mentioned in the 2006 ICH S8 Immunotoxicity Studies for Human Pharmaceuticals guidance document (ICH, 2006), a review of the data from standard toxicity studies, as well as pharmacologic properties of the drug, its targeting to immune cell types, and the immune status of the intended patient population should guide the decision to perform additional immunotoxicity testing. In this regard, additional assays may include measures of cellular immunity, such as delayed-type hypersensitivity (DTH). An advantage of the use of DTH as a parameter to gauge immunotoxicity of pharmaceuticals is that it can easily be adapted to humans and used as a relatively noninvasive immunotoxicity biomarker during clinical trials. Cellular immunity protects against intracellular bacteria, viruses and cancer, and is mediated by antigen-specific memory T lymphocytes. DTH is a measure of cellular immunity that relies on the generation of antigen (Ag)-specific memory T cells, which upon subsequent encounter with Ag, become activated, release inflammatory mediators, migrate, and recruit other cell types to the site of contact. The result is an inflammatory reaction and tissue injury. DTH can only be transferred from sensitized to normal individuals via lymphocytes (thus cell-mediated), not humorally (Ig-mediated), and often takes several days to develop. Thus, the study of DTH (as a follow-on when the weight-ofevidence review indicates additional immunotoxicity studies are warranted) in nonclinical immunotoxicity risk assessment can be a useful predictor of effects on cell-mediated immunity. Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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DTH is often studied in the skin due to its accessibility across species, especially in humans, and the availability of generally noninvasive end points for evaluation. Further, the skin’s role as a mechanical barrier to the environment has proven cutaneous immunity to be critical to host defense and immunosurveillance. DTH in human skin is often observed clinically as an erythematous, indurated, granulomatous lesion. Progression of DTH can lead to both clearance of infection and tissue damage (Kobayashi and Yoshida, 1996). Intradermal tuberculin (purified protein derivative, PPD) antigen testing (also known as the Mantoux test) is the most routine DTH skin test performed in humans to determine previous sensitization to mycobacterium tuberculosis (Poulter et al., 1982; Waldorf et al., 1991; Huebner et al., 1993; Vukmanovic-Stejic et al., 2006). The elicitation of DTH requires previous sensitization to an antigen. During the sensitization phase, antigens are recognized by antigen-presenting cells (APCs) including, but not limited to, Langerhans cells in the skin. These cells become activated and migrate to the draining lymph node where they present antigen to T cells in an MHC-restricted fashion. Antigen encounter and engagement of the T cell receptor in the presence of co-stimulatory signals results in T cell activation, clonal expansion, and the transition of naive T cells to memory/effector T cells (Figure 3.1.3-1). These T cells express skin homing receptors, which play an important role in recruitment to the site of antigen challenge (Fabbri et al., 2003; Roychowdhury and Svensson, 2005; VukmanovicStejic et al., 2006). Histologically, classical DTH responses are biphasic, composed of an initial nonspecific infiltration of primarily neutrophils at early time points (4–6 hours) followed by an influx of antigen-specific T cells (Boughton and Spector, 1963; Turk, 1980; Platt et al., 1983). Approximately 12 hours after challenge, T cells start to appear and by 48 hours, the majority of infiltrating cells are T cells and macrophages, which accumulate perivascularly although there are some diffused T cells in the epidermis and interstitium (Poulter et al., 1982; Gibbs et al., 1984; Kenney et al., 1987; Vukmanovic-Stejic et al., 2006). The DTH response is evoked primarily by CD4+ T-helper (Th)-1 cells, whereas CD8+ T cells mediate direct cell cytotoxicity. The initial encounter of naive CD4+ Th cells with antigen induces the differentiation of naive CD4 Th cells to Th-1 cells and eventually memory Th-1 cells (or TDTH cells) by cytokines including interleukin (IL)-12 and IL-18 derived from APCs and interferon (IFN)-γ produced from natural killer (NK) cells and Th-1 cells, and IL-2 produced from Th-1 cells (Trinchieri, 1995). IL-12 produced by APCs can stimulate production of IFN-γ, tumor necrosis factor (TNF)-α, and granulocyte–macrophage colonystimulating factor (GM-CSF), which causes further activation of macrophages, NK cells, and Th1 cells. Macrophages also play a pivotal role in successful elicitation of DTH reactions through production of chemokines such as IL-8, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α), which facilitate the recruitment of inflammatory cells (monocytes, neutrophils, lymphocytes) to the site of the DTH reaction. Secretion of proinflammatory cytokines such as IL-1, IL-6, TNF-α, and CC chemokines by macrophages and other APCs, in turn activates the recruited cells.


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Figure 3.1.3-1 Critical events involved in the elicitation of delayed-type hypersensitivity (DTH). DTH is a measure of cellular immunity that relies on the generation of antigen (Ag)-specific memory T cells, which upon subsequent encounter with Ag, become activated, release inflammatory mediators, migrate, and recruit other cell types to the site of contact. The result is an inflammatory reaction and tissue injury. Progression of DTH can lead to both clearance of infection and tissue damage. The elicitation of DTH requires previous sensitization to an antigen. During the sensitization phase, antigens are recognized by antigen-presenting cells (APCs) including, but not limited to, Langerhans cells in the skin. These cells become activated and migrate to the draining lymph node where they present antigen to T cells in an MHC-restricted fashion. The initial encounter of naive CD4+ Th cells with antigen and engagement of the T cell receptor in the presence of co-stimulatory signals induces the proliferation and differentiation of naive CD4 Th cells to Th-1 cells and eventually memory Th-1 cells (or TDTH cells) by cytokines including interleukin (IL)-12, IL-1, and IL-18 derived from APCs, interferon (IFN)-γ produced from natural killer (NK) cells and Th-1 cells, and IL-2 produced from Th-1 cells. Macrophages also play a pivotal role in the propagation of the process through production of chemokines such as IL-8, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and granulocyte–macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein 1α (MIP-1α), which facilitate the recruitment of inflammatory cells (monocytes, neutrophils, lymphocytes) to the site of the DTH reaction. Secretion of proinflammatory cytokines and chemokines by macrophages and other APCs, in turn activates the recruited cells, and influences the extent, quality, and duration of the cellular reaction. Illustration reproduced with the permission of the McGraw-Hill Companies.

Chemokines produced during the inflammatory process may determine the extent, quality, and duration of the cellular reaction, and may lead to significant accumulation of mononuclear cells (granuloma formation) (Kobayashi et al., 2001). Th1 cells also express CCR5 which binds several chemokines including

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MIP-1α, MIP-1β, and RANTES (Romagnani et al., 2000). In addition, adhesion molecules such as ICAM-1 are expressed in DTH lesions caused by mycobacteria and other organisms and may be involved in granuloma formation since recruited macrophages are tightly bound in the lesion (Sullivan et al., 1991). Contact hypersensitivity is a type of delayed hypersensitivity in which the antigen is exposed to the skin topically. The small haptens that can induce contact hypersensitivity would not normally be antigenic; however, in some cases these low-molecular-weight compounds can traverse the skin and then become conjugated, either covalently or noncovalently, to normal selfproteins. Such haptenized proteins become antigenic and invoke the immune response (Roitt et al., 2001). Since the DTH response is a measure of multiple cellular components involving several cell interactions, inflammatory mediators and trafficking proteins, and complex signaling cascades, several pharmacologic drug targets, especially those that suppress the immune system, have the potential to alter the magnitude and/or timing of the DTH response. Thus, DTH may be used as a model for assessing cell-mediated immunity, as well as general immune competence, in risk assessment. A brief overview of preclinical DTH models, and their advantages and limitations with respect to use in risk assessment during drug development is presented in this section. Drug hypersensitivity reactions, which can be immediate or delayed and usually involve bioactivation or covalent binding of drug to protein (Naisbitt, 2004; Bircher, 2005; Roujeau, 2005), are covered in Chapter 8 and will not be described in detail in this section. PRECLINICAL MODELS OF DELAYED-TYPE HYPERSENSITIVITY Models for DTH testing are available in many of the standard toxicology species including mice, rats, guinea pigs, and monkeys. Much less work has been reported in dogs, and this has usually been in the scope of veterinary monitoring for vaccine effectiveness (Miyomoto et al., 1992) or infection or exposure to pathogens (i.e., Leishmania) (Cardoso et al., 2007). In rodents, ear and footpad swelling methods are most common, whereas in guinea pigs and other non-rodents, the skin test is used routinely. There is growing interest in optimizing and improving DTH methods in monkeys since many biopharmaceuticals are not cross-reactive in non-primate species. Sensitization Phase In general, to assess the DTH response, animals that have been previously sensitized with an antigen or cocktail of antigens are challenged with a lower concentration of that antigen at a distal site, and the subsequent induration and erythema at the injection site are quantitated after 24–72 hours. To improve consistency of response and ensure exposure to selected antigens, animals can be manually sensitized to DTH antigens in advance. Sensitization to a neoantigen usually requires at least 100 to 200 μg of protein emulsified with an appropriate adjuvant, which is subsequently injected subcutaneously at a total

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volume of approximately 200 μL divided among multiple sites. In mice, the flanks and/or the base of the tail can be used (Sunday et al., 1980; Sinha et al., 1987; Coligan et al., 1994). In guinea pigs, dogs, and monkeys, sensitization in the upper back/shoulder region is typical (Coligan et al., 1994; Price et al., 2004). Although complete Freund’s adjuvant (CFA) has been used historically, more recent protocols have employed the use of other adjuvants, including incomplete Freund’s adjuvant (IFA), to avoid irritation, direct cytotoxicity, and antibody-mediated swelling associated with CFA (Sunday et al., 1980). In addition, use of CFA may sensitize animals to tuberculin proteins that may interfere with routine monitoring for tuberculosis infection (Bleavins and de la Iglesia, 1995). Bacterial antigens are often used for assessment of DTH response; although common immunogenic proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA) can also be employed. Dinitrochlorobenzene (DNCB) is commonly used to assess contact sensitivity. Certain antigens, such as KLH, have elicited DTH in rodents without use of a sensitization adjuvant (Exon et al., 1990). The amount of time required for sensitization can be dependent on a number of factors including the type of antigen and the species. The timing of the response can be influenced by the antigen and how well it is recognized and processed by APCs. In rodents and guinea pigs, positive footpad and/or skin DTH responses can be observed upon challenge within 6 to 14 days of sensitization (Sinha et al., 1987; Coligan et al., 1994). Generally 1 or 2 sensitizations are required. However, in monkeys, DTH responses have been much less consistent and more robust sensitization is needed. In recent experiments in our laboratory, monkeys were successfully sensitized with an antigen cocktail containing diphtheria (Dp;15 Lf), trichophyton, and C. albicans (6375 PNU) toxoids in IFA administered as five subcutaneous injections given 3 days apart (i.e., Days 1, 4, 7, 10, and 13) (Price et al., 2004). We and others have observed that at least 4 weeks between sensitization and challenge is required for elicitation of a classical skin DTH reaction in monkeys. Earlier attempts at DTH challenge (i.e., at 2 weeks post-sensitization) yielded erythematous and indurated clinical reactions, but a primarily neutrophilic response with little to no lymphocytic infiltrate was observed histologically. Challenge Phase In rodents, typical sites of challenge are in the footpads or pinnae (ear swelling assay). In guinea pigs, dogs, monkeys, and humans, the skin is usually the preferred site for DTH evaluation. A smaller quantity of antigen is used for challenge (generally ranging from 5 to 100 μg, but higher amounts can be used) in 20 to 50 μL. The vehicle or carrier for the antigen(s) is also injected or applied as a control. In rodents or guinea pigs, contact hypersensitivity can be induced by challenging the pinnae of animals previously sensitized with dinitrofluorobenzene (DNFB) or DNCB. DNFB/DNCB (100 μL of 1% w/v in acetone : olive oil [4 : 1]) is applied topically onto the shaved skin of the pinnae with a micropipette on Days 1 and 2 (Bloom and Chase, 1967; Turk, 1980; Thorne et al.,

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1991; Dhabar and McEwen, 1996). On Day 5, baseline pinna thickness is recorded for each ear using a micrometer, and subsequently animals are challenged on the dorsal surface of one ear with 50 μL of DNFB (0.5% in acetone : olive oil 4 : 1) on Day 6. The other ear is treated similarly with vehicle. The thickness of the right and left pinnae is measured with a micrometer in the same location at several time points over a 24- to 48-hour period. Pinnae can also be removed and examined histologically. Measurements should be made gently to avoid displacing and compressing edema fluid and increasing the variability in the measurements. The inflammatory response induced in this manner is characterized by swelling at the site of challenge and infiltration of macrophages and lymphocytes into the epidermis and dermis. Intradermal antigen challenge can also be performed in the pinnae, although technically more challenging (Sakato and Fujio, 1986). Alternatively, antigens that are not typical contact sensitizers (i.e., bacterial antigens including mycobacterium, OVA, KLH, BSA, hen egg white lysozyme, etc.) can be injected into the footpads of rodents. Typical protocols employ a sensitization with antigen as described above approximately 6–14 days prior to challenge. On the day of challenge, antigens solubilized in small volumes (20–50 μL) of vehicle without adjuvant are subcutaneously injected into the footpad, while the alternate footpad is challenged with vehicle. Footpad thickness is measured with a caliper-type micrometer before and at 24- to 72-hour post-challenge (Sinha et al., 1987; Exon et al., 1990; Li et al., 2006; Sun et al., 2006). Footpads can also be removed for histopathologic analysis or can be homogenized and supernatants monitored for cytokine levels. In a 2-week rat multiple immunotoxicity assay model, the same antigen (KLH) is used to induce both DTH and Tdependent humoral immune responses, and the timing of the antigen injections for DTH response (Days 1 and 8 for sensitization, Day 14 for challenge) is measured at the same time other assays are performed. A less common and less sensitive method for measuring DTH in rodents relies on the measurement of cellular infiltration, and relies on the concept that most cells entering the DTH lesion are derived from a pool of recently divided bone marrow cells (Vadas et al., 1975; Coligan et al., 1994). Antigen challenge is administered to the right or left pinna; the other pinna serves as the control. Rapidly dividing cells are labeled in 5-fluorodeoxyuridine-pretreated mice administered a thymidine analogue, 125I-5-iodo-2-deoxyuridine, 2 to 6 hours after antigen challenge. At 24 to 48 hours after challenge, both ears are removed for gamma scintillation counting, and the amount of radioactivity is proportional to the intensity of cellular infiltrate. DTH in Nonhuman Primates In primates, DTH assessment requires a more vigorous and multifaceted approach, due to the high inter-animal and inter-site variability observed, and the use of smaller numbers of animals in toxicity testing. An approach that was successful in our lab included the use of antigen cocktail containing


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diphtheria (Dp;3.75 Lf), trichophyton (625 PNU), and C. albicans (625 PNU) toxoids in IFA for sensitization (five subcutaneous injections on the dorsal thorax given every 3 days) followed by intradermal challenge, in an area distinct from the sensitization sites, with a cocktail of the same antigens at 4- to 10-fold lower amounts in phosphate-buffered saline (PBS) (Price et al., 2004). To determine the time course of cellular reaction, injections of antigen cocktail and the PBS vehicle were given at several replicate sites. Dermal responses (reaction diameter, induration, erythema) were measured at 24–96 hours after challenge and skin biopsies were obtained for histology and immunohistochemistry. Elicitation of DTH responses at 2 weeks post-sensitization was unsuccessful, complicated by a nonprogressive neutrophilic response that was only distinguishable microscopically. Classical DTH was observed at ≥4 weeks post-sensitization and sustained when challenges were administered for up to 24 weeks. Erythema preceded induration, which peaked at 72 hours. DTH cellularity was observed microscopically even if a clinical reaction was not, and was preceded by an influx of neutrophils and some macrophages at 24 hours (subacute), followed by angiocentric distribution of lymphocytes and macrophages (chronic inflammation) at 48–96 hours (Figure 3.1.3-2). Immunohistochemistry revealed an influx of macrophages and T cells (CD8 > CD4) but not B cells (Price et al., 2004). Immunomodulation of the DTH Response Successful elicitation of DTH relies on the proper functioning of numerous cell types including APCs and T cells, encompassing antigen processing and presentation, T cell recognition and activation, trafficking, and release of inflammatory mediators. Thus, there are many potential immunopharmacologic drugs that could modify or inhibit DTH. Some immunosuppressive molecules have an effect only if they are administered at the time of sensitization, whereas others can prevent recall and/or lymphocyte trafficking at the time of antigen challenge. Known immunomodulators with immune cell targets have been shown to affect DTH in various models; a few are summarized here. In rats, a footpad DTH response to KLH was significantly suppressed by cyclophosphamide (75 mg/kg) or dexamethasone (Dex; 1.5 mg/rat) administered repeatedly by subcutaneous injections during the sensitization and challenge phases, and this suppression correlated with a decrease in NK cell activity, Tdependent antibody production, and IL-2 synthesis (Exon et al., 1990). In previously sensitized (Ag cocktail of diphtheria [Dp], trichophyton, and C. albicans toxoids) cynomolgus monkeys, clinical signs of DTH challenge reactions following oral (1 week during challenge) Dex (2 or 20 mg/kg) or cyclosporin A (120 mg/kg) were not diminished, and humoral immunity to Dp was not suppressed. However, dose-related decrease in cellularity and subacute, rather than chronic, inflammation was observed in monkeys given Dex (Figure 3.1.3-3) (Price et al., 2004). Thus, clinical signs may not always predict classical DTH and microscopic confirmation is needed, particularly to monitor

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A 24 hr

48 hr

72 hr

96 hr

Figure 3.1.3-2 (A) Time course of the cellular DTH reaction in cynomolgus monkey skin. Cynomolgus monkeys, previously sensitized subcutaneously with an Ag cocktail containing diphtheria (Dp;15 Lf), trichophyton, and C. albicans (6375 PNU) toxoids in IFA on Days 1, 4, 7, 10, and/or 13, received intradermal challenge injections of Ag cocktail (containing 4–10× lower amounts of each antigen) at 2 to 24 weeks postsensitization. Dermal responses (reaction diameter, induration, erythema) were measured at 24–96 hours post-challenge and skin biopsies were obtained for histology and immunohistochemistry. Elicitation of DTH responses at 2 weeks post-sensitization was unsuccessful, complicated by a nonprogressive neutrophilic response that was only distinguishable microscopically. Classical DTH was observed at ≥4 weeks postsensitization and sustained for >24 weeks as indicated by histopathologic examination of infiltrating cells. At 24 hours post-challenge (upper left), an influx of neutrophils and some macrophages was observed (subacute inflammation), followed by angiocentric distribution of lymphocytes and macrophages (chronic inflammation) at 48 hours (upper right), and persistence of chronic inflammation at 72 and 96 hours (lower left and right). Immunohistochemistry revealed an influx of macrophages and T cells (CD8 > CD4) but not B cells. Clinically, induration at the antigen injection site peaked at 72 hours. (B) Sites of cellular inflammation were located primarily around blood vessels (indicated by arrows) and were absent in the saline control challenge sites.


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B Saline control

Antigen cocktail

Figure 3.1.3-2 Continued

Control - Ag cocktail

Dex, 20 mg/kg - Ag cocktail

120 micron

120 micron

Figure 3.1.3-3 DTH in cynomolgus monkey skin is suppressed by dexamethasone. Cynomolgus monkeys (n = 4/group), previously sensitized subcutaneously with an Ag cocktail containing diphtheria (Dp;15 Lf), trichophyton, and C. albicans (6375 PNU) toxoids in IFA on Days 1, 4, 7, 10, and/or 13, received intradermal challenge injections of Ag cocktail (containing 4–10× lower amounts of each antigen) at 4 to 7 weeks postsensitization. A dose-related decrease in cellularity and a delay in the progression of the DTH response from acute to chronic inflammation was observed in monkeys given dexamethasone at 2 (not shown) or 20 mg/kg (above right). Microscopically, the inflammatory changes in skin biopsies were characterized as acute or subacute at up to 72 hours post-challenge in monkeys receiving dexamethasone relative to the chronic inflammation observed at ≥48 hours in vehicle controls (above left); however, gross clinical reactions were not diminished in dexamethasone-treated monkeys. Thus, clinical reactions may not always predict classical DTH and microscopic confirmation is needed, particularly to monitor immunomodulation.

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immunomodulation. Others have shown similar immunosupression of the monkey DTH response with topical corticosteroids known to inhibit T cell function (0.05% diflorasone diacetate cream, given 3 times/day during the challenge phase) (Bleavins and de la Iglesia, 1995). DTH has also been used to assess affects on the developing immune system (see Chapter 9.1). However, other nonimmunologic effects can also influence DTH and you must be careful to distinguish between these and immunologic effects. For example, stress has also been shown to affect DTH cell-mediated immunity. Sprague-Dawley rats previously sensitized to DNFB and exposed to acute stressors (restraint stress confinement in a Plexiglass tube or shaking stress due to the placement of the animal’s cage on an oscillatory shaker) immediately before challenge of DNFB to the pinnae exhibited a statistically significant increase in the DTH response (ear thickness with histological confirmation) compared to unstressed control animals. Further, the time course of DTH was faster and attained a higher peak (Dhabar and McEwen, 1996). Oxidative stress, which increases with age, also interferes in Th-1 cell-mediated immunity, and thus, DTH. Glutathione depletion (redox disequilibrium), induced by intraperitoneal administration of diethyl maleate at 4.5 mmol/kg 1 hour before oxazalone (OXA) challenge to the pinnae of previously sensitized mice, significantly reduced the ear swelling DTH response and ear tissue IFN-γ message levels, suggesting effects on Th-1 helper cell differentiation/activation (Kim et al., 2007). Glutathione depletion in DNFB-pulsed dendritic cells also inhibited the DTH response in recipient mice upon adoptive transfer. In contrast, the glutathione precursor, N-acetyl cysteine, administration reversed the typical decline of DTH response in aged (19–21 months) mice, suggesting that glutathione repletion is capable of reversing decreased Th1 immunity with aging (Kim et al., 2007). DTH is used less commonly to study pharmacologic immunostimulation. However, it may be possible to modulate the response to achieve granuloma formation or eventual tissue damage. As shown by Tanaka et al. (2007), mice sensitized with 0.25 mg/body (two sites on abdomen) mBSA elicited a prolonged (for at least 7 days) footpad mBSA-specific DTH response (0.05 mg mBSA/footpad, sc) when injected intravenously with an anti-collagen type II monoclonal antibody (0.5 mg/footpad) 4 days after sensitization. In addition to classical DTH histopathology in the swollen footpad, the hindpaw was also swollen with joint inflammation and bone destruction. This model was henceforth termed DTH-arthritis and, through adoptive-transfer experiments of splenocytes from sensitized mice to SCID mice, was shown to be mediated by antigen-specific CD4+ T cells. In contrast, footpad challenge of mBSAsensitized mice with a neoantigen, KLH (0.05 mg/footpad), and the anticollagen antibody did not induce DTH arthritis (Tanaka et al., 2007). In monkeys, the addition of oligonucleotide CpG to the antigen cocktail during sensitization did not enhance the challenge DTH response, although it did augment humoral immunity to the challenge antigens (Price et al., 2004).

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DTH TESTING IN RISK ASSESSMENT DURING DRUG DEVELOPMENT: ADVANTAGES AND CONSIDERATIONS Since assays to measure cell-mediated immunity are not as well established and not as quantitative as those used to measure other immune end points, inclusion of the DTH test within a battery of other immune function assays or studies would be beneficial in determining the relative risk of immunotoxicity. A weight-of-evidence approach should be used when evaluating DTH responses to determine the biological and/or toxicological relevance for observed changes. The following points should be considered: (1) Are there statistically significant and/or biologically relevant changes in the magnitude, time course, or cell composition of the DTH reaction in drug-treated animals compared to vehicle-control animals? (2) Is there a dose-response in incidence and severity for the observed change? (3) Are there other changes in immune parameters (e.g., changes in hematology, organ weight, and/or histopathologic changes in lymphoid organs, effects on functional immune response, increased incidence of infections or tumors) in drug-treated animals? Evaluation of the incidence of individual drug-treated animals with changes in magnitude or kinetics of DTH response is especially important in nonrodent species due to the small number of animals typically evaluated and variability between animals. Like the T-dependent antibody response, elicitation of DTH requires proper functioning of several cell types and signal transduction cascades, and thus can be altered in a number of ways by investigational drugs. However, the redundancy of the immune system may also mask subtle immune toxicities and require potent immunomodulation to measure robust effects in the DTH models. It is important to recognize species differences within the context of DTH as well. In mice, the DTH response tends to be more neutrophil rich compared to humans (Crowle, 1975; Mestas and Hughes, 2004). Also, recall responses may occur by different mechanisms in mice and humans, involving draining of antigen to lymph nodes in mice compared with local antigen presentation by endothelial cells (EC) in humans. This is based on evidence that human EC can present antigen to and activate resting memory CD4+ cells; whereas in mice, EC cannot activate CD4+ T cells (Pober et al., 2001; Mestas and Hughes, 2004). The timing required between sensitization and challenge to elicit an optimal DTH response can vary from species to species, and can also be dependent on the sensitization antigen. In cynomolgus monkeys, at least 4 weeks is required following sensitization to elicit a measurable antigen-specific response to an intradermal antigen challenge. Attempts to challenge the animals prior to 4 weeks have yielded cellular responses composed primarily of neutrophils

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with fewer macrophages and no discernable T cell infiltrates. These early reactions are likely nonspecific and not mediated by T cells, but due to immunecomplex deposition and subsequent complement activation (Arthus reactions). Conversely, primates have been shown to remain sensitized for up to at least 24 weeks post-sensitization, and thus could potentially be reused for multiple evaluations. Due to the variability among animals and antigens, it is important to study as many parameters as possible within the context of a single study and use each animal as its own control. Since the DTH response is a measure of multiple cellular components, the clinical signs alone (scoring of redness/erythema and measurements of induration or swelling/thickness) may not always yield data in the most useful form. In primates in particular, clinical reactions have not been robust and the assays have shown a very high inter-animal and inter-site variability. In order to gauge the cellular response to a particular antigen and any effects of orally or parentally administered drugs on the DTH response, histopathologic analysis of a biopsy of the intradermal challenge site is usually warranted to confirm the nature of the response. The biopsied site should contain perivascular distribution of lymphocytes and macrophages with low numbers of neutrophils. Quantitation of specific cell types can be accomplished via immunohistochemistry; however, it may also be useful to assess local cytokine profiles via quantitation of protein or message expression. Serial biopsies collected over a time course can also aid in the relative timing of the antigen-specific cellular response. DTH models may also be used to study potential immunostimulation of a drug but again, due the variability of the response, a profound increase in severity would be needed to suggest a true drug effect. SUMMARY The incorporation of DTH testing into immunotoxicity assessment may provide supporting evidence of effects on cellular immunity. DTH data should be interpreted with respect to effects on other immune end points as part of a weight-of-evidence approach for assessing immunotoxicity. As DTH models are evolving rapidly and adapting to newer technologies (i.e., gene and protein expression profiling within biopsied tissue), they will likely become more quantitative, and thus more advantageous for assessing cell-mediated immunity in vivo. REFERENCES Bircher AJ. Symptoms and danger signs in acute drug hypersensitivity. Toxicology 2005; 209:201–207. Bleavins MR, de la Iglesia FA. Cynomolgus monkeys (Macaca fascicularis) in preclinical immune function safety testing: development of a delayed-type hypersensitivity procedure. Toxicology 1995;95:103–112.

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Bloom BR, Chase MW. Transfer of delayed-type hypersensitivity: a critical review and experimental study in the guinea pig. Prog Allergy 1967;10:151–255. Boughton B, Spector WG. Histology of the tuberculin reaction in guinea pigs. J Pathol Bacteriol 1963;85:371–381. Cardoso L, Schallig HDFH, Cordeiro-da-Silva A, Cabral M, Alunda JM, Rodrigues M. Anti-Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs. Vet Immunol Immunopathol 2007;117: 35–41. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current Protocols in Immunology, Vol 1. New York, NY: John Wiley & Sons, 1994. Crowle AJ. Delayed type-hypersensitivity in the mouse. Adv Immunol 1975;20:197. Dhabar FS, McEwen BS. Stress-induced enhancement of antigen-specific cell-mediated immunity. J Immunol 1996,156:2608–2615. Exon JH, Bussiere JL, Mather GG. Immunotoxicity testing in the rat: an improved multiple assay model. Int J Immunopharmacol 1990;12(6):699–701. Fabbri M, Smart C, Pardi R. T lymphocytes. Int J Biochem Cell Biol 2003;35: 1004–1008. Gibbs JH, Ferguson J, Brown RA, Kenicer KJ, Potts RC, Coghill G, Swanson Beck J. Histometric study of the localisation of lymphocyte subsets and accessory cells in human Mantoux reactions. J Clin Pathol 1984;37:1227–1234. ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006. Huebner RE, Schein MF, Bass JB. The tuberculin skin test. Clin Infect Dis 1993; 17(6):968–975. Kenney RT, Rangdaeng S, Scollard DM. Skin blister immunocytology. A new method to quantify cellular kinetics in vivo. J Immunol Methods 1987;97:101–110. Kim HJ, Barajas B, Chun-Fai Chan R, Nel AE. Glutathione depletion inhibits dendritic cell maturation and delayed-type hypersensitivity: implications for systemic disease and immunosenescence. J Allergy Clin Immunol 2007;119:1225–1233. Kobayashi K, Yoshida T. The immunopathogenesis of granulomatous inflammation induced by mycobacterium tuberculosis. Methods 1996;9(2):204–214. Kobayashi K, Kaneda K, Kasama T. Immunopathogenesis of delayed-type hypersensitivity. Microsc Res Tech 2001;53:241–245. Li J, Bracht M, Shang X, Radewonuk J, Emmell E, Griswold DE, Li L. Cell Immunol 2006;241:75–84. Mestas J, Hughes CW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–2738. Miyomoto T, Taura Y, Une S, Yoshitake M, Nakama S, Watanabe S. Changes in blastogenic responses of lymphocytes and delayed-type hypersensitivity responses after vaccination in dogs. J Vet Med Sci 1992;54(5):945–950. Naisbitt DJ. Drug hypersensitivity reactions in skin: understanding mechanisms and the development of diagnostic and predictive tests. Toxicology 2004;194: 179–196. Platt JL, Grant BW, Eddy AA, Michael AF. Immune cell populations in cutaneous delayed-type hypersensitivity. J Exp Med 1983;158:1227–1242.

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Pober JS, Kluger MS, Schechner JS. Human endothelial cell presentation of antigen and the homing of memory/effector T cells to skin. Ann N Y Acad Sci 2001; 941:12–25. Poulter LW, Seymour GJ, Duke O, Janossy G, Panayi G. Immunohistiological analysis of delayed-type hypersensitivity in man. Cell Immunol 1982;74(2):358–369. Price K, Mezza L, Diters R, Wells S, Devona D, Tzogas Z, Haggerty H. Development and immunomodulation of delayed-type hypersensitivity (DTH) in cynomolgus monkeys. (Abstract 2091) The Toxicologist 2004. Roitt I, Brostoff J, Male D, editors. Immunology, 6th ed. London, England: Mosby, 2001. Romagnani P, Annunziato F, Piccinni MP, Maggi E, Romagnani S. Cytokines and chemokines in t lymphocytes and T-cell effector function. Immunol Today 2000; 21:416–418. Roujeau JC. Clinical heterogeneity of drug hypersensitivity. Toxicology 2005;209: 123–129. Roychowdhury S, Svensson C. Mechanisms of drug-induced delayed-type hypersensitivity reactions in the skin. AAPS J 2005;7(4):E834–E846. Sakato N, Fujio H. Suppression of the delayed-type hypersensitivity response to hen egg white lysozyme (HEL) by HEL peptides in a genetically high-responder mouse strain: evidence for requirement of the loop structure for induction of suppressor T cells. Cell Immunol 1986;100:66–78. Sinha S, Sreevat SA, Gupta SK, Sengupta U. Comparative study of immunizing and delayed hypersensitivity eliciting antigens of Mycobacterium leprae, M tuberculosis, M vaccae, and M bovis (BCG). Int J Lepr Other Mycobact Dis 1987;5(1);42–53. Sullivan L, Sano S, Pirmez C, Salgame P, Mueller C, Hofman F, Uyemura K, Rea TH, Bloom BR, Modlin RL. Expression of adhesion molecules in leprosy lesions. Infect Immunol 1991;59:4154–4160. Sun JB, Cuburu N, Blomquist M, Li BL, Czerkinsky C, Holmgren J. Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+ CD25+ CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions. Scand J Immunol 2006;64:251–259. Sunday ME, Weinberger JZ, Benacerraf B, Dorf ME. Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. IV. Specificity of cutaneous sensitivity responses. J Immunol 1980;25:1601–1605. Tanaka D, Kagari T, Doi H, Shimozato T. Administration of anti-Type II collagen antibody sustains footpad swelling of mice caused by a delayed-type hypersensitivity reaction and induces severe arthritis. Clin Exp Immunol 2007;48:360–367. Thorne PS, Hawk C, Kaliszewski SD, Guiney PD. The non-invasive mouse ear swelling assay. I. Refinements for detecting weak contact sensitizers. Fundam Appl Toxicol 1991;17:790–806. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Ann Rev Immunol 1995;13:251–276. Turk JL. Delayed Hypersensitivity, 3rd ed. Amsterdam, the Netherlands: Elsevier, 1980.

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Vadas MA, Miller JFAP, Gamble J, Whitelaw A. A radioisotopic method to measure delayed-type hypersensitivity in the mouse. I. Studies in sensitized and normal mice. Int Arch Allergy Appl Immunol 1975;49:670–692. Vukmanovic-Stejic M, Reed JR, Lacy KE, Rustin MHA, Akbar AN. Mantoux test as a model for a secondary immune response in humans. Immunol Lett 2006;107: 93–101. Waldorf HA, Walsh LJ, Schechter NM, Murphy GF. Early cellular events in evolving cutaneous delayed hypersensitivity in humans. Am J Pathol 1991;138(2):477–486.

3.2 EVALUATION OF DRUG EFFECTS ON IMMUNE CELL PHENOTYPES Laurie Iciek

Lymphocyte immunophenotyping using flow cytometry can be a useful tool to gain information pertinent to the overall risk assessment of the immunotoxic potential of pharmaceuticals. Lymphocyte immunophenotyping provides information about the types of cells that are altered following exposure to a drug, which can aid in the design and selection of appropriate functional tests to incorporate into future studies. Reagents are now available to assess lymphocyte subpopulations in many of the standard toxicology species including mice, rats, beagle dogs, baboons, marmosets, and monkeys (cynomolgus and rhesus). The standard set of markers used for lymphocyte immunophenotyping generally includes markers for total T and B cells, CD4+ T-helper (Th) cells, and CD8+ T-cytotoxic/suppressor (Tcyt/sup) cells. In addition to these subsets, reagents are available to monitor natural killer (NK) cells in mice, rats, and monkeys. These same lymphocyte populations can also be monitored in the peripheral blood of patients in clinical trials, to help determine the relevance of animal findings for humans and to establish a biomarker for immunomodulatory effects. Much of the earlier work that showed a high concordance for the ability of lymphocyte subset enumeration to predict immunotoxicity (more specifically immunosuppression) was obtained from studies of environmental chemicals and pesticides in mice. More recently, a growing number of publications have also shown agreement between alterations in peripheral blood or splenic lymphocyte subsets and decreased T cell-dependent antibody responses and/ Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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or NK cell activity in rats, beagle dogs, monkeys, and baboons following administration of immunosuppressive pharmaceuticals (Descotes et al., 1996; Quesniaux et al., 1999; Jones et al., 2000; Smith et al., 2003; Roman et al., 2004; Vugmeyster et al., 2006; Amevive® BLA). A brief overview of the history behind the use of lymphocyte immunophenotyping in risk assessment, considerations and timing for inclusion of lymphocyte immunophenotyping in the risk assessment process, study and assay design, and relevance of animal data to effects in humans are presented in the sections that follow.

HISTORICAL PERSPECTIVE ON THE USE OF LYMPHOCYTE IMMUNOPHENOTYPING IN IMMUNOTOXICITY RISK ASSESSMENT FOR PHARMACEUTICALS In 1999, the European Committee for Proprietary Medicinal Products (CPMP) issued a Note for Guidance on Repeated Dose Toxicity that included lymphocyte immunophenotyping plus NK cell activity as a recommended tier one test that could be used to screen pharmaceuticals for immunotoxic potential (EMEA, 2000). The selection of this combination of tests stems from an extensive collaborative study of the effects of 51 chemicals and pesticides in mice conducted by NIEHS Immunotoxicology Laboratory, the National Toxicology Program sponsored laboratories, and the Cell Biology Department at the Chemical Industry Institute of Toxicology (Luster et al., 1988, 1992). The immunotoxic potential of chemicals was assessed using a “tier approach,” with the first tier of studies evaluating cell-mediated immunity, humoral immunity and immunopathology, and the second tier conducted to further define which populations of cells in the immune system were affected. The data were then analyzed to determine which testing paradigm could identify compounds that altered host resistance against pathogens or tumors and which test, or combination of tests, had the same outcome as conducting the full battery of tests. As a single test, determination of lymphocyte population numbers (lymphocyte immunophenotyping) via flow cytometry had an 83% concordance value for prediction of an immunotoxic outcome, whereas simply assessing leukocyte counts through standard hematology was only 43% predictive (Luster et al., 1992). When lymphocyte immunophenotyping was combined with NK cell activity, the concordance was increased to 90% (Luster et al., 1992). Together these findings suggested that effects on lymphocyte subpopulations were predictive for effects on host resistance against pathogens or tumors; however, the authors of the publication cautioned against overinterpretation of the findings because lymphocyte subpopulations were only measured for nine of the chemicals studied (Luster et al., 1992). Subsequent to the studies conducted in mice, a two-phase inter-laboratory study for quantification of rat splenic lymphocyte subpopulations using immunofluorescent staining and flow cytometry was conducted to determine if this method would be useful for immunotoxicology studies (Ladics et al., 1997,

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1998). The first phase of the study included analyses conducted in six independent laboratories that used a common protocol and the same reagents in order to determine baseline values for rat splenic lymphocyte populations, the variability of flow cytometry data within and between laboratories, and the value of single versus dual cell labeling and quadrant and histogram analysis procedures (Ladics et al., 1997). The conclusions from the first phase of the study indicated that intra-laboratory variability was similar across analysis and labeling procedures (Ladics et al., 1997). When absolute versus relative numbers of lymphocytes were compared, intra-laboratory variability was higher and inter-laboratory reference ranges were large (Ladics et al., 1997). In the second phase of the study, four independent laboratories evaluated rat splenic lymphocyte populations following exposure to the immunosuppressive agent cyclophosphamide (CY), to determine if data obtained from lymphocyte subset phenotyping could show effects of an immunosuppressive agent (Ladics et al., 1998). At the highest CY dose tested (10 mg/kg), each laboratory was able to detect immunosuppressive effects of CY on B and T cell phenotypic markers (Ladics et al., 1998).

CONSIDERATIONS AND TIMING FOR INCLUSION OF LYMPHOCYTE IMMUNOPHENOTYPING IN THE RISK ASSESSMENT PROCESS When determining the necessity for inclusion of lymphocyte phenotyping in the risk assessment process, the same considerations as outlined in the 2006 ICH S8 (Immunotoxicity Studies for Human Pharmaceuticals) guidance document should be applied (ICH, 2006). Assessment of lymphocyte subsets may provide beneficial information in the following cases: (i) data from standard toxicity studies indicate an effect on the immune system (e.g., alterations in peripheral blood lymphocytes; organ weight and/or histopathologic changes in lymphoid tissues); (ii) the intended pharmacologic action of the drug is to modulate the immune system; (iii) the intended patient population is immunocompromised due to disease or concurrent medications; (iv) the drug is structurally similar to known immunomodulators; (v) the drug accumulates in immune cells; and (vi) there is clinical information suggesting immunomodulation (e.g., increased incidence of infections or tumors). Although the ICH S8 guidance indicates that immunotoxicity studies should be conducted prior to exposure of a large population of patients (generally Phase 3 clinical trials) or at an earlier time point in development if the patient population is immunocompromised, it may be of value to include lymphocyte subset analysis in the early toxicity studies (e.g., 4-week studies) for drugs that are immunomodulatory and for drugs that will be administered to immunocompromised patient populations. An evaluation of lymphocyte subsets may help to determine populations of immune cells that are being altered which in turn could aid in the design of functional studies. If these studies are conducted early, then

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lymphocyte subset evaluations and appropriate screening for functional effects can be added into sub-chronic animal toxicity studies avoiding the need for separate studies. When appropriate, additional monitoring can be included in Phase 2 human clinical trials. If the pharmacologic action of the drug is immunomodulation and the drug will be administered to pregnant women or a pediatric patient population, then it may be useful to include an evaluation of lymphocyte subsets (along with immune function tests) in pre- and postnatal development animal studies or juvenile animal toxicity studies, respectively. The ICH S8 guidance does not include biologics; however, these same considerations should be used for biologics, many of which are designed to modulate the immune system or for use in immunocompromised patient populations. Designing Appropriate Studies for Risk Assessment As indicated in the ICH S8 guidance, the overall assessment of immunotoxicity risk is similar to the assessment of the toxicity risk for other organ systems and includes the following parameters: (i) statistical and biological significance of the changes; (ii) severity of the effects; (iii) dose/exposure relationship; (iv) safety factor above the expected clinical dose; (v) treatment duration; (vi) number of species and end points affected; (vii) changes that may occur secondarily to other factors; (viii) possible cellular targets and/or mechanism of action; (ix) doses that produce these changes in relation to doses that produce other toxicities; and (x) reversibility of effects (ICH, 2006). In order to have appropriate information to conduct a valuable immunotoxicity risk assessment, it is important that the overall study design addresses these parameters. Factors that can influence interpretation of the statistical and biological relevance of apparent drug-related changes in lymphocyte subpopulations include the number of animals available for evaluation in each dose group, both at the end of the dosing and recovery periods; the availability of historical ranges for the lymphocyte subsets being assessed; the availability of baseline lymphocyte immunophenotyping data; and the overall accuracy/precision of the immunophenotyping assay. For rodent studies, a minimum of 5 animals/ gender/dose group should be available for assessment at the end of the dosing period and at the end of the recovery period. For non-rodent studies, there are generally 5 animals/gender/dose group evaluated at the end of the dosing period and 2 animals/gender/dose group evaluated at the end of the recovery period. However, consideration may be given to the use of 6 animals/gender/ dose group which would allow for statistical evaluation of 3 animals/gender/ dose group at the end of the recovery period. This information may be particularly important if immunomodulatory effects have been shown in previous studies or other species. The availability of historical ranges for the applicable lymphocyte subpopulations can impact the overall data interpretation. Historical ranges can be used to determine if changes observed in drug-treated groups are due to outof-range control values (e.g., controls are higher or lower than historic range)


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and can determine if values are significantly increased or decreased but still within historical range, suggesting that there is no biological relevance for the observed change. In addition to these points, historical ranges can be used to evaluate baseline data and help to screen out animals that appear to have altered lymphocyte subset parameters which can have a great impact on data review, since the non-rodent animal studies generally have very few animals for assessment in each dose group and there is often a high degree of variability between animals. For nonhuman primates, there are demonstrated differences in various immune cell parameters depending on which geographic region (mainland versus island) the monkeys come from (Lappin and Thomas, 2001). Thus, it is advisable that all studies for one compound are conducted in monkeys from the same geographic region. Published data (Bleavins et al., 1993; Franch et al., 1993; Tryphonas et al., 1996; Morris and Komocsar, 1997; Byrne et al., 2000; Jones et al., 2000) for peripheral blood lymphocyte subset percentages and absolute numbers in rat, dog, monkey, and human are included in Tables 3.2-1 and 3.2-2. Caution should be taken when using published data for comparisons as multiple factors, including reagents, multiparameter settings, and animal age can influence lymphocyte subset ranges. Published literature is also available for lymphocyte subsets at various developmental stages and/or ages in rats, dogs, and monkeys (Baroncelli et al., 1997; Nam et al., 1998; DeMaria et al., 2000; Barrow and Ravel, 2005; Faldyna et al., 2005). When possible, historical ranges should be determined at the time of assay validation. Although this can readily be done for peripheral blood lymphocytes, it may not be feasible for lymphoid organs such as the spleen, which would require additional animal usage. To determine normal ranges for lymphocyte subsets in lymphoid organs, it may be necessary to collect data from vehicle control animals retrospectively. A statistician should be consulted to determine if it is appropriate to combine data sets obtained with different control vehicles. If the drug in question is active in both rodent and non-rodent species, and appropriate reagents are available, then lymphocyte immunophenotyping should be assessed in both species. Assessments should include pre-study (baseline), end of treatment, and recovery time points whenever possible. Although this may not be possible for small rodent species, at least one, but preferably two or even three (Lappin and Black, 2003), baseline assessments are critical for non-rodent animal species where individual variability may be large and the number of animals assessed is small. Baseline data can help to select animals with a normal lymphocyte subset profile (repeated measurements show that the profile is stable) and repeated measurements aid in the understanding of within animal variation, which can help to determine the biological relevance of changes observed within each individual animal following drug treatment. This approach allows for monitoring of effects in each individual animal, as well as overall group effects. Incorporation of Immunophenotyping into Standard Toxicity Studies. Lymphocyte immunophenotyping can be performed as part of the standard toxicity

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TABLE 3.2-1 Percentage of Peripheral Blood B, T, CD4+ T-Helper, and CD8+ T Cytotoxic/Suppressor Lymphocytes in Rat, Dog, Monkey, and Human Percentage Peripheral Blood Lymphocytes Species Sprague-Dawley rat

a

Wistar ratb Beagle dogc Beagle dogd Cynomolgus monkey (4–10 years)e Cynomolgus monkey (4.2–5 years)f Humang

Gender

B

T

CD4+ T

CD8+ T

NK Cells

M F F F M&F M F M&F

38.5 33.7 18.6 12.9 11.2 11.6 12.7 14.1

48.9 54.6 75.8 83.3 82.1 ND ND 63.5

33.3 35.8 59.0 45.0 57.9 28.1 30.4 33.9

16.5 21.8 24.5 28.8 20.4 55.8 53.8 46.8

ND ND ND ND ND ND ND 16.8

M F

12.6 12.7

ND ND

43.0 48.8

24.1 22.3

ND ND

ND = Not Determined; M = Male, F = Female. a Single- or two-color analysis; surface markers included CD45RA (B cells), CD3 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Morris and Komocsar, 1997). b Single-color analysis; surface markers included sIg (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Franch et al., 1993). c Single-color analysis; surface markers included CD21 (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Byrne et al., 2000). d Single-color analysis; surface markers included CD21 (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Jones et al., 2000). e Single-color analysis; surface markers included CD20 (B cells), CD4 (Th), CD8 (Tcyt/sup); (Bleavins et al., 1993). f Two- or three-color analysis; surface markers included CD20 (B cells), CD3 (T cells), CD4 (Th), CD8 (Tcyt/sup), CD16 (NK); (Tryphonas et al., 1996). g Single-color analysis; surface markers included CD20 (B cells), CD4 (Th), CD8 (Tcyt/sup); (Bleavins et al., 1993).

studies provided that sufficient data are available from dose range-finding toxicity studies to identify one or more doses that exhibit minimal systemic toxicity. This paradigm could be advantageous (for example, if the pharmacologic action of the drug is immunomodulation) to help define what lymphocyte populations are altered by the drug and help to design functional studies. If all doses produce systemic toxicity, then it may be difficult to determine whether effects on lymphocytes represent primary effects or if they are secondary to other systemic toxicities. Another confounding effect of systemically toxic doses is the induction of stress, which may lead to increased levels of glucocorticoids and subsequent secondary changes in lymphocyte subsets. Based on published data, rodent, dog, and human lymphocytes are sensitive to the immunomodulatory (immunosuppressive) effects of glucocorticoids (Franchimont, 2004; Igarashi et al., 2005; Ammersbach et al., 2006).

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TABLE 3.2-2 Absolute Number of Peripheral Blood B, T, CD4+ T-Helper, and CD8+ T Cytotoxic/Suppressor Lymphocytes in Rat, Dog, Monkey, and Human Absolute Number Peripheral Blood Lymphocytes (×109/L) Species Sprague-Dawley rata Wistar ratb Beagle dogc Beagle dogd Cynomolgus monkeye Humanf

B

Total T

5.0 M 3.4 F 0.65 F NR NR 0.8 M 0.9 F 0.3 M 0.3 F

6.2 M 5.2 F 2.51 F NR NR ND ND

CD4+ T

CD8+ T

CD4/CD8 Ratio

4.2 M 3.5 F

2.1 M 2.1 F 0.82 F NR NR 3.8 M 3.7 F 0.5 M 0.5 F

2.1 M 1.7 F 2.5 F 1.87 F 2.83 0.53 M 0.59 F 1.94 M 2.48 F

NR NR 1.9 M 2.1 F 0.9 M 1.2 F

ND = Not Determined; M = Male, F = Female; NR = Not Reported. a Single- or two-color analysis; surface markers included CD45RA (B cells), CD3 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Morris and Komocsar, 1997). b Single-color analysis; surface markers included sIg (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Franch et al., 1993). c Single-color analysis; surface markers included CD21 (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Byrne et al., 2000). d Single-color analysis; surface markers included CD21 (B cells), CD5 (T cells), CD4 (Th), CD8 (Tcyt/sup); (Jones et al., 2000). e Single-color analysis; surface markers included CD20 (B cells), CD4 (Th), CD8 (Tcyt/sup); (Bleavins et al., 1993). f Single-color analysis; surface markers included CD20 (B cells), CD4 (Th), CD8 (Tcyt/sup); (Bleavins et al., 1993).

Incorporation of Immunophenotyping into Immunotoxicity Studies. In cases where the standard toxicity studies indicate there may be effects on the immune system, lymphocyte immunophenotyping can be incorporated into an immunotoxicity study (Roth et al., 2006). This approach has been studied in 4-week rat toxicity studies for calcineurin inhibitors cyclosporine A (CsA), tacrolimus (FK506, Prograf), and pimecrolimus (ASM981), and for cyclophosphamide (Descotes et al., 1996; Richter-Reichhelm and Schulte, 1998; Smith et al., 2003; Roman et al., 2004; Ulrich et al., 2004; Kurogi et al., 2005). The parameters generally assessed include in-life parameters (clinical observations, body weight, and food consumption); hematology and clinical chemistry parameters; antibody response to a T cell-dependent antigen such as keyhole limpet hemocyanin (KLH), or another functional test such as NK cell activity, as appropriate; lymphocyte phenotyping in peripheral blood and/or thymus, spleen, and lymph nodes; macroscopic pathology; lymphoid organ weights; and histopathological evaluation of lymphatic organs/tissues and of the injection site following a secondary immunization with either the KLH or a neoantigen.

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The test item is administered to 5 or 10 rats/gender/dose group using the route of administration indicated for use in humans. Considerations for dose selection include use of at least one dose level that is not anticipated to have effects on the immune system, and one dose level that may have shown some effects on the immune system in standard 4-week or range-finding toxicity studies. When possible, at least one or more dose levels should also provide a safety margin above the anticipated efficacious clinical dose/exposures. There are generally five dose groups: nonimmunized placebo control, placebo control immunized with KLH, low-dose test item immunized with KLH, mid-dose test item immunized with KLH, and high-dose test item immunized with KLH. Although many of the published studies did not include recovery animals, reversibility (including functional test) should be assessed. If the known pharmacologic activity of the drug is immunomodulation or data from rangefinding studies suggest immunomodulation, a similar design could be used in a 4-week standard toxicity study in rats (Roth et al., 2006), provided that sufficient range-finding toxicity data are available for dose selection. Assessments of peripheral blood and/or lymphoid organ lymphocyte subsets and immune function testing (e.g., T cell-dependent antibody response and/or delayed-type hypersensitivity [DTH] reaction) have also been incorporated into standard toxicity studies in the monkey and baboon (Quesniaux et al., 1999; Vugmeyster et al., 2006; Amevive® BLA). This approach allows for direct comparisons between effects on lymphocyte subsets from both the peripheral blood and lymphoid tissues, and effects on immune system function. It also provides direct support for the use of lymphocyte immunophenotyping as a biomarker for potential effects in human clinical trials. Lymphocyte Phenotyping in Developmental and Juvenile Toxicity Studies Studies to assess immunotoxic potential should be considered for immunomodulatory drugs that will be administered to pregnant women and/or young patient populations (see Chapters 9.1 and 9.2). As part of this overall assessment, lymphocyte immunophenotyping may be added to pre- and postnatal toxicity studies if the concern is exposure in utero or via milk after birth or in juvenile toxicity studies if the drug is intended for use in a pediatric patient population. In the case of pre- and postnatal toxicity studies, it is important to include assessment at varying postnatal stages in order to determine if the drug simply delays development of the immune system or results in an irreversible alteration (e.g., immunosuppression). Immune function tests and histopathologic assessment of lymphoid organs are also usually included in the study design, allowing for direct comparisons between effects on lymphocyte subsets and immune function. In addition to the standard panel of markers, it may be important to monitor the CD4+CD8+ double positive T cell population in the periphery. Normally these immature cells are not released from the thymus so an increase in this population may be indicative of effects on thymic selection and/or maturation.

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Assay Design Surface Markers. Lymphocyte immunophenotyping analysis generally includes markers for assessment of total T and B cells, CD4+ T-helper cells, CD8+ T cytotoxic/suppressor cells, and NK cell populations (if reagents are available). The surface markers that are commonly used to define each of these cell populations are presented in Table 3.2-3. Commercial antibodies are available for use in mouse, rat, dog, monkey, marmoset, baboon, and chimpanzee (BD Biosciences catalog; Ab Direct catalog). Additional parameters may be assessed based on pharmacologic targets for the drug or microscopic evidence of drug effects in bone marrow, thymus, or spleen. For assessment of nonroutine parameters, the availability of commercial antibodies may be more limited for rat and dog; however, there are many antibodies commercially available for use in the mouse, monkey, and human (see Chapter 4.2). Although early studies conducted in rats utilized single parameter analysis, it was subsequently determined that T cell subpopulations should be further defined by labeling with a pan T cell marker (such as CD5) because CD4 and CD8 are also expressed on other cell types (e.g., macrophages and NK cells) that are present in the spleen (Ladics and Loveless, 1994). For this reason, current methods

TABLE 3.2-3 Markers Used for Standard Lymphocyte Subset Phenotyping Lymphocyte Population Species a

Mouse

Ratc Beagle dogd Common marmosete Cotton-Top Tamarinf Cynomolgus/Rhesus monkeyg Cacma baboonh Humani a

B Cells b

T Cells

CD4+ T

CD8+ T

NK Cells

sIg , CD45R/ B220 sIg, CD45RA CD21 CD20 CD20 CD20

Thy1.2

CD4

CD8

NK1.1, 3A4

CD5, CD3 CD5 CD3 CD3 CD3

CD4 CD4 CD4 CD4 CD4

CD8 CD8α CD8 CD8 CD8

CD161a NA CD16 NA CD56, CD16

CD20 CD20

CD3 CD3

CD4 CD4

CD8 CD8

CD56, CD16 CD56, CD16

Luster et al. (1988); BD Biosciences catalog. sIg = surface immunoglobulin; NA = Not Available. c Ladics et al. (1997, 1998), Morris and Komocsar (1997), Smith et al. (2003), Franch et al. (1993); BD Biosciences catalog. d Jones et al. (2000), Byrne et al. (2000); Ab Direct catalog. e Brok et al. (2001), Frings and Weinbauer (2003). f Brok et al. (2001). g Amevive® BLA; Bleavins et al. (1993), Tryphonas et al. (1996); BD Biosciences catalog. h Amevive® BLA; Quesniaux et al. (1999); BD Biosciences catalog. i Bleavins et al. (1993); BD Biosciences catalog. b

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generally include the use of antibodies targeted to two, three, or four different surface markers, each of which is labeled with a different fluorescent dye so that multiparameter analysis can be conducted (see Chapter 4.2). Blood and Tissue Samples. Immunophenotyping is routinely conducted with peripheral blood samples and/or spleen tissue; however, bone marrow, thymus, and lymph node tissue are sometimes used. There has been some debate over the ability of peripheral blood to accurately measure effects that are occurring in lymphoid organs (ITC, 2001). There are advantages for using peripheral blood lymphocytes including the ability to monitor changes in individual animals prior to and post administration of pharmaceuticals and the relevance of animal findings to humans, since this is the population of lymphocytes that is monitored in human clinical trials. The use of peripheral blood lymphocytes versus spleen tissue was assessed in outbred Wistar/Mol rats following administration of a single dose of the immunosuppressant cyclophosphamide (Nygaard and Løvik, 2002). The results of this study showed that there were more effects in peripheral blood lymphocytes than splenic lymphocytes; significant changes in lymphocyte populations were noted earlier in the peripheral blood as compared to the spleen, and blood volume was sufficient in the rat to perform repeated assessments in the same animal. Overall, splenic lymphocyte analysis did not provide any additional information compared to information obtained from peripheral blood. Lymphocyte immunophenotyping data from a study of three different immunosuppressive drugs (azathioprine, cyclophosphamide, and cyclosporine) in Fischer 344 rats showed that similar decreases in absolute B, T, CD4+, and CD8+ cells were detected in blood, spleen, and thymus (Smith et al., 2003). Together these data support the use of peripheral blood lymphocyte assessment in rats. The use of both peripheral blood and splenic lymphocyte immunophenotyping may be warranted based on the pharmacologic action of the drug. For example, if the drug targets a specific marker on B or T lymphocytes and causes depletion of that population in the peripheral blood, then assessment of lymphocyte phenotyping by flow cytometry or assessment of that lymphocyte population by immunohistochemistry is warranted. Assessment of peripheral blood and lymphoid organs in the preclinical animal model will help to determine if the peripheral blood lymphocytes are a good marker for changes in lymphoid organs which will help determine the relevance for monitoring peripheral lymphocyte populations in humans. If the drug targets molecules that are involved in lymphocyte trafficking/homing, then the peripheral blood population may not accurately represent the lymphocyte status in lymphoid organs, so studies for these types of drugs should include both peripheral blood and lymphoid organ assessments. Finally, if the drug preferentially affects memory or activated lymphocytes versus naive lymphocytes, then the assessments should include both peripheral blood and lymphoid organs because the populations of memory cells may be differentially affected in blood and lymphoid tissue.


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Assay Validation. In order to obtain meaningful data, it is important that the flow cytometry assay used for immunophenotyping is developed and/or validated. The reagents (antibodies) that will be used for immunophenotyping should be titrated to determine the appropriate concentration. The validation should include assessment of intra-assay and inter-assay repeatability using duplicate or triplicate samples from at least 5 animals/gender on the same day and on several days, respectively. Diluting lymphocytes and determining the minimal number of cells that will still provide in-range results can be used to determine assay sensitivity. Assay validation should also include an assessment of sample and reagent stability, which will allow for more flexibility when conducting large studies with multiple samples. Data Evaluation and Interpretation The data obtained from peripheral blood lymphocyte immunophenotyping should always include an assessment of percentages of lymphocyte populations, together with a more quantitative measurement of lymphocyte numbers. The importance of this has been illustrated in a study of immunosuppressant cyclophosphamide in dogs (Jones et al., 2000). In this study, when the data for peripheral blood lymphocyte phenotyping were expressed as a percentage of lymphocytes, there was a decrease in the percentage of B lymphocytes and a corresponding increase in the percentage of T lymphocytes. However, when the absolute number of B and T cells was calculated, the data show that both peripheral blood B and T cells are decreased following administration of cyclophosphamide. Both qualitative and more quantitative assessments should also be made when conducting immunophenotyping for lymphoid tissues. The lymphoid organs should be weighed. If immunophenotyping is conducted using a portion of lymphoid tissue (e.g., piece of spleen or thymus tissue), then the piece of tissue assessed should be weighed and data should be presented as both percentage of lymphocyte populations and as normalized values using the weight for the respective piece of tissue assessed. The CD4 : CD8 T cell ratio should also be calculated because early studies conducted by NTP have shown that this ratio was more informative and showed a higher concordance for predicting immunotoxic chemicals than quantitating total T (Thy 1.2+) or B (sIg+) cells (Luster et al., 1992). The CD4 : CD8 T cell ratio is also commonly used to assess immune status in humans (especially in HIV-infected patients) and can be used as a biomarker in human clinical trials. Data obtained in rodent studies are generally presented as lymphocyte subset percentage, absolute numbers (or normalized if lymphoid tissue is assessed), and CD4 : CD8 T cell ratio. Typically, results are expressed as percent change between the mean value of drug-treated animals and the mean value of vehicle control animals, and statistical analyses are conducted to evaluate significance of the changes. For non-rodent species, it is important to look at the changes in lymphocyte populations from baseline, since there is often variation both within an individual animal and between individual animals,

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and the number of animals in each dose group is usually small (N = 3 to 6). One method often used, is to calculate the percent change from baseline values (Lappin and Black, 2003; Amevive® BLA; Rituxan® BLA) in which the mean of baseline values from two or more pre-dose evaluations is compared to data obtained following dosing and following a recovery period. This method helps to normalize the individual variability for each animal. A weight-of-evidence approach should be used when evaluating lymphocyte immunophenotyping data to determine the biological and/or toxicological relevance for observed changes. The following questions should be addressed: (1) Are there statistically significant and/or biologically relevant changes in lymphocyte subpopulations in drug-treated animals compared to vehicle control animals? (2) Is there a dose-response for the observed change? (3) Is the change evident in both males and females? (4) Are the values (including baseline when available) obtained in individual vehicle control and drug-treated animals within normal historical ranges? (5) Are there other overt toxicities (e.g., severe weight loss, morbidity/ mortality) that suggest observed changes in lymphocyte subpopulations are secondary to overt systemic drug-related toxicities? (6) Is there a trend (although not statistically significant) toward changes in a given lymphocyte population in drug-treated animals? (7) Is there an increased incidence in the number of drug-treated animals with changes in lymphocyte subpopulations? Is there a dose-response for this incidence? (8) Are there other changes in immune parameters (e.g., changes in hematology, organ weight and/or histopathologic changes in lymphoid organs, effects on functional immune response, increased incidence of infections or tumors) in drug-treated animals? Evaluation of the incidence of individual drug-treated animals with changes in lymphocyte subpopulations is especially important in non-rodent species because the statistical analysis is generally not sufficiently powered due to a small number of animals evaluated and variability between animals. RELEVANCE OF LYMPHOCYTE IMMUNOPHENOTYPING FINDINGS IN PRECLINICAL ANIMAL STUDIES TO OVERALL HUMAN RISK ASSESSMENT To date, the greatest body of literature for effects of compounds on lymphocyte subpopulations focuses on immunosuppressive/immunomodulatory compounds, with much of the literature obtained from studies of chemicals or

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pesticides in rodents (mice and rats) and pharmaceuticals in rats. There are also a few published reports on the effects of immunosuppressive/immunomodulatory pharmaceuticals on lymphocyte subpopulations in dogs, baboons, and monkeys. A summary of effects of immunosuppressive and/or immunomodulatory pharmaceuticals on lymphocyte subsets and immune function tests (when available) is provided in Table 3.2-4. Based on the limited data

TABLE 3.2-4 Summary of Effects of Immunosuppressive/Immunomodulatory Pharmaceuticals on Lymphocyte Subsets and Immune Function Compound

Species

Abatacept (CTLA4-Ig)a

Mouse

AZAb

Fischer 344 female rat

CYb


Dose

Effects on Lymphocyte Subsets and Immune Function

• Decrease in percentage of splenic B cells (males) • Transient decreases in mean serum IgG, and inhibition of ex vivo B and T cell activation (males) 25 (reduced to • Significant decrease in 17) mg/kg/day, absolute number of oral, 30 days peripheral blood total T and B cells, CD4 T, and CD8 T cells; significant decrease in absolute number of splenic T cells, CD4 T, and CD8 T cells; significant decrease in absolute number of thymic total T cells, CD4 T, and CD8 T cells • Significant decrease in secondary antibody response to T cell-dependent antigen 10 mg/kg/day, oral, • Significant decrease in 30 days absolute number of peripheral blood and splenic total T and B cells, CD4 T, and CD8 T cells; significant decrease in absolute number of thymic total T cells, CD4 T, and CD8 T cells • Significant decrease in NK activity; significant decreases in ex vivo proliferative responses to ConA and LPS; significant decreases in primary and secondary antibody responses to T celldependent antigens 65 and 200 mg/kg/ week, subcutaneous, 26 weeks

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Continued Species

CsAb


CsAc

Wistar rat

CsAd

Wistar rat

Dose


25 mg/kg/day, oral, • Significant decrease in 30 days absolute number of peripheral blood total T and B cells, CD4 T, and CD8 T cells; significant decrease in absolute number of splenic T cells, CD4 T, and CD8 T cells; significant decrease in absolute number of thymic total T cells, CD4 T, and CD8 T cells • Significant decreases in ex vivo proliferative responses to ConA and LPS; significant decreases in primary and secondary antibody response to T cell-dependent antigens 20 mg/kg/day, oral, • Significant decrease in 4 weeks absolute number of peripheral blood T cells (CD3, CD4, and CD8) and B cells; and in percentage of CD3 T lymphocytes (associated with decrease in CD4 values) in spleen and (CD4 and CD8 values) in axillary lymph node • Decreases in IgM and IgG antibody response to KLH 1, 5, or 25 mg/kg/ • Dose-related decrease in day, oral, 4 splenic and mesenteric weeks lymph node T cells and CD4 T cells • Decreases in T-dependent antibody response at >5 mg/ kg/day and lymphoproliferative responses to mitogens (ConA, PWM) and alloantigens (MLR) at the high dose


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TABLE 3.2-4 Continued Compound

Species

Tacrolimus (FK506)c

Wistar rat

Pimecrolimus (ASM981)c

Wistar rat

Prednisonee

Dog (mixed breeds)

CYf

Beagle dog

FTY720g

Chacma baboon

Dose 3 mg/kg/day, oral, 4 weeks


• Significant decrease in absolute number of peripheral blood T cells (CD3, CD4, and CD8); and in percentage of CD3 T lymphocytes (associated with decrease in CD4 values) in spleen and (CD4 and CD8 values) in axillary lymph node • Decrease in IgG antibody response to KLH 10 or 30 mg/kg/ • Significant decrease in day, oral, 4 absolute number of weeks peripheral blood T cells (CD3, CD4, and CD8); and in percentage of CD3 T lymphocytes (associated with decrease in CD4 values) in spleen and (CD4 and CD8 values) in axillary lymph node at the high dose • Decrease in IgG antibody response to KLH 50 mg (1.66– • Decrease in leukocytes, total 2.44 mg/kg/day), T and B cells, CD4 Th and oral, 3 days CD8 Tcyt/sup cells in popliteal lymph node aspirate 20 mg/kg/day, • Decrease in percentage of intraperitoneal, peripheral blood B cells, 3 days increase in percentage of peripheral blood CD4 T and CD8 T, and CD5 panT cells; CD4/CD8 ratio unchanged; decrease in absolute number CD5-panT cells • Decrease in serum IgM following vaccination • Decrease in peripheral blood 0.3 or 0.1 mg/kg/ lymphocytes, total T and B day, oral, 3 days cells, CD4 and CD8 T cells 0.03 mg/kg/day, • Decreases in ex vivo oral, 10 days proliferative responses to ConA and PHA

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TABLE 3.2-4 Continued Compound

Species

Dose

BR3-Fc (soluble BAFF antagonist)h

Cynomolgus monkey

2 or 20 mg/kg, intravenous, once weekly for 13 or 18 weeks

Rituximab (anti-CD20 mAb)i

Cynomolgus monkey

Rituximab (anti-CD20 mAb)i

Human patients with recurrent B cell lymphoma

269 mg/m2, intravenous, once weekly for 4 weeks 125, 250, or 375 mg/m2, intravenous, once weekly for 4 weeks

Alefacept (fusion protein binding to CD2)j

Cynomolgus monkey

0.3, 1, 3, or 10 mg/ kg, intravenous, single dose


Baboon

0.05, 1, or 20 mg/ kg, intravenous, twice weekly for 13 weeks


Cynomolgus monkey

0.005, 0.1, or 1.0/5.0 mg/kg, intravenous, once weekly for 44 weeks

Effects on Lymphocyte Subsets and Immune Function • Significant decrease in peripheral blood B cell count • Decrease (modest but statistically significant) in IgG antibody response to tetanus toxoid • Decrease in peripheral blood B cells with recovery observed approximately 2 weeks after cessation of treatment • Decrease in peripheral blood B cells with recovery observed approximately 6 months after completion of treatment and return to normal levels between 9 and 12 months following cessation of treatment • Depletion of CD2, CD3, CD4, and CD8 peripheral blood T lymphocyte populations (effects mild at 0.3 mg/kg); decrease in CD20 B cells at 10 mg/kg • Decrease in absolute and relative number of CD2+, CD4+, and CD8+ peripheral blood lymphocytes at 1 or 20 mg/kg; CD4 lymphocytes 50–65% of baseline at end of 7-month recovery period; based on immunocytopathology, lymphoid depletion in CD2+, CD4+, and CD8+ T cell areas of selected lymph nodes and spleen • Dose-related decrease in all mature, peripheral blood T lymphocytes which correlated with slight depletions in T lymphocyte regions of lymph nodes and spleen • Mild reductions in primary and secondary humoral immune response to immunization with exogenous protein antigen


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TABLE 3.2-4 Continued Compound Alefacept (fusion protein binding to CD2)k,l

Species Human patients with chronic psoriasis

Dose 0.025, 0.075, or 0.150 mg/kg/ week, intravenous, 12 weeks

Effects on Lymphocyte Subsets and Immune Function • Decrease in peripheral blood CD4+ and CD8+ T lymphocytes; reduction predominantly affected the memory effector subsets of the T lymphocyte compartments (CD4+CD45RO+ and CD8+CD45RO+) with circulating naive T lymphocyte and NK cell counts minimally affected

AZA = azathioprine; CY = cyclophosphamide; CsA = cyclosporine A; ConA = concanavalin A; LPS = lippopolysaccharide; KLH = keyhole limpet hemocyanin; PWM = pokeweed mitogen; MLR = mixed-lymphocyte reaction; PHA = phytohaemagglutinin. a Orencia® BLA. b Smith et al. (2003). c Roman et al. (2004). d Descotes et al. (1996). e Ammersbach et al. (2006). f Jones et al. (2000). g Quesniaux et al. (1999). h Vugmeyster et al. (2006). i Rituxan® BLA. j Amevive® BLA. k Ellis and Krueger (2001). l Amevive® package insert.

available, changes in lymphocyte subpopulations did correspond with changes in immune function in preclinical animal studies. To better understand correlations between effects observed in animals and effects observed in humans, the PharmaPendium™ database that includes 60 immunomodulatory/immunosuppressive compounds was searched. Of the 60 compounds included in the search, only three reported effects on lymphocyte subpopulations in preclinical studies (Abatacept, CTLA4-Ig [Orencia® BLA]; Rituximab, anti-CD20 monoclonal antibody [Rituxan® BLA]; and Alefacept, fusion protein binding to CD2 [Amevive® BLA]), and only two reported effects on lymphocyte subpopulations in human clinical trials (Rituximab and Alefacept). For both Rituximab and Alefacept, the effects observed in preclinical studies (decrease in peripheral blood B cells and decrease in peripheral blood T cells/NK cells, respectively) were similar to the effects observed in clinical trials and were consistent with the desired pharmacologic profile,

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and were therefore expected findings for these immunosuppressive and/or immunomodulatory pharmaceuticals. SUMMARY Lymphocyte immunophenotyping does not provide a direct measurement of immunotoxicity risk for humans because it does not assess functional effects on the immune system. However, the available data from preclinical animal studies that included assessment of lymphocyte immunophenotyping and immune functional tests such as T cell-dependent antibody response, NK cell activity, and/or ex vivo proliferation to lymphocyte mitogens, suggest that changes in lymphocyte subpopulations correspond to changes in immune function. Thus, lymphocyte immunophenotyping may be useful as a biomarker for monitoring drug-related effects on the immune system in humans and for monitoring peripheral recovery from those effects. Such evaluations can help to determine if patients may be at risk for decreased response to a vaccination. It may also provide support for additional monitoring if the intended patient population has a pre-existing risk for altered lymphocyte subpopulations because of disease or genetic makeup. ACKNOWLEDGMENTS The author acknowledges Drs. Donna Davila, Guenter Blaich, Lori Gallenberg, and Klaus Krauser, and Ms. Anne-Illi-Love for their review of and helpful comments on this chapter. REFERENCES Ab Direct Catalog. Available at http://www.ab-direct.com Amevive® BLA. BLA 125036. Available at http://www.accessdata.fda.gov/scripts/cder/ drugsatfda/index.cfm Amevive® package insert. Available at http://www.fda.gov/cder/foi/lavel/2003/ alefbio013003LB.htm Ammersbach MAG, Kruth SA, Sears W, Bienzle D. The effects of glucocorticoids on canine lymphocyte marker expression and apoptosis. J Vet Intern Med 2006; 20:1166–1171. Baroncelli S, Panzini G, Geraci A, Pardini S, Corrias F, Iale E, Varano F, Turillazzi PG, Titti F, Verani P. Longitudinal characterization of CD4, CD8 T-cell subsets and of haematological parameters in healthy newborns of cynomolgus monkeys. Vet Immunol Immunopathol 1997;59:141–150. Barrow PC, Ravel G. Immune assessment in developmental and juvenile toxicology: practical considerations for the regulatory safety testing of pharmaceuticals. Regul Toxicol Pharmacol 2005;43:35–44.

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BD Biosciences Catalog. Available at http://www.bdbiosciences.com Bleavins MR, Brott DA, Alvey JD, de la Iglesia FA. Flow cytometric characterization of lymphocyte subpopulations in the cynomolgus monkey (Macaca fascicularis). Vet Immunol Immunopathol 1993;37:1–13. Brok HPM, Hornby RJ, Griffiths GD, Scott LAM, Hart BA. An extensive monoclonal antibody panel for the phenotyping of leukocyte subsets in the common marmoset and the cotton-top tamarin. Cytometry 2001;45:294–303. Byrne KM, Kim HW, Chew BP, Reinhart GA, Hayek MG. A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes. Vet Immunol Immunopathol 2000;73:167–182. DeMaria MA, Casto M, O’Connell M, Johnson RP, Rosenzweig M. Characterization of lymphocyte subsets in rhesus macaques during the first year of life. Eur J Haematol 2000;65:245–257. Descotes G, Pinard D, Gallas JF, Penacchio E, Blot C, Moreau C. Extension of the 4week safety study for detecting immune system impairment appears not necessary: example of cyclosporin A in rats. Toxicology 1996;112:245–256. Ellis CN, Krueger G. Treatment of chronic plaque psoriasis by selective targeting of memory effector T lymphocytes. N Engl J Med 2001;345(4);248–255. EMEA. European Medicines Agency. Note for guidance on repeated dose toxicity (CPMP/SWP/1042/99), 2000. Available at http://www.emea.eu.int/pdfs/human/swp/ 104299en.pdf Faldyna M, Sinkora J, Knotigova P, Leva L, Toman M. Lymphatic organ development in dogs: major lymphocyte subsets and activity. Vet Immunol Immunopathol 2005; 104(3–4):239–247. Franch A, Castellote C, Pelegrí C, Tolosa E, Castell M. Blood B, T, CD4+ and CD8+ lymphocytes in female Wistar rats. Ann Hematol 1993;67:115–118. Franchimont D. Overview of the actions of glucocorticoids on the immune response. Ann N Y Acad Sci 2004;1024:124–137. Frings W, Weinbauer GF. Assessment of the nonhuman primate immune system by immunophenotyping: comparison of two relevant species with human. Toxicol Lett 2003;144:s34. ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006. Igarashi H, Medina KL, Yokota T. Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids. Int Immunol 2005;17:501–511. ITC. Application of flow cytometry to immunotoxicity testing: summary of a workshop. Toxicology 2001;163:39–48. Jones RD, Offutt DM, Longmoor BA. Capture ELISA and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles. Toxicol Lett 2000;115:33–44. Kurogi K, Naohara K, Kouchi M, Aoki Y, Tanaka K, Yasuba M. Assessment of T-celldependent antibody response, hematology, pathology and immunophenotyping in the identical rat on immunotoxicology studies. J Toxicol Sci 2005;30(Suppl):S69. Ladics GS, Loveless SE. Cell surface marker analysis of splenic lymphocyte populations of the CD rat for use in immunotoxicological studies. Toxicol Methods 1994; 4(2):77–91.

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Ladics GS, Childs R, Loveless SE. Interlaboratory evaluation of the quantification of rat splenic lymphocyte subtypes using immunofluorescent staining and flow cytometry. Toxicol Methods 1997;7:241–263. Ladics GS, Smith C, Loveless SE, Green JW. Phase two of an interlaboratory evaluation of the quantification of rat splenic lymphocyte subtypes using immunofluorescent staining and flow cytometry. Toxicol Methods 1998;8:87–104. Lappin PB, Black LE. Immune modulator studies in primates: the utility of flow cytometry and immnohistochemistry in the identification and characterization of immunotoxicity. Toxicol Pathol 2003;31(Suppl):111–118. Lappin PB, Thomas CA. Subsets of peripheral blood lymphocytes differ in cynomolgus monkeys obtained from distinct geographical regions. Toxicologist 2001;60:262. Luster MI, Munson AE, Thomas PT, Holsapple MP, Fenters JD, White KL, Lauer LD, Germolec DR, Rosenthal GJ, Dean JH. Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program’s Criteria for Immunotoxicity evaluation in mice. Fundam Appl Toxicol 1988;10:2–19. Luster MI, Portier C, Pait DG, White KL, Gennings C, Munson AE, Rosenthal GJ. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam Appl Toxicol 1992;18:200–210. Morris DL, Komocsar WJ. Immunophenotyping analysis of peripheral blood, splenic, and thymic lymphocytes in male and female rats. J Pharmacol Toxicol Methods 1997;37:37–46. Nam KH, Akari H, Terao K, Itagaki S, Yoshikawa Y. Age-related changes in major lymphocyte subsets in cynomolgus monkeys. Exp Anim 1998;47(3):159–166. Nygaard UC, Løvik M. Blood and spleen lymphocytes as targets for immunotoxic effects in the rat—a comparison. Toxicology 2002;174:153–161. Orencia® BLA. BLA 125118. Available at http://www.accessdata.fda.gov/scripts/cder/ drugsatfda/index.cfm Quesniaux V, Fullard L, Arendse H, Davison G, Markgraaff N, Auer R, Ehrhart F, Kraus G, Schuurman HJ. A novel immunosuppressant, FTY720, induces peripheral lymphodepletion of both T and B cells and immunosuppression in baboons. Transplant Immunol 1999;7:149–157. Richter-Reichhelm HB, Schulte AE. Results of cyclosporin A ringstudy. Toxicology 1998;129:91–94. Rituxan® BLA. BLAs 97-0260 and 97-0244. Available at http://www.accessdata.fda. gov/scripts/cder/drugsatfda/index.cfm Roman D, Ulrich P, Paul G, Court M, Vit P, Kehren J, Mahl A. Determination of the effect of calcineurin inhibitors on the rat’s immune system after KLH immunization. Toxicol Lett 2004;149:133–140. Roth DR, Roman D, Ulrich P, Mahl A, Junker U, Perentes E. Design and evaluation of immunotoxicity studies. Exp Toxicol Pathol 2006;57:367–371. Smith HW, Winstead CJ, Stank KK, Halstead BW, Wierda D. A predictive F344 rat immunotoxicology model: cellular parameters combined with humoral response to NP-CγG and KLH. Toxicology 2003;194:129–145.

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PART IV EXTENDED IMMUNOTOXICOLOGY ASSESSMENT: EX VIVO MODELS

4.1 FUNCTIONAL CELLULAR RESPONSES AND CYTOKINE PROFILES Elizabeth R. Gore

Establishing the strategy, study design, and timing of immunotoxicity evaluations during preclinical drug development requires careful consideration of the pharmacologic properties and toxicity profile of the drug under investigation. Beyond the conventional immune parameters incorporated into general toxicology studies, i.e., lymphoid organ histopathology and hematology (see Chapter 2), there is a wide array of study designs, immune parameters, and analytical methods to select from to address specific questions/concerns of a drug’s immunotoxic potential. While regulatory guidelines on the development of pharmaceuticals for human use (ICH, 2006) offer recommendations on strategy and functional models according to well-established, predictive end points (Luster et al., 1992), customization and/or refinement of the models to address specific questions is essential and may require incorporation of additional end points for optimal identification and characterization of druginduced immunotoxicity. The objective of this section is to provide a description of several immune parameters and methodologies that are used in the preclinical setting to characterize immune hazards for human risk assessments. The utility of a standalone functional evaluation, specifically the rat T cell-dependent antibody response (TDAR) model developed for regulated immunotoxicity evaluations (Gore et al., 2004), is mainly used for hazard identification, i.e., unintended immunosuppression. This assay is based on an end point parameter that is the Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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final product of the immune response following exposure to an antigen (keyhole limpet hemocyanin, KLH) unassociated with human disease. However, hazard identification alone may be insufficient for risk assessment of novel drugs in development. Additional in vitro and/or ex vivo evaluations, incorporated into screening tests, can elucidate mechanisms of toxicity and provide critical information for adequate human risk assessment. Animal models that are representative of human disease (e.g., neoplasia, infection, autoimmunity), and associated with immune processes previously identified as drug targets, would enhance risk assessment, specifically by demonstrating the impact of an immune perturbation on the susceptibility to disease (Burleson, 2000; Germolec, 2004). Conducting such in vivo studies, however, may not be necessary if the mechanism of toxicity is well defined and there is sufficient information in the scientific literature to link the targeted effect to impaired host defense or self-tolerance. Additional parameters that are readily incorporated into a stand-alone immune function test such as the KLH-TDAR model include ex vivo lymphocyte proliferation, cytokine protein expression, and immunophenotype analysis; any or all of which can enhance hazard identification and characterization of a potential immunotoxicant. While the KLH-TDAR is an example of a combined immune function screen and mechanistic study, the ex vivo methodologies described herein are generally applicable to toxicology studies that do not include an immunization protocol. Moreover, the methodologies are not species-specific; however, responsiveness to various stimulants to induce ex vivo lymphocyte proliferation and cytokine production may differ across species and strain, requiring procedural optimization for a given species and ex vivo test.

LYMPHOCYTE PROLIFERATION Rationale and Experimental Design Lymphocyte proliferation is an essential component of an effective immune response in the adaptive arm of the immune system. Upon an encounter with their cognate antigen, naive T lymphocytes proliferate and differentiate into functionally active cells (e.g., helper, cytotoxic, regulatory). A subset of antigenspecific lymphocytes survives the original insult and differentiates into memory cells, which rapidly expand upon subsequent antigen exposure. The expanded population of antigen-specific T-helper cells orchestrates a series of events including cytokine synthesis and secretion, B cell differentiation and antibody production, and/or T cell-mediated activation of effector macrophages and cytotoxic T cells. In productive host defense responses, differential effector cell expansion is dependent on various factors, including the invading organism, its route of entry, as well as the immune bias of the host (i.e., epigenetic factors) (Ochsenbein et al., 2000; Kulkarni et al., 2003; Dudda et al., 2005).

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Nevertheless, lymphocyte proliferation is the basis of progression to an immunocompetent adaptive immune reaction; dysregulation of the proliferative response, either by enhancement or suppression, can lead to immunopathologies. Recent evidence suggests that contiguous interaction between innate (nonspecific) and adaptive immune responses elicited by ubiquitous antigens can result in chronic disease states, including allergy/asthma and inflammation associated with chronic obstructive pulmonary disease and inflammatory bowel disease (Das et al., 2006; Sabroe et al., 2007). Alternatively, druginduced suppression of lymphocyte function can lead to susceptibility to opportunistic infections and inefficient tumor surveillance as demonstrated in transplant patients undergoing chronic immunosuppressive therapy (Fishman and Rubin, 1998; Vial and Descotes, 2003). Evaluation of lymphocyte proliferation in preclinical toxicity investigations provides useful information about a drug’s immunotoxic potential. For example, drugs intended for long-term administration in HIV+ individuals (i.e., prophylactic-based therapies against secondary infections) have been evaluated preclinically in rats to determine effects on ex vivo lymphocyte proliferation and cytokine responses (Viora et al., 1996). Such information is essential in identifying the appropriate and safest treatment strategies for immunocompromised patients. Ex vivo lymphocyte proliferation assays have also been utilized in dog and rodent toxicology studies with azaspiranes to differentiate their targeted, immunomodulatory activity from generalized immunosuppression as an indicator of an improved safety profile over alternative therapies for chronic treatment of autoimmune diseases and transplantation (Kaplan et al., 1993; Badger et al., 1997). Moreover, assessment of ex vivo lymphocyte proliferation can contribute to an understanding of the mechanism of a drug’s activity as demonstrated with the anti-inflammatory statins, atorvastatin and lovastatin, in a rodent model of experimental autoimmune uveoretinitis (Kohno et al., 2007). While ex vivo proliferation may or may not afford the level of sensitivity as the more predictive functional tests (i.e., TDAR and NK cytotoxicity) (Luster et al., 1992; Dean et al., 1998; The ICICIS Investigators Group, 1998), concomitant alterations in complementary parameters, albeit not over the entire in vivo dose range, would provide a potential mechanism of the manifestation of an immune perturbation. This notion would support a single dose level (e.g., maximum exposure) to test ex vivo parameters, while the in vivo functional parameter with higher sensitivity would be performed over the entire dose range. Characteristic of immune function evaluations, there is considerable diversity in the approaches to proliferation assays, from the experimental design to the analytical method. Experimental procedures necessitate sterile technique and cell culture expertise to ensure accurate assessment of the cellular response to a particular stimulant. Culture conditions vary depending on the cell source and type of stimulant, but generally are conducted at 5% CO2, 37 °C for 48 to 96 hours. The source of lymphocytes may be from peripheral blood, spleen,

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and/or lymphoid cells driven by species, experimental objective, and/or previously identified target organ(s). When using peripheral blood, the choice of anticoagulant is important, as EDTA can inhibit cellular proliferation due to its calcium-chelating properties (Kozlov and Novikova, 1978); heparin is the anticoagulant most often used for functional evaluations. Whole blood, peripheral blood mononuclear cells (PBMC), or highly purified cell preparations (e.g., magnetic bead separation or fluorescent activated cell sorting) can be used to assess general or target-specific effects, respectively (Geiselhart et al., 1996; Stanciu et al., 1996; Fasanmade and Jusko, 1999; Gummert et al., 1999; Stanciu and Djukanovic, 2000; Langezaal et al., 2001; Barten et al., 2002; Chattopadhyay et al., 2006). Also, the choice of stimulant(s) may enable identification of a targeted effect on a particular cell population (Fecho et al., 1993; Tryphonas, 2001; Onlamoon et al., 2006). Plant lectins (e.g., concanavilin A [ConA]; phytohemagglutinin [PHA]; pokeweed mitogen [PWM]) and bacterial antigens (e.g., lipopolysaccharide [LPS]) are frequently used to differentiate effects on T and B lymphocytes, respectively. Receptor/co-receptor ligation (e.g., CD3/CD28) is another means of assessing proliferation of a targeted cell population, while phorbol esters in the presence of ionomycin bypass receptor signaling to measure proliferative capacity of lymphocytes non-discriminately. Recall responses to antigens can also be measured ex vivo to evaluate in vivo sensitization with a neoantigen (Franco et al., 1998). This requires in vivo “priming” with an antigen, ex vivo stimulation of peripheral or tissue lymphocytes with the same antigen, and a highly sensitive analytical method to measure the proliferative response given the discrete subset of antigen-specific lymphocytes. A mixed lymphocyte reaction (MLR) is another option and has been established as the in vitro correlate to cell-mediated immunity (Luster et al., 1988). This ex vivo test requires a source of allogenic cells (i.e., dissimilar histocompatibility antigens) pretreated with mitomycin C or radiation to ensure unidirectional response of the cell population of interest. This system is less practical than other proliferation assays due to a need for additional animal strains, cell inactivation procedures, and increased culture period (4 to 5 days). Analytical Methods. The conventional approach to evaluating in vitro or ex vivo lymphocyte proliferation is by 3H-thymidine uptake and liquid scintillation counting (Andersson et al., 1972). Dividing cells incorporate 3H-thymidine into DNA, and the radioactive signal is commensurate to the number of dividing cells. This method is especially suitable to whole blood preparations, as colorimetric and fluorescent-based approaches are compromised due to interference with red blood cells. Furthermore, 3H-thymidine uptake has a considerably higher signal-to-noise ratio (i.e., stimulation indices) relative to non-radioisotopic methods which is of particular importance when evaluating drug effects on lymphocyte responses to antigenic or allogenic stimuli. Colorimetric methods using oxidation–reduction indicators (e.g., tetrazolium salts) have demonstrated utility in evaluating cellular growth and

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expansion (Loveland et al., 1992; Wemme et al., 1992; de Fries and Mitsuhashi, 1995), as have bioluminescent assays using intracellular ATP as an indicator of lymphocyte responses to PHA (Sottong et al., 2000; Kowalski et al., 2006). However, the weaker stimulation indices of these alternative platforms relative to 3H-thymidine uptake, and the lack of specificity (i.e., increased metabolic activity and cellular expansion), have hindered greater application in the evaluation of ex vivo lymphocyte activity (Russell and Vindelov, 1998; Maghni et al., 1999). A more promising non-radioisotopic alternative is bromodeoxyuridine (BrdU) incorporation. Similar to thymidine, BrdU is incorporated into DNA for a direct and specific assessment of the proliferative response. BrdU incorporation, detected with fluorescent or enzyme-conjugated antibodies with specificity for BrdU, is adaptable to flow cytometric or ELISA methodologies, respectively (Wagner et al., 1999; Messele et al., 2000; Rosato et al., 2001; Smith et al., 2003).

CYTOKINE ANALYSIS Rationale and Experimental Design Cytokines are soluble mediators of the immune system that play a role in numerous processes including cellular growth, migration, development, and differentiation (Foster, 2001). Cytokine production and activity can be affected by direct toxicity to cytokine-producing cells, inhibition of cytokine production, induction of immunosuppressive factors, and alteration in cellular homeostasis (Descotes, 2006). The cytokine network is highly redundant and compensatory in nature, yet abnormal expression of key regulatory cytokines can have profound effects on health status as evidenced by widespread development of agonistic/antagonistic cytokine therapeutics for the treatment of various diseases including cancer, inflammation, and autoimmunity (Rosenblum et al., 2002; Moreland, 2004; Vilcek and Feldmann, 2004; Hasan, 2006; Chabalgoity et al., 2007). Thus, drug-induced alterations in cytokine homeostasis and/or cytokine induction via internal or external stimuli can signal a potential for increased susceptibility to infection and disease. This concept underscores the importance of measuring cytokines as part of preclinical immunotoxicity evaluations of new therapeutic candidates. In the majority of cases, cytokine activity occurs at the local level and direct measurements in biological samples, such as serum and plasma, may lack the sensitivity required to identify a cytokine as a drug target (House, 1999b). However, if a drug that is administered in vivo has a direct and lasting effect on immune cell population(s), then an ex vivo experiment utilizing peripheral or tissue (i.e., spleen, lymph node, thymus) lymphocytes might identify a drug effect that was missed or underestimated by direct serum/plasma analysis. There is considerable overlap in the culture conditions used for lymphocyte proliferation and cytokine induction; however, several important aspects are

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raised to address specific objectives. The use of whole blood for ex vivo cytokine induction has several advantages: a diverse cellular milieu, taking into account cell–cell interactions and signaling cascades; and a potential to assess direct drug effects representative of in vivo conditions that would not be possible with purified cell preparations (Barten et al., 2002; Agnello et al., 2004). The latter advantage is contingent upon timing of last dose and pharmacokinetic properties of the drug under investigation. The potential for a rapidly reversible effect has been demonstrated in ex vivo studies with human peripheral blood lymphocytes after in vivo treatment with cyclosporine, a known immunosuppressive agent. While profound decreases are observed after 4 hours of treatment, by 24 hours cellular responses return to baseline (Stein et al., 1999). In such situations, in vitro experiments may help to characterize mechanisms of immunotoxicity identified by in vivo parameters (i.e., histopathology, hematology) or functional tests (i.e., TDAR). Subtle changes in cytokine synthesis and/or secretion due to a highly specific and targeted drug effect can be masked under culture conditions maximizing the expression of numerous cytokines via diverse activation pathways (Lakew et al., 1997). Enriching for distinctive cell populations by density gradient centrifugation or cell sorting (e.g., phenotypic markers attached to magnetic beads or fluorescent probes) increases the sensitivity of the ex vivo assay in identifying drug targets associated with a specific functional response, i.e., pro-inflammatory or cell-mediated immunity. Durez et al. (1998), using a combination of in vivo and ex vivo investigations in mice that were administered the immunosuppressive drug, methotrexate, were able to demonstrate specific impairment of macrophage function in the absence of an effect on T cells. Effects on biomarkers of macrophage activity ex vivo (i.e., TNF-α, nitric oxide production) were consistent with decreased susceptibility of methotrexatetreated mice to a lethal challenge of LPS. In contrast, methotrexate did not inhibit TNF-α release in mice injected with ConA or anti-CD3 monoclonal antibody. Cytokines involved in immune processes characteristic of a pro-inflammatory or adaptive immune response have different kinetics of expression. Multiple sample collections post ex vivo stimulation are essential to identify and/or distinguish between drug targets of divergent pathways. For instance, mediators of pro-inflammatory responses (e.g., TNF-α, IL-1, IL-6) are up-regulated in cell culture supernatants as early as ∼4 to 6 hours after ex vivo stimulation, while cytokines driving cellular and humoral responses (i.e., IL-2, IFN-γ, IL-12, IL-4, IL-5, IL-10) generally peak between 24 to 48 hours. Screening early for drug targets by ex vivo cytokine analysis establishes a framework for follow-on in vitro studies that not only provide meaningful information with regard to dose relationships, molecular targets, and signaling events, but also serve as a bridge to in vitro human models for increased relevance to human risk assessment (Vandebriel et al., 1998a; Langezaal et al., 2002). Gene expression analysis provides a window into the molecular events that initiate cytokine synthesis in response to various stimuli encountered by

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peripheral or tissue leukocytes. While cytokine protein is a measure of the accumulation of cell “product” in biological fluid or culture supernatant over time, gene expression is a measure of events at a specific point in time with the potential to influence downstream events (e.g., protein synthesis, transport, excretion) necessary for effector cell activity and a protective immune response. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) has been used for targeted cytokine evaluation to assess the in vivo biologic activity of a protein therapeutic in the nonhuman primate (Herzyk et al., 2002). In addition, ex vivo cytokine mRNA expression in ConA-activated splenocytes from rats administered the immunotoxicant, tributylin oxide, demonstrated a dose-dependent decrease in IL-2 receptor expression and increases in IL-2 and IFN-γ expression. These findings suggested an early stage deviation with potential impediment to downstream events involved in thymocyte maturation culminating in thymic atrophy (Vandebriel et al., 1998b). Finally, an ex vivo, molecular-based approach has been used to characterize differential cytokine expression associated with diverse susceptibility to hypersensitivity reactions in rats (Sirois and Bissonnette, 2001). Characterizing cytokine bias at the molecular level, in conjunction with cellular frequencies of Th1/Th2 subpopulations, should contribute to a better understanding of the diversity of clinical manifestations of drug-induced hypersensitivity reactions, one of the leading reasons for taking drugs off the market (Gaspard et al., 1999; Lebrec et al., 2001; Ratajczak, 2004) (see Chapter 8). Analytical Methods. Multi-analyte profiling technology is a powerful beadbased platform enabling a comprehensive evaluation of cytokine profiles in normal, diseased, and drug-treated subjects (Vignali, 2000; Kellar et al., 2001; de Jager and Rijkers, 2006; Kofoed et al., 2006). The technology (e.g., xMAP®) is adaptable to numerous sample types (e.g., plasma, tissue homogenates, culture supernatants) and commercial assay kits are readily available for preclinical and clinical evaluations. One of the key advantages of this technology is the ability to measure up to 20 different analytes in a single sample without compromise to the precision and sensitivity observed with single-analyte platforms (Dupont et al., 2005; Giavedoni, 2005; de Jager and Rijkers, 2006). This has significant implications for mechanistic evaluations: drug targets, previously untested due to limited sample volumes, resources and/or scientific rationale, can now be readily detected in multiplex cytokine panels spanning the repertoire of soluble mediators that initiate, maintain, and/or regulate immune processes. These multi-analyte platforms (e.g., Luminex) consist of a benchtop flow cytometer/analyzer for the detection of cytokines captured onto microspheres (“beads”) with unique fluorescent intensities. The beads are covalently coupled to cytokine-specific antibodies so that cytokines present in biological fluids can be captured when mixed with the desired assortment of cytokine-specific beads. Detection antibodies carry the reporter molecule, phycoerythrin (PE), so that fluorescent signal is proportional to the amount of cytokine present in

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the sample. The unique fluorescent properties of the beads enable distinct mapping of the cytokine-specific complexes, so that PE intensities for each cytokine can be determined from a single well. Cytokine concentrations are quantified against calibration curves prepared in separate wells. In theory, hundreds of unique bead sets are available for multiple combinations of arrays; however, cross-reactivity of reagents and/or sample “matrix” effects can reduce assay sensitivity/selectivity and place multiplexing for evaluations in the realm of ∼20 to 30 analytes per sample. Complementary or alternative platforms for ex vivo cytokine protein analysis include bioassay, enzyme-linked immunosorbent assay (ELISA), ELISPOT, and electrochemiluminescent immunoassay (ECLIA). The bioassay approach has the distinct advantage of measuring both concentration of the cytokine and its biological activity. It is a cell-based system dependent upon the cytokine of interest to induce a measurable change (i.e., proliferation, cytotoxicity) (House, 1999a). Disadvantages of the bioassay include lack of specificity given overlapping functions of cytokines present in a biological sample and the inability to multiplex. The ELISA is similar to bead-based multiplex assays in that it utilizes antibody pairs for capture and detection of a specific cytokine. The key difference is that the assay is performed on a microtiter plate with the capture antibody fixed to the surface. In stepwise fashion, the cytokine binds to the capture antibody, unbound proteins are washed away, and a secondary antibody, conjugated to an enzyme (e.g., horseradish peroxidase, alkaline phosphatase), is added for detection. Addition of substrate results in a color reaction that can be measured on a spectrophotometer against a calibration curve for absolute or relative quantification. While this format has high specificity and sensitivity, the major drawback is the inability to multiplex and a potential to detect inactive analytes. Similarly, the ELISPOT is limited with regard to multiplexing; however, this technology combines ex vivo stimulation of cells and cytokine detection in a single assay for improved sensitivity (Czerkinsky et al., 1988). Enumeration of cytokine-secreting cells is determined by colored spot formation and image analysis, even when present at very low frequencies in the cellular population (e.g., 1 cytokine secreting cell/105 cells). This method is particularly advantageous for cytokines with weak systemic expression, or those that are bound to interfering proteins in plasma/serum samples precluding accurate and reliable assessment of drug-induced inhibition of cytokine synthesis and secretion by ELISA and multiplex platforms. Recent advances in ECLIA technology have combined high sensitivity of chemiluminescence with multiplexing capability (Debad et al., 2004; Fichorova et al., 2006; Samineni et al., 2006). A Multi-Spot®, microwell plate with distinct binding domains in a given well enables capture and detection of up to 10 different analytes per test sample using immunoassay methodology. A reporter molecule (Ru(bpy)32+), conjugated to the secondary (detection) antibody, emits light upon electrochemical stimulation at the electrode surfaces of the plate. Light intensity for each spot within a given well is measured by charge-

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coupled device (CCD) camera and the resulting signal is extrapolated to the calibration curve for quantification. The advantage of this platform is the wide dynamic range afforded by ECLIA and the capability to multiplex without the need for a flow cytometer which requires increased maintenance and more advanced technical expertise. Finally, real-time RT-PCR methodology is the frontrunner for cytokine gene expression analysis. This technique requires isolation of mRNA from cells and reverse transcription of mRNA to cDNA for subsequent amplification and detection of cytokine genes using specific primer/probe sets and instrumentation for real-time PCR cycling (Heid et al., 1996). Commercial cytokine arrays are available for pathway-focused investigations (e.g., inflammatory cytokines/receptors, Th1/Th2/Th3, chemokines/receptors) applicable to preclinical immunotoxicity assessments, especially in mechanistic studies. RT-PCR is extremely sensitive, measuring ∼5 to 10 copies of mRNA per reaction, and is well established as a research tool to evaluate differential expression patterns; however, standardization and validity of gene quantification remains to be determined (Bustin and Nolan, 2004). Furthermore, differences at the molecular level may not equate to meaningful changes in protein expression; RT-PCR as a stand-alone tool for preclinical immunotoxicity assessment is not recommended for this reason. SUMMARY Lymphocyte proliferation and cytokine production form the basis of progression to an immunocompetent response, and drug-induced alterations in either or both parameters can indicate a potential for impaired host defense or selftolerance. Furthermore, ex vivo functional assays have the potential to elucidate mechanisms of drug-induced immunotoxicity and may provide essential information to address human risk (i.e., relevant host defense models). Finally, state-of-the-art methodologies enable rapid, highly sensitive and comprehensive evaluations of leukocyte activity, and implementation in preclinical evaluations may assist in the identification and/or characterization of drug-induced immunotoxicity. REFERENCES Agnello D, Mascagni P, Bertini R, Villa P, Senaldi G, Ghezzi P. Granulocyte colonystimulating factor decreases tumor necrosis factor production in whole blood: role of interleukin-10 and prostaglandin E(2). Eur Cytokine Netw 2004;15:323–326. Andersson J, Moller G, Sjoberg O. Selective induction of DNA synthesis in T and B lymphocytes. Cell Immunol 1972;4:381–393. Badger AM, Newman-Tarr TM, Satterfield JL. Selective immunomodulatory activity of SK&F 106615, a macrophage-targeting antiarthritic compound, on antibody and cellular responses in rats and mice. Immunopharmacol 1997;37:53–61.

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4.2 APPLICATION OF FLOW CYTOMETRY IN DRUG DEVELOPMENT Padma Narayanan, Renold J. Capocasale, Nianyu Li, and Peter J. Bugelski

Flow cytometry is a technology that is well suited for analyzing and characterizing the effects of drugs on the immune system of animals and humans. What makes flow cytometry such an important tool is its ability to simultaneously and rapidly detect multiple objective measurements of structural and functional characteristics of individual cells. In comparison to traditional “bulk” methods, the lineage and functional phenotype of individual cells can be assessed in real time, providing a more accurate assessment of the cell populations comprising a given sample. Collectively these features coupled with the ability to expeditiously prepare, process, and store data, yield a multiparametric analysis with informational content that is readily applicable to immunotoxicology studies.

OVERVIEW OF FLOW CYTOMETRY TECHNOLOGY Instrumentation Flow cytometers distribute suspensions of cells into a single column by laminar flow and hydrodynamic focusing. Individual cells are then intercepted by a laser source, and the scattered light and fluorescence emission (when using Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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fluorescent probes) of each cell measured. The modern era of commercial flow cytometers began in the 1970s using air-cooled (rather than water-cooled lasers) for immunofluorescence measurements, decreasing the size, cost, and power consumption of the instruments (Shapiro, 2004). From the mid-1990s on, solid-state lasers have been increasingly incorporated into even smaller benchtop flow cytometers (Telford, 2004). These newest benchtop instruments can interrogate up to eight to nine colors simultaneously for analysis or sorting and can be placed in a biosafety cabinet for added operator safety. A typical benchtop flow cytometer is equipped with lasers with excitation maxima at 407 (Violet), 488 (Blue), and 635 (Red) nanometers (nm). Low-angle deflection of the beam, or forward light scatter, provides information on the relative size of the cell or particle. Reflection and refraction of the beam at high angle, referred to as orthogonal, side, or 90 ° scatter, discriminates intracellular complexity. The photons of light are collected by lenses, and separated by optical filters and dichroic mirrors. A great variety of optic filters are available for use in narrowing the portion of the electromagnetic spectrum seen by any particular detector. Dichroic mirrors reflect light below a specific wavelength and pass longer-wavelength light which is then captured by detectors or photomultiplier tubes (PMTs). PMTs measure integral or log intensities of fluorescence peaks at specific designated wavelengths. Conversion of photons to analog voltage signals takes place in photodetectors, and both the PMT and high-voltage amplifiers allow manual adjustment of the gain or sensitivity, thereby ensuring large dynamic range or good “signal-to-noise ratio.” It is important to set the detector voltage at a high enough level to bring the autofluorescence of negative cell or bead populations above the threshold of the instrument to ensure low-level sensitivity and the signal-to-noise ratio are not compromised. The analog signal is subsequently digitized into numerical values, and stored in computer memory on a cell by cell defined “listmode” basis. This permits re-analysis of the data based on desired characteristics following data collection. Increasingly, the internal electronics of a flow cytometer has become computer-based. The latest systems incorporate special purpose large-scale integrated circuits, micro-processors, micro-controllers, and digital signal processing chips. These digital systems offer great value with increased performance at prices most laboratories can afford (Shapiro, 2003). Utility of Flow Cytometry in Immunotoxicology The generation of monoclonal antibodies (mAbs) to antigens expressed by lymphocytes and hematopoietic cells has led to major advances in the fields of immunology, hematology, and immunotoxicology. The antigens recognized by mAbs have been refined into cluster designation or cluster of differentiation (CD) antigen groups, and the expression of these molecules by blood cells is demonstrated by a technique known as immunophenotyping. Differentiation of a cell’s lineage phenotype is predicated on the antigens expressed on the cell surface, and subsequently, intracytoplasmic and nuclear antigens.

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Flow cytometry has been at the forefront of immunophenotyping (see Chapter 3.2). Immunophenotyping is the term used to describe the application of fluorescently tagged antibodies to discriminate and quantitate the various constituent cells of the immune system by flow cytometry. Immunophenotyping cells on the basis of cell surface markers can be thought of as the first generation application of flow cytometry in immunotoxicology. Immunophenotyping relies on the well-established observation that expression of certain proteins on the cell surface can correlate with functional activity of the constituent cells of the immune system. It should be noted that most cell surface markers are markers for the lineage of the cells and do not necessarily correlate with the actual function of the cells. We thus draw distinction between markers of Lineage Phenotype and Functional Phenotype. By coupling fluorochromes to mAbs, the specific binding of antibody to antigen can be detected by the fluorescence emitted by the cell as it passes through the laser. The ability of a flow cytometer to rapidly process millions of cells, at a nominal rate of 300–30,000 s−1, and store the physical and fluorescence details of each cell in listmode data, illustrates the capacity of this technology to characterize constituent cell populations. Initially, the light source was restricted to an argon-ion laser and the choice of fluorochromes was limited. Analysis was therefore constrained to one mAb/fluorochrome combination (i.e., color) for each assay. Subsequently, the advent of new fluorochromes with different emission spectra, and multiple lasers on one platform has resulted in the simultaneous routine analysis of four colors. Despite its novelty, polychromatic flow cytometry (PFC) utilizing more than 9 colors has already made important implications in the diagnosis of leukemias and lymphomas and in the identification of antigen-specific T cells. Multiparameter analysis now appears to be only restricted by physical limitations of data representation, when confined to a two-dimensional paper document. Sample Preparation A fundamental requirement for immunotoxicity samples destined for flow cytometric analysis is the need for a single-cell suspension. Immunophenotyping of intact cells such as from spleen, thymus, lymph nodes, or tumors can be accomplished by both mechanical and enzymatic disaggregation techniques. The success of any disaggregation procedure can be evaluated by three important parameters: cell yield, cell viability, and baseline debris. Blood samples, collected into heparinized tubes, can be evaluated as whole blood or as a fraction, e.g., peripheral blood mononuclear cells (PBMCs) following red blood cell lysis and subsequent washing procedure. Erythrocyte lysing, sample fixation, and other sample preparation techniques used in flow cytometry are known to affect antigen stability or to activate blood cells, particularly leukocytes, leading to an alteration in surface glycoprotein expression and intracellular processes. Thus, appropriate control samples for the preparation procedures should be included in any flow cytometry analysis. Excellent

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reviews on permeabilization techniques, activation, and appropriate cytometric controls are reviewed elsewhere (Maino and Picker, 1998; Baumgarth and Roederer, 2000; Pala et al., 2000; Krutzik and Nolan, 2003).

Lineage Phenotype Marker Reagents Most reagents detecting surface markers on different cell populations consist of monoclonal antibodies coupled to one of a wide range of fluorochromes. Each of the reagents has particular performance and stability characteristics that must be evaluated individually and then in combination to assure adequate signal intensity across the range to be detected.

Relative Fluorochrome Intensity A prerequisite to the construction of reagent panels is knowledge of the relative intensities of each of the fluorochromes to be used. Most commonly used fluorochromes are illustrated in Figure 4.2-1. In general, phycoerythrin (PE) and the PE tandem fluorochromes (PE-Texas Red, PE-Cy-5, PE-Cy5.5, and PE-Cy-7) are the brightest, followed by allophycocyanin (APC) and the APC tandems APC-Alexa 700 and APC-Cy7. The remaining small molecule fluorochromes, including fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), Pacific Blue, and the Alexa series of dyes exhibit fluorescence approximately 1 log dimmer than the previously mentioned dyes which contributes to a lower signal-to-noise ratio.

Excitation (nm) Excitation (nm) Emission (nm) FACSScan FACSCaliFACSVanLSRII™ FACSAriaEPICS™XFC500 CyAn™ AMoFlo® Fluorochrome 360, 405, 407 377 420 Blue Blue Blue Blue Blue Cascade Blue® 401 360, 405, 407 421 Blue Blue Blue Blue Blue Alex Fluor 405 410 455 360, 405, 407 Pacific Blue Blue Blue Blue Blue Blue 488 495 Green 519 Green Green Green Green Green Green Green Alexa Fluor 488 Green 525 Green 493 488 FITC Green Green Green Green Green Green Green Green 496,565 488 575 Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow PE 613 Orange 488 496,565 PE/Texas-Red Orange Orange Orange Orange Orange Orange Orange 660 645 595, 633, 635, 647 Red APC (Allophycocyanin) Red Red Red Red Red Red 649 633, 635 666 Red Red Red Red Red Red Red Cy5 668 650 595, 633, 635, 647 Alexa Fluor 647 Red Red Red Red Red Red Red Red 488 670 496, 565 Red Red PE/Cy5 Red Red Red Red Red Red Red 675 482 488 Red Red PerCP Red Red Red Red 690 650 595, 633, 635, 647 Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red APC/Cy5.5 690 496, 565 488 Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red PE/Cy5.5 488 690 482 Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red Far-Red PerCP-Cy5.5 650 774 488 APC/Cy7 Infra-Red Infra-Red Infra-Red Infra-Red 496, 565 488 774 PE/Cy7 Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Infra-Red Red 546 488 647 Red Red Red Red Red Red Viability Probe (7-AAD) Red Red 305, 540 620 Orange 325, 360, 488 Propidium Iodide (PI) Orange Orange Orange Orange Orange Orange Orange Orange 488 519 493 CFSE Green Green Green Green Green Green Green Green Green Green Trademarks: BD FACScan™ , BD FACSCaliber™, BD FACSVantage™ SE, BD FACSCanto™, BD™ LSRII, BD FACSAria™ are trademarks of Becton, Dickinson and Company. Coulter® EPICS® XL™/XL-MCL™ are registered trademarks of Beckman Coulter, Inc. inFlux™ is a trademark of Cytopeia, Inc. CyAn™ ADP and MoFlo™ are trademarks of Dako Denmark A/S Cascade Blue®, Alexa Fluor® , Texas Red® are registered trademarks of Molecular Probes, Inc. Pacific Blue™ is a trademark of Molecular Probes, Inc.

Figure 4.2-1 Fluorochromes for flow cytometric analysis. Source: Modified from www.biolegends.com.

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Staining Controls Staining controls are important for all flow cytometry experiments and are critical in PFC experiments. As a general rule, the higher the number of fluorochromes used, the greater the risk for artifacts introduced by compensation errors (see below) and/or reagent interactions. Generally two types of controls should be included: those that address fluorochrome compensation and those that address biologically relevant negative or positive controls. Compensation is the analytical process by which the spectral overlap between different fluorochromes is mathematically eliminated. Typically, a linear algebraic algorithm is used. Compensation between detectors can be performed either by hardware post signal detection but before logarithmic conversion, or by software using digitization following collection. Adequate compensation is critical for proper analysis of PFC data. For each fluorochrome used in an experiment, a single-color stained sample for each fluorochrome should be collected. In general, the best permutation for these controls is in using the brightest case sample, i.e., the highest stimulating dose of a drug that stains the most cells possible. This method works particularly well with non-tandem dyes (FITC, PE, APC, AF dyes). When using tandem dyes (Cy5PE, Cy7PE, Cy7APC), lot-to-lot variations are common and so should mandate controls and samples derived from the same lots. Biological controls are also important in immunotoxicity testing by flow cytometry. Populations of cells known to express (or not express) a given marker should be included in parallel experiments as a check on the validity of the staining in the “unknown” samples. Perfetto et al. (2004) outline a protocol that includes a three-part quality assurance program to optimize, calibrate, and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (De Rosa et al., 2003). The initial steps of this program (system optimization) ensure that the instrument’s lasers, mirrors, and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of PMT voltages by determining the PMT voltage range and linearity, and validating the PMT voltage. Finally, to ensure consistent performance, Perfetto et al. (2004) describe procedures to monitor the precision, accuracy, and sensitivity of fluorescence measurements over time. The authors note that all three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer’s optical path. However, only a few of these procedures need to be carried out on a routine basis.

Novel Stains The fundamental advantage of flow cytometric technology in immunotoxicology testing is the identification, enumeration, and characterization of multiple

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cell types simultaneously. To do this, of course, requires simultaneous and independent measurement of many proteins through the use of many distinguishable fluorophores. While many organic fluorophores and chemically synthesized fluorescent dyes are currently available, their simultaneous use is complicated by relatively broad, overlapping emission spectra and the need for different excitation sources. To minimize these limitations requires multilaser, multi-detector instruments and relative expertise in compensation adjustment. The recent discovery of semiconductor nanoparticles or quantum dots has provided a significant alternative or complimentary technology to traditional fluorophores that alleviates compensation burden while increasing the multiplexing capability of traditional flow cytometry. Quantum dots are inorganic crystals of cadmium selenide (core) surrounded by a zinc sulfide shell (Bruchez et al., 1998). These structures offer several advantages over traditional organic fluorochromes. First, although the wavelength (or color) of light emitted by different quantum dots can vary, depending on the size of the nanocrystal core, they share similar biophysical properties including size and solubility, as well as fluorophore conjugation chemistries (Chan and Nie, 1998). Second, quantum dots do not lose fluorescence when exposed to light for long periods of time so they are photostable and less susceptible to metabolic degradation than organic fluorophores (Chan and Nie, 1998; Goldman et al., 2005). Third, quantum dots have relatively narrow and symmetrical emission spectra (Hotz, 2005), making them desirable for reducing the need of spectral overlap adjustments in multiplex immunophenotyping (Chattopadhyay et al., 2006). Data Generation The visualization and analysis of high-level flow cytometric data is complex, and improved software tools are needed to accomplish this effectively. Using current software, the most general data representations are either single-color histograms of target relative expression or a series of 2-parameter dot plots that includes all unique combinations of parameters analyzed. Discrete populations can be identified and their events gated and colored to permit visualization in each of the other dot plots. This process is one way of incorporating information from other parameters into the 2-parameter display of a single dot plot, thus creating a higher-level polychromic analysis. The inclusion of a density display further increases the informational content and in combination with multicolor gating provides a marked improvement in data visualization. The overall effect becomes an exercise in pattern recognition that can lend itself to algorithm gating. Data Analysis and Interpretation The primary goal in analyzing flow cytometric data is to classify the cells in the sample. This classification can be to a lineage phenotype, a functional

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phenotype or increasingly, to a combination to lineage and functional phenotype. Once a classification has been made, it is used to compare across the treatment groups to determine if there is a test article effect on one or more populations of interest. Numerical analysis of the classified populations is by and large, straightforward. Standard parametric or nonparametric tests, e.g., ANOVA or ANOVA on Ranks, coupled with the appropriate corrections for multiple comparisons are usually applicable. More challenging is making a meaningful classification in the first place and then interpreting the results as to their toxicological importance. As described in the preceding paragraph, data analysis and interpretation of immunophenotype data is typically conducted using what can be described as expert analysis. In expert analysis, the scientists conducting the analysis draw on their experience and a number of established (but often nonstandardized) rules for assigning a classification. Isotype controls, while sometimes essential, e.g., for semiquantitative analysis or if the primary antibody is of poor quality, may not be necessary as a routine for analysis of common subsets with high-quality antibodies (Sreenan et al., 1997). Similarly, crosslaboratory standardization, despite its seductivity may provide only limited benefit (Gratama et al., 1986). Expert analysis is usually conducted visually on the basis of unidimensional histograms, two-dimensional scatter plots or as a nested, sequential analysis of two or more two-dimensional scatter plots. For classification in routine lymphocyte subset analysis, expert analysis is often fully adequate. For polychromic flow cytometry, particularly when both lineage and functional phenotypes are being evaluated, more sophisticated analysis may be required. Indeed, more sophisticated analysis can define subpopulations that cannot be discriminated by any combination of twodimensional projections of multicolor data (Zamir et al., 2005). One of the first steps in enabling more sophisticated downstream analysis is ensuring acquisition of robust data. Some of the recently described analytical methods for high-dimensional flow cytometry data are listed in Table 4.2-1.

ADVANCED FLOW CYTOMETRY TECHNIQUES Functional Cell Phenotype Evaluation of T Cells. T cell immunity is one of the most important components of adaptive immune system. Unlike other hematopoietic cells, T cells are not generated in the bone marrow. T cell development consists of at least three major stages: first, migration of multipotent precursors from bone marrow to thymus; second, development of T cell in the thymus including selection and positive selection; and third, migration of mature T cells from thymus to periphery for continuous differentiation (for reviews, see Ellmeier et al., 1999; Rothenberg and Taghon, 2005). Defective T cell development and function may lead to increased propensity for infectious diseases or cancer (Holsapple

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TABLE 4.2-1 Examples of Analytical Methods Used with Multidimensional Flow Cytometry Data Method of Analysis Knowledge-guided cluster analysis Hierarchical clustering Multidimensional clustering Axes scaling Feedforward artificial neural network (ANN) Radial Basis Perceptron ANN

Fractal analysis (correlation dimension)

Application 2-color analysis of nonHodgkins lymphoma 8-color analysis of T cells 4-color analysis of bone marrow 11-color analysis of murine B cells Panel of 27 markers on leukemia cells 4-color analysis of lineage and functional phenotypes in murine bone marrow B cell immunoglobulin light chain utilization

Reference Barlage et al. (1999) Petrausch et al. (2006) Zamir et al. (2005) Tung et al. (2004) Kothari et al. (1996) Quinn et al. (2007)

Wongchaowart et al. (2006)

et al., 2004; Dietert and Holsapple, 2007). On the other hand, unregulated T cell activity may lead to autoimmune-related immunotoxicities (Pieters and Albers, 1999; Pieters et al., 2002). A variety of pharmaceuticals are known to cause immunotoxicity to T cells (Heo et al., 1997; Pillet et al., 2006; Dietert and Holsapple, 2007; Pieters, 2007). Therefore, characterization of T cell development and function is crucial for assessment of drug safety and chemical toxicity. Development and differentiation of T cells in thymus, spleen, or lymph nodes is assessed by T cell surface markers (see Chapters 2.3 and 3.2). Some T cell surface markers can also be used to examine T cell function. For example, the population of CD4positive/CD62L low/CD44high is considered memory T cells, whereas CD4positive/CD25positive T cells are considered regulatory T cells in the periphery. Ex vivo activation of T cells following isolation from drug-treated animals can be utilized to further estimate T cell function. The most commonly used T cell activators are anti-CD3 antibody, phorbol 12-myristate 13-acetate (PMA), and superantigen (SEB). Anti-CD3 binds to T cell receptor (TCR) and this cross-linking leads to TCR signaling. PMA mimics intracellular diaceglycerol, which activates protein kinase C. Superantigens are bacterial proteins that activate T cells through cross-linking major histocompatibility complex (MHC) class II molecules of antigen-presenting cells (APCs) with TCRs. Once T cells are activated in vitro, either surface markers or intracellular proteins can be stained and analyzed by flow cytometry to investigate T cell function. CD62L down-regulation and CD69 up-regulation are early markers for T cell


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activation (detected within several hours), whereas CD25 up-regulation is sustained and can be detected even 24 hours poststimulation. With the advent of PFC, leukocyte subset analysis (De Rosa et al., 2003) in toxicology species, especially nonhuman primates (NHPs), has also undergone tremendous modifications. Mattapallil et al. (2005) characterized T cell responses by multiparameter flow cytometry to extend the analysis of the cellular immune responses generated after immunization by ninecolor flow cytometry. Immunophenotyping and enumeration of the frequency of memory CD4+ and CD8+ T cells producing IL-2, IFN-γ, and TNF-α were performed after ex vivo stimulation. Such cytokines are important for sustaining memory (IL-2) and mediating effector function (IFN-γ and TNF-α) and, thus, provide an assessment of the quality of the response. CD4+ and CD8+ T cells were further separated into CD45RA+CD95– naive and CD45RA–CD95+ memory cells, respectively. Essentially, all cytokineproducing T cells were CD45RA–CD95+ memory cells, and this population was used to calculate the total magnitude and quality of the cytokine response. Within the gated CD4+ or CD8+ CD45RA–CD95+ memory T cell population, cells were then segregated into IFN-γ– or IFN-γ+ cells and further assessed for production of IL-2 or TNF-α, or both. This analysis revealed seven functionally distinct populations producing IL-2, IFN-γ, and TNF-α, individually or in any combination. Together, such populations comprise 100% of the total CD4 or CD8 memory cytokine response. Intracellular Staining for T Cell Subtype and Signaling While surface markers have been widely used in examining T cell development and function, they may not be sufficient to identify T cell subpopulations or reflect T cell function. One such example is enumeration of regulatory T cells. Although CD4+/CD25+ cells represent most of the regulatory T cells in animals, this population also includes activated CD4 T cells. In addition, it has been shown that there is a minor subset of CD25 low expressing regulatory T cells. Therefore, intracellular staining of “regulatory T cell-specific” transcription factor Foxp-3 is used for more accurate enumeration of regulatory T cells. Intracellular cytokine staining is also used to study T cell activation and T cell differentiation. Particularly, intracellular IL-2 and INF-γ expression are associated with Th-1 type activation, while intracellular IL-4 and IL-10 detection characterize Th-2 type activation. Recently, intracellular staining of IL-17 has been used for the identification of a newly discovered Th-17 cell population (Bettelli et al., 2006). Recently, phospho-specific antibodies have been used in flow cytometry for detecting intracellular phosphorylation process at single-cell level. This technique has been proven to be extremely valuable for studying T cell function. Several groups have successfully used a variety of phospho-specific antibodies to detect T cell activation in vitro and in vivo. Currently the most widely used

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phospho-specific antibodies for T cell function are antibodies targeting MAP kinase phosphorylation and STAT protein phosphorylation (for reviews, see Krutzik and Nolan, 2003; Perez and Nolan, 2006). Evaluation of Monocytes and Macrophages. Monocytes and macrophage are phagocytes in the body responsible for the clearing of cellular debris and pathogens. Monocytes are generated from bone marrow and circulate in blood. Mature monocytes migrate from the bloodstream into various tissues and differentiated into specific types of tissue macrophages. Flow cytometry has been used to evaluate immunophenotypic and functional characteristics of monocytes. Since most of the tissue macrophages are difficult to isolate, the most commonly assessed phagocytes in toxicological studies are monocytes from peripheral blood and alveolar macrophages. In peripheral blood, monocytes can be easily identified by their size (forwardangle scatter) and surface properties (side-angle scatter) on a flow cytometer. Sometimes surface markers can also be added to further confirm this population. Most commonly used surface markers for murine monocytes are CD11b (MAC-1) and MHCII. In human, CD14 and HLA-DR have been widely used to identify monocytes. Drugs such as glucocorticoids and imatinib inhibit monocyte growth/development or promote monocyte death, which could lead to depletion of monocytes from peripheral blood. One of the most important functions of phagocytes, monocytes, and macrophages, is their ability to engulf or phagocytose opsonized and un-opsonized particles, including pathogenic components (Lambrecht, 2006 and Chapter 3.1.2). It is clear that macrophages phagocytose opsonized particles through Fc and complement (C) receptors (Brown, 1991; Swanson and Baer, 1995). However, the receptors for un-opsonized particles are still elusive. For functional measurement of phagocytes, it is important to measure the phagocytosis of both opsonized and un-opsonized particles (Steinkamp et al., 1982; Lehnert et al., 1986). Currently, the most commonly used method is incubating macrophages with fluorescent probe-conjugated particles or heat-inactivated bacteria and then measuring phagocytosis by an increase in the fluorescence intensity of treated phagocytes. Evaluation of Natural Killer (NK) Cells. NK cells are lymphoid cells capable of mediating host resistance against neoplasia, especially metastases (Cederbrant et al., 2003 and Chapter 3.1.2). Traditionally, NK cells are defined by their ability to cause rapid lysis of sensitive target cells (e.g., human K562 cells and murine YAC-1 cells). Typically, CD56 is used as a lineage phenotype marker for NK cells. Although expression of CD56 indicates membership in the NK lineage, its presence does not ensure NK function and CD56+ cells are highly heterogeneous. In contrast, CD69 is a functional triggering molecule on activated NK cells and up-regulation of CD69 can be used as a marker of increased NK activity (Makar et al., 2005). Thus, semiquantitative analysis of CD69 will likely provide greater insight into NK function that a


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simple assignment to the lineage by CD56. Also, as reviewed by Cederbrant et al. (2003), a number of flow cytometry-based assays for NK function have been developed. These rely on pre-labeling the target cells with a “permanent” fluorescent dye, mixing the target cells with the effector cells, and then selectively labeling necrotic cells with membrane-impermeant dyes (e.g., octadecylamine-fluorescein isothiocyanate [F-18] and propidium idodide) for human whole blood NK cells or the membrane of apoptotic cells with annexin-V (Radosevic et al., 1990). Evaluation of Dendritic Cells. Dendritic cells (DCs) are a distinct group of potent antigen-producing cells (APCs) that are bone marrow–derived and found in the circulation and within lymphoid and non-lymphoid tissues. DCs have been associated with the initiation of primary immune responses, graft rejection, autoimmune diseases, and generation of T cell-dependent immune responses. DCs mediate allergic contact dermatitis, a delayed-type hypersensitivity reaction induced by small reactive chemicals. This stimulatory process can be studied through in vitro sensitization tests looking at co-stimulatory molecule up-regulation, proliferation, and the production of various cytokines or chemokines, e.g., TNFα, IL-1β, IL-6, IL-8, IL-10, and IL-12. DCs represent only a minute subpopulation of circulating peripheral blood cells, as well as the cellular populations of the lung, intestine, genito-urinary tissue, and lymphoid tissue. DCs are also present in the skin and mucous membranes. Langerhan cells are skin-derived DCs that migrate to regional lymphoid organs after taking up antigen and undergoing an activation and maturation step. DCs are optimized for antigen presentation in that they capture antigen with high efficiency (Steinman and Swanson, 1995), undergo recruitment and migration, express high levels of MHC and co-stimulatory molecules, and produce T cell regulatory cytokines, as well as chemokines. DC activation can occur upon contact with immunogenic drugs and activated DCs have up-regulated costimulatory molecules CD83 and CD86. They also secrete various cytokines such as IL-1β and down-regulate proteins involved in antigen uptake such as aquaporins. In parallel, activated DCs start to migrate from the epidermis into the draining lymph node, complete maturation and present fragments of the haptenated self-proteins to T helper cells resulting in antigen-specific immune responses (see Chapter 8). This process can be exploited in identifying perturbations of the DC compartment by staining for MHC Class I, CD54, CD83, and CD86 in response to activation. DC cells and their subtypes are increasingly analyzed by flow cytometry in mechanistic studies of immune responses. Cytokine Production Cytokines make up an extensive group of signaling proteins that are produced following cellular activation and which act as cellular modulators throughout developmental, homeostatic, or pathologic conditions (Dinarello, 2002). Flow

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cytometric cytokine analysis constitutes studying cytokine-producing cells at the single-cell level. This can be achieved by coupling phenotypic and other functional markers with intracellular cytokine staining (ICS). The use of protein transport inhibitors such as brefeldin A and monensin, which inhibit endoplasmic reticulum (ER) and Golgi complex trafficking, respectively, without effecting de novo protein synthesis, greatly increases the sensitivity of ICS (Corsetti et al., 1992; Jung et al., 1993). Flow cytometry has been used to great effect to study cytokine changes in infectious, neoplastic and inflammatory disease in humans (Lucey et al., 1996), and techniques are available for the study of both plasma membrane and intracellular cytokines (Jung et al., 1993). Changes in cytokine expression are frequently observed in human subjects or animals in response to toxic compound exposure. Tissues such as the lungs, liver, and skin appear to be particularly susceptible to immunotoxicity induced by any means and mediated through macrophages (Henson and Riches, 1994). Subsequent release of cytokines and reactive oxygen and nitrogen intermediates from these cells can exacerbate drug adverse events. Cytokines such as colony-stimulating factors, chemotactic factors, TNF-α, and IL-6, stimulate inflammatory cell infiltration, activation, and proliferation. It is well established that overproduction of IL-1 and TNF-α can lead to amplification of the inflammatory process (Laskin and Pendino, 1995). The ability of xenobiotics to induce (Dastych et al., 1999) or suppress (O’Keefe et al., 1992) cytokine expression has been proposed as a possible mechanism explaining their adverse effects on the immune system. To this end, ICS has been used to measure drug-induced inhibition of cytokine production, inhibition of cytokine release, induction of immunosuppressive cytokines, cytokine homeostatic maintenance, and cytokine-induced alterations in cellular activation or transcriptional mechanisms as reviewed by House (1999). The importance of cytokine release as an end point for immunotoxicity is underscored by the ultimate exacerbation of this phenomenon, termed “cytokine storm.” This condition is exemplified by the proliferation of cytokinespecific cells and subsequent release of a cascade of pro-inflammatory cytokines resulting in pain, fever, and life-threatening organ failure due to hypotension (Suntharalingam et al., 2006). An example is the case of a CD28-specific monoclonal antibody, TGN1412, with claimed therapeutic benefit for a number of diseases. Routine immunotoxicity testing in nonhuman primates failed to demonstrate the potential of this molecule to induce cytokine storm. Subsequently, severe adverse effects attributed to cytokine storm were seen in a Phase I clinical trial (Suntharalingam et al., 2006). The need to contain pharmaceutical development costs obviates the desire to perform relevant and predictive preclinical immunotoxicity studies that limit the use of live animals. Thus, increased focus on in vitro ICS performed with either human whole blood or PBMC has been evident in recent years (see reviews by Maino and Picker, 1998; Godoy-Ramirez et al., 2004).


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Neutrophil Oxidative Burst The neutrophil is a critical element in host defense against infection, as evidenced by the devastating outcomes that can ensue when a host’s neutrophils are numerically depleted or functionally defective. Phagocytosis and the subsequent production of reactive oxygen species (ROS) during oxidative burst represent a central element of the host defense system against pathogens and xenobiotics. Neutrophils undergoing oxidative burst produce superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl radical, peroxyl radical, and hypochlorous acid, along with reactive nitrogen species (RNS): nitric oxide (NO) and peroxynitrite (ONOO−). Of these ROS, O2− and H2O2 can be directly measured by flow cytometry using fluorescent probes such as hydroethidine, dichlorofluorescein diacetate, and dihydrohodamine. Conversion of dihydroethidine to ethidium bromide is the most sensitive assay to detect intracellular O2− production in neutrophils. In diverse inflammatory settings, neutrophils activation by host-derived inflammatory mediators can lead to tissue damage (Han et al., 2006). Hence most of the anti-inflammatory drugs developed thus far are geared toward inhibiting neutrophil function in such disease settings. Nonsteroidal antiinflammatory drugs (NSAIDs)—ibuprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, naproxen, and indoprofen—are among the most widely prescribed drugs for the treatment of pain, fever, and inflammation. Other drugs that inhibit neutrophil function are: antifungal agents, immunosuppressants—cyclosporine and corticosteroids; histaminergic drugs—loratidine and dithiaden; and chemically reactive metabolites of sulfamethoxazole. The assays described above fit into the host resistance experiment models and can be considered for immunotoxicology testing of anti-inflammatory drugs when data from a primary screen suggest alteration in immune parameters.

Cell Apoptosis and Necrosis A number of methods for the study of apoptosis and necrosis by flow cytometry have been developed (see review by Steensma et al., 2003). Unfortunately, none of the methods are rigorously specific for apoptosis and many show poor overall specificity for cell death or cannot discriminate between the terminal stages of apoptosis and necrosis. Moreover, some of the fluorochromes used to detect apoptosis have emission spectra that fully overlap the spectra of those typically used for immunophenotyping or have a broad emission spectrum that makes compensation in multi- or polychromatic flow cytometry difficult, if not impossible. Thus, the preferred method would be one that measures two aspects of apoptosis, can discriminate between apoptotic and dead cells, and uses fluorochromes that allow simultaneous immunophenotyping. One such method is the annexin-V/7-amino-actinomycin-D assay described by Merchant et al. (2001) that is available in kit from BD Biosystems (San

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Jose, CA). Recently, we have described the use of this method in four-color immunophenotyping of mouse bone marrow cells (Quinn et al., 2007).

PRACTICAL CONSIDERATIONS IN APPLYING FLOW CYTOMETRY TO IMMUNOTOXICOLOGY Despite the popularity of immunophenotyping, interpreting changes in the absolute or especially relative numbers of the various component cells of the immune system in the context of risk assessment remains problematic. As described in Chapter 3.2, the results of the workshop on application of flow cytometry in immunotoxicology (Immunotoxicology Technical Committee, 2001) indicated that “immunophenotyping had not been sufficiently validated for routine use in immunotoxicity hazard identification and is unlikely to be used by itself to predict the immunotoxic potential of a previously uncharacterized chemical.” Although 10 years have passed, the conclusions of the panel are still largely valid today. This conjecture is supported by recent work that showed no correlation was found between lymphocyte subset numbers and function in dimethylbenzathracene (DMBA) or N,N-diethylaniline-treated mice (Gao et al., 2005) or in rats treated with dexamethasone (Immunotoxicology Technical Committee, 2001; Dietert and Holsapple, 2007), or exposed to a mixture of N,N-dimethyl-m-toluamide (DEET)-pyrodostigmine bromide (PYR) and JP-8 jet fuel in an attempt to model Gulf War Syndrome (PedenAdam et al., 2001). Similarly, there was no correlation between lymphocyte subsets and survival in Toxoplasma gondii-infected mice treated with methylmercury (King et al., 2001). A number of factors may contribute to the lack of correlation between immunophenotyping and functional changes. Some factors that limit the predictive power of immunophenotyping are listed in Table 4.2-2. Although immunophenotyping is part of many immunotoxicologic evaluations, it is unlikely to ever be a stand-alone analysis in risk assessment because of limited applicability as a screen for immunotoxicity. Despite not being a stand-alone technique and with only a limited role in hazard identification, immunophenotyping remains an important tool in hazard characterization, primarily in understanding mechanisms of immunotoxicity.

SUMMARY In order for novel flow cytometry methodologies to gain acceptance in routine immunotoxicology testing, rigorous assay validation and establishment of a database for known immunotoxicants are needed prior to attempting to interpret data from new and unknown xenobiotics. Flow cytometry can be a hugely valuable tool for drug development if it is found to be predictive, sensitive,

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TABLE 4.2-2 Factors Limiting the Predictive Power of Immunophenotyping Limiting Factor Sample

Timing

Rationale

Example

Reference

The sample evaluated may not reflect the actual functional compartment The time point(s) selected may not be optimal

Differences in expression of cell surface markers in spleen, lymph node, and thymus Sequential effects on different subsets after exposure to cyclosporine CD45RA (naive) and CD45RO (memory) T cells respond differently to simulation by anti-CD3 Regulation of CD56 correlates with activation of NK activity

Vremec and Shortman (1997)

Antigen-specific cells constitute ∼1% of splenic B cells

Newman et al. (2003)

Parameters

Cell surface markers of cell lineage may not reflect functional status

Granularity

Simply assigning a cell as positive or negative for one of more surface markers may not reflect functional status The relative number of relevant functional cells may be too low to detect

Rarity

Huby et al. (1995)

Hall et al. (1999)

Wendt et al. (2006)

and specific in immunologic toxicity-based risk assessment of drugs leading to reduced cost of evaluation of drug candidates.

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PART V EXTENDED IMMUNOTOXICOLOGY ASSESSMENT: IN VIVO MODELS

5.1 ANIMAL MODELS OF HOST RESISTANCE Gary R. Burleson and Florence G. Burleson

The aim of all toxicity studies is to obtain data useful for safety assessment. In immunotoxicity safety testing, a major objective is to determine the significance of an immunotoxic effect with respect to increased susceptibility to infectious and neoplastic disease (Gleichmann et al., 1989). Immunotoxicity caused by a test compound may result in an impaired clearance of an infectious agent, increased susceptibility to opportunistic infections, prevention or ineffective immunization, exacerbation of latent viral infections, or unintended immunostimulation. Clearance of an infectious microorganism allows an assessment of immunocompetence and serves as a biomarker of net immunological health. An aggregate, integral, intact, and functional immune system that includes local and systemic, innate and acquired, as well as humoral and cell-mediated immunity is required to protect from and eliminate infectious and neoplastic disease. Host resistance models evaluate the functional integrity of the immune system and provide the only means to directly assess the functional immunological reserve. A small statistically significant decrease in several immunological functions may not result in increased disease susceptibility due to immunological reserve while several small, not statistically significant, decreases in one or more immunological function, may result in additive or synergistic effects that together affect susceptibility to opportunistic microorganisms (Burleson, 1996). The biological significance of “x” percent change in any immunological parameter is not known, and the percent change

Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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associated with an increased susceptibility to disease may vary, depending on a number of factors including the virulence of the infecting microorganism and the immunological reserve of the individual (Burleson and Dean, 1995). Screening assays to detect immunosuppression are surrogates for functional assays which are surrogates for host resistance assays. Luster’s group initiated a series of studies that form the basis of risk assessment in immunotoxicology evaluations. Luster et al. (1988, 1992a, 1992b, 1993) evaluated immunological assays that predicted immunotoxicity and reported concordance values using host resistance as the comparator, since host resistance assays are considered to be the ultimate predictor of adverse effects (Germolec, 2004). There are numerous host resistance models available for immunotoxicity testing. Some representative models are listed in Table 5.1-1. Immunotoxicity testing strategies may differ depending on the (a) data from standard toxicity tests, (b) pharmacological properties of the drug, (c) intended patient population, (d) structural similarities to known immunomodulators, (e) disposition of the drug, and (f) clinical information (Guidance for Industry, 2006). Further considerations for an immunotoxicity testing strategy include (a) pharmaceutical company policy, (b) budget, (c) timeline, and (d) the number of other therapeutics under development. Immunotoxicity testing should begin with standard toxicology tests that provide screening assays for immunotoxicity. Positive screening assays or drugs intended to target the immune system should evaluate immune function. The testing strategy may first evaluate immune function, and if positive, proceed to host resistance testing or may first evaluate host resistance concurrent with or followed by immune function testing to provide mechanistic insight. Testing immune

TABLE 5.1-1 Commonly Used Host Resistance Assays Representative Host Resistance Models Influenza virusa • Numerous strains have been used for immunotoxicity testing: Influenza A/PR8/34 (H1N1)a; Influenza A/Taiwan/1/64 (H2N2)a; Influenza/A HKx31 (H3N2)b, Influenza A/Hong Kong/8/68 (H3N2)a, and Influenza A/Port Chalmers/1/73 (H3N2)a. Murine Cytomegalovirus (primary and/or reactivation)a Rat Cytomegalovirus (primary and/or reactivation)a Streptococcus pneumoniaea Listeria monocytogenesa Pseudomonas aeruginosaa Candida albicansa B16F10 Syngeneic tumor cell assayc PYB6 Syngeneic tumor cell assayc a

Species Mouse and Rat

Mouse Rat Mouse and Rat Mouse and Rat Mouse Mouse Mouse Mouse

Strains available from the American Type Culture Collection (ATCC), Manassas, VA. Strain not available from the ATCC. c Cells available from the ATCC. b

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MODELS OF HOST RESISTANCE TO INFECTIONS

function prior to host resistance assay must evaluate all facets of the immunological armamentarium and include an evaluation of innate immunity, cellmediated immunity, as well as humoral-mediated immunity. The timing of testing may vary but should begin prior to or parallel to Phase 2 clinical trials, unless specific concerns are identified, such as indication, chemical class, molecular concerns, or inclusion of immunocompromised patients in Phase 1 studies.

MODELS OF HOST RESISTANCE TO INFECTIONS Influenza Host Resistance Assay The algorithm in Table 5.1-2 provides a path to ensure a thorough immunotoxicological safety evaluation for therapeutics. The influenza host resistance assay is a well-characterized model and can serve as a biomarker for evaluating the overall health of the immune system. Therapeutic agents can be evaluTABLE 5.1-2 Algorithm for Immunotoxicity Evaluation Using Host Resistance Assays: Generalized Assay to Evaluate the Overall Health of the Immune System Influenza virus host resistance model to test the overall health of the immune system: (A) Viral clearance (B) Mechanistic immune function assays associated with influenza host resistance assay that may or may not be included: • • • • • • • • •

Cytokines Interferon activity Macrophage activity NK cell activity CTL activity Influenza-specific IgM, IgG (IgG1 and IgG2a)—TDAR Secondary infection (vaccine response) Immunophenotyping Histopathology

MCMV Latent Virus Reactivation Model

➞ ➞

➞ ➞

Suppressed NK, CTL, TDAR, CD4+

Decreased MZB cells

S. pneumoniae systemic model for encapsulated bacteria; measure T-independent antibody response (TIAR)

NK: natural killer; CTL: cytotoxic T lymphocyte; TDAR: T-dependent antibody response; MCMV: murine cytomegalovirus; MZB cells: marginal zone B cells; TIAR: T-independent antibody response.

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ated for immunotoxicity using the influenza host resistance model in either mice or rats (Lebrec and Burleson, 1994; Burleson, 1995; Hammerbeck et al., 2007). Clearance of virus requires an intact and functional immune system that incorporates a cascade of immune responses including innate immunity (cytokine production, interferon activity, natural killer [NK] cell activity, and macrophage activity), as well as acquired or adaptive immunity (cytotoxic T lymphocyte [CTL] activity and antibody production). A vaccine response or effect on secondary infection, immunophenotyping, and histopathology evaluations may also be included in the immunotoxicity evaluation with this model. Influenza virus is a T-dependent antigen and formation of antibody to influenza virus requires functional T cells, B cells, and macrophage antigen processing and presentation activity. Therefore, measurement of influenza-specific IgM or IgG provides an evaluation of TDAR function. Clearance of an infectious agent is the ultimate measure of immunotoxicity and optional mechanistic immune functions are also available in the mouse and rat influenza model, including measurement of selected cytokines, interferon activity, macrophage function, NK activity, CTL activity, and TDAR (Table 5.1-3). The influenza virus host resistance model has been characterized in mice and rats, and has been widely used to evaluate the potential immunotoxicity of therapeutics. Influenza virus is used as the infectious challenge agent and administered intranasally in a 28-day repeat-dose study. Mice or rats are dosed for 7 days, infected and then dosed for an additional 21 days. Viral clearance is quantified by measuring infectious virus (plaque-forming units) at various times following infection. Dexamethasone may be used as a positive immunomodulatory control. This host resistance assay has been used in Balb/c, C57BL/6, and B6C3F1 mice and Fischer 344 (CDF), Brown Norway, and TABLE 5.1-3 Algorithm for Immunotoxicity Evaluation Using Host Resistance Assays: Targeted Host Resistance Assays to Evaluate Specific Immunotoxicity Questions Targeted Host Resistance Assays: Streptococcus pneumoniae pulmonary assay for innate immunity: • Therapeutics affecting neutrophils and/or macrophages • Anti-inflammatory therapeutics • Therapeutics targeting TNF-α Marginal zone B (MZB) cell assay: • Systemic Streptococcus pneumoniae assay to evaluate T-independent antibody response and bloodborne infections controlled by MZB cells Listeria monocytogenes systemic assay: • Intracellular Gram-positive bacterial assay to evaluate liver and splenic neutrophils and macrophages Murine cytomegalovirus (MCMV) latent viral reactivation assay: • Assay to evaluate reactivation of latent virus due to immunosuppression

TARGETED HOST RESISTANCE ASSAYS

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Sprague-Dawley (CD) rats. The mouse influenza host resistance model has been used as one of a battery of three host resistance assays to evaluate a small molecule therapeutic targeted for splenic tyrosine kinase (Syk) (Zhu et al., 2007), by Cowan et al. (2002), Olivier et al. (2007), Roque et al. (2006), and Miller et al. (2007). Human biologicals have been evaluated in the influenza host resistance assay. Recombinant TACI-Ig, a transmembrane activator, calcium-modulator and cyclophilin ligand interactor, has activity in rodents and was evaluated for immunotoxicity in the mouse influenza host resistance assay (Roque et al., 2006). TACI-Ig treatment resulted in a dose-dependent decrease in spleen weight and influenza-specific IgM and IgG in both the lungs and serum, thereby indicating a decreased TDAR response. Immunophenotyping revealed a decrease in B cells, but not T cells, in peripheral blood. However, there was no effect on viral clearance and thus no immunotoxicity as measured in this host resistance assay. This study demonstrates the importance of immunological reserve. Surrogate biologicals may be used for immunotoxicity testing of human biologicals that do not have activity in rodents. The surrogate hamster anti-mouse α1B1 integrin antibody had no effect on influenza viral clearance and was therefore not immunotoxic in the mouse influenza host resistance assay (Olivier et al., 2007). Regulation of osteoclast differentiation is mediated by the receptor activator of NF-κB (RANK) ligand (RANKL), and two receptors, osteoprotegrin and RANK. rRANK-Fc is a fusion protein containing the murine RANK extracellular domain with the C terminus of the Fc domain of murine IgG1. The murine surrogate biological fusion protein RANK-Fc was not immunotoxic in the mouse influenza host resistance assay since there was no effect on viral clearance, influenza-specific IgG (TDAR) and no effect on IL-1B, IL-6, or TNFα (Miller et al., 2007). The rat influenza host resistance model has also been used in numerous immunotoxicity evaluations (Steele et al., 2005). The therapeutic agent should be further tested in the murine cytomegalovirus (MCMV) latent virus reactivation model if results from the influenza host resistance assay indicate a decreased functional activity for either NK, CTL, or TDAR, or a decrease in CD4+ cells as observed by immunophenotyping. The MCMV latent virus reactivation model is discussed in detail below. The test article should be tested in the Streptococcus pneumoniae systemic model for encapsulated bacteria if immunophenotyping or histopathology, done in conjunction with the influenza host resistance model, reveals a decrease in the number of marginal zone B (MZB) cells. MZB cells are critical in host defense against bloodborne encapsulated bacteria and this host resistance assay is discussed in detail below.

TARGETED HOST RESISTANCE ASSAYS The algorithm in Table 5.1-3 lists targeted host resistance assays that are useful to evaluate specific immunotoxicity questions.

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Streptococcus Pneumoniae Pulmonary Assay for Innate Immunity Therapeutics Affecting Neutrophils and/or Macrophages. Streptococcus pneumoniae has been used in mice and rats as a pulmonary infection following intranasal infection (Gilmour and Selgrade, 1993; Gilmour et al., 1993; Burleson and Burleson, 2006). The Streptococcus pneumoniae host resistance model has been used with Balb/c and C57BL/6 mice and Fischer 344 (CDF), Lewis, and Sprague-Dawley (CD) rats. Animals are infected intranasally and bacterial clearance measured. Bacterial clearance is evaluated by determining the number of colony-forming units (CFU) per gram of lung tissue. Dexamethasone or cyclophosphamide is used as a positive immunomodulatory control as each has an immunosuppressive effect on innate immunity and delays bacterial clearance. Bacterial clearance is quantified before the specific, acquired adaptive immune system is operative. Macrophages were demonstrated to be important in the clearance of streptococci from the lungs of mice (Gilmour et al., 1993) and rats (Gilmour and Selgrade, 1993). Further studies by Gilmour and Selgrade (1993) demonstrated the importance of neutrophils in pulmonary streptococcal disease in rats by pretreatment with an antibody to neutrophils. Cytokines may also be measured in the streptococcal model. The Streptococcus pneumoniae host resistance model in mice has been used in numerous immunotoxicity evaluations, and was reported as one of a battery of three host resistance assays to evaluate a small molecule therapeutic targeted for splenic tyrosine kinase (Syk) (Zhu et al., 2007). The streptococcal host resistance model in rat has also been used in numerous immunotoxicity evaluations (Steele et al., 2005). Anti-Inflammatory Therapeutics. The Streptococcus pneumoniae pulmonary host resistance assay is recommended for anti-inflammatory therapeutics (Komocsar et al., 2007). The Streptococcus pneumoniae pulmonary host resistance model in Lewis rats was used to assess the effects of anti-inflammatory agents on innate immunity. The model was able to predict potential drug suppression of the innate immune response to Streptococcus pneumoniae. The authors stated the ability to rank order the severity of innate immune suppression with multiple test articles in the same study made this model effective in screening potential drug candidates. Therapeutic Agents Targeting TNF-a. The Streptococcus pneumoniae host resistance model is also valuable for evaluating the importance of macrophage cytokines on bacterial host resistance. Human biological therapeutics targeting inhibition of TNF-α have been used to treat inflammatory autoimmune diseases such as rheumatoid arthritis. Decreased TNFα as a result of treatment with monoclonal antibodies (mAb) to TNF-α has an effect on several biomarkers of infection (Takashima et al., 1997; van der Poll et al., 1997; Benton et al., 1998; O’Brien et al., 1999). These studies have reported that treatment of mice with a mAb to TNF-α results in altered levels of TNF-α in the lungs


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and serum, decreased neutrophils and increased numbers of bacteria (impaired bacterial clearance) with decreased survival in mice infected intranasally with Streptococcus pneumoniae. The streptococcal pulmonary host resistance model is thus an important means to assess the functional immunological capacity of macrophages and neutrophils, as well as macrophage cytokines. Therapeutic agents that target TNF-α may be tested using the Streptococcus pneumoniae pulmonary host resistance model and this host resistance assay may be used to choose a lead among compounds with equivalent therapeutic efficacy based on immunosuppression. Monoclonal antibody to TNF-α has a dramatic effect on bacterial clearance in this model. Pseudomonas aeruginosa can also be used as a pulmonary bacterial host resistance assay to evaluate the immunotoxicity of therapeutics when immunotoxicity is suspected in neutrophils, macrophages, and/or TNFα (Gosselin et al., 1995; Burleson and Burleson, 2006). TNFα also plays an essential role in preventing reactivation of latent tuberculosis (Mohan et al., 2001). Marginal Zone B (MZB) Cell Assay Bacteria encapsulated with a polysaccharide capsule such as Streptococcus pneumoniae present a different challenge to the immune system. Capsular polysaccharide antigens belong to a group of thymus-independent type 2 antigens (TI-2) (Mond et al., 1995; Pillai et al., 2005) and are highly dependent on the presence of a functional marginal zone (Amlot et al., 1985; Harms et al., 1996; Guinamard et al., 2000). MZB cells consist of a distinct naive B lineage, as well as memory B cells. It is clear that MZB cells in both humans and rodents are considered a critical host defense mechanism directed against encapsulated bloodborne pathogenic microorganisms. Therefore, immunotoxicity directed against MZB cells not only decreases protection against bloodborne pathogens but also results in a depletion of B cell memory. Thus, TI-2 antibody responses are decreased or ablated as a result of MZB cell immunotoxicity. The Streptococcus pneumoniae MZB cell model has been characterized in mice and Sprague-Dawley rats with a systemic bloodborne infection by intravenous inoculation. Bacteria are quantified by determining the number of CFU in the spleen, liver, lungs, and blood over a 2-week period. Cytokines, hematology, immunophenotyping, and anti-streptococcal antibody (TIAR) are also quantified in this model (Burleson and Burleson, 2006). Listeria Monocytogenes Host Resistance Assay The Listeria monocytogenes host resistance model is controlled primarily in the liver and spleen. The Listeria monocytogenes systemic infection assay is useful primarily to evaluate adverse effects on neutrophils and Kupffer cells of the liver and splenic macrophages and neutrophils. NK cells and T lymphocytes also play a role in bacterial clearance. The Listeria monocytogenes host

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resistance model has been used to evaluate mAbs directed against CD11b to determine whether inhibition of this adhesion molecule would enhance disease susceptibility to Listeria and therefore predict whether this anti-inflammatory therapeutic approach would enhance susceptibility to opportunistic infections in humans. CD11b/CD18 (Mac-1) is a leukocyte integrin that plays a critical role in neutrophil adhesion and the initiation of acute inflammatory processes and is therefore a therapeutic anti-inflammatory target. CD11b (alpha M integrin) complexes with CD18 (beta 2 integrin) to form complement receptor type 3 (CR3) heterodimer. Treatment with either monoclonal antibody NIMPR10 or 5C6, both directed against CD11b resulted in decreased clearance of Listeria in the liver and spleen with increased mortality (Conlan and North, 1992; Burleson and Burleson, 2006). Neutrophils and monocytes were decreased and mice were unable to control the infectious intracellular bacterial disease. Treatment of mice with a surrogate biological mAb designated NIMP-R10, directed against the CD11b polypeptide of the CD18/CD11b heterodimer, exacerbated listeriosis by preventing myelomonocytic cells from focusing at sites of infected hepatocytes in the liver. Under these conditions, an otherwise sublethal Listeria inoculum grew unrestricted within hepatocytes and caused death in 3 days (Conlan and North, 1992). The results obtained with NIMP-R10 are similar to those reported with a different anti-CD11b mAb (5C6) (Rosen et al., 1989; Conlan and North, 1991). Treatment with 5C6 inhibits the diapedesis of monocytes, macrophages, and neutrophils into the inflammatory lesions in the liver and spleen. Murine Cytomegalovirus (MCMV) Latent Virus Reactivation Model MCMV and rat cytomegalovirus (RCMV) are well-characterized models for human cytomegalovirus (CMV) disease. MCMV and RCMV have been used for immunotoxicity testing by evaluating the effect on clearance of the virus in a primary viral infection (Selgrade et al., 1988; Goettsch et al., 1994; Garssen et al., 1995; Selgrade and Daniels, 1995; Van Loveren, 1995; Ross et al., 1996, 1997). These viruses cause a primary infection with infectious virus detectable in a variety of organs (lung, liver, spleen, and salivary gland). After primary infection, virus is cleared and viral replication is terminated by immune control mechanisms. The virus can remain in a nonproductive latent state for a lifetime (Jordan et al., 1977; Shanley et al., 1979) unless viral reactivation or recrudescence occurs following intended immunosuppression in transplant patients or unintended immunosuppression in individuals receiving immunotoxic therapeutics, or in individuals immunosuppressed by infection with HIV or idiopathic immunosuppression. Reactivation of latent viral infection is a serious problem for immunosuppressed individuals and may be due to cytomegalovirus (CMV), herpesvirus (HSV), or the human BK and JC polyomaviruses. BK virus and JC virus were isolated in 1971—BK virus from the urine of a renal allograft patient who was treated with immunosuppressive therapy and JC virus from brain tissue of a patient with PML. BK virus, JC virus, CMV, and

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HSV infect the majority of humans in childhood with a mild primary infection that often then resides as a latent viral infection (Alford and Britt, 1990; Eckhart, 1990; Whitley, 1990). The MCMV latent viral model is an excellent model to assess reactivation of latent viral disease as a result of immunosuppression. There are many similarities between these viruses (CMV and HSV belong to the Herpesviridae virus family while BK virus and JC virus belong to the Papovaviridae virus family). All the viruses have double-stranded DNA (the human polyomaviruses are circular); all are ubiquitous in the human population; all cause mild primary infections followed by a latent viral infection; and immunosuppression, especially a suppressed CMI, results in reactivation of latent viral infection. The MCMV reactivation model may be used for any pharmaceutical agent that causes immunosuppression in CMI or HMI. Lymphocyte depletion studies revealed a hierarchy of immune control functions of CD8+, NK, and CD4+ cells. Reactivation was rare if only one cell type was deleted, but was evident after deletion of another cell type (Polic´ et al., 1998). This study demonstrates immunological reserve since depleting one immunological function such as NK activity reactivates virus in 5.6% of the mice, depleting either CD4 and NK or CD8 and NK activity reactivates either 25 or 80% of the mice respectively, while abrogation of CD4, CD8, and NK activity reactivates 100% of the mice (Table 5.1-4). The use of B cell-deficient mice for this model increases the sensitivity of detecting reactivated virus by 100 to 1000-fold (Polic´ et al., 1998). Suppression of HMI also results in an increased sensitivity to reactivated virus disease (Polic´ et al., 1998) and perhaps acts in a primary or secondary manner in the induction of reactivation disease. The US FDA issued an alert concerning spontaneous fatal PML due to JC polyomavirus in two patients with systemic lupus erythematosus (SLE) who had received treatment with Rituximab, a monoclonal antibody directed against CD20 on B cells (Fox, 2007). Natalizumab, a monoclonal antibody against alpha-4 integrins, has

TABLE 5.1-4 Immunological Reserve Demonstrated by Induction of Latent Virus Recrudescence Using Monoclonal Antibodies to NK, CD4, or CD8 Cells Either Singly or in Combinations (Polic´ et al., 1998) Treatment

Percent of Mice with Reactivated Virus (Salivary Gland)

Anti-NK antibody

5.6%

Anti-NK antibody Anti-CD4 antibody

25%

Anti-NK antibody Anti-CD8 antibody

80%

Anti-NK antibody Anti-CD4 antibody Anti-CD8 antibody

100%

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also been associated with the development of PML from reactivation of latent JC virus infection (Yousry et al., 2006). The MCMV reactivation model is recommended to test immunosuppressive therapeutics with the potential to cause serious recrudescence of latent viral disease. Other Models of Host Resistance to Infections There have been numerous other host resistance models used for immunotoxicity testing and these have been discussed and reviewed by Burleson and Burleson (2007). The Candida albicans fungal model (Herzyk et al., 2001) is another important model and may be used instead of or in addition to the Listeria monocytogenes model. Candida-specific IgG and cytokines may also be quantified. Other bacterial host resistance models include Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae; parasitic host resistance models including malaria and Trichinella spiralis; and viral host resistance models including encephalomyocarditis (EMC) virus, vesicular stomatitis virus (VSV), and reovirus.

HOST RESISTANCE AGAINST TUMORS B16F10 and PYB6 Host Resistance Assays Tumor host resistance models including the B16F10 and PYB6 models in mice have been used to evaluate immunotoxicity (McCay, 1995). The B16F10 melanoma and the PYB6 models are syngeneic for the C57BL/6 mouse. Studies using these syngeneic tumor cell models have also been performed successfully in C67BL/6 and B6C3F1 mice. B6C3F1 mice are the F1 generation of a cross between C57BL/6 × C3HHeN mice. The B16F10 melanoma model is based on the early studies of Fiedler (Fiedler, 1973; Fiedler et al., 1978) taking a B16 spontaneous tumor and developing a selection of B16F1 through B16F10 to derive the B16F10 cell line. B16F10 tumor cells are injected intravenously and tumor nodules counted visually on the surface of the lungs that have been removed and placed in Bouin’s solution. An alternative method for the B16F10 model utilizes the incorporation of 125IUDR in the lungs as a measure of tumor growth. The PYB6 fibrosarcoma tumor cell model (Dean et al., 1982) involves the subcutaneous injection of tumor cells. The number of mice with tumors (tumor incidence) is quantified in order to determine the effect of a test article compared to the vehicle control.

SUMMARY The influenza host resistance assay is able to evaluate the overall health of the immune system, as well as to evaluate mechanistic immunological function of

REFERENCES

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the innate, cell-mediated immunity (CMI) and humoral-mediated immunity (HMI) of the immune system. Host resistance assays measure the functional integrity of the immune system and are able to evaluate functional immunological reserve. The immunotoxicity safety assessment is difficult to assess when the biological significance of “x” percent change in a particular immune function is not known. Functional assessment must evaluate all facets of the total immune system and include innate immunity, CMI, and HMI. Targeted host resistance assays are available to evaluate specific immunotoxicity concerns including therapeutics affecting neutrophils and/or macrophages, antiinflammatory therapeutics, as well as therapeutics targeting TNFα in the treatment of autoimmune disorders such as rheumatoid arthritis and Crohn’s disease. Targeted host resistance assays also include evaluations of therapeutics affecting splenic marginal zone B (MZB) cells. Immunotoxicity of MZB cells may result in an increase in the number of infections with encapsulated bacteria, bloodborne infections, and bacterial pneumonias. A whole cadre of bacterial infections is possible if the antibody response to T-independent antigens is depleted or suppressed. Listeria monocytogenes in a targeted host resistance assay is available to evaluate systemic immunotoxicity involving Kupfer cells and neutrophils of the liver and splenic neutrophils and macrophages. Therapeutics that suppress CMI or HMI should be further evaluated with a latent virus reactivation model. Suppression of CMI can result in recrudescence of latent viral disease with resultant serious herpesvirus disease, cytomegalovirus disease, or reactivation of JC virus causing PML. Suppression of antibody production may lower one’s threshold and make one more sensitive to reactivated virus disease if concomitant suppression of CMI has resulted in reactivation of latent virus. Suppression of the humoral arm of the immune system may not only affect susceptibility to opportunistic infections, but also may result in ineffective immunizations which can also be tested in host resistance models. There are several strategies to evaluate immunotoxicity of therapeutic compounds. However, host resistance assays allow the evaluation of immunotoxicity in an aggregate functional immune system, utilize sufficient numbers of animals to assure the statistical power to detect immunotoxicity, and include the use of positive controls necessary to confirm negative findings. It is crucial to evaluate all arms of the functional immune response in order to derive data that are useful in performing a meaningful immunotoxicity safety assessment.

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5.2 APPROACHES TO EVALUATION OF AUTOIMMUNITY Danuta J. Herzyk

Autoimmunity is broadly defined as the reaction of the immune system involving subsets of cells (autoreactive T lymphocytes) or cell products (autoantibodies) against the organism’s own tissue components called autoantigens. Such a reaction can be transient and part of the physiological immune response or can be prolonged and lead to pathological processes described as autoimmune diseases. A broad range of autoimmune disorders, characterized by the excessive and abnormal immune response against autoantigens, are associated with chronic inflammation, tissue destruction, and functional changes of multiple organs (Rose, 2002). They are classified mainly as systemic diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis) or organ-specific diseases (e.g., diabetes mellitus type 1, Graves’ disease, Crohn’s disease, multiple sclerosis, myasthenia gravis, psoriasis). The common feature of autoimmune diseases is the presence of circulating autoantibodies (e.g., antinuclear, anti-dsDNA, anti-thyroglobulin, antiplatelet, islet cell antibodies, rheumatoid factor). The etiologies and pathophysiological mechanisms involved in the development of autoimmune diseases are only partially understood. The immune response can be initiated by stimulation with a foreign antigen (e.g., infectious agent) or an autologous antigen (e.g., platelet surface molecule, collagen, myelin base protein). Typically, homeostatic mechanisms are able to control immune responses to antigens and prevent pathological effects. The control mechanisms include both the central regulation of production of T and B Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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lymphocytes in the thymus and bone marrow, respectively, and peripheral interactions of activated or self-reactive T and B cells. Recent immunological research is focused on dendritic cells (DCs) as important component of the control of different mechanisms of immunity and tolerance (Steinman, 2004; Trombetta and Mellman, 2005). The role of DC as tolerogens is viewed to be critical in balancing their role as immunogens. On one hand trough tolerance, DCs influence the lymphocyte repertoire to diminish the danger of selfreactivity. On the other hand, upon encountering an infectious agent, DCs direct immunity toward the microbe. Another currently emphasized facet of tolerance and regulation of immune responses by DC involves T regulatory cells (“Tregs”) that are very effective in suppressing immunity in vivo (Watanabe et al., 2005). Profound dysregulation of any control mechanism as a consequence of either intrinsic (e.g., genetic predisposition, endocrine changes, age) or extrinsic factors (e.g., exposure to infections or chemicals) most likely causes the onset of autoimmune diseases (George et al., 1996). Some data indicate that chemically induced autoimmune-like responses include medicines used in therapeutic treatments (Olsen, 2004). While the association of drugs with adverse immune-mediated reactions is documented (Bachot and Roujeau, 2001; Demoly and Bousquet, 2001), clinical data indicating therapeutic agents as causes of autoimmune disease are sparse and often derived from case studies, indicating a low percentage of a patient population affected by these reactions. Furthermore, the adverse effects mediated by immune responses to drugs involve mainly hypersensitivity reactions. Despite some overlapping mechanisms, hypersensitivity needs to be distinguished from chemically induced autoimmune diseases. Thus, described herein approaches to the assessment of potential autoimmunity induction by new therapeutic agents are separated from the section on immunotoxicology testing for hypersensitivity (see Chapter 8). Concerning the role of therapeutics in the etiology of autoimmune diseases, a few mechanisms have been postulated. One hypothesis implies an association between genetic polymorphisms of xenobiotic-metabolizing enzymes (P450) with drug-induced autoimmunity (Griem et al., 1998; Naisbitt et al., 2001). Another points to a drug’s ability to stimulate MHC class II expression on cells that do not normally express class II molecules and consequently to enhance immune reactivity (Vial et al., 2002). These two scenarios relate to an important distinction between different types of drugs. The majority of small molecule drugs, metabolized by P450 enzymes, are not designed to interact with the immune system. On the other hand, some potent immunostimulatory agents, such as recombinant proteins IL-2 and IFN-α (cleared by protein catabolism) increase immune responses in patients as their therapeutic activity. While a prolonged use of immunostimulatory agents may be associated with a wide range of autoimmune disorders, a non-immunostimulatory drug may lead to a specific type of autoimmune response. In the latter case, the

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response can mimic the spontaneous, e.g., organ-specific, autoimmune disease; however, cessation of the drug treatment should result in the progressive recovery of manifestations of the disease. Evaluation of potentially drug-associated autoimmunity is very challenging not only because of the complicated etiology but also due to great difficulties in their diagnoses. Because of the relative low incidence and the involvement of multiple factors, including the duration of treatment and patient’s medical history and/or concurrent therapies, autoimmune disorders are often referred to as idiosyncratic. Sometimes they are described as autoimmune-like diseases or autoimmune syndromes (e.g., lupus syndrome) as the presentation and characteristics differ from the spontaneous diseases (Sarzi-Puttini et al., 2005). Several examples of drugs associated with autoimmunity and the diagnostic terms used for its description are presented in Table 5.2-1. The association between the indicated drugs and autoimmunity is based on clinical experience. Routine toxicology studies with these drugs did not detect signals for potential autoimmunity in animals. Therefore, many of the listed drugs were tested retrospectively in various experimental systems designed specifically to address the question if autoimmune reactions could be detected in animals. To date, only a few drugs (e.g., Hydralazine, D-Penicillamine, Procainamide) ascribed to the autoimmune diseases in humans have been shown to induce or exacerbate the autoimmune responses in animals, mainly rodent species genetically prone to autoimmunity. While identification of autoimmunity hazard in preclinical safety assessment studies during the drug development seems not feasible, there is growing concern about drug-induced autoimmune diseases in human medicine. The gap between the lack of reliable experimental systems to identify such hazard and the increased focus on potential drug-induced autoimmunity has been recognized in the immunotoxicology community.

TABLE 5.2-1 Examples of Therapeutic Agents Associated with Human Autoimmune Disorders Wide Range Autoimmune Diseases

Lupus Syndrome

rIFN-α

Acebutolol

Carbimazole

rIL-2

Chlorpromazine Hydralazine Isoniazid Procainamide

Propylthiouracil Hydralazine

Vasculitis with ANCA

Hemolytic Anemia

Autoimmune Hepatitis

Dermatomyositis

Alphamethyldopa Fludarabine Nomifensine

Dihydralazine

Penicillamine

ANCA: anti-neutrophil cytoplasmic antibodies; rIFN-α: recombinant interferon alpha, rIL-2: recombinant interleukin-2.

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STRATEGY FOR EVALUATION OF AUTOIMMUNITY POTENTIAL IN DRUG DEVELOPMENT In the absence of an established model to assess the autoimmunogenic potential of new drugs in development, a strategy for such an evaluation has to be based on alternative and feasible approaches. The strategy may include consideration of two aspects: (1) specifically focused review of animal data generated in routine, especially chronic, toxicology studies for potential signals; and (2) an application of specialized models when a signal is suspected or detected in any phase of drug development (“fit-for-purpose” approach). Such approaches may be considered to address very specific questions about novel drugs, but should not be viewed as broadly applicable. Specifically Focused Review of General Toxicology Data Each new drug candidate is extensively studied typically in two animal species (commonly in the rat and the dog) to characterize its toxicity, toxicokinetic, and drug metabolism profile. The preclinical data are used for the evaluation of safety of new drug candidates and decision making about proceeding or not to clinical trials. The adequate assessment of toxicity is mainly based on wellcontrolled and statistically powered studies to detect changes (clinical signs, hematology, clinical chemistry, tissue morphology) in drug-treated animals compared to control animals and the correlation between observed changes and exposure to a drug. These established toxicological methods are quite effective in finding systemic and/or organ-specific toxicity of drug candidates. However, such studies may miss “idiosyncratic” events of low incidence that could be potentially immune-mediated, e.g., sporadic inflammatory responses in some tissue(s) in an individual animal, especially non-rodent, on a repeatdose study. This type of finding may possibly be excluded from the interpretation of a body of data as outliers. Perhaps a higher scrutiny and potential follow-up evaluation of such “outliers” seen occasionally in animal studies could provide additional information about the individual animal responses to a drug and enable addressing a question if an “idiosyncratic” finding could be a signal for development of autoimmunity. Fit-For-Purpose Approach The concept of “fit-for-purpose” paradigm for testing immunoregulatory agents, particularly ones that can activate the immune system, is aligned with the broader strategy in immunotoxicologic assessment of all new drugs according to the weight-of-evidence approach recommended by ICH S8 guidance (ICH, 2006). When a new drug candidate has characteristics indicating a possible reactivity with the immune system, either as part of pharmacologic or off-target activity, its evaluation for potential autoimmunity induction by using a specific

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animal model could be considered. In this case, a model of either normal or autoimmune-prone animals should be selected based on scientific rationale and specific questions that could be answered by using a proposed model. In addition to a drug activity and model characteristics, relevant route of administration and adequate systemic exposure in a selected animal species, most likely different from the conventional toxicology species, need to be taken into consideration. Most of the time, pilot studies would be needed prior to conducting an autoimmune assessment study with a drug candidate in development to establish its tolerability and/or suitability of a proposed model for the evaluation, including reliable measurement of relevant end points (in serum, urine, and/or tissue histopathology). Since a considerable effort has to be undertaken in this approach, it is important to apply it when the probability of accomplishing the purpose of a study and learning about drug’s safety and clinical relevance is reasonably high. When deciding to conduct a study using an animal model of autoimmune disease, the objective would be to determine and evaluate the dose-response relationship between the estimated therapeutic dose and an adverse dose that causes exacerbation of the disease (accelerated onset and/or increased severity). As a result, the established no-observable-adverse-effect (NOAEL) in the general toxicology studies may be different from the NOAEL derived in the autoimmune disease model. Consequently, such an outcome would shed a new light on projected therapeutic window of a drug. In some cases however, a drug dose-response may not be achievable in an autoimmune disease model and the study would provide only “yes or no” answer for drug’s exacerbation of autoimmunity. The evaluation of autoimmunity potential of drug candidates in animal studies would have value if the results could make an impact on the conduct of clinical trials during drug development, e.g., influence selecting patient population, clinical monitoring, dose selection and/or escalation, stopping criteria, or other safety decisions.

EXPERIMENTAL TEST SYSTEMS FOR EVALUATION OF AUTOIMMUNITY While more research is needed to develop novel experimental approaches, the utility of the existing models for the evaluation of drugs potential to cause autoimmunity has been explored in immunotoxicology testing. There are numerous animal, especially mouse, models of autoimmune diseases used to study mechanisms and/or pharmacological potential of drug candidates to treat autoimmune diseases. They represent both systemic and organ-specific autoimmune diseases. In the various models of autoimmunity, diseases are developed either spontaneously in genetically predisposed animals or by induction with specific antigens that include infectious pathogens or endogenous molecule, usually combined with an adjuvant.

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A few examples of animal models used in immunotoxicologic testing of drugs include Brown Norway rat and Lewis rat models, which are described in detail below. Other established mouse models of autoimmune diseases, e.g., systemic lupus erythematosus, collagen-induced arthritis, pristane-induced arthritis and systemic scleroderma, may be considered for potential application in immunotoxicology evaluation. Brown Norway Rat Model Brown Norway rat strain is known as genetically prone to autoimmunity based on the clinical and immunological phenotype characterized as Th2 immune response, including production of a high level of immunoglobulins, especially IgE and autoantibodies, and increased polyclononal lymphoproliferation (Hirsch et al., 1982). The Brown Norway rat strain has been used to evaluate various therapeutics that cause adverse immune responses in some patients. In studies with D-Penicillamine (Donker et al., 1984; Tournade et al., 1990; Masson and Uetrecht, 2004), the majority of D-Penicillamine-treated rats developed the expected autoimmune disease, indicating that this model can be used for the detection of drugs with potential to induce autoimmunity. However, studies with other drugs demonstrated to be involved in autoimmune responses in humans were negative in Brown Norway rats (e.g., Captopril; Donker et al., 1984). Taken together, the Brown Norway rat model may have some utility in the hazard identification of autoimmune-related effects of novel drugs, but cannot be viewed as a universal and reliable system. Lewis Rat Model The Lewis rat strain has been used to study immune dysregulation caused by Cyclosporine, a known immunosuppressive drug. Cyclosporine, a therapeutic agent for treatment of organ transplant patients and also patients with rheumatoid arthritis, is rarely thought of as an autoimmunity inducer. Nevertheless, Lewis rats subjected to lethal X-irradiation followed by autologous bone marrow transplantation develop autoimmune disease (Damoiseaux, 2002). In this model, Cyclosporine-induced disease in the first acute phase is characterized as graft versus host response with erythroderma, dermatitis, and alopecia, which in the chronic phase develops scleroderma-like skin pathology. Cyclosporine effects are most likely related to the suppression of both autoreactive and regulatory T cells, and to thymic lymphocyte selection and migration processes. This model illustrates a situation when the widely immunosuppressive drug (in animal and human studies) can induce autoimmunity in genetically prone animals under certain (i.e., irradiation) conditions. While the model is technically challenging, theoretically, such an approach may be applied to evaluate immunoregulatory drugs suspected to have a potential to induce or exacerbate autoimmune responses based on the mechanism of action and/or indication for a specific patient population.

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Mouse Models of Autoimmune Diseases Systemic Lupus Erythematosus (SLE) Models. A few specific strains of mice, including NZBxNZW/F1, NZBxSWR/F1, MRL/lpr/lpr/MP, NZM, AKR, BWF1, have genetic predisposition to spontaneous, time-dependent onset of disease resembling human SLE (Pollard et al., 1999; Burnett et al., 2004). In this model, progression of disease is characterized by the development of antiDNA antibodies, proteinuria and glomerulonephritis. Recently reported work was focused on the adaptation of the polygenic SLE model in female (NZBxNZW)F1 mice to immunotoxicological evaluation of pharmaceuticals when needed to address drug-specific concerns for autoimmune reactions (Keegan et al., 2005). Based on this report, the disease-related parameters can be reliably monitored in mice between 4 and 9 months of age, and include altered balance of IL-10 and IFN-γ production, the increased anti-DNA antibodies in plasma associated with the increased proteinuria that correlates with histological changes in the kidney during disease progression. This model of the systemic autoimmune disease is viewed as suitable for immunotoxicology studies with novel drugs; however, such studies would require long duration (up to 6 months) of treatment and extensive monitoring. Collagen-Induced Arthritis (CIA) Model. The CIA mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis can be induced in various strains of genetically susceptible mice by immunization with an emulsion of complete Freund’s adjuvant and type II collagen (CII). Among many protocols applied in the CIA model and described in literature, a recent review paper is focused on the selection of mouse strains, the preparation of CII, proper immunization technique, and evaluation of the arthritis incidence and severity (Brand et al., 2007). Typically, the clinical signs of arthritis appear in this model 21–28 days after immunization and the pathology of the disease is well established. The CIA model is viewed as highly reproducible and often used to test pharmacological activity of novel antiarthritic drugs. Because of the rapid onset and high severity of the disease, the utility of the CIA model in the context of immunotoxicology evaluation for potential exacerbation of autoimmune response is very limited. Pristane-Induced Arthritis (PIA) Model. In one strain of mice, DBA/1, injection of a mineral oil pristane (2,6,10,14-tetramethylpentadecane) into joint regions induces arthritic disease associated with a broad spectrum of autoantibody production, including rheumatoid factor, anti-collagen and antiheat shock protein antibodies, and polyclonal T cell activation (Wooley and Whalen, 1991). Later work showed the ability to modulate the disease by immunoregulatory agents, e.g., administration of IL-12 cytokine (Beech et al., 1997). However, this disease-induction model appears less frequently used, likely due to lower reproducibility between laboratories, and potential

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application of the PIA model in immunotoxicology testing of drugs could be challenging. Systemic Scleroderma (SSc) Model. Many animal models of diffuse systemic sclerosis have been studied, including genetic models in mice, challenge with chemicals such as bleomycin or vinyl chloride to induce fibrosis, and models of graft-versus-host (GVH)-induced disease using certain strains of mice with differences in minor histocompatibility loci. Systemic scleroderma is characterized by excessive dermal fibrosis with later progression to internal organs. Other components of the disease include vascular changes and immune dysregulation evidenced by inflammatory cells in affected tissues and production of autoantibodies. In one of the described models of scleroderma, the disease was induced by GVH response involving injection of spleen cells from B10. D2 mice into BALB/c mice deficient in mature T and B cells (via recombination-activating gene 2, RAG-2) (Ruzek et al., 2004). The recipient mice developed dermal thickening, particularly in the extremities, progressive fibrosis of internal organs, vasoconstriction and altered expression of vascular markers in skin and internal organs, early immune activation, inflammation in skin and internal organs, and autoantibody generation. This apparently highly reproducible model with clinically relevant features has been developed to evaluate drug candidates as therapeutics for treatment of the SSc disease. Its potential utility in immunotoxicology studies of drugs that may be of concern for exacerbation of autoimmunity would have to be evaluated. Such an assessment should address if time course and severity of the disease provide sufficient window for potential exacerbation by treatment with a suspected drug. Based on the short overview of the known animal models relevant to autoimmune diseases, it is quite clear the task of hazard identification and risk assessment for potential drug-induced autoimmunity is presently very difficult. There is no single in vivo model that can be used routinely to evaluate new drugs for autoimmunity induction. Nevertheless, some of the models could possibly be applied to test therapeutics with specific properties (e.g., off-target immunoregulatory activity) and concerns (e.g., patient population) in order to gain additional perspective and understanding of their role in autoimmune responses.

SUMMARY Induction of autoimmune diseases by new therapeutics in humans is very rare. Similarly, drug-mediated autoimmunity in normal animals used in preclinical safety studies is hardly observed. In addition, due to a lack of predictive experimental systems (either in vitro or in vivo) as screening tools, presently the drug-induced autoimmunity hazard cannot be specifically identified. However, some input into risk assessment regarding the potential exacerbation of autoimmunity might be attainable for specific drugs. When possible signals are

REFERENCES

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suspected or observed, either as unexpected findings in general toxicology studies or in specialized animal models, these signals need to be further examined and understood in the context of drug properties and intended therapeutic use. While the ability of prospective detection and prediction of drug-induced autoimmunity in preclinical studies is not evident to date, a more targeted evaluation may help to answer the question whether or not animal studies can aid in the assessment of autoimmunity potential of novel therapeutics. REFERENCES Bachot N, Roujeau JC. Physiopathology and treatment of severe drug eruptions. Curr Opin Allergy Clin Immunol 2001;1:293–298. Beech JT, Siew LK, Ghoraishian M, Stasiuk LM, Elson CJ, Thompson SJ. CD4+ Th2 cells specific for mycobacterial 65-kilodalton heat shock protein protect against pristane-induced arthritis. J Immunol 1997;159:3692–3697. Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc 2007; 2:1269–1275. Burnett R, Ravel G, Descotes J. Clinical and histopathological progression of lesions in lupus-prone (NZB × NZW) F1 mice. Exp Toxicol Pathol 2004;56:37–44. Damoiseaux JG. Cyclosporin A-induced autoimmunity in the rat: central versus peripheral tolerance. Int J Immunopathol Pharmacol 2002;15:81–87. Demoly P, Bousquet J. Epidemiology of drug allergy. Curr Opin Allergy Clin Immunol 2001;1:305–310. Donker AJ, Venuto RC, Vladutiu AO, Brentjens JR, Andres GA. Effects of prolonged administration of D-penicillamine or captopril in various strains of rats. Brown Norway rats treated with D-penicillamine develop autoantibodies, circulating immune complexes, and disseminated intravascular coagulation. Clin Immunol Immunopathol 1984;30:142–155. George J, Levy Y, Shoenfeld Y. Immune network and autoimmunity. Intern Med 1996; 35:3–9. Griem P, Wulferink M, Sachs B, González JB, Gleichmann E. Allergic and autoimmune reactions to xenobiotics: how do they arise? Immunol Today 1998;19:133–141. Hirsch F, Couderc J, Sapin C, Fournie G, Druet P. Polyclonal effect of HgCl2 in the rat, its possible role in an experimental autoimmune disease. Eur J Immunol 1982; 12:620–625. ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006. Keegan S, Nadwodny K, Speal B, Herzyk D, Soos J. Adaptation of the systemic lupus erythematosus prone (NZB X NZW)F1 mouse strain for autoimmune toxicology evaluation. The Toxicologist CD 84:S-1, Abstract 910, 2005. Masson MJ, Uetrecht JP. Tolerance induced by low dose D-penicillamine in the brown Norway rat model of drug-induced autoimmunity is immune-mediated. Chem Res Toxicol 2004;17:82–94. Naisbitt DJ, Gordon SF, Pirmohamed M, Burkhart C, Cribb AE, Pichler WJ, Park BK. Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabo-

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lism-dependent haptenation and T-cell proliferation in vivo. Br J Pharmacol 2001; 133:295–305. Olsen NJ. Drug-induced autoimmunity. Best Pract Res Clin Rheumatol 2004; 18:677–688. Pollard KM, Pearson DL, Hultman P, Hildebrandt B, Kono DH. Lupus-prone mice as models to study xenobiotic-induced acceleration of systemic autoimmunity. Environ Health Perspect 1999;107(Suppl 5):729–735. Rose NR. Mechanisms of autoimmunity. Semin Liver Dis 2002;22:387–394. Ruzek MC, Jha S, Ledbetter S, Richards SM, Garman RD. A modified model of graftversus-host-induced systemic sclerosis (scleroderma) exhibits all major aspects of the human disease. Arthritis Rheum 2004;50:1319–1331. Sarzi-Puttini P, Atzeni F, Capsoni F, Lubrano E, Doria A. Drug-induced lupus erythematosus. Autoimmunity 2005;38:507–518. Steinman RM. Dendritic cells: from the fabric of immunology. Clin Invest Med 2004; 27:231–236. Tournade H, Pelletier L, Pasquier R, Vial MC, Mandet C, Druet P. D-penicillamineinduced autoimmunity in Brown-Norway rats. Similarities with HgCl2-induced autoimmunity. J Immunol 1990;144:2985–2991. Trombetta ES, Mellman I. Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol 2005;23:975–1028. Vial T, Choquet-Kastylevsky G, Descotes J. Adverse effects of immunotherapeutics involving the immune system. Toxicology 2002;174:3–11. Watanabe N, Wang YH, Lee HK, Ito T, Cao W, Liu YJ. Hasall’s corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature 2005;436:1181–1185. Wooley PH, Whalen JD. Pristane-induced arthritis in mice. III. Lymphocyte phenotypic and functional abnormalities precede the development of pristane-induced arthritis. Cell Immunol 1991;138:251–259.

PART VI IMMUNOTOXICITY TESTING IN BIOPHARMACEUTICAL DEVELOPMENT

6.1 DIFFERENTIATING BETWEEN DESIRED IMMUNOMODULATION AND POTENTIAL IMMUNOTOXICITY Jeanine L. Bussiere and Barbara Mounho

Biotherapeutics often target the immune system and the desired pharmacologic effect is to modulate the immune response in various disease states. However, due to the complexity of the immune system and the pleiotrophic nature of many mediators, there can also be undesirable effects on the immune system either due to extended or exaggerated pharmacology, which would be considered immunotoxicity. In general, the major concerns for risk associated with long-term use of biologic immunomodulators are acute reactions or cytokine release syndrome, or chronic immunosuppression leading to opportunistic infection, chronic infection, and malignancy. The question then becomes, can we modulate the immune system to block the harmful effects without blocking the beneficial effects?

IMMUNOTOXICITY SPECTRUM OF IMMUNOMODULATORY AGENTS Monoclonal Antibodies Modulating T Cell Receptors During the clinical testing of a novel superagonist anti-CD28 monoclonal antibody, TGN1412, six healthy male volunteers suffered from a systemic Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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inflammatory response characterized as a “cytokine storm” and became critically ill (Suntharalingam et al., 2006), which had not been predicted from the preclinical studies in nonhuman primates (Hopkin, 2006). Despite the use of a battery of murine, nonhuman primate (NHP) studies and even ex vivo human cell assays (Wing et al., 1995), the immunological models used in TGN1412 preclinical testing were of insufficient predictive power to anticipate the serious adverse events in humans (Schneider et al., 2006). Cytokine release syndrome (CRS; defined as headache, chills, fever, nausea, vomiting or myalgia) has been seen with other monoclonal antibodies such as OKT3, a murine antiCD3 monoclonal antibody (Abramowicz et al., 1989). This anti-CD3 monoclonal antibody (muromonab) is designed to inhibit T cells in the treatment of acute cellular rejection following solid organ transplantation. In addition to this intended pharmacology, the clinical use of muromonab is limited because of CRS (Sgro, 1995). This immunotoxic effect of muromonab may contribute to the pathogenesis of transient acute tubular necrosis and renal dysfunction seen after the prophylactic use of muromonab in cadaveric renal allograft transplantation (Olyaei et al., 1999). A humanized anti-CD3 antibody, HuM291, with a modified Fc 28 domain was tested to see if the CRS could be eliminated (Hsu et al., 1999). Both antibodies were tested in chimpanzees and there was no clinical evidence of CRS, although substantial cytokine secretion was detected. Clinical testing of HuM291 also showed that humans still experienced mild to moderate CRS. This suggests that even chimpanzees may not predict the clinical effects of cytokine release (or the side effects associated with anti-CD3 therapy in humans may not necessarily result directly from cytokine secretion) (Norman et al., 2000). Evidence in vitro has also implicated involvement of cell surface molecules CD16 and leukocyte function antigen-1 (LFA-1) on NK cells as additional contributors to CRS (Wing et al., 1996). New in vitro techniques are currently being developed in attempt to better predict this risk of CRS of immunomodulatory agents (Stebbings et al., 2007). The differentiation between immunopharmacology and immunotoxicity is further highlighted with the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antagonist of CD28 co-stimulation (abatacept; Orencia®). Abatacept is a recombinant human fusion protein comprising of the extracellular domain of CTLA-4 linked to the modified Fc portion of human IgG1 and is indicated for reducing signs and symptoms in adult patients with moderately to severely active rheumatoid arthritis (Orencia package insert, 2007). Abatacept blocks the engagement of CD28 with its ligands CD80 and CD86, and inhibits full activation of T cells by (1) inhibiting the early phases of T cell activation, including progression into cell cycle, effector differentiation and cell survival; and (2) promoting passive cell death and limiting the clonal expansion of antigen-reactive T cells (Linsley et al., 1991; Bluestone et al., 2006). However, other in vivo effects have been described such as increasing production of an intracellular enzyme that suppresses T cell activation, and under certain circumstances, can augment immunity by blocking the

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negative regulator CTLA-4 and having a direct effect on reducing regulatory T cells, which are important in the control of autoimmunity. Similar to the other immunomodulators described above, there may be an increased risk of infection in patients administered Orencia (Nogid and Pham, 2006; Orencia package insert, 2007). The potential of increased risk for reactivation of latent Mycobacterium tuberculosis is also a concern for patients receiving abatacept; however, administration of abatacept to mice did not exacerbate chronic Mycobacterium tuberculosis infection in mice (although the clinical significance of this mouse study has not yet been determined) (Bigbee et al., 2007). The effect of inhibiting T cell activation by abatacept on the development of malignancy is not understood at this time. In a mouse carcinogenicity study, administration of weekly injections of abatacept for up to 84 (males) and 88 (females) weeks resulted in increases in the incidence of malignant lymphomas (all doses) and mammary gland tumors (mid and high doses in females) (Orencia, FDA Review, Application Number 125118/000, approval 12/23/2005; Orencia package insert, 2007). These mice were positive for murine leukemia virus and mammary tumor virus, and the lymphomas and mammary tumors observed in this study were considered to be secondary to long-term induced immunomodulation in the presence of these viruses. The clinical relevance of these findings, however, remains to be determined, and monitoring of the patient data over years will be necessary to understand the risk to humans. Inhibitors of Tumor Necrosis Factor The tumor necrosis factor (TNF) inhibitors are a case in point for understanding the effects of chronic immunomodulation. TNF affects many organs, has a variety of actions, and is expressed by a variety of immune cells (Aggarwal et al., 2001). Normally it is present in nanomolar concentrations and is believed to be essential for protection against bacterial, fungal, parasitic, and perhaps even viral infections. However, TNF has also been implicated in many pathological inflammatory diseases such as rheumatoid arthritis, multiple sclerosis (MS), diabetes, Graves’ disease, allergic asthma, Crohn’s Disease, etc. TNF-α inhibitors such as Enbrel® (etanercept), Remicade® (infliximab), and Humira® (adalimumab) are all designed to block or reduce the effects of TNF-α; however, their structural differences confer distinct properties that impact the safety and efficacy of these drugs (Weaver, 2003). For example, they vary with regard to how they block cytokine activity (i.e., as receptors or as anti-cytokine antibodies), with regard to mode of administration, and with regard to molecular characteristics that result in differences in pharmacokinetics which may influence their safety profiles (Kohno et al., 2007). Although these TNF-α inhibitors have been successful in treating a variety of diseases (i.e., rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, Crohn’s disease, ulcerative colitis), there have also been many inflammatory diseases associated with effects of TNF-α that do not respond to a

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similar degree to these therapies (i.e., Wegener granulomatosis, MS, congestive heart failure, sarcoidosis) (Calabrese, 2006). Thus, the role of TNF-α in these various diseases is not well understood and the pharmacologic activity of these drugs does not produce the same outcome in different diseases with inflammatory and autoimmune components. Since TNF-α plays a critical role in host defenses against mycobacteria and other pathogens, patients administered TNF-α inhibitors may be at risk for increased incidence of infections. TNF inhibitors are remarkably well tolerated with the most common adverse event being injection site reactions. Serious side effects appear rare, but include bacterial sepsis, tuberculosis, opportunistic infections, multiple sclerosis, drug-induced lupus, and precipitation of cardiac failure (Roberts and McColl, 2004). It is uncertain to what extent therapy with these TNF inhibitors might be associated with an increase in serious infections. This uncertainty is based on the difficulties that generally emerge from the analysis and interpretation of sparse adverse event data derived from randomized controlled trials, which have not been powered to detect these rare adverse events. In addition, post-licensure observational studies usually lack an adequate control group, which leaves the question whether the events are associated with the therapeutic agent or with the disease itself. In a meta-analysis of anti-TNF antibody therapies in randomized controlled clinical trials, there was evidence of an increased risk of serious infections in patients with rheumatoid arthritis regardless of their treatment (Bongartz et al., 2006). In vitro studies with various TNF inhibitors do show decreases in interferon-gamma production between infliximab and adalimumab compared to etanercept, and differences in intracellular growth of Mycobacterium tuberculosis between adalimumab and etanercept (Wallis, 2007). This difference in interferon production between infliximab and etanercept remained whether one compared equal or peak therapeutic drug concentrations, suggesting a relationship to mechanism of action rather than pharmacokinetics. Thus, understanding the immunotoxicity versus the immunopharmacology of TNF-inhibitors is very molecule dependent, which adds to the challenge of identifying a risk for therapy-related recurrent tuberculosis vis-à-vis the multiple roles that TNF-α plays in tuberculosis (Lin et al., 2007). Monoclonal Antibodies Modulating Adhesion Molecules Another example of an immunomodulator showing immunotoxicity is Tysabri® (natalizumab). Natalizumab is a monoclonal antibody against α4integrin that inhibits binding of lymphocytes and monocytes to adhesion molecules on endothelial cells, thereby preventing inflammatory cells from trafficking into the brain and other tissues (Tysabri® package insert, 2008). The rationale for natalizumab therapy in multiple sclerosis is the reduction of leukocyte extravasation into the central nervous system. However, three patients who were treated with natalizumab developed progressive multifocal leukencephalopathy (PML), which was thought to be due to reactivation of

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latent JC virus as a result of diminished viral immunosurveillance in the brain (Engelhardt and Briskin, 2005; Khalili et al., 2007). JC virus had been identified as a major opportunistic infection following immunosuppression in acquired immunodeficiency syndrome (AIDS), occurring in up to 5% of patients (Koralnik, 2006). Distinguishing between the immunopharmacology of natalizumab and the immunotoxicity is further complicated by the fact that the biological half-life of natalizumab far exceeds its pharmacokinetic half-life (Sheremata et al., 1999). Although this immunotoxic event of PML is rare and the mechanism not completely understood, it highlights the dichotomy involved in modulation of the immune system. Efalizumab (Raptiva®), a monoclonal antibody against CD11a (the alpha subunit of leukocyte function antigen-1 [LFA-1], which is expressed on all leukocytes), inhibits the binding of LFA-1 to intracellular adhesion molecule-1 (ICAM-1), and is indicated for the treatment of adult patients with chronic plaque psoriasis (Raptiva package insert, 2005). Efalizumab inhibits multiple key pathogenic steps seen in psoriasis patients such as T cell activation, cutaneous T cell trafficking and T cell adhesion to keratinocytes (Menter et al., 2005). However, this immunomodulatory therapeutic also causes cytokine release syndrome that occurred on the day of injection or the following 2 days. This incidence was higher with the initial injection and decreased with each subsequent injection; by the third dose, the incidence was similar to that observed in the placebo-treated group (Leonardi, 2003; van de Kerkhof, 2006). Since efalizumab is an immunosuppressive agent, there is the potential risk of increased infection and reactivation of latent infections in patients. Serious infections such as cellulites, pneumonia, and bronchitis have been reported in patients receiving efalizumab, and it is advised that caution be exercised when considering the use of efalizumab in patients with chronic or recurrent infections (Raptiva package insert, 2005; Scheinfeld, 2006). Although malignancies may be another unintended toxicity following immunosuppression, efalizumaband placebo-treated patients had similar incidence rates of malignancy including lymphoproliferative disease, solid tumor, malignant melanoma and non-melanoma skin cancer (Leonardi et al., 2006; Scheinfeld, 2006). Treatment with efalizumab can also cause rare but serious hematological side effects including immune-mediated thrombocytopenia and hemolytic anemia (Scheinfeld, 2006). Again, this shows the difficulty in achieving a balance between immunopharmacology and immunotoxicity likely related to predispositions of individual patients.

SUMMARY The complexity of the immunomodulatory biotherapeutics demonstrates how difficult it can be to distinguish between the intended pharmacology desirable for treating the disease and any unintended immune effects leading to immunotoxicity. Despite the risks of infection and other toxicities with some of these

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immunomodulators, these risks need to be interpreted in the context of the benefits of treatment. The currently available biotherapeutics have been used successfully in the treatment of severe diseases in many patients and the benefits generally outweigh the risk of their rare adverse events.

REFERENCES Abramowicz D, Schandene L, Goldman M, Crusiaux A, Vereerstraeten P, DePauw L, Wybran J, Kinnaert P, Dupont E, Toussaint C. Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation 1989;47:606. Aggarwal BB, Samanta A, Feldmann M. TNF-α. In: Cytokine Reference: A Compendium of Cytokines and Other Mediators of Host Defense, edited by Oppenheim JJ, Feldmann M, pp. 413–434. San Diego, CA: Academic Press, 2001. Bigbee CL, Gonchoroff DG, Vratsanos G, Nadler SG, Haggerty HG, Flynn JL. Abatacept treatment does not exacerbate chronic Mycobacterium tuberculosis infection in mice. Arthritis Rheum 2007;56:2557–2565. Bluestone JA, St. Clair EW, Turka LA. CTLA4Ig: Bridging the basic immunology with clinical application. Immunity 2006;24:233–238. Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL, Montori V. Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. JAMA 2006;295:2275–2285. Calabrese L. The yin and yang of tumor necrosis factor inhibitors. Cleveland Clinic J Med 2006;73:251–256. Engelhardt B, Briskin MJ. Therapeutic targeting of a4-integrins in chronic inflammatory diseases: tipping the scales of risk towards benefit? Eur J Immunol 2005; 35:2268–2273. Hopkin M. Can super-antibody drugs be tamed? Nature 2006;440:855–856. Hsu D, Shi JD, Homola M, Rowell TJ, Moran J, Levitt D, Druilet B, Chinn J, Bullock C, Klingbeil C. A humanized anti-CD3 antibody, HuM291, with low mitogenic activity, mediates complete and reversible T-cell depletion in chimpanzees. Transplantation 1999;68:545–554. Khalili K, White MK, Lublin F, Ferrante P, Berger JR. Reactivation of JC virus and development of PML in patients with multiple sclerosis. Neurology 2007;68: 985–990. Kohno T, Tam LT, Stevens SR, Louie JS. Binding characteristics of tumor necrosis factor receptor-Fc fusion proteins vs anti-tumor necrosis factor mAbs. J Invest Dermatol Symp Proc 2007;12:5–8. Koralnik IJ. Progressive multifocal leukencephalopathy revisited: has the disease outgrown its name? Ann Neurol 2006;60:162–173. Leonardi CL. Efalizumab: an overview. J Am Acad Dermatol 2003;49:S98–S104. Leonardi CL, Toth D, Cather JC, Langley RGB, Werther W, Compton P, Kwon P, Wetherill G, Curtin F, Menter A. A review of malignancies observed during efalizumab (Raptiva®) clinical trials for plaque psoriasis. Dermatology 2006;213:204–214.

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Lin PL, Plessner HL, Voitenok NN, Flynn JL. Tumor necrosis factor and tuberculosis. J Invest Dermatol Symp Proc 2007;12:22–25. Linsley PS, Brady W, Urnes M, Grosmaire LS, Damle NK, Ledbetter JA. CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med 1991;174: 561–569. Menter A, Gordon K, Carey W, Hamilton T, Glazer S, Caro I, Li N, Gulliver W. Efficacy and safety observed during 24 weeks of efalizumab therapy in patients with moderate to severe plaque psoriasis. Arch Dermatol 2005;141:31–38. Nogid A, Pham DQ. Role of abatacept in the management of rheumatoid arthritis. Clin Therap 2006;28:1764–1778. Norman DJ, Vincenti F, DeMattos AM, Barry JM, Levitt DJ, Wedel NI, Maia M, Light SE. Phase 1 trial of HuM291, a humanised anti-CD3 antibody, in patients receiving renal allografts from living donors. Transplantation 2000;70:1707–1712. Olyaei AJ, de Mattos AM, Bennett WM. Immunosuppressant-induced nephropathy. Drug Safety 1999;21:471–488. Orencia® approved package insert. 2007. Available at http://packageinserts.bms.com/ pi/pi_orencia.pdf Orencia®, FDA Review, Application Number 125118/000, approval 12/23/2005. Raptiva® approved package insert. 2005. Available at http://www.gene.com/gene/ common/inc/pi/raptiva.jsp Roberts L, McColl GJ. Tumour necrosis factor inhibitors: risks and benefits in patients with rheumatoid arthritis. Intern Med J 2004;34:687–693. Scheinfeld N. Efalizumab: a review of events reported during clinical trials and side effects. Expert Opin Drug Saf 2006;5:197–209. Schneider CK, Kalinke U, Löwer J. TGN1412—a regulator’s perspective. Nature Biotechnol 2006;24:493–496. Sgro C. Side effects of a monoclonal antibody, muromonab CD3/Orthoclone OKT3: Bibliographic review. Toxicology 1995;105:23–29. Sheremata WA, Vollmer TL, Stone LA, Willmer-Hulme AJ, Koller M. A safety and pharmacokinetic study of intravenous natalizumab in patients with MS. Neurology 1999;52:1072–1074. Stebbings R, Findlay L, Edwards C, Eastwood D, Bird C, North D, Mistry Y, Dilger P, Liefooghe E, Cludts I, Fox B, Tarrant G, Robinson J, Meager T, Dolman C, Thorpe SJ, Bristow A, Wadhwa M, Thorpe R, Poole S. “Cytokine storm” in the Phase 1 trial of monoclonal antibody TGN1412: better understanding the causes to improve preclinical testing of immunotherapeutics. J Immunol 2007;179: 3325–3331. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, Panoskaltsis N. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. New Engl J Med 2006;355:1–11. Tysabri® approved package insert. 2008. Available at http://www.tysabri.com. van de Kerkhof PCM. Consistent control of psoriasis by continuous long-term therapy: the promise of biological treatments. JEADV 2006;20:639–650. Wallis RS. Reactivation of latent tuberculosis by TNF blockade: the role of interferon gamma. J Invest Dermatol Symp Proc 2007;12:16–21.

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Weaver AL. Differentiating the new rheumatoid arthritis biologic therapies. J Clin Rheumatol 2003;9:99–114. Wing MG, Waldmann H, Isaacs J, Compston DAS, Hale G. Ex-vivo whole blood cultures for predicting cytokine-release syndrome: dependence on target antigen and antibody isotype. Therap Immunol 1995;2:183–190. Wing MG, Moreau T, Greenwood J, Smith RM, Hale G, Isaacs J, Waldmann H, Lachmann PJ, Compston A. Mechanism of first-dose cytokine-release syndrome by CAMPATH 1-H: involvement of CD16 (FcγRIII) and CD11a/CD18 (LFA-1) on NK cells. J Clin Invest 1996;98:2819–2826.

6.2 RELEVANT IMMUNE TESTS ACROSS DIFFERENT SPECIES AND SURROGATE MODELS Jeanine L. Bussiere

The species specificity of biopharmaceutical products such as human proteins and monoclonal antibodies generally means that evaluating toxicity is restricted to nonhuman primates (NHPs) as the only pharmacologically relevant species. These human proteins may not be pharmacologically active in rodents or may be immunogenic, such that rodents develop neutralizing antibodies to the drug, precluding evaluation or interfering with interpretation of results. The general guidance for toxicity assessment of biological therapeutics is the ICH S6 document (1997). Although the ICH S8 guidance (2006) on “Immunotoxicity Testing for Human Pharmaceuticals” does not specifically apply to biopharmaceuticals, the same principals for understanding immunotoxicity can be applied. However, regulatory agencies should continue to treat the immunotoxicity testing of biological therapeutics on a case-by-case basis. Additionally, for human biopharmaceuticals, the immune system is often the intended target of the therapy and the immunotoxicity observed may be exaggerated pharmacology. In this case, the immune tests have been selected based on the known immunomodulatory properties of the drug. These assays have also been used as pharmacodynamic markers of drug activity or efficacy for these immune modulators. It is important to distinguish between immunopharmacology, where the immune system is the target organ of the therapeutic effect, immunotoxicity, where nontarget immune effects such as autoimmunity or immunosuppression may be observed and immunogenicity, which represents an Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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immune response to the drug. Adverse events can also result from the intended immunomodulatory mechanism of action. For example, excessive downregulation of the immune system can result in recrudescence of a previously inactive virus.

IMMUNOTOXICITY TESTING IN NONHUMAN PRIMATES Immunotoxicity testing guidelines exist for small molecules where the toxicology is largely unpredictable and rodent species are typically used. For human biotherapeutics, NHPs are generally used and the immune tests need to be selected based on the known immunomodulatory properties of the drug. These assays can also be used as pharmacodynamic markers of drug activity or efficacy for these immune modulators. Several important factors should be considered when including immunotoxicity testing into GLP (Good Laboratory Practice) toxicology studies, especially if they are conducted in NHPs. These include (1) whether the assays have been validated; (2) whether to use the main study animals or a satellite group; and (3) the timing of these tests within the context of the GLP toxicology study. The advantages of using the main study animals for immunotoxicity testing are reduced animal use and correlation of any immunotoxicity findings with other toxicities seen in those same animals. The disadvantage of using main study animals is that the additional manipulations for immune testing (e.g., injection of an antigen for determining antibody response) may influence the toxicity or immunogenicity of the therapeutic agent. It is very important to include several baseline measurements because of the variability seen between animals, and even in the same animal over time. Because of the small number of NHP animals per group, it is important to reduce the variability in the assays as much as possible with regard to antigen source, technique, etc.

Immunotoxicity Assays Generally, specialized immunotoxicity tests would not be conducted unless an effect on the immune system was seen in the general toxicity studies, or if there is a known pharmacologic effect of the test agent on the immune system. Additional immunotoxicity testing would be conducted if there was a cause for concern. In these cases, immunotoxicity testing is generally included in the IND-enabling NHP toxicology studies. The current regulatory guidelines recommend that immunopathology be used as the initial screen to detect immunotoxicity, since standard hematology and histopathology may often be sufficient to detect immune system alterations. Immunopathology can include total and differential white blood cell counts, and evaluation of the histopathology of lymphoid organs such as the thymus, spleen, lymph nodes, gutassociated lymphoid tissue (GALT), and the bone marrow (see Chapters 2.1

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and 2.2). In addition, more detailed measurements of any change in size and cellularity of immune cells, germinal center development, cortex:medulla ratio of the thymus, and immunohistochemistry of the lymphoid organs may be included. Flow cytometry can be included in the GLP toxicology study in monkeys to evaluate changes in lymphocyte subsets, including T cells (CD4+, CD8+), B cells (CD20+), NK cells (CD16+), and monocytes (CD14+). These assays are typically conducted using peripheral blood, which allows for repeated sampling over time within the same animal (see Chapters 3.2 and 4.2). However, immunophenotyping can also be conducted on tissues to determine if there are effects on lymphocyte trafficking, although time points are limited to study termination unless serial biopsies can be performed (i.e., on lymph nodes). Serial biopsies may be difficult because they cannot be performed by all laboratories, and potential infections or other effects on the animals can affect data interpretation. Flow cytometry can also be used for more functional end points of immune competence including lymphocyte activation, cytokine release, phagocytosis, apoptosis, oxidative burst, natural killer (NK) cell activity, etc. These can be added if the mechanism of action of the drug suggests involvement of a particular function or type of immune cells. In NHPs, the assay most commonly used to assess the ability to mount a T cell-dependent antibody response (TDAR) is immunization with keyhole limpet hemocyanin (KLH) or tetanus toxoid (TT), and measurement of circulating antigen-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) methods. Several immunization protocols have been applied in NHP-TDAR tests. The most comprehensive approach involves the evaluation of immune responses to two different antigens over the course of treatment with a novel biopharmaceutical. In such a study, immunization with one antigen (e.g., KLH) should occur prior to drug treatment, and then during drug treatment to assess the effects on the secondary (anti-KLH) antibody response (i.e., first immunization 7 days prior to dosing start and second immunization 14 days later, on Day 7 of the dosing period). The other antigen (e.g., TT) can be injected after 2 weeks of treatment to determine the effect on the primary (anti-TT) antibody response 7 to 10 days later. This immunization regimen allows for the assessment of both the primary and the secondary T cell-dependent antibody response within the 1-month GLP toxicology study. For studies of longer duration, a booster immunization can be given at a later time point to assess the effect on the memory response, or to see if an altered response returns to normal during the recovery period. Other immune parameters can be measured in the NHP, including cytokine measurements (see Chapter 4.1) and delayed-type hypersensitivity test (see Chapter 3.1.3). For cytokine measurements in the NHP, many human ELISA kits can be used, although it is very important to determine if the reagents in these kits do truly cross-react with NHP cytokines. Many of the human reagents do cross-react, but exceptions exist, and these need to be tested prior to use on a toxicology study.

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Assessment of Immunomodulation Although immunomodulation can be assessed in the NHP, the assays are less well characterized than those used in the rodent, particularly in mice. One issue is the lack of consistent protocols, and the timing of incorporating these assays into standard GLP toxicology studies varies. More historical control data are needed, and many assays have not been tested with an immunomodulatory control to confirm the level of sensitivity of the assay for detecting a mild/moderate immune modulator (both immunoenhancing and immunosuppressive activity). Inherently, greater variability is seen in NHP than in inbred rodents, and the animal number per group is generally much smaller than in rodent studies. It is critical to find ways of reducing the variability in the assay to allow for more meaningful data interpretation. These can include decreasing the inter-animal variability (using animals from the same source and of similar ages, decreasing stress during the study, increasing the number of baseline samples, etc.) and decreasing assay variability (standardizing the antigen source, assay technique, timing, etc.). In addition, building a database with sufficient data regarding which assays are the most useful in predicting immunomodulatory effects in humans is highly warranted. Assay methods need to be standardized so that we can truly compare the data to make that determination. Comparing data from the NHP with the immunotoxicity data in rodents would also be useful to evaluate whether the NHP is more predictive of the human response. However, immune testing in NHP for biotherapeutics goes beyond the estimation of immunotoxicity and can be very valuable for understanding the pharmacology of an immune modulator and can help to establish pharmacodynamic markers that can then be used in clinical trials. Combining all of the available data in monkey studies will allow for an improvement in the models and a better understanding of the value of these data. In addition, differences have been seen in immune parameters (especially immunophenotyping) between cynomolgus monkeys from different geographical locations. It is therefore very important to keep the same source of animals for toxicology studies throughout the drug development program. Recent adverse events reported with immunomodulatory monoclonal antibodies, such as Tysabri and TGN1412, highlight the need to improve the predictivity of immune effects in humans. During the clinical testing of a novel superagonist anti-CD28 monoclonal antibody, TGN1412, six healthy male volunteers suffered from a systemic inflammatory response characterized as a “cytokine storm” and became critically ill (Suntharalingam et al., 2006), which had not been predicted from the preclinical studies in monkeys (Hopkin, 2006). Despite the use of a battery of murine, NHP studies and even ex-vivo human cell assays (Wing et al., 1995), the immunological models used in TGN1412 preclinical testing were judged by some to be of insufficient predictive power to anticipate the serious adverse events in humans (Schneider et al., 2006). Cytokine release syndrome (CRS) has been seen with other monoclonal antibodies such as OKT3, a murine anti-CD3 monoclonal anti-

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body (Abramowicz et al., 1989). A humanized anti-CD3 antibody, HuM291, with a modified Fc 28 domain was tested to see if the CRS could be eliminated (Hsu et al., 1999). Both antibodies were tested in chimpanzees and there was no clinical evidence of CRS, although substantial cytokine secretion was detected. Clinical testing of HuM291 also showed that humans still experienced mild to moderate CRS. This suggests that even chimpanzees may not be the best model for assessing the clinical effects of cytokine release (or the side effects associated with anti-CD3 therapy in humans may not necessarily result directly from cytokine secretion) (Norman et al., 2000). Evidence in vitro has also implicated CD16 and LFA-1 on NK cells in CRS (Wing et al., 1996). Natalizumab (Tysabri), an anti-α4 integrin monoclonal antibody approved for the treatment of multiple sclerosis, was recently withdrawn from the market temporarily due to cases of progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system associated with immunosuppression (Sheriden, 2005). These cases highlight our incomplete understanding of the immune system and the translation of preclinical results to humans. What could be done to better assess immunomodulatory effects in NHP that will be predictive of the outcome in humans? One important consideration is the “relevance” of the animal species and the appropriateness and relevance of the immune assays being utilized.

IMMUNOTOXICITY TESTING WITH SURROGATE MOLECULES One alternative to using NHPs (or if the biological therapeutic does not crossreact with the target in NHPs) is to use a surrogate molecule. This would be the homologous protein (i.e., the murine protein for use in mice) or, for a monoclonal antibody therapeutic, an antibody that cross-reacts with the rodent target. This allows for the safety testing of the pharmacologic activity of the drug, but does not allow for testing of the clinical candidate itself. For a surrogate molecule to be relevant, the pharmacological mechanism should be as similar as possible to the clinical candidate. This not only includes similar epitope for binding, similar binding affinity, and in vitro functional activity, but if possible, similar in vivo functional activity as well. This pharmacologic data can then be used to select doses with the surrogate that will be relevant to human exposure with the clinical candidate. Having similar pharmacokinetics is less important for comparability, but is important in determining the appropriate dosing schedule with the surrogate molecule. Tissue cross-reactivity studies can also show that binding of the surrogate to the relevant target in rodents is similar to the binding of the clinical candidate to human tissues. In addition, the surrogate molecule should resemble the clinical candidate as much as possible with regard to the production process and range of impurities/contaminants.

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The decision to develop a surrogate molecule should occur early in the program since a lot of preparation is needed to characterize the surrogate prior to use in toxicology studies. Making and characterizing a second molecule, along with the clinical drug candidate, results in the use of a great deal of additional resources. Not only does this take manufacturing capacity away from another clinical drug candidate, but there is also the characterization of the surrogate molecule needed to demonstrate pharmacologic comparability to the clinical candidate, and additional assays to be developed for assessing PK, as well as for antidrug antibodies. In addition, assays and resources are necessary to characterize (i.e., potency, presence of aggregates, host cell protein contaminants, etc.) and to monitor the stability of the surrogate material used in the toxicology studies. There can still be challenges associated with developing a surrogate molecule to assess toxicity of a clinical drug candidate. There are no established criteria to understand whether differences in the pharmacological and toxicological activities of the surrogate, or differences in the material characterization could affect the utility of the surrogate molecule. It is also important to understand that just as a human biotherapeutic can be immunogenic in humans, a surrogate molecule can still be immunogenic in the homologous species. However, in spite of the limitations of using a surrogate molecule (it is not the clinical candidate), these efforts can allow for a greater understanding of the potential toxicities of the therapeutic candidate, and allow for immunotoxicity testing in the standard rodent models.

IMMUNOTOXICITY TESTING WITH TRANSGENIC AND KNOCKOUT MICE Another alternative to testing immunotoxicity in NHPs is to evaluate the toxicity in a knockout (KO) or transgenic (Tg) mouse model (also see Chapter 10.1). Knockout mice have been used to assess drug specificity, to investigate mechanisms of toxicity, immunotoxicity, and to screen for mutagenic and carcinogenic activities of therapeutic candidates. Generation of viable and fertile animals with null mutations for a potential target protein implies that pharmacological inhibition of the molecule in vivo will elicit no major adverse effects. Particular emphasis in future pharmacology and toxicology studies will be directed toward conditional knockout mice (to evaluate the impact of chemically mediated inhibition of a particular gene product at the relevant stage of life) and “humanized” knock-in animals (in which the endogenous mouse gene is replaced with the homologous human gene to examine its role in disease or drug metabolism). “Humanized” mice are of particular importance as these animals can be employed to evaluate the efficacy and toxicity of human biopharmaceutics that are not pharmacologically active in normal rodents or that induce a neutralizing antibody response that limits long-term exposure. One particular criticism is that “humanized” mice manufacture one

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or a few human proteins of interest, but other proteins that interact with the human molecules are still of mouse origin. The physiological effect of human– mouse protein interactions may differ slightly—or substantially—from that of the normal human–human association. Toxicity bioassays are often performed in knockout animals for some specific purposes (e.g., mutagenicity and carcinogenicity). With the increasing number of biotherapeutics on the market, these data become even more important to demonstrate that the knockout mice are a viable alternative to testing in NHPs and are relevant to the findings seen in humans.

Case Study: Immunomodulatory Assessment of sIL-1R Type II Interleukin-1 receptor type II (IL-1RII) is a soluble decoy receptor that binds to IL-1β and prevents it from binding to IL-1RI, which is the signaling receptor. IL-1RII was in development as a potential therapeutic to block the effects of IL-1 in inflammatory diseases such as rheumatoid arthritis (Irikura et al., 2002). Human sIL-1R type II does not bind to rodent IL-1, so all safety assessment studies to support the development of this molecule were conducted in cynomolgus monkeys. Because of the difficulties in assessing effects on the immune system in NHPs as mentioned above, an IL-1R KO mouse was used to assess the immunomodulatory potential of this molecule. The intent was to show that this molecule was not a general immune suppressant, but a specific, targeted immune modulator. Various immune function assays were incorporated into a 1- and 9-month toxicity study in cynomolgus monkeys. These included DTH, TDAR, immunophenotyping, and immunohistochemistry (IHC). However, one potential liability of blocking IL-1 is an increased risk of infection. Since there is no model for host resistance in NHPs, the receptor knockout mice were tested in a host resistance model of influenza. Host resistance models provide the opportunity to directly assess the functional reserve of the immune system. Clearance of virus requires an intact and functional immune system that incorporates a cascade of immune responses including cytokine production, natural killer cell activity, macrophage activity, cytotoxic T lymphocyte (CTL) activity, and antibody production. Influenza virus is a T-dependent antigen (see Chapter 5.1). As such, formation of antibody to influenza virus requires intact and functional T cells, B cells, and macrophage antigen processing and presentation activity. Dexamethasone, a corticosteroid that exerts its anti-inflammatory and immunologically mediated effects through specific ubiquitously distributed cellular receptors, was used as a positive immunosuppressive control. Corticosteroid therapy has been reported to inhibit T cell function, as well as B cell produced serum immunoglobulin levels (Marino et al., 1996). Corticosteroid use has been associated with increased incidence of both viral and bacterial infections, as well as an increased exacerbation of opportunistic infections (Marino et al., 1996).

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Adaptation of Murine Influenza Model. Because the KO mice were a C57BL/6 origin and this host resistance model had typically been tested in BALB/c mice, a pilot study was conducted to ensure that this strain of mice would not behave differently in response to the viral challenge. Enhanced mortality was observed in dexamethasone (DEX)-treated, influenza-infected C57BL/6 mice when compared to historical data in BALB/c mice or vehicle controls. Therefore, two lower challenge doses of infectious virus (4 × 103 or 4 × 104 PFU) and two lower doses of DEX (1 mg/kg/day or 5 mg/kg/day) were evaluated. DEX at 5 mg/kg/day caused a decreased ability to clear or eliminate viral infection at both high- and low-dose virus challenge and also decreased influenza-specific IgG concentration in lung homogenate. Survival rate in mice receiving 5 mg/kg/d of DEX and 4 × 104 PFU of influenza virus, was suboptimal; survival rates in the remaining groups were acceptable. The viral challenge dose of 4 × 103 PFU and 5 mg/kg/day of DEX was considered optimal in the C57BL/6 mice. Overall, the study demonstrated that C57BL/6 mice were more susceptible to the combination of influenza virus infection and DEX treatment in this mouse influenza model than BALB/c mice. Establishment of strain-specific characterization is critical for accurate assessment of a potential immunotoxic effect. The Influenza Host Resistance Study in Knockout Mice. C57BL/6 wild-type mice treated with DEX and a KO line of mice which lacks the type I receptor for IL-1 (IL-1R KO), were inoculated with influenza virus titrated in the pilot study. Analyses included the measurement of the clearance of infectious influenza virus, body weight, lung weight, spleen weight, cytokine production in the lung (IL-1β, IL-6, and TNFα), and influenza-specific IgG in the lung at six time points: Days 2, 6, 8, 10, 14, and 21 post virus challenge. Additionally, baseline values for the cytokines and influenza-specific immunoglobulins were obtained on Day 0 from naive C57BL/6 and the KO mice. There was no effect on viral clearance, body, lung or spleen weight, or cytokine production for the IL-1R KO mice. Interestingly, the IL-1R KO mice had enhanced influenza-specific IgG levels. A deficiency of IL-1 receptor has no deleterious effect on the ability to clear an intracellular viral infection; thus, blocking the activity of IL-1 with the sIL-1RII therapeutic does not alter immune competence and is not likely to cause an increase in this type of infection.

SUMMARY Immunotoxicity testing of biopharmaceuticals can be very challenging, since rarely can the clinical drug candidate be tested in the standard rodent models. These assays are either conducted (when appropriate) in NHPs or in a rodent with a surrogate molecule, or in a rodent with the target knocked-out or overexpressed. All of these options have their own issues and caveats which pres-

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ents a challenge for assessing the effect of a biotherapeutic on the immune system.

REFERENCES Abramowicz D, Schandene L, Goldman M, Crusiaux A, Vereerstraeten P, DePauw L, Wybran J, Kinnaert P, Dupont E, Toussaint C. Release of tumor necrosis factor, interleukin-2, and gamma-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation 1989;47:606–608. Hopkin M. Can super-antibody drugs be tamed? Nature 2006;440:855–856. Hsu D, Shi JD, Homola M, Rowell TJ, Moran J, Levitt D, Druilet B, Chinn J, Bullock C, Klingbeil C. A humanized anti-CD3 antibody, HuM291, with low mitogenic activity, mediates complete and reversible T-cell depletion in chimpanzees. Transplantation 1999;68:545–554. ICH (International Conference on Harmonization). Guidance for Industry. S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, July 1997. Available at http://www.ich.org ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006. Available at http://www.ich.org Irikura VM, Lagraoui M, Hirsh D. The epistatic interrelationships of IL-1, IL-1 receptor antagonist, and the type I IL-1 receptor. J Immunol 2002;169:393–398. Marino C, McDonald E, Romano J. Corticosteroid use in HIV disease. The AIDS Reader 1996;6(1):29–32. Norman DJ, Vincenti F, DeMattos AM, Barry JM, Levitt DJ, Wedel NI, Maia M, Light SE. Phase 1 trial of HuM291, a humanised anti-CD3 antibody, in patients receiving renal allografts from living donors. Transplantation 2000;70:1707–1712. Schneider CK, Kalinke U, Löwer J. TGN1412—a regulator’s perspective. Nat Biotechnol 2006;24:493–496. Sheriden C. Third Tysabri adverse case hits drug class. Nat Rev 2005;4:357–358. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, Panoskaltsis N. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. New Engl J Med 2006;355:1–11. Wing MG, Waldmann H, Isaacs J, Compston DAS, Hale G. Ex-vivo whole blood cultures for predicting cytokine-release syndrome: dependence on target antigen and antibody isotype. Ther Immunol 1995;2:183–190. Wing MG, Moreau T, Greenwood J, Smith RM, Hale G, Isaacs J, Waldmann H, Lachmann PJ, Compston A. Mechanism of first-dose cytokine-release syndrome by CAMPATH 1-H: involvement of CD16 (FcγRIII) and CD11a/CD18 (LFA-1) on NK cells. J Clin Invest 1996;98:2819–2826.

6.3 ANTIDRUG ANTIBODY RESPONSES IN NONCLINICAL STUDIES AND THEIR IMPLICATIONS Barbara Mounho

Therapeutic proteins (biopharmaceuticals, biotherapeutics) are fundamentally different from conventional, small-molecule pharmaceuticals because they are derived from living organisms (mammalian, bacterial, or fungal) using genetic recombination (versus chemical synthetic processes), and are generally complex, large-molecular-weight (≥1000 Dalton) molecules. Biopharmaceuticals encompass a wide range of polypeptide or protein products including monoclonal antibodies, recombinant human proteins, and fusion proteins (peptide fused to human IgG Fc) and vaccines (Terrell and Green, 1994; Dempster, 2000; Sims, 2001; Tsang and Beers, 2003; Brennan et al., 2004). Immunogenicity is an inherent property of biotherapeutics that distinguishes these molecules from traditional drugs. It is generally accepted that immune responses against a therapeutic protein (antidrug antibody response) can occur in animals or humans that have been administered the product (Wierda et al., 2001; Koren et al., 2002; Schellekens, 2002; Chamberlain and Mire-Sluis, 2003; Mire-Sluis et al., 2004; Frost, 2005; Kessler et al., 2006). While an immune response is the desired pharmacological response of vaccines (see Chapter 7.2), immunogenicity is not the intended effect for the majority of therapeutic proteins (see Chapter 10.3). Most biopharmaceuticals are human proteins that are highly targeted to a human receptor or antibodies specific for a human target protein (soluble or membrane bound). Thus, it is not unexpected that the administration of a Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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therapeutic protein to animals in nonclinical repeated-dose toxicology studies (particularly long-term studies) results in the production of antibodies against the product (Working, 1992; Dempster, 1995; Cavagnaro, 2002). As a general rule, the greater the dissimilarity between the sequence of the human recombinant protein and the sequence of the animal counterpart (endogenous) protein, the more likely the animal’s immune system will produce an antibody response to the recombinant product (Weirda et al., 2001; Bugelski and Treacy, 2004). Although the sequence homology of a protein target is generally more similar between human and nonhuman primates compared to other animal species such as rodents and dogs, antibody responses to the biopharmaceutical can develop in nonhuman primates (Zwickl et al., 1991; Gunn, 1997).

IMPACT OF DRUG IMMUNOGENCITY IN TOXICOLOGY STUDIES Antibody responses can impact the outcome of nonclinical toxicology studies by altering the pharmacokinetics, tissue distribution, or pharmacological activity of the biotherapeutic product, and consequentially result in misleading interpretations of the toxicity data (Working, 1992; Terrell and Green, 1994; Serabian and Pilaro, 1999; Wierda et al., 2001; Koren et al., 2002). Therefore, measuring antibody responses in repeat-dose toxicity studies is currently expected by regulatory authorities (ICH S6, 1997; Griffiths and Lumley, 1998; Dempster, 2000; Sims, 2001; Koren et al., 2002; Brennan et al., 2004; Shankar et al., 2006, 2007). Although the development of antidrug antibodies in some animals in a toxicology study does not necessarily invalidate the study, in some instances it is important to measure the presence of antibodies and determine if the antibody responses correlate with the pharmacology, pharmacokinetics, and toxicity of the therapeutic protein (ICH S6, 1997; Shankar et al., 2006).

TYPES OF ANTIDRUG ANTIBODIES Various types of antibody responses can develop in animals in nonclinical toxicology studies that can potentially alter interpretation of the study: (i) antibodies that are clearing or sustaining; (ii) antibodies that neutralize the pharmacological activity of the biological; and (iii) antibodies that neutralize natural, endogenous counterparts (Dempster, 2000; Wierda et al., 2001; Koren et al., 2002). Clearing antibodies bind to the biotherapeutic resulting in increased plasma clearance of the drug (Gunn, 1997; Wang et al., 2001). Increased plasma clearance ultimately results in a decrease in the distribution and exposure of the drug to target organs, which can confound interpretation of the toxicology study results (Working, 1992). For example, Figure 6.3-1 illustrates the effect of clearing antibodies on serum concentrations of a monoclonal antibody in cynomolgus monkey. In this study, cynomolgus monkeys were intravenously

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Figure 6.3-1 The effect of clearing antibodies on serum concentration levels of a monoclonal antibody administered to cynomolgus monkeys. Cynomolgus monkeys were administered the antibody on Days 1, 8, 15, and 22. The expected decrease in serum concentration levels of the monoclonal antibody over time is observed in all monkeys after the first dose on Day 1. On Day 28 (after four weekly doses), a rapid decrease in serum concentration levels is observed in monkeys 1, 2, 3, and 5, all of which developed antibodies (positive) against the monoclonal antibody.

administered a monoclonal antibody (drug) once weekly on Days 1, 8, 15, and 22. After the first dose on Day 1, the expected serum levels and clearance profile of the monoclonal antibody over time were observed in all monkeys. However, after four weekly doses, four monkeys (1, 2, 3, and 5) developed clearing antidrug antibodies (ADAs), and a rapid decrease in serum concentrations of the monoclonal antibody was observed in these four ADA-positive monkeys compared to monkeys that were ADA negative. In the case of sustaining ADA, they also bind to the biotherapeutic, but slow the rate of plasma clearance of the product resulting in prolonged exposure of the drug to the animal test species (Rosenblum et al., 1985; Pendley et al., 2003). For example, intravenous administration of recombinant hirudin (CGP 39 393) for 3 months resulted in the development of anti-hirudin antibodies; the hirudin-antibody complexes accumulated in the plasma leading to an extended plasma half-life of recombinant hirudin (Gygax et al., 1996). Neutralizing ADA can bind to or near the target-binding domain of the biopharmaceutical product and interfere with its ability to bind to its target receptor, thus resulting in a partial or complete reduction in the pharmacological activity of the drug (Working, 1992; Gunn, 1997; Dempster, 2000).

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When conducting nonclinical toxicology studies with therapeutic proteins, the primary concern for the development of clearing or neutralizing antibodies in animals is the potential for lower exposure of the pharmacologically active drug to target organs, resulting in fewer treatment-related toxicities and an underestimation for the potential for human toxicity (ICH S6, 1997). In addition to binding and neutralizing the pharmacological action of the drug, ADA can also bind and neutralize the biological function of the endogenous counterpart of the therapeutic protein, which ultimately, can result in toxicity. For example, the subcutaneous administration of recombinant human thrombopoietin (rhuTPO) to rhesus monkeys produced binding ADA that not only neutralized the pharmacological activity of rhuTPO, but also the biological function of the monkeys’ endogenous thrombopoietin, which consequently resulted in reduced platelet counts and thrombocytopenia in several monkeys (Hardy et al., 1997; Koren, 2002). In another example, administration of recombinant human erythropoietin (rhuEPO) to dogs resulted in the development of antibodies which neutralized the bioactivity of rhuEPO, as well as the animal’s endogenous erythropoietin, subsequently resulting in several animals developing life-threatening red cell aplasia (Cowgill et al., 1998). Clinically, the development of antibodies that neutralize the biological activity of an endogenous protein that mediates a critical biological function is a significant safety concern for manufacturers, clinicians, and regulatory authorities because of the potential life-threatening consequences (Li et al., 2001; Basser et al., 2002; Casadevall et al., 2002; Chamberlain, 2002; Kessler et al., 2006). The formation of antibody-antigen (immune) complexes can be another potential consequence of ADA that can affect the outcome of a toxicology study. Antibody-antigen complex reactions can form in the blood, which can then deposit in various tissues such as blood vessels and kidney, and ultimately lead to immune complex-mediated toxicity (Working, 1992; Dempster, 2000). Intramuscular administration of recombinant human interferon-γ (rHuIFN-γ) to cynomolgus monkeys resulted in the development of anti-rHuIFN-γ antibodies. Glomerulonephritis, which morphologically resembled immunecomplex glomerulitis, was also observed in these monkeys, and may have been secondary to the deposition of anti-rHuIFN-γ antibody complexes in renal glomeruli (Terrell and Green, 1993).

SUMMARY Immunogenicity remains a challenge in the development of therapeutic proteins when conducting repeated-dose toxicity studies in animals, particularly in terms of maintaining exposure of pharmacologically active drug throughout the duration of the study. There are some approaches, however, to manage and mitigate the development of clearing and/or neutralizing antibodies in toxicology studies. For example, one potential solution is to increase the dose levels

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administered in the toxicology study to saturate or “dose through” the ADA response to allow exposure of free (unbound) drug throughout the study. More frequent dosing (e.g., twice weekly rather than once weekly) is another approach to overwhelm the antibody response and maintain drug exposure. A drawback to these approaches, however, is an increase in material demand, which is often not feasible, especially for early stage development programs. ACKNOWLEDGMENT The author would like to thank Peggy Lum for the contribution of Figure 6.3-1. REFERENCES Basser RL, O’Flaherty E, Green M, Edmonds M, Nichol J, Menchaca DM, Cohen B, Begley CG. Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor. Blood 2002;99:2599–2602. Brennan FR, Shaw L, Wing MG, Robinson C. Preclinical safety testing of biotechnologyderived pharmaceuticals: understanding the issues and addressing the challenges. Mol Biotechnol 2004;27:59–74. Bugelski PJ, Treacy G. Predictive power of preclinical studies in animals for the immunogenicity of recombinant therapeutic proteins in humans. Curr Opin Mol Ther 2004;6:10–16. Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, Michaud P, Papo T, Ugo V, Teyssandier I, Varet B, Mayeux P. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 2002;346:469–475. Cavagnaro JA. Preclinical safety evaluation of biotechnology-derived pharmaceuticals. Nat Rev Drug Discov 2002;1:469–475. Chamberlain P. Immunogenicity of therapeutic proteins, Part 1: causes and clinical manifestations of immunogenicity. Reg Rev 2002;5:4–9. Chamberlain P, Mire-Sluis AR. An overview of scientific and regulatory issues for the immunogenicity of biological products. In: Immunogenicity of Therapeutic Biological Products, edited by Brown F, Mire-Sluis, pp. 3–11. Basil, Switzerland: Karger AG, 2003. Cowgill LD, James KM, Levy JK, Browne JK, Miller A, Lobingier RT, Egrie JC. Use of recombinant human erythropoietin for management of anemia in dogs and cats with renal failure. J Am Vet Med Assoc 1998;212:521–528. Dempster AM. Pharmacological testing of recombinant human erythropoietin: implications for other biotechnology products. Drug Dev Res 1995;35:173–178. Dempster AM. Nonclinical safety evaluation of biotechnologically derived pharmaceuticals, Biotechnol Annu Rev 2000;5:221–258. Frost H. Antibody-mediated side effects of recombinant proteins. Toxicology 2005; 209:155–160.

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Griffiths SA, Lumley CE. Non-clinical safety studies for biotechnologically-derived pharmaceuticals: conclusions from an international workshop, Hum Exp Toxicol 1998;17:63–83. Gunn H. Immunogenicity of recombinant human interleukin-3. Clin Immunol Immunopathol 1997;83:5–7. Gygax D, Botta L, Ehrat M, Graf P, Lefèvre G, Oroszlan P, Pfister C. Immunoassays in monitoring biotechnological drugs. Ther Drug Monit 1996;18:405–409. Hardy L, Rogers B, Thomas D, Ryan A, Peterson M, Koren E, Rowell T, Fuller B, Hobson W. Thrombocytopenia and antigenicity assessment of thrombopoietin treated chimpanzees and rhesus monkeys. The Toxicologist 1997;36:277. ICH S6. Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, July 1997. Retrieved on August 11, 2005. Available at http://www.ich.org Kessler M, Goldsmith D, Schellekens H. Immunogenicity of biopharmaceuticals. Nephrol Dial Transplant 2006;21(Suppl 5):v9–v12. Koren E. From characterization of antibodies to prediction of immunogenicity. Dev Biol 2002;109:87–95. Koren E, Zuckerman LA, Mire-Sluis AR. Immune responses to therapeutic proteins in humans—clinical significance, assessment and prediction, Current Pharm Biotechnol 2002;3:349–360. Li J, Yang C, Xia Y, Bertino A, Glaspy J, Roberts M, Kuter DJ. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 2001;98: 3241–3248. Mire-Sluis AR, Barrett YC, Devanarayan V, Koren E, Liu H, Maia M, Parish T, Scott G, Shankar G, Shores E, Swanson SJ, Taniguchi G, Wierda D, Zuckerman LA. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods 2004;289:1–16. Pendley C, Schantz A, Wagner C. Immunogenicity of therapeutic monoclonal antibodies. Curr Opin Mol Ther 2003;5:172–179. Rosenblum MG, Unger BW, Gutterman JU, Hersh EM, David GS, Frincke JM. Modification of human leukocyte interferon pharmacology with a monoclonal antibody. Cancer Res 1985;45:2421–2424. Schellekens H. Immunogenicity of therapeutic proteins: clinical implications and future prospects. Clin Ther 2002;24:1720–1740. Serabian MA, Pilaro AM. Safety assessment of biotechnology-derived pharmaceuticals: ICH and beyond. Toxicol Pathol 1999;27:27–31. Shankar G, Shores E, Wagner C, Mire-Sluis A. Scientific and regulatory considerations on the immunogenicity of biologics. Trends Biotechnol 2006;24:274–280. Shankar G, Pendley C, Stein KE. A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs. Nat Biotechnol 2007; 25:555–561. Sims J. Assessment of biotechnology products for therapeutic use. Toxicol Lett 2001; 59:120–166. Terrell TG, Green JD. Comparative pathology of recombinant murine interferon-γ in mice and recombinant human interferon-γ in cynomolgus monkeys. In: International Review of Experimental Pathology: Cytokine-Induced Pathology Part A: Interleukins

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and Hematopoietic Growth Factors, edited by Richter GW, Solez K, Ryffel B, pp. 73–101. San Diego, CA: Academic Press, 1993. Terrell TG, Green JD. Issues with biotechnology products in toxicologic pathology. Toxicol Pathol 1994;22:187–193. Tsang L, Beers D. Legal and scientific considerations in nonclinical assessment of biotechnology products. Drug Info J 2003;37:397–406. Wang DS, Ohdo S, Koyanagi S, Takane H, Aramaki H, Yukawa E, Higuchi S. Effect of dosing schedule on pharmacokinetics on alpha interferon and anti-alpha interferon neutralizing antibody in mice. Antimicrob Agents Chemother 2001;45:176–180. Wierda D, Smith HW, Zwickl CM. Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology 2001;158:71–74. Working PK. Potential effects of antibody induction by protein drugs. In: Protein Pharmacokinetics and Metabolism, edited by Ferraiolo BL, Mohler MA, Gloff CA, pp. 73–92. New York, NY: Plenum Press, 1992. Zwickl CM, Cocke KS, Tamura RN, Holzhausen LM, Brophy GT, Bick PH, Wierda D. Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys. Fundam Appl Toxicol 1991;16:275–287.

PART VII DEVELOPMENT OF VACCINES

7.1 PHARMACOLOGICAL IMMUNOGENICITY AND ADVERSE RESPONSES TO VACCINES Mary Kate Hart, Mark Bolanowski, and Robert V. House

Vaccines are administered to induce specific immune responses that protect against death or illness following exposure to a pathogen or its toxin. Vaccines may be formulated as live attenuated organisms, killed organisms, recombinant proteins, vectored recombinants that express proteins from the pathogen, or as plasmids. Live attenuated organisms generally induce long-lasting immunity after a single exposure, but have a risk of being insufficiently attenuated. Killed organisms and protein-based vaccines may require periodic boosters to maintain immunity and, for killed vaccines, complete inactivation of the organism must be ensured to avoid infecting the recipient. Some vaccines are administered with an adjuvant to improve the magnitude of the immune responses or to elicit a specific type of immune response. The historic objective of vaccination has been to induce long-lasting immunity against a disease for which the recipient is at risk. The most successful vaccination campaign eradicated smallpox as a public health problem. Particularly, vaccination against common infectious agents, including poliomyelitis, measles, mumps, and rubella, over the last 50 years has led to effective prevention of many diseases. Short-term immunity may be achieved by “passive” vaccination, or the transfer of protective antibodies into the recipient. Vaccination is currently being explored as a therapeutic approach for inducing needed immune responses in individuals who have acquired a disease (e.g., cancer or Alzheimer’s disease) Immunotoxicology Strategies for Pharmaceutical Safety Assessment, edited by Danuta J. Herzyk and Jeanine L. Bussiere Copyright © 2008 John Wiley & Sons, Inc.

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or following a known potential exposure to a pathogen such as might occur following a needlestick. Vaccines are tested preclinically for toxicity and immunogenicity before they are evaluated in clinical trials, and vaccine licensure by the Food and Drug Administration (FDA) requires the demonstration of safety, immunogenicity, and efficacy. Careful design of the preclinical animal studies is important to minimize the chances of administering a product to humans that may cause pathology. Typically, preclinical safety evaluation of vaccines includes acute toxicity and repeat-dose toxicity studies before entering clinical testing. Reproductive toxicity studies are performed while the vaccine is being evaluated in clinical trials. Immunogenicity, and in some cases protective efficacy, may be evaluated in animals if models exist for the disease. More extensive animal studies need to be performed for vaccines pursuing licensure using the “Animal Rule,” for diseases in which it is not feasible to evaluate efficacy in humans. However, these studies will not always successfully predict the human response to vaccine candidates (Lebron et al., 2005). As true for other biopharmaceuticals, vaccines must be evaluated in clinical trials, including Phase 1 studies conducted to assess a candidate vaccine’s safety. If Phase 1 trials demonstrate acceptable safety profiles, the efficacy and safety of vaccines are further tested in Phase 2 and Phase 3 clinical trials. Vaccines that obtain licensure continue to be monitored for adverse events during the post-marketing phase, as events with lower frequencies may not be captured during clinical trial observations.

IMMUNOTOXICOLOGY EVALUATION OF VACCINES The administration of a vaccine may induce beneficial, neutral, or harmful immune responses in the recipient. Beneficial responses include protective pathogen or toxin-neutralizing antibodies and cell-mediated responses that are capable of clearing the pathogen or toxin before irreparable harm is done to the host. Neutral responses are neither protective nor harmful (e.g., nonneutralizing, non-enhancing antibodies). Harmful responses may include antibodies or cell-mediated responses that cross-react with self-antigens that potentiate disease following exposure to the pathogen, or that induce immune complex formation or inflammation. Such responses may result from the vaccine antigen, materials the vaccine antigen is formulated in or which derive from the manufacturing process, and/or the adjuvant. For over three decades, there has been an increasing understanding that exposure to human therapeutics, including immunotherapeutics, can produce adverse changes in the human immune response (House and Luebke, 2006). While this has historically been associated primarily with immunosuppression, more recently the potential deleterious effects of immunostimulation (whether inadvertent or deliberate) have received increased attention (Shankar et al., 2006). Although the methods and tools for assessing pathology associated with

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immunosuppression are by now well established, determining the long-term consequences of immunostimulation is in its infancy (House and Hastings, 2004). Paradoxically, the potential toxicity of the clearly intentional antigenspecific immunostimulation produced by vaccination is even less well understood than many of the newer therapies for nonspecific immune augmentation. A better understanding of real risks associated with vaccination is necessary to combat the burgeoning anti-vaccine lobby that is developing (Bonhoeffer and Heininger, 2007).

Possible Adverse Reactions to Vaccination Hypersensitivity. Hypersensitivity reactions, considered to be vaccine-related based on the timing and specificity of the reactions, were reported to the United States’ Vaccine Adverse Event Reporting System (VAERS) following vaccination for Lyme disease and subsequently evaluated (Burmester et al., 1995; Lathrop et al., 2002). Other reported immune system-related events to vaccines included rheumatoid arthritis, immune system disorders, detection of antinuclear antibodies, lupus syndrome, and lymphocytosis (Zhou et al., 2003). These were very rare events, with each condition comprising 0.2% or fewer of the total reports. Anaphylactic responses to vaccines were also rare and were estimated at less than one case per million administered vaccine doses (Bohlke et al., 2003). A number of studies evaluated anaphylactic responses to the measles-mumps-rubella (MMR), hepatitis B, diphtheria or tetanus vaccines with similar findings (Dobson et al., 1995; D’Souza et al., 2000; Patja et al., 2000; Pool et al., 2002). However, some of the vaccine-induced hypersensitivity reactions are attributed to components of the formulation, such as gelatin or egg, rather than the antigen itself (Patja et al., 2001; Pool et al., 2002). Hypersensitivity reactions (for more detail, see Chapter 8) were induced following vaccination with the human diploid cell vaccine (HDCV) for rabies. Booster vaccinations for people who previously experienced hypersensitivity reactions to HDCV were recommended to be administered cautiously based on Morbidity and Mortality Weekly Report (MMWR, 1999a). By the time of the report, HDCV had been administered to approximately 100,000 people, most of whom received it following exposure to the virus. During a 46-month period, 108 reports of hypersensitivity to some component of the vaccine (MMWR, 1984) were received. Type I hypersensitivity reactions, which are IgE-mediated and occur soon after exposure, were described in nine cases. The majority of the cases of hypersensitivity were presumed to be Type III and occurred 2 to 21 days following vaccination. These responses are mediated by IgG and/or IgM antibodies that form immune complexes that are deposited in tissues and which then induce inflammatory responses at their location. Type III responses were usually observed in people receiving a recommended booster at 2-year intervals for continued risk of exposure, although a few reports were received during the primary series.

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The clinical presentations included a generalized rash or urticaria sometimes accompanied by nausea, malaise, vomiting, fever, arthralgias, and angioedema. Serious adverse events, defined as death, life-threatening illness requiring hospitalization, or resulting in persistent or significant disability or birth defect (FDA 21 CFR Part 600.80) comprised 14% of the VAERS reports. Linking the reported events to a vaccine in a cause-and-effect relationship is difficult and may require extensive investigation. Investigations were performed on all VAERS-reported deaths during this period; after review by the FDA and the Institute of Medicine of 206 deaths reported between 1990 and 1991, the only one that was subsequently concluded to be vaccinerelated occurred in a 28-year-old woman who developed Guillain-Barré syndrome after tetanus vaccination (Stratton et al., 1994). Guillain-Barré syndrome involves an inflammatory demyelination of peripheral nerves, and is treated with intravenous administration of immunoglobulin. Increased risk of developing Guillain-Barré syndrome has been associated with swine influenza vaccination (Langmuir et al., 1984; Safranek et al., 1991). Following reports of a possible association between vaccination and the onset of Guillain-Barré syndrome, a history of the syndrome was added as a contraindication for the Menactra vaccine for meningococcus except in cases of elevated risk (MMWR, 2006a; MMWR, 2006b; MMWR, 2005).

Response to Replicating Vaccines. Live vaccines, such as the smallpox vaccine (vaccinia virus), can induce pathologies resulting from replication in tissues and the ability to spread beyond the site of inoculation. This risk has been realized with the smallpox vaccine. The vaccine is administered by scarification and causes a pustule to form at the site. Vaccinia can be transmitted from the recipient to others through close contact and can induce the same adverse events in those people. Recipients are advised to keep the area dry and to disinfect their hands after changing bandages to avoid spreading the virus to the eyes (ocular vaccinia) and other areas of the body (generalized vaccinia). People with a history of atopic dermatitis are at risk of potentially life-threatening eczema vaccinatum. These patients often become systemically ill and require treatment with vaccinia immunoglobulin and/or antiviral drugs. Progressive vaccinia is a rare but often fatal disease that can occur in immunodeficient people and results in necrosis at the vaccination site that may spread to other sites. This form of disease requires intensive medical treatment and support. A recent program to protect healthcare workers from a possible bioterrorist attack with smallpox reported cases of myocarditis and pericarditis following vaccination (MMWR, 2003), and other studies provided evidence that these effects might be associated causally with vaccinia vaccination (Feery, 1977; Halsell et al., 2003) and may have caused the death of one vaccine recipient (Finlay-Jones, 1964).

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The Myth of “Antigen Overload” In recent years, a number of unsubstantiated concerns have surfaced regarding the ability of multivalent vaccines, or the administration of multiple monovalent vaccines, to somehow “overload” the immune system, particularly in juveniles. In theory, the simultaneous onslaught of many different immune targets depletes the body’s reserve of immune mediators, leading to an inability to respond to natural infections. This fallacy was refuted in detail by Offit et al. (2002). In short, their calculations demonstrate that the functional reserve of the human immune system is theoretically capable of responding to approximately 10,000 vaccines simultaneously with no appreciable “depletion” of the self-replenishing leukocyte stores. Moreover, the total number of antigens contained in modern vaccines is far less than in the past; for example, in 1900 the smallpox vaccine contained approximately 200 antigens by itself, whereas in 2000 the total antigen burden in the 11 principal childhood vaccines was approximately 125. The myth of antigen overload has likewise been discounted by careful evaluation of clinical history (Miller et al., 2003). Nevertheless, this concern continues to be one reason that parents hesitate to vaccinate children even today (Hilton et al., 2006). Other Adverse Conditions Possibly Associated with Vaccination Intussusception and Rotavirus Vaccine. Following reports of intussusception, a form of bowel obstruction, in 15 infants who received a licensed rhesushuman reassortant vaccine for rotavirus, the Centers for Disease Control recommended suspension of the vaccine in 1999 (MMWR, 1999b). The manufacturer of the vaccine voluntarily discontinued distribution of the vaccine because of its association with intussusception (MMWR, 2004). The risk of intussusception was highest within 3 to 14 days of the primary vaccination, although increased risk was also observed after a second dose. A newer vaccine, based on human–bovine reassortant rotaviruses, was licensed in 2006 following three Phase 3 clinical trials. The risk of intussusception was evaluated in more than 70,000 recipients in the Phase 3 trials, and was not observed to differ significantly from the placebo group (MMWR, 2006c). The Advisory Committee on Immunization Practices currently recommends routine vaccination of infants with the human–bovine vaccine, except in those with known serious allergic reactions to any vaccine components or that resulted from a prior dose of vaccine. Potentiated Disease. Vaccination can induce immune responses that establish a condition that potentiates disease upon subsequent exposure to the pathogen. Preclinical studies to evaluate adjuvanted protein-based vaccines comprising helper T cell and/or B cell epitopes from Plasmodium berghei indicated that vaccinated mice died earlier of malaria than unvaccinated control mice, a finding attributed to the immune response induced against the T cell epitope and a nonspecific effect of the adjuvant (Reed et al., 1997).

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NOVEL TECHNOLOGIES IN VACCINE DEVELOPMENT New vaccine technologies may present potential risks that need to be evaluated in nonclinical studies. Plasmid vaccines, for example, pose the potential risks (reviewed in Robertson and Griffiths, 2006) of integrating into the host chromosome and altering the normal replication of the cell. If integration activates an otherwise dormant oncogene, uncontrolled cellular division could result. Although the risk of this occurring is low, careful design of the nonclinical studies supporting the vaccine is necessary, especially when the vaccines are delivered in ways that can enhance the uptake of DNA into cells. As this technology is tested in more nonclinical studies and clinical trials, the data obtained will provide an indication of whether concerns are warranted regarding the induction of anti-DNA antibodies following administration of plasmid vaccines. Similarly, these studies will address the concerns that immunopathology or tolerance may result from the overexpression or continued expression of the vaccine antigen and/or cytokine or immunostimulatory molecules. The nonclinical studies should be designed to evaluate both local and systemic responses over time. Another approach requiring careful safety studies is replication-competent vaccines derived from viruses with known pathology. Vesicular stomatitis virus (VSV) is currently being evaluated as a vaccine vector, either by replacing its glycoprotein with the glycoprotein of another virus or through the expression of foreign proteins. A recent study evaluating the neurovirulence of rVSV in nonhuman primates indicated that, although significantly attenuated compared to wild-type VSV, neurovirulence was observed in a route-dependent fashion (Johnson et al., 2007). Modeling Immune Responses to Vaccines Methods for assessing or modeling of the responses of the human immune system to vaccines are critical components of any effort to understand the relationship between immunogenicity and either a positive or a negative outcome of vaccination. Decades of research have yielded many useful in vitro methods that enable the isolation and molecular dissection of selected components (modulators or cell types) of an immune system, whether mouse or human. While of limited scope biologically, these systems have elucidated the modulators and cell types responsible for certain facets of humoral or cellular immunity. These systems have also been employed to define the roles that modulators and specific cell types play in particular aspects of an immune response. Missing, until recently, was an in vivo setting in which to study the human immune system. The discovery and characterization of the severe combined immunodeficient (SCID) mutation two and a half decades ago opened up the possibility of engrafting the human immune system in animals lacking a functional endogenous immune system. The ability of SCID mice to be engrafted with the

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human immune system from the bone marrow to circulating mature and competent B and T cells (Barry and Haynes, 1992) was enhanced by combining the SCID mutation with either the NOD or Rag-2–/– mouse strain, coupled with the targeted deletion of the interleukin-2 receptor γ chain locus (for a recent review, see Shultz et al., 2007a). The NOD-SCID-ILrg–/– and other strains are being employed to model the responses of the human immune system to vaccines and therapeutic proteins (biopharmaceuticals), as well as in autoimmune disease (Macchiarini et al., 2005; Shultz et al., 2007b). In addition to these models, human HLA transgenic, immunocompetent mice serve as useful models for defining T cell epitopes that are critical for cell-mediated immune responses. Another approach to risk mitigation is to refine the mode in which the antigen is presented to the host. Several recent technological advances offer promising alternatives. In addition to simply augmenting the intensity or duration of immune responses to vaccination, novel adjuvants such as CpGcontaining oligonucleotides (CpGs) and immune-stimulating complexes (ISCOMs) can drive the mouse immune system toward a Th1 response which can be critical for eliciting protection against certain pathogens. This ability to influence the type of immune response by activating one or more Toll-like receptors (TLRs) during vaccination suggests that in the future, adjuvants could be used to elicit a protective response without stimulating any other component of the immune system. For example, Liu et al. (2007a) co-administered stimulatory modulators such as secondary lymphoid tissue cytokine (SLC), fas ligand (fasL), CD-137 ligand (4-1BBL), and TNF-related activation-induced cytokine (TRANCE) along with the vaccine, while this same group reported success using dendritic cells (DCs) coated with SLC, 4-1BBL, and TRANCE (Liu et al., 2007b). Furthermore, Cui et al. (2004) showed that HIV-tat-coated nanoparticles elicited Th1 responses in mice in contrast to the non-protective Th2 response normally observed following vaccination with tat adjuvanted with alum. Other approaches have sought to deliver antigens and epitopes directly to DC via the targeting of DC-specific cell surface receptors (Bonifaz et al., 2004) or by using microparticles, nanoparticles, and liposomes (see Reddy et al., 2006). Lastly, though not robust enough yet, mathematical modeling may prove to be a viable approach to defining the mechanisms of immunity and how we may improve vaccine efficacy. Morel et al. (2006) are developing models to glean information about the immune system from microarray data. In addition, the field of immunoinformatics seeks to expand this effort to incorporate data from all sources with the goal of developing in silico models of the immune system and how it works (Petrovsky and Brusic, 2006).

SUMMARY The use of vaccines has arguably resulted in some of the greatest improvements in overall human health of any medical treatment in history. However,

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like all other biopharmaceuticals, vaccines must be judiciously and thoroughly evaluated for human safety. This is particularly true since vaccines, unlike most other medical treatments, are routinely administered to large numbers of otherwise healthy humans. It is increasingly clear that vaccines can exert toxicity not only directly via the antigen and other vaccine components, but as a result of the immunological reaction to the antigen itself. Future safety evaluations of vaccines must consider these potential interactions as part of the overall development plan.

REFERENCES Barry TS, Haynes BF. In vivo models of human lymphopoiesis and autoimmunity in severe combined immune deficient mice. J Clin Immunol 1992;12(5):311–324. Bohlke K, Davis RL, Marcy SM, Braun MM, DeStefano F, Black SB, Mullooly JP, Thompson RS; Vaccine Safety Datalink Team. Risk of anaphylaxis after vaccination of children and adolescents. Pediatrics 2003;112(4):815–820. Bonhoeffer J, Heininger U. Adverse events following immunization: perception and evidence. Curr Opin Infect Dis 2007;20(3):237–246. Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii S, Soares H, Brimnes MK, Moltedo B, Moran TM, Steinman RM. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J Exp Med 2004;199(6):815–824. Burmester GR, Daser A, Kamradt T, Krause A, Mitchison NA, Sieper J, Wolf N. Immunology of reactive arthritides. Annu Rev Immunol 1995;13:229–250. Cui Z, Patel J, Tuzova M, Ray P, Phillips R, Woodward JG, Nath A, Mumper RJ. Strong T cell type-1 immune responses to HIV-1 Tat (1-72) protein-coated nanoparticles. Vaccine 2004;22(20):2631–2640. D’Souza RM, Campbell-Lloyd S, Isaacs D, Gold M, Burgess M, Turnbull F, O’Brien E. Adverse events following immunisation associated with the 1998 Australian Measles Control Campaign. Commun Dis Intell 2000;24(2):27–33. Dobson S, Scheifele D, Bell A. Assessment of a universal, school-based hepatitis B vaccination program. JAMA 1995;274(15):1209–1213. FDA 21 CFR Part 600.80. Post-marketing reporting of adverse drug experiences. Federal Register 1997;62:52252–52253. Feery BJ. Adverse reactions after smallpox vaccination. Med J Aust 1977;2(6): 180–183. Finlay-Jones LR. Fatal myocarditis after vaccination against smallpox. Report of a case. N Engl J Med 1964;270:41–42. Halsell JS, Riddle JR, Atwood JE, Gardner P, Shope R, Poland GA, Gray GC, Ostroff S, Eckart RE, Hospenthal DR, Gibson RL, Grabenstein JD, Arness MK, Tornberg DN, Department of Defense Smallpox Vaccination Clinical Evaluation Team. Myopericarditis following smallpox vaccination among vaccinia-naive US military personnel. JAMA 2003;289(24):3283–3289.

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Hilton S, Petticrew M, Hunt K. Combined vaccines are like a sudden onslaught to the body’s immune system: parental concerns about vaccine ‘overload’ and ‘immunevulnerability’. Vaccine 2006;24(20):4321–4327. House RV, Hastings KL. Multidimensional immunomodulation. J Immunotoxicol 2004;1:123–129. House RV, Luebke RW. Immunotoxicology: thirty years and counting. In: Immunopharmacology and Immunotoxicology, 3rd eds., edited by Luebke R, House R, Kimber I, pp. 3–20. Boca Raton, FL: CRC Press, 2006. Johnson JE, Nasar F, Coleman JW, Price RE, Javadian A, Draper K, Lee M, Reilly PA, Clarke DK, Hendry RM, Udem SA. Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates. Virology 2007; 360(1):36–49. Lathrop SL, Ball R, Haber P, Mootrey GT, Braun MM, Shadomy SV, Ellenberg SS, Chen RT, Hayes EB. Adverse event reports following vaccination for Lyme disease: December 1998-July 2000. Vaccine 2002;20(11–12):1603–1608. Langmuir AD, Bregman DJ, Kurland LT, Nathanson N, Victor M. An epidemiologic and clinical evaluation of Guillain-Barré syndrome reported in association with the administration of swine influenza vaccines. Am J Epidemiol 1984;119(6): 841–879. Lebron JA, Wolf JJ, Kaplanski CV, Ledwith BJ. Ensuring the quality, potency and safety of vaccines during preclinical development. Expert Rev Vaccines 2005;4(6): 855–866. Liu S, Breiter DR, Zheng G, Chen A. Enhanced antitumor responses elicited by combinatorial protein transfer of chemotactic and costimulatory molecules. J Immunol 2007a;178(5):3301–3306. Liu S, Foster BA, Chen T, Zheng G, Chen A. Modifying dendritic cells via protein transfer for antitumor therapeutics. Clin Cancer Res 2007b;13(1):283–291. Macchiarini F, Manz MG, Palucka AK, Shultz LD. Humanized mice: are we there yet? J Exp Med 2005;202(10):1307–1311. Miller E, Andrews N, Waight P, Taylor B. Bacterial infections, immune overload, and MMR vaccine. Arch Dis Child 2003;88:222–223. MMWR, 1984;33:185–187. MMWR, 1999a;48:1–21. MMWR, 1999b;48:1007. MMWR, 2003;52(27):639–642. MMWR, 2004;53:786–789. MMWR, 2005;54:1023–1025. MMWR, 2006a;55:1120–1124. MMWR, 2006b;55:364–366. MMWR, 2006c;55(RR12):1–13. Morel PA, Ta’asan S, Morel BF, Kirschner DE, Flynn JL. New insights into mathematical modeling of the immune system. Immunol Res 2006;36(1–3):157–165. Offit PA, Quarles J, Gerber MA, Hackett CJ, Marcuse EK, Kollman TR, Gellin BG, Landry S. Addressing parents’ concerns: do multiple vaccines overwhelm or weaken the infant’s immune system? Pediatrics 2002;109(1):124–129.

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Patja A, Davidkin I, Kurki T, Kallio MJ, Valle M, Peltola H. Serious adverse events after measles-mumps-rubella vaccination during a fourteen-year prospective follow-up. Pediatr Infect Dis J 2000;19(12):1127–1134. Patja A, Mäkinen-Kiljunen S, Davidkin I, Paunio M, Peltola H. Allergic reactions to measles-mumps-rubella vaccination. Pediatrics 2001;107(2):E27. Petrovsky N, Brusic V. Bioinformatics for study of autoimmunity. Autoimmunity 2006; 39(8):635–643. Pool V, Braun MM, Kelso JM, Mootrey G, Chen RT, Yunginger JW, Jacobson RM, Gargiullo PM, VAERS Team. US Vaccine Adverse Event Reporting System. Prevalence of anti-gelatin IgE antibodies in people with anaphylaxis after measles-mumps rubella vaccine in the United States. Pediatrics 2002;110(6):e71. Reddy ST, Swartz MA, Hubbell JA. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol 2006;27(12):573–579. Reed RC, Verhuel AF, Hunter RL, Udhayakumar V, Louis-Wileman V, Jennings VJ, Jue DL, Wohlhueter RM, Lal AA. Rapid onset of malaria-induced mortality by immunizations with lipo-peptides: an experimental model to study deleterious immune responses and immunopathology in malaria. Vaccine 1997;15(1):65–70. Robertson JS, Griffiths E. Assuring the quality, safety, and efficacy of DNA vaccines. Methods Mol Med 2006;127:363–374. Safranek TJ, Lawrence DN, Kurland LT, Culver DH, Wiederholt WC, Hayner NS, Osterholm MT, O’Brien P, Hughes JM. Reassessment of the association between Guillain-Barré syndrome and receipt of swine influenza vaccine in 1976-1977: results of a two-state study. Expert Neurology Group. Am J Epidemiol 1991;133(9): 940–951. Shankar G, Shores E, Wagner C, Mire-Sluis A. Scientific and regulatory considerations on the immunogenicity of biologics. Trends Biotechnol 2006;24(6):274–280. Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol 2007a;7:118–129. Shultz LD, Pearson T, King M, Giassi L, Carney L, Gott B, Lyons B, Rossini AA, Greiner DL. Humanized NOD/LtSz-scid IL2 receptor common gamma chain knockout mice in diabetes research. Ann N Y Acad Sci 2007b;1103:77–89. Stratton KR, Howe CJ, Johnston RB. Adverse events associated with childhood vaccines other than pertussis and rubella. Summary of a report from the Institute of Medicine. JAMA 1994;271(20):1602–1605. Zhou W, Pool V, Iskander JK, English-Bullard R, Ball R, Wise RP, Haber P, Pless RP, Mootrey G, Ellenberg SS, Braun MM, Chen RT. Surveillance for safety after immunization: Vaccine Adverse Event Reporting System (VAERS)-United States, 19912002. MMWR 2003;52/SS-1:1–24.

7.2 IMMUNOTOXICOLOGICAL CONCERNS FOR VACCINES AND ADJUVANTS Catherine Kaplanski, Jose Lebron, Jayanthi Wolf, and Brian Ledwith

The nonclinical safety assessment of vaccines includes an assessment of toxicity in at least one relevant species, where relevance is based on observation of the expected immune response. Toxicity studies of vaccines generally include similar toxicological assessments to those performed for pharmaceuticals, including observations of general toxicity (mortality, physical signs, body weight, food consumption), ophthalmic examinations, hematology, serum biochemistry, and complete necropsy with gross and histological examination of a broad panel of tissues (EMEA, 1997; WHO, 2003; Lebron et al., 2005). Therefore, an evaluation of immunotoxicity, per se, is typically performed for vaccines in a comparable manner to that recommended as the first tier assessment of immunotoxicity for pharmaceuticals in ICH S8 guidance (ICH, 2006): i.e., examination of relevant parameters within the routine repeated-dose toxicity studies. The difference with vaccines of course is that immune stimulation is an intended pharmacological effect, and thus effects on various immune system parameters are expected and desirable. Such effects may include changes in hematology (various white blood cell types) and serum biochemistry (e.g., protein and globulin) parameters, local irritation and inflammation at the injection site, lymphoid enlargement and hyperplasia, and spleen weight increases. These effects are generally modest and reversible, and, as conse-


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quences of the intended pharmacological activity of the vaccine, are usually not considered adverse (Lebron et al., 2007). Immunosuppression is generally not a relevant concern for vaccines. Rather, theoretical concerns of immunotoxicity by vaccines are generally related to the potential for hyperstimulation of the immune system. This section will discuss three topics in which stimulation of the immune system could theoretically result in adverse effects: (1) Adjuvants and the Risk of Systemic Inflammatory Response; (2) Therapeutic Vaccines and Risk of Cell-Mediated Toxicity; and (3) Therapeutic Cancer Vaccines and The Risk of Autoimmunity.

ADJUVANTS AND RISK OF SYSTEMIC INFLAMMATORY RESPONSE Regulatory guidelines (WHO, 2003; EMEA, 2005) discuss the theoretical potential for hyperstimulation of the immune system by adjuvants. Since traditional adjuvants such as alum have predominantly functioned via local rather than systemic mechanisms, a systemic inflammatory response has generally not been a concern. However, the theoretical concern of a systemic inflammatory response may be heightened for some new types of experimental adjuvants (Lindblad, 2007), particularly for “molecular” adjuvants such as Toll-like receptor (TLR) agonists (Johnson and Baldridge, 2007) and cytokines (Portielje et al., 2005; Egilmez, 2007) that can be soluble (with greater potential for systemic exposure) and potent activators of the immune system. While the risk of a systemic inflammatory response should be manageable with considerations of dose response and route of administration, the potential for a systemic inflammatory response should be assessed within the nonclinical toxicology study (e.g., by measurement of pro-inflammatory cytokines such as IL-6 in serum or plasma; Lebron et al., 2007), and the potential for differences in sensitivity between humans and the animal species used for toxicity testing should be considered (EMEA, 2007).

THERAPEUTIC VACCINES AND RISK OF CELL-MEDIATED TOXICITY The goal of therapeutic vaccination is to harness the power of the immune system to treat complex diseases such as cancer, autoimmunity, obesity, and Alzheimer’s disease. The basic principle in the design of a therapeutic vaccine is to identify the self-protein that causes a disease, and then try to elicit an immune response against the protein, by breaking immune tolerance, in order to remove the protein from the body and alleviate the symptoms of the disease. Once immune tolerance is broken, it leads to the destruction of the targeted self-protein; thus, the balance between safety and efficacy for therapeutic vaccines needs to be carefully assessed.

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The patient population targeted for therapeutic vaccination can be immunocompromised (e.g., cancer patients and elderly patients), hence various immunostimulation techniques need to be investigated in an effort to bolster the immune system and to overcome immune tolerance to self-antigens. Various strategies to stimulate antigen presentation are under investigation, including the use of novel adjuvants. The stimulation of the immune response needs to be carefully tempered to avoid overactivation of cytotoxic T cells that could be more destructive than intended. An example of unwanted T cell-mediated toxicity induced by a therapeutic vaccine occurred with a vaccine for Alzheimer’s disease. Alzheimer’s disease appears to be an excellent target for therapeutic vaccination since it is thought that the abnormal accumulation of amyloid-β into extracellular plaques is responsible for the neurodegeneration and resulting dementia in Alzheimer’s disease. Preclinical studies have shown that immunization against amyloid-β in transgenic mouse models can provide protection against and reversal of the pathology of Alzheimer’s disease in animal models (Schenk et al., 1999). After evaluating the preclinical safety and efficacy data in several species (including mice, rabbits, guinea pigs, and monkeys), an amyloid-β vaccine, AN1792, that consisted of Aβ1-42 with the QS-21 adjuvant entered clinical studies for possible treatment of Alzheimer’s disease (Schenk, 2002). A small, single-dose, Phase 1 study in 24 patients, and a subsequent multiple-dose Phase 1 study with over 70 patients showed good tolerability and immunogenicity. A Phase 2a trial was initiated with 372 patients; however, signs and symptoms of meningoencephalitis in 6% of the patients were observed and dosing was halted. The nervous system inflammation after vaccination with AN1792 could have been due to activation of T lymphocytes, since T cell infiltrates in the brain were observed in two encephalitis cases when compared to one case that did not exhibit encephalitis and also lacked T cell infiltrates (Schenk et al., 2004). This suggests that the encephalitis might have resulted from a T cell response again Aβ1-42. The full-length Aβ1-42 peptide contains both B cell and T cell epitopes, and would be expected to produce both B and T cell responses. Antibody production is the desired response in therapeutic Alzheimer’s disease vaccination, and requires the activation of Th2 memory effector cells. In the elderly, however, the predominant T cell population are Th1 cells, which produce pro-inflammatory cytokines such as interferon-gamma (IFN-γ) when stimulated (Münch and Robinson, 2002). IFN-γ in turn activates microglia, which secrete neurotoxic factors including tumor necrosis factor-alpha (TNFα) and nitric oxide which cause tissue damage (McMillian et al., 1995). In addition, the Aβ-specific T cell line repertoire contains a high percentage of CD8+ cytotoxic T cells which could lyse neurons and astrocytes that present the Aβ sequence (Grubeck-Loebenstein et al., 2000). When activated, these cells could enter the brain and cause tissue damage. The inclusion of the QS-21 adjuvant in the AN1792 vaccine might have also contributed to the Th1 response, since studies in transgenic mice with an Aβ1-42 vaccine showed that QS-21 containing formulations induced significant IFN-γ,

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IL-4, and TNF-α expression (Cribbs et al., 2003). Comparison of T cell reactivity in patients enrolled in either the Phase 1 or 2a clinical trials demonstrates a shift toward a predominantly Th1 response in the Phase 2a trial (Pride et al., 2004). The formulations used in these trials differ only by the inclusion of polysorbate 80 (PS-80) in the Phase 2a formulation, and highlight the possible importance of the vaccine formulation in the delicate balance between safety and efficacy of therapeutic vaccines (Brown et al., 2005). Future vaccines for Alzheimer’s disease will need to be carefully designed to avoid a T cell response. One possibility is to use shortened amyloid-β peptides which contain B cell epitopes but not T cell epitopes, and link these short peptides to a non-amyloid-β T-helper cell epitope (Maier et al., 2006). A shortened amyloid-β peptide vaccine has been tested in a transgenic mouse model and resulted in a lack of T cell responses against full-length amyloid-β, but still produced an antibody response with production of Th2 isotypes (IgG1 and IgG2b) that reduced amyloid-β plaque levels (Maier et al., 2006). Using this type of approach, the T cell-mediated toxicity associated with Alzheimer’s disease therapeutic vaccines might be avoided.

THERAPEUTIC CANCER VACCINES AND THE RISK OF AUTOIMMUNITY One of the approaches being developed for the treatment of cancer is the use of therapeutic cancer vaccines. This approach is still experimental and involves the use of peptides, proteins, antigen-expressing vectors, or primed cells to induce an active immune response directed against tumor antigens, the majority of which consists of shared antigens (Tabi and Man, 2006). Given that these non-tumor-specific antigens are self-antigens that are preferentially expressed, overexpressed, or re-expressed in cancer cells, mounting an immune response against them involves breaking tolerance. The correlate to breaking tolerance against a self-antigen is inducing autoimmunity. Thus, in order for this type of vaccines to be used clinically, they must achieve an acceptable balance between inducing an autoimmune response that can control and/or destroy the targeted tumor cells, and not inducing a pathological autoimmune response that would result in detrimental tissue damage. The potential risk of autoimmune pathology associated with tumor immunotherapy using cancer vaccines has been examined extensively during the past decade (Gilboa, 2001; Rodriguez-Lecompte et al., 2004). It is indeed theoretically possible to break self-tolerance and induce an antitumor response due to the presence of functionally competent autoreactive T cells in the periphery. Such cells have escaped thymic deletion (T cells of low avidity that did not go through thymic negative selection and low- to high-avidity T cells specific for tissue antigens that are not expressed in the thymus) and can be activated under appropriate conditions.

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Animal studies have explored the efficacy and the adverse effects of tumor vaccination using various types of tumor antigens. These studies have shown the feasibility of tumor vaccination, i.e., tolerance to self-antigens expressed by tumor cells can indeed be broken, leading to protection against tumor challenge and in some cases, to tumor eradication. Although autoimmunity should be expected when vaccinating with shared antigens, pathological autoimmune reactions have occurred infrequently and when observed, they were of mild intensity and well tolerated. For example, immunotherapy with melanoma cancer vaccines in mice resulted in the induction of autoimmune depigmentation (vitiligo) associated with rejection of tumors (Overwijk et al., 1999; van Elsas et al., 1999). When melanoma cancer vaccines were further evaluated in humans, some autoimmune toxicity including depigmentation, colitis, dermatitis, and uveitis were observed and were also usually associated with partial or complete clinical responses (Attia et al., 2005). These studies showed that tumor tissue is more susceptible to an immune response against a shared antigen than normal tissue, thus indicating that potent antitumor immunity could be achieved in the absence of widespread autoimmune pathology. The differential susceptibility of normal versus tumoral tissue is far from being understood, but might involve the overexpression of the targeted antigen in tumor cells, as well as distinct cytokine/chemokine environments. Severe autoimmune reactions (diabetes, arteritis, myocarditis, dilated cardiomyopathy) were observed when mice expressing a model tumor antigen as a transgene in the pancreatic β-islet cells or in the cardiomyocytes and arterial smooth muscle cells were transplanted with antigen-expressing tumors, and then vaccinated with repetitive infusions of dendritic cells presenting the same antigen (Ludewig et al., 2000). It appears that the choice of the shared antigen targeted for tumor vaccination is of key importance, with the best choices being antigens that are either expressed in immuno-privileged sites (testes for example), or are fetal/embryonic antigens re-expressed in cancer cells, but normally not expressed or expressed at low levels in adult tissues. A good illustration of the key importance of antigen selection is the oncofetal antigen carcinoembryonic antigen (CEA), which belongs to the group of the safest shared antigens to be targeted for tumor vaccination with a minimal risk of induction of autoimmunity and the highest chances of efficacy. Indeed, immunization of CEA transgenic mice (expressing human CEA) with a recombinant vaccinia-CEA virus induced protective immunity against CEA-expressing colon adenocarcinoma cells with no apparent autoimmune response against CEA-positive normal tissue (intestinal tract, esophagus, stomach) (Kass et al., 1999). In this murine transgenic model, the expression of CEA is very similar to that seen in normal human tissues. More precisely, CEA has been detected in the gastrointestinal tract (with the highest expression in the colon) and at even lower intensity in the epithelium of the esophagus, trachea, lung, prostate, testes, uterus, and breasts (Eades-Perner and Zimmermann, 1995; Zhang et al., 1998). Since CEA can be detected in normal human tissues, the potential induction of an autoimmune response against normal CEA-expressing tissues is

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usually assessed when developing a CEA-based cancer vaccine. It should be noted that CEA has been extensively studied in human subjects with no evidence of untoward autoimmune reaction (Hörig et al., 2000; Marshall et al., 2000; Knutson et al., 2001; Conry et al., 2002; Disis et al., 2002, 2004). A cancer antigen more recently targeted for a cancer vaccine is the human telomerase reverse transcriptase (hTERT). The protein component of hTERT is considered a very attractive tumor antigen for the immunotherapy of cancer. Telomerase is a ribonucleoprotein in which the protein component, hTERT, uses its RNA as template for addition of telomeric repeat sequences to the ends of chromosomes. hTERT is shut down in most somatic tissues but reactivated in the 85% of tumors during the oncogenic transformation process (Shay and Wright, 2002). The expression of telomerase is essential to the transformation process by permitting unlimited replicative potential to tumor cells. Several groups have shown that it is possible to obtain in vitro human CD8 T cells against hTERT, and that these T cells are able to kill in vitro a variety of tumor cell lines. Moreover, more recently the possibility of breaking tolerance and inducing an anti-hTERT cellular-mediated immune response has been demonstrated in nongenetic hTERT-based vaccine clinical trials (Frolkis et al., 2003; Vonderheide et al., 2004; Huo et al., 2006). Additionally, there was no evidence of untoward autoimmune reactions associated with vaccination with hTERT (Vonderheide et al., 2004; Danet-Desnoyers et al., 2005; Su et al., 2005). In conclusion, therapeutic cancer vaccines carry the potential risk of inducing pathological autoimmune reactions based on the fact that they rely on inducing a response against a self-antigen through the breaking of tolerance. Studies have shown that the choice of the tumor antigen for vaccination is critical in preventing these types of reactions. When targeting antigens of choice (i.e., present in immuno-privileged sites, or are fetal/embryonic antigens re-expressed in cancer cells, but normally not expressed or expressed at low levels in adult tissues), pathological autoimmune responses were observed only infrequently, and in such cases were generally well tolerated. Given the positive therapeutic effects that cancer vaccines promise, the induction of mild adverse effects related to autoimmune response in cancer patients is likely to be outweighed by its benefits.

SUMMARY The described three cases address stimulation of the immune system by vaccines and/or adjuvants that could theoretically lead to adverse effects, including the potential induction of a systemic inflammatory response by new molecular adjuvants, T cell-mediated cytotoxicity by therapeutic vaccines, and autoimmunity by therapeutic cancer vaccines. In each case, the risk can be evaluated via appropriate assessments in nonclinical toxicity studies and by considerations of the relevance of the animal models to humans.

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PART VIII TESTING FOR DRUG HYPERSENSITIVITY

8.1 SYSTEMIC HYPERSENSITIVITY Raymond Pieters

Hypersensitivity is defined as a condition in which a defined stimulus causes objectively reproducible symptoms at a dose that is tolerated by normal (most) subjects (Johansson, 2001). Accordingly, hypersensitivity includes both allergic and non-allergic hypersensitivity and hence both immune and nonimmune mechanisms. Here hypersensitivity is used in the context of adverse hyper-reactive immunological condition (both true and pseudo allergy) that may result in a clinical condition upon administration of a therapeutic drug. This chapter is focused on approaches for preclinical testing of immunemediated hypersensitivity, in particular systemic hypersensitivity. It may result from parenteral and oral, but also from topical (Stoney et al., 2004) and inhalatory exposure. Other terms used in this context are drug hypersensitivity, defined as an immune-mediated response to a drug agent in a sensitized patient, and drug allergy, by some restricted to IgE-mediated responses (Riedl and Casillas, 2003). For many years, allergic responses to drugs have also been classified according to the Gell and Coombs classification, and this has now been further subclassified for skin manifestations, based on recent findings (Lerch and Pichler, 2004). Drug hypersensitivity responses lacking a (proven) role of adaptive immune responses are called pseudo-allergic reactions. Pseudo-allergy may comprise up to 77% of all hypersensitivity reactions (Demoly et al., 1999). In general, drug hypersensitivity reactions are relatively rare (occurrence 1 in 1000 to 1 in 10,000 patients). However, some drugs cause immune-related


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drug reactions in 5–10% of patients (Demoly and Bousquet, 2001; Alvestad et al., 2007). In addition, because a wide variety of different pharmaceuticals can cause these effects and some of these drugs are so widely used the occurrence of these reactions is a serious concern to physicians, regulators (such as US-FDA, European EMEA, Japanese PMDA), and pharmaceutical companies (Adkinson et al., 2002). The FDA recently reported a marked increase (3 to 4 times) in reported deaths and serious injuries associated with drug therapy over the period 1998–2005 (Moore et al., 2007). Although they did not clearly distinguish immune-mediated causes, it was stated that 4 out of the 15 most frequently named drugs had primary effects on the immune system. Conceivably, early withdrawal from the market due to drug hypersensitivity results in loss of large investments of pharmaceutical companies. Adverse hypersensitivity may start 1 to 8 weeks after start of drug use and clinically display as anaphylaxis, fever, rash and various cutaneous reactions (exanthema’s, bullous responses, etc.), blood dyscrasias and involve multiple internal organs, including liver, kidney and lungs (Sullivan and Shear, 2001; Gomes and Demoly, 2005). Occasionally drug hypersensitivity results in very serious and life-threatening conditions, such as Steven Johnsons Syndrome (SJS) and toxic epidermal necrolysis (TEN). Drug-induced hypersensitivity reactions are sometimes compared with a graft-versus-host disease (GVHD) in which a similar clinical pattern occurs and also multiple organs are affected. As for GVHD drug-specific hypersensitivity responses may spread to encompass hypersensitivity responses to self-molecules (e.g. DNA, collagen), and become autoimmune disease-like. Intriguingly, drug hypersensitivity symptoms may also deteriorate or even start after cessation of drug use. The reason for this latter effect is unknown but can be due to immunological mechanisms such as cross-reactivity, or/and immunoregulatory phenomena (Depta and Pichler, 2003; Shiohara et al., 2006, 2007). Because of its partly idiosyncratic nature, systemic drug hypersensitivity is often regarded as unpredictable or at best poorly predictable. One could however also argue that its unpredictability is rather due to a lack of mechanistic understanding as to how several not yet fully defined inherent and unexpected environmental factors interplay with complex immune systemrelated processes. Among the predisposing inherent factors MHC-haplotype, metabolic polymorphisms, and gender are considered most important, whereas environmental factors include ongoing infections or diseases, and nutritional factors (Riedl and Casillas, 2003). In particular, interactions with virus infections have received much attention in this context (Wong and Shear, 2004; Shiohara et al., 2006, 2007). Predisposing for pseudo-allergic responses are atopic diseases, in which patients may have high number of mast cells or basophils that can be directly triggered for instance by NSAIDs (Fahrenholz, 2003).

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EVALUATION OF HYPERSENSITIVITY POTENTIAL IN DRUG DEVELOPMENT It is important to note that at present no validated preclinical models are available to predict a drug’s potential to sensitize the immune system, let alone the potential to predict any of the clinical appearances of drug hypersensitivity. One of the reasons that systemic hypersensitivity responses (including clinical outcomes) are not easily detected, or else rather late, in the preclinical phases of drug development is because the etiology of disease resulting from drug hypersensitivity is so complex. In addition, generally accepted toxicity studies (such as 28-day toxicity studies) are not designed to detect exceptional and genetically determined adverse outcomes, in particular because outbred strains of animals are used. Some inbred strains of rats (i.e., Brown Norway rat) or mice (autoimmune-prone animals) may be well suited as starting point to develop disease models (see Chapter 5.2), but probably one model is sufficient to predict all possible clinical outcomes of drug hypersensitivity or predict these outcomes for all drugs. The FDA guidelines for preclinical testing recommend using topical exposure or inhalation exposure models before human studies are done, and it also is recommended that any sign of potential drug hypersensitive potential (e.g., activation of immune cells, indications of an inflammation, but also depletions of certain blood leukocytes) should be an alert for further evaluation (Bala et al., 2005). The general approach in pharmaceutical industry that is currently used includes all kinds of rationalistic methods to “de-risk” compounds in development (structural alerts, reactive metabolite screens, covalent binding assays) or straightforward animal models such as local lymph node assays. These de-risking methods are based on existing mechanistic knowledge on sensitization by low-molecular-weight drugs. Mechanistic Background on Immunization by Low-Molecular-Weight Compounds Fundamental immunological processes described for immunization to compounds via topical or respiratory routes of exposure to low-molecular-weight compounds apply to a large extent also to drug-induced systemic hypersensitivity reactions (Griem et al., 1998). Mechanistic steps include processes such as bioactivation, conjugation to proteins resulting in hapten-carrier formation, non-covalent interactions, induction of adjuvant signals, antigen presentation to lymphocytes, activation of (innate) effector mechanisms, and modulation of immune responses (Figure 8.1-1). Over the past years, three non-mutually exclusive hypotheses or concepts have been presented in literature: the hapten hypothesis, the danger hypothesis, and the p-i-concept. The hapten-hypothesis proposes that sensitizing low molecular weight (i.e. too small to be recognized by T cells directly) drugs conjugate to proteins that

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Haptenization

protein-X

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antigen processing

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X

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Clinical effects CK

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Effector cells e.g. B cell/Cytotoxic T Lymphocyte/ Inflammatory cells (Mast-cells, Eosinophils Neutrophils, Macrophages)

CK, cytokines LMWC, low molecular weight compound (X) PRR, pattern recognition receptors

Figure 8.1-1 Simplified view of how low-molecular-weight compounds (LMWC, i.e., a drug designated as compound X) may induce sensitization of T cells and subsequent activation of effector mechanisms. Compound X may: (i) conjugate to proteins or MHC-peptide complexes (hapten hypothesis); (ii) bind to pattern recognition receptor (PRR), and cause cell stress or death, or interact with a signal pathway, resulting in up-regulation of co-stimulatory signals (receptors and/or cytokines) (danger hypothesis); or (iii) bind directly (non-covalently) to T cell receptor (TCR) or MHC and activate T cells (p-i-concept). Compounds may also interact with regulatory mechanisms (Tregs activation or inhibition) or with effector cells such as mast cells. See text for additional information.

subsequently will be presented to T cells in the context of MHC. It is also shown that compounds directly bind to MHC molecules (previously referred to as altered-self hypothesis). This hapten-hypothesis implies that compounds need to chemically interact with a protein and therefore often need to be bioactivated, for instance by hepatocytes, keratinocytes, or neutrophils. An extension of the hapten hypothesis is the release of so-called cryptic epitopes, i.e., parts of self-proteins that are normally not presented to the immune system and to which no tolerance exists. Chemical modification may cause altered processing of proteins and presentation of such neo-epitopes (Griem et al., 1998). Based on recent concepts that immune responses are triggered in response to dangerous insults instead of to nonself-infectious agents (Matzinger, 1994, 2002), the danger hypothesis has been brought forward for chemical allergens as well

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(Seguin and Uetrecht, 2003). This hypothesis states that a sensitizing chemical activates the innate immune system so that co-stimulatory adjuvant signals are provided to adaptive immune cells. Innate immune activation can result from a direct or an indirect effect on innate immune cells such as dendritic cells (DCs). Direct DC activation may for instance result from ligation of innate receptors on these cells (also named pattern recognition receptors, PRRs) such as Toll-like receptors (TLRs). Imidazoquinolones (imiquod, R-848) are well-known examples of compounds that can directly activate DC via TLR, in this case TLR7. Interestingly in view of the hapten-hypothesis which may involve chemical reactivity, stressed cells (e.g., by induction of oxidative stress [Mizuashi et al., 2005]) or cell remnants (either from apoptotic [Janssen et al., 2006] or necrotic cells [Gallucci et al., 1999; Shi et al., 2003]) also stimulate DC to up-regulate co-stimulatory signals. So, reactive compounds that can form hapten-carrier conjugates additionally may indirectly stimulate DC by causing cell toxicity. The importance of the danger (or adjuvant) hypothesis extends also to modulation of ongoing responses, as illustrated for instance by the finding that poly I:C (synthetic viral double-stranded RNA) or LPS can increase autoimmune responses induced by D-penicillamine in Brown Norway rats (Sayeh and Uetrecht, 2001) or by HgCl2 in DBA mice (Abedi-Valugerdi et al., 2005), respectively. The p-i-concept is based on recent findings that provided indications that induction of drug-specific responses not always requires covalent binding and antigen processing, but that chemically inert drugs can directly link TcR and MHC (through hydrogen bonds or van der Waals interactions) and thus activate T cells. The corollary of the p-i concept is that drugs may stimulate T cells without the requirement of metabolic activation, co-stimulation and subsequent hapten-carrier formation (Pichler, 2002). The hapten and danger hypotheses are linked by the general immunological concept that T cell sensitization relies on an epitope-specific signal (TcR-MHC interaction, leading to signal activation through TcR, also called signal 1) and on sufficient adjuvant signals (resulting in proper co-stimulatory signals, or signal 2) (Weaver et al., 2008). The p-i concept is based on the existence of specific, possibly cross-reactive, T cells and therefore less dependent on costimulation at the time of drug exposure. However, based on recent studies (Nierkens et al., 2002, 2005a; Sanderson et al., 2007) and the hapten-danger concept, signal 2 (in particular co-stimulatory receptors such as CD40, CD80, and CD86) may in many instances be regarded as the decisive signal for the initiation of drug sensitization. A mechanism distinct from the above-mentioned involves drug interference at central tolerance level in the thymus. The particular example that supports this mechanism shows that intrathymic injection of a reactive metabolite of procainamide, procainamide-hydroxylamine (PAHA), causes the appearance of chromatin-specific T cells in the spleen (Kretz-Rommel et al., 1997; Kretz-Rommel and Rubin, 1999). Mechanisms of pseudo-allergy or anaphylactoid reactions do not involve adaptive immunity, but rather innate effector components such as complement

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and mast cells or basophils. The pharmacological effect of the drug may be crucial for the adverse effects, for instance in the case of aspirin and NSAIDs that interfere directly with leukotriene synthesis in mast cells. Radiocontrast media may also directly activate mast cells, or the complement cascade (e.g., C3a and C5a) (Szebeni, 2005). Interaction with Regulatory Mechanisms Innate as well as adaptive immune processes are continuously subjected to all kinds of regulatory mechanisms. During the last 5–10 years, in particular regulatory T cells (Tregs), as well as dendritic cells, have received much (partly renewed) attention. Various subsets of regulatory T or T-like cells (Bach, 2003) can be identified and certain DCs (Thompson and Thomas, 2002) have been found to be in control of these Tregs. These regulatory mechanisms are known to have a profound role in oral tolerance induction, and hence are expected to be involved in oral exposures to drugs as well. Their relevance to hypersensitivity responses to low-molecular-weight drugs and other compounds can be assessed by transfer studies or by depletion or specific deactivation (e.g., by using monoclonal antibodies). One clear example showing that regulatory mechanisms are important comes from studies on D-penicillamine-induced adverse effects in Brown Norway (BN) rats. The antirheumatic drug D-penicillamine induces autoimmune-like effects in BN rats that resemble those observed in patients undergoing adverse effects (Tournade et al., 1990). Phenomena include transient increases in T and B cell numbers, enhanced serum IgE levels, IgG deposition in glomeruli, and skin reactions. Masson and Uetrecht (2004) found that only 60–80% of all treated BN rats develop the autoimmune disease at 20 mg/day via drinking water. Interestingly, low-dose pretreatment (5 mg/day, for 14 days) with the D-penicillamine protected animals to subsequent autoimmunogenic doses (Donker et al., 1984; Masson and Uetrecht, 2004). The transience of the adverse effects and the low-dose tolerance appeared to be mediated by IFNγ-producing CD8 cells. In addition, low doses of D-penicillamine appeared to stimulate the formation of TGF-β- and IL-10-expressing CD4+ and CD8+ cells. But depletion of macrophages also inhibited tolerance induction and transfer studies with non-T cell fractions of tolerant animals appeared to confer tolerance to naive animals as well (Seguin et al., 2004). So, tolerance in BN rats to low doses of D-penicillamine appears to result from a complex mechanism including various T cell subsets but also non-T cells, possibly antigenpresenting cells. In mouse studies, blockade of a CTLA-4, which is a regulatory molecule that is constitutively expressed on some regulatory T cells, caused an increased production of IgE antibodies in response to D-penicillamine (Nierkens et al., 2005a). This mouse study was done using the RA-PLNA (see also later), indicating that this assay may be used to study tolerance mechanisms as well. Gutting et al. (2003) have also used the PLNA to investigate oral tolerance to diclofenac and oxazolone.

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Although mechanisms are discussed here more or less separately, it should be stressed that a single allergenic drug may act via multiple mechanisms and on multiple stages during the onset or progression of an immune response. All kinds of other drug-targeted processes that may result in immunomodulatory effects, for instance related to neuroendocrine system, are not even considered here. Possible Alerts of Hypersensitivity Induction Based on the above-mentioned theoretical considerations, possible alerts can be defined to flag a drug as potentially immunogenic and possibly allergenic. In Figure 8.1-2, alerts are incorporated in a multistep tiered approach that may be useful to investigate preclinically whether a newly synthesized molecular entity has the potential hazard to induce systemic hypersensitivity. Drugs with Potential to be Metabolized and Form Hapten-Carrier Conjugates. The importance of bioactivation of certain drugs, which are regarded as so-called prohaptens, is extensively reviewed (see Riley and Leeder, 1995; Park et al., 2005). Metabolism of a new chemical entity is generally determined in conjunction with data on ADME (absorption, distribution, metabolism, and excretion). In toxicological literature, many methods have been described to assess metabolic conversion by intestines, liver, skin, or leukocytes, and these include isolated cells and enzymes, but also tissue slices. Bioactivation of procainamide has been studied using activated human neutrophils (Rubin and Curnutte, 1989) and the same has been done for

Tiered Strategy to Hazard Identification of IDHR Information on chemical structure, metabolism, (non-)covalent binding and potency to induced cell stress/death, basophil activation test, stimulation of patients’ T cell clones Any sign of inflammation, hyperplasia, dyscrasias (in general 28 day tox-study)

Initial Screening in Local Lymph Node Tests RA-PLNA, ear-injection, LNPA focus on sensitization potential

Assessment of Systemic Hypersensitization oral sensitization models (e.g., with RA)-clinical disease models (e.g., BN rat)

Figure 8.1-2 Mechanism-based translational approach composed of preclinical tests that may help to assess potential risk of immune-mediated drug hypersensitivity reaction (IDHR).

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propylthiouracil using PMA-activated neutrophil or a mixture of MPO/H2O2/ Cl– (to mimic leukocyte-mediated metabolism) (Waldhauser and Uetrecht, 1991). The interesting aspect of using leukocyte-mediated metabolism of compounds is that it may link metabolic activation directly to the sites where inflammation may take place. Many methods (including measurements of metabolic enzymes such as P450, various transferases, etc.) have been described to further study metabolism of compounds (see for instance Bus et al., 2006). Closely linked to bioactivation is the formation of hapten-carrier complexes. Generally the binding capacity of reactive compounds to model carrier peptides or nucleophilic amino acids is determined. For this also a number of assays are available for instance using HPLC or LC, in combination with MS or NMR (Singh et al., 2004; Ahlfors et al., 2005). If the new chemical can be radiolabeled, binding to peptides can be easily detected. In cases where specific antibodies to the compound or to structurally related compounds are available, immunochemistry techniques such as Western blotting or even immunohistology can be applied. Drugs with Potential to Activate According to the P-I Concept. To assess whether a new drug candidate can activate T cells according to the p-i concept, a battery of T-cell clones derived from drug allergic patients may be used in so-called lymphocyte transformation tests (LTTs) (Beeler and Pichler, 2007). Since the affinity involved in these interactions is very low, LTTs would also allow identifying cross-reactivity. Drugs with Potential to Activate Dendritic Cells and Act as Adjuvant. DC cultures from different species (human, mice) are already in development to assess of allergic potential of contact allergens (Ryan et al., 2007). Focus is on expression of co-stimulatory molecules (CD80, CD86, CD40) and production of cytokines (Type 1 IFN, IL-10, IL-12), or stress signals (oxidative stress, NFκB activation, etc.). Cells stress, mild cell death (including apoptosis) or necrosis, for instance caused by newly formed reactive drug derivatives, may add to adjuvant potential of the drug. A recent study by Sanderson et al. (2007) shows the optimal use of DC cultures to address mechanisms of sulfamethoxazole-induced immunosensitization, including DC-mediated bioactivation and up-regulation of co-stimulatory signals. Drugs with Potential to Activate Basophils. Drug-induced stimulation of basophils can be assessed in vitro for instance using the flow cytometric basophile activation test (BAT), which uses surface expression of CD63 or CD203 as signatures of degranulation and/or activation (reviewed in Sanz et al., 2007). This test may be in particular useful to determine whether a compound can directly activate basophils and hence induce pseudo-allergy. In case the test would be used in preclinical testing, of which no data are available

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in literature, preloading of basophils with specific IgE and relevant antigen may be required. Local Lymph Node Assays The popliteal lymph node assay (PLNA) is an assay that is often used to assess whether a drug can induce sensitization. The assay basically determines changes in the paw-draining lymph node induced by subcutaneous injection of a chemical into the hind footpad and hence is a straightforward, robust and fast (the assay lasts 6 to 8 days) test that links lymph node reactions directly to the injection of a compound of interest. The PLNA is mostly performed in mice (Bloksma et al., 1995; Goebel et al., 1996) but also rats (Descotes, 1992) have been used. The PLNA can be easily modified in order to answer specific questions, and in particular immune system-related parameters such as leukocyte characterization, cytokine production (Choquet-Kastylevsky and Descotes, 2004), immunohistology (De Bakker et al., 1990), and responses to co-injected bystander antigens (also called reporter antigens, RA, see later) have been demonstrated to improve to predictive value of the assay. To assess T cell specificity, transfer studies have been done, i.e., spleen cells from sensitized mice have been adoptively transferred to naive recipient mice that subsequently received a paw injection of a non-sensitizing challenge dose of the same or a related compound (e.g., a metabolite). Metabolizing systems (S9 mixes [Patriarca et al., 1993], neutrophils, peritoneal macrophages [KubickaMuranyi et al., 1993; Goebel et al., 1995, 1999]) have also been used in conjunction with the PLNA to assess the role of bioactivation. The most recent modification of the PLNA, the RA-PLNA, was designed to assess immunosensitizing versus adjuvant potential (Albers et al., 1997; Gutting et al., 1999). The specific antibody-forming cells (AFCs) to RA can be determined by ELISPOT-assay and the specific immune requirements needed for an antibody response to the particular RA is indicative of the mechanism by which a compound may cause T-cell sensitization. For instance, when a compound is co-injected together with TNP-Ficoll, which is a T cellindependent antigen that is susceptible to neo-antigen-specific T cell help, an increase of TNP-specific AFC of the IgG isotype indicates that the compound induces T-cell sensitization. When a regular T cell-dependent antigen like TNP-OVA is used, increases in the number of TNP-specific IgG-AFC indicate that the chemical has at least adjuvant activity (but not necessarily the ability to stimulate neo-antigen-specific T cells). In all, this immunology-based readout parameter (RA response) improves the predictability of the PLNA and can be used to further study fundamental aspects of chemical-induced immune effects (Albers et al., 1998; Nierkens et al., 2002, 2005c). Over 130 compounds, including a substantial number of structural homologues of phenythoin (around 50) (Kammüller and Seinen, 1988) and zimeldine (around 10) (Thomas et al., 1990), have been tested in the PLNA up till now.

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Importantly, chemicals with known immunostimulating activity in man were detected correctly (Pieters and Albers, 1999). However, compounds (such as procainamide) that require metabolic conversion turned out to be false negative unless metabolic systems (see above) were added as well (Patriarca et al., 1993). Alternatively, metabolites have been tested and appeared positive, whereas their precursor compounds were negative (Goebel et al., 1995; Popovic et al., 2004). Recently, a prevalidation study (Weaver et al., 2005) was carried out to evaluate the predictive value of local lymph node approach for the immunosensitizing potential of drugs. Since footpad injection sometimes induces more or less severe paw inflammation, the use of the PLNA may raise ethical concerns. Therefore, the prevalidation study was done using subcutaneous injection on the head combined with local lymph node effects. The head injection protocol (designated lymph node proliferation assay, or LNPA) showed that of the 11 drugs tested, 7 were detected correctly (5 as positive and 2 as negative). Of the 4 compounds that were false negative, 2 are known to require metabolic activation; the other 2 were dose-limited due to toxicity. In a study using the RA protocol with TNP-Ficoll as RA, ear injection and subsequent detection of specific antibody formation with a range of eight pharmaceuticals showed comparable results with those previously obtained in the footpad RAPLNA (Nierkens et al., 2004). Over the years, the PLNA has provided much basic knowledge with regard to mechanisms of drug-induced sensitization and is a very useful preclinical screening assay for the assessment of sensitizing potential of candidate drugs. However, subcutaneous exposure is an irrelevant route with regard to normal human intake of pharmaceuticals and therefore limits the use of PLNA outcomes as stand-alone data in further risk assessment. But, PLNA outcomes may be very useful as first alerts to perform further animal testing, for instance using oral exposure models. Oral and Other Systemic Exposure Models Oral or systemic exposure studies with sensitizing drugs are scarce in literature. Only a few drugs have been studied more (D-penicillamine, procainamide, nevirapine) or less (diphenylhydantoin) extensively. D-penicillamine has been shown to induce anti-ssDNA and anti-insulin antibodies in C57BL/Ks (H2d) and C3H/He (H2k) but not in BALB/c (H2d) or C57BL/6 (H2b) mice after subcutaneous exposure for 4 weeks (Brik et al., 1995). Also after oral treatment (for 7 to 8 months, in the drinking water) D-penicillamine (as well as quinidine) resulted in increased levels of autoantibodies in A.SW/Sn (H2s) mice (Monestier et al., 1994). Administration of diphenylhydantoin (via drinking water for 6 months) to genetically predisposed mice (C57BL/6-lpr/lpr strain) rather depressed the levels of anti-nuclear antibodies (ANA) (Bloksma et al., 1994). In another study (Okada et al., 2001), the effect of a 4-week exposure to diphenylhydantoin on KLH sensitization was studied. It was found that the drug caused an

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increase in KLH-specific IgE serum levels and production of IL-4 by splenocytes, whereas the IFN-γ production by spleen cells was reduced. Proliferative responses of spleen cells to KLH, mitogens (ConA, LPS) or anti-CD3, as well as NK cell activity, were also reduced. Function of adherent cells (production of IL1-β) isolated from spleen was also lowered by diphenylhydantoin treatment. Procainamide has been found to induce an increase in ANA in A/J mice after 8 months of exposure via the drinking water (Layland et al., 2004). This increase appeared to be mediated by CD25−CD4+ T cells and regulated by CD25+CD4+ Tregs. A recent study was aimed at applying RA in systemic exposure models. These studies demonstrated that oral exposure to D-penicillamine or diclofenac and intraperitoneal exposure to nevirapine stimulated the responses to systemically applied TNP-OVA (Nierkens et al., 2005b). Moreover, oral exposures to D-penicillamine (Nierkens et al., 2005b) and diclofenac (Gutting et al., 2002; Nierkens et al., 2005b) have also been found to stimulate compound-specific anamnestic responses that upon challenge with non-stimulating doses of the compounds facilitated non-cognate stimulation of IgG1 production to TNP-Ficoll. This is indicative of induction of neo-antigen (possibly compound-specific) T cell responses. So, the RA approach, including local readout of immune responses, may be useful in combination with oral drug exposures. Rats, in particular BN rats, have been frequently used to study drug hypersensitivity, but again to only very limited number of drugs (Balazs, 1987). Dpenicillamine has been studied most extensively in BN rats (Donker et al., 1984; Tournade et al., 1990; Seguin et al., 2003, 2004; Masson and Uetrecht, 2004). Recently, nevirapine has been found to cause skin rash in 100% of highexposed (150 mg/kg by oral gavage) female BN rats (Shenton et al., 2003). Female Sprague-Dawley rats were less sensitive (21% of rats showed a rash), and male BN, Sprague-Dawley rats, and female Lewis rats were resistant. Nevirapine-induced disease was shown to be immune-mediated because upon rechallenge with nevirapine, the rash developed faster in previously exposed rats, and because skin reactions were transferable by splenocytes from nevirapine-treated to naive animals (Shenton et al., 2003).

SUMMARY Preclinical validated models to assess drug hypersensitivity do not exist at the time. Considering the complex multifactorial etiology of immune-mediated adverse drug reactions and the limited efforts to study this aspect of immunotoxicology, this is not so surprising. However, although knowledge on the mechanisms is limited, based on the information that exists, a rationalistic set of tests can be used to obtain alerts for further testing or increase cautiousness in drug evaluation. This set of tests could be used in a tiered approach that

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8.2 NONCLINICAL MODELS TO ASSESS RESPIRATORY HYPERSENSITIVITY POTENTIAL Curtis C. Maier

Drugs that are intended for delivery by the inhalation route may require an evaluation for their potential to induce respiratory hypersensitivity. This is generally considered under guidance for local tolerance testing, but has also been explicitly requested in the FDA Guidance Document on Immunotoxicology Evaluation of Investigational New Drugs (Center for Drug Evaluation and Research, 2002) and publications from the Division of Pulmonary Drug Products of the FDA (DeGeorge et al., 1997). Of particular concern are drugs that have the potential to elicit immediate-type hypersensitivity reactions and allergic asthma; however, delayed-type hypersensitivity reactions in the lung can also lead to serious adverse pathophysiology (hypersensitivity pneumonitis). Since a standardized validated nonclinical model for evaluating respiratory hypersensitivity potential of drugs does not exist, stepwise approaches borrowed from the field of occupational asthma testing are being employed to identify and characterize risk. In general, the most common strategy for pharmaceuticals is to first identify immune sensitization hazard potential using tests validated for contact hypersensitivity testing (e.g., local lymph node assay). If the test article is positive in a skin sensitization test, then it can be considered to have the necessary chemical attributes to elicit an immune response, though it may not necessarily produce pulmonary allergies in patients. The route and level of exposure are critical factors in induction of respiratory


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hypersensitivity and positive skin sensitizers may be subsequently tested in a nonclinical inhalation challenge model to better characterize potential risk to humans. This chapter will focus primarily on models for testing allergenicity potential of low-molecular-weight drugs rather than biotechnology-derived pharmaceuticals since immune responses to recombinant human therapeutic proteins in preclinical animal species are generally not predictive of responses in humans (Bugelski and Treacy, 2004).

RESPIRATORY ALLERGIC REACTIONS Respiratory hypersensitivity reactions are adverse immune reactions generally localized to the airways or alveolar interstitium that are provoked by inhaled allergens. The terms hypersensitivity and allergy are used interchangeably in this chapter and imply a latency period of subclinical immune sensitization followed by a robust immunopathologic reaction elicited upon re-exposure to the sensitizing agent. The concern for the potential to develop respiratory hypersensitivity to inhaled drugs is inferred from allergic occupational respiratory diseases. In the workplace, allergic asthma and hypersensitivity pneumonitis are serious respiratory hypersensitivity diseases associated with high levels of morbidity that can develop following repeated exposure to respiratory allergens, including pharmaceutical products such as beta lactam antibiotics (Beckett, 2000). Over 300 low-molecular-weight (LMW) and high-molecular-weight (HMW) agents have been identified that cause immunologic occupational asthma (Chan-Yeung and Malo, 1994; Mapp et al., 2005). Some of the most common agents include isocyanates, acid anhydrides, aldehydes, certain metals and reactive dyes, commercial enzymes, animal products, as well as latex and dusts from flour and grains. For HMW agents (arbitrarily >5000 daltons), asthma is generally induced by an antigen-specific IgE-dependent mechanism. Specific IgE antibodies have also been detected against certain LMW agents, such as trimellitic anhydride and platinum salts (Pepys et al., 1972; Zeiss et al., 1977). While the presence of allergen-specific IgE antibodies does not always accompany respiratory disease, they have generally been helpful in diagnostic tests for occupational asthma (Baur and Czuppon, 1995). For other LMW agents, such as diisocyanates or plicatic acid, specific IgE antibodies are not consistently detected and there is a poor association with disease status (Cullinan, 1998; Bernstein et al., 2002; Sastre et al., 2003). However, for all known LMW respiratory allergens, specific IgE has been detected in at least some symptomatic patients. The lack of detectable IgE in other patients may be associated with technical limitations, in that inappropriate hapten conjugates may have been used in detection assays or possibly screening was conducted after specific IgE had cleared (Kimber and Dearman, 2002). LMW occupational respiratory allergens have highly reactive electrophilic groups that can covalently couple with hydroxyl, amino, or thiol functional

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groups on host macromolecules (e.g., proteins) to create haptens in an antigenspecific immune response (Agius et al., 1991; Karol, 2001). In the induction phase of sensitizing the immune system, these haptenated proteins are taken up by professional antigen-presenting cells (APCs), such as dendritic cells, which then migrate to lung draining lymph nodes and present the epitopes via MHC class II to naive T cells. A number of factors, including differential expression of co-stimulatory molecules and regulatory cytokines, contribute to the differentiation of Th1 or Th2 effector cell phenotypes that orchestrate the immune-mediated process, eventually resulting in pathological inflammatory respiratory diseases during the elicitation phase. In the case of allergic asthma, it is generally presumed that the T cell response is polarized toward a Th2 phenotype, producing IL-4, IL-5, and IL-13, which promotes B cell IgE production, mast cell growth, and eosinophilia (Mapp et al., 2005). Upon subsequent antigen exposure, IgE bound to mast cells is cross-linked and induces the release of preformed mediators (e.g., histamine and proteases). These mediators initiate an immediate phase reaction of bronchoconstriction, airway edema, and mucus secretion. Mast cells also synthesize and release leukotrienes, prostaglandins, and cytokines (e.g., TNFα and IL-5) that produce a late-phase airway inflammation response several hours later characterized by infiltration of lymphocytes, neutrophils, monocytes, and eosinophils. The eosinophils release major basic protein and eosinophil cationic protein, in addition to other toxic mediators damaging airway epithelium and causing airway constriction. The infiltrating lymphocytes have an activated status and perpetuate the inflammatory response by secreting additional cytokines and chemokines. Prolonged repeated allergic reactions can result in airway wall remodeling, characterized by hypertrophy of smooth muscles, goblet cell hyperplasia, and thickening of basement membranes. Allergic asthma is diagnosed by measuring airflow obstruction (e.g., peak expiratory flow) or bronchial hyper-responsiveness upon nonspecific challenge (e.g., methacholine). Airway hyper-responsiveness can also be induced by IL13 independently of IgE and eosinophilia (Kay, 2006). Bronchoconstriction or changes in breathing patterns can also be induced by high concentrations of chemical irritants, and these nonimmunologic asthmatic reactions are distinguished from allergic asthma by the absence of a latency (sensitization) period. Another serious occupational allergic respiratory disease is hypersensitivity pneumonitis, also known as extrinsic allergic alveolitis, which is characterized by inflammation in the alveoli, conducting bronchioles and lung interstitium (Mohr, 2004). The interstitial inflammatory cell infiltrates are predominantly lymphocytes and macrophages. Poorly formed, noncaseating granulomas and multinucleated giant cells are frequently present. The disease is progressive and chronic forms can result in debilitating pulmonary fibrosis. Unlike allergic asthma, hypersensitivity pneumonitis is not associated with IgE or eosinophilia, but instead is generally considered a type IV (delayed-type) cellmediated allergic reaction that is dependent on IFN-γ (Gudmundsson and

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Hunninghake, 1997). Upon allergen challenge, neutrophils are initially recruited to the alveoli, followed by lymphocytes, predominantly CD8+ T cells. Antigen-specific IgG antibodies may also contribute to the acute inflammatory reaction. Occupational allergens that induce hypersensitivity pneumonitis overlap with those inducing allergic asthma (e.g., diisocyanates, trimellitic anhydride) but also include a number of animal products and microorganisms not associated with allergic asthma, such as fungal spores, moldy hay, and bird droppings (Beckett, 2000).

RISK FACTORS The induction of respiratory hypersensitivity depends on many factors inherent to the chemical structure of the allergen and exposure, including route, aerosol concentrations, frequency, duration and lung deposition, as well as host genetic and health factors (Baur et al., 1998; Arts et al., 2006). As mentioned above, occupational LMW respiratory allergens are highly reactive chemicals that haptenate host proteins and hypersensitize the immune system, but not all reactive chemicals are allergens presumably because either they do not engage the immune system or they induce immunotolerance. Furthermore, many reactive chemicals that are immune sensitizers produce contact hypersensitivity, such as dinitrochlorobenzene (DNCB) or oxazolone, but do not cause respiratory hypersensitivity (Botham et al., 1989; Dearman and Kimber, 1991; Blaikie et al., 1995; Arts et al., 1998). The reason for this is poorly understood but is in part attributed to nature of the T cell response (e.g., Th1, Th2, or regulatory T cells), where haptens that induce Th1, Th2, or Treg responses would likely promote contact allergy, respiratory allergy, and immunotolerance, respectively (Dearman and Kimber, 2001; Hawrylowicz and O’Garra, 2005; Ahern and Robinson, 2007). In addition to chemical reactivity (or perhaps bioactivation), the deposition of the chemical in the airways is also a critical element in immune sensitization and intensity of allergic reactions (Pauluhn et al., 2000). Deposition is associated with particle size and characteristics of the aerosol. Particles that are ≥10 μm in diameter are predominantly deposited in the nose and pharynx with limited deposition in the lower respiratory tract, while particles 1 to 5 μm in diameter can penetrate the lower airways and alveoli, and have the potential to produce respiratory hypersensitivity reactions. Particles smaller than 1 μm often remain suspended in air and most are carried out of the respiratory tract in exhaled air. For LMW agents that are respiratory allergens, airborne concentrations appear to be a very important determinant in influencing severity and type of symptoms (Arts et al., 2006). The tendency to develop allergic asthma was shown to be more dependent on intensity of exposure than on weekly duration in occupational settings (Nieuwenhuijsen et al., 2003). Furthermore, the dose necessary for sensitization appears to be at least 10-fold higher than the dose necessary to provoke an allergic attack in a sensitized individual (Swanson,

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2003). In experimental models of occupational asthma using trimellitic anhydride (TMA) as a prototypical respiratory allergen, Brown Norway (BN) rats produce TMA-specific IgE, and inhalation challenge elicits some pulmonary functional and histological changes characteristic of asthma (early- and latephase airway responses, airway hyper-responsiveness, perivascular and peribronchiolar eosinophilic infiltrates, goblet cell hyperplasia and hypertrophy, and mucous production) (Arts et al., 1998, 2003, 2004; Pauluhn et al., 2002; Zhang et al., 2004). Dose-response relationships of either the sensitizing dose or challenge dose have been evaluated in these TMA models, and concentration-dependent incidence and severity of findings of both pulmonary functional changes and inflammation have been characterized (Pauluhn et al., 2002; Arts et al., 2004; Zhang et al., 2004). Importantly for drug development and risk assessment of hypersensitivity potential, no effect levels (NOELs) have been identified as well. Individual health and genetic predisposition also contributes to the risk of developing respiratory hypersensitivity. Atopy is an important risk factor for allergic asthma to HMW agents, but this does not appear as important for LMW agents (Heederik, 1999). Genetic risk factors that have been associated with occupational asthma include specific HLA alleles and mutations in the glutathione S-transferase (GST) family; however, these associations are poorly characterized to date (Mapp et al., 2005). In preclinical testing, TMA produces very different responses in rats, depending on the strain used. As mentioned above, atopic BN rats develop allergic asthma-like reactions to TMA, while outbred strains, such as Wistar and Sprague-Dawley (SD) rats, which are not prone to atopic IgE reactions, also develop respiratory hypersensitivity reactions to TMA, but the reactions have a different nature. In Wistar rats, the reactions are characterized by laryngeal inflammation and pulmonary hemorrhages, while cell infiltrates in the lung are mononuclear, rather than eosinophils, and specific IgE is not detected (Arts et al., 2004). Furthermore, sensitization-dependent pulmonary functional changes are not detected in Wistar rats. Respiratory hypersensitivity reactions to TMA in SD rats tend to be intermediate between BN and Wistar rats in that there is some specific IgE and eosinophilic infiltration, as well as laryngeal inflammation and pulmonary hemorrhages, but the predominant lung reaction resembles allergic pneumonitis-type reactions, characterized by proliferation of bronchial associated lymphoid tissue (BALT) and lung-draining lymph nodes, and pulmonary histiocytosis accompanied by mononuclear peribronchiolar and perivascular inflammation (Leach et al., 1987; Maier et al., 2006). Presumably the differences in responses to the same chemical allergen, TMA, are related to genetic host factors, though these factors are poorly understood. There appears to be differences in sensitivity to the allergen concentration required to elicit respiratory hypersensitivity reactions in different animal strains or species, but this is very difficult to compare because of considerable differences in experimental designs. The minimal effect TMA atmospheric concentration for eliciting a reaction in sensitized BN rats was 2 to 5 mg/m3

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and the NOEL was 0.2 mg/m3, as either a dry powder aerosol or nebulized solution (mass median aerodynamic diameter [MMAD] between 0.5 to 4 μm) (Arts et al., 2004; Zhang et al., 2004). In Wistar rats, effects were observed at the lowest concentration tested (15 mg/m3) (Arts et al., 2004). In SD rats, effects were seen at concentrations of 0.005 to 0.01 mg/m3 (dry powder aerosol, MMAD 1 to 3 μm) and a NOEL was not identified (Leach et al., 1987; Maier et al., 2006). However, the total challenge dose (concentration × time) was higher in SD rats (1- to 6-hour challenge exposures) than BN and Wistar rats (7 to 10 minutes), so further studies are necessary to clarify relative sensitivities of the different strains. Guinea pigs respond to TMA inhalation challenges with immediate allergic reactions, represented by bronchoconstriction, and eosinophilic infiltration (Botham et al., 1989; Blaikie et al., 1995). Moderate breathing pattern changes (increase in respiration rate and decrease in tidal volume, or rapid, shallow breathing) were observed at challenge concentrations of 7 mg/m3 for 15 minutes, which increased in incidence and severity (rapid decrease in respiration rate to ≤70% of normal background rate) with increasing concentrations (Blaikie et al., 1995). However, a NOEL was not identified and a comparison of sensitivity to rat cannot be determined.

NONCLINICAL MODELS FOR EVALUATING RESPIRATORY HYPERSENSITIVITY POTENTIAL OF INHALATION DRUGS Several LMW pharmaceuticals have been implicated in occupational asthma including beta lactam antibiotics (penicillins and ampicillin), cephalosporins, spiramycin, tetracycline, cimetidine, and methyldopa (Chan-Yeung and Malo, 1994; Sastre and Quirce, 2007). However, there is only anecdotal information on pulmonary allergic reactions in patients treated with inhaled pharmaceuticals (primarily found in product labels), and validated nonclinical models to test respiratory hypersensitivity potential have not been developed. Strategies for determining hypersensitivity potential have focused on hazard identification by measuring sensitization potential or more complex inhalation challenge models to evaluate dose-response relationships. Local Lymph Node Assay for Identifying Sensitization Potential Initially, LMW inhalation drugs can be screened for their ability to sensitize the immune system. A contact sensitization model, the local lymph node assay (LLNA), has been validated for the identification of agents that cause skin sensitization (Kimber et al., 1991). In this model, the test article is prepared in a vehicle, such as olive oil/acetone, and applied to both ears of mice for three consecutive days, and on the sixth day, proliferation in draining lymph node is determined (Gerberick et al., 2000). Test articles that produce a stimulation index greater than three times vehicle control are considered positive contact sensitizers. To date, most if not all LMW respiratory sensitizers tested

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in the LLNA have been positive, so this appears a reasonable approach to screen for sensitizing potential of inhaled LMW drugs (Kimber et al., 1996). Furthermore, respiratory allergic reactions can be elicited by inhalation challenge in animals previously sensitized by topical application, suggesting that this route of initial exposure has some relevance in induction of respiratory allergies (Arts et al., 1998; Pauluhn, 1999; Pauluhn et al., 2002; Vohr et al., 2002; Zhang et al., 2004; Pauluhn and Mohr, 2005). Modifications to the assay to determine immune responses polarized to Th2 or IgE production have shown promising results for distinguishing respiratory allergens from contact allergens; however, the optimal study design and validation of end points is still an active area of research (Potter and Wederbrand, 1995; Hilton et al., 1996; Dearman et al., 1998, 2002; Manetz and Meade, 1999; Dearman and Kimber, 2001; Ulrich et al., 2001; Van Och et al., 2002; Vanoirbeek et al., 2003; Goutet et al., 2005; Selgrade et al., 2007). For drugs intended for delivery via the inhalation route, it is unclear whether these additional end points added to the dermal model are sufficient for human risk assessment. The LLNA is a rapid, relatively cost-effective screen that is recognized as a useful first step in the nonclinical safety evaluation of respiratory hypersensitivity potential (Center for Drug Evaluation and Research, 2002). It has also been recommended that the LLNA be conducted prior to first in human trials (Holsapple et al., 2006). However, because the route of administration is topical rather than inhaled, and because a topical dose inducing a positive signal cannot be extrapolated to inhalation exposures, and because the vehicle is invariably different than that used in the inhaled product, then other studies may be needed to assess risk of hypersensitivity potential of the clinical product. Theoretically, a drug that is positive in a skin sensitization test may not test positive in an inhalation study because the maximum tolerated inhalation dose may fall below the sensitization threshold (e.g., due to irritation at higher concentrations), or the route of exposure and antigen presentation (e.g., Langerhans cells versus dendritic cells or alveolar macrophages) may influence the outcome of the immune response (Tournoy et al., 2002, 2006; Arts et al., 2006). Furthermore, negative skin sensitization tests cannot completely rule out sensitization potential via inhalation due to potential inefficient skin penetration or differences in local metabolism (Weaver et al., 2005). Inhalation Challenge Models for Risk Characterization Inhalation challenge elicitation models can provide the most informative nonclinical data for assessing LMW respiratory hypersensitivity potential. Many different models, mostly using guinea pigs and rats, have been published and reviewed extensively, particularly with the intention of identifying and characterizing agents that induce occupational asthma, but none of these models completely reproduce the pathophysiology of allergic asthma reactions seen in humans exposed to respiratory LMW allergens (Briatico-Vangosa et al., 1994; Karol, 1995; Pauluhn, 1996; Pauluhn and Mohr, 2005; Arts and Kuper,

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2007). Furthermore, there have been no rigorous cross-validation studies conducted, so a standardized nonclinical model for testing hypersensitivity potential of LMW inhalation drugs does not exist. The general features of study designs of inhalation challenge models include a sensitization phase by single or repeated exposures (e.g., up to 14 doses), followed by an off-treatment rest period (approximately 2 weeks), then inhalation challenge exposure to elicit the allergic reaction. This induction/challenge design utilizes test article control groups that are sensitized only or challenged only, as well as vehicle control groups, making for rather large studies. The FDA Guidance on Immunotoxicology Evaluation of Investigational New Drugs (Center for Drug Evaluation and Research, 2002) specifically mentions a guinea pig model described by Karol (1995), in which animals are sensitized during daily inhalation exposures to aerosols on Days 0 to 5 (chamber concentration and particle size are determined), followed by an inhalation challenge on Day 21, and appropriate experimental end points (e.g., plethysmography, drug-specific antibody production) are evaluated. Adaptations to the models are acceptable but should be tested with positive control test articles (e.g., ovalbumin, diisocyanate, or TMA). While several models use topical administration during the sensitization phase, it is recommended that hypersensitivity models for testing investigational drugs use a route of administration intended for clinical use (DeGeorge et al., 1997). This is particularly important when considering how the physical characteristics of the test article, including particle size and excipients, and dose can influence the outcome of induction and elicitation of respiratory hypersensitivity reactions. The species and strains used in inhalation elicitation challenge models are generally selected because they are prone to develop immediate hypersensitivity reactions, though other factors should be considered as well, such as historical experience with the species or strain. Since no model completely recapitulates the pathophysiology of human respiratory diseases, advantages and disadvantages using a particular species or strain to test respiratory hypersensitivity potential must be considered. For example, guinea pigs develop asthma-like early and late bronchial spasms and eosinophil infiltrates into the lungs upon antigen challenge and are also very sensitive to nonspecific airway hyper-responsiveness (Karol, 1995; Pauluhn, 1996). However, anaphylactic reactions in guinea pigs usually involve IgG1 rather than IgE and tend to be lethal so that animals must routinely be protected with antihistamines prior to challenge. Brown Norway rats are prone to Th2 responses and produce antigen-specific IgE and histopathologic changes in lung similar to human allergic asthma (Arts et al., 1998; Pauluhn and Mohr, 2005). However, rats are weak bronchoconstrictors relative to guinea pig, and background pulmonary lesions (granulomas in lung) in BN rats can confound interpretation of test article-related histopathology (Arts et al., 1998). Sprague-Dawley rats occasionally develop immediate hypersensitivity reactions to inhaled allergens but are more prone to allergic pneumonitis-type reactions (Leach et al., 1987;

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Maier et al., 2006). For testing respiratory hypersensitivity of investigational drugs, using a rodent species and strain consistent with the rest of the general toxicology package (e.g., Sprague-Dawley rat) is practically useful in selecting doses and integrating findings with other studies. End points for respiratory tract allergic reactions generally include measuring changes in breathing patterns, nonspecific hyper-responsiveness, cell differentials by bronchoalveolar lavage (BAL), inflammation and histomorphologic changes, and if available, drug-specific IgE. Plethysmography can be used to measure changes in respiratory rate, tidal volume, or peak expiratory flow. Nonspecific airway hyper-responsiveness to stepped increases of methacholine or acetylcholine can also be measured approximately 24 hours after antigen challenge (during inflammatory infiltration, particularly eosinophils) and expressed as enhanced end-expiratory pause (Penh), an indirect indicator of airway resistance. However, these breathing pattern changes can be rather variable and confounded by similar changes induced by irritation (even in the absence of bronchoconstriction). To this end, doses selected for induction/challenge should avoid levels identified in general toxicology inhalation studies that cause dose-limiting irritation (Arts et al., 2004). Additional end points, such as histological examination of the entire respiratory tract including lungdraining lymph nodes, and evaluation of total protein and cell differentials by BAL, can be used to support findings from pulmonary function tests (Pauluhn and Mohr, 2005; Arts and Kuper, 2007), or can potentially be stand-alone indicators of allergic reactions in rats (Leach et al., 1987; Maier et al., 2006). The presence of pulmonary functional changes or increased severity/incidence of histologic changes in the induced/challenge group relative to appropriate control groups (e.g., challenge only) is the strongest diagnostic criteria for sensitization. Structure–Activity Relationship and Screen Assays Recent advances using in silico structure–activity relationship (SAR) algorithms show promise in identifying substructures of LMW chemicals that are associated with respiratory hypersensitivity (Sarlo and Clark, 1992; Graham et al., 1997; Cunningham et al., 2005; Jarvis et al., 2005). However, developing and validating SAR models for respiratory sensitizers are challenging due to the limited number of known respiratory allergens included in the learning set. Similarly, there is progress in developing in vitro cell-based assays measuring activation of APCs following incubation with sensitizers; while most of the work so far in this field has primarily focused on contact sensitizers (Arrighi et al., 2001; Boisleve et al., 2004; Gildea et al., 2006; Rodriguez-Pena et al., 2006), there has been some effort on respiratory allergens (Toebak et al., 2006). Further evaluation of these models is necessary before they can be considered useful in predicting human safety, though they may have application in early screening for selection of chemical leads.

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SUMMARY Specialized immunotoxicity testing is useful for inhalation drugs to evaluate hypersensitivity potential. This is generally done by first screening for contact sensitization potential using relatively simple and validated contact hypersensitivity tests such as the LLNA. If an inhaled drug is found to have contact sensitization potential, then further characterization of respiratory hypersensitivity potential should be considered rather than abandoning development of the drug. Nonclinical inhalation induction/challenge studies are specifically designed to determine hypersensitivity potential by a relevant route of administration and can be used to evaluate a dose–response relationship. At this time, there is no standardized, validated nonclinical model for testing respiratory hypersensitivity, so careful consideration of the test system and end points, as well as inclusion of appropriate controls, is suggested.

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PART IX TESTING FOR DEVELOPMENTAL IMMUNOTOXICITY

9.1 DEVELOPMENTAL IMMUNOTOXICITY IN RODENTS Rodney R. Dietert and Leigh Ann Burns-Naas

When the immune system encounters an infectious agent, this generally leads to immunity and thus a robust, but generally less severe reaction to the same agent later in life. Interestingly, it is well established that compared with adults, common infectious diseases occur more frequently and are often more severe in very young children. In some cases, age-related differences in the developing immune system are responsible for this apparent increase in susceptibility. Additionally, children with severe immunodeficiency diseases show increased frequency and severity of common and opportunistic infections while those with less severe forms of the disease are more prone to respiratory and ear infections than their age-matched controls in the general population. Though there are many intrinsic factors that influence susceptibility, in recent years it has been suggested that exposure to certain environmental chemicals and therapeutic agents may exacerbate some of these immune-related diseases (Weisglas-Kuperus et al., 1995; Thurston et al., 1997; Timonen and Pekkanen, 1997; von Ehrenstein et al., 2000; Akinbami and Schoendorf, 2002; Smyth, 2002). Because of some of these observations, a concern has been raised regarding the possibility that children may have weaker responses to some childhood vaccinations following perinatal exposure to chemicals or drugs that impair the immune system in some way.


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DEVELOPMENTAL IMMUNOTOXICITY IN RODENTS

THE UNIQUE NATURE OF THE DEVELOPING IMMUNE SYSTEM The interest in developmental immunotoxicology (DIT) is predicated around the possibility that the immune system may exhibit greater susceptibility to chemical perturbation during ontological development that may not be detected if immune function is only evaluated in adult animals. This greater susceptibility of the young versus the adult may be manifested as qualitative, quantitative, or temporal differences (Figure 9.1-1). The recent literature is replete with examples of compounds that can alter the structure and/or function of the developing immune system (reviewed in Dietert and Piepenbrink, 2006a; Luebke et al., 2006; Dietert and Dietert, 2007; Burns-Naas et al., 2008). It is also apparent that some of these observed changes can be subtle in terms of routine nonfunctional assessment end points (Dietert and Piepenbrink, 2006a, 2006b). But why is there a difference between adults and children? The development of the mammalian immune system comprises a sequence of events that are exquisitely timed and coordinated, begin very early in fetal life, and continue through early postnatal development. The key developmental processes are the same and define the “critical windows” of development where the relative risks associated with exposure to immunotoxicants are likely to be different. In order to examine immune sensitivity differences across age-groups, immune development has been examined and critical windows of immune development have been defined and compared for rodents, canines, nonhuman primates, and/or humans (Dietert et al., 2000; Holladay and Smialowicz, 2000; Landreth, 2002; Holsapple et al., 2003; Dietert and Piepenbrink, 2006a; Burns-Naas et al., 2008). A time-normalized comparison

A.

B. Child

C. Child

Exposure Ends

Adult

Effect

Effect

Effect

Child

Adult Adult

Time

Dose

Dose

Figure 9.1-1 A depiction of how children may show greater susceptibility to toxicants than adults. (A) A temporal difference may occur when a toxicant produces a more persistent effect in younger animals than in adults; (B) a qualitative difference, in that the toxicant may affect the developing immune system without affecting that of the adult; or (C) a quantitative difference such that the toxicant may affect the developing immune system at lower doses than the adult immune system. Adapted from BurnsNaas et al. (2008).

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Gestation

Maximal TDAR Adult level IgG

Juvenile

Gestation

Adult level IgG

Lactation Parturition (Week 36)

Young Adult (42-49 Days)

Weaning (PND 21)

Splenic germinal centers Maximal TDAR Maximal TIAR

Splenic demarcation (red & white pulp)

Splenic mito-responsiveness MLR

Small numbers of T and B cells

Thymic primoidia

Maximal TIAR

Lactation Parturition (GD 22)

B.

Splenic germinal centers Detectable TDAR

Splenic demarcation (red & white pulp) DTH

Small numbers of T and B cells

Selective mito-responsiveness

Thymic primoidia

A.

MLR

of immune development in rodents and humans is presented in Figure 9.1-2. The earliest hematopoietic progenitors are derived from uncommitted stem cells that migrate to the fetal liver and fetal spleen where they begin their differentiation into hematopoietic lineages. Macrophages begin seeding the various tissues, and these and other cells begin developing the primary (thymus, bone marrow) and secondary (spleen, lymph nodes) immune organs. Within the thymus, T cell education and selection and the formation of regulator T cells (Tregs) are critical early life events. Additionally, there are key dendritic cell changes occurring. Lineage-restricted differentiation continues and the immune system begins to mature in its ability to respond to internal and external stimuli. During this time (and until parturition), the immune system is skewed toward a Th2 bias in order to protect the pregnancy (Lim et al., 2000), a condition that will be considered in more detail later in this chapter.

Juvenile Weaning (Week 104)

Young Adult (18 Years)

Figure 9.1-2 Comparison of developmental landmarks in the developing immune systems of the rodent (A) and human (B). Data have been normalized to three key developmental phases—gestation, lactation, and maturation to young adulthood, and clearly demonstrate that the development of the immune system of the rodent is delayed compared to that of the human, a fact that should be considered in the design and interpretation of DIT studies. Adapted from Holsapple et al. (2003) and BurnsNaas et al. (2008).

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The final stage of development is the maturing of functional responses to the adult level and the development of immunologic memory. Each of these critical pieces of the development puzzle represents a potential target for xenobiotic-induced alterations. Because the developing immune system represents a moving target for immunotoxicity, it is hardly surprising that when compared, immunotoxic risk differs—sometimes greatly—across age-groups. Figure 9.1-3 illustrates seven key immune maturation events in rodents between conception and birth, including sample xenobiotics reported to disrupt those specialized early-life processes. Many of those developmental immunotoxicants are known to have adverse effects at sufficient doses in the adult. Therefore, it is not surprising that low-dose, xenobiotic-induced disruption of key maturation events is likely to result in a significantly greater health risk when compared with adult exposures to the same xenobiotics. While DIT studies in rodents date to at least the 1970s (Vos and Moore, 1974; Luster et al., 1978, 1979; Faith et al., 1979), the vast majority of the studies have been conducted during the past 10–15 years and have focused primarily on industrial and environmental chemicals. However, the number of drugs examined in rodents and reported in the peer-reviewed literature now includes a wide array of potential immunotoxicants (Holladay, 2005). They include acyclovir (Stahlmann et al., 1992), atrazine (Rooney et al., 2003; Rowe et al., 2006), cyclosporin A (Hussain et al., 2005), dexamethasone (Dietert et al.,

Macrophage seeding of tissues

X

Dendritic cell maturation

Thymocyte selection

Lead LPS Ozone

X

DEX

X Nicotine

TCDD

Fetal macrophage maturation

X

Alcohol

Lead?

Conception

Birth

X

PAHs T-2 toxin TCDD

Seeding of thymus and thymopoiesis

X CsA CY Treg generation and seeding

Lead Mercury Kynurenines

X

Th1 response capacity

Figure 9.1-3 Key in utero immune maturation events and toxicant disruption. Seven in utero critical immune maturation events in the rodent are shown with developmental immunotoxicants known to disrupt those processes. In some cases, the processes are blocked by the xenobiotics while in others the integrity of the process may be compromised by early-life exposure. Arrows are not designed to indicate precise in utero timing but rather general periods of prenatal development. CsA = cyclosporin A; CY = cyclophosphamide; DEX = dexamethasone; LPS = lipopolysaccharide. Adapted from Dietert and Piepenbrink (2006a).

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2003), diazepam (Schlumpf et al., 1989, 1994; Burgi et al., 2000), and diethystilbesterol (Holladay et al., 1993; Fenaux et al., 2004). Limitations of Extrapolating Adult-Exposure Data to Early Life Developmental immunotoxicity testing has gained increasing attention with the recognition that for most drugs compared to date, when immunomodulatory effects are observed, the developing immune system is more sensitive than that of the adult. Therefore, safety limits for exposure of non-adults can be difficult to predict in the absence of age-relevant exposure assessment. Of equal concern is the fact that the nature and duration of immune alterations following adult versus non-adult exposure can be quite distinct. Depending upon the age of exposure, the spectrum of effects can vary significantly (Bunn et al., 2001a, 2001b, 2001c; Dietert et al., 2003). Limited exposure during different critical windows of development may produce qualitative differences in immunotoxic outcomes. Therefore, a particular immunotoxic alteration seen following a late gestational or early postnatal exposure may be quite different from one resulting from an early gestation exposure event. Clearly, this can complicate predictability when attempting to extrapolate risk across ages and particularly if adult exposures are used to model early-life immunotoxic risk. Another age-related difference in immunotoxicity concerns, recently reviewed by Luebke et al. (2006), is the persistence of immunotoxic effects (Figure 9.1-1A). In many cases, immunotoxicants will produce only transitory immune effects following adult exposure but can cause persistent immune alterations after early-life exposure. For example, cyclosporin A (CsA) produced only transitory immunosuppression when administered to adult CD strain rats. Given a sufficient recovery time without the drug, the adult rats mounted normal immune responses and had normal immune cell phenotypes. However, similar dosing of CsA in utero produced persistent immunotoxicity following this early-life exposure (Hussain et al., 2005). Therefore, toxicant exposures that might produce only transitory immune effects in adults have the potential to result in persistent immunotoxicity following early-life exposure. Clearly such age-related differences in outcome would result in differential health risks. Additionally, some immunotoxic effects following early exposure may only become apparent during conditions of postnatal stress or immune challenge (Lee et al., 2002). Still others may alter the physiological balance in such a way that the immune system responds unpredictably following apparently innocuous adult exposures to drugs. Fenaux et al. (2004) showed that early-life exposure of mice to DES preprogrammed the thymus for aberrant adult responses to subsequent low doses of endocrine-altering xenobiotics. Such examples of latent adult-onset immunotoxicity arising from early-life xenobiotic exposures are a concern. Much of the existing database for age-based immunotoxicity comes from rodents and was recently reviewed by Luster et al. (2003), Dietert and

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Piepenbrink (2006a), Luebke et al. (2006), Dietert and Dietert (2007), Dietert and Holsapple (2007), Holsapple et al. (2007), and Smialowicz et al. (2007). If one examines the nature of unique or novel events associated with rodent and human immune development (Dietert et al., 2000; Dietert and Piepenbrink, 2006a) and the capacity of diverse toxicants to disrupt these maturation processes, it is not surprising that immunotoxic risk based on adult-exposure assessment has limited utility when considering pediatric and in utero drug safety. Information on immunotoxic safety for non-adults can be important in extending the use of a pharmacologic agent across a broader spectrum of agegroups. Additionally, while many drugs may be developed with the adult as the primary target, unintended in utero exposure can still occur if a drug is prescribed to women of childbearing age. Since it is difficult to completely ensure that such exposure would never occur, it may be important to understand the likely risks arising from such exposures. Accurate estimation of health risks associated with early-life exposures is the central element driving DIT assessment. Because DIT predicts health risk pertaining to a potentially more sensitive subpopulation of humans than adult immunotoxicity assessment, it seems redundant for DIT testing to be performed subsequent to a positive adult immunotoxicity finding. The default should be that the xenobiotic is likely to be a developmental immunotoxicant and may be problematic at lower doses or may produce more severe outcomes. In those instances, the only benefit of additional DIT data would be to establish new safety levels pertaining to the non-adult that cannot be accurately extrapolated from adult immunotoxicity data. Considering the reciprocal relationship of safety testing, if DIT data exist, then adult immunotoxicity assessment may be unnecessary (Dietert and Piepenbrink, 2006a; Dietert and Holsapple, 2007; Holsapple et al., 2007). Dietert and Piepenbrink (2006a) have also suggested that it may be more cost-effective and protective in some circumstances to substitute DIT testing in place of adult-exposure immunotoxicity evaluation. The following section of this chapter will review strategies for DIT testing in the context of improved safety assessment across life stages.

EVALUATION OF DEVELOPMENTAL IMMUNOTOXICITY OF PHARMACEUTICALS Recent Regulatory Perspectives Consistent with the approach taken for assessment of immunotoxicity in adult animals, which is based on consideration of a weight-of-evidence review of available data and key potential triggers (ICH, 2006), global regulatory agencies have not routinely required assessment of developmental immunotoxicity during drug development. Rather, these assessments are driven by “triggers” (cause for concern) from a weight-of-evidence evaluation of data in adult animals. A number of criteria have been proposed as possible triggers that

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might result in further immune evaluation involving the developing immune system (Holsapple et al., 2003). These include class-specific effects, and structural similarity of a new drug to known immunotoxicants. Studies in adult animals that show the immune system as a potential target organ may suggest the need for an assessment of DIT as the data to date show that all compounds that are immunotoxic in adults have been shown to be immunotoxicants in the developing immune system when evaluated, often with differential sensitivity between the two age-groups. Potential signs of immunotoxicity could be structural or functional. Other potential triggers are the intended patient population and the potential for neonatal exposure. For example, drugs used to treat immunocompromised individuals, including children, may warrant consideration for DIT testing; and if pregnant or lactating women are to be treated, and the drug may cross either the placenta or into breast milk (or this information is unknown), sponsors may wish to consider the need for DIT testing. Decisions to conduct DIT studies should be based on sound science. That is, if it is known that in utero exposure is very low or nonexistent, then performing a DIT assessment may not provide information that would significantly aid in assessing the overall risk of the drug. Global regulatory authorities generally concur that evaluation of potential adverse effects of human pharmaceuticals on the immune system should be incorporated into the standard drug development process (ICH, 2006), though this specifically applies to the adult animal. In the United States, the Food and Drug Administration first addressed the potential need for DIT testing in their guidance for industry on the nonclinical evaluation of immunotoxicity for new chemicals (FDA, 2002). In it, the Agency notes that a DIT assessment should be considered if the drug is “expected to be used in pregnant women and has been shown to induce immunosuppression in adults, . . .” The European Committee for Human Medicinal Products (CHMP) has taken a similar approach in its most recent draft guidance on nonclinical testing to support pediatric drug development (EMEA, 2005). CHMP recognizes that major developmental differences exist between the immune systems of human neonates/infants and adults, and considers that these developmental differences are generally apparent until age 12. CHMP also intimates that pre- and postnatal exposure can potentially result in all types of immunotoxicity in the offspring, including immunosuppression, hypersensitivity, allergy, and autoimmune disease. Juvenile studies have been suggested to be performed “on a case-by-case basis and only after a careful consideration of the available data and the age and duration of treatment of the intended paediatric population” but that “if any of the major functional systems are shown to be potential targets, either from human or from nonclinical studies, studies in juvenile animals should be considered.” Further, “immunotoxicity studies are only required if the chemical/pharmacological class of compound or previous studies in humans or animals gives cause for concern for the developing immune system.” FDA’s juvenile testing guidance (FDA, 2006) also recognizes the immune system as one that continues to mature after birth (until approximately 5–12

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years of age) and may well be a novel target for drug-induced toxicity. Like its 2002 guidance on immunotoxicity testing, and the CHMP draft guidance on nonclinical juvenile testing, FDA considers that DIT evaluations may be warranted on a case-by-case basis with consideration given to “(1) the intended of likely use of the drug in children; (2) the timing of dosing in relation to phases of growth and development in pediatric populations and juvenile animals; (3) the potential differences in pharmacological and toxicological profiles between mature and immature systems; and (4) any established temporal developmental differences in animals relative to pediatric populations.” The Agency also considers that “the greatest concern is with chronic, long-term therapy,” and thus “the duration of anticipated treatment of the pediatric population should be considered in relation to the duration of developmentally sensitive phases.” As is noted in subsequent sections describing recently published age-based comparisons of rodent immunotoxicity (Luebke et al., 2006), the adult immune system appears to be relatively insensitive for immunotoxicity compared with that of earlier life stages. Based on these recent conclusions, use of an adult immunotoxicity indicator as a trigger for developmental immunotoxicity assessment is likely to be ineffective (Dietert and Piepenbrink, 2006a). Rodents versus Non-Rodents for Assessment Rodents have been a predominant model of choice for DIT studies based on the published literature. Historically, the significant knowledge of the developing and mature immune system in rodents, as well as the availability of reagents for rodent immune assessment, helped to spur the use of the mouse and rat for immunotoxicologic research and testing, including DIT of drug candidates. More recently rodents, particularly the rat, were designated by a consensus panel of immunotoxicologists as the preferred test system for DIT assessment (Holsapple et al., 2005) with the exception of nonhuman primates used in testing of biopharmaceuticals (see Chapter 9.2). The rat was seen as having an advantage for DIT assessment based on the fact that early-life exposure assessment protocols measuring developmental and reproductive toxicity already employ the rat. If DIT testing protocols could be merged with developmental and/or reproductive assessment, this would be advantageous from both an animal use and a cost standpoint. While rodents probably serve as the most utilized safety testing animal model for human health protection, there are limitations that are important to recognize. Beyond fundamental xenogenic differences with humans that can result in species differences in pharmacokinetics and actions of certain chemicals, rodent and human immune development does not proceed on an identical timeline. Landreth (2002) and Landreth and Dodson (2005) have shown that some events that occur gestationally in humans happen postnatally in rodents. This may be a consideration if limited exposure windows are utilized or if the maternally transferred exposure in rodents could not simulate the appropriate human fetal exposure. Therefore, knowledge of drug metabolism and the

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capacity of the drug and/or its metabolites to cross the placenta, as well as to be transferred during lactation, may be an important consideration in designing the most appropriate exposure regime. One advantage of rodents, beyond the exceptionally well-characterized immune system and availability of reagents, is the fact that DIT assessment may be able to be dovetailed into existing developmental and reproductive assessments. Certainly DIT testing using other mammalian species may be appropriate under some circumstances, but the opportunity to examine developmental immunotoxicity, developmental neurotoxicity, and reproductive toxicity using a common model suggests that the rodent is likely to remain the standard model of choice. This conclusion was also reached by various consensus panels (Holsapple et al., 2005). A Predominant DIT Phenotype? It is recognized that the developing immune system undergoes a myriad of maturational steps occurring in the bone marrow, thymus and extra-thymically such as in the Peyer’s patches, GALT and BALT. Because different drugs can disrupt different processes during maturation, there is an almost endless combination of potential immunotoxic outcomes. However, based on the literature to date, many immunotoxicity profiles seem to follow a predominant mode or theme among the diversity of immunotoxic possibilities. The apparent predominant DIT phenotype seems likely to be associated with the unique nature of the developing fetus–maternal interaction. Many xenobiotic exposures in utero seem to interfere with necessary perinatal adjustments to immune capacity. A frequent outcome of DIT seems to be an immune functional capacity that is frozen in a fetal state. This outcome was recently reviewed in Dietert and Piepenbrink (2006a) and Dietert and Dietert (2007). Successful in utero development to parturition requires that the developing immune capacity of the fetus, as well as that of the mother, avoids problematic Th1-dependent allogeneic reactions. For this reason, Th2-associated immune capacities seem to predominate in the late-term fetus, while an acceptable balance of Th1/Th2 is only achieved during and immediately after the birth process. Part of this conversion from a Th2-biased capacity to an appropriate neonatal Th1/Th2 balance requires a series of changes among accessory cells such as dendritic cells and macrophages. Not surprisingly, drug interference during the perinatal period frequently has the effect of freezing immune maturation in a Th2 preferred state. In fact, for low-dose in utero immunotoxicant exposures, population of immune organs can often be normal but immune function capacity is skewed such that disease risk is increased. Because lowdose in utero or neonatal exposure may not always lead to severe thymic atrophy or other profound histological alterations, functional balance appears to be a critical factor in DIT evaluations. Other postnatal compound exposures can also be a help or hindrance in achieving a balanced immune capability. Some of these factors were recently

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reviewed in the context of asthma (Selgrade et al., 2006; Yeatts et al., 2006). The take-home conclusion from a comparison of several different DIT versus adult-exposure assessments and from recent multi-endpoint DIT evaluations is that information on the range and extent of immune functional capacity is critical for an effective evaluation of DIT (Dietert and Holsapple, 2007). In fact, advocacy for immune functional assessment for DIT has extended beyond rodent models to direct assessment in humans. Luster et al. (2005) advocated the need for functional immune-response measures in evaluating potential immunotoxicity in children. This translational aspect will be considered briefly at the end of this chapter.

METHODS FOR DIT ASSESSEMENT Like the prior National Toxicology Program (NTP) Immunotoxicity testing panel used for the adult mouse (Luster et al., 1992), several parameters appear to be useful in delineating the identification and extent of DIT. These include cell population analysis via flow cytometry, cytokine production analysis, and immune organ histology. However, as with the NTP comparisons established in prior decades, functional assessment appears to be essential for DIT hazard identification and risk assessment. CHMP has recognized this in their draft juvenile guidance document indicating a requirement for functional assessment, as well as histopathology (FDA only suggests immunopathology). Importantly, however, no single functional assay appears capable of detecting a commonly observed outcome of DIT—changes in immune functional balance. Since skewed immune functional balance, not comprehensive immune deficiency, is an outcome that must be detected with DIT assessment, combinations of parameters designed to survey the spectrum of immune functions have proven most useful.

The T-Dependent Antibody Response (TDAR) Although detailed descriptive immunopathology has been widely employed for detecting early signs of drug-induced immunomodulation in adult rodents (Haley et al., 2005), immunopathology in juvenile animals is less established; therefore, evaluation for DIT hazard identification and risk assessment in the non-adult should include immune function evaluation (Holsapple et al., 2005; Dietert and Holsapple, 2007). Not surprisingly, the TDAR is particularly important as an end point. Because of its broad range of assessment (antigen presentation, T cell help, and B cell effector function) and its significant utility in adult immunotoxicity assessment, as well as the information provided in Table 9.1-1 for DIT, it has been concluded that the TDAR probably represents the single best functional test of immunocompetence for DIT. There are several caveats to its use, however. First, the ability to measure a TDAR in non-adult

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METHODS FOR DIT ASSESSEMENT

TABLE 9.1-1 Comparisons of DIT Evaluations in Rodents among Recent Studies* Assay Assay Used/ Total DIT Studies in Rodents Positives/Total Assessments

TDAR

DTH or CTL or Flow TDAR + CS NK Cytokines Cytometry Histopathology CMI

9/16

8/16

9/16

5/16

10/16

5/16

15/16

7/9

7/8

6/9

4/5

8/10

2/5

15/15

* Modified from Dietert and Holsapple (2007). Results are from 16 recent DIT studies in rodents appearing in the published literature. Single-assay results are presented for the T-dependent antibody response (TDAR), delayed-type hypersensitivity response (DTH), contact sensitivity (CS), cytotoxic T cell response (CTL), natural killer cell assay (NK), cytokine production measurements, flow cytometry of leukocyte populations, histopathology of lymphoid organs, and the combination of TDAR plus CMI (DTH, CS, CTL, or NK). In this case, positives represented a positive determination that the chemical or drug was a developmental immunotoxicant using this evaluation measure (or the combined TDAR plus CMI). Note all chemicals and/or drugs were concluded to be developmental immunotoxicants in rodents based on the full spectrum of tests performed

rodents is limited as virtually no response is detectable at PND 10 and only a suboptimal response is detectable at weaning (PND 21) (Kimura et al., 1985; Ladics et al., 2000). This is consistent with the development of the immune system in the species (Figure 9.1-2). The maximal response appears to occur at the point in which rodents reach young adulthood (PND 42–49). Thus, unless exposure is continued through young adulthood (Figure 9.1-4B), it is possible that an early DIT effect may be missed. Additionally, because skewing of the immune system in perinatal development frequently involves functional shifts, a multi-isotype TDAR has the advantage of comparing antigen-specific isotype production and thereby helping to examine concerns of Th balance (Dietert et al., 2003; Hussain et al., 2005; Dietert and Holsapple, 2007), something that is not routinely done for adults. Despite the reasonable predictivity of the TDAR, especially if conducted at the optimal time and using multiple isotype assessment, it is still considered that this assay needs to be combined with other functional and/or structural assessments to give the best overall assessment of true DIT risk. Limitation in the TDAR as a single-parameter predictor for immunotoxicity in rodents is not a new issue. Furthermore, no other single functional measure appears adequate for a DIT determination either. Table 9.1-1 summarizes results of a recent comparison of 16 DIT studies employing different combinations of assessment parameters (Dietert and Holsapple, 2007). While the ability to detect the TDAR exists during the early juvenile period (PND 21), the relative sensitivity of the assay at this time point (e.g., the ability to detect subtle changes) is unclear, and negative data may be misleading. If anything, extension of information well beyond the TDAR appears to be even more important for DIT assessment using rodents. This conclusion is based on the increasing recognition that several developmental immunotoxicants among

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A. Gestation

Lactation

Parturition (GD 22)

Juvenile

Weaning (PND 21)

Culling (PND 4)

Young Adult (PND 42-49)

Immunopathology

Immunopathology

B. Gestation

Lactation

Parturition (GD 22) Culling (PND 4) Immunopathology

Juvenile

Young Adult (PND 42-49)

Weaning (PND 21) Immunopathology CMI Assay

and/or

Immunopathology CMI Assay TDAR

Figure 9.1-4 A proposed testing scheme for DIT assessment of rodents. The designs are derived from the standard ICH peri- and postnatal developmental and reproductive toxicity study (ICH S5 Section 4.1.2) and recommendations from FDA (2002, 2006), EMEA (2005), Ladics et al. (2005), and a consensus workshop on development of a testing framework for DIT (Holsapple et al., 2005). (A) In the conventional periand postnatal study, maternal exposure would begin on GD 6 and continue through PND 21. Immunopathology (organ weights, hematology, and histopathology) are recommended by regulators and could be performed on culled pups (PND 4) or on a subset of pups at weaning (PND 21). Evaluation of pups at an older time is possible, but not recommended here because the potential for exposure stops at weaning. (B) This proposal depicts a “flexible, alternative peri- and postnatal design” or a standalone DIT assessment, and is the more rigorous of the two schemes presented here. In it, pups (or a subset thereof) are continually dosed throughout all critical windows of development via maternal exposure or direct dosing. (The decision when to begin direct dosing should be made based on information regarding the ability of the drug to cross into breast milk in the rodent.) In addition to the current regulatory recommended options for immunopathology, functional assessment of the immune system may be made at PND 21 (cellular assay) and PND 42 (TDAR; cellular assay). Assessments at all time points are not advocated for screening purposes, unless appropriate for the drug being investigated. Adapted from Burns-Naas et al. (2008). Black lines = maternal dosing; hatched lines = direct dosing of pups.


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different categories of toxicants negatively impact cellular immunity, and particularly Th-dependent cellular immune responses, but may produce little change in antibody production. Cellular Immunity (DTH Response, CTL Assay, and NK Activity) For the reasons noted above, cytotoxic T lymphocyte (CTL) assays, delayedtype hypersensitivity (DTH) assays, and the natural killer (NK) cell assay are likely to be important in the overall assessment of DIT. This is consistent with the findings of the prior consensus panel (Holsapple et al., 2005) and recent reviews (Dietert and Holsapple, 2007; Holsapple et al., 2007; Burns-Naas et al., 2008) that stressed the need to broaden assessment beyond just the TDAR. Because several important categories of developmental immunotoxicants failed to alter the TDAR while significantly altering cellular immunity (Miller et al., 1998; Gehrs and Smialowicz, 1999; Bunn et al., 2001a, 2001b; Karrow et al., 2004), protocols using the TDAR alone or lacking functional measures were not recommended as effective screens for DIT (Dietert and Holsapple, 2007). A majority of recent DIT studies have successfully employed the TDAR in conjunction with cellular immune measurements. As a second assay option, DTH to KLH or BSA, CTL activity, and NK cell assays have been most frequently used, although challenges with infectious agents have also been used (Voderstrasse et al., 2006). These results led Dietert and Holsapple (2007) to propose that a two-function assay minimum be considered for any DIT assessment regime. Clearly, a starting point is the TDAR, a cellular immune evaluation and immunopathology (organ weights, hematology, and histology) as suggested in Figure 9.1-4B. The importance of including a cellular assay in a full DIT assessment is twofold: (1) the assay measures Th1 responses (TDAR is more focused on Th2 responses); and (2) because of the timing of immune development in rodents, an accepted cellular assay can (unlike the TDAR) probably be effectively performed earlier in the developmental period. Though CTL and NK assays have been used to assess cellular immunity in DIT studies (reviewed in Dietert and Holsapple, 2007), studies comparing these assessments across early life stages are generally lacking. The DTH is the assay used most frequently to date (see Chapter 3.1.3). It is important to consider, however, that whatever immunogen is utilized (e.g., KLH, BSA, whole cells), the investigator should assure that the end point being evaluated represents a “true” DTH response and is not confounded by the production of antibody. Thus far, antigens typically used to evaluate the DTH response in DIT assessments do have the potential to produce an antibody response. To date there is no consensus on the best methodology (including immunogens) and standardization/validation of these assays is an issue for use in regulatory DIT assessment (reviewed in Burns-Naas et al., 2008). Nevertheless, it is clear that inclusion of an assay to assess cellular immunity is important in the overall assessment of DIT risk.

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Immunopathology (Organ Weights, Hematology, Histology) The value of including immunopathology, i.e., hematology, organ weights, and histopathology of lymphoid tissues, in assessment of DIT in fetal, neonatal, and juvenile rodents has been a matter of debate. Histological examination of specimens from nonclinical toxicology studies may reveal reductions in cell populations associated with reduced immune responsiveness but would not reveal increased or misdirected immune responsiveness unless the immune system alteration results in structural damage to another organ or tissue (Burns-Naas et al., 2008). Consensus from a workshop to evaluate the state of the science of DIT concluded that immunopathology is important as a secondary (supportive) end point to be used in conjunction with a functional assessment, but may not be the best “stand-alone” means to assess DIT (Holsapple, 2002; Dietert and Holsapple, 2007; and Burns-Naas et al., 2008). This concern centers on the sensitivity of immunopathology relative to functional immune evaluation. Recent evaluation of DIT studies shows that histologic evaluation could explain some of the functional changes observed in young animals (Dietert and Holsapple, 2007). However, Hussain et al. (2005) have also demonstrated that functional impairment of the developing immune system does occur in the absence of histologic alteration. The latter observation specifically contradicts the initial screening approach recommended by most regulatory agencies (e.g., EPA, FDA, etc.), which suggests immunopathology as an initial screen with the potential for follow-up DIT investigations upon a positive finding. The suggestion that immunopathology may not be a sensitive indicator of DIT for screening purposes could be because historically speaking, most pathologists have much less experience with fetal and neonatal immunohistology than they do with the adult rat. Also, it is probable that most of the studies conducted to date have not utilized the full spectrum of available techniques (Haley, 2003; Holsapple et al., 2003), especially for an organ system that undergoes such remarkably dynamic changes over the course of days and weeks (Burns-Naas et al., 2008). While the histologic features of immune system organs in adult laboratory animals are well known (Dunn, 1954; Haley, 2003), these are not as well defined in neonatal animals. Burns-Naas et al. (2008) assert that early studies of the developing immune system suggest that there is little potential for histopathologic detection of chemically mediated alterations in the developing immune system of laboratory rats at or prior to gestation Day 15 (GD 15). However, there is a remarkable progression in the histologic appearance of fetal organs including organs of the immune system, which takes place between GD 15 and GD 20. At GD 15, the only visceral organ with histologic similarity to the mature counterpart is the liver, which consists of approximately equal hepatocellular and hematopoietic elements. However, by GD 20 the rat fetus has easily recognized internal organs, including thymus, spleen, and mesenteric lymph nodes. Major perturbations (e.g., failure in the formation of immune system organs) could be detected by


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standard histopathologic examination by GD 20. The immune system of the developing rat is anatomically intact by postnatal day (PND) 22, perhaps earlier, but histological features at PND 22 suggest relative inactivity (lack of stimulation by exogenous antigens). Despite the need for additional information, it is clear that presently immunopathology of the developing immune system is certain to provide valuable information when combined with functional assessments. However, it is important to consider a few specific aspects during the evaluation. Changes in immune organ weights are often observed with immunosuppressive drugs and can be complicated by conditions where release of corticosteroids occurs (e.g., stress). As would be expected, weighing the developing immune organs is made more difficult by their small size, as well as the normal variability that can occur during a typical necropsy (e.g., trimming, fluids in tissue, etc.). It is recommended (Burns-Naas et al., 2008) that changes in organ weights consider absolute and relative (body and brain) weights (Schwartz et al., 1973; Scharer, 1977) and careful attention to other environmental stressors (e.g., food consumption, maternal effects) that could indicate nondrug-related effects. Hematology can be a reasonable indicator of immune effects and should be given a priority over other clinical pathology end points when sample size is small. Supportive Analyses (Cytokines, Flow Cytometry, Immunomics, Host Resistance) It is clear that there is support within the immunotoxicology community for the use of the TDAR and a cellular assay to evaluate DIT in rodents. Similarly, there is agreement that classical host resistance assays, relying on morbidity and mortality measures, are not appropriate for frontline screens. These evaluations are typically a “final tier” assessment and are only conducted when data from initial screening studies indicate functional immune alterations, there is some understanding of the mechanism of immunomodulation that can drive the selection of the appropriate host resistance model, and animal welfare concerns are not a significant factor (Holsapple, 2002; reviewed in Burns-Naas et al., 2008). However, it is recognized that many infectious agents represent potent stimulators of immune response and can serve as useful targets in the context of evaluating TDAR and cellular immune assays such as the cytotoxic T lymphocyte (CTL) assay. What is less clear, at present, is the role that other traditional immune evaluations may play in DIT assessment, including cytokine evaluation, flow cytometry, and immunomics. Based on the literature to date and the specific protocols employed, cell population changes, cytokine production alterations, dendritic cell and/or macrophage assessment, and immune histologic manifestation seem to be accessory parameters that are supportive of functional changes but not of themselves sentinel predictors of DIT. As described for adult animals (see Chapter 4), cytokine measurements in serum or peripheral blood may potentially be performed as a “functional” means to assess

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immunomodulation. Because cytokines have been shown to have patterns indicative of Th1 and Th2 profiles, and clearly a shift from Th2 to Th1 in early development is important to the overall health of the individual, it is intriguing to consider the potential utility cytokine measurements may play. This could be particularly useful in considering the impact of a drug on the development of atopy and/or asthma later in life. So far, though, cytokine measurements as screening biomarkers of DIT should not be used because of lack of baseline data on “normal” levels (and their daily and age-related temporal variability), the lack of standardization of both in vitro and in vivo methods, and a lack of understanding of the time- and dose-dependent kinetics of these profiles relative to known developmental immunotoxicants in peripheral blood and immune organs. Likewise, there are similar limitations on the use of immunomic data at this time (Burns-Naas et al., 2006; Luebke et al., 2006). Flow cytometry has been used to investigate xenobiotic-induced DIT, and as in adult animal immunotoxicity studies (see Chapters 3.2 and 4.2), its use seems to be best positioned as an investigational tool examining whether alterations observed in first line studies may result in changes in cell populations (Dietert and Holsapple, 2007). Ladics et al. (2000) did compare phenotypic profiles at PND 10 and PND 21 and, not surprisingly, reported that levels of B cells and T cells (CD4+, CD8+, and CD3+) were 50% higher. It has been demonstrated that adding some combination of flow cytometric evaluations, dendritic cell/ macrophage measures, and/or cytokine measurements can further buttress functional changes in immune responses (Dietert and Dietert, 2007; Dietert and Holsapple, 2007). Development of a Testing Framework for DIT Incorporation into Existing Developmental and Reproductive Toxicity Studies. There is consensus that, wherever possible, methods to evaluate the developing immune system be incorporated into existing developmental and reproductive toxicology (DART) protocols (Holsapple et al., 2003; Ladics et al., 2005; Burns-Naas et al., 2008). The FDA has stated that, “If a drug has been shown to have immunosuppressive potential in adult animal studies, determination of potential developmental immunosuppression should be incorporated into an ICH Stage C to F reproductive toxicology study (ICH, 1994). At a minimum this would include determination of clinical and anatomical pathology parameters indicative of immunosuppression (e.g., effect of maternal drug exposure on lymphoid system organ weights, histology and hematology in the F1 generation offspring) should be included in the terminal examination” (FDA, 2002). A general depiction of this study design is shown in Figure 9.1-4A. No recommendation was made in the guidance regarding functional assessment of the developing immune system, probably because there was no full consensus at that time regarding which functional assays would be most appropriate.


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During a panel discussion for a scientific session on including DIT into existing DART assessments (Ladics et al., 2005), the panel agreed that there was a need to recognize that maternal toxicity, particularly with the use of a maximum tolerated dose, can have a significant impact on the various end points of DIT, as well as DART studies. Carney et al. (2004) reached a similar conclusion for DART and immunotoxicity end points using feed restriction as a surrogate for maternal toxicity. It should be emphasized, however, that the ability to implement DART-DIT combination end point study design should not be viewed as an endorsement of the approach as the study design for the future. Rather, flexibility to alter existing testing paradigms to meet the needs of the evaluated drug candidate is important. An Alternative DART Design or Stand-Alone DIT Study in Rodents. Of notable importance, incorporation of DIT into the existing peri- and postnatal study design (Figure 9.1-4A) can be problematic in that exposure generally is stopped on PND 20, while postnatal developmental assessments (neurobehavioral) continue through approximately PND 80. Full functional evaluation at PND 42 (e.g., TDAR + cellular assay) would occur following approximately 3 weeks of non-exposure allowing for the possibility of recovery of effects and thus, a potential for false-negative assessments. There is consensus that there should be flexibility to alter existing testing paradigms and this is presented in Figure 9.1-4B. In it, exposure/dosing is continued through PND 42–49 and full assessment is made at that time (functional and structural). This is also the recommended approach and general strategy for a “stand-alone” DIT study (Holsapple, 2002; Luster et al., 2003; Holsapple et al., 2005; Dietert and Holsapple, 2007; Burns-Naas et al., 2008). Triggers have been proposed to determine the need for a DIT study (Holsapple et al., 2005), including the following: structure–activity relationships and analogs of known immunotoxicants, findings from standard toxicity studies preclinical tests, the intended use of the drug, and the potential for pediatric and/or maternal exposure (FDA, 2002). As previously mentioned it is unclear, though, whether the proposed triggers are adequate or whether sufficient data would be available early enough in product development to indicate when a DART-DIT combination end point study would be needed. Gender and DIT: Sex-Specific Differences Are Common While gender-based differences in immunotoxic responses following adult exposures have been reported, the incidence of sex-specific outcomes for developmental immunotoxicants is unexpectedly high. In fact, sex-specific outcomes have been reported for a majority of developmental immunotoxicants examined in rodents where both sexes have been tested. Such sex-based skewing of responses might be expected for endocrine-disrupting compounds. However, sex-based differences in DIT have been reported for a variety of toxicants many of which are not presently classified as endocrine disruptors.

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TABLE 9.1-2 Examples of Development Immunotoxicants Producing Sex-Based Differences in Rodents Xenobiotics Showing Sex-Based Differences in Rodent DIT* Atrazine (rat) Cadmium (rat) Chlordane (mouse) Cigarette smoke (mouse) Diazepam (rat) Diethylstilbesterol (mouse) Ethanol (rat) Genistein (rat) Heptaclor (rat) Lead (rat) Mercury (mouse) Methoxychlor (rat) Nonyphenol (rat) Particulate matter (mouse) Trichloroethylene (mouse)

Reference(s) Rooney et al. (2003); Rowe et al. (2006) Pillet et al. (2005) Blyler et al. (1994) Ng et al. (2006) Burgi et al. (2000) Fenaux et al. (2004) Redei et al. (1993) Guo et al. (2002) Smialowicz (2002) Miller et al. (1998); Bunn et al. (2001c) Silva et al. (2005) Chapin et al. (1997); White et al. (2005) Karrow et al. (2004) Drela and Zesko (2003) Peden-Adams et al. (2006)

* The species examined for each xenobiotic is presented in parentheses.

Table 9.1-2 lists 15 different chemicals, drugs, and environmental contaminants for which males and females differed in DIT among rodents. In some cases, the differences were qualitative for the assays examined with only one gender showing alteration among the tests employed. In other cases, the genders differed in the spectrum and/or dose sensitivity for the DIT alterations. These results have ramifications for testing considerations since they suggest that single-gender assessments may not be widely applicable for the alternate sex. Both genders need to be examined to ensure that important immunotoxic sensitivities are not missed in screening. Key Factors to Consider in Study Design DIT assessment has recently been considered by several consensus panels (Luster et al., 2003; Holsapple et al., 2005), as well as in recent reviews (Dietert and Piepenbrink, 2006a; Dietert and Holsapple, 2007; Dietert and Dietert, 2007; Holsapple et al., 2007). Key factors to consider when designing studies to assess DIT are summarized here: • Development of the immune system of rodents is delayed compared to that of the human (Figure 9.1-2) and, therefore, the design and interpretation of results (relative to the clinical situation) should take this into account. • Males and females should be examined.


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• The litter should be the experimental unit, where feasible. • Exposure duration • As an initial screen, the best approach is to assess all of the critical windows at once by assuring that exposure to the developing organism occurs in utero, during lactation, and during juvenile development. This generally means assuring exposure from GD 6 through PND 42 (Figure 9.1-4B). • At this time it is unclear the relative sensitivity of conducting a DIT assessment only through PND 21 (Figure 9.1-4A), though this is an alternative design currently suggested by regulatory authorities evaluating pharmaceuticals. • An understanding of exposure during development (e.g., the ability of the drug to cross the placenta or into breast milk) is important. If it is not known whether the drug crosses into breast milk, the investigator should consider direct dosing of pups as soon as possible (e.g., PND 7). • Reversibility should be considered. There is no consensus on an appropriate duration, though 2–4 weeks has been suggested (Holsapple, 2002). • Dose Selection • Pharmacokinetic and pharmacodynamic data should be taken into account in study design and selection of doses. Consider that PK/PD may be different in the non-adult and a range-finding PK study may be needed prior to DIT assessment. A negative study in the absence of exposure would not be considered valid. • Maternal toxicity can result in secondary immune effects arising from a stress response (e.g., not a direct effect of the drug on the immune system). This should be factored into dose selection and data interpretation. • End points • Should include multifunctional, as well as structural assessments • Should be predictive of underlying immune system status or capacity, sensitive, able to be performed with reliability, and well understood biologically • Should be selected to provide the broadest assessment of immunocompetence (e.g., TDAR + cellular assay + immunopathology) • Should be assessed immediately following cessation of dosing • Should include immune functional measures when infectious agentbased approaches are employed Translational Medicine—Evaluating DIT in Humans Ultimately, the most appropriate model for evaluating DIT in humans is, of course, humans. Our ability to do this presently is an area that needs further

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research (Luster et al., 2005). The use of T cell receptor rearrangement excision circles (TRECs), immunophenotyping, the evaluation of cytokines and serum immunoglobulin (Ig), and the quantification of the immune response to childhood vaccines have been suggested as possible end points or biomarkers. None of these has been evaluated to an extent that reasonable recommendations can be made, and some (serum Ig and phenotyping) have been considered relatively insensitive markers in the adult and thus may (or may not) also have questionable value for assessing the developing immune system. Clearly more research is needed and it is recommended that the reader continue to follow the developing literature on this over time.

SUMMARY Rats and mice provide excellent test models to identify developmental immunotoxicity (DIT) relative to pharmaceutical evaluation when nonhuman primates are not required. The database for DIT is more extensive in rodents than for any other mammals. It is sufficiently extensive for certain patterns to have emerged particularly for low-level xenobiotic exposures in early life. The most commonly observed DIT alteration is a shift in functional capacity (frequently including targeted immunosuppression) and not pervasive immunosuppression/immune deficiency. Since functional shifts are a major hallmark of DIT, immune challenge either via immunization(s) or introduction of an infectious agent is essential for assessment. Additionally, immune tests covering the spectrum of critical functions are needed for such immune functional shifts to be detectable. This means that assays like the TDAR should be used in combination with other functional assessments such as a cellular evaluation, and immunopathology for the most rigorous detection of potential effects of drug candidates on development of the immune system. Finally, assessment of DIT may be merged with developmental and reproductive toxicity studies if possible to maximize efficiency of testing, thus minimizing animal usage. However, stand-alone DIT assessment may be needed if modified development and reproductive toxicity protocols do not include juvenile (postweaning) exposure and assessment.

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9.2 DEVELOPMENTAL IMMUNOTOXICITY IN NONHUMAN PRIMATES Pauline L. Martin and Eberhard Buse

For pharmaceuticals intended to treat patient populations that include women of childbearing potential, developmental toxicity studies are generally required during drug development or prior to registration. The intention of these studies is to evaluate any potential adverse effects on the embryo/fetus from unintentional exposure to the pharmaceutical. Regulatory guidance regarding the evaluation of reproductive and developmental toxicity is provided in the ICH S5A guidance document (www.fda.gov). For most pharmaceuticals, developmental toxicity studies are conducted in rodents and rabbits. However, for certain pharmaceuticals, the nonhuman primate is the only relevant species in which developmental toxicity studies can be conducted. This is particularly the case for many human therapeutic proteins that bind only to human and nonhuman primate receptors or antigens, and consequently developmental studies conducted in other species are not relevant for assessing human risk (see Chapter 6). Therefore, in order to evaluate potential adverse effects of these human therapeutic proteins on reproduction and development, nonhuman primate models have been developed that can address various aspects of the reproductive process (Vogel and Bee, 1999; Hendrickx et al., 2002, 2005; Weinbauer, 2002). The nonhuman primate most frequently used in developmental toxicity testing is the macaque. In the macaque (rhesus and cynomolgus), the gestation


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period is approximately 165 days and the period of organogenesis is from gestation day (GD) 20 through GD 50. The macaque has many physiological and endocrinological similarities to humans and therefore is a good model for human development. The design of the macaque developmental studies has been adapted from the rodent study designs that are well validated for risk assessment of potential developmental effects from exposure to drugs and chemicals. However, there are some major differences in the timing of the immune system development between primates and rodents, and the routes of exposure to pharmaceuticals that need to be taken into consideration when designing a developmental toxicity study in nonhuman primates versus rodents (Haley, 2003; Colucci et al., 2003; Holsapple et al., 2003). At the time of birth, macaques and humans have a fully developed immune system. This contrasts with rodents that have an immature immune system at the time of birth (Holladay and Smialowicz, 2000). This difference alone does not preclude the use of the rodent in developmental toxicity studies, so long as there is adequate species cross-reactivity of the test agent and the animals are dosed appropriately during critical periods in immune system development. Because a considerable amount of extrauterine immune system development occurs in the mouse versus the human or nonhuman primate, it may be necessary to dose the rodent pups during the postnatal period in order to mimic the human situation. This is not necessary for nonhuman primates where intrauterine immune system development mimics the human situation. The extrauterine exposure to drug in rodents may differ from that of intrauterine exposure in nonhuman primates because metabolic mechanism could differ in the extrauterine situation especially if the drug is being administered orally, and physiological influences will differ between the intrauterine and extrauterine situation, for example, pulmonary versus placental oxygen supply, nutrient supply, or different kidney function. To fully understand the critical periods of susceptibility for toxicity to the primate immune system, an understanding of the timings of the development of each of the major immune system organs is required. The following narrative briefly describes the development of the macaque immune system and discusses the relevance of these timings to developmental toxicity study designs. PRENATAL DEVELOPMENT OF THE NONHUMAN PRIMATE IMMUNE SYSTEM The embryonic immune organs are initiated as anlages, prior to their histological appearance. As described in Chapter 2.2, the key organs of the immune system are thymus, lymph nodes, spleen, bone marrow, and gastric-associated lymphoid tissue (GALT). Each of the lymphoid organs is characterized by a variety of specific cells including cells of the lymphoid (T and B lymphocytes and NK cells) and of the hematopoietic lineage (monocytes/interdigitating cells) (Figure 9.2-1).

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Pluripotent stem cell Myeloid stem cell (CFU-GEMM) BFUE

CFUEo

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3. Proliferation, maturation

CFU-M CFU-G Monocyte lineage

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Fetal Immune Organs

1. Matrix formation Entoderm Mesoderm

Figure 9.2-1 Fetal immune organs and involved cell lineages. The immune organ generation starts initially with formation of a mesodermal or entodermal matrix (1). Such represents the organ bin for stationary cells and free cells, and supplies the base for later organ function. In a second step, free cells invade from the myeloid (monocyte) and from the lymphoid stem cell lineage (2). The third step is that of proliferation and maturation of the specific immune cells within their specific immune organ (3). Such is differential in terms of cell types and needs fully functioning at birth. CFB = colonyforming unit; BFU = burst-forming unit.

The hematopoietic lineage, which includes the myeloid/myelomonocyte stem cell, appears to be expressed earlier than the lymphoid lineage. Initial hematopoietic activity is located in the aorta-gonad-mesonephros region but it becomes relocated into the liver in more advanced embryos. Because of the early key role of the hematopoietic system in myelopoietic activity, the liver is additionally mentioned as an early immune cell-generating organ. The time frame of immune organ generation in the cynomolgus monkey is illustrated in Figure 9.2-2 and the timing of the appearance of the immune cells within the immune organs is illustrated in Figure 9.2-3. The Liver and the Hematopoietic System The first functional organ generating free cells of the immune system is the hematopoietic system. In the cynomolgus monkey, hematopoiesis starts in week 2, i.e., prior to gestational day 14 (GD 14) while the conceptus is still at a pre-organogenetic phase. The liver bud becomes visible by GD 20–28 (weeks 3 to 4). Soon thereafter, hematopoiesis is taken over by the liver (in humans: GD 42–49 [from week 6 onwards]; Hinrichsen, 1990). This includes the supply of pluripotential stem cells that are the source for both lymphoid (T, B, NK cells) and myelomonocyte (macrophages, interdigitating cells) stem cells. Pluripotent stem cells and macrophages have been observed in the liver on GD

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GALT Bone marrow Lymph nodes Spleen Thymus Liver Extrahepatic Hematopoiesis 35 40

50

Organ bud/matrix formed Organ in structural differentiation Organ populating with specific immune cells

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85 100 Gestation Day (GD)

Figure 9.2.2 Time frame of immune organ generation in the macaque.

40 using antibodies toward CD117 (pluripotent stem cells) and CD68 (macrophages), whereas the lymphoid stem cell is not thought to be present at this stage. By GD 35, the liver is well developed and highly occupied with blood cell generation. The Thymus The thymus primordium is evident by GD 35 at which time it has formed as an epithelial bud in a medio-sagittal position cranial to the thoracic aperture. The thymus primordium elongates and lobulates, and by GD 40 forms a parenchyme meshwork with an endothelial ensheathment. By GD 50, the primordial thymus contains a polymorphic cell population and is on an obvious differentiation into cortex and medullary zones. The first T-like cells can be identified histologically by GD 50 but do not show CD3positive immunoreactivity (Buse et al., 2006). The first cells that have been identified immunohistochemically are those expressing Human Leukocyte Antigen-D Region (HLA-DR of the major histocompatibility complex, class II). These cells are found scattered throughout the medullary area (thymic primordium and mesenchyme). By GD 60, the medullary and cortical regions can easily be distinguished microscopically. The medulla contains primarily CD3-positive cells and immature Hassall’s bodies. The main zone of proliferation is the peripheral cortex. The identification of CD3 immunoreactivity signifies the onset of the prenatal thymus function of educating lymphocytes to discriminate self versus foreign proteins. From GD 70 onwards, macrophages (CD68) are observed in minute numbers evenly distributed over both cortex and medulla. By GD 85, B lym-

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=

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Lymph nodes

CD 138 CD 35 CD 4 CD 2

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Figure 9.2-3 Early evidence of cells identified with CD antibodies and with HLA-DR antibodies in the different immune organs and in the liver. All antibodies are claimed to be reactive in humans and hence, cross-reactive with cynomolgus monkey cells (see Table 9.2-1).

phocytes (CD20) are first observed at the cortico-medullary border. At later stages, the CD20-positive B cells increasingly populate the medulla. CD56 immunoreactive cells (natural killer cells) appeared faintly stained and are present in low numbers. Immunohistochemical analysis of GD 100 thymic tissues from cynomolgus macaques failed to identify T-helper cells (CD4) and T-suppressor cells (CD8) (Buse et al., 2006). However, CD4+ and CD8+ cells were identified from Hendrickx et al., (2002) in GD 80–145 fetuses by means of flow cytometry analysis of thymus tissue from rhesus macaques.

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The Spleen On GD 40, the spleen primordium is visible and consists of a minute mesenchyme cell accumulation. By GD 65, the spleen is heavily vascularized and the splenic matrix contains HLA-DR-immune-reactive cells. Most of the cells in the spleen by GD 65 are reported to be CD20-positive B cells and are dispersed throughout the spleen occasionally forming small loose aggregates. Relatively few cells are CD3-positive T cells. At this time, a distinct white pulp has not yet formed (Makori et al., 2003). Macrophages, identified by CD68-positive immune reactivity are scattered throughout the spleen. By GD 85, the organ displays both distinct red and white pulp areas, and an equal number of T and B cells is present. Through GD 100, there is an expansion of the white pulp. The lymphocyte distribution pattern is characterized by a white pulp predominantly made of T cells and a peripherally surrounding cover of B cells. There are no primary follicles at this stage. This distribution pattern remains preserved throughout the remainder of gestation with the additional positive immune reaction with dendritic cells that are located in the while pulp (Makori et al., 2003). Flow cytometry analysis of splenic tissues from rhesus macaques identified CD4+ and CD8+ cells in GD 80–145 fetuses with an increase in both subsets from GD 80 through GD 145 (Hendrickx et al., 2002).

The Lymph Nodes The description of the development of the lymph nodes focuses primarily on the mesenteric lymph node since this is the most commonly collected lymph node in toxicity studies. However, the development of the different lymph nodes does not occur simultaneously. The first lymphatic tissue primordium becomes evident as a number of inter-mesenchymal cavernae in the caudothoracic para-aorta mediastinum in the 35- to 40-day-old embryo. It is clearly identified as primordial lymphoid tissue by the presence of intermingled HLADR immunoreactive cells on GD 50 and is considered to, at least in part, give rise to the chylic cisterne. Further cavernae in the cervical, axillary, and inguinal region are apparent from GD 45 onwards. The sacculi of prospective nodes in the various body regions become filled up with reticulum tissue within the following 10 days or so. Early lymph node formation begins with the population of HLA-DR-positive cells followed by small numbers of CD3 (T lymphocytes) and CD20 (B lymphocytes) and a few macrophages (CD68) through GD 70. By GD 100, there is a distinct T cell differentiation toward CD2, CD3, and CD4 (T-helper cells) antigen expression. B cells have formed a thin line on the outer aspect of the T cell cortical region. Further B cell differentiation is apparent by CD35 (activated B cells) and CD138 (plasma cells) immune reactivity. The appearance of these cells correlates well with the presence of serum immunoglobulins such as IgM.

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The perinatal nodes from cynomolgus macaques contain primary follicles. T cells are centro-follicularly positioned whereas the B cell population is located toward the periphery. Macrophages are present in the medulla and dendritic cells are present in the paracortex (Makori et al., 2003). The Bone Marrow On GD50, the first bone cavity forms in the fetal clavicle. It displays a high prevalence of CD68-immunoreactive chondroclasts, which are an attributed subgroup of macrophages. From GD 60 onwards, a faint mesenchymal network fills the bone cavity with HLA-DR-positive matrix cells. Further bone cavities open up in the following weeks; for example, the femur is open before GD 85. The marrow on GD 85 has obviously taken over activity of hematopoiesis and in particular of B cell generation, as is evident from a small number of intensely immune-reactive B cells (CD20). B cells and in addition CD117 immune-reactive stem cells become increasingly prevalent until GD 100 and birth, whereas all subtypes of T cells and NK cells (CD56) remain absent. The Gut-Associated Lymphoid Tissues (GALTs) The intestine contains a variety of immune tissue, predominantly but by no means solely of follicular organization. On GD 60, disseminated HLA-DR immunoreactive cells are distributed within the mucosal lamina. On GD 70, there are HLA-DR immune-positive spots that are interpreted to be follicle predecessors, the earliest being observed in the area of the gastro-duodenal junction. Peyer’s patches are present on GD 100 and are characterized by a population of HLA-DR-, CD3-, CD20-, and CD68-immune-reactive cells. Prenatal Immunoglobulin Formation Activated B cells transform into plasma cells and thus become the source of immune globulin (Ig) production. Small numbers of immunoglobulin-secreting cells have been identified in the spleen from GD 80 through GD 145 fetuses (Makori et al., 2003). The number of IgM-secreting cells in the spleen exceeds the number of IgG-secreting cells. In contrast, the gut contains large numbers of IgG- and IgA-secreting cells and few IgM-secreting cells. IgG and IgM are detectable in fetal blood from GD 92 onwards. The IgM levels remain fairly constant throughout the later part of gestation and during a 6 months postnatal period (Buse, 2005). Since IgM does not readily cross the placenta, the IgM is considered to be of fetal origin. In contrast, IgG is well known to cross the placental barrier and therefore the increase in IgG levels from GD 92 onwards is most likely primarily of maternal origin. POSTNATAL DEVELOPMENT OF THE IMMUNE SYSTEM Although there is considerable information on the postnatal development of the rodent immune system, much less is known about the postnatal

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development of the nonhuman primate system (Holsapple et al., 2003; Hendrickx et al., 2005). However, based upon the prenatal developmental similarities of the macaque and the human immune system, it can be assumed that the postnatal development of the macaque immune system will also be similar to that of humans. At the time of birth, the primate immune system is fully developed but is devoid of antigenic exposure. The development of functional immune competence in primates requires exposure to specific antigens during the postnatal period. At the time of birth, macaques have IgG concentrations that approach that of the mothers (Fujimoto et al., 1983; Coe et al., 1993, 1994). As mentioned previously, although macaques have immunoglobulin-secreting cells from GD 80 onwards, most of the IgG in the neonatal blood at the time of birth is considered to be primarily of maternal origin. In humans, IgG concentrations in the neonates at the time of birth exceed that of the mothers suggesting active transport of IgG from mothers to fetus (Malek et al., 1996; Firan et al., 2001). The efficiency of IgG transport across the human placenta is IgG1 > IgG4 > IgG3 > IgG2 (Malek et al., 1994). It is this maternally derived repertoire of IgG molecules that affords the neonate with protection against infection during the first 6 months of life. IgA molecules secreted in the breast milk also provide protection against infection particularly from microbes in the gastrointestinal tract (Van de Perre, 2003). Therefore, maternal influences play a major role in the postnatal functioning of the immune defense. Only when these maternal influences have been eliminated after about 6-month postpartum can the independent functioning of the neonatal immune system be fully appreciated. The amount of antibody that is transferred across the nonhuman primate placenta is lower for new world primates than for old world primates (Coe et al., 1994). Therefore, if a new world primate is used for reproductive toxicity testing of a human monoclonal antibody, the fetal exposure may be less than that seen in old world primates or in humans. Based upon the preceding descriptions, and of descriptions of rodent and human immune system development, landmarks of particular vulnerability to immunotoxicity can be identified. These landmarks include organ anlagen and formation of primordia, migration and invasion of immune cells into organs, and differentiation and maturation of the immune organs in the developing organism (Dietert et al., 2000; West, 2002; Bommhardt et al., 2004). Therefore, study designs for evaluating developmental immunotoxicity should take these critical periods into consideration. For example, treatment-related interference in the process of T cell selection for discriminating self versus nonself could have serious adverse consequences for the offspring. With the development of novel immune-modulating therapeutic agents, which may affect aspects of the immune system at periods not traditionally defined as a critical period of susceptibility, novel methods of evaluation may need to be developed on a case-by-case basis to identify developmental immunotoxicity risk with these agents.

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METHODS FOR EVALUATING DEVELOPMENTAL IMMUNOTOXICITY IN NONHUMAN PRIMATES Methods for evaluating immunotoxicity in rodents are well established and are the basis of the ICH S8 guidance (ICH, 2006). Many of these methods have been adapted for use in developmental immunotoxicity studies in rodents (Dietert and Holsapple, 2007). Immunotoxicity testing in nonhuman primates, and in particular developmental immunotoxicity, has been requested by regulatory agencies for immunobiotherapeutics but the methods to date are not well established for nonhuman primates and vary to some extent from study to study. Study Design Considerations For nonhuman primates, a segmental approach to developmental testing using standard protocols may not be the most appropriate use of animals and may not provide the most meaningful data for assessing human risk. A more appropriate approach is to design each study on a case-by-case basis depending upon the mechanism of action of the test agent and the type of molecule being tested. The ICH-S5A regulatory guidance document “Detection of Toxicity to Reproduction for Medicinal Products” states that nonhuman primates “are best used when the objective of the study is to characterize a relatively certain reproductive toxicant, rather than to detect hazard.” The small group sizes in primate developmental studies, and the even smaller number of F1 animals available for evaluation, preclude any definitive assessment of hazard identification. However for biotherapeutics that cross-react only with nonhuman primates, the only relevant species is the nonhuman primate. In these instances, the studies need to be carefully designed so that the most relevant end points are incorporated. It should be noted that for many biopharmaceuticals, the intended pharmacological effect is a targeted modulation of the immune system. Therefore, the methods used to evaluate the developing immune system may be adapted from the methods used to identify biological effects in the adults. A number of protocols have been developed for the evaluation of developmental toxicity in nonhuman primates (Henck et al., 1996; Buse et al., 2003). For the evaluation of embryofetal development, confirmed pregnant macaques are treated with the test agent from GD 20 through GD 50 (period of organogenesis). As described in the previous section, the primordia of all of the major organs are present by GD 50. In embryofetal developmental toxicity studies, the pregnancies have previously been terminated by cesarean section on GD 100, at which time adequate anatomical evaluation for external, skeletal, and visceral malformations can be made. The fetuses are examined macroscopically for any major abnormalities. This basic study design, which has been adapted from the rodent and rabbit teratogenicity study designs, can identify maternal toxicity and teratogenic effects of small-molecular-weight drugs but

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may not be adequate for evaluating immunotoxins and in particular biopharmaceutical immunomodulators. This type of study design was, however, able to identify defects in thymus developed due to 13-cis-retinoic acid treatment (Hummler et al., 1990; Korte et al., 1993; Makori et al., 2002) and was also able to identify the abortifacient effects of treatment with recombinant forms of human interferon (Henck et al., 1996). Although the major organ systems are established by the end of organogenesis, the immune system continues to mature throughout the remainder of the gestation period and from birth through adolescence (Hendrickx et al., 2002; Buse, 2005; Buse et al., 2006). Therefore, the immune system is susceptible to modification by therapeutics throughout all stages of development according to its respective differentiation status in the different organs. Small-molecular-weight drugs have the potential to diffuse across the placenta and can therefore affect embryofetal development at all stages of gestation. The extent of exposure depends upon the physicochemical properties of the molecule (Van der Aa et al., 1998). For these molecules, if a nonhuman primate study is conducted, it is advisable to continue dosing throughout the embryonic and fetal periods to ensure that any potential effects on immune system development and maturation are detected. Conversely, large-molecular-weight drugs and proteins are too large to diffuse across the placenta and therefore direct developmental effects of these agents are rare. For example, certain human cytokines have been shown not to cross the human placenta (Reisenberger et al., 1996; Gregor et al., 1999; Aaltonen et al., 2005). For largemolecular-weight molecules, any effects seen in the developmental studies are likely to be maternal effects rather than effects resulting from fetal exposure. The one notable class exception, regarding molecular size and placental transfer, is that of antibodies that are transported across the placenta from mothers to fetuses (Simister, 2003). Binding of the constant region (Fc) of the antibody to a receptor on the placenta is a prerequisite for placental transfer of IgG molecules (Firan et al., 2001). In humans and nonhuman primates, this transfer occurs mostly during the third trimester (Figure 9.2-4). Therapeutic antibodies are transported across the placenta in a similar manner to naturally acquired antibodies. Fab molecules that lack the Fc portion of IgG are not transported across the placenta (Miller et al., 2003). The greatest period of susceptibility for Fc containing therapeutic molecules is therefore during the fetal period when maturation of the immune system occurs rather than the embryonic period when organogenesis occurs. Therefore, for antibody therapeutics, the basic embryofetal development study (teratogenicity study) where animals are treated only during organogenesis, is not an optimal study design. A more appropriate design would be to continue treatment until the time of the cesarean section, thereby increasing exposure during the fetal period. Continuing the pregnancy beyond GD 100 to allow additional maturation of the immune system to occur could also be considered. However, it should be noted that the macaque has two periods of susceptibility to pregnancy loss.


Fetal/Maternal Ratio

1.25

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Rhesus Cynomolgus

1.00 0.75 0.50 0.25

Birth

organogenesis

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Gestation Day Figure 9.2-4 Schematic representation of the ratio of fetal/maternal IgG concentrations throughout gestation in the macaque and relationship of the fetal antibody exposure relative to the period of organogenesis. Serum IgG concentrations shown in the graph have been adapted from data from Coe et al. (1993, 1994) and Fujimoto et al. (1983).

The first period is early in gestation when 30% or more of pregnancies may be lost due to spontaneous abortions (Hendrie et al., 1996). The second period is late in gestation when an additional 5–10% of pregnancies may be lost. Therefore, continuing treatment through to full-term pregnancy may result in fewer fetuses being available for evaluations than if the pregnancies were terminated at GD 100 through GD 120. For a therapeutic that has a long serum half-life, maternal exposure may persist for some time during the fetal period even when dosing is discontinued at the end of the embryonic period. However, the maternal serum concentrations will be decreasing during the fetal period and consequently the fetal exposure will be limited. Maintaining the serum concentrations at steady state during the embryonic and fetal period is optimal. Also for therapeutics with a long serum half-life, it may take many weeks for the drug to obtain steady state. In this case, a loading dose may be considered to ensure adequate maternal exposure during the early stages of pregnancy. This is particularly important for biotherapeutics that are administered subcutaneously because the Cmax may not occur until 2–3 days after the first dose by which time a critical period of susceptibility may be missed. For most biotherapeutics that do not cross-react with rodents or rabbits, an embryofetal development study in macaques is generally conducted during clinical development to support the inclusion of women of childbearing potential in clinical trials. However, postnatal development studies in nonhuman primates have not routinely been conducted. Consequently, the protocols and the end points are less well established for evaluation of postnatal effects on the immune system.

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End Points for Nonhuman Primate Developmental Immunotoxicity Assessment Many of the end points that have been validated for use in rodent immunotoxicity (ICH, 2006) assessment have been adapted for use in developmental immunotoxicity studies in nonhuman primates (Buse et al., 2003). Histopathology. Rodent embryofetal developmental toxicity studies (teratogenicity studies) do not routinely include histopathology as an end point. In embryofetal development studies, the fetuses are generally examined macroscopically and only those tissues with macroscopic observations are examined histopathologically. However, for therapeutics that are known to modulate the adult immune system, additional histopathological end points should be incorporated into the development studies. When the therapeutic cross-reacts only with the primate and the only study that will be conducted is a single developmental study, then it is important to include histopathology of lymphoid tissues (thymus, spleen, lymph nodes, bone marrow) as an end point if the pregnancies are not going to be carried through to full term. In addition to examination by light microscopy using standard hematoxylin and eosin (H&E) staining, immunohistochemical staining of lymphoid tissues can also be conducted to characterize observed or suspected changes in the immune system. It is obvious from the previous description of the development of the lymphoid tissues that the immune system interacts with a large number of cell types that cannot be fully discriminated with H&E stain alone. Thus, to address the timing of the appearance of the specific cell types and their distribution pattern within the developing immune system, an in-depth examination of the lymphoid tissues using antibodies directed toward cell specific antigens is required. The evaluation of the immune system in primate reproductive toxicity studies should include the identification of specific immune cell populations, taking into consideration the specificity of the detection antibodies for their target antigens in nonhuman primates and the commercial availability of the antibodies. Because of the close genetic similarities between humans and macaques, many of the human-specific antibodies show good cross-reactivity with macaque antigens. These human targeted antibodies may not necessarily cross-react with marmosets or rodent antigens. A few examples of commercially available antibodies used in macaque developmental studies are listed in Table 9.2-1. However, increasingly more specific questions have to be addressed in monkey studies with the introduction of therapeutics with novel structures and novel mechanisms of action. This may require new specifically constructed reagents (antibodies or lectins) for the evaluation of the immune system. The pluripotential stem cells may become a candidate of special interest for immunohistopathological evaluation in the near future. The immune system is characterized by high functional and regenerative capacity following


311

TABLE 9.2-1 Antibodies with Positive Immune Reactivity to Macaque Antigens Used to Identify Specific Cell Population in Macaque during Embryofetal Development Studies Antibody CD 2 CD 3 CD 4 CD 8 CD 20 CD 35 CD 56 CD 68 CD 117 CD 138 HLA-DR

Characteristic Cell Type

Supplier

T cells (pan) T cells (pan) T-helper cells T suppressor cells (cytotoxic T cells) B cells Activated B cells Natural killer cells Macrophages Stem cells Plasma cells Interdigitating cells

Neo Maker; Fremont Dako; Hamburg Zytomed; Berlin Medak; Wedel Biocare Medical; Concord Dako; Hamburg Dako; Hamburg Dako; Hamburg Dako; Hamburg Dako; Hamburg Dako; Hamburg Dako; Hamburg

immunologic challenge. Stem cells have regenerative capacity and therefore need to remain unaffected by drug treatments. Therefore, these cells should be a focus of attention in developmental immunotoxicology studies. One antibody of particular relevance is the CD117 antibody that can identify the basic stem cells of both hematopoietic (myeloid stem cell) and lymphoid lineage (lymphoid stem cell) (Hibi et al., 1991; Matsuda et al., 1993; Michie et al., 2000). The CD117-antibody listed in Table 9.2-1 has shown crossreactivity to fetal and adult cynomolgus monkeys in liver, lymph nodes, and bone marrow. This antibody, which also shows cross-reactivity with mouse (Michie et al., 2000) and human (Zuniga-Pflücker, 2004) antigens, could be a candidate for routine immunohistopathology. Flow Cytometry Analysis. Flow cytometry analysis of blood and tissues has the advantage of allowing enumeration of lymphocyte subsets (see Chapters 3.2 and 4.2). Standard hematology parameters and enumeration of lymphocyte subsets can be measured in serial blood samples from the dams and neonates and from the fetus at the time of cesarean sectioning. Figure 9.2-5 shows an example of lymphocyte subsets in dams throughout pregnancy and in fetuses (cord blood). At GD 100, the percent of T and B lymphocytes in the blood is similar in the dams and the fetuses, but the ratio of CD4+ to CD8+ lymphocytes is greater in the fetuses than in the dams. The greater proportion of CD4+ T cells versus CD8+ T cells in blood is consistent with the previously published immunophenotypic studies of the macaque spleen but not of the thymus (Hendrickx et al., 2002, 2005). In cynomolgus macaque blood there is no change in the percent of CD4+/CD8+ cells from 2 months of age postpartum to 6 months of age, and the ratios are similar to those seen in the adult (Martin et al., 2007).

312

DEVELOPMENTAL IMMUNOTOXICITY IN PRIMATES 100

% CD20+ B cells

% CD3+ T Cells

100

75

50

25

0

75

50

25

0 GD20

GD35

GD52

GD100

Fetal

GD20

Maternal

GD52

GD100

Fetal

GD100

Fetal

Maternal 100

% CD8+ T Cells

100

% CD4+ T Cells

GD35

75

50

25

0

75

50

25

0 GD20

GD35

GD52

Maternal

GD100

Fetal

GD20

GD35

GD52

Maternal

Figure 9.2-5 Percent of B and T lymphocytes determined by flow cytometry analysis in pregnant cynomolgus macaque and fetal (cord) blood. Each bar represents the mean ± SEM of data from 14 pregnant macaques and six fetuses.

Immune Function Testing. The advantage of continuing the pregnancies through to full term is that the neonates can be evaluated for functional competence of the immune system for a time after birth. The immune function tests that have been applied to neonates have been adapted from immune function tests that have been conducted in adult animals. There currently is no standard approach to immune function testing in the primate and consequently the type of test conducted varies from laboratory to laboratory. Evaluation of a T cell-dependent antibody response (TDAR) is a critical component of the evaluation of immune function in the neonates. In this assay, neonates are injected with a T-dependent antigen, usually keyhole limpet hemocyanin (KLH) or tetanus toxoid (TTX), and the development of antibodies is measured over time. In monkeys there can be high inter-animal variability so the collection of serial blood samples is important and the data can be expressed as the area under the antibody titer versus time curve. Figure 9.2-6 shows examples of TDAR results in cynomolgus macaques following immunization with KLH or TTX. The antigens for immunization in this study were administered on one occasion and in the absence of adjuvant. Alternate immunization procedures have involved single or multiple injections of the antigen in incomplete Freund’s adjuvant. The presence of the adjuvant results

313

SUMMARY

Anti-KLH Titer IgM IgG

Anti-TTX Titer

Anti-KLH Titer

100

Anti-TTX Titer 1000

10

1

0

IgM IgG

100 10 1 0

140

150

160

Day After Birth

170

140

150

160

170

Day After Birth

Figure 9.2-6 Anti-KLH and anti-TTX antibody titers in infant macaques. Macaques were injected with KLH (2 mg/kg subcutaneously) and TTX (6 Lf intramuscularly) at 140 days of age. Anti-KLH and anti-TTX antibodies were measured using an ELISA.

in a more robust antibody response than when the antigen is administered alone. Assays to measure cell-mediated immunity (e.g., DTH) in macaques have produced inconsistent results. The antigens used in these studies and the concentrations of antigens used have varied between laboratories and the results have been highly variable. Most laboratories have used a cocktail of antigens that includes diphtheria, trichophyton, and Candida albicans in incomplete Freund’s adjuvant (Price et al., 2004 and Chapter 3.1.3).

SUMMARY The macaque has proven to be a valuable species for the evaluation of developmental immunotoxicity. The development of the macaque immune system is similar to that of humans with the exception that after the first 35 days of development, it proceeds at an accelerated rate relative to human immune development. At the time of birth, the macaque, like the human, has a fully developed immune system. Immunotoxicity in the macaque can be evaluated by immunohistochemistry, immunophenotyping, in vivo functional immune testing, and by a variety of ex vivo functional immune tests (Buse et al., 2003). Because of the phylogenic similarities between humans and nonhuman antigens (Mestas and Hughes, 2004), many of the antibodies developed against human targets show good cross-reactivity with nonhuman primates, especially macaques, and are therefore valuable tools for use in immunohistological and immunophenotyping studies in fetal and adult tissues and blood. For therapeutics that do not cross-react with rodent or rabbits, the only suitable alternate may be the nonhuman primate. In these cases, the study

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designs need to be carefully considered in order to maximize the amount of useful information that can be obtained per animal while minimizing the use of animals. Study designs that involve evaluation of additional immunotoxic end points or evaluating different durations of dosing may need to be considered on a case-by-case basis depending upon the molecule. Small-molecular-weight drugs that can diffuse across the placenta and can be secreted in milk have the potential to affect all aspects of development. In this case, the nonhuman primate may be used to define the critical window for toxicity. For monoclonal antibodies or fusion proteins that contain the Fc portion of human immunoglobulins, the major period of exposure will be during the fetal period and therefore, study designs that focus more on preand postnatal development rather than on embryonic development may be more appropriate. For other large-molecular-weight proteins that do not diffuse across the placenta and are not transported across the placenta, for example certain cytokines and Fab molecules, any effects are likely to be maternal effects rather than direct fetal effects and therefore studies, if conducted, need to be designed accordingly. Clearly the nonhuman primate can provide valuable and relevant information on the effects of human therapeutics on the development of the immune system. In order to optimize the use of animals and to obtain the most meaningful information for the human therapeutic, each study may need to be designed on a case-by-case basis. Nevertheless, there is a need for greater standardization of the tests used to evaluate immunotoxicity in nonhuman primates.

REFERENCES Aaltonen R, Heikkinen T, Hakala K, Laine K, Alanen A. Transfer of proinflammatory cytokines across the term placenta. Obstet Gynecol 2005;106:802–808. Bommhardt U, Beyer M, Hünig T, Reichardt HM. Molecular and cellular mechanisms of T-cell development. Cell Mol Life Sci 2004;61:263–280. Buse E. Development of the immune system in the cynomolgus monkey: the appropriate model in human targeted toxicology? J Immunotoxicol 2005;2:211–216. Buse E, Habermann G, Osterburg I, Korte R, Weinbauer GF. Reproductive/developmental toxicity and immunotoxicity assessment In the nonhuman primate model. Toxicology 2003;185:221–227. Buse E, Habermann G, Vogel F. Thymus development in Macaca fascicularis (Cynomolgus monkey): an approach for toxicology and embryology. J Mol Hist 2006; 37:161–170. Coe CL, Kemnitz JW, Scneider ML. Vulnerability of placental antibody transfer and fetal complement synthesis to disturbance of the pregnant monkey. J Med Primatol 1993;22:294–300.

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Coe CL, Lubach GR, Izard KM. Progressive improvement in the transfer of maternal antibody across the order primates. Am J Primatol 1994;32:51–55. Colucci F, Caligiuri MA, DiSanto JP. What does it take to make a natural killer? Nat Rev Immunol 2003;3:413–425. Dietert RR, Holsapple MP. Methodologies for developmental immunotoxicity (DIT) testing. Methods 2007;41:123–131. Dietert RR, Etzel RA, Chen D, Halonen M, Holladay SD, Jarabek AM, Landreth K, Peden DB, Pinkerton K, Smialowicz RJ, Zoetis T. Workshop to identify critical windows of exposure for children’s health: Immune and Respiratory Systems Work Group summary. Environ Health Perspect 2000;108:483–490. Firan M, Bawdon R, Radu C, Ober RJ, Eaken D, Antohe F, Ghetie V, Ward ES. The MHC class I-related receptor FcRn, plays and essential role in the maternofetal transfer of γ-globin in humans. Int Immunol 2001;13:993–1002. Fujimoto K, Terao K, Cho F, Honjo S. The placental transfer of IgG in the cynomolgus monkey. Jpn J Med Sci Biol 1983;36:171–176. Gregor H, Egarter C, Levin D, Sternberger B, Heinze G, Leitich H, Reisenberger K. The passage of granulocyte-macrophage colony-stimulating factor across the human placenta perfused in vitro. J Soc Gynecol Invest 1999;6:307–310. Haley PJ. Species differences in the structure and function of the immune system. Toxicology 2003;188:49–71. Henck J, Hilbish KG, Serabian MA, Cavagnaro JA, Hendrickx AG, Agnish ND, Kung AHC, Mordenti J. Reproductive toxicity testing of therapeutic biotechnology agents. Teratology 1996;53:185–195. Hendrie TA, Peterson PA, Short JJ, Tarantal AF, Rothgarn E, Hendrie MI, Hendrickx AG. Frequency of prenatal loss in a macaque breeding colony. Am J Primatol 1996;40:41–53. Hendrickx AG, Makori N, Peterson P. The nonhuman primate as a model of developmental immunotoxicity. Hum Exp Toxicol 2002;21:537–542. Hendrickx AG, Peterson PE, Makori NM. The nonhuman primate as a model of developmental immunotoxicity. In Developmental Immunotoxicology, edited by Halladay SD, pp. 117–136. Boca Raton, FL: CRC Press, 2005. Hibi K, Takahashi T, Sekido Y, Ueda R, Hida T, Ariyoshi Y, Takagi H, Takahashi T. Coexpression of stem cell factor and c-kit genes in small-cell lung cancer. Oncogene 1991;6:2291–2296. Hinrichsen KV. Intestinaltrakt. Human-Embryologie. Berlin, Germany: Springer-Verlag, 1990. Holladay SD, Smialowicz RJ. Development of the murine and human immune system: differential effects of immunotoxicants depend on the time of exposure. Environ Health Perspect 2000;108:463–473. Holsapple MP, West LJ, Landreth KS. Species comparison of anatomical and functional immune system development. Birth Defects Res 2003;68:321–334. Hummler H, Korte R, Hendrickx AG. Induction of malformations in the cynomolgus monkey with 13-cis retoic acid. Teratology 1990;42:263–272. ICH (International Conference on Harmonization). Guidance for Industry. S8 Immunotoxicity Studies for Human Pharmaceuticals, 2006.

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Korte R, Hummler H, Hendrickx AG. Importance of early exposure to 13-cis retinoic acid to induce teratogenicity in the cynomolgus monkey. Teratology 1993; 47:37–45. Makori N, Peterson PE, Lantz K, Hendrickx AG. Exposure of cynomolgus monkey embryos to retinoic acid causes thymic defects: effects on peripheral lymphoid organ development. J Med Primatol 2002;31:91–97. Makori N, Tarantal AF, Lü FX, Rourke T, Mathas ML, McChesney MB, Hendrickx AG, Miller CJ. Functional and morphological development of lymphoid tissues and immune regulatory and effector function in Rhesus monkeys: cytokine-secreting cells, immunoglobulin-secreting cells, and CD5+ B-1 cells appear early in fetal development. Clin Diag Lab Immunol 2003;10:140–153. Malek A, Sager R, Schneider H. Maternal-fetal transport of immunoglobulin G and its subclasses during the third trimester of human pregnancy. Am J Reprod Immunol 1994;32:8–14. Malek A, Sager R, Kuhn P, Nicholaides KH, Schneider H. Evolution of maternofetal transport of immunoglobulins during human pregnancy. Am J Reprod Immunol 1996;36:248–255. Martin PL, Oneda S, Treacy G. Effects of an anti-TNFα monoclonal antibody, administered throughout pregnancy and lactation, on the development of the macaque immune system. Am J Reprod Immunol 2007;58:138–149. Matsuda R, Takahashi T, Nakamura S, Sekido Y, Nishida K, Seto M, Seito T, Sugiura T, Ariyoshi Y, Takahashi TRU. Expression of the c-kit protein in human solid tumors and in corresponding fetal and adult normal tissues. Am J Pathol 1993; 1:339–346. Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;224:2731–2738. Michie AM, Carlyle JR, Schmitt TM, Ljutic B, Cho SK, Fong Q, Zuniga-Pflücker JC. Clonal characterization of a bipotent T cell and NK cell progenitor in the mouse fetal thymus. J Immunol 2000;164:1730–1733. Miller RK, Mace K, Polliotti B, DeRita R, Hall W, Treacy G. Marginal transfer of ReoPro (abciximab) compared with immunolglobulin G (F105), Inulin and water in the perfused human placenta in vitro. Placenta 2003;24:727–738. Price KD, Mezza L, Diters R, Wells S, DeVona D, Tzogas Z, Haggerty H. Development and immunomodulation of delayed-type hypersensitivity (DTH) in cynomolgus monkeys. 2004;78:431. Reisenberger K, Egarter C, Vogl S, Sternberger B, Kiss H, Husslein P. The transfer of interleukin-8 across the human placenta perfused in vitro. Obstet Gynecol 1996; 87:613–616. Simister NE. Placental transport of immunoglobulin G. Vaccine 2003;21:3365–3369. Van de Perre P. Transfer of antibody via mother’s milk. Vaccine 2003;21:3374–3376. Van der Aa E, Copius Peereboom-Stegeman JHJ. Noordhoek J, Gibnau WJ, Russel FGM. Mechanisms of drug transfer across the human placenta. Pharm World Sci 1998;20:139–148. Vogel F, Bee W. Reproduction toxicology in primates: an overview of methods and techniques. In: Reproduction in Nonhuman Primates, edited by Weinbauer GF, Korte R, pp. 95–110. Münster, Germany: Waxmann Press, 1999.

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PART X NEW METHODS IN ASSESSING IMMUNOMODULATION, IMMUNOTOXICITY, AND IMMUNOGENICITY

10.1 ALTERNATIVE ANIMAL MODELS FOR IMMUNOMODULATION AND IMMUNOTOXICITY Peter J. Bugelski

Studies in normal intact animals, especially in rodents, play a critical role in hazard identification and characterization and risk assessment of drugs with immunomodulatory or immunotoxic activity. Most xenobiotics (the term is used to identify small molecule hydrocarbon agents 1000 Daltons MW such as peptides, proteins, and nucleic acids) are immunoactive in nonhuman primates. However, they may have little effect in rodents, e.g., the difference in the structure–activity relationship for CpG oligonucleotides in primates and rodents (Verthelyi and Klinman, 2003) and the failure of many human directed monoclonal antibodies to cross-react in rodents. Moreover, there are important limitations on the data that can be obtained from conventional laboratory animals as described in Table 10.1-1. Many biopharmaceuticals are active only in primates. In this case, there is no scientific rationale to study immunoactivity in rodents and one is limited to studies in primates. This circumstance brings another set of limitations. These include immunogenicity of human proteins in nonhuman primates; availability of adequate numbers of age-matched, mature test animals; imprac-


321

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ALTERNATIVE ANIMAL MODELS

TABLE 10.1-1 Limitations on the Data on Immunoactive Agents Obtained from Conventional Laboratory Animals Theme Species Differences in ADME

Topic Bioavailability/ Clearance Phase I and Phase II metabolism

Affinity for FcRN

Immunogenicity

Natural antibodies

Seroconversion

Immune tolerance

Species Differences in Immune Function

Immunodominance

Different T Cell subsets T Cell Receptor utilization

MHC Class I and Class II Immunoglobulin light chain variable sequence

Evidence

Reference

Large cross species differences in exposure Differences in expression and catalytic activity of drug metabolic enzymes across species Differential binding of human IgG to human and murine FcRN Naturally occurring heterophillic antiimmunoglobulin antibodies Human proteins are commonly immunogenic in laboratory animals PEG conjugated uricase highly tolerogenic in mice, still immunogenic in rabbits Differences in immunodominant epitopes of botulinum toxin across mice, horses, and humans CD4-CD8 double-positive lymphocytes common in monkeys BC1-BJ1 common in a new world monkey (Callithrix jacchus) compared to BC2-BJ2 in humans Limited homology in monkeys at the DNA level Some differences despite high degree of homology between human and cynomolgus monkey

Sakai et al. (1989) Gonzalez and Yu (2006)

Vaccaro et al. (2006) Hennig et al. (2000)

Bugelski and Treacy (2004) Savoca et al. (1984)

Atassi et al. (1996)

Zuckermann (1999) Uccelli et al. (1997)

Mwenda et al. (1997) Lewis et al. (1993)

ticality of conducting studies with infectious organisms; and the unavailability of a model of host defense against neoplasia. When a biopharmaceutical is active only in primates (in some cases only in chimpanzees), an alternative approach is to create a rodent homologue (or

323

GENE DEFECT AND KNOCKOUT RODENTS

TABLE 10.1-2 Potential Issues with the Applicability of Homologous Immunomodulatory Biopharmaceuticals Attribute Efficacy

Role of target in immune function Fc functionality Stability Purity Immunogenicity

Potential Issue The epitope bound by the rodent homologue monoclonal antibody may not express the same functional activity as the human biopharmaceutical The therapeutic target may not subserve the same function in rodents and humans The rodent homologue may not express the same Fc functionality as the human biopharmaceutical The rodent homologue may not have sufficient stability to allow testing It may be impractical to produce the homologue with sufficient purity The rodent homologue may be immunogenic in rodents

surrogate) biopharmaceutical that is active in rodents and use the standard models for immunotoxicity testing as discussed previously (see Chapter 6.2). However, in addition to the limitations listed in Table 10.1-1, the use of homologues presents additional issues that are listed in Table 10.1-2. Taken together, the issues raised in the preceding paragraphs suggest that with some immunoactive xenobiotics and biopharmaceuticals, there is a need for alternate approaches to evaluating immunomodulation or immunotoxicity. Some of the limitations of conventional models are unlikely to ever be resolved by any feasible alternative, e.g., drug clearance is inherently quicker in smaller animals. Others, e.g., T cell receptor-dependent effects, may be addressable by genetic manipulation or by transplantation or reconstitution of rodents with human cells. The methods of addressing these limitations will be a major thrust of this chapter.

GENE DEFECT AND KNOCKOUT RODENTS Gene defect and knockout are typically defined as homologous recombinations that result in disruption of a gene and thus, lack of expression of the gene product. In this chapter, the term will be used more generally for any recombinant event that down-regulates gene expression in mice. Such mice have proven to be very important in aiding our understanding of the role of the various elements of the immune system in host defense and autoimmunity. Genetic alterations can arise spontaneously (e.g., MRL-lpr mice; Tsubata, 2005), can be the result of chemical mutagenesis (e.g., N-ethyl-N-nitrosourea; Hoebe and Beutler, 2005), or the result of site-directed genetic manipulation (e.g., glucocorticoid receptor knockouts; Reichardt, 2004). In the context of

324


immunotoxicology, gene defect and knockout mice can provide an important “proof of concept” that an agent that acts by a given mechanism may exert an immunotoxic effect (Wang et al., 2003). However, observations made with such mice need to be approached with caution as both false-positive and false-negative results may be obtained. A variety of compensatory or adaptive changes are possible. For example, although severe combined immunodeficient (SCID) mice lack functional B and T cells, it is well known that they express good natural killer (NK) and macrophage (mφ) activity, and are thus resistant to metastases (Bancroft and Kelly, 1994). Similarly, although beige (bg/bg) mice are deficient in NK activity, they can mount a heightened T cell response to lymphocytic choriomeningitis virus (LCMV) (Biron et al., 1987) but show increased susceptibility to Candida albicans, probably through a defect in granulocyte function (Ashman and Papadimitriou, 1991). Moreover, as the defect is absolute, it may overpredict the effects of an agent that demonstrates a dose-dependent effect. A wide variety of gene defect and knockout mice have been described and a number of examples of relevance to immunotoxicology can be found. Beige (bg/bg) mice, a model of Chediak-Higashi Syndrome have a autosomal defect in chromosome 13 that codes for a 3801 amino acid highly conserved protein that results in impairment of NK and granulocyte function (Shiflett et al., 2002). As shown in Table 10.1-3, bg/bg mice show increased susceptibility to a number of types of infectious organisms and can be used to demonstrate the effects of immunoactive agents. The beige defect has also been described in rats but data in this model are much more limited (Nishimura et al., 1989). Nude mice (nu/nu) have a defect in expression of forkhead transcription factor FoxN1 that results in impairment of T cell development (Balciunaite et al., 2002). As shown in Table 10.1-4, nu/nu mice show increased susceptibility to a number of infectious organisms and tumors and decreased effectiveness

TABLE 10.1-3 Examples of the Application of Beige Mice Observations Increased susceptibility to Pseudomonas aeruginosa Increased susceptibility to Cryptosporidium Increased susceptibility to Coccidioides immitis Increased susceptibility to Cryptococcus neoformans Inhibition of tumor necrosis factor alpha further increases susceptibility Mycobacterium avium complex Loxoribine fails to enhance NK activity IL-10 fails to inhibit metastases of Lox human melanoma Interleukin-12 expresses prophylactic and therapeutic activity in herpes simplex virus infection

Reference Tanaka et al. (1994) Enriquez and Sterling (1991) Clemons et al. (1985) Marquis et al. (1985) Bala et al. (1998)

Pope et al. (1994) Zheng et al. (1996) Carr et al. (1997)

325

GENE DEFECT AND KNOCKOUT RODENTS

TABLE 10.1-4 Examples of the Application of Nude Mice Observations Efficacy and toxicity of antimelanoma immunocytokine scFvMEL/TNF in melanoma-bearing mice Exacerbation of viral hepatitis by hexachlorobenzene Decreased suppression of the humoral response to dinitrophenol (a T-independent antigen) by 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin Increased susceptibility to Coccidioides immitis Increased susceptibility to systemic Cryptococcus neoformans 7-Allyl-8-oxoguanosine (loxoribine), a di-substituted guanine ribonucleoside, mediates B cell proliferation IL-10 inhibits metastases of B16-F10 and Lox human melanoma IL-12 expresses reduced antitumor effects against B16F10 melanoma, M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma

Reference Liu et al. (2006) Carthew et al. (1990) Kerkvliet and Brauner (1987) Clemons et al. (1985) Salkowski and Balish (1990) Pope et al. (1994) Zheng et al. (1996) Brunda et al. (1993)

TABLE 10.1-5 Examples of the Application of Severe Combined Immunodeficiency Mice Observations huKS1/4-IL2 (antibody-cytokine fusion protein) inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma 7-Allyl-8-oxoguanosine (loxoribine), a di-substituted guanine ribonucleoside, enhances NK activity Efficacy and toxicity of proteasome inhibitor (benzyloxycarbonylleucyl-leucyl-phenylalaninal) in Burkitt’s lymphoma-bearing mice

Reference Dolman et al. (1998) Pope et al. (1994) Orlowski et al. (1998)

of immunotherapy. Nude rats have also been described, but as with beige rats, data are more limited (Krueger et al., 1985; Noble and Norbury, 1986). More recently, a model that lacks functional T and B cells (SCID mice) has been described. SCID mice have defective DNA recombinase that results in an inability to make functional antigen receptors (Kotloff et al., 1993; Løvik, 1995). As shown in Table 10.1-5, SCID mice have been used in a variety of test systems where a profound immunodeficiency is required. To further impair immune function, the defects described above have also been combined through selective crossbreeding, resulting in beige-nude and SCID-beige mice. These mice lack T and NK cells and T, B and NK cells, respectively. Examples of how these mice have been used are shown in Tables 10.1-6 and 10.1-7. In addition to broad cellular defects described above, a number of other immunodeficient mice created by genetic engineering have been studied.

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TABLE 10.1-6 Examples of the Application of Beige-Nude Mice Observations Efficacy and toxicity of murine anti-VEGF monoclonal antibody in A673 human rhabdomyosarcoma-bearing mice Increased susceptibility to Plasmodium falciparum Increased susceptibility to systemic Cryptococcus neoformans Increased susceptibility to Candida albicans NK activity cannot be enhanced by poly I:C; failure to proliferate in response to concancavalin A have an impaired response to lipopolysaccharide CpG-oligodeoxynucleotide 1826 (CpG) induced antitumor effects in B16 melanoma

Reference Mordenti et al. (1999) Moreno Sabater et al. (2005) Salkowski and Balish (1990) Cantorna and Balish (1990) Pflumio et al. (1989) Buhtoiarov et al. (2007)

TABLE 10.1-7 Examples of the Application of SCID-Beige Mice Observations IL-10 inhibits metastases of Lox human melanoma Increased susceptibility to Sendai virus Increased susceptibility to hepatitis C virus Transfection with interferon-γ protects from lethal mycobacterial infection Increased susceptibility to Yersinia pestis Rapamycin increases survival in mice bearing a renal adenocarcinoma (renal cancer) of BALB/c origin and T24 human bladder transitional cell carcinoma Persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in SCID-bg mice, depleted of granulocytes

Reference Zheng et al. (1996) Percy et al. (1994) Walters et al. (2006) Xing et al. (2001) Green et al. (1999) Luan et al. (2002) Dolman et al. (1998)

In Table 10.1-8, a number of knockouts of cell surface differentiation markers (Cluster of Differentiation, CD) are listed while in Table 10.1-9, a number of cytokine and miscellaneous knockouts are listed.

RECONSTITUTED MODELS Some of the first attempts in generating murine models that would more closely mimic human immune responses were made in irradiated mice trans-

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TRANSGENIC AND KNOCK-IN ALTERNATIVE MODELS

TABLE 10.1-8 Examples of CD Knockouts Knockout

Observations

CD1 CD2 CD4 CD8 CD9 CD14 CD24 CD25 CD28 CD34

Decreased immunogenicity of lipoid antigens Decreased T cell function Decreased T-helper function Decreased T-suppressor/killer function Decreased granuloma formation Decreased macrophage signaling Decreased function of CXCR4 Decreased regulator T cell function Decreased antiviral host defense Impaired bone marrow stem cell homing

CD38 CD40 CD44

Decreased NADP Increased susceptibility to Pneumocystis cariini Enhanced T cell response to conventional and superantigens Increased signaling Decreased airway hyper-responsiveness Altered susceptibility to malaria sporozoites Decreased airway hyper-responsiveness Renal failure Decreased cell-mediated immunity

CD45 CD80 CD81 CD86 CD151 CD154

Reference Hong et al. (1999) Ding et al. (1996) Senaldi et al. (1999) Senaldi et al. (1999) Yamane et al. (2005) Perera et al. (1997) Schabath et al. (2006) Sharma et al. (2007) Fang and Sigal (2006) Gangenahalli et al. (2006) Chini et al. (2002) Furuta et al. (2001) McKallip et al. (2002) Alexander (2000) Mathur et al. (1999) Silvie et al. (2006) Mathur et al. (1999) Sachs et al. (2006) Grewal and Flavell (1998)

planted with human bone marrow stem cells. Although met with some success, issues with difficulty in obtaining suitable bone marrow samples, reproducibility, and a relative immunodeficiency (compared to normal humans or mice) render such models of limited practicality in an immunotoxicology setting. More recently, in addition to the applications of gene defect and knockout mice for proof of concept and for dissection of the role of specific cells or gene products in immune function described above, these mice are also being used as the “hosts” for reconstitution of the immune system with human cells. Some examples are listed in Table 10.1-10. TRANSGENIC AND KNOCK-IN ALTERNATIVE MODELS Transgenic and knock-in technologies permit the addition of relevant human genes or the replacement of mouse genes by their human ortholog (Løvik, 1997; Pascolo, 2005). By these processes, it is possible to produce mice in which some aspects of drug metabolism or immune function are mediated by the relevant human gene product. These model systems are being used with increasing frequency for evaluation of immunoactive agents and human

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TABLE 10.1-9 Examples of Cytokine and Miscellaneous Knockouts Knockout Interferon-γ Interferon-γ

IL-10

IL-12 Immunoglobulin heavy and light chain alpha1,3Galactosyltransferase Glucocorticoid receptor Annexin 1 (lipocortin 1) Id (inhibitor of differentiation) proteins Toll-like receptors

Observations Increased susceptibility to Francisella tularensis Increased susceptibility to Mycobacterium avium. No additional effect of anti-asialoGM1 Increased susceptibility to Mycobacterium avium. Not protected by G-CSF Increased susceptibility to Francisella tularensis Lack of functional B cells Production of anti-Gal antiblood group antibodies Numerous effects in various cell types Exaggerated inflammatory response Numerous effects in various cell types Increased susceptibility to various pathogens

Reference Duckett et al. (2005) Florido et al. (1997)

Goncalves and Appelberg (2001) Duckett et al. (2005) Lonberg and Huszar (1995) Galili (2004) Reichardt (2004) Roviezzo et al. (2002) Sugai et al. (2004) Akira and Takeda (2004)

biotherapeutics. A variety of human genes coding for proteins important immune function have also been engineered into mice. Some examples are listed in Table 10.1-11. Although not all human gene products will subserve their function in mice, some are functional and can essentially replace the murine protein.

MULTI-GENE ENGINEERING AND CONDITIONAL KNOCKOUTS As described above, a wide variety of gene defect, knockout and knock-in mice and rats are being used to evaluate immune function. By and large, these are single gene effects, either eliminating expression of a target gene or adding expression of another. In the early days of transgenic animals, the goal was to ensure high-level expression of the transgene gene product. To achieve this, many investigators incorporated very potent promoter sequences into the transgene vector, e.g., simian virus-40 (SV-40) promoter. Although this usually resulted in high-level gene expression, it also could lead to ectopic gene expression, e.g., in unnatural locations (cells or tissues) or at inappropriate times in ontogeny. This issue has largely been eliminated by selecting more “natural”

329

MULTI-GENE ENGINEERING AND CONDITIONAL KNOCKOUTS

TABLE 10.1-10 Examples of Reconstituted Systems Useful for the Study of Immune Function System SCID mice engrafted with human fetal thymus and liver tissue fragments Fetal guinea pig liver, thymus, spleen in SCID-bg mice SCID-beige mice as an experimental animal system for the acceptance of human leukocyte xenografts

Human NK cells in irradiated SCID-bg mice Human NK cells in SCID-bg mice Beige/nude/xid mice carrying human lymphoid xenografts Rag2(−/−)gamma(c)(−/−) mice that are neonatally injected with human CD34+ cells

Observations Toxicity of 2-acetyl-4(5)-(1,2,3,4tetrahydroxybutyl)-imidazole and the organotin compound, di-nbutyltin dichloride Presence of guinea pig IgG in serum

Presence of human macrophages (CD68+), T cells (CD43+), and B cells (CD20+) in lung, spleen, lymph node, and thymus. Functional immune system was demonstrated by the ability to respond to immunization with KLH Protection from intravenously (i.v.) injected K562 leukemia cells Inhibition of growth of Hsp70expressing and non-expressing CX(+) and CX(−) tumor cells Exogenous but not endogenous EBV induces lymphomas Human T, B, and dendritic cells are present in peripheral blood, thymus, spleen, and lymph nodes. CCR5 and CXCR4 are expressed on human immature and mature T cells. DKO-hu HSC mice allow efficient HIV-1 infection with plasma high viremia

Reference de Heer et al. (1995)

Shibata et al. (1997) McBride et al. (1995)

Guimaraes et al. (2006) Multhoff et al. (2000) Dosch et al. (1991) Zhang et al. (2007)

promoters to associate with the transgene. Single transgene systems are beginning to give way to more complex systems where in addition to knocking out selected genes, multiple transgenes are being engineered in to give a more faithful representation of human immune function. Some examples are listed in Table 10.1-12. Another area where transgenic mice of greater sophistication are coming on line is conditional knock-ins and conditional knockouts. In conditional knock-in systems, expression of the transgene is under the control of a promoter that functions only when the animal is exposed to a pharmacologic

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TABLE 10.1-11 Examples of Transgenic/Knock-in Systems Useful for the Study of Immune Function Type of Gene Product

Transgene/ Knock-In

Immunoglobulin

Human germline heavy- and kappa lightchain minilocus transgenes in KO mice

Cytokine

Human TNF-α (Tg197) transgenic mice MHC I peptide epitopes

Histocompatibility Antigens

HLA-B27

Cluster of Differentiation Antigens

HuCD1d

HuCD2

HuCD3

HuCD4

HuCD8

HuCD46

Observations The transgenes rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation in response to antigen stimulation Express polyarthritis and bone marrow dysplasia Comparison between mouse and human MHC I antigen processing Spondyloarthropathy arises spontaneously in HLAB27 transgenic rats. In contrast, HLA-B27 transgenic mice have usually remained healthy Expression of hCD1d on thymocytes but not on APCs in MuCD1d KO, was sufficient to select Valpha14i NKT Used to confirm role of FcγR in activity of alphacept Decreased cytokine storm from Hu/Mu chimeric antiCD3-IgM compared to murine antiCD3 Human CD4 mediates rescue of the CD4 lineage and restoration of normal helper cell functions Increased responsiveness to MHC Class I antigens Human-like cytokine response to N. meningititis

Reference Lonberg and Huszar (1995)

Capocasale et al. (2007) Pascolo (2005) Breban et al. (2004)

Schumann et al. (2005)

da Silva et al. (2002) Choi et al. (2002)

Killeen et al. (1993) LaFace et al. (1995) Johansson et al. (2005)

331

MULTI-GENE ENGINEERING AND CONDITIONAL KNOCKOUTS

TABLE 10.1-12 Selected Examples of Multiple Transgene Mice Type of Gene Product

Transgene/ Knock-In

Observations

Cluster of Differentiation

HuCD46/HuCD150

Show human-like infections with measles virus

T cell receptor (TCR) + Ig

2D2 myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor crossed with MOG-specific IgH knock-in HLA-DQ-HuCD4

Spontaneously develop a severe form of experimental autoimmune encephalomyelitis (EAE)

MHC Class II + CD

HLA-B27-HuCD8

MHC Class II + CD + TCR

HLA DR4-DQ3 haplotype transgenic crossed to HuCD4 Tg mice and endogenous class II knockout mice HLA-DQ8, HLADQ6, and CD4 KI HLA-DR2-T cell receptor (TCR) specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the HuCD4

Human-like response to streptococcal pyrogenic exotoxin A (SpeA) HLA restricted cytotoxic lymphocyte response to measles virus Reconstituted the CD4 T cell compartment, in both the thymus and the periphery, and mediate thymic selection of a broad range of Vbeta families. Eosinophilia in response to cockroach antigen Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination and clinical manifestations resembling multiple sclerosis

Reference Shingai et al. (2005) Bettelli (2007)

Welcher et al. (2002) Tishon et al. (2000) Chen et al. (2006)

Papouchado et al. (2001) Madsen et al. (1999)

agent, e.g., tetracycline or doxycycline, progesterone, ecdysone and polycyclic aromatic hydrocarbons (see review by Bockamp et al., 2002). Similarly, in conditional knockouts, expression of the gene is suppressed in the presence of the agent. As is the usual case, application of these advanced models in a toxi-

332


cology setting tends to lag behind more conventional systems. However, use of conditional transgenic and multiple transgenic mice will likely increase.

ADVANTAGES AND LIMITATIONS OF ALTERNATIVE MODELS The role of immunotoxicology in the development of new therapeutics can be thought of in three parts: Hazard Identification, Hazard Characterization, and Risk Assessment. In hazard identification and hazard characterization, there is a clear role for alternate systems and they can be of great advantage over conventional systems (Table 10.1-13). Some of the more obvious advantages of alternate systems is that they can allow testing of “human-specific” drugs in a system that requires small amounts of material, in large numbers of agematched animals of both genders. They also allow conduct of experiments that would be impossible or grossly impractical in nonhuman primates, e.g., host defense against infectious organisms or neoplasia and provide a ready supply of cells and tissues for in vitro and ex vivo testing of function and histopathologic examination. Other advantages of specific types of alternate systems are listed in Table 10.1-13. When considering conducting an immunotoxicologic investigation in an alternative model system, it is important to not lose sight of the limitations on these systems. As shown in Tables 10.1-13 and 10.1-14, many of the same limitations relevant to testing a rodent homologue in an intact rodent (Table 10.1-2) are also relevant to the gene defect, transgenic, and human reconstituted systems. In addition, there are issues of particular importance when testing human protein therapeutics in a rodent system. While none of these issues will necessarily preclude testing, they should be addressed in preliminary experiments designed to qualify the alternate system for hazard identification and characterization. Knock-in mice are of particular concern. Whether one is testing an inhibitor (e.g., a monoclonal antibody) or an agonist (e.g., a human cytokine) in a receptor knock-in mouse, care must be taken to ensure that the ligand-receptor combination expresses in mice at least some of the functions expressed in the fully human system. An example of this is the MuCD4KO-HuCD4/Tg mice, where human CD4 appears to express essentially normal function in mice (Killeen et al., 1993). These mice have been used to support the discovery and development of two anti-CD4 monoclonal antibodies (keyliximab and clenoliximab) that bound human and chimpanzee CD4, but not the CD4 of other nonhuman primates (Davis et al., 1996; Bugelski et al., 2000; Sharma et al., 2000; Podolin et al., 2000; Herzyk et al., 2001, 2002). This, however, appears to be a unique example of the full application of a transgenic system in immunotoxicology and preclinical development of therapeutic proteins. Expression of one immune function in a transgenic system, however, does not guarantee faithful expression of all functions. Even if the transgene product appears to have functional activity, it is important to remember that the

Pharmacologic activity

Human reconstituted

Knock-ins

Human reconstituted All rodent systems Homologous systems

Transgenics

Knockouts and genetic defects

Genetic defects

Immune function

Enable dissection of the role of a particular cell type in immune function Small size and available in large numbers Enable testing hypotheses that may not be testable with a human-specific drug in nonhuman primates, e.g., host defense, in an in vivo setting Enable testing hypotheses that may not be testable with a human-specific drug in nonhuman primates, e.g., host defense, in an in vivo setting Enable testing activity on human cells in a quasi-in vivo setting, e.g., host defense

Enable testing activity on human cells in a quasi-in vivo setting, e.g., tumor xenografts Provide the “ultimate” test of the potential adverse effect of inhibition of a function free of the constraints imposed by pharmacokinetics and pharmacodynamics Enable dissection of the role of a particular gene product in immune function

Potential Limitation

The human target may not express the same functional activity when expressed in rodents and thus over- or underestimate pharmacologic activity Reconstitution is rarely complete and thus may overestimate the impact of a test article on immune function

Insertion of the transgene(s) may disrupt expression of adjacent genes in the rodent genome causing overt or occult additional functional deficit(s) The human target may not express the same functional activity when expressed in rodents The human drug may be immunogenic in rodents The murine homologous target may not express the same functional activity in rodents and thus over- or underestimate pharmacologic activity

Loss of function/expression of one gene may lead to increased expression of other genes as an adaptive response Because they are usually complete, knockouts/ gene defects may correctly identify a hazard, but may overestimate the impact of the test article on immune function

Alternate System

Application

Advantages

Potential Limitations on the Use of Alternate Models in Hazard Identification

TABLE 10.1-13

ADVANTAGES AND LIMITATIONS OF ALTERNATIVE MODELS

333

334


TABLE 10.1-14 Potential Limitations on the Use of Alternate Models in Hazard Characterization Type of Alternate System Homologous systems Knockouts/gene defects Knock-ins

Human reconstituted

Potential Limitation The human drug may show a dramatically different dose–response relationship in rodents Background loss of function may underestimate hazard posed by a test article in models of host defense The transgene product may not be expressed by the same cells, at the same level of expression or at the same time (i.e., during ontogeny or during an immune response) as the human gene in humans The rodent Fc receptors (FcR) may not bind the human drug in a similar fashion as human FcRs Human pathogens may not show the same immunopathology in rodent models Limited number of human donors will limit MHC class II repertoire and thus may under- or overestimate hazard

immune system functions as a network, with multiple cell types expressing multiple receptors working in concert. This is illustrated by studies conducted in human TNF-α transgenic mice. Although these mice are a useful model of arthritis (Keffer et al., 1991) and bone marrow dysplasia (Capocasale et al., 2007), and have been used to demonstrate the beneficial effects of anti-TNF-α antibodies (Shealy et al., 2002), they cannot express the full spectrum of activity of human TNF-α. This is because there are two receptors for TNF-α, p55 and p75, and the murine p75 receptor is highly selective for murine TNF-α and fails to mediate signaling from human TNF-α (Lewis et al., 1991). Thus, all of the effects seen in these mice must be due to signaling from the p55 receptor and they have no value for evaluating effects mediated by p75. Thus, since there are no practical models of rheumatoid arthritis in nonhuman primates, while these mice are of demonstrated advantage in demonstrating the pharmacologic activity of anti-TNF-α antibodies and can elucidate some of the mechanisms at play, they are limited in their usefulness for the study of the full range of potential adverse effects. From the many exemplars listed in the tables of this chapter, it is clear that alternate systems are playing an important role in demonstrating the pharmacologic activity of immunoactive drugs. And, when an immunotoxicity has already been identified, either by conventional preclinical test systems or in clinical trials, alternate systems can enable in-depth characterization of the hazard posed by a therapy. Their role in the final step of immunotoxicology, Risk Assessment, however, is less clear. As shown in Tables 10.1-13 and 10.1-14,

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there are limitations that must be understood when using alternate systems in immunotoxicology. These limitations make it unlikely, at least for the foreseeable future, that alternated systems can serve as a stand-alone measure of risk. This is not to say that there is no role for alternate systems in risk assessment and in many ways, this is no different from the approach that must be used when interpreting any preclinical data in terms of risk assessment. However, because application of all alternate systems to immunotoxicology is relatively new and there is only a limited knowledge base (particularly with highly novel systems), an extra measure of caution should be applied when interpreting these data. When combined with all other preclinical and clinical data on the hazards and benefits of a therapy, alternate systems can play an important role in the overall risk assessment.

SUMMARY Limitations on conventional systems have lead researches to explore alternative models of immune function. Ever more sophisticated models, either reconstituted with human cells or bearing multiple human knock-in genes are on line for basic research and are beginning to be used in immunotoxicology. However, as is natural, although use of these complex models in immunotoxicology lags behind their application in basic research, and qualifying these mice for use in immunotoxicology requires extensive work, it is anticipated that they will play an ever-greater role in hazard identification and characterization and ultimately risk assessment.

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10.2 ANIMAL MODELS FOR PRECLINICAL COMPARATIVE IMMUNOGENICITY TESTING Daniel Wierda

The opinion of how useful animal models are for characterizing the immunogenic potential of a new therapeutic protein is influenced by the involvement in one of two different areas of biopharmaceutical research and development, namely work on the utility of the protein as a human therapeutic (discussed in Chapter 6) or a human vaccine (discussed in Chapter 7). For this discussion, a biopharmaceutical is any therapeutic biological entity that is not used as a vaccine. In the discovery and development of vaccines, animals are used to test the potency or efficacy of different protein formulations to optimize for immunogenic potential and to determine the relative safety of the vaccine. In many cases, laboratory animals in well-controlled studies may actually be preferable to human models in predicting the relative effectiveness, i.e., desired immunogenicity, of a molecule due to the variety in any given individual’s immune status (age, disease, nutrition, etc.). On the other hand, in the development of human biopharmaceuticals (for which immunogenicity is undesired), animal models inconsistently predict human immunogenic potential, for example, when immunogenicity is evaluated during standard preclinical toxicity tests. A direct prediction of immunogenicity potential in humans (the incidence and/or severity) based on animal studies using a therapeutic dosing regimen with a biopharmaceutical (not designed to invoke a strong immune response) is very challenging. However, there is the need for a more reliable way to


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meaningfully determine how engineered changes to the amino acid structure of a biopharmaceutical, glycosylation, or formulation changes, may influence the immunogenic potential of the intended human therapeutic protein. The purpose of this chapter is to show that in vivo animal models can be very informative in addressing the question of how inherently immunogenic a molecule is in relation to a comparator protein of known immunogenicity. That this approach is feasible is supported by the science of immunobiology which has for decades relied upon studies in animals in order to understand the relationships between antigen structure and immune responsiveness. In fact, the immune system can be exquisitely sensitive in distinguishing differences in molecular epitopes as originally demonstrated by Landsteiner’s work with haptens (Landsteiner, 1962). Early immunological research in this area utilized many different animal models, including Guinea pigs, rabbits, rodents, and chickens. The key is to choose the most appropriate model for characterizing the immunogenic properties of a newly synthesized biopharmaceutical and determine to what extent they differ from the immunogenic properties of the wild-type or native molecule. Thus, to minimize the immunogenic potential of a biopharmaceutical candidate, comparative animal studies could be utilized in the early drug discovery and lead optimization phase. Product comparability tests typically consist of the characterization of protein solubility, stability, and degradability to confirm similarity between different batches or lots of a manufactured biopharmaceutical. This can be accomplished because the proteins being tested have specific physicochemical properties that can be defined. Similarly, each protein molecule also possesses specific properties that determine its potential to induce an immune response, or in other words, its immunogenic properties. A favorable bioanalytical characterization does not guarantee the stability or degradation rate of a molecule after it is administered to a human subject or patient, but we know from experience that the likelihood of having these problems is minimized based on selecting for these bioanalytical criteria. By analogy, characterizing the immunogenic properties of a biopharmaceutical, and selecting molecules in a series with the most favorable immunogenic characteristics in animals, should help to lessen its immunogenic potential when administered to humans.

PROTEIN IMMUNOGENICITY AND ANTIGENCITY Immunogenicity refers to the inherent properties of a molecule to stimulate an immune response, whereas antigenicity refers to the properties of a molecule that allow it to react with antibodies (Golub, 1987). For biopharmaceuticals, there are several product-specific factors of immunogenicity which are frequently associated with the “foreignness” or structure of the molecule, such as species homology (human or nonhuman), specific T cell epitopes, degree of glycosylation, pegylation, hydrophilicity, conformational changes, and neoepitopes due to creation of fusion proteins. Pharmacodynamic and pharma-

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cokinetic factors also influence immunogenicity including dose, frequency, route of administration, and target disposition (cellular or soluble). Additionally, product formulation can impact immunogenicity based on the degree of product aggregation, degradation, or impurities. Lastly, the host’s overall immune status, which is affected by genetics (MHC phenotype), health, nutrition, and concurrent medications, plays a significant role in the ultimate generation of an immune response to biopharmaceuticals. The terms antigenicity and immunogenicity have in the past been used somewhat interchangeably when referring to the measurement of serum antibodies to a drug or a biopharmaceutical. This has frequently caused confusion about whether reference is being made to specific tests in Guinea pigs, previously required by regulatory groups (e.g., in Japan) to determine if a test material produced anaphylaxis via reagenic (IgE) antibodies, or to the actual quantitation of antidrug antibodies. Practically speaking, most proteins possess both antigenic and immunogenic properties, and so a test for antigenicity could also incorporate an immunoassay for immunogenicity (the detection of antidrug antibody [ADA]), especially if there is prior knowledge that would exaggerate the concern about the potential for a protein therapeutic to induce anaphylaxis. Over the years, however, it has become the convention to refer to ADA measurements as immunogenicity testing and to drug allergy screening in Guinea pigs as antigenicity testing. Further complicating the vocabulary in this area is the fact that the ICH S6 (1997) guidance document refers to immunogenicity testing under the heading of Immunotoxicity. This is not to be confused with the preclinical immunotoxicity tests that are performed to characterize the effect of test drugs on functional immune responsiveness. While with an intended immunomodulatory biopharmaceutical, toxicology studies may include functional immunotoxicity tests, the evaluation of immunogenicity potential (via antidrug antibody assays) should be considered for all biopharmaceuticals in the broader context of toxicokinetic and toxicity data.

IMMUNE RESPONSES IN ANIMALS AND HUMANS Interspecies differences among the immune systems of animals and of humans are always an overlying consideration in biomedical research and especially in the area of preclinical immunogenicity (Gordon et al., 2001; Mestas and Hughes, 2004; Loisel et al., 2007). There can be significant differences between animals and humans in the anatomical location of lymphoid cells, as well as in immune reactions to pathogens (Haley, 2003). Additionally, there are speciesrelated differences in the developmental stages of the immune system (Holsapple et al., 2003). Together, this information could support the argument that testing for those antigenic characteristics that define the immunogenicity of a protein in animals is unproductive. However, the point of this chapter is to provide evidence that animals can be used to model human immunology

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as long as there is awareness of the caveats associated with these models in any experimental study (Mestas and Hughes, 2004). Although the molecular biology of immune responses to antigens can be very finely dissected with modern science in both human and animal models, it is a fundamental principle that all vertebrates will respond to a particular antigen with specific cellular responses that will result in the production of antibodies. Given the universal nature of the response, it follows that regardless of species differences, one can utilize an experimental in vivo platform to query the antigenic characteristics of proteins in general, based on the type and magnitude of the subsequent antibody response. That this is a tenable approach is supported by those investigations showing that the immune system can detect structural differences in various antigens. For example, as previously mentioned, experiments by Landsteiner and others demonstrated the exquisite ability of antibodies generated in animals immunized with stereoisomeric compounds to react specifically based on the structural differences of the immunogen (reviewed in Landsteiner, 1962). Many other studies, in a variety of animal species, have repeatedly confirmed the utility of antibodies for characterizing the antigenicity of haptenated, conjugated, or structurally altered molecules (Benacerraf et al., 1963; Pinchuck and Maurer, 1965; Wood and Kabat, 1981; Zwickl et al., 1991; Braden and Poljak, 1995; Tsujihata et al., 2001). The formation of T cell peptide-MHC II complexes after antigen intake by antigen-presenting cells (APCs) is a key mechanism involved in the majority of immunogenicity responses to biopharmaceuticals or vaccines (Stevanovic, 2002). This is supported by the presence of high-affinity, class-switched IgG antibodies detected to all classes of therapeutic proteins (Amin and Carter, 2004). B cells can only produce high-affinity antibodies when they are stimulated by cytokines from T-helper cells. If this interaction between APC and T cell could be avoided, immunogenicity would also be eliminated (reviewed in De Groot et al., 2003; Chirino et al., 2004; Jones et al., 2005; Koren et al., 2007). Because of this T cell dependency, the process of antigen recognition requires the binding of TCRs with proteins presented on MHC I or II molecules by APCs. Central to this interaction are the inherent antigenic properties of the protein whose amino acid structure can be strongly correlated with its propensity to bind to MHC molecules. Highly immunogenic proteins contain many T cell epitopes that bind strongly to MHC II whereas nonimmunogenic proteins contain fewer T cell epitopes (De Groot and Moise, 2007). Hence the density of T cell epitopes identified in the amino acid sequence of a biopharmaceutical is a physicochemical property known to correlate with a molecule’s antigenicity (Tong et al., 2006). Incorporating this information with an understanding of the genetic differences in MHC alleles between a particular animal model, such as mice and humans, should be useful then for understanding the relative immunogenicity potential of any given protein. Whether animal or human, the generation of an immunogenic antibody response to an antigen is driven by T cells after presentation of MHC-bound

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peptides on APCs. The higher the density of T cell epitopes on a protein, the more immunogenic the molecule is. In general, nonhomologus human proteins that are administered to monkeys, for example, will tend to have more T cell epitopes than if an endogenous monkey protein was given. Similarly, the same nonhomologus human protein should be even more immunogenic if given to rodents. As discussed later in this chapter, one can take advantage of this function of the immune system to query the relative immunogenic properties of different therapeutic biopharmaceuticals.

ANIMAL MODELS FOR VACCINE IMMUNOGENICITY Optimizing the antigenicity of protein or peptide immunogens by modifying specific amino acid residues involved in MHC binding (T cell epitopes) is a method used in the development of various vaccines (De Groot et al., 2003; De Groot, 2006; Palena et al., 2006; Tong et al., 2006; De Groot and Moise, 2007). This process involves using computational methods to guide the engineering of peptide sequence changes in silico, followed by in vitro assays, such as T cell stimulation or MHC binding, to verify an increase in binding affinity and, presumably, a corresponding enhancement in its immunogenic potential. Ultimately, the experimental vaccine must be tested in an animal model. The issues that must be addressed by animal testing depend, to some extent, on the type of vaccine and its intended use, but can include evaluations for optimizing the immunogenicity (efficacy) of the vaccine by exploring different routes of administration, adjuvants, vectors, frequency of administration, or coadministration with other therapies. Subsequent testing for safety of the vaccine product also relies heavily on studies in nonhuman primates. In general, animal models are heavily relied upon to predict both efficacy and safety for human therapy (Descotes et al., 2002; and Figure 10.2-1). The choice of an animal model for vaccine development depends not only on the ability to generate an antibody response to a protein antigen or vaccine, but also on the relevance of the model that is pertinent to the human disease for which the vaccination therapy is targeted. For this reason, rabbits and cattle are deemed the best models for evaluating the efficacy of vaccines to human tuberculosis because of similar lung pathology, while the mouse is a better model to test effects on the immune response itself even though the lung pathology caused by the tuberculosis pathogen is different than in humans (Orme, 2005). In fact, several tuberculosis vaccines progressing in clinical trials have used the mouse species to test efficacy (Sander and McShane, 2006). An interesting history is provided by Garg and Dube (2006) on the use of different animal models employed over the last 60 years in unsuccessful attempts to develop anti-leishmaniasis vaccines. These authors also emphasize that an animal model for vaccine development must mimic the human pathogenesis and the immunological responses to the disease. Although none of the models accurately reproduced the human response, the collective research produced

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Figure 10.2-1 Maximizing the benefit-to-risk in vaccine development. No vaccine can be administered to humans without solid data showing safety. Product development and safety testing for these products relies very heavily on animal studies which are relied upon to predict both efficacy and safety. Toxicity assessments for vaccines are challenging because, in general, vaccines trigger complex immune reactions. There must be a balance between desired immunogenicity and unwanted adverse side effects.

a detailed knowledge of the immune mechanisms associated with vaccineinduced immunity in animals and thereby has provided further clues for continued work in this area. Similarly, research on potential vaccines against human immune deficiency virus has relied principally on the use of nonhuman primates (Haigwood, 2004; Staprans and Feinberg, 2004). The development of vaccines remains a highly experimental process with many different and novel strategies employed to induce an effective immunogenic response to pathogens or tumors (Levine and Sztein, 2004). Although new technology, along with clinical experience, has improved the ability to manufacture more effective immunogens for vaccines, testing in animal models remains the primary means to characterize the tolerability and efficacy of the therapy. This approach is unlikely to change in the near future because the intrinsically complex immune response can only be evaluated in an intact system offered by mammals (van Regenmortel, 2001), and because these models have, overall, been useful in identifying effective immunization strategies in animals, and humans. For example, the influence of such factors as route of administration, dose, frequency, duration, adjuvants, and degree of aggregation, on the formation of an antibody response in animals generally correlates

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with these same factors in humans (Bugelski and Treacy, 2004). For the foreseeable future, animal models will continue to be very important in the development of vaccines, especially for predicting the most optimum formulation and dosing schedule for maximum immunogenicity.

COMPARATIVE IMMUNOGENICITY OF BIOPHARMACEUTICALS In contrast to the need to optimize the immunogenicity of vaccines, immunogenicity should be minimized for therapeutic biopharmaceuticals (see Chapter 10.3). One way to reduce immunogenicity is the re-engineering of these protein molecules to remove “unwanted” T cell epitopes, a process now referred to as de-immunization (De Groot, 2006). Although sophisticated in silico and in vitro technologies employed are gaining acceptance, verification that the biopharmaceutical is not immunogenic emerges only after it has been administered to a sufficient number of human subjects and a thorough follow-up analysis of immunogenicity conducted (Locatelli and Roger, 2006). An intermediate surrogate in vivo test system would seem to be very desirable, especially to interrogate the effect on immunogenicity after re-engineering resulting in a change in the degree of glycosylation, fucosylation, amino acid sequences, or hydrophilicity and any other formulation characteristics of the biotherapeutic. Despite efforts to eliminate immunogenicity, the ultimate outcome will be influenced by the route of administration, frequency, and duration, as well as the animal model chosen for the evaluation. In light of the fact that animal models have been used to optimize the antigenic properties of vaccines, it seems that they should also have value in minimizing the immunogenicity of pharmaceuticals. Immunogenicity of Biopharmaceuticals Dependent on Protein Structural Characteristics In vivo assessment of immunogenicity represents the best opportunity to evaluate the antigenic characteristics of a molecule in the context of the immune system as a whole. For biopharmaceuticals, the nonhuman primate has been the animal of choice for characterizing immunogenicity in a safety setting primarily because the sequence homology between human product and nonhuman native molecule is most similar (Wierda et al., 2001). In some cases, rhesus or cynomolgus monkeys have been used in studies to compare the relative immunogenicity of structurally different human recombinant proteins (antigens) in a class (Zwickl et al., 1991, 1995, 1996). Collectively, these studies demonstrated that differences in amino acid sequences between analogs (insulin, tissue plasminogen activator, leptin, and growth hormone) can be detected and a rank order of immunogenicity assigned to the different proteins (reviewed in Smith and Wierda, 2005). In several cases, the more immunogenic recombinant proteins (e.g., leptin) in monkeys were also more immunogenic

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in human subjects compared to the native protein. However, in all but one case, the immunogenic response in humans did not result in any adverse safety or pharmacodynamic effects. An alternative model to nonhuman primates for a comparative evaluation of the immunogenic differences between recombinant proteins and different variants are transgenic mice expressing the human protein (reviewed in Chapter 10.1) in question. Such a model was used to explore the immunogenic differences of structural changes in tissue plasminogen activator, human insulin (Stewart et al., 1989), interferon-alpha2a, (Ottesen et al., 1994; Palleroni et al., 1997; Hermeling et al., 2006), interferon-beta1a (Jaber et al., 2007), and Fas ligand inhibitory protein (FLINT) (Smith and Wierda, 2005). The advantage of a transgenic mouse model is that the animal is immunologically tolerant to the expressed human proteins. To generate an immune response to a protein antigen, this tolerance must be broken and the resulting degree of immunogenicity (incidence and antibody titer) becomes an indication of the relative immunogenic potential of each analog. Interestingly, when mice transgenically expressing human FLINT were used to analyze the anti-FLINT antibody response to amino acid variants of this human protein, the responses correlated with the number of amino acid substitutions (Smith and Wierda, 2005). None of the mice generated antibody against the protein with native human sequence or with the 1 amino acid variant. But the incidence for mice immunized with the 2-, 3-, or 5-amino acid variants was 33%, 17%, and 100%, respectively. These results lend further support to the idea that the immune system can discriminate differences in the antigenic structure of proteins. Another transgenic model, first used to investigate mechanisms underlying autoimmune disease and subsequently to optimize T cell epitopes, is the human histocompatibility leukocyte antigen (HLA) transgenic mouse. HLA molecules are pivotal for proper thymic development of T cells and it is the MHC II alleles that determine the T cell repertoire via the presentation of selfpeptides. Mice with a single HLA transgene have been used to investigate the breakdown in tolerance to self-antigens in diseases such as diabetes, multiple sclerosis, and rheumatoid arthritis (Taneja and David, 1998; Boyton and Altmann, 2002; Blancou et al., 2007). Mice with double and triple transgenes in the HLA system (HLA-DR, HLA-DQ, and human CD4+) were subsequently developed to further explore mechanisms associated with autoimmune diseases, as well as to identify T cell epitopes for cancer vaccines and infectious diseases (Men et al., 1999; Sonderstrup et al., 1999; Taneja and David, 1999; Rojas et al., 2005; Horton et al., 2007; Depla et al., 2008). The utility of these transgenics in vaccine development suggests that these animals are not tolerized to human proteins and maybe good in vivo models for identifying specific immunogenic epitopes relevant to humans. Importantly, these studies also demonstrate that the human class II transgenes expressed on murine cells can pair with class II molecules on mouse APCs and effectively stimulate mouse CD4 T cells (Taneja and David, 1999).

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Although the binding of peptides to MHC molecules is highly correlated with an immunogenicity response, peptide analogs can be made that carry amino acid substitutions at sites other than the main MHC anchor points and are considerably more immunogenic in both HLA transgenic mice and in humans (Tangri et al., 2001). This phenomenon is termed “heteroclitic activity” because the immunogenicity deviated from the common, or expected, norm. A panel of nine predicted, heteroclitic protein analogs (based on ability to stimulate T cell interferon responses) were administered to HLA2.1/Kb transgenic mice resulting in identification of three analogs that were more immunogenic than the native peptide. These analogs were also recognized by human T cells in vitro. The mechanism responsible for the enhanced immunogenic response of the analogs may be due to increases in TCR-binding affinity of the peptide-MHC complex caused by a subtle change in peptide conformation. Aside from the exquisite sensitivity to detect structural changes demonstrated by this transgenic model, these results also exemplify how a single transgenic strain of mouse may allow for the systematic evaluation of immunogenicity studies of biopharmaceuticals, such as the relative characterization of manufacturing changes or biosimilars. Data from Depla et al. (2008) suggest that HLA transgenic mice respond to approximately 70% of known human epitopes due to a more restricted T cell receptor variability in inbred mice and, therefore, assessments of immunogenic epitopes with these models may underestimate the expected results for humans. Despite this limitation, these same authors point out that HLA transgenic mice can be useful for generation of comparative immunogenicity data with synthetic peptide analogs by providing a reference incidence and antibody response profile against which other protein constructs can be compared (Depla et al., 2008).

Manufacturing and Immunogenic Properties The above discussion highlights that a structure-dependent immunogenic potential of biotherapeutics can be detected in animals. This approach can also be used to determine if changes to manufacturing or formulation impact the relative immunogenic characteristics of biopharmaceutical materials. As previously pointed out above, there is ample precedent for this approach in optimization of immunogenic properties for vaccines. Changes introduced during the production process, including those associated with glycosylation, deamination, oxidation, aggregation, pegylation, host cell proteins, as well as leachates or extractables from packaging materials, can affect immunogenicity (reviewed in Chirino and Mire-Sluis, 2004; Hermeling et al., 2004; Rosenberg, 2006; Sharma, 2007). Studies reported by Jaber et al. (2007) provide an excellent example of how nonclinical evaluations can lead to the selection of a less immunogenic, and more tolerable formulation of Rebif®, a therapeutic human interferon beta-1a (IFNβ-1a) biopharmaceutical. Following genetic engineering efforts to improve

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cloning efficiency and changes in vehicles and formulations, several different IFNβ-1a molecules were systematically tested in Balb/c mice. It was determined that one formulation produced neutralizing antibodies more slowly and at lower titers than other formulations given at equivalent dose levels. In Phase I clinical trials, this same formulation also proved superior causing fewer adverse events such as injection site pain and erythema when compared to a second formulation or to the current IFNβ-1a therapeutic (Jaber et al., 2007). These results build on the now classic study of Braun et al. (1997) 10 years earlier, and a more recent study with human IFNα-2b (Hermeling et al., 2006), with both normal mice and mice transgenically expressing human IFNα. These studies demonstrated that problematic immunogenicity in humans treated with recombinant therapeutic IFNα-2b correlated with the degree of aggregates present in the formulations. A comparative immunogenicity approach was recently taken in the author’s laboratory to address a safety question related to the contamination of a therapeutic monoclonal antibody formulation with eukaryotic host cell protein (unpublished data). Specifically, was the host cell protein immunogenic and if so, would an immunogenic response be adverse? To test this hypothesis, we chose a nonhuman primate model because of existing information from nonclinical safety studies with the test monoclonal antibody, as well as extensive pharmacokinetic data for comparison. The basic design involved twice weekly, intravenous administration of two different dose levels of a humanized monoclonal antibody to cynomolgus monkeys for 6 weeks. Serum samples were subsequently analyzed for the presence of antidrug (monoclonal) antibody and for anti-host cell protein antibody. A key feature of this study was the inclusion of a group of monkeys that were hyperimmunized with purified host cell protein with adjuvant to stimulate a maximum level of high-affinity antihost cell protein antibody response. This positive control group thus represented a potential worst-case scenario and provided a standard for which anti-host cell protein antibody responses from the treatment groups could be compared with. Similarly, a second hyperimmunized, positive control group was included and consisted of the formulated product that was also administered in adjuvant. The remaining two treatment groups (low and high dose) received formulated product but without adjuvant. The results from this study are summarized in Table 10.2-1. As expected, both positive control groups produced high-titer antibody responses in all monkeys, but importantly, there were no adverse toxicological effects associated with either an anti-host cell protein or an anti-monoclonal antibody immune response in the hyperimmunized groups. In contrast, when the final formulation was given without adjuvant for up to 6 weeks, no detectable antibody could be measured (with a drug tolerant radioimmunoassay) against either host cell protein or against the monoclonal antibody itself. This latter finding was considered significant because the difference in homology between the humanized IgG1 monoclonal antibody and its endogenous counterpart in the cynomolgus monkey would be expected to produce at least some immunogenic responses. The formulation

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SUMMARY

TABLE 10.2-1 Testing Impact of Host Cell Protein Contamination on Immunogenicity of Biotherapeutic Drug in Cynomolgus Monkeys Adjuvant

Antibody to Contaminant

Antibody to Drug

Toxicity (at 6 weeks)

Contaminant1 (positive control)

Yes

Tested positive

Tested negative

Not detectable

Biotherapeutic drug1 (positive control)

Yes

Tested negative

Tested positive

Not detectable

Biotherapeutic drug2,3 (clinical material)

No

Tested negative

Tested negative

Not detectable

Test Material

1

Administered in hyperimmunization protocol Administered by therapeutic route 3 Contains host cell protein contaminant 2

has subsequently been tested in Phase I clinical trials with similar results being observed as reflected by low immunogenicity and no adverse effects.

SUMMARY In summary, animal models have proven to be very useful for probing the antigenic and immunogenic properties of biopharmaceuticals. A key consideration in the design and use of these models is the inclusion of the appropriate controls, for example the native human molecule, or a well-known human therapeutic analog such as the case for the insulins, to allow for a degree of standardization and provide for a means of interpretation based on comparability. Another critical factor is the use of an animal species that has the ability to generate an immune response to the antigen in question as some rodent strains can be unresponsive to various proteins (e.g., Fisher 334 rats do not immunologically recognize some parathyroid hormone variants; unpublished data). Therefore, careful research and deliberation up front on the many factors that influence immunogenicity, including dosing regimen, route of administration, and other factors discussed in this chapter, are important in the design of comparative immunogenicity studies. Understanding the impact that each variable can have on the ultimate immune response is paramount for ensuring the generation of useful information, such as the relative rank order of immunogenicity for several, or a series, of biopharmaceutical analogs. As with all models, there are limitations and this approach may not always predict the incidence or magnitude of an immunogenic response to a single biopharmaceutical in humans, but the evidence would suggest that animal testing can identify immunogenic properties across a class of biopharma-

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ceuticals. Thus, by employing the available new technologies, such as de-immunization methods, in conjunction with nonclinical in vivo screening, we can provide data that will aid in the selection of the least immunogenic biopharmaceuticals.

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Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–2738. Orme IM. The use of animal models to guide rational vaccine design. Microbes Infect 2005;7:905–910. Ottesen JL, Nilsson P, Jami J, Welguny D, Duhrkop M, Bucchini D, Havelund S, Fogh JM. The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model. Diabetalogia 1994;37:1178–1185. Palena C, Abrams SI, Schom J, Hodge JW. Cancer vaccines: preclinical studies and novel strategies. Adv Cancer Res 2006;95:115–145. Palleroni AV, Aglione A, Labow M, Brunda MJ, Pestka S, Sinigaglia F, Garotta G, Alsenz J, Braun A. Interferon immunogenicity: preclinical evaluation of interferonalpha 2a. J Interferon Cytokine Res 1997;17:S23–S27. Pinchuck P, Maurer PH. Antigenicity of polypeptides (poly alpha amino acids). XV. Studies on the immunogenicity of synthetic polypeptides in mice. J Exp Med 1965;122:673–679. Rojas JM, McArdle SEB, Horton RBV, Bell M, Mian S, Li G, Selman AA, Rees RC. Peptide immunization of HLA-DR-transgenic mice permits the identification of a novel HLA-DRb1*0101-and HLA-DRb1*401-restricted epitope from p53. Cancer Immunol Immunother 2005;54:243–253. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J 2006;8:E501–E507. Sander C, McShane H. Translational mini-review series on vaccines: development and evaluation of improved vaccines against tuberculosis. Clin Exp Immunol 2006;147: 401–411. Sharma B. Immunogenicity of therapeutic proteins. Part 3. Impact of manufacturing changes. Biotechnol Adv 2007;25:325–331. Smith H, Wierda D. Preclinical immunogenicity testing for recombinant therapeutic proteins. J Immunotoxicol 2005;2:1–8. Sonderstrup G, Cope AP, Pate S, Congia M, Hain N, Hall FC, Parry SL, Fugger LH, Michie S, McDevitt J. HLA class II transgenic mice: models of the human CD4+ Tcell immune response. Immunol Rev 1999;172:335–343. Staprans SI, Feinberg MB. The roles of nonhuman primates in the preclinical evaluation of candidate AIDS vaccines. Exp Res Vaccines 2004;3:5–32. Stevanovic S. Structural basis of immunogenicity. Transplant Immunol 2002;10:133– 136. Stewart TA, Hollingshead PG, Pitts SL, Chang R, Martin LE, Oakley H. Transgenic mice as a model to test immunogenicity of proteins altered by site-specific mutagenesis. Mol Biol Med 1989;6:275–281. Taneja V, David CS. HLA transgenic mice as humanized mouse models of disease and immunity. J Clin Invest 1998;101:921–926. Taneja V, David CS. HLA transgenic mice as models of human diseases. Immunol Rev 1999;169:67–79. Tangri S, Ishioka GY, Huang X, Sidney J, Southwood S, Fikes J, Sette A. Structural features of peptide analogs of human histocompatibility leukocyte antigen class I epitopes that are more potent and immunogenic than wild-type peptide. J Exp Med 2001;194:833–846.

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Tong JC, Tan TW, Ranganathan S. Methods and protocols for prediction of immunogenic epitopes. Brief Bioinform 2006;8:96–108. Tsujihata Y, So T, Hashimoto Y, Ueda T, Imoto T. A single amino acid substitution in a self protein is sufficient to trigger autoantibody response. Mol Immunol 2001;38:375–381. van Regenmortel MHV. Antigenicity and immunogenicity of synthetic peptides. Biologicals 2001;29:209–213. Wierda D, Smith HW, Zwickl CM. Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology 2001;158:71–74. Wood C, Kabat EA. Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid. I. Antigenicity of the glycolipids and the production of specific antibodies in rabbits. J Exp Med 1981;154:432–449. Zwickl CM, Cocke KS, Tamura RN, Holzhausen LM, Brophy GT, Bick PH, Wierda D. Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys. Fundam Appl Toxicol 1991;16:275–287. Zwickl CM, Smith HW, Zimmerman JL, Wierda D. Immunogenicity of biosynthetic human LysPro insulin compared to native-sequence human and purified porcine insulins in rhesus monkeys immunized over a 6-week period. Arzneimittelforschung 1995;45:524–528. Zwickl CM, Hughes BL, Piroozi KS, Smith HW, Wierda D. Immunogenicity of tissue plasminogen activators in rhesus monkeys: antibody formation and effects on blood level and enzymatic activity. Fundam Appl Toxicol 1996;30:243–254.

10.3 T CELL EPITOPES AND MINIMIZATION OF IMMUNOGENICITY Harald Kropshoffer and Thomas Singer

Immunogenicity relates to the property of a molecule to provoke an adaptive immune response, encompassing both the cellular and humoral branch of the immune system. When therapeutic proteins are administered, e.g., intravenously, subcutaneously, or intranasally, the natural response of the immune system may be the formation of anti-protein antibodies. To address this phenomenon adequately, the current regulatory standard for immunogenicity assessment during development of biotherapeutic drugs is screening for antidrug antibodies (ADA). Although in many cases, the incidence and clinical consequences of immunogenicity are limited, a high incidence or severity of ADA may still contribute to failure of therapeutic proteins in clinical testing, e.g., due to neutralizing ADA-mediated efficacy loss or to severe adverse events (AE) (Koren et al., 2002; Haselbeck, 2003; Locatelli and Del Vecchio, 2003). Numerous cases have been reported where ADA does neither impair efficacy nor give rise to AE.

THE IMMUNOLOGY BEHIND IMMUNOGENICITY There are at least three immunological mechanisms by which therapeutic proteins are supposed to trigger ADA formation. (i) The classical antibody


361

362

T CELL EPITOPES AND IMMUNOGENICITY

response which may explain the majority of ADA cases, at least in humans, is based on the recognition of amino acid stretches of a therapeutic protein entering the human body for the first time (Figure 10.3-1, pathway 1). This type of immune response normally depends on a preceding priming event that relies on the presence of specialized interleukins, such as IL-4 and/or IL-13. As IL-4 and IL-13 are selectively secreted by activated CD4+ helper T cells, this type of immunogenicity is T cell dependent. In the absence of CD4+ T cells, ADA responses are expected to be limited to the IgM type of antibodies with low to moderate avidity and a rapid decline in titers. (ii) A second mechanism for the induction of antibodies, which often takes far longer than the former, is based on breakage of B cell tolerance (Hermeling et al., 2005): the therapeutic protein of question, often a homolog or mimic of a human selfprotein, is thought to bind directly to a surface IgM or IgG molecule, also denoted as B cell receptor (BcR), of B cells and activates them without the

Aggregated Therapeutic Protein B Therapeutic Protein

2. IgG against Therapeutic Protein CD4

1.

CD4 CD4 +

A n t i g en Pr es en t i n g C el l

Pl as m a c el l

B

IL-4 IL-13

CD4

3.

T cell Epitope

Aggregated Therapeutic Protein

CD4

B

Figure 10.3-1 Mechanisms leading to the formation of antibodies against therapeutic proteins. In the majority of cases, B cells are thought to need the help of activated CD4+ T cells recognizing a T cell epitope in the context of MHC II molecules on antigen-presenting cells that have internalized the therapeutic protein (pathway 1). Alternatively, oligomerized or aggregated therapeutic protein may directly bind to the B cell receptor on B cells. Multivalent binding may lead to activation and differentiation to a plasma cell without prior help by CD4+ T cells (pathway 2). As a third alternative, B cells stimulated by aggregates of a therapeutic protein still need to interact with CD4+ T cells which, however, do not need to be activated by antigen-presenting cells before (pathway 3).

DRUG AND DRUG TARGET-RELATED MODULATORS

363

involvement of T cells (Figure 10.3-1, pathway 2). Small subsets of randomly aggregated or otherwise di- or oligomerized protein molecules are believed to trigger this type of B cell-mediated immunogenicity. (iii) The third alternative is a hybrid between the latter two: an aggregated (multivalent) protein stimulates a B cell in such a way that the B cell needs to present a peptide to a CD4+ T cell and requires T cell help; however, this T cell no longer needs to be pre-activated by an antigen-presenting cell (Figure 10.3-1, pathway 3). According to most recent findings, multivalent ligation of the BcR promotes presentation of antigenic peptides by B cells to CD4+ T cells, whereas monovalent ligation fails to do so, thereby maintaining B cell tolerance (Kim et al., 2006). The underlying mechanism of multivalent ligation is reminiscent of how bacteria or some viruses trigger B cell responses: the organization in molecular arrays consisting of the same immunodominant cell wall or capsid protein readily leads to BcR clustering followed by proliferation and differentiation of the respective B cell. Another mechanism which can never be excluded, may involve crossreactivity: the protein therapeutic may be recognized by memory CD4+ T cells and/or B cells which have originally been generated against an unrelated foreign or self-protein sharing structural similarity with the therapeutic protein.

DRUG AND DRUG TARGET-RELATED MODULATORS OF IMMUNOGENICITY Apart from patient-related factors, such as age, disease type, co-medication, or genotype, several drug-related aspects determine the immunogenic potential of biotherapeutics (Figure 10.3-2). A critical if not the most important criterion is the primary structure or amino acid sequence of a protein drug. Protein drugs that carry peptide stretches able to bind to molecules of the human MHC II appear to be prone to induction of immunogenicity in humans, as will be outlined below. In addition to the amino acid sequence, post-translational modifications of proteins, such as glycosylation, are thought to play a role (De Groot et al., 2005). On the one hand, glycosylation may impose steric hindrance and thereby may prevent proteins from aggregation and proteolysis leading to lowered incidence of immunogenicity. On the other hand, modifications in the glycosylation pattern of therapeutic proteins may increase their affinity to particular Fc receptors on antigen-presenting cells (APCs), thereby increasing their immunogenic potential (Figure 10.3-2). Pegylated proteins have been seen to be less immunogenic than non-pegylated counterparts, albeit this may not apply to all pegylated biologics (Kronman et al., 2007). In contrast, the presence of nonhuman glycans, such as α1,3-galactose, upon incorporation into oligosaccharide chains by nonhuman production cell lines, is expected to increase the immunogenicity risk as a high percentage of circulating B cells in human blood are recognizing the α1,3-galactose moiety (Galili et al., 1993).

364


Protein Sequence

Modifications (Glycosylation, PEGylation etc.

Pre- and CoMedication

Genotype of Patient

Uptake by immune cells

Route of Administra tion s.c. > i.v.

Formulation & Dosing

Figure 10.3-2 Drug- and patient-related criteria modulating the capacity of a biotherapeutic drug to become immunogenic in man.

Furthermore, particular formulations that may favor formation of protein aggregates are thought to contribute to immunogenicity by breaking B cell tolerance (Hermeling et al., 2005). Similar effects may be obtained in the presence of certain excipients, in case they activate co-stimulatory molecules on immune cells—a characteristic typically observed with adjuvants (Manjili et al., 2006). The same rationale may explain why biotherapeutics, which are directed against cell surface-associated rather than soluble target molecules, tend to bear an intrinsically higher immunogenicity risk (Gilliland et al., 1999). In case the target molecule is expressed on immune cells, binding of a therapeutic protein may directly induce a co-stimulatory event. In case the target molecule is expressed on a cell unrelated to the immune system and the drug is a therapeutic antibody that binds to multiple copies of its surface target molecule at the same time, the decorated target cell may trigger co-stimulatory signals in trans on Fc receptor-bearing immune cells, such as monocytes, macrophages, or dendritic cells, via the Fc portion of clustered therapeutic antibody molecules. Along the same lines of reasoning, protein therapeutics that are efficiently taken up by and/or accumulate within professional APC—due to structural features recognized by receptors on APCs, e.g., Fc, C-type lectin, or cytokine receptors (cf. Figure 10.3-2)—may be more potent in triggering a humoral antidrug immune response (Vogt et al., 2005). T Cell Epitopes Similar to other exogenous proteins, biotherapeutic proteins can be taken up by professional APCs, such as macrophages and/or dendritic cells, residing in


365

the blood, the skin, the lung, or another target tissue. These APCs are specialized in digesting exogenous proteins, irrespective of whether they represent “self” or “foreign,” and loading the resulting peptide fragments onto MHC II molecules, provided that they fulfill the requirements for binding into their antigenic peptide-binding groove (Vogt et al., 2005). In contrast to threedimensionally folded epitopes recognized by antibodies, denoted as “B cell epitopes,” MHC II-restricted peptides recognized by CD4+ helper T cells are unfolded, linear entities containing about 12–25 amino acids. In the majority of cases, these peptides need to be presented by MHC II molecules on dendritic cells (DCs) in order to be immunogenic, as it is essentially DCs that prime naive T cells against protein or peptide antigens presented to the immune system for the first time. Activation of T cells appears to be key, as only activated CD4+ helper T cells secrete the cytokines IL-4 and/or IL-13 which mediate ADA formation through inducing differentiation of B cells. Antigenic peptides which successfully activate T cells are termed “T cell epitopes.” T cell epitopes derived from different antigenic proteins share similar sequence features when they bind to the same type of MHC II molecule (Figure 10.3-3). They display three to four amino acids at defined positions within a 9-mer core region. These amino acids form MHC allele-specific binding motifs and are termed “anchor” residues, which fit into corresponding specificity pockets of the peptide-binding groove (Kropshofer and Spindeldreher, 2005). The interspersed non-anchor residues are candidates for recognition by TcR, with the precise choice of residues determining the TcR specificity. The stability of MHC II-peptide complexes and, thus, the composition of the set of peptides actually available for T cell activation, is not simply defined by the sum of anchor residue-specific incremental energies but also by the combination of anchor residues and side chains at non-anchor positions: e.g., proline, glycine,

C

P K

V Y

N

K Q

P4

L K T

P6

A

Residues to contact the T cell receptor

L Anchor residues contacting HLA pockets P1-P9 P9

P1 a

b

Figure 10.3-3 Structural features of a peptide critical for functioning as a T cell epitope. Schematic view on how peptides bind into the antigenic cleft of a heterodimeric MHC II molecule (black) via contacts of anchor residues (in one-letter code, above the arrows) with specificity pockets (P1-P9). Residues potentially interacting with the T cell receptor of a CD4+ T cell are shown in white letters.

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or charged residues may prevent peptides from binding to human MHC II HLA-DR molecules—even in the presence of appropriate anchor residues (Kropshofer and Spindeldreher, 2005). Moreover, formation of MHC IIpeptide complexes is a multistep process that follows the principle of an induced-fit mechanism—giving rise to several conformers. Consequently, the thermodynamically stable conformer, reflected by X-ray structures, may not disclose all structural features of a peptide critical for kinetically stable binding into the MHC II groove, which is a prerequisite for T cell epitopes. These arguments provide a serious challenge for in silico algorithms that aim at predicting T cell epitopes by employing available X-ray structures or molecular modeling tools. HLA Polymorphism and Diversity in Immunogenicity in Humans Classical MHC II molecules have evolved to bind and present peptide fragments from pathogen-derived foreign proteins. To cope with the high variability of bacterial and viral proteins, the loci coding for the human MHC II genes, termed human leukocyte antigens (HLA-) DR, DP, and DQ, display the highest degree of polymorphism in the human genome. The locus encoding the HLA-DR beta chain, representing both the most thoroughly explored and the most relevant HLA class II isotype with respect to immunogenicity, comprises more than 400 allelic variants (Vogt et al., 2005). Structurally, the polymorphism is restricted to those regions that constitute the single peptide-binding site of HLA molecules in order to accommodate a large variety of potential T cell epitopes across a population. As a consequence, for the assessment of immunogenicity risks, the ligand-binding prerequisites of those HLA alleles have to be considered primarily that are most abundantly represented in the respective patient population. Within the Caucasian population, the DRB1 supertypes *0101, *0301, *0401, *0404, *0701, *0801, *1101, *1301, and *1501 provide a coverage of roughly 85%. Noteworthy, particular HLA alleles function as susceptibility factors in autoimmune diseases leading to their enrichment in the respective autoimmune disease population: e.g., the allele DRB1*1501 attains a frequency of 11% in the Caucasian population, whereas in multiple sclerosis patients the same DRB1 allele attains a frequency of almost 20%. The potential relevance of the HLA genotype of patients for the evaluation of the risk of a patient as to developing neutralizing antibodies against the drug of choice has been demonstrated in a recent study (Barbosa et al., 2006). The study describes a link between the HLA class II allele DRB1*0701, present in more than 20% of multiple sclerosis patients studied, and the incidence of ADA after treatment with the interferon-beta drug Betaseron. Almost 50% of the patients that developed neutralizing antibodies against Betaseron carried the HLA-DRB1 genotype *0701. Moreover, it could be shown that a single T cell epitope within the Betaseron sequence binds to HLA-DRB1*0701 molecules and activates autologous CD4+ T cells. Likewise, the HLA haplo-

367


Japanese

15.0 Gene Frequency (%)

Caucasian

10.0

5.0

0101

0401

0405

0701

0802

0901 1101

1302 1501

1602

HLA-DRB1 alleles Figure 10.3-4 Differences in the gene frequency of prominent HLA-DRB1 allotypes in the Caucasian (black) versus Japanese (blue) population. As particular DRB1 alleles may confer increased immunogenicity risk for a particular drug, the incidence of immunogenicity of a particular drug may vary widely in patient populations of different ethnic groups.

type DRB1*0701/1501 was associated with the highest T cell and antibody response in a clinical trial where 76 healthy human subjects were treated with a fusion protein consisting of two identical, biologically active, peptides attached to human Fc fragment (Koren et al., 2007). Importantly, the HLADRB1*0701 allotype varies widely in its frequency across different populations: it is quite frequent among Caucasians (around 15%), whereas it is very rare in the Japanese population (

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