Biologics in General Medicine

W.-H. Boehncke · H.H. Radeke (Eds.) Biologics in General Medicine W.-H. Boehncke · H.H. Radeke (Eds.) Biologics in G...

Author: W.-H. Boehncke (Editor) | H.H. Radeke (Editor)

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W.-H. Boehncke · H.H. Radeke (Eds.) Biologics in General Medicine

W.-H. Boehncke · H.H. Radeke (Eds.)

Biologics in General Medicine With 51 Figures in 95 Parts and 23 Tables

Prof. Dr. med. Wolf-Henning Boehncke MA Department of Dermatology Clinic of the Johann-Wolfgang-Goethe-University Theodor-Stern-Kai 7 60590 Frankfurt Prof. Dr. med. Heinfried H. Radeke Dr.-Hans-Schleussner-Foundation Immune Pharmacology pharmazentrum frankfurt, Bldg. 75, Room 103 Johann-Wolfgang-Goethe-University Theodor-Stern-Kai 7 60590 Frankfurt

ISBN 978-3-540-29017-9

Springer-Verlag Berlin Heidelberg New York

Library of Congress Control Number: 2006938888 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media http://www.springer.com ˇ Springer-Verlag Berlin Heidelberg 2007 Printed in Germany The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about the application of operative techniques and medications contained in this book. In every individual case the user must check such information by consulting the relevant literature. Editor: Dr. Ute Heilmann Desk Editor: Dörthe Mennecke-Bühler Copy-editing: WS Editorial Ltd, Shrewsbury, UK Production Editor: Joachim W. Schmidt Cover design: eStudio Calamar, Spain Typesetting: FotoSatz Pfeifer GmbH, D-82166 Gräfelfing Printed on acid-free paper – 21/3150 – 5 4 3 2 1 0

Preface

The idea for this book was born during the symposium on biologics organized by ZAFES (Center for Drug Research, Development and Safety at the University of Frankfurt am Main) in September 2005. Highly distinguished researchers specializing in the field of biologics had gathered together to exchange information on this relatively new subject. Realizing that this symposium was one of the few sources of condensed information on biologics, it became obvious that we had to create a means of informing an interested wider circle of scientists and especially general clinicians. Therefore, the editors of this book suggested to the researchers at the symposium and also to prominent scientists and clinicians involved in the development and application of biologics as their major field of interest the idea of assembling this compendium. We received an overwhelmingly positive response – thankfully also from the publisher – most being more than willing to support this innovative project with highly relevant chapters on the latest state of the art. As we were eager to fill the information gap with up-to-date knowledge, the project had to be finished within the shortest time possible. To all experienced with editing this was obviously a challenge and we are very thankful to all the contributors that our timeline had to be extended by only a few months. This book represents a collection of the most recent knowledge on biologics written by people who have been active in the field for many years. Wolf-Henning Boehncke Heinfried H. Radeke

Contents

1 1.1 1.2

Introduction: Definition and Classification of Biologics W.-H. Boehncke, H.H. Radeke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

I

Development and Pre-clinical Pharmacology of Biologics

2

Infliximab: From the Idea to the Product M. Wiekowski, Ch.E. Antoni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Characteristics and Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Antibody Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Other Mechanisms of Infliximab Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Therapeutic Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 Pharmacokinetics in Rheumatoid Arthritis Patients . . . . . . . . . . . . . . . . . . . 2.4.2 Pharmacokinetics in Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3 Pharmacokinetics in Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4 Pharmacokinetics in Pediatric Crohn’s Patients . . . . . . . . . . . . . . . . . . . . . . . 2.5 Relationship Between Infliximab Concentration and Clinical Response . . . 2.6 Antibody Formation Against Infliximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1 HACA Formation and Clinical Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Infusion Reactions/Delayed Hypersensitivity Reactions . . . . . . . . . . . . . . . . 2.8 Alternative Routes of Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.3 3.3.1

Adalimumab J. Salfeld, H. Kupper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adalimumab Comparisons with Infliximab and Etanercept . . . . . . . . . . . . . Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pivotal Studies in Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adalimumab Plus Methotrexate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 5 5 5 6 6 7 7 9 9 9 10 10 11 11 11 12 12

14 15 15 16 16 16 18 18 19

VIII

Contents

3.3.2 Monotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Adalimumab Plus Traditional DMARDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4 Early Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Pivotal Studies in Psoriatic Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Pivotal Study in Ankylosing Spondylitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Future Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1 Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2 Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19 22 22 23 25 25 25 25 26 27 28

4

Etanercept Ch.T. Molta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Metabolism and Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4 Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5 Gender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.6 Patients with Renal or Hepatic Insufficiency . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.7 Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Pharmacodynamics in Disease States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32 32 32 32 34 34 34 34 34 35 35 35 36 38 40 40 40

5

Efalizumab: Antibody Characteristics, Mode of Action and Preclinical Development S. Jahn, K. Schmitt-Rau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Development and Characterization of the Antibody . . . . . . . . . . . . . . . . . . . 5.3 Efalizumab: From Mode of Action to the Treatment of Psoriasis . . . . . . . . . 5.3.1 Psoriasis: Prevalence, Characteristics and Therapeutic Options . . . . . . . . . 5.3.2 Pathogenesis of Psoriasis: Targets for Efalizumab . . . . . . . . . . . . . . . . . . . . . 5.3.3 Efalizumab: Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Pharmacology and Toxicology of Efalizumab . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Preclinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Indication(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42 42 43 44 44 45 46 47 47 47 48 49 49 49

Contents

6

Monoclonal Antibody Targeted Radiation Cancer Therapy L.M.M. Keller, C.A. Boswell, D.E. Milenic, Erik D. Brady, Martin W. Brechbiel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Introduction and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 The Radioisotope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Linking the Radionuclide to Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 The Protein Vehicle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50 50 50 51 54 56 57 57

7

The Production of Biopharmaceuticals B. Hughes, L.E. Hann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 The Success of Modern Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 The Science and Technology Behind Modern Biopharmaceuticals . . . . . . . 7.4 Process Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Biopharmaceutical Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Quality Assurance and Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Facility Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 Biosimilar Products (or Follow-on Biologics) . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Full Reference List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II

59 59 60 61 62 63 64 64 65 65 65 66

Disease-Specific Applications and Clinical Trials

8

Treating Autoimmune Bullous Skin Disorders with Biologics R. Eming, A. Niedermeier, M. Pfütze, A. Jacobi, M. Hertl . . . . . . . . . . 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.1 Autoimmune Bullous Skin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2 Immune Pathogenesis of Bullous Autoimmune Disorders . . . . . . . . . . . . . . 8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Biological Activity of Rituximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2 Clinical Experience with Rituximab Therapy . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3 Rituximab in Pemphigus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.4 Rituximab in Epidermolysis Bullosa Acquisita . . . . . . . . . . . . . . . . . . . . . . . . 8.2.5 Toxicity of Rituximab Treatment and Adverse Effects . . . . . . . . . . . . . . . . . . 8.2.6 Contraindications for Treatment with Rituximab . . . . . . . . . . . . . . . . . . . . . 8.3 Inhibitors of TNF- [ in the Treatment of Autoimmune Bullous Skin Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 Central Role of TNF- [ in Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 Inhibition of TNF- [ by Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.3 Inhibition of TNF- [ in Pemphigus Vulgaris . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.4 Inhibition of TNF- [ in Bullous Pemphigoid . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 69 69 71 72 72 73 73 74 76 76 76 76 77 78 79 79 80

IX

X

Contents

9

Biologics in Psoriasis W.A. Myers, W.-H. Boehncke, A.B. Gottlieb . . . . . . . . . . . . . . . . . . . . 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Mechanism of Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Etanercept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 Structure and Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Pharmacokinetics and Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5 Off-Label Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Efalizumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.1 Structure and Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Pharmacokinetics and Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . 9.3.3 Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.5 Off-Label Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Alefacept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.1 Structure and Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2 Pharmacokinetics and Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . 9.4.3 Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.5 Off-Label Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Infliximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Adalimumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81 81 81 85 86 86 86 87 88 89 89 89 89 90 91 92 92 92 93 93 94 95 95 95 96

10

Biologic Agents in Psoriatic Arthritis Ph. Mease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Classification and Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Genetic Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Immunopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Outcome Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8 Biologic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8.1 Etanercept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8.2 Infliximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8.3 Adalimumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9 Other Biologic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9.1 Alefacept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9.2 Efalizumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9.3 Abatacept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.10 Other Potential Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.11 Cost-Effectiveness Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.12 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97 97 97 97 98 98 101 101 102 102 103 104 105 105 106 106 106 107 107 108

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11

Biologic Therapies for Rheumatoid Arthritis Targeting TNF-␣ and IL-1 P.C. Taylor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 11.2 Biologic Therapies Targeting TNF- [ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 11.2.1 Rationale for TNF Blockade in the Treatment of Rheumatoid Arthritis 111 11.2.2 Clinical Studies of Anti-TNF Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 11.2.3 Safety of Biologic TNF Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 11.2.4 Infectious Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 11.2.5 Congestive Cardiac Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 11.2.6 Solid Tumours and Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 11.2.7 Other Toxicity Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 11.2.8 Injection Site Reactions or Infusion-Related Reactions . . . . . . . . . . . . . . 117 11.2.9 Mechanism of Action of TNF Blockade . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 11.3 Targeting IL-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 11.4 Combination Anti-cytokine Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 11.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 12

Biologics in Crohn’s Disease and Ulcerative Colitis: Focus on Tumor Necrosis Factor Antagonists J. Salfeld, P. Rutgeerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Clinical Features of Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Pathogenesis of Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Biologics for Use in Crohn’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 TNF Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.2 Selective Adhesion Molecule Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.3 Anti-IL-12/IL-23 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.4 Anti-IFN- * Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.5 Anti-IL-6 Receptor Monoclonal Antibody . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.6 Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Biologics in Ulcerative Colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124 124 124 126 130 134 135 136 136 137 137 137 138

13

Multiple Sclerosis: New Immunobiologics R. Gold, R. Hohlfeld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 Immunopathogenesis of Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Prominent Failure of TNF- [ Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4 Adverse Reactions in Highly Efficacious Anti- [ 4-Integrin Therapy with Natalizumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Currently Investigated Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . 13.5.1 Anti-CD52 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.2 Anti-CD25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.3 Anti-CD20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.4 Other Therapeutic Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 13.6 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 141 141 142 143 144 144 144 145 145 145 146

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XII

Contents

14

Biologics in Cutaneous Lymphoma M. Beyer, Ch. Assaf, W. Sterry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Cutaneous T-Cell Lymphomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.1 Cutaneous B-Cell Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Biologics in the Treatment of CTCL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.1 DAB389-Interleukin-2 (DAB389IL-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.2 Alemtuzumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.3 Rituximab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.4 90Y-Ibritumomab Tiuxetan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.5 Histone Deacetylase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147 147 148 149 149 149 150 150 151 152 152

15

Biologics in Targeted Cancer Therapy D. Schrama, J.C. Becker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Chemoimmunoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Immunotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4 Antibody-Cytokine Fusion Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 Evolving Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III

153 153 154 156 157 160 162 163

Safety and Perspectives

16

Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies Ch.K. Schneider, J. Löwer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 16.2 Intervention with Pleiotropic Cytokine Pathways . . . . . . . . . . . . . . . . . . . 170 16.3 Intervention with Adhesion Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 16.4 Intervention with Growth Factor Receptors . . . . . . . . . . . . . . . . . . . . . . . . 172 16.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 17

New Biological Therapeutics in the Genome Age T.N.C. Wells, S. Schnieper-Samec . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 17.2 Early Biotechnology Production of Human Cytokines and Hormones . . 176 17.3 Finding New Cytokine Orphans in the Human Genome: Early Excitement from Expressed Sequence Tags . . . . . . . . . . . . . . . . . . . . . . . . 177 17.4 Assembling the Complete Protein Collection: The Serono Secretome . . 177 17.5 Moving from the Protein to the Biological Activity: The Post-Genome Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178 17.6 Strategies for Blocking Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 17.6.1 Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 17.6.2 Receptor Fusion Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 17.6.3 Protein Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 17.6.4 Small Molecules: The Convenience of an Oral Medicine . . . . . . . . . . . . . 180 17.7 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

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18

Evidence Based Medicine’s Perspective on Biologics B. Rzany, A. Nast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.1 What is EBM? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 EBM Steps to Treating an Individual Patient . . . . . . . . . . . . . . . . . . . . . . . . 18.3 German S3 Guideline for the Treatment of Plaque Psoriasis . . . . . . . . . . . . 18.4 EBM and Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5 Where Do Biologics Stand Among Other Systemic Treatments of Psoriasis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

184 184 184 185 185 186 186

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

XIII

List of Contributors

Christian E. Antoni, MD Department of Clinical Immunology, Schering-Plough Research Institute 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA Chalid Assaf, MD Cutaneous Lymphoma Section, Department of Dermatology and Allergy, Skin Cancer Center Charité, Charité-Universitätsmedizin Berlin Fabeckstr. 60 – 62, 14195 Berlin, Germany Jürgen C. Becker, Prof. Dr. med., PhD Department of Dermatology, University of Würzburg Josef-Schneider Str. 2, 97080 Würzburg, Germany Marc Beyer, Dr. Department of Dermatology and Allergy, Skin Cancer Center Charité, CharitéUniversitätsmedizin Berlin, Fabeckstr. 60 – 62, 14195 Berlin, Germany Wolf-Henning Boehncke, Prof. Dr. med., MA Department of Dermatology, Johann-Wolfgang-Goethe-University Theodor-Stern-Kai 7, 60590 Frankfurt, Germany C. Andrew Boswell, PhD Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Bethesda, MD 20892, USA Erik D. Brady, PhD Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Bethesda, MD 20892, USA Martin W. Brechbiel, PhD Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Building 10, Room 1B40, Bethesda, MD 20892-1088, USA Rick Davis, MS, RPh Complete Healthcare Communications, Inc., One Dickinson Drive Chadds Ford PA 19317, USA

XVI


Rüdiger Eming, MD Department of Dermatology and Allergology, University Hospital, Philipps-University of Marburg, Deutschhausstraße 9, 35033 Marburg, Germany Ralf Gold, MD Experimentelle und Klinische Neuroimmunologie, Institut für MS-Forschung, Bereich Humanmedizin der Universität Göttingen und Gemeinnützige Hertie-Stiftung, Waldweg 33, 37073 Göttingen, Germany Alice B. Gottlieb, MD, PhD Department of Dermatology, Tufts-New England Medical Center 750 Washington Street, Boston, MA 02111, USA Louane E. Hann, PhD Wyeth BioPharma, One Burtt Road, Andover, MA 01810, USA Michael Hertl, MD Department of Dermatology and Allergology, Philipps University Deutschhausstraße 9, 35033 Marburg, Germany Reinhard Hohlfeld, Prof. Institute for Clinical Neuroimmunology, Klinikum Großhadern Ludwig Maximilians University, Marchioninistraße 15, 81366 Munich, Germany Brendan Hughes, PhD The Wyeth BioPharma Campus at Grange Castle, Grange Castle International Business Park, Clondalkin, Dublin 22, Ireland Arnd Jacobi, MD Department of Dermatology and Allergology, Philipps University Deutschhausstraße 9, 35033 Marburg, Germany Sigbert Jahn, MD, PhD Serono GmbH, Freisinger Str. 5, 85716 Unterschleißheim, Germany Lanea M.M. Keller, MS Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Bethesda, MD 20892, USA Hartmut Kupper, Dr. Abbott GmbH & Co., Knollstraße 50, 67061 Ludwigshafen, Germany Johannes Löwer, Prof. Dr. med. Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe (Federal Agency for Sera and Vaccines), Paul-Ehrlich-Straße 51 – 59, 63225 Langen, Germany Philip J. Mease, MD Division of Rheumatology Research, Swedish Medical Center; University of Washington School of Medicine, Seattle, WA, USA; Seattle Rheumatology Associates, 1101 Madison St., Suite 1000, Seattle, WA 98104, USA Diane E. Milenic, MS Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Bethesda, MD 20892, USA


Charles T. Molta, MD Wyeth Pharmaceuticals, 500 Arcola Road, Collegeville, PA 19426, USA Wendy A. Myers, MD UMDNJ-Robert Wood Johnson Medical School, Psoriasis Center of Excellence One Robert Wood Johnson Place, PO Box 19, New Brunswick, NJ 08903-0019, USA Alexander Nast, Dr. med. Division of Evidence-Based Medicine (dEBM), Klinik für Dermatologie, Charité-Universitätsmedizin Berlin, Campus Charité Mitte Schumannstr. 20/21, 10117 Berlin, Germany Andrea Niedermeier, MD Department of Dermatology and Allergology, Philipps University Deutschhausstraße 9, 35033 Marburg, Germany Martin Pfütze, MD Department of Dermatology and Allergology, Philipps University Deutschhausstraße 9, 35033 Marburg, Germany Heinfried H. Radeke, Prof. Dr. med. Dr.-Hans-Schleussner-Foundation Immune Pharmacology, pharmazentrum frankfurt, Bldg. 75, Room 103, Johann-Wolfgang-Goethe-University Theodor-Stern-Kai 7, 60590 Frankfurt, Germany Berthold Rzany, Prof. Dr. med., ScM Division of Evidence-Based Medicine (dEBM), Klinik für Dermatologie, Charité-Universitätsmedizin Berlin, Campus Charité Mitte Schumannstr. 20/21, 10117 Berlin, Germany Paul Rutgeerts, Prof. University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium Jochen Salfeld, Dr. Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA Karlheinz Schmitt-Rau, PhD Serono GmbH, Freisinger Str. 5, 85716 Unterschleissheim, Germany Christian Schneider, Dr. Mono- and Polyclonal Antibodies Section, Paul-Ehrlich-Institut, Bundesamt für Sera und Impfstoffe (Federal Agency for Sera and Vaccines) Paul-Ehrlich-Straße 51 – 59, 63225 Langen, Germany Sonia Schnieper-Samec, PhD Serono International, 15bis ch. des Mines, 1211 Geneva, Switzerland David Schrama, PhD Department of Dermatology, University of Würzburg Josef-Schneider Str. 2, 97080 Würzburg, Germany Wolfram Sterry, Prof. Department of Dermatology and Allergy, Skin Cancer Center Charité Charité-Universitätsmedizin Berlin, Fabeckstr. 60 – 62, 14195 Berlin, Germany

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XVIII List of Contributors Peter C. Taylor, MA, PhD, FRCP The Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, London, W6 8LH, UK Timothy N.C. Wells, PhD Serono International, 15bis ch. des Mines, 1211 Geneva, Switzerland Maria Wiekowski, PhD Department of Immunology, Schering-Plough Research Institute 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA

Chapter 1

Introduction: Definition and Classification of Biologics W.-H. Boehncke, H.H. Radeke

1.1 Aims Many chronic inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, and Crohn’s disease to name just a few are caused by an overreactive immune system. Carrier substances that falsely inform the body about an ongoing infection are produced in large quantities. The immune system answers by starting a strong immune response. The body’s own tissues are attacked and continuously destroyed. Specific models of reaction have to be understood to find a possible key to altering this perpetual process of destruction. On the other hand, the immune system may overlook the destructive process of cancer by neglecting its surveillance task, by allowing itself to be fooled by the tumor cell’s deceiving tactics. Here specific biologic reagents are needed to jump-start the immune reaction or simply for highly specific targeting of drugs to kill specifically the tumor. The development of biologics has both of these aims in mind. By definition biologics are proteins and/or derivatives thereof that modulate the immune system, downregulate the inflammatory response or support tumor specific defenses. Biologics – also known as “biologicals” or “recombinant therapeutics” – do not represent one homogeneous group of drugs. Monoclonal antibodies, fusion proteins (along with other proteins, toxins and radionucleotides) and recombinant proteins, growth factors, anti- and pro-angiogenic factors, and expression vectors generating proteins in situ may all be included as members of this class of pharmaceuticals. These drugs are designed to resemble or exploit substances that occur naturally in the body and are thereby able to influence the immune response. Advances in biotechnology and molecular biology have made their

production possible and further advances are necessary to widen their field of use. Part I of the book describes the process of development of biologics, starting with the rationales of the research labs of public institutions and also of the pharmaceutical industry. As we have limited our focus to drugs having reached clinical use, the pre-clinical part is also biased towards the success stories: It starts by defining this class of medications, which are not always recognizable as a group. The development and preclinical pharmacology of biologics are exemplified by looking at infliximab, adalimumab, etanercept and efalizumab. Part I also provides introductions to monoclonal antibody targeted radiation cancer therapy and the details of the production process, safety precautions and – last but not least – the profit margins necessary for the manufacture of biopharmaceuticals. In order to understand the background and possible problems with biologics at a more detailed level, the pre-clinical descriptions in Part I are followed by details of their clinical use in Part II. This part describes clinical studies and practical experience by presenting disease-specific applications and clinical trials. Biologics today have a broad use in medicine mainly in a group of diseases referred to as “immune-mediated inflammatory diseases” (IMIDs), comprising psoriasis and psoriatic arthritis, rheumatoid arthritis, colitis and multiple sclerosis, but also in the treatment of tumors of the skin and other organ systems. Moreover, they continue to find their way into other medical fields. Biologic therapies for rheumatoid arthritis targeting TNF- [ and IL-1 are extensively covered, and the treatments of psoriasis and psoriatic arthritis are also examined in detail. Close attention is then given to the use of biologics such as anti-TNF and the new anti-IL12p40 antibody in gastroenterology. A growing and promising use of biologics is coming up in neurology,

1

2

1 Introduction: Definition and Classification of Biologics

especially for the treatment of multiple sclerosis. Finally, more and more biologics are being successfully applied in the fight against cancer. In some types of cancer, biologics already have a solid basis as treatment, and other types of cancer are still being evaluated to identify the most suitable molecular target to start biologic therapy. Part III of this book highlights safety aspects and future perspectives, attempting to look into the future by defining possible new targets for biologic therapy. New biological therapeutic strategies in the genome age are discussed and suggested improvements for the safety of biologics are outlined. As biologics are proteins they are theoretically able to activate the immune system themselves and might cause adverse reactions such as antibody formation, which in turn may lead to allergies or neutralization of the drug. Finally, we provide a distillation of the evidence from the point of view of evidence based medicine.

1.2 Perspectives The development of biologics is still a young branch of the medical and pharmaceutical field. Further research and a wider usage of these substances will be necessary to fully explore their potential. Only by continuously expanding our knowledge about disease driving molecules and the pathomechanisms involved will science be able to extend the field of biological therapy. In fact, the application of biologics has in itself already unraveled new insights into disease processes. Clearly it is not within the scope of this book to evaluate the chances of even more advanced drug systems on the verge of clinical use, e.g., immune liposomes carrying highly active small molecules to the targets presently covered by biologics or nanoparticles targeting gene activity modifiers. Biologics are already widely recognized as milestones in the history of pharmacology. In the near future we will learn whether they will accompany us for long, as many think, or – as others feel – they will soon leave the stage like dinosaurs and be succeeded by those small molecules. More likely, particularly successful biologics will be applied more widely, will become more affordable and will be here to stay. In this case, this kind of textbook will be only the first of a continuing series....

Part I

Development and Pre-clinical Pharmacology of Biologics

I

Chapter 2

Infliximab: From the Idea to the Product M. Wiekowski, Ch. E. Antoni

2.1 Characteristics and Biological Activity 2.1.1 Antibody Characteristics Infliximab is a monoclonal antibody that neutralizes the cytokine tumor necrosis factor (TNF)- [ by binding selectively and with high affinity to soluble and membrane-bound TNF- [ . Infliximab does not bind to TNFq (lymphotoxin [ ), a related cytokine that utilizes the same receptors as TNF- [ . Thus infliximab was developed as a therapeutic agent for various inflammatory chronic diseases that are believed to be driven by the pro-inflammatory cytokine TNF- [ . Infliximab was developed by fusing the TNF- [ binding site of the murine antibody A2 to the constant region of human IgG1 κ immunoglobulin. This created a chimeric antibody with an acceptable immunogenic and pharmacokinetic profile (Knight et al. 1993). Infliximab binds the trimeric form of soluble TNF- [ with an affinity of Kd 100 pM but also binds to its monomeric form (Scallon et al. 1995, 2002). Although the trimeric form of TNF- [ is the bioactive form, binding of TNF- [ monomers might be clinically important by slowing down or even preventing the formation of trimeric TNF- [ . When testing the binding affinity of infliximab on a cell line that expresses only membrane-bound recombinant human TNF- [ , the affinity of the antibody to TNF- [ was about twofold higher when compared to the affinity to soluble TNF- [ (Kd 46 pM) (Scallon et al. 1995). In addition, the binding affinities of the dimeric F(ab’)2 fragment were 50-fold higher than that of the monomeric FAB fragment (Scallon et al. 1995). This verified that the strong avidity of the antibody binding to TNF- [ is in part based on the bivalent interaction of the antibody with its ligand.

The stability of the infliximab-TNF- [ complex was confirmed by its slow dissociation rate. In fact, no dissociation of infliximab was observed within 4 h from soluble or within 2 h from membrane-bound TNF- [ in an in vitro assay (Scallon et al. 2002). 2.1.2 Other Mechanisms of Infliximab Activity Binding and neutralization of soluble TNF- [ is probably not the only activity mediated by infliximab. Indeed, in vitro assays have shown that infliximab also binds to membrane-bound TNF- [ and induces cell death (Scallon et al. 1995). However, clinical observations suggest that infliximab mediated cytotoxicity in vivo is limited. Infliximab infusions are very well tolerated and patients do not experience a cell lysis syndrome (Scallon et al. 1995). Similarly, the number of circulating mononuclear cells does not decrease following infliximab infusions. In contrast, programmed cell death (apoptosis) has been observed in Crohn’s patients treated with infliximab within 24 h of a single infusion. In these patients a significant increase in the number of apoptotic T cells was noted in the lamina propria. Interestingly, infliximab dependent apoptosis of T cells appears to be restricted to activated T cells since in an in vitro experiment infliximab only lysed activated, but not resting, Jurkat (a T-cell line) cells (ten Hove et al. 2002). These experiments suggest that the efficacy of infliximab observed in chronic inflammatory conditions is not only mediated through the neutralization of TNF- [ , but also through other activities like induction of cell death in selected cells.

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2 Infliximab: From the Idea to the Product

infliximab

membrane-bound forms of TNF-α

soluble TNF-α

Fig. 2.1. Bivalent interaction of infliximab with its ligand cross-links soluble and membrane-bound TNF- [ (modified from Scallon B et al. 2002. J Pharmacol Exp Ther 301:418 – 426, with permission)

soluble TNF-α

membrane-bound TNF-α

2.2 Administration Infliximab is commercially formulated for intravenous infusions. It is administered to patients as an induction therapy with infusions at weeks 0, 2 and 6 followed by maintenance therapy with infusions every 8 weeks. Infusions are performed over a 2-h period.

2.3 Therapeutic Indications TNF- [ antagonists are used in inflammatory conditions that are characterized by elevated TNF- [ levels at the site of inflammation as well as systemically increased levels. For instance, increased TNF- [ expression has been detected in lesional skin of psoriatic patients (Johansen et al. 2006) or in the inflamed intestine in Crohn’s disease patients (Raddatz et al. 2005). Thus, treatment of these inflammatory conditions with TNF- [ antagonists results in the binding, neutralization and elimination of surplus TNF- [ from the blood circulation and from sites of inflammation and translates into clinical benefit for the patient. Infliximab is the only TNF- [ antagonist that is administered directly into the circulation by intravenous (i.v.) infusions; other TNF- [ antagonists like etanercept and adalimumab are administered by subcutaneous (s.c.) injections. While subcutaneous injections may be more convenient to the patient, subcutaneously administered drugs have to be absorbed from the injection site into the circulation before they can reach sites

of inflammation and can become active. Thus, based on the different routes of administration, TNF- [ antagonists present with different pharmacokinetics characterized by either a more even steady-state concentration of the subcutaneous drugs adalimumab or etanercept, or a serum profile characterized by peaks and troughs as for infliximab. The presence of peak concentrations of TNF- [ antagonist in the serum following infusions raises concern about the increased susceptibility of patients to infections by eliminating TNF- [ below a perceived safety window for an undefined time. However, accumulated data from registries that follow patients for extended period of times do not show differences in the infection rates between subjects receiving TNF- [ blockers by either s.c. or i.v. administration (Wolfe et al. 2006). Rather, increased rates of serious infections appear to be linked to elevated susceptibility of patients due to their disease (Askling et al. 2005) or concomitant medications (Wolfe et al. 2006; Westhovens et al. 2006; Lichtenstein et al. 2006). In contrast, high concentrations of TNF- [ antagonist in serum and peripheral tissue following administration might provide the opportunity for a rapid clinical response. Indeed, a clinical response to infliximab treatment was detected as early as 2 weeks following the first infusion in patients suffering from rheumatoid arthritis, Crohn’s disease and ulcerative colitis (Maini et al. 1999; Rutgeerts et al. 2005; Targan et al. 1997; Kavanaugh et al. 2000). Infliximab is currently (2006) approved for the treatment of six chronic inflammatory conditions:

2.4 Pharmacokinetics

1. For the reduction of signs and symptoms as well as improvement in physical function in patients with rheumatoid arthritis (RA) 2. For severe and active Crohn’s disease in patients who do not respond to corticosteroid or immunosuppressant therapy or those with fistulating, active Crohn’s disease 3. For ulcerative colitis in patients who do not respond to conventional therapy 4. For ankylosing spondylitis in patients who have severe axial symptoms, elevated serological markers of inflammatory activity and who have responded inadequately to conventional therapy 5. For active and progressive psoriatic arthritis in combination with methotrexate 6. For moderate to severe plaque psoriasis in patients who are intolerant to other systemic therapies

2.4 Pharmacokinetics The pharmacokinetics of infliximab has been determined in clinical studies in a variety of indications. In general, maximal or trough serum concentrations of infliximab are proportional to the dose of antibody administered independent of the inflammatory condition of the patient (summarized in Table 2.1). The volume of distribution is independent of the dose and indicates that infliximab is primarily distributed in the vascular space. The terminal half-life of infliximab is 8.0 – 9.5 days and the antibody is still detectable in the serum 12 weeks after the last infusion (Kavanaugh et al. 2000; Centocor 2006; Cornillie et al. 2001). 2.4.1 Pharmacokinetics in Rheumatoid Arthritis Patients The pharmacokinetics of infliximab has also been determined in patients with active RA. Patients with active rheumatoid who were on a stable dose of methotrexate received single infusions of 5, 10 or 20 mg/kg infliximab. Following infusions serum concentrations for infliximab peaked between 1- and 4- h post infusion and then declined exponentially from day 3 through week 12. The mean terminal half-life for infliximab was 9 – 12 days in doses of 5 – 20 mg/kg. Maximal serum concentrations (Cmax) as well as overall infliximab concentrations over time (area under the curve, AUC)

increased proportionally with an increase in the dose. Clearance, volume of distribution, mean residence time and terminal half-life were relatively constant for all three doses. The value of Cmax and the volume of distribution suggest that the total dose of infliximab distributes into the vascular space. Regardless of the dose, infliximab was still detectable in most patients at week 10 after the infusion. In this study, after having received only one dose of infliximab, most patients experienced a clinical response by week 1 or 2 and maintained improvement through week 10 – 12. The ACR20 response rates were similar for all three doses, with 81 % of patients responding at week 1 and 52 % maintaining the response at week 12. More patients in the higher dose groups achieved an ACR50 response at week 1 and more patients in the 20-mg/kg group achieved an ACR50 response at week 12 when response was compared across all dose groups. Of note is the rapid onset of a clinical response in this study as more than half of the patients on infliximab experienced a clinical response as early as week 1 after the first infusions (Kavanaugh et al. 2000). In the extension of this study, patients received 10 mg/kg infliximab infusions every 8 weeks. In these patients the serum infliximab concentrations stayed constant, suggesting that repeated administrations of this antibody did not substantially change its pharmacokinetics (Table 2.1). Most patients still had detectable serum inflixmab concentrations 12 weeks after the last infusion. Fifty-three percent of the patients in the extension study maintained an ACR20 response through week 40 (Kavanaugh et al. 2000). Dose and dosing frequency of infliximab and their effect on pharmacokinetics, efficacy and safety were also explored over a 30-week trial in rheumatoid arthritis patients. Patients who had active rheumatoid arthritis and remained on a stable dose of methotrexate received 3-mg/kg or 10-mg/kg infliximab infusions at weeks 0, 2 and 6, and then every 4 or 8 weeks thereafter. The trough infliximab serum concentrations at week 30 were determined [mean (SD) 1.5 (1.6) μg/ml for 3 mg/kg every 8 weeks, 9.7 (8.6) μg/ml for 3 mg/kg every 4 weeks, 8.9 (8.1) μg/ml for 10 mg/kg every 8 weeks and 35.8 (23.7) μg/ml for a 10-mg/kg dose every 4 weeks]. In all groups, over 50 % of patients experienced an ACR20 response as early as 2 weeks after the first infusion; this proportion of responders increased to 90 % at

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2 Infliximab: From the Idea to the Product Table 2.1. Pharmacokinetics of infliximab Dose

Times of dosing

Trough concentration (μg/ml)

Clinical response

Ref.

Rheumatoid arthritis

3 mg/kg 3 mg/kg 10 mg/kg 10 mg/kg Placebo

3 mg/kg

Week 0, 2, 6, then every 4 weeks 8 weeks 4 weeks 8 weeks

Week 0, 2, 6, 14

Maini et al. 1999 Mean (SD) 9.7 (8.6) 15.0 (1.6) 35.8 (23.7) 8.9 (8.1)

At week 30

At week 2

22.3 μg/ml (15.3 – 29.4) 14.5 μg/ml (7.3 – 22) 2.8 μg/ml (0.6 – 6.8)

DAS 28 response at week 14 in 78 % of patients

8.5 μg/ml (2.6 – 19.6) 7.9 μg/ml (0 – 20.3) 9.9 μg/ml (0.3 – 23.5)

At week 40

At week 6 At week 14 10 mg/kg

5, 10 or 20 mg/kg At week 20 at week 0 then At week 28 10 mg/kg at week 12, 20 and At week 36 28

ACR50 29 %** 27 %** 26 %** 31 %** 5%

ACR70 11 %** 8 %** 11 %** 18 %** 0% **p e 0.002

At week 30

Wolbink et al. 2006

Non-responders had significantly lower trough than responders 0.5 (0.2 – 2.2) vs 3.6 (1.4 – 8.2) μg/ml ACR20 58 %

ACR50 37 %

Kavanaugh et al. 2000

Psoriasis At week 1 Week 0, 2, 6 5 mg/kg 10 mg/kg

Gottlieb et al. 2003

0.67 μg/ml (< 0.1*–6.09) 7.11 μg/ml (2.38 – 15.13)

*2/9 patients had levels < 0.1

5 mg/kg

Week 0, 2, 6 then every 8 weeks

Crohn’s disease 10 mg/kg Every 8 weeks

Week 10 2.8 – 3.7 μg/ml between weeks 22 IFX Placebo and 46 Week 24 IFX Placebo Week 50 IFX

Week 20 Week 28 Week 36 Week 44

7.9 μg/ml 10.0 μg/ml 8.1 μg/ml 8.0 μg/ml

week 6 or 4 weeks following the second infusion. The total response rate was maintained at week 30 between 50 % and 60 %. The ACR50 response at week 30 ranged

PASI75

PASI90

80 % 3%

57 % 1%

82 % 4%

58 % 1%

61 %

15 %

Maintenance of remission At week 44 Placebo 35 % IFX 60 %

Reich et al. 2005

Rutgeerts et al. 1999

from 26 % to 31 %, and the ACR70 response from 8 % to 18 % across all dose groups with no apparent dose response (Table 2.1) (Maini et al. 1998).

2.4 Pharmacokinetics

2.4.3 Pharmacokinetics in Psoriasis

Patients with active Crohn’s disease were originally randomized to receive placebo or a single infusion of 5, 10 or 20 mg/kg infliximab. If they did not show a response by week 4, they received an additional dose of 10 μg/ml infliximab. Eight weeks later responders were eligible to enter an extension study where they were randomized to either placebo or 10-mg/kg infusions of infliximab. Patients were retreated every 8 weeks and evaluated at week 40. In these patients the analysis of the infliximab serum concentrations before an infusion every 8 weeks showed that median trough concentrations remained stable over time at about 8 – 10 μg/ml between weeks 20 and 44 (Table 2.1). Most patients had still detectable concentrations of serum infliximab 12 weeks after the final infusion (median 2.2 μg/ml). Patients who continued infliximab treatment during the extension phase were able to maintain the initial clinical benefit (Rutgeerts et al. 1999). In another study in Crohn’s disease patients, again dose-dependent serum concentrations were observed in Crohn’s disease patients after single infusions of 5, 10 or 20 mg/kg infliximab. At the recommended 5-mg/kg dose, the maximal serum concentration was 118 μg/ml and the median half-life 9.5 days. In this study at week 12 no antibody levels were detected in the serum after the 5-mg/kg infusion (Cornillie et al. 2001).

The pharmacokinetics of infliximab was determined in psoriasis patients in a phase 2 clinical trial (Gottlieb et al. 2003). Patients received doses of 5 mg/kg or 10 mg/ kg infliximab at weeks 0, 2 and 6. The highest serum concentrations of infliximab were observed in patients at week 2 immediately after the second dose: median 158.14 and 298.89 μg/ml for the 5and 10-mg/kg dose groups, respectively. The concentrations were directly proportional to the administered dose. The lowest serum concentrations of 0.67 μg/ml were observed at week 14 for the 5-mg/kg group (range < 0.1 – 6.09 μg/ml) while at week 14 the serum levels for the 10-mg/kg group were 7.11 μg/ml (Fig. 2.3). The median half-life of infliximab was determined as 7.62 days (interquartile range 6.62 – 10.15 days) for the 5-mg/kg dose group and 9.97 days (interquartile range 6.17 – 10.14 days) for the 10-mg/kg dose group (Gottlieb et al. 2003).

1000

In pediatric Crohn’s patients (mean age 10.5 ± 3.3 years), the levels of serum infliximab were determined after infusions of 5 mg/kg at week 2 and at week 6, 4 weeks after the second infusion. The trough infliximab concentration was 16.7 + 7.3 μg/ml at week 2 and 1000

100

100

1

2

6

10

14

18

1

0.1 Week

0

2

6

10

14

Infusion

Infusion 5 mg/kg Infliximab (n = 28)

10

5421

10

0.1 Week 0

2.4.4 Pharmacokinetics in Pediatric Crohn’s Patients

Infliximab serum concentration (μg/mL)

Infliximab serum concentration (μg/mL)

2.4.2 Pharmacokinetics in Crohn’s Disease

10 mg/kg Infliximab (n = 30)

Fig. 2.2. Profile of serum infliximab concentrations vs time for fistula patients who received three infusions of 5 or 10 mg/kg infliximab at weeks 0, 2 and 6 (arrows). Serum samples were obtained before and after each infusion and at the indicated time points after the third infusion. Each point represents the median result for that time period and dose group

5 mg/kg Infliximab

10 mg/kg Infliximab

Fig. 2.3. The median half-life of infliximab was determined as 7.62 days (interquartile range 6.62 – 10.15 days) for the 5-mg/ kg dose group and 9.97 (interquartile range 6.17 – 10.14) days for the 10-mg/kg dose group (Gottlieb et al. 2003)

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8.8 ± 7.1 μg/ml at week 6. This translates into a terminal half-life of 9.5 days and a mean residence time of 13 days. No infliximab (detection limit 1.8 μg/ml) was detected in serum of children at week 8 after sequential treatment with 5-mg/kg doses (Candon et al. 2006).

2.5 Relationship Between Infliximab Concentration and Clinical Response The relationship between serum infliximab concentrations and clinical response was tested in the ATTRACT trial for rheumatoid arthritis. Doses of 3 mg/kg or 10 mg/kg infliximab resulted in maximal maximum serum concentrations that were directly proportional to the intravenous dose; serum concentrations of 68.8 μg/ml and 219.1 μg/ml were detected 1 h post-infusion (St Clair et al. 2002). The trough concentration was also dependent on the amount and frequency of dose. Patients receiving 10 mg/kg every 4 weeks had the highest trough levels, while patients receiving 3 mg/kg every 8 weeks had the lowest trough levels. The median trough levels were comparable in patients receiving 10 mg/kg every 8 weeks and 3 mg/kg every 4 weeks (Kavanaugh et al. 2000; St Clair et al. 2002). When the trough serum levels of infliximab at week 54 were correlated with clinical response it became apparent that a higher ACR-N response was significantly associated with a higher trough serum concentration of infliximab. While no significant difference in the ACR20 response was detected for patients receiving 3 mg/kg or 10 mg/kg infliximab at either 4- or 8-week intervals, the ACR50 response rates were significantly lower in the group receiving 3 mg/kg every 8 weeks compared to the other groups. This suggested that clinical response relates to trough serum concentrations of infliximab. Indeed, when clinical responses were correlated with trough serum concentrations, most patients with a response of less than ACR20 had undetectable trough serum levels of infliximab, while the highest proportion of ACR50 and ACR70 responders had the highest trough serum levels (St Clair et al. 2002). In another open label study the relationship between infliximab levels and clinical response to infliximab treatment in RA was confirmed. Patients received 3mg/kg inflixmab infusions at weeks 0, 2, 6 and 14. At week 2 the median serum trough level for inflixi-

mab was 22.3 (15.3 – 29.4) μg/ml, at week six 14.6 (7.2 – 22) μg/ml, and at week fourteen 2.8 (0.6 – 6.8) μg/ ml. While trough serum concentrations varied considerably between patients, a better clinical response was associated with higher median trough serum concentrations (Wolbink et al. 2006). In general, in RA, a trough concentration of > 1.0 μg/ml is associated with a good therapeutic response (St Clair et al. 2002) and the clinical response declines rapidly after serum infliximab levels drop below this threshold (Markham and Lamb 2000). The relationship between trough infliximab serum concentrations and clinical response was also determined in a 1-year psoriasis study. Patients were receiving 5 mg/kg inflixmab treatment every 8 weeks after induction therapy. Between week 22 and week 46 the median pre-infusion inflixmab concentration stabilized between 2.8 μg/ml and 3.7 μg/ml. Patients who maintained their clinical response through week 50 had trough infliximab serum concentrations above 1 μg/ml, while in about 25 % of patients who lost the response the median pre-infusion infliximab concentrations dropped below 1 μg/ml (Reich et al. 2005).

2.6 Antibody Formation Against Infliximab The presence of antibodies to infliximab in patients is determined by an enzyme linked immunosorbent assay (ELISA). However, the detection of antibodies to infliximab can be hindered by the presence of infliximab in the serum. Thus, since infliximab can remain in the circulation for at least 4 – 12 weeks after infusion, serum samples are usually collected for analysis 12 weeks or later after the last infusion. When the formation of antibodies against infliximab (HACA) was determined 12 weeks after the last infusion in RA patients, HACA were detected in about 17 % of all treated patients. The rate of HACA responses was inversely proportional to the dosage; HACA formation occurred in 53 %, 21 % and 7 % of patients treated with 1, 3 and 10 mg/kg infliximab, respectively. Concomitant treatment with methotrexate decreased the rate to 15 %, 7 % and 3 % for the three respective dosages. The smaller proportion of patients on higher doses of infliximab who are developing HACA suggest that anti-TNF- [ treatment induces a phenomenon resembling tolerance (Maini et al. 1998).

2.8 Alternative Routes of Administration

A significantly higher incidence of antibodies to infliximab was detected in Crohn’s patients who received episodic treatment, i.e., they received one dose of infliximab at the beginning of the trial, but then only placebo until week 14 or later until the disease worsened. While 30 % of patients on this treatment schedule developed HACA, only 8 % of patients who received infliximab infusions every 8 weeks developed HACA. In addition, the incidence of HACA was higher in patients who did not receive immunomodulators compared to those who did (18 % vs 10 %, p = 0.02) (Hanauer et al. 2004). This suggests that higher doses of infliximab at scheduled (every 8 weeks) infusions and perhaps in combination with an immunomodulator are the best treatment paradigm to reduce the possibility of developing HACA. 2.6.1 HACA Formation and Clinical Response Administration of a chimeric human-mouse antibody can lead to the formation of antibodies against the chimeric protein and the formation of immune complexes. The presence of antibodies directed against therapeutic antibodies in patients can lead to adverse reactions upon administration or loss of efficacy due to accelerated elimination of the therapeutic antibody from the circulation. Preclinical studies in cynomolgus monkeys provided information on the formation and size of immune complexes between infliximab and anti-inflixmab antibodies, the distribution of these complexes in the whole body, and the rate and mechanism of their elimination. Cynomolgus monkeys received infusions of 1.74 mg/kg inflixmab followed by either an intravenous bolus of 0.5 mg/kg radio-labeled rhesus monkey anti-infliximab IgG (test) or radio-labeled rhesus monkey nonimmune IgG (control antibodies). In these monkeys the immune complexes between infliximab and the anti-infliximab antibodies formed quickly within 5 min of administration, while no complexes were formed between infliximab and the control antibodies. The terminal half-life of the anti-infliximab immune complex was approximately 38 h compared to 86 h for the control antibody, thus implying an about twofold accelerated clearance of infliximab in complex with an antibody (Rojas et al. 2005). In a cohort of infliximab treated Crohn’s disease patients, titers of anti-infliximab antibodies were cor-

related with clinical response. Again, anti-infliximab antibody concentrations of 8 μg/ml or greater were positively correlated with a higher risk for infusion reactions and a decreased duration of a therapeutic response to infliximab (Baert et al. 2003). These studies suggest that scheduled treatment in combination with an immunosuppressive drug carries the least risk of antibody formation against infliximab and offers the best probability to maintain the response.

2.7 Infusion Reactions/Delayed Hypersensitivity Reactions The highest frequency of adverse events in response to infliximab treatment are infusion reactions occurring within 2 h of the infusion. They typically consist of fever, shills, nausea, dyspnea, and headaches, and symptoms can be controlled with drug treatment (e.g., antihistamines). Infusion reactions led to discontinuation of treatment in approximately 3 % of patients, and were considered serious in less than 1 % of patients (St Clair et al. 2004; Sands et al. 2004; Lipsky et al. 2000; Gottlieb et al. 2004). Delayed reactions like myalgias, arthralgias, fever, rash, pruritus, facial, hand or lip edema, dysphagia, urticaria, sore throat and headache may occur within 3 – 12 days following the infusion (Lipsky et al. 2000). However, while the rate of infusion reactions positively correlated with the development of HACA, just the presence of HACA antibodies in the serum of patients proved to be poorly predictive for these events (Hanauer et al. 2004; Baert et al. 2003).

2.8 Alternative Routes of Administration For the treatment of rheumatoid arthritis, infliximab is approved in combination with methotrexate for intravenous dosing at 3 mg/kg every 8 weeks. As an alternative to the intravenous dosing route, an experimental formulation for subcutaneous and intramuscular (i.m.) dosing was evaluated in RA patients who are refractory to methotrexate therapy in a phase I clinical study. Fifteen patients were randomized to receive s.c. doses at 0.5 mg/kg, 1.5 mg/kg and 3 mg/kg. After evaluation of

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the pharmacokinetic data from this group, a 100-mg s.c. dose was chosen to treat patients either s.c. at weeks 0, 2 and 4, or after 3-mg/kg i.v. infusions of Remicade at weeks 0 and 2 with s.c. injections at weeks 4, 6 and 8. Another group of patients received 100-mg i.m. injections at weeks 0, 2 and 4. After a single s.c. injection at doses ranging from 0.5 to 3 mg/kg, the median half-life ranged from 8.0 to 8.4 days and the Cmax and AUC increased in a roughly dose-proportional manner across these dose groups. At day 7, a 6.7-fold higher Cmax and a 6.3-fold higher AUC were determined in the 3-mg/kg dose when compared to the 0.5-mg/kg group. Subjects receiving three doses of 100 mg s.c. every 4 weeks had trough concentrations of infliximab above or equal to 1 μg/ml. Over 80 % of subjects receiving multiple doses of 100 mg infliximab s.c. achieved an ACR20 response when evaluated 2 weeks after the last dose of infliximab. In contrast, the response to i.m. infliximab administration appeared to be delayed since at 2 weeks post-treatment ~57 % of patients achieved an ACR20 response, while at 4 weeks post-treatment 83 % or five out of six patients achieved an ACR20 response. Individual patients in this study achieved higher clinical responses even after a single s.c. administration, and the majority after multiple s.c. injections achieved an ACR50 response, as did about one-third of patients in the i.m. group. Irrespective of s.c. or i.m. administration, 34 out of 43 patients experienced transient AEs of mild to moderate severity. This included ten subjects having mild to moderate infections. There were no serious injection site reactions. In addition, the humoral response to either route of administration was not suppressed. Thus this exploratory study described alternative routes of administration for infliximab that resulted in trough levels above or equal to 1 μg/ml at week 4, which is considered the serum concentration required to achieve a clinical response. However, this study is too small to draw general conclusions regarding safety or efficacy (Westhovens et al. 2006).

2.9 Summary Intravenous administration of infliximab results in serum levels that are proportional to the dose in patients suffering from a variety of inflammatory diseases. Infliximab treatment is characterized by a clini-

cal response that occurs as early as 2 weeks following the first infusion. The maintenance of the clinical response correlates with the presence of detectable infliximab concentrations of > 1 μg/ml in the serum at trough which can be maintained by infusions every 8 weeks in most patients. Formation of antibodies against infliximab has been observed in patients, and can lead to infusion reactions and faster elimination of the antibody from the circulation. While routes of administration other than infusion have been demonstrated to result in clinical benefit for patients, infliximab is commercially available only in the intravenous formulation.

References Askling J, et al. (2005) Risk and case characteristics of tuberculosis in rheumatoid arthritis associated with tumor necrosis factor antagonists in Sweden. Arthritis Rheum 52(7): 1986 – 1992 Baert F, et al. (2003) Influence of immunogenicity on the longterm efficacy of infliximab in Crohn’s disease. N Engl J Med 348(7):601 – 608 Candon S, et al. (2006) Clinical and biological consequences of immunization to infliximab in pediatric Crohn’s disease. Clin Immunol 118(1):11 – 19 Centocor BV, Product Information (EU) [online] (2006) Available from URL: http://www.emea.eu.int/humandocs/Humans/EPAR/remicade/remicade.htm [accessed July 2006] Cornillie F, et al. (2001) Infliximab induces potent anti-inflammatory and local immunomodulatory activity but no systemic immune suppression in patients with Crohn’s disease. Aliment Pharmacol Ther 15(4):463 – 473 Gottlieb AB, et al. (2003) Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-alpha monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris. J Am Acad Dermatol 48(1):68 – 75 Gottlieb AB, et al. (2004) Infliximab induction therapy for patients with severe plaque-type psoriasis: a randomized, double-blind, placebo-controlled trial. J Am Acad Dermatol 51(4):534 – 542 Hanauer SB, et al. (2004) Incidence and importance of antibody responses to infliximab after maintenance or episodic treatment in Crohn’s disease. Clin Gastroenterol Hepatol 2(7):542 – 553 Johansen C, et al. (2006) Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPKactivated protein kinase 2. J Immunol 176(3): 1431 – 1438 Kavanaugh A, et al. (2000) Chimeric anti-tumor necrosis factor-alpha monoclonal antibody treatment of patients with rheumatoid arthritis receiving methotrexate therapy. J Rheumatol 27(4):841 – 850 Knight DM, et al. (1993) Construction and initial characterization of a mouse-human chimeric anti-TNF antibody. Mol Immunol 30(16):1443 – 1453

References Lichtenstein GR, et al. (2006) Serious infections and mortality in association with therapies for Crohn’s disease: TREAT registry. Clin Gastroenterol Hepatol 4(5):621 – 630 Lipsky PE, et al. (2000) Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group. N Engl J Med 343(22):1594 – 1602 Maini RN, et al. (1998) Therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis. Arthritis Rheum 41(9):1552 – 1563 Maini R, et al. (1999) Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. ATTRACT Study Group. Lancet 354(9194):1932 – 1939 Markham A, Lamb HM (2000) Infliximab: a review of its use in the management of rheumatoid arthritis. Drugs 59(6): 1341 – 1359 Raddatz D, Bockemuhl M, Ramadori G (2005) Quantitative measurement of cytokine mRNA in inflammatory bowel disease: relation to clinical and endoscopic activity and outcome. Eur J Gastroenterol Hepatol 17(5):547 – 557 Reich K, et al. (2005) Infliximab induction and maintenance therapy for moderate-to-severe psoriasis: a phase III, multicentre, double-blind trial. Lancet 366(9494):1367 – 1374 Rojas JR, et al. (2005) Formation, distribution, and elimination of infliximab and anti-infliximab immune complexes in cynomolgus monkeys. J Pharmacol Exp Ther 313(2): 578 – 585 Rutgeerts P, et al. (1999) Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn’s disease. Gastroenterology 117(4): 761 – 769 Rutgeerts P, et al. (2005) Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med 353(23): 2462 – 2476

Sands BE, et al. (2004) Infliximab maintenance therapy for fistulizing Crohn’s disease. N Engl J Med 350(9):876 – 885 Scallon BJ, et al. (1995) Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNFalpha and activates immune effector functions. Cytokine 7(3):251 – 259 Scallon B, et al. (2002) Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther 301(2):418 – 426 St Clair EW, et al. (2002) The relationship of serum infliximab concentrations to clinical improvement in rheumatoid arthritis: results from ATTRACT, a multicenter, randomized, double-blind, placebo-controlled trial. Arthritis Rheum 46(6):1451 – 1459 St Clair EW, et al. (2004) Combination of infliximab and methotrexate therapy for early rheumatoid arthritis: a randomized, controlled trial. Arthritis Rheum 50(11):3432 – 3443 Targan SR, et al. (1997) A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease. Crohn’s Disease cA2 Study Group. N Engl J Med 337(15):1029 – 1035 ten Hove T, et al. (2002) Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn’s disease. Gut 50(2):206 – 211 Westhovens R, et al. (2006) The safety of infliximab, combined with background treatments, among patients with rheumatoid arthritis and various comorbidities: a large, randomized, placebo-controlled trial. Arthritis Rheum 54(4):1075 – 1086 Wolbink GJ, et al. (2006) Development of antiinfliximab antibodies and relationship to clinical response in patients with rheumatoid arthritis. Arthritis Rheum 54(3):711 – 715 Wolfe F, Caplan L, Michaud K (2006) Treatment for rheumatoid arthritis and the risk of hospitalization for pneumonia: associations with prednisone, disease-modifying antirheumatic drugs, and anti-tumor necrosis factor therapy. Arthritis Rheum 54(2):628 – 634

13

Chapter 3

3 Adalimumab J. Salfeld, H. Kupper

Adalimumab (Humira, Abbott Laboratories, Abbott Park, IL, USA) is the first fully human recombinant IgG1 monoclonal antibody that acts by inhibiting tumor necrosis factor-alpha (TNF- [ or TNF) (Fig. 3.1) (Abbott Laboratories 2006). In contrast to infliximab, a chimeric antibody comprising both mouse and human domains that may elicit immune responses that potentially limit its long-term use in patients with chronic conditions such as rheumatoid arthritis, adalimumab is fully human, lowering the potential for immunogenicity (van de Putte et al. 2003). Adalimumab is composed of human-derived heavy and light chain variable regions and human IgG1:κ

Fig. 3.1. Three-dimensional structure of adalimumab

constant regions, engineered through phage display technology (Abbott Laboratories 2006). Phage display technology is designed to recapitulate the physiological antibody generation process in the laboratory. Comparable to the natural selection process for the B cell displaying the appropriate antibody, phage display allows for the selection of a fully human antibody specific for an antigen, in this case TNF, from a large repertoire of antibodies. If the desired antibody is felt to be rare in the repertoire, a variant of the technology allows for more rapid “guided selection” in a two-stage process. Therefore the first step in the generation of adalimumab was a guided selection approach using the murine anti-human TNF antibody MAK195 in order to isolate a human antibody that recognized the same neutralizing epitope as MAK195. MAK195 is a potent neutralizing monoclonal antibody, which has a high affinity and a low off-rate constant for human TNF. The MAK195 VH and VL were paired with human cognate repertoires and these phage antibody libraries underwent antigen binding selection using recombinant human TNF as the antigen. The selected human VH and VL genes were then combined to generate a fully human anti-TNF antibody. Early human anti-TNF antibodies were optimized in a second phase mirroring the natural process for antibody optimization. The final antibody, adalimumab, is a full-length immunoglobulin (IgG1) molecule with optimized heavy and light chain characterized by high specificity, affinity, and potency (van de Putte et al. 2003). It is produced in a Chinese hamster ovary host cell that is transfected with a plasmid vector containing the expression cassettes for adalimumab heavy and light chains. Adalimumab is produced by a standard, well controlled fermentation and purification process. Each batch of adalimumab is characterized rigorously in a series of biochemical and biophysical characteriza-

3.1 Pharmacology

tion assays in order to meet pre-specified release criteria. Adalimumab is indistinguishable in structure and function from naturally occurring human IgG1 and has a comparable terminal half-life (approximately 2 weeks). Adalimumab was designed as a fully human anti-TNF monoclonal antibody to have the following characteristics: ) High selectivity and affinity for TNF ) Suitability for long-term chronic administration with a low degree of immunogenicity, with or without concomitant use of methotrexate (MTX) ) A low incidence of allergic reactions ) A half-life comparable to that of human IgG1 resulting in infrequent dosing for patient convenience

shown to decrease disease activity (Cooper et al. 1992; Williams et al. 1992). Adalimumab is highly specific and does not bind to or inhibit other forms of TNF, such as lymphotoxin- [ (LT [ , previously called TNF- q ). Upon binding to TNF, adalimumab neutralizes the biologic activities of this cytokine by blocking its interaction with the TNF-RI/II cell surface receptors and modulating biologic responses that are induced or regulated by TNF (Fig. 3.2) (Salfeld et al. 1998; Lee and Kavanaugh 2005). Adalimumab also binds to and neutralizes the cell membrane-associated form of TNF, which may play a role in disease (Georgopoulos et al. 1996).

3.1 Pharmacology 3.1.1 Mode of Action TNF has been implicated in the pathogenesis of many chronic autoimmune and inflammatory diseases, in particular rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis, ankylosing spondylitis (AS), and Crohn’s disease. Among its diverse pathologic effects, TNF triggers the production of collagenases and other proinflammatory cytokines such as interleukin (IL)-1, IL-6, and granulocyte-macrophage colony stimulating factor (Brenner et al. 1989; Lee and Kavanaugh 2005); stimulates the expression of endothelial adhesion molecules (e.g., endothelial leukocyte adhesion molecule1, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1), attracting leukocytes into affected joints; upregulates matrix metalloproteinase synthesis by synovial macrophages, fibroblasts, osteoclasts, and chondrocytes; and inhibits proteoglycan synthesis in cartilage (Weinblatt et al. 2003). High concentrations of TNF are found in the synovial fluid of patients with RA (Saxne et al. 1988) and PsA (Partsch et al. 1997); in psoriatic lesions of patients with psoriasis (Ritchlin et al. 1998); in the joints of patients with AS (Braun et al. 1995); and in the stool, mucosa, and blood of patients with Crohn’s disease (Braegger et al. 1992; Murch et al. 1991, 1993). In animal models of inflammatory arthritis, TNF has been shown to accelerate disease activity, and anti-TNF antibodies have been

Fig. 3.2. Tumor necrosis factor (TNF) cascade in rheumatoid arthritis. DC dendritic cells, EC endothelial cells, Fib fibroblasts, GM-CSF granulocyte-macrophage colony-stimulating factor, IL interleukin, Ly lymphocytes, Mac macrophages, MMP matrix metalloproteases, ROI reactive oxygen intermediates

15

16

3 Adalimumab

3.1.2 Pharmacodynamics In patients with RA, a rapid decrease in levels of acute phase reactants of inflammation, C-reactive protein, and fibrinogen, as well as the serum cytokines, IL-1 q and IL-6, and IL-1 receptor antagonist is observed following treatment with adalimumab (Abbott Laboratories; Barrera et al. 2001). Serum levels of matrix metalloproteinases-1 and -3 and other markers of synovial cell activation and cartilage erosion are also decreased following adalimumab administration (Abbott Laboratories 2006; Weinblatt et al. 2003). An immunology study assessing the efficacy of adalimumab plus MTX in patients with RA demonstrated that normal immune function is preserved during adalimumab therapy (Kavanaugh et al. 2002). Adalimumab treatment did not significantly alter the numbers of peripheral blood natural killer cells, monocytes/macrophages, B cells, or major T-cell subsets; moreover, in vitro lymphocyte proliferation, delayed-type hypersensitivity reactivity, and antibody responses to pneumococcal antigen vaccination were maintained during therapy (Kavanaugh et al. 2002). 3.1.3 Pharmacokinetics Adalimumab has linear pharmacokinetic properties throughout the clinical dose range. In healthy adults, the absolute bioavailability of adalimumab following a single 40-mg subcutaneous (s.c.) dose is 64 %, and maximum plasma concentrations (4.7±1.6 μg/ml) are achieved at 131±56 h. Concentrations of adalimumab in the synovial fluid from RA patients range from 31 % to 96 % of those in serum (Abbott Laboratories 2006). Adalimumab 40 mg s.c. every other week (e.o.w.) produces steady-state serum trough concentrations of 4 – 8 μg/ml, which are 3 – 7 times higher than the effective concentration (0.8 – 1.4 μg/ml) in 50 % of RA patients (Granneman et al. 2003). Concomitant MTX treatment does not significantly alter adalimumab pharmacokinetics. The presence of MTX does not adversely affect adalimumab serum concentrations. At the recommended dose of 40 mg e.o.w., mean steadystate serum concentrations are 5 μg/ml without MTX and 8 – 9 μg/ml with MTX. MTX reduced adalimumab apparent clearance after single and multiple dosing by 29 % and 44 %, respectively. Pharmacokinetic studies

have not been conducted in children or in patients with hepatic or renal impairment (Abbott Laboratories 2006). 3.1.4 Adalimumab Comparisons with Infliximab and Etanercept A number of structural and functional features of adalimumab distinguish this agent from the other registered TNF antagonists, infliximab and etanercept (Fig. 3.3). A hallmark of monoclonal antibodies is their exquisite specificity for a target antigen. Adalimumab is highly specific for TNF (in both soluble and membrane-bound forms), whereas etanercept binds and neutralizes LT [ as well as TNF. Adalimumab binds to TNF with very high affinity (Kd ~85 pM) (Kaymakcalan et al. 2002), and potently neutralizes TNF in bioassays (IC50 ~130 pM) (Salfeld 1998). The kinetic binding parameters of adalimumab and infliximab are similar, but etanercept dissociates from TNF much more rapidly than adalimumab or infliximab (Kaymakcalan 2002). These differences may relate to the fundamental structural differences between antibodies and receptors, since adalimumab and infliximab are high-affinity antibodies and etanercept is a TNF receptor fusion protein. Immunogenicity is another parameter that is directly related to the structure of protein therapeutics and can result in diminished efficacy as well as increased side effects. Adalimumab is a fully human monoclonal IgG1 antibody, indistinguishable from naturally occurring IgG1. Antibodies to adalimumab have been observed in a small proportion of patients (Abbott Laboratories 2006). This low level of immunogenicity is reflective of the natural process of anti-idiotypic antibody formation to endogenous antibodies characteristic of the natural immune network (Jerne 1974). Infliximab is a chimeric antibody, a recombinant fusion of murine and human antibody components, and is immunogenic in a high proportion of patients unless given in combination with immunosuppressive drugs (Anderson 2005; Baert 2003; Maini et al. 1998). Etanercept is comprised of human protein components, but in an artificial construct. Low levels of immunogenicity have been reported with etanercept in patients, but considerable variability was seen, depending on the assays used to detect anti-etanercept antibodies (Anderson 2005). The pharmacokinetics of the three TNF antagonists also differ significantly, and these differences may

3.1 Pharmacology

Fig. 3.3. Adalimumab structure in comparison to other TNF-antagonists. IgG1 immunoglobulin G1, TNF tumor necrosis factor. (Adapted with permission from Anderson 2005)

relate to pharmacodynamic differences underlying the differential efficacy seen across indications. Adalimumab has a significantly longer plasma half-life than etanercept (10 – 20 days vs 3 – 5.5 days, respectively), which enables less frequent dosing of adalimumab to maintain efficacious steady state drug levels. In contrast to adalimumab and etanercept, infliximab is given by intravenous injection, resulting in maximal plasma concentrations of infliximab 10- to 30-fold higher than for adalimumab or etanercept, and trough concentrations of infliximab that fall below those of adalimumab or etanercept (Granneman et al. 2003; Maini et al. 1998; Zhou et al. 2004). The impact on efficacy or safety of the large range of plasma concentrations of infliximab during treatment is not known. The comparative efficacy of the three TNF antagonists was evaluated in an animal model of rheumatoid arthritis driven by a human TNF transgene (Kaymakcalan 2002). Adalimumab was more potent than infliximab or etanercept in preventing the development of arthritis and in the suppression of histopathological

evidence of synovial inflammation, vascularity, cartilage erosion, or bone erosion. Furthermore, the clearance of human TNF from serum was significantly slower after etanercept treatment than after adalimumab or infliximab treatment, suggesting that TNF-etanercept complexes persist longer in circulation. In human clinical studies, the efficacy of adalimumab is comparable to that of infliximab or etanercept in RA, but infliximab and adalimumab appear to be significantly more efficacious than etanercept in Crohn’s disease and psoriasis. The mechanistic basis for these differences is unclear at the present time, but it is possible that antibodies have better tissue penetration and/or effector function than etanercept in these disease states. All three agents bind to membrane TNF on transfected cells, but there are conflicting in vitro data on binding and initiation of Fcmediated effector functions such as complement activation or antibody-induced cellular cytotoxicity (ADCC) by normal human cells (Scallon 1995, van den Brande 2003). Other effector mechanisms, such as the induction of apoptosis in T cells, monocytes, or other

17

18

3 Adalimumab

cells, may be mediated by TNF antagonists by “reverse signaling” following binding to membrane TNF. However, several in vitro studies, particularly with infliximab and etanercept, have yielded conflicting results regarding the induction of apoptosis in T cells and monocytes by TNF antagonists (Catrina et al. 2005; Mitoma et al. 2005; Shen et al. 2005; van den Brande et al. 2003), and the relevance of these in vitro studies to the efficacy or safety of these agents in vivo remains to be determined.

3.2 Indications Adalimumab is indicated for the treatment of moderate to severe active RA in adult patients when the response to disease-modifying antirheumatic drugs (DMARDs) including MTX has been inadequate, and for the treatment of severe, active, and progressive RA in adults not previously treated with MTX. It also is indicated for the

treatment of active and progressive PsA in adults when the response to previous DMARD therapy has been inadequate. Most recently, regulatory agencies in both the United States and European Union added an indication for AS to the adalimumab labeling (EU Humira SPC 2006; US Humira PI 2006; Abbott Laboratories 2006) (Table 3.1). Adalimumab can be used as monotherapy or in combination with MTX. Future indications currently under investigation include psoriasis (Langley et al. 2005), Crohn’s disease (Hanauer et al. 2006; Sandborn et al. 2005a), and juvenile idiopathic arthritis (Lovell et al. 2004).

3.3 Pivotal Studies in Rheumatoid Arthritis The efficacy and safety of adalimumab were assessed in five randomized, double-blind, placebo-controlled pivotal studies in patients aged 18 years or older with

Table 3.1 Approved indications and indications under investigation for adalimumab (Abbott Laboratories 2006; Hanauer et al. 2006; Langley et al. 2005; Lovell et al. 2004; Sandborn et al. 2005a; van der Heijde et al. 2006) Approved indicationa

Dose

Rheumatoid arthritis Humira (adalimumab) (Abbott Laboratories, Abbott Park, IL, USA), in combination with MTX, is indicated for: ) The treatment of moderate to severe active RA in adult patients when the response to DMARDs including MTX has been inadequate ) The treatment of severe, active and progressive RA in adults not previously treated with MTX

Adalimumab 40 mg e.o.w.; in combination with MTX or as monotherapy May increase to 40 mg weekly if not receiving MTX

Humira can be given as monotherapy in case of intolerance to MTX or when continued treatment with MTX is inappropriate Humira has been shown to reduce the rate of progression of joint damage as measured by radiograph and to improve physical function when given in combination with MTX

a

Psoriatic arthritis Treatment of active and progressive PsA in adults when the response to previous DMARD therapy has been inadequate

Adalimumab 40 mg e.o.w.

Ankylosing spondylitis (van der Heijde et al. 2006) Reducing signs and symptoms in patients with active AS


Indications under investigation

Dose

Psoriasis (Langley et al. 2005) Crohn’s disease (Hanauer et al. 2006; Sandborn et al. 2005a) Juvenile idiopathic arthritis (Lovell et al. 2004)

Under investigation (phase III) Under investigation (phase III) Under investigation (phase III)

Based on approved labeling from the European Agency for Evaluation of Medicinal Products; indications in other areas may differ AS ankylosing spondylitis, DMARDs disease-modifying antirheumatic drugs, EMEA European Agency for the Evaluation of Medicinal Products, e.o.w. every other week, FDA Food and Drug Administration, MTX methotrexate, PsA psoriatic arthritis, RA rheumatoid arthritis

3.3 Pivotal Studies in Rheumatoid Arthritis

active RA diagnosed according to American College of Rheumatology (ACR) criteria (Table 3.2). Adalimumab was administered s.c. in combination with MTX (12.5 – 25 mg), as monotherapy, or with other DMARDs (Abbott Laboratories 2006). 3.3.1 Adalimumab Plus Methotrexate The ARMADA (Anti-TNF Research Study Program of the Monoclonal Antibody Adalimumab) trial was a randomized, double-blind, placebo-controlled, multicenter trial that evaluated 271 patients with active RA who had failed therapy with one to four DMARDs and had an inadequate response to MTX. Patients received MTX plus adalimumab 20, 40, or 80 mg or MTX plus placebo e.o.w. for 24 weeks. Significant improvement in disease activity was demonstrated in patients receiving adalimumab plus MTX compared with those receiving placebo plus MTX (see Table 3.2). The addition of adalimumab to MTX substantially improved measures of functional parameters, fatigue scales, and quality of life scores in RA patients not adequately responding to MTX alone, as evidenced by the approximately 35 – 40 % reduction with the addition of adalimumab in the Disability Index of the Health Assessment Questionnaire (HAQ), clinically meaningful ( & 10 point changes) in 6 domains of the Short Form-36 (SF-36) for adalimumab plus MTX as compared with 2 domains for placebo plus MTX, and clinically important differences between adalimumab plus MTX and placebo plus MTX in Functional Assessment of Chronic Illness Therapy fatigue scores. Levels of acute-phase reactants (e.g., C-reactive protein) also were markedly reduced (Weinblatt et al. 2003). The open-label extension of this study is ongoing. Of the patients completing 5 years of treatment, clinical efficacy was sustained, with 76 %, 64 %, and 39 % of patients achieving ACR20, 50, 70 responses; 52 % achieving clinical remission; and 28 % having no physical limitations (Weinblatt et al. 2004, 2005). Furthermore, the majority of these patients were able to reduce corticosteroid and/ or MTX dosages without adversely affecting long-term efficacy (Weinblatt et al. 2005). DE019 was a randomized, double-blind, placebocontrolled, multicenter study assessing the ability of adalimumab to inhibit radiographic progression and reduce disease activity in 619 RA patients with active disease despite therapy with MTX. Patients received MTX plus either adalimumab 40 mg e.o.w., adalimu-

mab 20 mg weekly or placebo for 52 weeks. ACR20, 50, and 70 responses were significantly higher among patients treated with adalimumab plus MTX at Week 52 (see Table 3.2). Importantly, statistically significantly less radiographic progression occurred, as measured by the change in Total Sharp Score (TSS), in patients receiving 40 mg e.o.w. adalimumab plus MTX versus the placebo group, with a mean progression of 2.7 Sharp units seen in the placebo group at 1 year, and 0.1 Sharp units in the adalimumab-treated group. Additionally, at 1 year, 72 % of treated patients receiving adalimumab plus MTX had no radiographic progression from baseline. Functional improvement and QOL, as measured by the HAQ and SF-36, were also significantly improved in the combination group versus placebo, with approximately 40 % decreases in HAQ scores with the combination versus 17 % with placebo and clinically meaningful improvement in the SF-36 for the combination versus placebo (Keystone et al. 2004a). In an OLE of this study, 40 mg adalimumab was administered e.o.w. to 457 of the patients who completed DE019. At 3 years, 61 % of patients had no radiographic disease progression; moreover, significant clinical responses were sustained with 58 %, 42 %, and 23 % of patients achieving ACR20, 50, and 70 responses, respectively. Patients treated with placebo in the randomized controlled trial who had significant disease progression at 52 weeks experienced inhibition of radiographic progression and improved clinical responses when treated with adalimumab during the OLE (Keystone et al. 2004b, 2005). 3.3.2 Monotherapy The use of MTX in RA has limitations in certain patients, and many patients are unable to tolerate a high enough dose to achieve an optimal therapeutic benefit. Thus, for patients who do not tolerate MTX, monotherapy with adalimumab may be appropriate. DE011, a 26-week, randomized, double-blind, placebocontrolled, multicenter trial, evaluated the efficacy and safety of adalimumab monotherapy in 544 severely active RA patients for whom previous DMARD treatment had failed. Patients were randomized to placebo or monotherapy with 20 or 40 mg of adalimumab weekly or e.o.w. Time to clinical improvement with adalimumab was as early as 2 weeks. After 26 weeks, patients treated with adalimumab 20 or 40 mg weekly or e.o.w.

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20

3 Adalimumab Table 3.2. Summary of primary and selected secondary endpoints from pivotal and other key trials of adalimumab in patients with rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, psoriasis, or Crohn’s disease Trial name

Treatmenta

Rheumatoid arthritis Adalimumab (20, 40, ARMADA 80 mg) + MTX (Weinblatt Placebo + MTX et al. 2003)

DE019 (Keystone et al. 2004a)

Study duration

69, 67, 73 24 weeks

Primary endpoint(s)

Secondary endpoints

ACR20: 48 %, 67 %, 66 % vs. 15 %

ACR50: 32 %, 55 %, 43 % vs. 8% ACR70: 10 %, 27 %, 19 % vs. 10 % 2 HAQ: –0.54, –0.62, –0.59 vs. –0.27

62

207, 212 Adalimumab (40 mg e.o.w., 20 mg weekly) + MTX Placebo + MTX 200

52 weeks

2 mTSS: 0.1, 0.8 vs. 2.7 ACR20 at Week 24: 63 %, 61 % vs. 30 % 2 HAQ: –0.59, –0.61 vs. –0.25

ACR20: 59 %, 55 % vs. 24 % ACR50: 42 %, 38 % vs. 10 % ACR70: 23 %, 21 % vs. 5 %

106, 112, 113, 103

26 weeks

ACR20: 36 %, 39 %, 46 %, 53 % vs. 19 %

ACR50: 19 %, 21 %, 22 %, 35 % vs. 8 % ACR70: 9 %, 10 %, 12 %, 18 % vs. 2 % Moderate EULAR response: 42 %, 48 %, 56 %, 63 % vs. 26 % Good EULAR response: 7 %, 10 %, 9 %, 14 % vs. 4 % 2 HAQ: –0.29, –0.39, –0.38, –0.49 vs. –0.07

24 weeks

AEs: no statistically significant ACR20: 53 % vs. 35 % differences between adalimu- ACR50: 29 % vs. 11 % ACR70: 15 % vs. 4 % mab and placebo group in serious AEs; severe or lifethreatening AEs; AEs leading to withdrawal; rates of infections or serious infections

268 274 257

2 years

ACR50 at 1 year: 62 %, 41 %, vs. 46 % 2 mTSS at 1 year: 1.3, 3.0, vs. 5.7

At year 2: ACR20: 69 %, 49 % vs. 56 % ACR50: 59 %, 37 % vs. 43 % ACR70: 47 %, 28 % vs. 28 % ACR90: 27 %, 9 % vs. 13 % 2 mTSS: 1.9, 5.5 vs. 10.4 DAS28 < 2.6: 49 %, 25 % vs. 25 % ACR70 maintained 6 months: 49 %, 25 % vs. 27 %

151 162

24 weeks

ACR20 at Week 12: 58 % vs. 14 % 2 mTSS at Week 24: –0.2 vs. 1.0

At Week 24: ACR20: 57 % vs. 15 % ACR50: 39 % vs. 6 % ACR70: 23 % vs. 1 % PASI 50: 75 % vs. 12 % PASI 75: 59 % vs. 1 % PASI 90: 42 % vs. 0 % PGA clear or almost clear: 71 % vs. 12 % 2 HAQ: –0.4 vs. –0.1

DE011 (van Adalimumab (20 mg de Putte et al. e.o.w., 20 mg weekly, 40 mg e.o.w., 40 mg 2004) weekly) Placebo

STAR (Furst et al. 2003)

N

Adalimumab + DMARDs Placebo + DMARDs

Early rheumatoid arthritis Adalimumab + MTX PREMIER (Breedveld Adalimumab et al. 2005, MTX 2006)

Psoriatic arthritis Adalimumab ADEPT (Mease et al. Placebo 2005a, 2005b, 2005c)

110

318 318

3.3 Pivotal Studies in Rheumatoid Arthritis Table 3.2. (Cont.) Trial name

Treatmenta

570 (Genove- Adalimumab Placebo se et al. 2005)

Ankylosing spondylitis ATLAS (data Adalimumab Placebo on file; van der Heijde et al. 2006) Psoriasis 528 (Langley Adalimumab (40 mg e.o.w., 40 mg weekly) et al. 2005; Wallace et al. Placebo 2005b; 2005c; 2005d)

529 (Langley et al. 2005; Wallace et al. 2005b; 2005c; 2005d)

Adalimumab (40 mg e.o.w., 40 mg weekly, placebo switched to 40 mg e.o.w.)

Crohn’s disease Adalimumab (160/ CLASSIC-I (Hanauer et 80 mg, 80/40 mg, 40/ 20 mg) al. 2006) Placebo CLASSIC-II Adalimumab (Sandborn et al. 2005a)

a

N

Study duration

Primary endpoint(s)

Secondary endpoints

51 49

12 weeks

ACR20: 39 % vs. 16 %

ACR50: 25 % vs. 2 % ACR70: 14 % vs. 0 % PGA clear or almost clear: 41 % vs. 7 % 2 TL: –47 % vs. –1.6 % 2 HAQ: –0.3 vs. –0.1

208 107

12 weeks

ASAS20: 58 % vs. 21 %

BASDAI 50: 45 % vs. 16 % ASAS40: 41 % vs. 14 % ASAS5/6: 49 % vs. 13 % Partial remission: 21 % vs. 4 %

45, 50

12 weeks

PASI 75: 53 %, 80 % vs. 4 %

PASI 50: 76 %, 88 % vs. 17 % PASI 90: 24 %, 48 % vs. 0 % PGA clear or almost clear: 49 %, 76 % vs. 2 % DLQI of 0: 33 %, 42 % vs. 2 % 2 SF-36 PCS: 3.9, 5.9 vs. 0.8 2 EuroQOL-5D: 0.25, 0.22 vs. 0.04

42, 44, 46 60 weeks

PASI 75: 67 %, 73 %, 50 %

PASI 50: 76 %, 75 %, 78 % PASI 90: 36 %, 55 %, 41 % PGA clear or almost clear: 63 %, 79 %, 66 % DLQI of 0: 34 %, 58 %, 45 % 2 SF-36 PCS: 3.4, 6.6, 2.9 2 EuroQOL-5D: 0.21, 0.22, 0.17

76, 75, 74 4 weeks

Clinical remission (CDAI 10 times the clinical dose) with tetanus toxoid produced an impaired antibody response compared with control animals. However, there have been no animal reproduction studies and long-term carcinogenicity studies conducted with efalizumab.

5.4.2 Pharmacodynamics The pharmacodynamic properties of efalizumab were investigated in several Phase I and Phase II studies following intravenous and subcutaneous administration, either as a single dose or repeated weekly administration. In the single-dose intravenous study, doses of 0.03 – 10 mg/kg were given. Within 24 h, treatment with efalizumab reduced the level of CD11a expression on T cells to 25 % of pretreatment levels. This suppression persisted as long as efalizumab was present in the circulation. In the above mentioned study (i.v., single dose), CD11a expression returned to baseline within 7 – 10 days following clearance of efalizumab, without showing any signs of lymphocyte depletion. Total white blood cell (WBC) count was slightly increased within about 8 h of efalizumab administration; circulating lymphocyte counts were increased by day 7. Following multiple weekly dosing, lymphocytes remained elevated but returned to baseline after efalizumab clearance. This elevation of lymphocyte count is probably due to demargination – blocked entry of efalizumab-bound cells to tissues. To achieve the full pharmacodynamic effect, intravenous doses of above 0.3 mg/kg were necessary. Complete saturation and maintenance of CD11a binding site down-regulation on lymphocytes required weekly intravenous doses of 0.6 mg/kg, which corresponds to an efalizumab plasma concentration of 5 μg/ml. Several histological changes were observed in psoriatic plaques following efalizumab administration. A marked reduction of keratin-16, corresponding to decreased disease activity, was noted. Keratinocyte ICAM-1 levels were also reduced, indicating reduced cytokine-mediated inflammation. Furthermore, a significant thinning of the epidermis and restoration of normal skin was observed after 28 days of treatment, in concordance with reductions of over 50 % in cutaneous T-cell infiltration and reduced CD11a availability. These data demonstrate that by reducing CD11a on the surface of circulating and cutaneous T cells, efalizumab is able to reverse both the histological signs of inflammation and the pathological hyperplasia characteristic of plaque psoriasis. In general, the effects of subcutaneous efalizumab on lymphocytes were comparable to those observed after intravenous dosing. Subcutaneous doses of

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5 Efalizumab: Antibody Characteristics, Mode of Action and Preclinical Development

1 mg/kg/week or above produced the required efalizumab plasma concentrations of 5 μg/ml for binding site down-regulation and saturation. No additional clinical benefits of higher doses (e.g. doses of 2 mg/kg/week and 4 mg/kg/week) were observed. In addition to reduced CD11a expression on the surface of CD3+ T cells, binding of efalizumab also causes a reduced expression of other adhesion molecules, such as CD11b, L-selectin or q 7 integrin. The down-modulation of these adhesion molecules likely contributes to the anti-adhesive effects of efalizumab. There is also a decrease of [ q + T-cell receptors and of TCR-associated co-receptors, such as CD4, CD8 or CD2. Inhibition of TCR-mediated activation therefore seems to also play a role in efalizumab’s mode of action. 5.4.3 Pharmacokinetics The pharmacokinetic properties of subcutaneous efalizumab were determined in an open, multicentre, Phase I study of 70 patients suffering from moderate-tosevere plaque psoriasis. Patients received weekly doses of either 1 mg/kg (n = 33) or 2 mg/kg (n = 37) efalizumab for 12 weeks subcutaneously. 5.4.3.1 Absorption After subcutaneous administration of efalizumab, peak plasma concentrations are reached after 2 – 3 days. The average estimated bioavailability was about 50 % at the recommended dose level of subcutaneous efalizumab, 1.0 mg/kg/week. 5.4.3.2 Distribution Steady-state serum concentrations of efalizumab were achieved after four doses of weekly efalizumab, 1 mg/ kg, and after 8 weeks in patients receiving 2 mg/kg. At this dose level (with an initial dose of 0.7 mg/kg in the first week), the mean efalizumab plasma trough values were 9.1 ± 6.7 μg/ml in the 1 mg/kg group and 23.5 ± 12.2 μg/ml in the 2 mg/kg group. Volumes of distribution of the central compartment after single intravenous doses were 110 ml/kg at dose 0.03 mg/kg and 58 ml/kg at dose 10 mg/kg.

5.4.3.3 Biotransformation The metabolism of efalizumab is through internalization followed by intracellular degradation as a consequence of either binding to cell surface CD11a or through endocytosis. The expected degradation products are small peptides and individual amino acids which are eliminated by glomerular filtration. Cytochrome P450 enzymes, as well as conjugation reactions, are not involved in the metabolism of efalizumab. 5.4.3.4 Elimination Efalizumab is cleared by dose-dependent non-linear saturable elimination. Mean steady-state clearance is 24.3 ± 18.5 and 15.7 ± 12.6 ml/kg/day for the 1 mg/kg/ week and 2 mg/kg/week groups, respectively. The elimination half-life was about 6.21 ± 3.11 days for the 1 mg/ kg/week group and 7.4 ± 2.5 days in the 2 mg/kg/week group. Tend at steady state is 25.5 ± 1.6 days at 1 mg/kg/ week and 44 ± 10 days at 2 mg/kg/week. Efalizumab shows dose-dependent non-linear pharmacokinetics, which can be explained by its saturable specific binding to cell surface receptors CD11a. Clearance was more rapid at lower doses, suggesting a receptor-mediated mechanism at drug levels below 10 μg/ml. In a population pharmacokinetic analysis of 1,088 patients, body weight was found to be the most significant covariate affecting efalizumab clearance. Other covariates such as baseline Psoriasis Area and Severity Index (PASI), baseline lymphocyte count and age had modest effects on clearance; gender and ethnic origin had no effect. Additional pharmacokinetic data are available from an open-label extended treatment trial in which patients who responded to an initial treatment of efalizumab, 2 mg/kg, for 12 weeks, received the drug in a maintenance phase for up to 33 months at a dose of 1 mg/kg. Pharmacokinetic analysis of each 12-week treatment period for up to 15 months showed that steady-state trough levels remained constant during continuous efalizumab dosing. There was no evidence of efalizumab accumulation or alteration of the pharmacokinetic profile of efalizumab during long-term continuous dosing.

References

5.5 Indication(s) The European Medicines Agency (EMEA) approved efalizumab (Raptiva) for the treatment of adult patients with moderate to severe chronic plaque psoriasis who have failed to respond to, or who have a contraindication to, or are intolerant to, other systemic therapies including cyclosporine, methotrexate and PUVA. The Food and Drug Administration (FDA) approved efalizumab for the treatment of adult patients ( & 18 years old) with chronic moderate to severe plaque psoriasis who are candidates for systemic therapy or phototherapy. Efalizumab was tested in a phase II study to treat psoriasis arthritis. However, there was no significant clinical improvement detected in treated patients in comparison with the placebo group. Case reports were published about the use of efalizumab in patients with dermatomyositis, palmo-plantar pustulosis or atopic dermatitis.

5.6 Summary Efalizumab is a humanized monoclonal antibody binding to lymphocyte function-associated antigen (LFA-1). LFA-1 belongs to the family of the q 2 integrins and is expressed on the surface of T cells (CD4+ cells, T-helper cells). It is involved in several T-cell activities, such as T-cell activation and migration, as well as T-cell adhe-

sion during cellular interactions that are important for induction and maintenance of immune-mediated inflammatory processes. Efalizumab (Raptiva) is indicated for treatment of adult patients with moderate to severe chronic plaque psoriasis who have failed to respond to, or who have a contraindication to, or are intolerant to, other systemic therapies including cyclosporine, methotrexate and PUVA.

References Dustin ML, Bivona TG, Philips MR (2004) Membranes as messengers in T cell adhesion signaling. Nature Immunol 5: 363 – 372 Hildreth JEK, August JT (1985) The human lymphocyte function-associated (HLFA) antigen and a related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies. J Immunol 134:3272 – 3280 Jullien D, Prinz JC, Langley RGB, Caro I, Dummer W, Joshi A, Dedrick R, Natta P (2004) T-cell modulation for the treatment of chronic plaque psoriasis with efalizumab (Raptiva™): mechanisms of action. Dermatology 208:297 – 306 Krueger JG (2002) The immunologic basis for the treatment of psoriasis with new biologic agents. J Am Acad Dermatol 46: 1 – 23 Schön MP, Boehncke W-H (2005) Medical progress: psoriasis. N Engl J Med 352:1899 – 1912 Werther WA, Gonzalez TN, O’Connor SJ, McCabe S, Chan B, Hotaling T, Champe M, Fox JA, Jardieu PM, Berman PW, Prestal LC (1996) Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1. J Immunol 157:4986 – 4995

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Chapter 6

6 Monoclonal Antibody Targeted Radiation Cancer Therapy L.M.M. Keller, C.A. Boswell, D.E. Milenic, E.D. Brady, M.W. Brechbiel

6.1 Overview Rituximab (Rituxan), a monoclonal antibody (mAb) against CD-20, and trastuzumab (Herceptin), a mAb against HER2, have shown efficacy in clinical trials and have gained approval for therapeutic use from the Food and Drug Administration (FDA). Mylotarg, an antiCD33 mAb conjugated with the exceedingly cytotoxic antibiotic calicheamicin, has also proven effective for treating patients with acute myeloid leukemia (AML) and it has also received FDA approval. A major area of development in mAb therapeutics involves the use of radionuclides to augment the inherent mAb activity and to exploit specific targeting properties. Zevalin, an antiCD20 mAb armed with 90Y, and Bexxar, an anti-CD20 mAb armed with 131I, are two radionuclide-bearing mAbs that have recently been approved by the FDA. This chapter presents the background and strategies pertaining to radiolabeled monoclonal antibody therapy.

6.2 Introduction and Background The advent of the hybridoma, described by Kohler and Milstein, resurrected the concept put forth by Ehrlich a century ago: antibodies might serve as “magic bullets” (Ehrlich et al. 1904). Their seminal publication described the fusion of a plasmacytoma with spleen cells and the subsequent isolation of hybrids that secreted monoclonal antibodies of a pre-defined specificity. Such work was a clear step towards the development of targeted antibody therapy (Kohler and Milstein 1975). In the 1980s much interest was focused on the generation of murine mAbs against tumor-associated antigens (TAAs). Multitudes of pre-clinical studies followed,

which provided proof-of-concept for the potential application of mAbs in chemotherapeutics. However, inherent limitations of these models also demonstrated discordance in predictability of therapeutic efficacy. Preclinical and clinical investigations with murine mAbs illustrated several constraints that required attention before any degree of success could be achieved in cancer therapy. Foremost of these was the seemingly inevitable production of human anti-murine immunoglobulin antibodies (HAMAs) after one to three treatments (Schlom 1990). Other factors limiting treatment included: (1) insufficient tumor penetration with resulting inadequate therapeutic dose delivered to tumor lesions; (2) insufficient activation of effector function(s); (3) slow blood compartment clearance; (4) low mAb affinity and avidity; and (5) trafficking to or targeting of normal organs (Schlom 1990). Some of these limitations were addressed by chemical modification of the mAb, but most of these challenges have been addressed with genetic engineering techniques (Milenic 2000). This effort has primarily been applied to eliminating HAMAs by the production of chimeric mAbs, complementaritydetermining region (CDR) grafting, or complete humanization of the protein (Milenic 2000). Current advances have reached the stage where investigators are finally able to fully explore the real therapeutic potential of radiolabeled mAbs (Fig. 6.1). With the elimination of many obstacles and a better understanding of the inherent limitations of mAbs, coupled with interest and support from industry, several radiolabeled mAbs have been and are currently being evaluated in Phase III clinical trials (Table 6.1). With the groundbreaking FDA approval of two radiolabeled mAbs, Zevalin and Bexxar, for the treatment of nonHodgkin’s lymphoma (NHL), additional targeted radiation therapy products seem very probable (Srivastava and Dadachova 2001).

6.3 The Radioisotope

Fig. 6.1. Monoclonal antibodies are linked to radionuclides through methods based on the chemical characteristics of that element. Halogens, such as 131I, are routinely introduced by direct halogenation of tyrosine residues of the protein. Metallic radionuclides, such as 111In or 90Y, require chelation of the metal through a suitable ligand. This chelating agent frequently targets N-terminal and 5 -amines of lysine residues. Linking moieties include isothiocyanates, bromoacetamides, maleimides (post-thiolation of the protein), and active esters. Variations of these moieties are also employed for the indirect introduction of radio-halogens (Milenic et al. 2004) Table 6.1. Selection of monoclonal antibodies in advanced radioimmunotherapy clinical trials (Milenic et al. 2004)

CLL chronic lymphocytic leukemia, NHL non-Hodgkin’s lymphoma, PEM polymorphic epithelial mucin, CEA carcinogenic embryonic antigen, HLA human leukocyte antigen, mu murine, hu humanized

Antibody

Antibody form

Radio- Antigen Disease nuclide

Clinical trial

Bexxar

mu IgG2a

131I

CD20

NHL

Lymphocide hu IgG1 (LL2) CEA-cide hu IgG1

90

CD22

NHL

FDA approved Phase III

90Y

CEA

Cotara

hu IgG

90Y

DNA

Oncolym

hu IgG1

131I

Theragyn Zevalin

mu IgG1 mu IgG1

90

HLADR10 PEM CD20

Colorectal, breast, lung, pancreatic, stomach carcinoma Glioblastoma multiforme, anaplastic astrocytoma NHL, CLL

Y

Y

90Y

6.3 The Radioisotope The size and presentation of the disease are both critical considerations when evaluating the most appropriate radionuclide for potential treatment. As disease does not present in an exclusive form, fractionation of

Ovarian, gastric carcinoma NHL

Phase III Phase II/III Phase II/III Phase II/III FDA approved

both chemotherapeutics and external beam radiation are routine regimens. It logically follows that no single radionuclide is likely to address every therapeutic need. Unfortunately, isotope selection is also often driven by economic rather than biomedical considerations, which could negatively impact both pre-clinical and clinical trials. Ultimately, the limitations of the tar-

51

52

6 Monoclonal Antibody Targeted Radiation Cancer Therapy Radionuclide 90

Y

131I 177Lu 153Sm 186

Re Re 67Cu 225Ac 213 Bi 212Bi 211At 212Pb 125 I 123I 67 Ga 195m Pt 188

Type

Half-life

Emax (MeV)

q

2.7 days 8.0 days 6.7 days 2.0 days 3.8 days 17.0 h 2.6 days 10 days 45.7 min 1.0 h 7.2 h 10.6 h 60.1 days 13.2 h 3.3 days 4.0 days

2.3 0.81 0.50 0.80 1.1 2.1 0.57 5.83 5.87 6.09 5.87 0.57 0.35 0.16 0.18 0.13

q, * q, * q, * q, * q, * q, * [, q [ [ [ q Auger Auger Auger, q , * Auger

Mean range (mm) 2.76 0.40 0.28 0.53 0.92 2.43 0.6 0.04 – 0.1 0.04 – 0.1 0.04 – 0.1 0.04 – 0.1 0.6 0.001 – 0.02 0.001 – 0.02 0.001 – 0.02 0.001 – 0.02

Imageable

Table 6.2. Therapeutic radionuclides (Milenic et al. 2004)

No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No

Fig. 6.2. A periodic table highlighting elements of interest for nuclear medicine and radiation oncology applications color coded by emission type. With the exception of the halogens, the majority of the medically relevant radionuclides require chelating chemistry for attachment to proteins or other targeting vehicles. Specific radionuclides from cyclotrons or reactors may be obtained in pure form for longer-lived isotopes, or as the products of generator elutions for short-lived isotopes (Milenic et al. 2004)

geting vehicles and radionuclides will be more clearly defined and a rational plan for future clinical trials will follow. Some of the critical considerations for successful targeted radiation are those variables pertaining to the radiation including emission type, energy and range of emission, and the radionuclide half-life. A sampling of available isotopes (Table 6.2) results in several options comprising (Fig. 6.2) three types of emission: [ - or q -particles, or Auger electrons (Srivastava and Dadachova 2001). Historically, q -emitters have received the greatest attention. The emission path lengths of q -emitters are relatively long, yet sparse, with an average range of 275 nm and

a maximum range of 500 – 600 nm (for 90Y). q -emitters have a relatively low linear energy transfer (LET) and thus energy deposition takes place some distance from the actual decay event (Humm 1986). Therapeutic benefit results from “crossfire” where the targeted cell is not necessarily the effective target of the decay event as there is a substantial amount of irradiation of neighboring receptor-negative tumor cells as well as potentially normal cells. As such, some of the limitations of q -emitters are clearly illustrated: q -emitters cannot adequately address the treatment of small tumor burdens including single cell metastatic disease (Humm 1986). However, one great advantage of q -emitters includes the ability to bypass tumor antigen heteroge-

6.3 The Radioisotope

neity through the differential penetration of the labeled mAb. The ability to uniformly target an entire lesion becomes possible when the particle emission range exceeds the radius of the targeted lesion (O’Donoghue et al. 1995). Convenience, availability, and familiarity with radiolabeling chemistry have traditionally supported the use of the iodine isotopes such as 131I. Other clinically relevant q -emitters include 90Y, 67Cu, 186Re, and more recently 177Lu, with others having either been investigated or proposed (vide infra). Discussion of these will be limited here except to state that emission energy and half-life requirements can be met with a small cross-section of isotopes that are already available (Humm 1986). Direct radio-iodination of tyrosine moieties of the mAb has dominated this field. However, the convenience of this method is overshadowed by the rapid deiodination of the protein post-cellular internalization – a characteristic bypassed when radio-metals are used (Mattes et al. 1997; Zhang et al. 2001). 90Y has a pure q -emission which delivers ~4.5 times more radiation per mCi to a tumor than does 131I. The greater emission range (typically 1 – 10 mm) of 90Y means that the majority of the decay energy is deposited in tumors with a diameter of at least 1 cm. Unlike 131I, 90Y lacks an imageable * emission, thereby requiring dosimetry with 111In for * -scintigraphy and single photon emission computed tomography (SPECT). However, 90Y and 111 In have different chemistries and as such this approach may not prove sufficiently accurate. Furthermore, the longer emission range associated with 90Y may result in a significant degree of irradiation of normal surrounding tissue. Notably myelosuppression, with a nadir at approximately 2 weeks post-radioimmunotherapy (RIT), due to longer range q -emissions that occur during circulation, is a consistent dose limiting toxicity (Knox and Meredith 2000). 177Lu and 67Cu, lower-energy q -emitters with much shorter ranges, may offer distinct advantages for treating smaller lesions and micrometastases. 177Lu and 67Cu have both been evaluated in clinical trials for therapeutic efficacy (Mulligan et al. 1995; Alvarez et al. 1997; O’Donnell et al. 1999). Both possess imageable * -emissions permitting determination of disease extent, calculation and prediction of dosimetry, and monitoring of therapeutic efficacy. It should be noted that this same * -emission contributes to normal tissue toxicity, illustrating the desires and compromises that must be balanced in radionuclide selection. Scientists are also

beginning to consider the fact that different q -particle combinations may work synergistically. Notably, a successful 177Lu/90Y combination radionuclide therapy has been reported (de Jong et al. 2005). Despite these concerns and limitations, use of q -emitters continues to dominate pre-clinical and clinical trials. Therefore, it is of no surprise that Zevalin and Bexxar, the first radiolabeled monoclonal antibodies to receive FDA approval, are both armed with q -emitters. The list of [ -emitting radionuclides qualified for targeted radiation therapy is short owing largely to half-life constraints. Presently 212Bi, 213Bi, and 211At are actively being studied (McDevitt et al. 1998; Hassfjell and Brechbiel 2001). Additionally, 225Ac (Table 6.2) has shown promise despite concerns about the lengthy half-life and trafficking of free decay products in vivo (McDevitt et al. 2001). The bismuth radioisotopes, 212Bi and 213Bi, are available from generators based on 224Ra and 225Ac, respectively, and decay via branched pathways that results in both [ - and q -emissions. Thirtytwo percent of 212Bi decay includes a high energy * emission which 213Bi lacks. As such 213Bi is generally considered a more attractive candidate for RIT (Geerlings 1993). Protocols analogous to radio-iodination chemistry were initially applied for 211At. However, this has been supplanted with linking reagents that address the inherent instability of direct tyrosine protein labeling with this isotope (Vaidyanathan and Zalutsky 1996). As a group, the [ -emitters have high-energy particles (4 – 9 MeV), which travel relatively short distances (40 – 100 μm). They are characterized by dense emission path lengths of high LET which is approximately 400 times greater than that of q -emitters (80 vs 0.2 keV/μm), with energy deposition taking place immediately at the decay site (Fig. 6.2) (Humm 1986). Alpha-particle radiation is exquisitely cytotoxic at a dose rate of 1 cGy/h (Kurtzman et al. 1988). The short emission range limits their use to a complementary scale of disease. Most, if not all, of the therapy results from the direct emission of the [ -particle, which makes the targeted cell and immediate neighboring cells the effective targets. In contrast to q -emitters, a very low number of nuclear traversals (approximately 1 – 3) are all that is required to kill a cell with an [ -emitter, as seen in Fig. 6.3 (Humm 1986). The inherent physical limitation of [ -emitters is their relatively short physical half-life. This, coupled with a short emission path length, has generally been thought to limit their use to

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6 Monoclonal Antibody Targeted Radiation Cancer Therapy

Fig. 6.3. A comparison of the path lengths and density of emission tracks of [ - vs q –-particle emissions is depicted. The q –-particle emissions occur in a spectrum of path lengths. The sparse energy track from these emissions is deposited over many cell diameters some distance from the decay event. The [ -particle emissions occur at a discrete energy and path length resulting in high linear energy transfer. The dense energy track from these emissions is deposited directly from the decay event over only a few cell diameters, 50 – 80 μm in tissues (Milenic et al. 2004)

leukemias, highly vascularized tumors, and micrometastatic disease in which optimal access, targeting time, and appropriate disease size converge. Avenues for future work include bone marrow purging for transplant conditioning and selective vasculature tumor targeting with the ultimate goal of total tumor eradication (Bethge et al. 2003; Thorpe and Derbyshire 1997). Adjuvant therapies and combination with q -emitters may ultimately prove viable (Hassfjell and Brechbiel 2001; Hartmann et al. 1994). Auger emitters such as 67Ga, 195mPt, 123I, and 125I (Table 6.2) have received the least attention due to the accepted premise that their extreme cytoxicity is limited by the prerequisite for the emissions to occur within the cell nucleus (Kassis et al. 1999). Auger electrons are extremely low-energy atomic orbital electrons that are emitted as an alternative to X-ray emission following electron capture, a form of q – decay (Kassis et al. 1987; Kassis 2004). Auger-electron therapy is a useful strategy for specific tumor cell killing originating from subcellular (nm) ranges and highly localized energy deposition (106 – 109 cGy) in an extremely small volume (several nm3) around the decay site (Goddu et al. 1994). Auger electron emitters produce an array of reactive radicals (e.g., OH, H, e–aq,) similar to [ -emitters. The estimated absorbed dose rate at the center of a cell delivered by 99mTc, 123I, 111In, 67Ga, and 201Tl is respectively 94, 21, 18, 74, and 76 times higher if the radioactivity is localized within the nucleus versus being on the cell membrane (Faraggi et al. 1994). In a revealing study, the therapeutic effects of an internalizing monoclonal antibody labeled with 125I, 131I, 111In, or 90Y were directly compared (Behr et al. 2000). Both Auger emitters (125I and 111In) showed

better therapeutic results than the q –-emitters. In addition, a trend towards better therapeutic results with the radiometals compared to radioiodine was demonstrated. The latter finding rationalized by the fact that radiometals attached to antibodies are residualized intracellularly while radioiodinated antibodies undergo lysosomal degradation to mono- or di-iodotyrosine that is rapidly released from cells. Despite the apparent limitation posed by a nuclear localization requirement, future studies may demonstrate that Auger emitters have a significant role in chemotherapeutics (Makrigiorgos et al. 1990; Michel et al. 2003).

6.4 Linking the Radionuclide to Protein Research has sought to balance the conditions required to achieve a radiolabeled product with adequate stability of the resulting complex. However, such scales must be balanced within the constraints imposed by isotope chemistry and half-life. As such, the options of realistic isotopes and chelating agents have become narrowed and focused as this field has matured. However, refinement of bifunctional chelating agents (BCAs) remains an active area of endeavor (Packard et al. 1999). All metallic radionuclides require chelation chemistry for attachment to a mAb (Fig. 6.4). BCAs are chelates possessing specific functional groups that permit the conjugation of proteins to stable metallic radionuclide complexes. Since suitable radio-metals are diverse in their properties and coordination chemistry, no single BCA is

6.4 Linking the Radionuclide to Protein Fig. 6.4. A general view of the conjugation of a bifunctional chelating agent to a monoclonal antibody. Specifically, a bifunctional chelating agent possesses two functionalities. One portion binds (crab = chelos = chelate) metallic radionuclides while the other portion bearing a reactive functional group reacts and covalently binds to N-terminal and 5 -amines from lysine on the protein. Generally, the metallic radionuclide is added last in this sequence prior to purification of the final product; however, a pre-formed radio-metal complex can also be conjugated to protein (Milenic et al. 2004)

suitable for all (Packard et al. 1999). A selection of examples is provided in Fig. 6.5. The laudable goal of “instant” radionuclide complex formation with infinite stability has proven non-trivial. Numerous chemical criteria must be considered in the choice of chelating agent including its design and actual use. Characteristics of the metal, such as coordination number, ionic radius, binding character (hard vs soft), and reactivity (hydrolysis vs complexation) must also be considered with respect to chelate design (Packard et al. 1999). A BCA may form and maintain an adequately stable metal complex, but the formation kinetics may render a BCA impractical for an intended radionuclide. For example, DOTA (1, 4, 7, 10-tetra-azacylcododecane-N, N’, N’’, N’’’-tetraacetic acid) forms highly stable and kinetically inert complexes with 212Bi and 213Bi (Michel et al. 2003). However, Bi(III) complexation kinetics with DOTA require 15 – 45 min for reaction completion. The half-lives of the radionuclides are 60 or 46 min, respectively, making this particular combination wasteful and highly impractical (Ruegg et al. 1990). Higher temperatures traverse this in part, but are limited by denaturation of the protein vehicle. In contrast to macrocyclic BCAs, acyclic BCAs tend to possess far faster complex formation rates (Fig. 6.5). However, these are not quite as stable, representing another forced compromise. The acyclic CHX-A’’, a cyclohexylDTPA (diethylenetriamine pentaacetic acid) (Fig. 6.5), has been shown to be a viable alternative to DOTA for labeling of mAbs with Bi(III) isotopes (Brechbiel and Gansow 1992; Milenic et al. 2001). This BCA complexes bismuth “instantaneously” (t1/2 = 0.27 s) and is sufficiently stable for clinical trials (Jurcic et al. 2002). In addition, it was reported to have similar stability with the q -emitter 177Lu versus DOTA and PA-DOTA, the lat-

ter of which has been employed in clinical trials in combination with 177Lu (Mulligan et al. 1995; Alvarez et al. 1997; Schott et al. 1994). In summary, this ligand not only provides considerable versatility for radiolabeling mAbs with the [ -emitters 213Bi and 212Bi, but also with q -emitters, such as 90Y and 177Lu, permitting a wide range of clinical applications (Milenic et al. 2001; Roselli et al. 1999; Milenic et al. 2002). Although 67Cu copper isotopes have been investigated, the choice of BCA remains an open and unresolved topic (Novak-Hofer and Schubiger 2002). Several different macrocyclic chelating agents have been reported as stable and inert with 67Cu, despite reports of its transchelation to superoxide dismutase and detection in patients’ ceruloplasmin and liver (DeNardo et al. 1998; Bass et al. 2000; Rogers et al. 1996). 67Cu remains an interesting candidate for therapy with regards to emission energy, half-life, and imageable emissions (Novak-Hofer and Schubiger 2002). Production and availability may limit 67Cu and as such 64Cu may eventually be deemed more viable (Wu et al. 2000). The chemistry for linking 211At to proteins has been dominated by aryl active ester reagents that have advanced to clinical trials (Vaidyanathan and Zalutsky 1996; Zalutsky et al. 2002). Issues of inadequate in vivo stability for general application in clinical settings are as yet unresolved. Such concerns may be addressed with a better understanding of the chemistry of 211At itself.

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6 Monoclonal Antibody Targeted Radiation Cancer Therapy

Fig. 6.5. Selected examples of acyclic and macrocyclic bifunctional chelating agents that have been or are in use in antibody targeted radiation therapy clinical trials (Milenic et al. 2004)

6.5 The Protein Vehicle Despite the fact that the protein vehicle is present in low concentrations in most targeted radiation therapies, its contribution to therapeutic efficacy must not be overlooked or discounted. Direct tumor cell killing via the protein vehicle may be achieved by two separate pathways: antibody-dependent cell cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). ADCC is triggered when the Fc * receptor on effector cells engages the Fc region of an engaged antibody and tumor cell death proceeds through an effector cell-dependent mechanism. CDC is induced when complement component C1q binds to the Fc region of an engaged antibody with resultant activation of the complement cascade and tumor cell death via an effector cell-independent mechanism. Cellular receptors are integral in all aspects of cellular function including proliferation, migration, and communications. Binding of cellular membrane receptors with a mAb can induce either cell growth or death (Clynes et al. 2000; Johnson and Glennie 2003). Preclinical experiments have demonstrated a reduced therapeutic effect against breast cancer and lymphoma, by rituximab and trastuzumab, respectively, when the Fc portion of the antibody is absent (Trauth et al. 1989; Baselga and Mendelsohn 1984). The chimeric antiCD20 mAb, rituximab, approved for therapy of NHL appears to invoke a series of signaling events including

increased phosphorylation, phospholipase C * activation, c-myc up-regulation and induction of apoptosis in B lymphocytes (Cragg et al. 1999; Deans et al. 1993; Maloney et al. 1998). Trastuzumab (Herceptin), which binds HER2 and has efficacy against adenocarcinomas which overexpress HER2 as determined by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), has been postulated to have a direct antiproliferative signaling effect by blocking receptorligand interactions and by causing down-regulation of the HER2 receptor (Sarup et al. 1991; Uchiyama et al. 1981). In addition, anti-Tac (daclizumab), which recognizes the 55-kDa [ -chain of the interleukin (IL)-2 receptor (also known as CD25), achieves therapy through interference of cellular signaling by blocking IL-2 binding and thereby inhibiting lymphoid lineage tumor cell proliferation (Goldenberg 1999). A mAb that modifies cell signaling may result in synergistic effects when used in conjunction with chemo- or conventional radiotherapy. Studies in animal models have served to confirm this, as anti-HER2 mAbs in combination with external beam radiation have resulted in therapeutic efficacy in model systems where radiation or mAb alone had minimal effect on tumor xenografts (Pietras et al. 1999). Also notable is that trastuzumab combined with paclitaxel or doxorubicin enhanced both rates of response and duration of response in patients with metastatic breast cancer (Hortobagyi and Perez 2001).

References

6.6 Conclusions An overview of radiolabeled mAb directed approaches with an emphasis on the components (protein, radionuclide, chemistry) has been presented. After greater than two decades, mAb targeted therapies are generally recognized as making a significant impact in chemotherapeutics. The FDA approvals of Zevalin and Bexxar have fueled renewed enthusiasm for developing mAb directed therapies. As such, their full potential is only beginning to be appreciated and understood. However, despite the wealth of knowledge and capability in antibody engineering, the first two approved radiolabeled mAbs are murine in nature and subject to all of the limitations therein including immunogenicity and short biological half-lives. In many aspects actual clinical knowledge pertaining to the use of radiolabeled mAbs remains in its infancy. This is particularly true in regards to therapies beyond hematological diseases, including fractionated dosing schema and the rational construction of drug combination cocktail therapies in efforts to functionally integrate targeted radiation therapy with established chemotherapies and external beam therapies. Clear evidence exists that valuable results may be achieved by execution of these strategies. Dominance of mAb therapies for the lymphohematopoietic malignancies and their success therein reflects inherent accessibility and radiosensitivity of these cancers. Literature consensus appears to support mAb-based therapies of solid tumor applied in the treatment of minimal residual micrometastatic disease and as an adjuvant component of a multi-modality treatment regimen. However, limitations were recognized through the investigations with less than optimal targeting agents, suboptimal chemistry, incorrect radionuclide choice, and a less than rational experimental design. As such there remains a continuing effort to refine and optimize all of the components to improve efficacy and minimize toxicity. The future will prove exciting due to the rational exploration and application of the cumulative knowledge towards making targeted radiation therapy a reality and mainstream component for the treatment and management of cancer. Acknowledgements. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, and Center for Cancer Research.

References Alvarez RD, et al. (1997) Intraperitoneal radioimmunotherapy of ovarian cancer with 177Lu-CC49: A Phase I/II study. Gynecol Oncol 65:94 – 101 Baselga J, Mendelsohn J (1984) Receptor blockade with monoclonal antibodies as anti-cancer therapy. Pharmacol Ther 64:127 – 154 Bass LA, et al. (2000) In vivo transchelation of copper-64 from TETA-octreotide to superoxide dismutase in rat liver. Bioconjugate Chem 11:527 – 532 Behr TM, et al. (2000) Therapeutic advantages of auger electron- over q -emitting radiometals or radioiodine when conjugated to internalizing antibodies. Eur J Nucl Med 27(7): 753 – 765 Bethge WA, et al. (2003) Selective T-cell ablation with bismuth213 labeled anti-TCR [ q as nonmyeloablative conditioning for allogeneic canine marrow transplantation. Blood 101: 5068 – 5075 Brechbiel MW, Gansow OA (1992) Synthesis of C-functionalized trans-cyclohexyldiethylene-triamine-pentaacetic acids for labelling of monoclonal antibodies with the bismuth-212 [ -particle emitter. J Chem Soc Perkin Trans 11173 – 11178 Clynes RA, et al. (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nature Med 6:443 – 446 Cragg MS, et al. (1999) Signaling antibodies in cancer therapy. Curr Opin Immunol 11:541 – 547 Deans JP, et al. (1993) Association of tyrosine and serine kinases with the B cell surface antigen CD20. Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2. J Immunol 151:4494 – 4504 DeNardo GL, et al. (1998) Maximum tolerated dose of 67Cu2IT-BAT-LYM-1 for fractionated radioimmunotherapy of non-Hodgkin’s lymphoma: A pilot study. Anticancer Res 18:2779 – 2788 Ehrlich P, et al. (1904) Ueber einige Verwendungen der Naphtochinonsulfos/iure. Z Physiol Chem 61:379 – 392 Faraggi M, et al. (1994) The influence of tracer localization on the electron dose rate delivered to the cell nucleus. J Nucl Med 35(1):113 – 119 Geerlings MW (1993) Radionuclides for radioimmunotherapy: criteria for selection. Int J Biol Markers 8:180 – 186 Goddu SM, et al. (1994) Multicellular dosimetry for micrometastases: dependence of self-dose versus cross-dose to cell nuclei on type and energy of radiation and subcellular distribution of radionuclides. J Nucl Med 35(3):521 – 530 Goldenberg MM (1999) Trastuzumab, a recombinant DNAderived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin Ther 21:309 – 318 Hartmann F, et al. (1994) Radioimmunotherapy of nude mice bearing a human IL2R-expressing lymphoma utilizing the [ -emitting radionuclide-conjugated monoclonal antibody 212Bi-anti-Tac. Cancer Res 54:4362 – 4370 Hassfjell S, Brechbiel MW (2001) The development of the [ particle emitting radionuclides 212Bi and 213Bi for therapeutic applications. Chem Rev 101:2019 – 2036

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6 Monoclonal Antibody Targeted Radiation Cancer Therapy Hortobagyi GN, Perez EA (2001) Integration of trastuzumab into adjuvant systemic therapy of breast cancer: ongoing and planned clinical trials. Semin Oncol 28:41 – 46 Humm JL (1986) Dosimetric aspects of radiolabeled antibodies for tumor therapy. J Nucl Med 27:1490 – 1497 Johnson P, Glennie M (2003) The mechanisms of action of rituximab in the elimination of tumor cells. Semin Oncol 30:3 – 8 Jurcic JG, et al. (2002) Targeted alpha particle immunotherapy for myeloid leukemia. Blood 100:1233 – 1239 Kassis AI (2004) The amazing world of auger electrons. Int J Radiat Biol 80(11 – 12):789 – 803 Kassis AI, et al. (1987) Kinetics of uptake, retention, and radiotoxicity of I-125 UDR in mammalian cells – implications of localized energy deposition by auger processes. Radiat Res 109(1):78 – 89 Kassis AI, et al. (1999) Comparison of strand breaks in plasmid DNA after positional changes of auger electron-emitting iodine-125. Rad Res 151:167 – 176 Knox SJ, Meredith RF (2000) Clinical radioimmunotherapy. Semin Radiat Oncol 10:73 – 93 Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495 – 497 Kurtzman SH, et al. (1988) 212Bismuth linked to an antipancreatic carcinoma antibody: model for alpha-particle-emitter radioimmunotherapy. J Natl Cancer Inst 80:449 – 452 Makrigiorgos G, et al. (1990) Auger electron emitters: insights gained from in vitro experiments. Rad Environ Biophys 29: 75 – 91 Maloney DG, et al. (1998) IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed lowgrade non-Hodgkin’s lymphoma. Blood 90:2188 – 2195 Mattes MJ, et al. (1997) The advantage of residualizing radiolabels for targeting B-cell lymphomas with a radiolabeled anti-CD-22 monoclonal antibody. Int J Cancer 71:429 – 435 McDevitt MR, et al. (1998) Radioimmunotherapy with alphaemitting nuclides. Eur J Nucl Med 25:1341 – 1351 McDevitt MR, et al. (2001) Tumor therapy with targeted atomic nanogenerators. Science 294:1537 – 1540 Michel RB, et al. (2003) A comparison of radionuclides conjugated to antibodies for single-cell kill. J Nucl Med 44:632 – 640 Milenic DE (2000) Radioimmunotherapy: designer molecules to potentiate effective therapy. Semin Radiat Oncol 10:139 – 155 Milenic DE (2002) Monoclonal antibody-based therapy strategies: Providing options for the cancer patient. Curr Pharm Design 8:1749 – 1764 Milenic DE, et al. (2001) In vivo evaluation of bismuth-labeled monoclonal antibody comparing DTPA-derived bifunctional chelates. Cancer Biotherap Radiopharm 16:133 – 146 Milenic DE, et al. (2002) In vivo comparison of macrocyclic and acyclic ligands for radiolabeling of monoclonal antibodies with 177Lu for radioimmunotherapeutic applications. Nucl Med Biol 29:431 – 442 Milenic DE, et al. (2004) Antibody targeted radiation cancer therapy. Nat Rev Drug Discov 6:488 – 499 Mulligan T, et al. (1995) Phase I study of intravenous Lulabeled CC49 murine monoclonal antibody in patients with advanced adenocarcinoma. Clin Cancer Res 1:1447 – 1454

Novak-Hofer I, Schubiger PA (2002) Copper-67 as a therapeutic nuclide for radioimmunotherapy. Eur J Nucl Med 29: 821 – 830 O’Donnell RT, et al. (1999) A clinical trial of radioimmunotherapy with 67Cu-2IT-BAT-Lym-1 for non-Hodgkin’s lymphoma. J Nucl Med 40:2014 – 2020 O’Donoghue JA, et al. (1995) Relationships between tumor size and curability for uniformly targeted therapy with betaemitting radionuclides. J Nucl Med 36:1902 – 1909 Packard AB, et al. (1999) Metalloradiopharmaceuticals. In: Clarke MJ, Sadler PJ (eds) Metalloradiopharmaceuticals II: diagnosis and therapy. Springer-Verlag, New York, pp 45 – 116 Pietras RJ, et al. (1999) Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene. Cancer Res 59:1347 – 1355 Rogers BE, et al. (1996) Comparison of four bifunctional chelates for radiolabeling monoclonal antibodies with copper radioisotopes: biodistribution and metabolism. Bioconjugate Chem 7:511 – 522 Roselli M, et al. (1999) In vivo comparison of CHX-DTPA ligand isomers in athymic mice bearing carcinoma xenografts. Cancer Biother Radiopharm 14:209 – 220 Ruegg CL, et al. (1990) Improved in vivo stability and tumor targeting of bismuth-labeled antibody. Cancer Res 50:4221 – 4226 Sarup JC, et al. (1991) Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth. Growth Regulat 1:72 – 82 Schlom J (1990) Monoclonal antibodies: They’re more and less than you think. In: Broder S (ed) Molecular foundations of oncology. Williams and Wilkins, Baltimore, MD, pp 95 – 134 Schott ME, et al. (1994) Biodistribution and preclinical radioimmunotherapy studies using radiolanthanide-labeled immunoconjugates. Cancer 73:993 – 998 Srivastava S, Dadachova E (2001) Recent advances in radionuclide therapy. Semin Nucl Med 31:330 – 341 Thorpe PE, Derbyshire EJ (1997) Targeting of vasculature of solid tumors. J Control Release 48:277 – 288 Trauth B, et al. (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301 – 305 Uchiyama T, et al. (1981) A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. II. Expression of Tac antigen on activated cytotoxic killer T cells, suppressor cells, and on one of two types of helper T cells. J Immunol 126:1398 – 1340 Vaidyanathan G, Zalutsky MR (1996) Targeted therapy using alpha emitters. Phys Med Biol 41:1915 – 1931 Wu AM, et al. (2000) High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment Proc Natl Acad Sci USA 97:8495 – 8500 Zhang Y, et al. (2001) Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody. J Nucl Med 42:1538 – 1544 Zalutsky M, et al. (2002) Astatine-211 labeled human/mouse chimeric anti-tenascin monoclonal antibody via surgically created resection cavities for patients with recurrent glioma: Phase I study. Neuro-oncol 4:S103

Chapter 7

The Production of Biopharmaceuticals B. Hughes, L.E. Hann

7.1 Introduction The term biologics refers to a broad class of medicinal products that share a number of common features. Unlike traditional medicines that are made by chemical synthesis, biologics are made by biosynthesis in living cells. Biologics are generally much larger than traditional synthetic medicinal products and range from highly complex inactivated vaccines and plasmaderived factors to highly purified, well characterised recombinant therapeutic proteins. As new biological therapies come to market, the term biologics may encompass a diverse portfolio and include therapeutic options such as gene and cellular therapies, therapeutic vaccines, and nucleic acid preparations. The scope of this chapter focuses primarily on therapeutic proteins produced in mammalian cell culture processes. The use of therapeutic proteins as the treatment of choice for certain unmet medical needs was enabled by the convergence of two emerging technologies in the 1970s: genetic engineering and the science of cell culture. These technologies provided researchers with the ability to create specific recombinant DNA molecules encoding specific proteins and the methodology to introduce these recombinant DNA molecules into bacterial or animal cells that synthesised the protein. Further advances in cell culture technology permitted the development of high-viability, high-density cell cultures and the ability to scale cultures to larger volumes. Cell cultures, maintained in large, computer-controlled, stainless steel bioreactors enabled large-scale protein production. An interesting illustrative case history in the development of a biologic can be seen with the medicinal product alpha-interferon. In the early 1970s, interferons were heralded as promising therapeutics for a vari-

ety of disease conditions from viral infections to cancer. Initially, alpha-interferon was produced by purification of the active protein from human white blood cells. As cell culture technology advanced, a number of groups were successful in producing alpha-interferon in vitro, from cultures of transformed human lymphoblastoid cells that spontaneously produced a range of endogenous interferons. The advent of recombinant DNA technology enabled the creation of DNA vectors containing the alpha-interferon gene and the successful expression of the gene in bacterial cells. In 1986, both nonrecombinant and recombinant alpha-interferons gained regulatory approval. The introduction of recombinant expression systems cleared the way for several major protein products to be launched as therapeutics. Peptide hormones (erythropoietin, growth hormone, beta-interferon, reproductive hormones) (Chu and Robinson 2001; Lubiniecki and Lupker 1994; Simson 2002; Walsh 2003a) and enzymes (tissue plasminogen activator) (Walsh 2003a, 2003b) were produced. These molecules were used as “replacement therapies” to treat patients with diseases caused by the deficiency of specific molecules; supplementation of endogenous protein levels with the recombinant product provided a therapeutic benefit. Frozen cell banks, containing recombinant cells producing these replacement proteins, provided a readily available supply of the required factor that was not dependent on rare and potentially hazardous raw materials such as human blood and tissue. The next generation of protein therapeutics moved beyond the established strategies of managing disease states by restoring or supplementing endogenous proteins. Recombinant proteins emerged in the 1990s that included antibodies designed to bind to specific antigens or the cells they were attached to, permitting the removal or destruction of the antibody-bound moiety

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7 The Production of Biopharmaceuticals

by the immune system or via toxic molecules attached to the antibodies. Antibodies targeting tumor markers [alemtuzumab (CamPath 2005), gemtuzumab ozogamicin (Mylotarg 2006) and trastuzumab (Herceptin 2005)] and markers of inflammatory disease [antitumour necrosis factor (TNF) antibodies for rheumatoid arthritis (Humira 2005, Remicade 2005) and antiIgE for asthma (Xolair 2005)] were successfully developed and deployed in the clinic, having a profound impact on a range of diseases. Additionally, the ability to screen patients and identify those who would respond to a particular therapy added a further refinement in treatment of various diseases. The trastuzumab molecule (Herceptin 2005), targeted toward the human epidermal growth factor receptor 2 (HER2) antigen present on certain tumor cells, has been described as an early example of “patient-directedmedicine”. In this model, the patient is first assessed for the presence and level of a specific cancer antigen, allowing for treatment with an antibody that binds to that specific antigen and recruits the immune system to attack the tumor cells. Additional mechanisms are suspected in the case of trastuzumab. Fusion proteins such as etanercept (Enbrel 2005), an anti–TNF-targeted therapy, joined the arsenal of therapeutic proteins in the late 1990s. Etanercept contains a portion of the human endogenous TNF receptor fused to the constant region (Fc) of an immunoglobulin molecule; the therapeutic effect of the molecule is to bind and sequester the proinflammatory cytokine, TNF. The development of biologics for therapeutic purposes has shown a rapid series of advances over the past 25 years from the extraction of endogenous human proteins to the development and manufacture Products (company) Erypo/Procrit (Johnson & Johnson) Epogen (Amgen) Enbrel (Amgen/Wyeth) Aranesp (Amgen) Remicade (Johnson & Johnson/ Schering-Plough) Mabthera/Rituxan (Roche) Neulasta (Amgen) Avonex (Biogen Idec) Neupogen (Amgen/Roche) Lantus (Aventis) Total top 10 Global biotech market

2004 sales Percent ($US millions) change

of specifically designed molecules targeting specific mediators of disease processes. “Designer” antibodies, containing significant modifications and specialization, add an even further level of complexity: some antibodies that target tumor cells contain a covalently bound toxin or radionuclide to replace or supplement the potency of the immune system. Additionally, advances in formulation science have allowed the development of liquid formulations that have improved patient convenience, compliance, and persistence with treatment.

7.2 The Success of Modern Biotechnology The contribution of biotechnology to medical practice and the pharmaceutical industry can be evaluated by reviewing medical advances and product revenues, as well as looking at pipeline compositions and recent approvals. Biopharmaceuticals are a growing part of research and development pipelines across the pharmaceutical industry, with an ever increasing percentage of discovery stage candidates being described as large molecules. Biologics that have gained regulatory approval over the past 10 years include molecules that offered new approaches to treating a range of diseases, allowing physicians to intervene close to the root cause of the disease rather than alleviating symptoms. Antibodies have been developed for the treatment of infectious disease (Synagis 2004), anemia (Epogen 2005), and allergenic asthma (Xolair 2005), and a number of anticancer antibodies have been added to the options available to oncologists (Walsh 2003a). Additionally, Annual 2004 market growth (%)a share (%)

3,989 2,897 2,578 2,569 2,506

–4.2 –3.8 58.8 77.9 19.8

23.0 14.1 42.1 N/A 130.8

2,192 1,873 1,383 1,344 1,014 22,346 44,353

24.4 52.1 16.4 –6.8 80.9 20.7 17.0

62.7 N/A 18.1 2.5 N/A 31.3 21.6

9.0 6.5 5.8 5.8 5.6 4.9 4.2 3.1 3.0 2.3 50.4 100.0

Table 7.1. Top ten biopharmaceuticals by global sales [from IMS Health reproduced in Lawrence S (2005) Biotech drug market steadily expands. Nat Biotechnol 23:1466]

a

Compound annual growth 1999 – 2003 N/A not available

7.3 The Science and Technology Behind Modern Biopharmaceuticals

specific anti-TNF therapies have made major contributions to the treatment of inflammatory disease. Data published in December 2005, based on 2004 sales of biotechnology drugs (excluding vaccines), showed that the global market for the top ten recombinant protein therapeutics was in excess of $44 billion (Table 7.1) (Lawrence 2005). In these reports, biopharmaceuticals constituted 5 of the top 20 best-selling drugs, and monoclonal antibody sales grew by 52 %. Moreover, biotech molecules comprised 12 of 64 FDA new molecular entity approvals in 2003 and 2004 (F-D-C Reports 2005).

7.3 The Science and Technology Behind Modern Biopharmaceuticals Heterologous gene expression is possible in a variety of systems including bacteria, yeast and animal cells, as well as in transgenic animals and plants. However, to date, every commercial therapeutic protein produced utilises either a mammalian or a microbial cell culture expression platform (Walsh 2005). The first licensed human biotechnology product, recombinant human insulin (Humulin), was produced using the bacterium Escherichia coli (Johnson 1983). Today, 30 – 40 % of all approved biopharmaceuticals are made in E. coli (Walsh 2005). The small size and relatively simple structure of insulin permitted successful manufacture in E. coli. However, insolubility issues necessitating expensive refolding steps, often associated with a reduction in bioactivity, have hampered the widespread use of bacterial manufacturing platforms. Many of today’s biopharmaceuticals are more complex recombinant replacement proteins (e.g. factor VIII for haemophilia A) or monoclonal antibodies that require post-translational modifications, such as glycosylation, for biological activity and stability. While proteins expressed in yeast and transgenic systems are glycosylated, N-glycans produced by yeast and plants are different than those present in humans, thus creating immunogenicity concerns (Wurm 2004). Recent advances in yeast glycoengineering, utilizing humanised Pichia pastoris, have demonstrated the ability to produce therapeutic glycoproteins containing nearly homogeneous human glycoforms (Gerngross 2004), paving the way for the future production of glycosylated biopharmaceuticals in nonmammalian

expression systems. As of this writing, transgenic animal systems remain commercially unproven; currently, mammalian expression systems remain the platform of choice for the manufacture of high-fidelity, soluble glycoproteins. At present, about 60 – 70 % of all licensed biopharmaceuticals are produced using mammalian cell processes. For an excellent review on protein production in mammalian systems see Wurm (2004). The majority of mammalian cell culture processes utilise Chinese hamster ovary (CHO) cells, although alternative cell lines have been used successfully including mouse myeloma (NSO), baby hamster kidney (BHK), human embryonic kidney (HEK-293), or human retina derived (PER-C6) (Butler 2005). CHO cells are the dominant platform due to their ability to grow rapidly in single-cell suspension cultures. CHO cells produce glycan structures similar, but not identical, to those found in humans due to the absence of several enzymes present in the human glycosylation pathway (Jenkins and Curling 1994). However, unlike mouse cells that generate glycan structures that are highly immunogenic in humans (Jenkins et al. 1996), nonhuman glycoforms produced by CHO cells are generally not immunogenic (Butler 2005). Suspension cultures offer advantages in terms of scale-up. Currently, stainless steel bioreactors as large as 20,000 litres are used for the manufacture of biopharmaceuticals (Thiel 2004). In 2001, 70 % of licensed processes for the production of recombinant proteins utilised stirred-tank bioreactors. While animal sera were required to support the growth of high-density cell cultures in the past, approximately 50 % of current manufacturing processes employ serum-free cell culture medium. The most common production processes for the manufacture of biopharmaceuticals are perfusion and fed-batch cultures (Hu and Aunins 1997). Perfusion culture systems allow for the continuous or semicontinuous removal of medium containing accumulated inhibitory metabolic waste products such as ammonia and lactate and add an equal volume of fresh nutrients (Griffiths 2001). Fed-batch processes utilise slow feeding of key nutrients to maintain a low concentration and steady level of primary carbon sources. This results in a more efficient primary metabolism that generates lower ammonia and lactate levels (Butler 2005). Fed-batch processes are cost-effective and have greatly increased yields. With the ability to control gas and nutrient levels as well as inhibitory waste products, cell densities of greater than 107 cells per millilitre can

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be achieved (Butler 2005). Currently, high producing processes are in the range of 3 – 6 grams per litre (Winder 2005; Wurm 2004). Cell culture conditions can affect product quality. Therefore, identification and control of critical process parameters are essential to ensure performance consistency from batch to batch. For example, the glycosylation profile of a particular recombinant protein can vary depending upon the culture duration, nutrient level, growth state of the cells, pH, temperature, and dissolved gas. Thus, process control and accurate analytical methods to monitor process controls can greatly affect manufacturing process robustness and product quality. Future technological advances will significantly influence the biopharmaceutical industry’s approach to manufacturing. Presently, mammalian cell culture processes remain competitive relative to other seemingly less expensive platforms due to the proven ability of mammalian processes to deliver high-fidelity, soluble, efficacious proteins. It is expected that further improvements, such as increases in overall productivity through increased volumetric productivity and increased yields via streamlined chromatographic purification trains, will enable mammalian expression systems to remain economically competitive with other expression platforms. However, as alternative manufacturing platforms demonstrate production and economic advantages through higher yields and shorter production times, while maintaining product safety and efficacy, these new production platforms may offer a competitive advantage. The development and application of new technologies to current manufacturing paradigms will be key in determining operational flexibility of manufacturing facilities, influencing future capital investments, overall manufacturing costs, and ultimately patient access to important new therapeutic proteins.

7.4 Process Development The delivery of a new biopharmaceutical to the marketplace requires an extensive and extended period of process development involving the application of advanced techniques in molecular biology, cell culture, separation technology, and formulation science, taking several years to complete. Prior to regulatory approval,

development of the manufacturing process consumes considerable resources in terms of equipment and material as well as the people that are needed to prepare and characterise the clinical trial material. Once the manufacturing process is finalised, the process details are transferred to the manufacturing facility where the material is to be made. Details of the manufacturing process and the characterisation of the material produced are provided to the regulatory agencies to provide evidence of a stable and reproducible process, molecule, and product. Process changes after regulatory approval require additional process and molecular analyses as well as additional filings in many cases. The creation of the Master Cell Bank (MCB) is a critical milestone in biopharmaceutical process development. The MCB is a cryopreserved, long-term store of recombinant cells, either bacterial or mammalian, containing the gene that encodes the desired protein. Following transfection of host cells with a DNA plasmid containing the desired gene, the cells are subjected to a cloning procedure to ensure genetic uniformity and then are screened to identify clones that have a stable, high-level expression of the desired protein. Once identified, the desired clone is expanded and cryopreserved in vials, creating the MCB. The MCB is the source of all cells used to manufacture the medicinal product, either directly by thawing MCB vials or via an intermediate working cell bank (WCB) derived from the MCB. Cell banks are usually laid down as hundreds of vials and stored at multiple redundant locations to ensure security of supply. These cell banks are extensively tested and characterised to ensure that they are fit for purpose, stably express the desired protein over the manufacturing period, and do not contain microbiological contaminants. Another major component of process development is defining the cell culture process that expands the small number of cells in the MCB or WCB vials into the large volumes of cells required to produce economically viable amounts of proteins in a production facility. For example, the process may require expanding 3 million cells in a 1-millilitre vial to a 20,000-litre volume in a stainless steel bioreactor to achieve 10 million cells per millilitre. The cell culture development process establishes the appropriate nutrient media and specific physiological conditions for cell growth including O2 and CO2 levels and the pH of the medium, as well as any specific manipulations required to achieve high levels of protein production. Processes may be separated into

7.5 Biopharmaceutical Manufacturing

Fig. 7.1. Overview of the production process for a biopharmaceutical product. [Reprinted with permission from Walsh, G (ed) (2003) Biopharmaceuticals: Biochemistry and biotechnology, 2nd edn. John Wiley & Sons, Chichester]

two phases including an early rapid cell growth phase to maximise the number of cells available to make protein and a second production phase to maximise protein output. The phase transition can be triggered by changes in bioreactor conditions or by the addition of certain induction molecules to the cell culture medium. The cell culture process (”upstream process”) presents the expressed protein to the “downstream process” for further processing to a pure active ingredient (Fig. 7.1). This downstream processing is usually composed of a harvest step that separates the cells from the protein product as well as a number of further separation steps that purify the protein product from the remaining cell culture-derived impurities. The process development team evaluates a wide range of separation technologies and experimental conditions to determine the optimal conditions for separating the protein product from the process impurities to ensure that all products made using the process will meet predetermined quality specifications. An important activity in most process development projects is the definition of the formulation in which the protein is delivered to the clinic or marketplace. Chemical solutions are assessed for their ability to maintain stable, intact, and biologically active protein for the desired shelf life of the biopharmaceutical. Biopharmaceuticals generally are relatively fragile at room temperature and require cold chain transport and storage. Long-term stability studies (several years in duration) are carried out on the protein using a range of temperatures followed by detailed characterisation to detect any changes in chemical structure or biological activity.

7.5 Biopharmaceutical Manufacturing Over the past 2 decades, the manufacture of biopharmaceuticals has progressed dramatically to the highly complex, state-of-the-art operations that epitomise the industry today. Modern biopharmaceutical production facilities comprise multiple departments that function together to produce, test, and assure the quality of the biological drug (usually called the drug substance) before release to a fill/finish facility for drug product manufacture. Drug product manufacturing involves the preparation of the final, sterile presentation of the biological drug substance and can include lyophilised or liquid products in vials or syringes. The drug product is packaged before ultimate delivery to the patient. Biopharmaceutical manufacturing is carried out under the philosophy and approach of good manufacturing practice (GMP). GMP is an aspect of quality assurance that ensures that the biological drug is consistently produced and controlled to a standard appropriate for its intended use. The tight controls present in modern biopharmaceutical manufacturing plants ensure consistency in the manufacture of the biological drug. Raw materials such as media, water, and gases are tested against multiple specifications before being released for use in the process. Cleaning procedures are validated to ensure that process residues or by-products are removed from equipment between successive batches. Sterilisation procedures are verified for all equipment, such as bioreactors and automated process equipment, governed by a central or distributed con-

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trol system, which helps to remove human error in the process step execution. Air quality is regulated by environmental control systems to maintain pressure differentials and high-quality, low-particulate air in processing zones that require clean operations such as latestage purification operations. Taken together, these steps assure that all aspects of manufacture are tightly controlled to deliver a biological drug that meets a predefined series of pharmaceutical biochemical and functional specifications. No aspect of the manufacturing process may be changed without proper technical assessment of its impact by authorised groups within the manufacturing organisation, and where relevant, regulatory authorities. This process, known as change control, is overseen by the quality organisation. During its manufacture, the biological drug is transferred between stages of the manufacturing process (e.g., drug substance to drug product manufacturing) only after all quality control test criteria are met and a quality assurance-led batch release process is executed.

7.6 Quality Assurance and Quality Control Quality systems, including Quality Control (QC) and Quality Assurance (QA), ensure that patients receive a safe, pure, potent, and stable product. While there are considerable similarities between biopharmaceuticals and chemically synthesised drug products, the QA/QC issues and challenges are very different in some respects. Raw materials of biological origin are carefully selected and extensively tested to minimise the risk of microbial contamination of the cell culture systems. Although biopharmaceuticals can be characterised chemically to varying degrees, it is generally accepted that the structure-function relationships and key determinants of activity for biologics are not fully determined. For these reasons, most biopharmaceuticals require some element of biological testing in addition to chemical testing, as part of routine quality control processes. Immunological, biochemical, or bioassay techniques are often used to determine the concentration and activity of the active ingredient. There is heavy reliance on in-process sampling and testing as well as testing of the final drug substance and drug product due to the complexity of the molecules. As most biopharmaceuticals are provided as injectable solutions

and are administered parenterally, careful attention is paid to the maintenance of sterility of the final product.

7.7 Facility Considerations The continued development of the biopharmaceutical industry has resulted in a significant increase in demand for manufacturing capacity. The construction of a biopharmaceutical manufacturing facility is a major undertaking, even for large, established pharmaceutical companies. Wyeth’s Grange Castle facility in Dublin, Ireland, is an example of a modern integrated biopharmaceutical campus. For smaller start-up companies, the risk level and levels of human and financial capital investment required to build a manufacturing facility can be prohibitive. A large biopharmaceutical plant with the ability to generate bulk drug substance and manufacture final drug product can take up to 5 years to build and can cost up to $1 billion. Due to timeline considerations, the construction of these expensive facilities is often underway before the efficacy of the medicine or the marketplace demand is known. Because of the inherent risk and high cost associated with building a manufacturing facility, many companies decide to partner with another company with existing capacity or use a contract manufacturer. There is a complex interplay between facility design, process development, and market assessment. It is not unusual for market volume predictions to vary by 50 – 100 % or more during the development of an innovative new biopharmaceutical. For new facilities, where emerging market data exceed the initial design capacity, a difficult series of decisions must be made. A change to the facility design during the detailed planning phase will usually incur cost and time penalties. A similar change once construction has commenced can involve major increases in project cost and timelines. In this environment, the value of an effective process development unit and a sophisticated approach to project management are invaluable. An innovative process development group can help mitigate extra expense by increasing process output or recovery, in the bioreactor or downstream process respectively, increasing plant output without additional capital spending.

Key References

7.8 Biosimilar Products (or Follow-on Biologics) The era of follow-on biopharmaceuticals has arrived. A number of early biopharmaceuticals that lost patent protection in 2006 are already in production by noninnovator companies (Walsh 2003a). Some of the follow-on biologics currently on the market include human growth hormone and alpha-interferon in eastern Europe, and colony stimulating factor in China (Walsh 2003a). The terminology used for generic biopharmaceuticals is complex, with different terms having different regulatory implications. The terms “generic biopharmaceuticals”, “biogenerics”, or “generic biologics” are very similar to the small-molecule generic paradigm and suggest no requirement for clinical trials. Regulatory agencies in the United States (Food & Drug Administration, FDA) and Europe (European Medicines Agency, EMEA) have adopted the terms “followon biologic” and “biosimilar” respectively. Implied in this terminology is the understanding that clinical trials would be required to assess safety and efficacy prior to approval. The EMEA has already issued guidelines for regulatory approval of biosimilars where limited clinical trials would be required for simple, less complex products, and more extensive clinical trials required for more complex products. While numerous stakeholders have asked that the FDA develop guidance paving the way for approval of certain “generic” biologics, as of this writing, the agency has yet to offer guidelines for generic biopharmaceutical production. Many believe that the paradigm for chemical identity between biopharmaceuticals and their generic counterparts is different from small molecule drugs. For small-molecule generics, pharmaceutical and bioequivalence predict an equal therapeutic equivalence. This certainly may not be the case for many generically manufactured biopharmaceuticals. Hurdles will be high for a generic biopharmaceuical to be approved as interchangeable with an innovator’s product, especially in terms of safety and efficacy profiles: the use of a different cell line for the manufacture of a generic biopharmaceutical may result in a different product profile with respect to heterogeneity, impurities, and glycosylation, raising potential immunogenicity and other concerns. The increased immunogenicity observed with reformulated erythropoietin (Eprex) is a cautionary example illustrating how changes in stabilisers, storage, and route of administration can influence

human clinical immunogenetic responses, experiences, and safety (Casadevall and Rossert 2005). The probability that follow-on biologics or biosimilars will require limited clinical trials to prove “comparability” will make the cost of these molecules higher than their small-molecule generic counterparts. While the targets for biosimilars are known and proven, the development of generic versions of biopharmaceuticals will take longer and cost more than small-molecule generics. Sales and marketing costs are likely to be incurred, as most biosimilars will not be approved as interchangeable with the innovators’ product. However, even with these costs, it is likely that the price of biosimilars may be lower than innovators’ biopharmaceuticals and the savings may be significant enough for healthcare payers to exert pressure or offer incentives to switch to the lower cost alternative. Safety issues such as immunogenicity, which are difficult to predict from animal models, remain a concern and may potentially affect the widespread use of biosimilars.

7.9 Conclusion Biopharmaceuticals have become well established in the treatment of serious diseases. Hundreds of molecules, targeting a range of diseases, are being evaluated in the academic research community and in the pipelines of the pharmaceutical industry. In parallel, the technology for manufacturing these molecules is advancing. Higher capacity processes with better yields and reliability are beginning to make an impact on the high cost of producing these medicines, allowing more efficient use of costly manufacturing plants. Further progress in the technologies of biopharmaceutical discovery, development, and manufacturing is likely to increase the supply of important medicines to the patients who need them.

Key References Chu L, Robinson DK (2001) Industrial choices for protein production by large scale cell culture. Curr Opin Biotechnol 12:180 – 187 Thiel KA (2004) Biomanufacturing, from bust to boom...to bubble? Nature Biotechnol 22:1365 – 1372 Walsh G (2003a) Biopharmaceutical benchmarks – 2003. Nature Biotechnol 21:865 – 880

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7 The Production of Biopharmaceuticals Walsh G (ed) (2003b) Biopharmaceuticals: Biochemistry and biotechnology, 2nd edn. John Wiley & Sons, Chichester Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393 – 1398

Full Reference List Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68:283 – 291 CamPath® (alemtuzumab) (2005) Full prescribing information. Genzyme, Cambridge, MA Casadevall N, Rossert J (2005) Importance of biologic followons: experience with EPO. Best Pract Res Clin Haematol 18:381 – 387 Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12:180 – 187 Enbrel® (etanercept) (2005) Full prescribing information. Immunex Corporation, marketed by Amgen and Wyeth Pharmaceuticals, Thousand Oaks, CA Epogen® (epoetin alfa) (2005) Full prescribing information. Amgen, Thousand Oaks, CA F-D-C Reports (2005) FDA clears 31 new molecular entities in 2004, up 10 from previous year. Pharmaceuticals Approvals Monthly 10 (January 15):3 – 5 Gerngross TU (2004) Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol 22:1409 – 1414 Griffiths B (2001) Scale-up of suspension and anchoragedependent animal cells. Mol Biotechnol 17:225 – 238 Herceptin® (trastuzumab) (2005) Full prescribing information. Genentech, South San Francisco, CA Hu WS, Aunins JG (1997) Large-scale mammalian cell culture. Curr Opin Biotechnol 8:148 – 153 Humira® (adalimumab) (2005) Full prescribing information. Abbott Laboratories, North Chicago, IL

Jenkins N, Curling EM (1994) Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 16:354 – 364 Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14:975 – 981 Johnson IS (1983) Human insulin from recombinant DNA technology. Science 219:632 – 637 Lawrence S (2005) Biotech drug market steadily expands. Nat Biotechnol 23:1466 Lubiniecki AS, Lupker JH (1994) Purified protein products of rDNA technology expressed in animal cell culture. Biologicals 22:161 – 169 Mylotarg® (gemtuzumab ozogamicin) (2006) Full Prescribing Information. Wyeth Pharmaceuticals, Philadelphia, PA Remicade® (infliximab) (2005) Full prescribing information. Centocor, Inc., Malvern, PA Simson H (2002) Growth hormone replacement therapy for adults: into the new millennium. Growth Hormone IGF Res 12:1 – 33 Synagis® (palivizumab) (2004) Full prescribing information. MedImmune, Inc., Gathersburg, MD Thiel KA (2004) Biomanufacturing, from bust to boom...to bubble? Nat Biotechnol 22:1365 – 1372 Walsh G (2003a) Biopharmaceutical benchmarks – 2003. Nat Biotechnol 21:865 – 870 Walsh G (ed) (2003b) Biopharmaceuticals: Biochemistry and biotechnology, 2nd edn. John Wiley & Sons, Chichester Walsh G (2005) Current status of biopharmaceuticals: approved product and trends in approvals. In: Knablein J (ed) Modern biopharmaceuticals. John Wiley & Sons, Chichester, pp 1 – 34 Winder R (2005) Biomanufacturing. Cell culture changes gear. Chemistry Industry 20:18 – 20 Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393 – 1398 Xolair® (omalizumab) (2005) Full prescribing information. Genentech, South San Francisco, CA

Part II

Disease-Specific Applications and Clinical Trials

II

Chapter 8

Treating Autoimmune Bullous Skin Disorders with Biologics R. Eming, A. Niedermeier, M. Pfütze, A. Jacobi, M. Hertl

8.1 Introduction

the specificity of the targeted antigens, several clinically and immune serologically distinct bullous disorders have been defined (Fig. 8.2).

8.1.1 Autoimmune Bullous Skin Disorders Autoimmune bullous skin disorders represent a group of severe, chronic skin diseases which are characterized by the presence of autoantibodies targeting distinct adhesion molecules of the epidermis and dermoepidermal basement membrane zone leading to a loss of adhesive function of the target antigen(s) (Fig. 8.1). The appearance of blisters and erosions of the skin and/ or mucous membranes is the leading clinical sign of autoimmune bullous skin disorders (Fig. 8.2). While histopathology reveals the location of the blister formation, the detection of tissue bound autoantibodies by immunofluorescent staining of uninvolved perilesional skin biopsies is mandatory for diagnosing autoimmune bullous skin disorders. Circulating autoantibodies can be visualized by indirect immunofluorescence using tissue substrates such as monkey esophagus and sodium chloride-split human skin. Based on

a

Fig. 8.1. Schematic overview of desmosomal and hemidesmosomal autoantigens in autoimmune bullous skin disorders. Shown are the major components of desmosomes which have been identified as autoantigens in the different clinical variants of pemphigus (a)

8.1.1.1 Pemphigus Desmogleins (Dsg) are transmembranous components of desmosomes, adhesion units specialized in conferring epidermal keratinocyte adhesion, and are linked to intercellular molecules of the desmosomal plaque which in turn interact with components of the cytoskeleton (Fig. 8.1a). Among several forms of pemphigus, pemphigus vulgaris (PV) and pemphigus foliaceus (PF) represent the major subtypes. IgG autoantibodies against Dsg3 in PV and Dsg1 in PF lead to loss of desmosomal adhesion of epidermal keratinocytes and intraepidermal blister formation (Fig. 8.2A – C). In PV patients suffer from flaccid blister/erosions of the mucous membranes, primarily the oral mucosa and the skin. PF is characterized by crusted painful erosions typically of the seborrheic areas such as scalp, face,

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8 Treating Autoimmune Bullous Skin Disorders with Biologics Fig. 8.1. (cont.) The integrity of epidermal cohesion is largely dependent on desmosomes, plaque-like intercellular adhesion structures that connect transmembranous adhesion molecules such as the desmogleins and desmocollins with keratins of the cytoskeleton through interaction with intracellular components of the desmosomal plaque, such as desmoplakin, plakoglobin, plakophilin, envoplakin and periplakin. In addition to desmoglein 3 and 1, which are autoantigens in pemphigus, all the other aforementioned components of desmosomes have been identified as autoantigens of different clinical variants of pemphigus (b). Components of hemidesmosomes and the basement membrane are autoantigens of the pemphigoids and epidermolysis bullosa b acquisita. Basal keratinocytes adhere to the basement membrane zone by the interaction of cytoplasmic and transmembranous components of hemidesmosomes, such as BP180, BP230 and q 4 integrin, respectively, with ligands such as laminin 5 located in the dermoepidermal basement membrane zone. The intracellular hemidesmosomal components BP230 and plectin are linked to keratins of the cytoskeleton and interact with the cytoplasmic domains of BP180 and [ 6 q 4 integrin which in turn interact with laminin 5 via their ectodomains. Type VII collagen is the major component of anchoring fibrils which link the basement membrane to the dermis by direct interaction with laminin 5 in the lamina densa of the basement membrane (modified from Hertl et al. 2006) Fig. 8.2. Diagnostic and clinical characteristics of autoimmune bullous skin disorders. Pemphigus is characterized by the presence of IgG (and occasionally IgA) specific for desmosomal target antigens (as shown here by direct immunofluorescence, (A) resulting in a loss of intraepidermal adhesion (as shown by histopathology, (B) and blisters/erosions of the mucous membranes and skin (C). In the pemphigoids, including linear IgA bullous dermatosis, IgG (or IgA) autoantibodies bind to antigens of hemidesmosomes and the dermoepidermal junction (D), resulting in a loss of subepidermal adhesion (E) and tense blisters (F). Epidermolysis bullosa acquisita is associated with IgG (and sometimes IgA) binding to the anchoring fibrils underneath the dermoepidermal junction (G), resulting in a subepidermal loss of adhesion (H) and tense blisters with a tendency to scarring and milia formation (I). Dermatitis herpetiformis is associated with IgA deposits in the papillary dermis (the dotted line indicates the dermoepidermal junction) reactive with epidermal transglutaminase (K), a subepidermal loss of adhesion (L), and herpetiform blisters or pruritic papules (M)

8.1 Introduction

chest and upper back whereas mucosal lesions are absent in PF. In general, the treatment of pemphigus is based on immunosuppressive treatment regiments consisting of high dose glucocorticoids and immunosuppressive “steroid-sparing” adjuvants, including azathioprine or mycophenolate mofetil. 8.1.1.2 Bullous Pemphigoid In the pemphigoids, IgG autoantibodies against components of the dermoepidermal basement membrane, such as bullous antigen 180 (BP180/BP antigen 2/type XVII collagen), BP antigen 230 (BP230/BP antigen 1), and laminin 5 interfere with the adhesion of basal epidermal keratinocytes to the dermoepidermal basement membrane zone (Fig. 8.1b). BP180 and BP230 are transmembranous and intracellular components, respectively, of hemidesmosomes of basal epidermal keratinocytes while laminin 5 is a ligand of BP180 located in the lamina lucida of the basement membrane zone (Fig. 8.1b). Bullous pemphigoid (BP) is the most common autoimmune blistering disease in adults. Prodromal, non-bullous periods of BP may occur in which patients demonstrate pruritic eczematiform papules or urticarial plaques. The bullous manifestations of BP present as tense, fluid-filled vesicles and bullae on normal or erythematous skin in combination with urticated plaques (Fig. 8.2D – F). Involvement of the oral mucosa is rarely observed in about 10 – 30 % of patients. In contrast to pemphigus, the use of potent topical corticosteroids such as clobetasol propionate has been shown to be effective even in the treatment of generalized BP. Nevertheless, BP patients with extensive disease are usually treated with systemic prednisone at doses of 0.5 – 1.0 mg/kg/day, which is then progressively tapered. 8.1.1.3 Epidermolysis Bullosa Acquisita Epidermolysis bullosa acquisita (EBA) is a rare chronic subepidermal bullous disease of the skin and mucous membranes characterized by the presence of autoantibodies against type VII collagen, a major component of anchoring fibrils. There is a great diversity in the clinical presentation of the disease. Generally, it can be differentiated into the mechanobullous or classic form of EBA and inflammatory variants, resembling BP, Brun-

sting-Perry pemphigoid, mucous membrane pemphigoid or linear IgA bullous dermatosis. Mechanobullous EBA presents as a non-inflammatory disease with an acral distribution and skin fragility over trauma prone surfaces. The blisters and erosions heal with scarring and milia formation (Fig. 8.2G – I). Especially the mechanobullous form of EBA often reveals itself refractory to high doses of systemic glucocorticoids combined with immunosuppressive adjuvants. Other treatment options include the use of colchicines, high dose immunoglobulins or immunoadsorption. 8.1.1.4 Dermatitis Herpetiformis Duhring Dermatitis herpetiformis (DH) represents a bullous or pruritic autoimmune disorder with subepidermal blister formation which is considered to be a specific cutaneous manifestation of celiac disease, although most DH patients do not present gastrointestinal symptoms. The autoantigen of dermatitis herpetiformis, epidermal transglutaminase, is targeted by autoantibodies of the IgA class. Immunofluorescent staining of uninvolved perilesional skin biopsies reveals granular IgA deposition in the papillary dermis (Fig. 8.2K – M). DH patients show an intense pruritus with eruption of erythematous papules and herpetiform vesicles distributed symmetrically on the extensor surfaces. In addition to a gluten-free diet, the sulphonamide diamino-diphenyl sulfone (dapsone) is most commonly used in the treatment of DH. 8.1.2 Immune Pathogenesis of Bullous Autoimmune Disorders Pemphigus and pemphigoid are considered to be prototypic bullous disorders based on their well-characterized immune pathogenesis. Apart from pemphigus and BP, there is only circumstantial evidence that autoreactive T cells are present and involved in the pathogenesis of the autoimmune bullous disorders epidermolysis bullosa acquisita and dermatitis herpetiformis. In PV and BP, autoreactive CD4+ T lymphocytes that are presumably crucial in initiating the autoimmune response recognize distinct epitopes of the extracellular portions of Dsg3 and BP180, components of desmosomal and hemidesmosomal adhesion complexes of human skin, respectively. Dsg3- and BP180-reactive T cells produce T-helper 2 (Th2) cytokines, such as IL-4,

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8 Treating Autoimmune Bullous Skin Disorders with Biologics Fig. 8.3. Schematic overview of the immune pathogenesis of pemphigus vulgaris. Pemphigus vulgaris (PV) is the prototype of an autoantibody-mediated immunobullous skin disorder and is characterized by a loss of intraepidermal adhesion primarily caused by IgG autoantibodies specific for desmoglein (Dsg)3 and Dsg1, components of the desmosomal adhesion complex of epidermal keratinocytes that are connected to the keratin cytoskeleton through interaction with the intracellular plaque proteins plakoglobin (PG) and desmoplakin (DP) (inset). IgG production by the autoreactive B cells is presumably regulated by Dsg3- and Dsg1-reactive Th1 and Th2 cells

IL-5 and IL-13, and presumably foster the production of autoantibodies of the Th2-dependent IgG4 subtype which are preferentially seen in active stages of these disorders (Fig. 8.3).

8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders 8.2.1 Biological Activity of Rituximab Rituximab is a chimeric human/mouse IgG1 monoclonal antibody with human constant regions and variable murine regions derived from a murine anti-CD20 antibody (IDEC 2B8). Rituximab is directed toward CD20, a pan B-cell glycoprotein which is expressed on B lymphocytes from the pre-B-cell stage to the pre-plasmacell stage. CD20 is a four-transmembrane glycoprotein that is involved in B-cell differentiation and activation, although its exact physiological function is not yet fully understood. Several characteristics make the CD20 antigen attractive for immunotherapy; since CD20 does not circulate in the plasma, it is neither internalized nor downregulated and there is no evidence that antibody binding leads to shedding from the cell sur-

face of the CD20+ target B cells. Among several mechanisms involved in B-cell killing by rituximab, its B-cell cytolytic activity mainly depends on antibody-dependent cell-mediated cytotoxicity (ADCC). Polymorphisms of the Fc * RIII receptor which mediates ADCC seem to influence the response to rituximab. Other mechanisms such as complement-mediated lysis and induction of apoptosis may also contribute to the Bcell-depleting activity of rituximab. Interestingly, there is recent evidence suggesting that the mechanism of Bcell killing by rituximab depends on the tissue microenvironment (Fig. 8.4). In vivo studies using hCD20 BAC (bacterial artificial chromosome) transgenic mice demonstrate that depletion of B lymphocytes varies among different B-cell compartments. In these mice, over 90 % of the circulating B cells in the peripheral blood are depleted within minutes. In lymph nodes and the spleen, B-cell depletion occurs within 24 h after application of rituximab and within 7 days in the peritoneal cavity where significant B-cell depletion is delayed. Even though more than 90 % of follicular B cells in the spleen are depleted within 2 days after rituximab application, germinal center B cells, particularly marginal zone B cells, appear to be more resistant to killing. These differences in resistance to rituximab are neither due to lack of CD20 expression nor due to differences in drug bioavailability. Thus, the

8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders

Fig. 8.4. Mode of action of rituximab. B-cell killing is largely mediated by antibody-dependent cell mediated cytotoxicity (ADCC). Recent data indicates that the patient’s Fc * RIII allotype seems to influence the response to rituximab. In vitro studies have shown that rituximab effectively induces complement-dependent cytotoxicity (CDC) against B-cell lines and lymphoma cells. Induction of apoptosis may also contribute to B-cell killing

differences in kinetics and sensitivity to the B-cell cytolytic activity of rituximab seem to depend on protective microenvironmental factors. The expression of the CD20 antigen is not restricted to B cells. A small number of T cells and NK cells also express low levels of CD20. A recent study with 24 rheumatoid arthritis (RA) patients showed that these cell populations disappear for a mean of 5 months after rituximab therapy. The authors did not find significant changes in the total number or frequency of other T-cell subpopulations in the peripheral blood. 8.2.2 Clinical Experience with Rituximab Therapy In December 1997 the US Food and Drug Administration (FDA) approved rituximab for the treatment of relapsing or refractory indolent CD20+ B-cell nonHodgkin’s lymphoma (NHL). More recently, rituximab was also approved for the treatment of aggressive NHL in combination with standard chemotherapy in the US and in Europe. The use of rituximab in the treatment of RA was endorsed by the FDA approval in March 2006.

The current application of rituximab in autoantibody mediated autoimmune diseases was catalyzed by findings that treatment of B-cell NHL improved symptoms of lymphoma-associated autoimmune phenomena, since malignant B-cell clones are capable of secreting low-affinity self-reactive antibodies, for example autoantibodies specific for self antigens on red blood cells leading to autoimmune hemolytic anemia (Boye et al. 2003). Moreover, the rationale for applying rituximab in primary autoimmune disorders is the long-term depletion of pathogenic, autoantibody-secreting B-cell clones, thus restoring tolerance. Follow-up of rituximab-induced B-cell depletion in 24 patients with active RA showed that B-cell repopulation in the peripheral blood occurred around 8 months after rituximab treatment. Increased numbers of na¨ıve and immature B cells (CD19+, IgD+, CD38high, CD10low, CD24high) were identified during repopulation in the peripheral blood similar to the B-cell populations found following bonemarrow transplantation. In contrast, patients who experienced a relapse of RA tended to show higher numbers of memory B cells during repopulation (Edwards et al. 2006). Another report by Rouziere et al. (2005) demonstrated changes in the immunoglobulin heavy-chain repertoire after rituximab treatment, suggesting that new clones of B cells emerged from the bone-marrow dominating the repopulation process. In most clinical studies treating autoimmune disorders, the oncological regimen consisting of four weekly i.v. doses of 375 mg/m2 rituximab is adopted. However, different treatment protocols are reported with two single infusions, multiple courses including four weekly infusions or single doses with up to 1 g rituximab. Besides its use in RA, rituximab is being investigated in numerous autoimmune disorders including idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, lupus erythematosus, myasthenia gravis, Sjögren’s syndrome and, finally, autoimmune bullous disorders. Within the latter group of disorders, mainly severe cases of paraneoplastic pemphigus, pemphigus vulgaris, pemphigus foliaceus and, to a lesser extent, epidermolysis bullosa acquisita have been successfully treated with rituximab. 8.2.3 Rituximab in Pemphigus There is a body of evidence demonstrating that rituximab treatment is highly beneficial in the treatment of

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8 Treating Autoimmune Bullous Skin Disorders with Biologics

recalcitrant clinical variants of pemphigus. As reported for lymphoma patients and other autoimmune diseases, four i.v. treatments with rituximab at 375 mg/m2 over 4 weeks induced a complete and sustained B-cell depletion in the peripheral blood which usually lasted for 6 – 9 months. As recently reviewed, all clinical studies and case reports using rituximab in severe recalcitrant pemphigus demonstrated good clinical responses with remissions lasting from several months to 2 years. A recent open-label study including one PF and four PV patients with therapy resistant disease showed that clinical improvement correlated with persistent B-cell depletion. However, clinical remission was not accompanied by a significant decrease of desmoglein (Dsg)specific autoantibodies in all the patients (Arin et al. 2005). Another ongoing clinical study including a total of ten PV patients receiving the standard dose (four weekly doses of 375 mg/m2) demonstrated a decrease of Dsg1-/Dsg3-specific autoantibodies by 70 – 80 % of the initial titers within 2 – 3 months after rituximab treatment and a further decrease by 40 – 50 % of the initial autoantibody levels 6 months after therapy, correlating with a good clinical response (Fig. 8.5). In contrast, only minor changes were detected in anti-tetanus toxoid IgG antibodies, suggesting that autoreactive B cells and their corresponding plasma cells tended to have short life spans whereas allo-reactive plasma cells (as for post-vaccination immunity) were more resistant to rituximab and had longer life spans. Compared to other autoimmune diseases such as RA and systemic lupus erythematosus, in pemphigus as an autoantibody mediated autoimmune disorder, the major autoantigens (Dsg1 and Dsg3), pathogenic autoantibodies and autoreactive, Dsg3-specific CD4+ T cells have been identified and functionally characterized. As

recently discussed for RA by Edwards and Cambridge (2006), the two-way interaction between autoreactive B- and T-cell clones seems to be crucial for both the initiation and perpetuation of these autoantibody driven autoimmune diseases. B cells have been shown to activate T cells through antigen presentation, and CD4+ T cells provide “help” to B cells through the delivery of cytokines and cell surface ligands, thus leading to a positive feedback-loop. First evidence for this immunological hypothesis in pemphigus is provided by a recent study investigating the frequency of Dsg3-specific autoreactive CD4+ T cells in the peripheral blood of PV patients undergoing rituximab therapy. In a group of 11 PV patients receiving the standard dose of rituximab, the frequencies of Dsg3-reactive CD4+ T cells were determined by MACS cytokine secretion assay. Frequencies of IL-4- (Th2 cells), IFN * - (Th1 cells) and IL10-producing type 1 regulatory T cells (Tr1 T cells) were determined before and up to 12 months after rituximab therapy. In all patients the frequencies of Dsg3-reactive CD4+ T cells decreased significantly compared to the pre-treatment values. The frequencies of tetanus toxoid-specific T cells in these patients were not affected by B-cell depletion and showed only minor changes in frequency. These results suggest that activation of autoreactive T cells in pemphigus largely depend on continuous antigen presentation by autoreactive B cells delivering activation signals. 8.2.4 Rituximab in Epidermolysis Bullosa Acquisita A recent case report described an inflammatory variant of EBA with lesions on the trunk, the hands and the oral mucosa. The patient received four courses of rituximab

Fig. 8.5. Clinical response in a patient with mucosal pemphigus vulgaris (PV) to treatment with rituximab. The PV patient with extensive oral erosions shown here was refractory to immunosuppressive treatment including mycophenolate mofetil (3 g/day) and azathioprine (1.75 mg/kg/ day), respectively, in combination with methylprednisolone (0.5 – 1.5 mg/kg/day) for 6 months (A, before rituximab treatment). Rituximab treatment led to a rapid clinical response (B, 2 months after rituximab treatment)

8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders

Fig. 8.6. Clinical response of mechanobullous epidermolysis bullosa acquisita to rituximab. Patient 1 showed blisters and erosions on his hands (a, left), feet and the oral mucosa before rituximab treatment. Marked improvement of the lesions on the hands was noted 15 weeks after completion of therapy (a, right), while erosions of the oral mucosa remained largely unaffected. Patient 2 (b, left) showed extensive blisters and erosions of the trunk and hands. Nine months after completion of rituximab therapy, an almost complete clinical remission with postinflammatory atrophic hyperpigmentations on the chest and a few crusty erosions was visible (b, right)

(i.v., 375 mg/m2) as an adjuvant treatment in weekly intervals. Seven weeks after the completion of rituximab therapy, the patient was in complete remission and received tapering doses of azathioprine (175 mg/ day), colchicine (160 mg/day) and prednisolone (80 mg/day). As an unusual adverse event a deep venous thrombosis of the lower leg developed between the first and second infusion of rituximab. In contrast, administration of rituximab in patients with mechanobullous EBA shows a different clinical response. In our own experience, an EBA patient with erosive lesions of the oral mucosa, the esophagus and the nasopharynx and tense blisters on the hands, lower legs and feet showed only a partial clinical response to rituximab. Subsequently, he received adjuvant therapy

with rituximab 375 mg/m2 × 4 over a period of 4 weeks. Concomitant medication with mycophenolate mofetil 3 g/day was maintained during and after B-cell depletion therapy. Within 15 weeks gradual improvement of the lesions on the hands was perceived, whereas the oral lesions and the lesions on the soles showed only slight improvement (Fig. 8.6). A second patient with mechanobullous EBA refractory to treatment to a variety of systemic immunosuppressive drugs in the past, who had lesions that mainly affected the trunk and the hands, was treated in the same way as the previous patient with rituximab. Within 8 months his disease was well controlled on monotherapy with rituximab with only very few residual crusty erosions and atrophic lesions. The excellent clinical response was accom-

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panied by a decline in titres of circulating autoantibodies, which were undetectable & 9 months after completion of rituximab therapy. Complete B-cell depletion persisted for & 9 months. In both patients rituximab was well tolerated with no side effects. 8.2.5 Toxicity of Rituximab Treatment and Adverse Effects In general, rituximab is well tolerated in both patients with malignancies and those with autoimmune bullous disorders. Except for two reports of serious infections including septic arthritis, Pneumocystis carinii pneumonia and pneumonia after treatment of pemphigus patients with rituximab, the most common adverse events are infusion-related events occurring during or shortly after the first infusion. Larger studies with lymphoma patients demonstrated that about 95 % of the infusion-related symptoms such as chills, fever, headache, rhinitis, pruritus or vasodilatation were mild to moderate. Most of these symptoms could be avoided by pre-treatment with antihistamines, antipyretics and steroids. Another report showed that a severe cytokine release syndrome more frequently occurred in lymphoma patients with a higher tumor burden in their peripheral blood which might be correlated with increased serum levels of tumor necrosis factor-alpha (TNF- [ ) and interleukin 6 (IL-6) after infusion of rituximab. At present, severe cytokine release syndromes have not yet been reported in patients with bullous autoimmune disorders. Hematological toxicity has been reported in about 10 % of patients including a temporary reduction of platelets or neutrophils. Despite the depletion of B cells for several months the risk for infectious diseases does not seem to be significantly higher in patients treated with rituximab. A recent study in RA patients demonstrated a significant decrease of serum rheumatoid factor (IgM, IgG and IgA isotypes) over several months, whereas total immunoglobulin levels decreased by only 25 % for IgG and IgA and by 43 % for IgM and the titers for pneumococcal polysaccharide-reactive antibodies remained unchanged. Another recent report showed unaffected levels of anti-tetanus toxoid antibodies in patients treated with rituximab. These observations help to explain why, despite complete depletion of B cells for several months, the risk for secondary infections does not seem to be significantly higher in patients treated with rituximab. As rituximab is a chimeric antibody with

reduced immunogenicity, the presence of human antichimeric antibodies (HACA) is a rare event which has been reported in less than 1 % of treated patients with NHL. In patients with HACA impairing the function of rituximab, a newly designed humanized monoclonal anti-CD20 antibody (hCD20) might be effective. 8.2.6 Contraindications for Treatment with Rituximab Ongoing or recurrent infectious disorders including clinically apparent bacterial or viral infections exclude the application of rituximab. Patients with a history of malignancies or immunodeficiency syndromes, as well as pregnant or lactating women, should not undergo rituximab therapy. Patients with cardiac disorders should be closely monitored during therapy with rituximab. A history of anaphylactic hypersensitivity reactions against mouse proteins also excludes treatment with rituximab.

8.3 Inhibitors of TNF-␣ in the Treatment of Autoimmune Bullous Skin Disorders 8.3.1 Central Role of TNF-␣ in Inflammation Tumor necrosis factor- [ is a proinflammatory cytokine which plays a key role in most of the inflammatory processes as well as in immune responses to infections and tumor antigens. TNF- [ is largely produced and released by macrophages and monocytes. It exists as a soluble 17-kDa sized protein made of three subunits. The cytokine stands at the beginning of a cascade of pro-inflammatory cytokines and triggers off proinflammatory signals at the target cell by binding to membrane-based TNF receptors, e.g., on T cells and macrophages. Furthermore, TNF- [ causes a rise in body temperature as well as the induction of the acute phase proteins, an increased migration of dendritic cells from the periphery to regional lymph nodes and an activation of neutrophils. In inflammatory diseases, TNF- [ possesses pleiotropic effects and leads, among other factors, through the activation of a pro-inflammatory cytokine cascade, to an induction of adhesion molecules on endothelial cells (E-selectin, ICAM-1), which leads to an increased migration of leukocytes. Furthermore, TNF- [ induces the secretion of metallo-

8.3 Inhibitors of TNF-␣ in the Treatment of Autoimmune Bullous Skin Disorders

proteinases and the release of pro-inflammatory cytokines (IL-1, IL-6, IL-8, GM-CSF). TNF- [ -conveyed induction of pro-inflammatory cytokines, leukocyte chemotaxis and angiogenesis possibly play a fundamental role in autoimmune diseases of the skin, presumably diseases which are characterized by elevated TNF- [ serum concentrations, fever and an increase of acute phase proteins. Elevated serum levels of TNF- [ are detectable in many autoimmune diseases including RA, psoriasis and Crohn’s disease. 8.3.2 Inhibition of TNF-␣ by Biologics A new class of TNF- [ inhibitors are the so-called “biologics,” which are either recombinant monoclonal antibodies or soluble TNF receptor fusion proteins. These proteins can be either isolated from animal tissues or commonly synthesized by biotechnological methods. The more defined understanding of the pathophysiology of autoimmune diseases has led to the therapeutic use of biologics in chronic inflammatory disorders (Scheinfeld 2004). At present, a few uncontrolled case reports suggest that the therapeutic blockade of TNF- [ may be a novel option for the short-term control of otherwise recalcitrant autoimmune bullous skin disorders (Table 8.1). Infliximab is a chimeric monoclonal antibody consisting of a murine anti-TNF- [ Fab fragment and the

constant region (Fc) of human IgG1. Infliximab binds with high specificity and affinity to free and membrane-bound TNF- [ , which is expressed at the surface by activated T cells and macrophages. Besides the blocking of TNF- [ , the lysis of the target cell may also occur through activation of complement. This probably accounts for the antibody conveying a cytotoxic effect of infliximab. Inhibition of the pro-inflammatory effects of TNF- [ results through the formation of stable complexes. In addition, the migration of leukocytes and the release of TNF- [ -dependent proinflammatory cytokines like IL-1 and IL-6 is inhibited. Adalimumab is a human monoclonal IgG1 antibody containing only human peptide sequences. It binds with high specificity and affinity to soluble and membrane-bound TNF- [ and blocks its interaction with the p55 and p75 cell surface TNF receptors, thereby neutralizing the biological activities of this cytokine. Adalimumab also modulates TNF-induced or regulated biological responses, such as changes in the levels of adhesion molecules responsible for leukocyte migration. Etanercept is a recombinant human fusion protein which consists of two soluble p75 TNF receptors and the Fc portion of human IgG1. Etanercept possesses a dimeric structure with high affinity to TNF- [ , and the linkage to the Fc portion of human IgG produces a longer half-life. Etanercept neutralizes TNF- [ better than the monomeric soluble p75 receptor. By blocking the

Table 8.1. TNF- [ inhibitors and application in autoimmune bullous dermatoses Infliximab (Remicade␤)

Adalimumab (Humira␤)

Etanercept (Enbrel␤)

Molecule

Chimeric (murine-human) monoclonal antibody (IgG1)

Fully human monoclonal IgG1 antibody

Human receptor fusion protein (p75-TNF-receptor dimer and IgG1)

Target structure

Free and membrane-bound TNF- [

Free and membrane-bound TNF- [

Free TNF- [ and lymphotoxin- [ (TNF- q )

Dosage

3 – 5 mg/kg/day intravenous infusion at weeks 0, 2 and 6; then every 8 weeks

40 mg every other week subcutaneously

25 mg (children 0.4 mg/kg/day) twice weekly subcutaneously; alternative 50 mg twice weekly at first 12 weeks

Efficacy in autoimmune bullous skin disorders

Pemphigus vulgaris Jacobi et al. 2005 Pardo et al. 2005

IgA pemphigus Howell et al. 2005

Pemphigus vulgaris Berookhim et al. 2004 Lin et al. 2005 Mucous membrane pemphigoid Sacher et al. 2002 Bullous Pemphigoid Yamauchi et al. 2006

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circulating TNF- [ trimers the interaction of TNF- [ with the cell bound receptors is prevented and the proinflammatory cytokine cascade interrupted. Etanercept, in addition, binds lymphotoxin (TNF- q ). Cells with membrane-bound TNF- [ bind etanercept; lysis due to ADCC of these cells does not occur in contrast to the binding of infliximab (Table 8.1). 8.3.3 Inhibition of TNF-␣ in Pemphigus Vulgaris In pemphigus vulgaris (PV), autoantibodies against Dsg1 and Dsg3 have been shown to induce loss of keratinocyte adhesion upon binding to desmosomal target autoantigens leading to the release of TNF- [ and IL-1 from epidermal keratinocytes, which presumably enhance the process of blister formation. In vivo, an increased expression of TNF- [ and IL-1 is found in the direct environment of the intra-epidermal loss of adhesion. In vitro, cultures of human keratinocytes are resistant to the acantholytic effect of pathogenic, Dsg3specific autoantibodies upon pre-treatment with antiTNF- [ antibodies. This protective effect of blocking TNF- [ can be reproduced in vivo. Feliciani et al. (2000) showed that the injection of anti-Dsg3 IgG in TNF receptor 1/2 deficient mice led more rarely to blister formation than the injection of the identical autoantibodies in syngeneic “wild type” mice with an intact TNF receptor. Based on these observations, the potent TNF- [ inhibitor, infliximab, was used in a 43-year-old male with a therapy refractory PV with a history of pulmonary embolism, arterial hypertension, steroid-induced osteoporosis and diabetes mellitus who presented with disseminated cutaneous blisters and extensive erosions of the oral mucosa (Jacobi et al. 2005). The patient was given three doses of infliximab (5 mg/kg/day i.v.) over

6 weeks at weeks 0, 2 and 6. Leflunomide (20 mg/day) and prednisone (0.25 mg/kg/day) were continued during the infliximab regimen as adjuvant treatment and with the aim of preventing formation of anti-infliximab antibodies (HACA). Within 48 h of the first dose of infliximab, blister formation ceased, and within the first 4 days cutaneous blisters and mucosal erosions gradually disappeared (Fig. 8.7). Infliximab infusions were discontinued 8 weeks after initiation when the patient developed varicella zoster of the left S1/2 dermatome, which was treated successfully with i.v. acyclovir. Of note, the patient’s clinical status remained stable with few cutaneous erosions during the previously insufficient therapy with leflunomide (20 mg/d) and prednisone (0.25 mg/kg/day). After a relapse 4 months later the disease was controlled by several cycles of immunoadsorption therapy in combination with mycophenolate mofetil (3 g/day). Further case reports confirm the successful management of severe pemphigus with infliximab. A 62-yearold male with PV and generalized bullous lesions and a severe oral stomatitis was managed with systemic corticosteroids for 6 years, mycophenolate mofetil, cyclosporin A, azathioprine, cyclophosphamide, methotrexate, thalidomide and plasmapheresis without much success. As a last resort, the patient received infliximab (5 mg/kg/d i.v.) at 0, 2 and 6 weeks and then every 8 weeks. No other drugs were needed as concomitant medication. The patient improved dramatically after five infusions leading to total clearance of active lesions at week 22 without severe side effects. After the 13th infusion, treatment with infliximab discontinued because new cutaneous and oral lesions had appeared. The disease was eventually controlled with oral corticosteroids and rituximab. There is a report on the effective treatment of PV with the TNF- [ antagonist, etanercept, in a 62-year-old

Fig. 8.7. Immediate clinical response of pemphigus vulgaris (PV) to treatment with the TNF inhibitor infliximab. Cutaneous bullae in a patient with recalcitrant PV before (A) and 2 days after (B) the first infliximab infusion

8.4 Summary

woman with long-standing cutaneous PV and concomitant seronegative arthritis. Upon initiation of treatment with etanercept for arthritis, the cutaneous blisters gradually disappeared. Lin et al. (2005) reported the successful treatment of recalcitrant PV and pemphigus vegetans with etanercept and carbon dioxide laser. The human anti-TNF- [ monoclonal antibody, adalimumab, has in combination with mycophenolate mofetil been successfully employed in the subcorneal pustular dermatosis subtype of IgA pemphigus. A 41-yearold male with IgA pemphigus failed multiple treatments, including acitretin, broadband ultraviolet B therapy, dapsone, methotrexate and oral steroids. He showed improvement to alefacept, but treatment was discontinued because of a low peripheral CD4+ T-cell count. Cyclosporine was also effective, but was discontinued secondary to elevated creatinine levels. Adalimumab (40 mg s.c. every other week) was started, and after the third dose his skin completely cleared. Mycophenolate mofetil (1 g/day) was used as concomitant medication. The patient has been symptom-free for 5 months, besides the occasional occurrence of a few pustules. 8.3.4 Inhibition of TNF-␣ in Bullous Pemphigoid There are a few reports on the effect of TNF- [ antagonists in clinical variants of BP, an autoimmune bullous skin disorder associated with IgG autoantibodies against components of the dermo-epidermal basement membrane zone, such as BP180 and BP230. In the clinical variant mucous membrane pemphigoid, a disorder which is characterized by chronic blistering of the mucous membranes with secondary scarring, the TNF[ blocker etanercept has been applied with great success. A 72-year-old woman with long-standing mucous membrane pemphigoid and acute exacerbation of oral lesions had already been treated with prednisone (1 mg/kg/day) in combination with azathioprine (100 mg/day) and mycophenolate mofetil (2×1 g/day) over a year, leading to a moderate clinical response. Etanercept (25 mg s.c. ×2/week) in combination with prednisone (initially 60 mg/day) led after the third cycle to the disappearance of newly developed blisters. Even though the corticosteroids were gradually tapered, the patient was symptom-free after a total of six etanercept injections and clinical remission was

maintained for more than 8 months with low-dose prednisone treatment (1 mg/day). There is also a report about the treatment of coexisting BP and psoriasis with the TNF- [ antagonist, etanercept. A 64-year-old man with a long history of plaque-type psoriasis developed acute symptoms of BP. Initial therapy consisted of mycophenolate mofetil (2 g/ day), which had no effect on the clinical activity of BP. Mycophenolate mofetil was discontinued, and he was started on prednisone (60 mg/day). The cutaneous bullae disappeared almost completely by the 10th day and also the plaque psoriasis improved. To prevent rebound of psoriasis and BP during the tapering phase of prednisone, etanercept (50 mg s.c. weekly) was added to the treatment regimen. At a dose of 20 mg/day prednisone, new cutaneous blisters developed. Etanercept was increased to 50 mg twice weekly and the blisters subsequently resolved. When prednisone treatment was eventually discontinued, the clinical activity of BP remained silent; no adverse events occurred.

8.4 Summary Rituximab has clearly evolved as a novel therapeutic option in refractory autoimmune bullous skin disorders such as severe pemphigus vulgaris and epidermolysis bullosa acquisita. The mode of action suggests that not only the production of autoantibodies is inhibited by depletion of autoreactive B cells but also the critical interaction of autoreactive T and B cells, which is essential for the perpetuation of the ongoing autoimmune response. Treatment of pemphigus with rituximab has been shown to be highly effective in refractory mucocutaneous pemphigus with a prolonged biological activity due to the long-term depletion of autoreactive B cells and a lack of major side effects. In addition, single case reports suggest that TNF- [ blockers may be a therapeutic option in pemphigus and mucous membrane pemphigoid with life-threatening or therapy refractory disease course, particularly as a short-term intervention therapy. However, potential side effects due to the immunosuppressive potential of TNF- [ antagonists such as severe bacterial infections, viral infections and tuberculosis should be carefully considered and constitute a serious hazard in patients on chronic immunosuppressive therapy.

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References Arin MJ, Engert A, Krieg T, Hunzelmann N (2005) Anti-CD20 monoclonal antibody (rituximab) in the treatment of pemphigus. Br J Dermatol 153: 620 – 625 Berookhim B, Fischer HD, Weinberg JM (2004) Treatment of recalcitrant pemphigus vulgaris with the tumor necrosis factor alpha antagonist etanercept. Cutis 74(4):245 – 247 Boye J, Elter T, Engert A (2003) An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab. Ann Oncol 14:520 – 533 Edwards JCW, Cambridge G (2006) B-cell targeting in rheumatoid arthritis and other autoimmune diseases. Nature Rev Immunol 6:394 – 403 Feliciani C, Toto P, Amerio P, Pour SM, Coscione G, Shivji G, Wang B, Sauder DN (2000) In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis. J Invest Dermatol 114(1):71 – 77 Hertl M, Eming R, Veldman C (2006) T cell control of autoimmune bullous skin disorders. J Clin Invest 116:1159 – 1166 Howell SM, Bessinger GT, Altman CE, Belnap CM (2005) Rapid response of IgA pemphigus of the subcorneal pustular dermatosis subtype to treatment with adalimumab and mycophenolate mofetil. J Am Acad Dermatol 53(3):541 – 543

Jacobi A, Manger B, Schuler G, Hertl M (2005) Rapid control of therapy-refractory pemphigus vulgaris by treatment with the tumour necrosis factor-alpha inhibitor infliximab. Br J Dermatol. 153(2):448 – 449 Lin MH, Hsu CK, Lee JY (2005) Successful treatment of recalcitrant pemphigus vulgaris and pemphigus vegetans with etanercept and carbon dioxide laser. Arch Dermatol 141(6): 680 – 682 Pardo J, Mercader P, Mahiques L, Sánchez-Carazo JL, Oliver V, Fortea JM (2005) Infliximab in the management of severe pemphigus vulgaris. Br J Dermatol 153(1):222 – 223 Rouziere AS, Kneitz C, Palanichamy A, Dorner T, Tony HP (2005) Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody. Arthritis Res Ther 7(4):R714 – 724 Sacher C, Rubbert A, Konig C, Scharffetter-Kochanek K, Krieg T, Hunzelmann N (2002) Treatment of recalcitrant cicatricial pemphigoid with the tumor necrosis factor alpha antagonist etanercept. J Am Acad Dermatol 46(1):113 – 115 Scheinfeld NA (2004) Comprehensive review and evaluation of the side effects of the tumor necrosis factor alpha blockers etanercept, infliximab and adalimumab. J Dermatol Treat 15:280 – 294 Yamauchi PS, Lowe NJ, Gindi V (2006) Treatment of coexisting bullous pemphigoid and psoriasis with the tumor necrosis factor antagonist etanercept. J Am Acad Dermatol 54(3 Suppl 2):S121 – 122

Chapter 9

Biologics in Psoriasis

9

W.A. Myers, W.-H. Boehncke, A.B. Gottlieb

9.1 Introduction

9.1.1.1 Plaque Psoriasis

As a better understanding of cutaneous diseases has been gained, more specific, targeted therapies have emerged. More recently, many of the T-cell immune mediated inflammatory changes seen in psoriasis have been elucidated, leading to the development of biologics that specifically act on immunological mechanisms, which are thought to be pathogenic in psoriatic lesions. By acting on specific immunological actions in the large cascade that results in psoriasis, many of the systemic toxicities that accompanied older treatments such as methotrexate and cyclosporine may be avoided. Currently, there are three biologics approved in the United States for the treatment of psoriasis including alefacept, efalizumab, and etanercept, with several others currently under clinical investigation. A summary of the biologics currently used for the treatment of psoriasis can be found in Table 9.1.

Plaque psoriasis or psoriasis vulgaris is the most common form of psoriasis, occurring in more than 80 % of cases. Clinical features include sharply demarginated, erythematous plaques with non-adherent, silvery scales (Fig. 9.1A). Pain, itching, and cracking of the skin may be prominent as well. These lesions most typically affect the elbows, knees, scalp, lumbar area, umbilical area, and gluteal cleft. A characteristic sign called the Auspitz sign results from the mechanical removal of the non-adherent scale, resulting in pinpoint bleeding. Another additional finding seen in patients with psoriasis, called the Koebner phenomenon, is the predilection for new lesions to appear at sites of previous trauma such as burns, infections, or vaccinations.

9.1.1 Psoriasis Psoriasis is a very common and chronic papulosquamous skin disease that affects approximately 0.5 – 4.6 % of the total population, depending on race and country. The National Psoriasis Foundation states that 4.5 million people in the United States are affected by psoriasis and an additional 150,000 – 260,000 are newly diagnosed each year. It is diagnosed in patients of all ages, while the median age of onset is 29.1 years of age. It has been found that women have an earlier age of onset, but no difference in the prevalence between sexes has been observed.

9.1.1.2 Guttate Psoriasis Guttate psoriasis is often a form that begins in childhood or early adulthood. A variety of conditions have been known to precipitate guttate psoriasis including infections, such as streptococcal infections, as well as stress, injury to the skin, or certain medications. Strep throat is a common trigger, and can be associated with flares as well, even if the infection is not clinically evident. Guttate psoriasis appears as an eruption of scattered 0.1 – 1 cm “drop” shaped, erythematous, scaling papules of the trunk and extremities primarily (Fig. 9.1B). This type of psoriasis often has a more rapid response to therapy than other forms of psoriasis.

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9 Biologics in Psoriasis Table 9.1. Summary of biologics used for psoriasis Drug

Indication (USA)

Mechanism of action

Dose

Alefacept

Moderate to severe chronic plaque psoriasis

Inhibits T-cell acti- 15 mg intramuscu- PASI-75 results of phase III IM study: vation and prolifer- lar injection once weekly for 12 weeks 33 % patients on alefaation; induces cept 15 mg/week vs. selective T-cell apo13 % receiving placebo ptosis (at any time)

Similar to placebo, injection site pain/inflammation; no increased incidence of infections

Etanercept

RA, polyarticularcourse RA, AS, psoriatic arthritis, moderate to severe psoriasis

Dimeric TNF- [ receptor antagonist competitively inhibits interaction of TNF with cellsurface receptors

50 mg twice weekly by subcutaneous injection for 3 months followed by reduction to maintenance dose of 50 mg weekly

PASI-75 in one phase III study on 50 mg twice weekly: 47 % vs. 4 % in placebo after 12 weeks; after 24 weeks, PASI-75 in 50 mg twice weekly group was 54 %

Well tolerated in clinical trials: extended use does not result in cumulative toxicities. Most common adverse event is injection site reaction. Avoid in patients with demyelinating disease, congestive heart failure and active, significant infection, e.g., tuberculosis

Efalizu- Chronic mab moderate to severe plaque psoriasis

Monoclonal antibody which binds CD11a, blocking interaction with ICAM-1 resulting in decreased T-cell activation, adhesion, and trafficking

First dose 0.7 mg/ kg, then 1.0 mg/kg subcutaneous injection once weekly

At 12 weeks of therapy, Most common include heada phase III study found aches, myalgia, pain, and fever that 27 % of patients receiving efalizumab achieved PASI-75 vs. 4 % in placebo

Infliximab

RA, Crohn’s disease, AS, moderate to severe plaque psoriasis, psoriatic arthritis, and ulcerative colitis

Chimeric monoclonal antibody which binds with high affinity to free and membrane bound TNF, and inhibits its binding to its receptors

Studies examining infliximab for psoriasis have dosed using 3 or 5 mg/kg intravenous infusion at weeks 0, 2, and 6

A phase III study found at 10 weeks, 80 % of subjects achieved PASI75 on 5 mg/kg (weeks 0, 2, and 6 dosing) vs. 3 % in placebo

Well tolerated. Headache, nausea, upper respiratory infections seen. Avoid in patients with deymyelinating disease, congestive heart failure and active, significant infection, e.g., tuberculosis

Adalimumab

RA, psoriatic Monoclonal antiarthritis body which binds and inhibits TNF- [

40 mg subcutaneous injection every other week

53 % of patients in a phase II study achieved a PASI-75 with 40 mg every other week compared to 4 % in placebo

Rates of adverse events comparable between adalimumab and placebo. Avoid in patients with deymyelinating disease, congestive heart failure and active, significant infection, e.g., tuberculosis

9.1.1.3 Erythrodermic Psoriasis Erythrodermic psoriasis is a very inflammatory psoriasis that affects most of the body. It presents as generalized indurated erythema with diffuse exfoliation of fine scales, often accompanied by severe itching and pain (Fig. 9.1C). The patients may also present with fever, chills, rigors, arthralgias, and trouble maintaining core body temperature. Triggers for an episode of erythrodermic psoriasis include severe stress, discon-

Efficacy

Safety

tinuation of a systemic medication such as methotrexate, cyclosporine, or oral corticosteroids, diffuse phototherapy burns, and infections. Reports of patients with erythrodermic psoriasis suffering staphylococcal sepsis have been reported, and inpatient management with blood cultures and systemic antibiotics should be considered accordingly. Special attention must be paid to maintaining the appropriate fluid status in these patients, as they are highly susceptible to insensible losses.

9.1 Introduction

Fig. 9.1. Clinical features of psoriasis. The typical psoriatic lesion is a sharply demarked erythematous plaque covered by silvery white scales, often appearing on the extensor sites of the extremities (A). Initial eruptions of psoriasis may exhibit a guttate distribution pattern and are often triggered by streptococcal infections (B). In a dark-skinned patient, erythrodermic psoriasis (C), a clinical subtype of the disease, affects the entire body surface. If the scalp is involved, the lesions typically extend a short distance beyond the region covered by terminal hair (D). Inverse psoriasis (E) is located at intertriginous areas and usually lacks scaling. Pustular forms of psoriasis also exist (F, G). Localized forms of psoriasis include palmo-plantar psoriasis (H) and acrodermatitis continua suppurativa, or Hallopeau’s disease, leading to severe dystrophy (I) or even loss of nails. Joint involvement (psoriatic arthritis) is frequently observed (J). Mild cases of nail involvement are characterized by small pits and yellowish discoloration of the nail plate (K), which were also created in a wax-model moulage, manufactured around 100 years ago (L, item 1766 from the collection of the Johann Wolfgang Goethe University, Frankfurt am Main). (Reproduced with permission from: N Engl J Med 2005; 352:1901)

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9.1.1.4 Pustular Psoriasis

9.1.1.7 Flexural Psoriasis

Pustular psoriasis is a rare form of psoriasis that typically affects adults. Sterile, white pustules in areas of erythema and scale characterize this form of psoriasis (Fig. 9.1F, G). There are several subtypes of pustular psoriasis including von Zumbusch pustular psoriasis, palmoplantar pustular psoriasis, and acropustulosis (acrodermatitis continua of Hallopeau). Von Zumbush pustular psoriasis is characterized by abrupt waves of widespread, erythematous patches of skin, which become painful and sore. Typically within a few hours, pustules appear, which then peel off after 1 – 2 days. These waves of pustules may last days to weeks. Patients may also report fever, chills, muscle weakness, and weight loss. Von Zumbusch pustular psoriasis may be triggered by infections, withdrawal of topical mediations, pregnancy, and certain medications such as lithium and some hypertension medications.

Also known as inverse psoriasis, this form of psoriasis is typically localized to the axilla, submammary folds, genitocrural area, and neck. These lesions usually have no scale and appear as well-demarcated, salmon red plaques that can fissure (Fig. 9.1E). The lesions can be very painful as perspiration becomes trapped in the skin folds, causing irritation and maceration of the tissue. Lesions tend to be difficult to treat because of their location and a tendency for superimposed fungal infections to occur.

9.1.1.5 Palmoplantar Pustulosis This is a condition characterized by erythematous and scaly plaques studded with sterile pustules on the palms and soles, typically the insteps, sides, and back of the heel as well as the thenar and hypothenar eminences of the hands. This type of psoriasis affects patients between the ages of 20 and 60 years, and commonly affects females more than males. Topical treatments are typically prescribed first but this form of psoriasis can be very difficult to manage, and systemic treatments are often necessary. 9.1.1.6 Acropustulosis (Acrodermatitis Continua of Hallopeau) This rare form of pustular psoriasis is often localized to the tips of digits and occasionally toes. There is usually scaly inflammation and sterile pustules at the tip of the digit, typically after a recent injury or infection to the digit (Fig. 9.1H). This form of psoriasis can often be disabling, with secondary nail changes including separation from the nail plate, ridging, crumbling, and total destruction of the nail. Although acropustulosis can affect all age groups, it is typically seen in adults.

9.1.1.8 Palmoplantar Psoriasis Palmoplantar psoriasis, as its name indicates, is a form of psoriasis that affects the palms and soles, presenting as discrete, erythematous, scaling patches and plaques. These lesions are usually bilateral, and involvement of the palms typically stops at the wrist-palm junction, but the dorsal aspect of the hand can be involved. Patients often find this form of psoriasis very disabling from fissuring that makes walking painful, as well as embarrassing, due to prominent scaling on very publicly visual areas such as the hands. In addition to the above types of psoriasis, the scalp and nails may also be affected (Fig. 9.1D, J, K). Approximately 79 % of patients with psoriasis have involvement of the scalp. Nail changes can occur in up to 50 % of people with psoriasis and up to 80 % of people with psoriatic arthritis. The proximal nail fold, nail matrix, nail bed, and hyponychium can be involved with nail psoriasis. Patients may see discoloration of the nail with yellowbrown changes, pitting, nail thickening, and separation of the nail from the nail bed (onycholysis). 9.1.1.9 The PASI Score The PASI score (Psoriasis Area Severity Index) is a common tool in clinical trials used to assess psoriasis activity and to follow response to treatment. The PASI evaluates the erythema, scaling and thickness of psoriatic plaques, as well as assessing the area of involvement in the four areas of the body (head, trunk, upper, and lower extremities). Scores range from 0 to 72, providing a subjective estimate of disease activity.

9.1 Introduction

9.1.2 Mechanism of Disease The pathogenesis of psoriasis has undergone several revisions over the past several decades. The most recent evidence has suggested that psoriasis is due to activated T cells present in the psoriatic plaques that produce proinflammatory cytokines and mediators that produce the changes seen in psoriasis (Fig. 9.2). Today’s newest therapies for psoriasis target specific steps in this cascade of events to specifically inhibit the reactions necessary for the inflammation changes seen in psoriasis. There are two subsets of T cells that are differentiated by the type of cytokines they release: T1 (type 1) and T2 (type 2). T1 cells produce interleukin (IL)-2 and IFN- * , are in part responsible for cell-mediated immunity and are inflammatory in nature. T2 cells release IL-4, IL-5, and IL-10, which enhance the humoral

Fig. 9.2. Putative T-cell responses in the pathogenesis of a psoriatic lesion. To generate a cutaneous T-cell response, antigen-presenting cells take up and process antigens and migrate to the regional lymph nodes. There, they come in contact with na¨ıve T cells. Within an immunologic synapse (inset), molecular interactions result in T-cell activation. Following the activation signals, T cells differentiate into memory T-cells and re-enter the circulation, where they extravasate at sites of cutaneous inflammation. In the skin, on encountering the respective antigen, T cells exert their effector functions, which include the secretion of pro-inflammatory cytokines. Psoriasis is characterized by a chronically persisting response in effector T-cells. (Reproduced with permission from: N Engl J Med 2005: 352:1905)

immune system and are anti-inflammatory in nature. It is believed that psoriasis is a T1 mediated process, as suggested by the increased levels of IFN- * in psoriatic skin, lesional and non-lesional. IFN- * also induces macrophages to secrete high levels of inflammatory cytokines such as TNF- [ , which is present in high levels of psoriatic plaques and synovium of patients with psoriatic arthritis. T2 cytokines levels, on the other hand, have been shown to be low in psoriatic patients. There are many steps required before the phenotypic production of psoriasis is expressed. Mehlis and Gordon break this process down into three steps: (1) the activation of T cells, (2) the migration of T cells into the skin, (3), and the effector function of T cells or the induction of T cells by the secretions of inflammatory cells. The newer biologic therapies have targeted these specific steps in order to provide specific and less toxic therapy for psoriasis.

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T-cell activation is a multi-step process that begins with binding to an antigen-presenting cell (APC) through interactions between LFA-1 (leukocyte function associated antigen), ICAM-1 (intercellular adhesion molecule), and LFA-3. Once bound, the T cell becomes activated by two signals, antigen (it is currently not known which antigen is responsible) bound to class I or II MHC on the APC, and another signal supplied by a number of different cell surface molecules including LFA-1, ICAM-1, CD-2, and LFA-3 (Gottlieb 2003). Alefacept is a fusion protein that consists of the extracellular domain of LFA-3 fused to hinge sequences of IgG1. This biologic binds CD-2 on T cells, which results in the inhibition of T-cell costimulation and a reduction in memory effector cells. By binding CD-2 on memory effector T cells, alefacept facilitates apoptosis of the cell. T cells must also be present in the skin to produce the inflammatory changes, and therefore must migrate from the circulation. The activated T cell must slow and then bind the endothelium before it can enter the affected tissue. Several surface molecules are responsible for this process including CLA (cutaneous lymphocyte antigen) on the T cell and E selectin on the endothelium. This interaction slows the cell along the endothelial surface. The interaction between LFA-1 and ICAM and VLA and VCAM allows the T cell to bind to the endothelium. Once bound, the cell can cross the endothelium. Efalizumab is a monoclonal antibody that binds LFA-1 and blocks its interaction with ICAM, and inhibits migration and possibly activation. Once in the skin, T cells and the inflammatory changes they can induce, alter keratinocytes. As previously mentioned, the cytokines released in psoriatic plaques are primarily T1, and include TNF- [ , which in turn increases the production of other inflammatory cytokines such as IL-1, IL-6, and IL-8. Therapies such as etanercept, infliximab, and adalimumab work to block the effects of TNF- [ and therefore its actions including the increased production of pro-inflammatory cytokines, adhesion molecules, vascular endothelial growth factor, and keratinocyte hyperproliferation.

9.2 Etanercept Etanercept was first used in human clinical trials in 1992, and in 1995 studies on the use of etanercept for

rheumatoid arthritis were initiated. Currently, etanercept is approved for the treatment of rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and moderate to severe psoriasis. 9.2.1 Structure and Mode of Action Etanercept is a fully human, soluble TNF- [ receptor dimeric fusion protein produced by recombinant technology using Chinese hamster ovary cells. It consists of two molecules of the p75 TNF- [ receptor linked to the constant Fc portion of IgG1.The dimeric structure of etanercept permits the molecule to bind to two molecules of TNF- [ , either free or membrane bound, simultaneously, and thereby neutralizing TNF- [ ’s proinflammatory actions. The dimeric structure of etanercept also causes it to have a 50- to 1,000-fold higher affinity for TNF- [ than the naturally occurring monomeric soluble form of the receptor. 9.2.2 Pharmacokinetics and Pharmacodynamics Once administered by subcutaneous injection, etanercept is slowly absorbed in both healthy volunteers and in patients with psoriasis, with time to peak serum concentrations in excess of 50 h. In addition, the drug appears to be widely distributed, including in the synovium, and is likely metabolized by proteolytic processes before recycling or elimination in the bile or urine. Elimination half-lives are similar for etanercept in patients with psoriasis and rheumatoid arthritis, with 68 h being seen in healthy volunteers and 102 h in patients with rheumatoid arthritis. In addition, steadystate pharmacokinetic properties of etanercept administered twice weekly are similar to those in patients with rheumatoid arthritis. Lastly, studies examining intermittent and continuous etanercept administration have found no differences in the pharmacokinetic profiles of the two dosing regimes. Most data on the biological effects of etanercept have been conducted in those patients with rheumatoid arthritis. In this population, etanercept therapy has been found to reduce plasma levels of IL-6 and matrix metalloproteases (MMP), and the immunohistochemical staining of CD3+ T cells, CD 38+ T cells, IL-1 q , and vascular cell adhesion molecule (VCAM). In addition,

9.2 Etanercept

long-term treatment reduces the numbers of TNF- [ and IL-1 producing cells to the numbers seen in healthy controls. Gottlieb and colleagues studied histological response, inflammatory gene expression, and cellular infiltration in psoriatic plaques of patients receiving etanercept, 25 mg subcutaneously twice weekly for 6 months. After 6 months of treatment with etanercept, nine out of the ten patients treated had thinning of the epidermis and normalization of keratinocyte differentiation, and eight of the ten displayed an absence of keratin 10 (K 16), indicating normalization of keratinocyte differentiation and proliferation. A rapid and complete reduction of both IL-1 and IL-8 were observed, with maximal suppression seen by 1 month of treatment. Unlike IL-1 and IL-8, which are early TNF- [ induced genes, most other inflammatory genes, such as STAT-1, inducible nitric oxide synthase (iNOS), IL-23, and IP-10 (IFN- * -inducible protein-10, CXCL 10), showed a more gradual response and generally were most suppressed at 6 months. Slower reductions in infiltrating myeloid cells (CD11c+ cells) and T lymphocytes were also observed. In another study, NF-κB, a nuclear transcription factor central in the cell stress response and keratinocyte differentiation, was found to be upregulated in the epidermis of normal epidermis of psoriasis patients, and even more so in the plaques of these patients. Treatment with etanercept correlated with downregulation of phosphorylated NF-κB as well as decreases in epidermal thickness, return of normal markers of keratinocyte differentiation, and lastly clinical outcomes. 9.2.3 Efficacy The efficacy of etanercept for the treatment of plaque psoriasis has been evaluated in four placebo-controlled studies, one of which evaluated psoriasis in the setting of psoriatic arthritis. This study by Mease and colleagues which evaluated etanercept in the treatment of psoriatic arthritis also examined a subset of 38 patients who had more than 3 % of their body surface area (BSA) covered with plaque psoriasis. Of the 38 patients, 19 received etanercept, 25 mg subcutaneous twice weekly, while the remaining 19 received placebo. After 12 weeks of therapy, 26 % of those subjects receiving etanercept achieved PASI-75 compared to 0 % of those receiving placebo. A larger, phase 2, randomized, double-blind and placebo controlled study by Gottlieb and colleagues (Gottlieb, Chaudhari, et al. 2003) examined the efficacy

and safety of etanercept, 25 mg subcutaneously twice a week compared to placebo, as monotherapy for moderate to severe plaque psoriasis. After 12 weeks of therapy, 30 % of patients achieved a PASI-75 compared to 2 % in the placebo group. After 24 weeks of continuous therapy, 56 % of patients receiving etanercept achieved PASI-75 compared to 5 % in the control group. In addition, by 24 weeks, psoriasis was clear or minimal by the physician’s global assessment in more than 50 % of patients who received etanercept. Leonardi and colleagues later conducted a phase 3, placebo-controlled, double blind study that evaluated etanercept for psoriasis. Patients with moderate to severe psoriasis who were not receiving any other therapies including systemic, phototherapy or topical treatments, were enrolled. Six hundred and seventy-two patients underwent randomization and 652 received either placebo or received etanercept subcutaneously at low dose (25 mg once weekly), medium dose (25 mg twice weekly), or high dose (50 mg twice weekly). After 12 weeks, patients who were in the placebo group began twice weekly treatment with 25 mg of etanercept. After 12 weeks of therapy, 4 %, 14 %, 32 %, and 47 % of patients achieved a PASI-75 on placebo, 25 mg once weekly, 25 mg twice weekly, and 50 mg twice weekly, respectively. After 24 weeks of continuous therapy, the PASI-75 score in the low dose group was 21 %, 41 % in those receiving 25 mg twice weekly, and 54 % in those receiving the high dose of 50 mg twice weekly. A second large, phase 3, double-blind study evaluating etanercept for moderate to severe plaque psoriasis was conducted in the US, Europe, and Canada by Papp and colleagues. The study involved three groups of patients, each receiving placebo twice weekly, etanercept 25 mg twice weekly, or etanercept 50 mg twice weekly, for the first 12 weeks. After 12 weeks, all three groups were continued on etanercept 25 mg twice weekly for 12 weeks. After the first 12 weeks of therapy, 3 %, 34 %, and 49 % of patients receiving placebo, 25 mg twice weekly, and 50 mg twice weekly, achieved PASI-75 respectively, findings consistent with those results seen in the US studies. During the second 12-week period during which all patients received 25 mg, those patients who were previously receiving 25 mg twice weekly continued to improve, with 45 % achieving PASI-75 at week 24. The high dose group (50 mg twice weekly) who were then placed on 25 mg twice weekly maintained their previous improvements, with 54 % achieving PASI-75 at week 24, and of those patients who were

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previously receiving placebo, 28 % achieved a PASI-75. These finds suggested that induction with high-dose etanercept can then be maintained with a lower dose and still preserve PASI-75 scores. 9.2.4 Safety In psoriasis clinical trial experience, etanercept has been well tolerated. The most common adverse event in patients receiving placebo or any dose of etanercept was injection site reaction, where rates in the previously mentioned two phase 3 trials ranged from 6 % to 18 %. These reactions typically occur 2 – 3 weeks into treatment and consist of erythema, pain, itching, and/ or swelling, and typically resolve in 3 – 5 days. In addition, upper respiratory tract infections (5 – 11 %) and headache (3 – 12 %) were also seen. In the study by Leonardi and colleagues, serious infectious adverse events were infrequent and were not more frequent in the high dose etanercept groups when compared to the placebocrossover group of lower dose groups. In placebo-controlled trials for all uses of etanercept, the most common type of adverse event was an upper respiratory tract infection, which occurred in between 12 % and 20 % of patients, but not at an increased frequency when compared with placebo groups. 9.2.4.1 Serious Infections In rheumatoid arthritis patients in whom there is more long-term data, it appears that etanercept may increase the risk of serious infection. In clinical trials, the rates of infection that required hospitalization or parental antibiotic therapy were 0.04 per patient-year in etanercept treated groups, which is very similar to the total population. In post marketing data on the use of etanercept, serious infections and sepsis were reported in patients using etanercept, but most of these cases were in patients receiving concomitant immunosuppressive therapies. Great care should be practiced when placing a patient on multiple immunosuppressive therapies, and they should be monitored closely. Rare cases of reactivation of tuberculosis have been noted in patients receiving etanercept, and consideration should be given to performing a purified protein derivative (PPD) skin test prior to initiation of treatment, especially if geographic location makes a patient more at risk.

9.2.4.2 Malignancy The rates of malignancy in patients with psoriasis do not appear to be increased in those receiving etanercept. In placebo-controlled, randomized studies, 8 of 933 etanercept treated patients were diagnosed with malignancy whereas 1 in 414 patients receiving placebo were diagnosed with malignancy. The rate of lymphoma was threefold greater in patients receiving etanercept than in the general population. A cohort study by Gelfand though found that both rheumatoid arthritis and psoriasis patients are at a threefold increased risk of developing lymphoma. Taking this into account, analysis of the effects of etanercept or any other immunosuppressive therapy must consider the inherited risk of lymphoma to that specific disease population. 9.2.4.3 Demyelinating Disease TNF- [ inhibition should not be initiated in patients with a history of demyelinating disorders such as multiple sclerosis (MS). Post-marketing surveillance has reported rare incidences of demyelinating disorders or exacerbations of pre-existing multiple sclerosis in patients receiving etanercept. In addition, early studies examining the use of a TNF- [ inhibitor, lenercept, in the treatment of multiple sclerosis found increased numbers of MS exacerbations compared to placebo as well as MS exacerbations that occurred earlier compared to placebo. Physicians should be wary of newonset neurological symptoms in patients receiving etanercept treatment, and a good neurological history should be obtained before commencing therapy. 9.2.4.4 Autoimmunity Approximately 6 % of patients with RA, psoriatic arthritis, ankylosing spondylitis, or plaque psoriasis developed non-neutralizing antibodies to the TNF receptor (package insert). Antinuclear antibodies develop in some patients receiving etanercept, but most typically this finding has no clinical significance. There have been rare cases of systemic and cutaneous lupus associated with etanercept use.

9.3 Efalizumab

9.2.4.5 Congestive Heart Failure Etanercept and infliximab were evaluated for their use in patients with congestive heart failure (CHF), but the studies were terminated early due to lack of efficacy. One of the studies actually suggested a higher mortality rate in patients with CHF who received treatment with etanercept. In addition, there have been case reports describing new onset CHF in patients receiving etanercept who had no previous symptoms and were under the age of 50. Physicians should proceed with caution when prescribing etanercept for patients with a history of heart failure. 9.2.5 Off-Label Use Clinical studies have been performed to investigate the use of etanercept for other dermatological conditions. A small open-label study was conducted in patients with early stages of diffuse progressive systemic sclerosis, which found that 25 mg of etanercept subcutaneously provided improvement in skin symptoms as well as functional status. Also there have been numerous reports on the successful use of etanercept in the treatment of the following diseases: multicentric reticulohistiocytosis, erythroderma-associated pruritus, palmoplantar and pustular psoriasis, pyoderma gangrenosum, alopecia areata, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid, cutaneous sarcoidosis, erythema annulare, mixed connective tissue disease, recurrent aphthous stomatitis, refractory hidradenitis suppurativa, Beh¸cet’s disease, and Sweet’s syndrome.

9.3 Efalizumab 9.3.1 Structure and Mode of Action Efalizumab (Raptiva, Genentech, Inc., South San Francisco, CA) is a recombinant, humanized, monoclonal IgG1 antibody whose action affects several steps of the T-cell inflammatory cascade. The humanized version of the murine antihuman CD11a Mab, MHM24, was made by grafting the complementary-determining regions, or hypervariable region, from murine antibody to the human framework. Efalizumab binds to

CD11a, the [ -subunit of leukocyte-associated antigen (LFA-1). LFA-1 is a cell-surface glycoprotein of the integrin family that has been shown to promote intercellular adhesion of inflammatory cells, as well as play a role in T-cell activation. By binding with CD11a, efalizumab blocks the interaction between LFA-1 and the cell surface molecule, intracellular adhesion molecule 1 (ICAM-1), which is present on antigen-presenting cells (APCs), as well as endothelial cells and keratinocytes that are involved in psoriatic plaques. Research into the LFA-1/ICAM-1 interaction has found that the interaction is necessary for T-cell activation by activating the T-cell costimulatory pathways, as well as inhibiting the binding of T cells to endothelial cells and trafficking of inflammatory cells into the dermis (Lebwohl et al. 2004). Efalizumab is believed to act by inhibiting these reactions, thus decreasing the release of inflammatory mediators into the skin that cause the phenotypic features of psoriasis. 9.3.2 Pharmacokinetics and Pharmacodynamics The pharmacokinetic profile of subcutaneous efalizumab was described by Gottlieb, Miller et al. (2003) in a Phase 1, open-label, escalating dose study, in which it was found that peak efalizumab plasma concentrations were achieved in approximately 1 – 2 days following injection, and that efalizumab was detectable in the serum for 3 – 5 weeks following the final injection. Patients receiving efalizumab 0.5 – 1.0 mg/kg SC weekly had an average T1/2 of 4 days, whereas those receiving 1.0 – 2.0 mg/kg had a T1/2 of 6 days. Subcutaneous doses administered weekly gave peak plasma concentrations and efalizumab exposure of approximately one-third to one-half of the equivalent intravenous dose. A study examining the pharmacodynamic profile of subcutaneous efalizumab found that CD11a expression on circulating lymphocytes rapidly decreased to 15 – 30 % of pretreatment levels and remained at this suppressed level until efalizumab was cleared from the plasma (Wellington and Perry 2005). Within 7 – 10 days of efalizumab clearance, CD11a expression returned to baseline. Following subcutaneous efalizumab administration of 1 mg/kg/week, CD11a expression is reduced within 1 – 2 days and is maintained with weekly administration. In addition, phase 2 studies evaluating SC efalizumab have noted histologically that epidermal thickness was reduced in the 0.3 mg/kg group com-

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pared to placebo. This treatment group, 0.3 mg/kg efalizumab, also noted a reduction in the number of CD3+ T cells in skin biopsy specimens with a concurrent increase in circulating lymphocytes. 9.3.3 Efficacy The efficacy of efalizumab for the treatment of adults with moderate to severe psoriasis was evaluated in three large phase 3 studies. Of these studies, two also had extension phases lasting an additional 12 weeks. In addition, an open-label study investigating the long-term efficacy over 3 years is ongoing. The phase 3 study by Lebwohl and colleagues was divided into three phases: a treatment phase from weeks 0 to 12, an extended treatment phase from weeks 13 to 24, and lastly a follow-up phase from weeks 25 to 36. In two of these studies, one by Lebwohl et al., the other by Leonardi and colleagues, subjects received efalizumab 1 or 2 mg/kg or placebo subcutaneously, after a first dose of 0.7 mg/kg to reduce first dose adverse events. In the other phase 3 study by Gordon et al., subjects received efalizumab 1.0 mg/kg subcutaneously or placebo. In the 3-year long term study, patients were randomized to receive 12 weeks of open-label efalizumab 2.0 mg/kg once weekly with or without topical fluocinolone ointment (0.025 %) during weeks 9 through 12. After these 12 weeks, patients with a PASI score reduction of 50 % (PASI-50) were then scheduled to receive efalizumab 1.0 mg/kg once weekly for up to 33 months. Patients who relapsed during the maintenance phase were switched to once weekly dosing of 2.0 mg/kg for 12 weeks or 4.0 mg/kg for 4 weeks. In the extension studies by Menter et al. (Menter et al. 2005), all the patients who finished the initial 12-week doubleblind phase received efalizumab 1.0 mg/kg/week for a further 12 weeks. In another extension phase by Leonardi et al., patients who received efalizumab previously with PASI score reductions of less than 75 % were then re-randomized to receive placebo or continue on their previously administered dose of efalizumab at 1.0 or 2.0 mg/kg/week. All of the phase 3, double-blind studies used PASI score reductions of 75 % (PASI-75) after 12 weeks of treatment as the primary efficacy endpoint. In the longterm study by Gottlieb et al. (2004), in addition to PASI75, PASI-50 and PASI-90 scores were examined. The study by Gordon and colleagues found that all efalizumab treated patients experienced statistically

significant improvement on all end points compared with those patients receiving placebo. Twenty-seven percent of patients receiving efalizumab achieved PASI-75 versus 4 % of the placebo group. In addition, 95 % of efalizumab treated patients achieved PASI-50 compared to 14 % of those receiving placebo. With regard to patient reported outcomes, at week 12, patients treated with efalizumab had a greater mean percentage improvement in Dermatology Life Quality Index (DLQI) with 47 % compared to 14 % in the placebo group. Efalizumab treatment also produced a 38 % improvement in Itching Visual Analog Score (VAS) compared to placebo. Lastly, efalizumab treated patients had a statistically significant improvement in Psoriasis Symptom Assessment (PSA), both frequency and severity subscales (48 % vs. 18 % and 46 % vs. 17 %, respectively), compared to placebo. In the extension study published by Menter and colleagues, of the 342 subjects who received and completed the 12-week course of efalizumab treatment, 342 entered an open-label treatment period for an additional 12 weeks, receiving 1 mg/kg/week. In addition, 174 subjects who completed a 12-week course of placebo were scheduled to receive 12 weeks of efalizumab at the same dose. As the duration of treatment continued, PASI indexes continued to improve. At week 24, 66.6 % of the previously efalizumab-treated patients achieved a PASI-50 response, and 43.8 % achieved a PASI-75 response. The percentage of patients who achieved a static Physician’s Global Assessment (sPGA) of minimal or clear increased from 25.7 % to 35.9 % from week 12 to week 24. For those subjects who received placebo followed by efalizumab, 28.7 % achieved an sPGA rating of minimal or clear after 12 weeks. In addition to physician-assessed parameters, there was a statistically significant improvement after 12 weeks of efalizumab treatment in Dermatology Life Quality Index (DLQI), Itching scale, and Psoriasis Symptom Assessment (PSA) frequency and severity. In a study by Lebwohl and colleagues, patients receiving 1 mg/kg of efalizumab per week achieved PASI-75 in 22 % of the subjects and in 28 % of those subjects receiving 2 mg/kg per week, compared to 5 % of those subjects receiving placebo. In the extended treatment phase, those subjects achieving PASI-75 or PASI-50 were randomly assigned to continue receiving 2 mg/kg of efalizumab weekly or every other week or placebo. Those subjects not attaining at least PASI-50 were randomly assigned to either an increased dose of

9.3 Efalizumab

4 mg/kg of efalizumab weekly or placebo. It was found in the extended treatment phase that of the efalizumabtreated subjects who initially achieved a PASI-75, a greater proportion of the subjects who received further treatment with efalizumab maintained a PASI-75 compared to those receiving placebo (p < 0.001). Of those subjects who did not achieve a PASI-50 on initial efalizumab treatment, an improvement of 75 % or more was achieved in 40 % of those subjects receiving efalizumab 4 mg/kg per week, compared to 15 % in the placebo group (p = 0.02). At the 36-week follow-up, 12 weeks after the discontinuation of study treatment, it was found that in subjects who received at least 50 % improvement in their PASI index at week 24, the time to relapse (loss of at least 50 % of the improvement in the PASI index that had been achieved between base line and week 24) was approximately 84 days. Leonardi and colleagues assessed short term and extended treatment efficacy and safety of efalizumab in another phase 3 study. The study was divided into three 12-week treatment periods, the first from weeks 1 to 12, and retreatment or extended treatment periods during weeks 13 – 24, with two observation periods, with subjects receiving an initial treatment of efalizumab 1 mg/ kg/week, 2 mg/kg/week, or placebo. During the first treatment week after 12 weeks, significantly more patients receiving 1 mg/kg and 2 mg/kg achieved a PASI-75 (39 % and 27 %, respectively) compared with those subjects receiving placebo (2 %). Those efalizumab-treated subjects who did not achieve PASI-75 were re-randomized at week 12 to receive efalizumab or placebo for an extended 12-week period. At week 24, 20.3 % of subjects who received an additional 12 weeks of efalizumab achieved a PASI-75 compared to 6.7 % of those receiving placebo. Gottlieb and colleagues assessed long term, continuous therapy with efalizumab in a multicenter, openlabel, phase 3 study in patients with moderate to severe chronic plaque psoriasis. Preliminary data regarding the first 15 months of this 3-year-long study showed once weekly subcutaneous efalizumab maintains sustained efficacy without toxicity. Patients were randomized to receive 12 weeks open-label subcutaneous efalizumab 2.0 mg/kg/week with or without topical fluocinolone during weeks 9 – 12. After the 12th week, patients were then scheduled to receive efalizumab, 1.0 mg/kg/week for up to 33 weeks, if they received at least a PASI-50 during the first 12 weeks of treatment. If

a patient relapsed, therapy was increased to 2.0 mg/kg/ week for 12 weeks or 4.0 mg/kg/week for 4 weeks. Concomitant topical corticosteroids and UVB phototherapy were also permitted. PASI improvement was maintained throughout the 15-month period. 9.3.4 Safety Once weekly injections of efalizumab, 1 mg/kg, were generally well tolerated for 12 weeks to 15 months. In published clinical trials, between 3 % and 6 % of subjects withdrew due to adverse events of efalizumab compared to 1 – 3 % in the placebo groups. The most common adverse events seen in clinical trials included a first dose complex consisting of headache, nausea, myalgia, fever, and chills that typically developed within 2 days after the first two injections. After the third dose, these reactions diminished, with similar incidence in both efalizumab and placebo groups. These reactions were typically well managed with acetaminophen or nonsteroidal anti-inflammatory drugs. Serious adverse events were uncommon. In the three 12-week studies, 2 % of efalizumab-treated patients (1 mg/kg/ week) had a serious adverse event during treatment. Withdrawals from the studies due to these adverse events were rare as well, with a total of 3.5 % of efalizumab (1 mg/kg/week) treated patients withdrawing from treatment due to adverse events in these same studies, whereas 2.1 % of placebo treated patients withdrew because of adverse events. Long-term treatment with efalizumab, examined in the study by Gottlieb and colleagues, was not associated with an overall increased incidence of adverse events. Those events noted were similar in nature to those documented in short-term trials. There was no evidence of cumulative toxicity noted. Two serious adverse events that were determined by the investigator to be drug related included arthritis and gastrointestinal carcinoma. 9.3.4.1 Infections In the phase 3 study by Gordon and colleagues, infections were present in 27 % of efalizumab treated patients compared to 23 % of those receiving placebo. Among these subjects, there was no increased susceptibility to any specific pathogen determined upon analy-

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sis. In all three phase 3 studies by Gordon et al., Lebwohl et al. and Leonardi et al., no statistically significant increased risk of infection was found in the efalizumab treated patients compared to those subjects receiving placebo. 9.3.4.2 Malignancy There were two cases of malignancy in one clinical trial by Gordon and colleagues, which were determined not to be related to efalizumab secondary to the time line of drug initiation and identification of malignancy. According to company generated information, of the 2,762 patients who received efalizumab for a mean duration of 8 months, the incidence of malignancies of any kind was 1.8 per 100 patient-years with efalizumab and 1.6 per 100 patient-years with placebo. 9.3.4.3 Psoriasis Flare During clinical trials, 19 of 2,589 patients experienced worsening (past baseline) of their psoriasis during or after treatment with efalizumab. The worsening involved new plaques, as well as different forms of their psoriasis, including pustular and erythrodermic psoriasis. Some patients required hospitalization and alternate psoriasis treatments were administered. 9.3.4.4 Arthritis Clinical and post-marketing data have included reports of arthritis, including new onset as well as recurrent, severe arthritis. Joint pain was noted during treatment as well following discontinuation of efalizumab, and typically resolved after discontinuation of efalizumab and without other therapies. 9.3.4.5 Hematologic Complications Platelet counts at or below 52,000 cells/μl were observed in eight subjects during clinical trials. Seven (one patient was lost to follow-up) were treated with systemic corticosteroids, with resolution. Post-marketing surveillance has reported cases of severe thrombocytopenia as well, and physicians should monitor plate-

let count closely. Patients experiencing thrombocytopenia while taking efalizumab should discontinue treatment (Raptiva package insert, 2005). Hemolytic anemia, usually 4 – 6 months after the initiation of therapy, was noted, and treatment with efalizumab should be stopped if this develops. 9.3.5 Off-Label Use Currently, efalizumab is approved only for the treatment of patients with chronic, moderate-to-severe plaque psoriasis. It has been used with success in the treatment of disseminated granuloma annulare. Besides this dermatologic condition, efalizumab is currently being studied for use in renal transplant patients for prevention of acute rejection, as well as in asthmatics as a possible agent to reduce the late asthmatic response in patients with mild allergic asthma.

9.4 Alefacept Psoriasis is an inflammatory disorder mediated primarily by T cells and the number of inflammatory cytokines that are released. Specific steps in the inflammatory cascade that cause psoriasis targeted by biologics include reduction of specific disease causing T-cell populations, inhibition of T-cell activation, prevention of T-cell trafficking, or specific inhibition of inflammatory cytokine release in psoriatic plaques. 9.4.1 Structure and Mode of Action Alefacept (Biogen, Inc., Cambridge, MA) is a fully human lymphocyte function-associated antigen 3/ immunoglobin 1 (LFA-3/IgG1) fusion protein, which consists of the first extracellular domain of LFA-3, fused to the hinge, CH2 and CH3, domains of human IgG1. The LFA-3 domain of the drug binds CD2 on T cells, thereby blocking T-cell activation and proliferation of memory effector cells (CD4+CD45+RO+ and CD8+CD45+RO+ T cells). This action results in decreased release of cytokines and less inflammation. CD2 is upregulated on memory T cells and therefore alefacept selectively targets and reduces a population of cells specific for the pathogenesis of psoriasis. In addi-

9.4 Alefacept

tion, the IgG1 domain of alefacept interacts with Fc * RIII receptors on natural killer cells and macrophages, leading to apoptosis of memory effector T cells. 9.4.2 Pharmacokinetics and Pharmacodynamics The pharmacokinetics of single and multiple dose intravenous alefacept were studied in healthy Caucasian and Japanese volunteers as well as Caucasian patients with chronic plaque psoriasis. The elimination half-life of IV alefacept was found to be 890 h or approximately 5 weeks. In healthy Caucasian and Japanese volunteers, the half-life of alefacept was found to be similar, 202 and 198 h, respectively. Also, the relative bioavailability of IM to IV infusion was approximately 60 %. After IM absorption was complete, the rate of elimination from the serum was consistent with that of IV administration, approximately 12 days. An intramuscular dose of approximately 150 – 200 % of the IV dose is an appropriate alternative for the IV dose. Multiple phase III studies examining patients with moderate-to-severe psoriasis found that alefacept treatment consistently reduced total lymphocyte and circulating memory-effector T cells. Specifically, Ellis and Kreuger found that during treatment with intravenous alefacept, there was a dose-dependent reduction in peripheral blood CD4+ memory cells (CD45RO+), but not in CD4+ na¨ıve cells (CD45RA+). These dose related reductions in memory T cells correlated with, but were not predictive of, clinical response. A similar study by Lebwohl and colleagues examining intramuscular alefacept provided consistent data, with similar memory T-cell subset reductions. 9.4.3 Efficacy The efficacy of alefacept is now well known after several phase II and III studies. In a phase II, randomized, placebo-controlled, dose-response trial of alefacept in subjects with chronic plaque psoriasis, patients received a single course of alefacept at 0.025, 0.0.75, or 0.15 mg/kg as an IV bolus injection weekly for 12 weeks with a 12-week follow-up period. Two weeks after completion of treatment, the mean PASI scores were 38 %, 53 %, and 53 % lower than the baseline groups, who received 0.025, 0.075, and 0.15 mg/kg alefacept, respectively, compared to 21 % lower in the placebo group

(p < 0.001). At both 2 weeks and 12 weeks after treatment completion, the PASI-50 and PASI-75 scores were significantly higher in the alefacept treatment groups than in the placebo group. For example, 2 weeks after treatment, 36 %, 60 %, and 56 % of the subjects receiving 0.025, 0.075, and 0.15 mg/kg alefacept achieved a PASI-50, compared to 27 % in the placebo group. Two weeks after treatment completion, 21 %, 33 %, and 31 % of subjects in the three-alefacept treatment groups achieved a PASI-75, whereas only 10 % in the placebo achieved such a score. A phase 3 intravenous study of alefacept consisted of two treatment courses, each with a 12-week treatment period followed by a 12-week treatment free period. There were three separate cohorts: cohort 1 received alefacept 7.5 mg IV in the first and second course of treatment; cohort 2 received alefacept in the first course and placebo in the second; and cohort 3 received placebo in the first course and alefacept in the second. The primary efficacy endpoint was the percentage of patients with a 75 % reduction in PASI at 2 weeks after the last dose of course 1, as well as an overall response rate defined as the percentage of people achieving “clear” or “almost clear” by the Physician’s Global Assessment (PGA). A significantly higher percentage of patients in the combined alefacept group (cohorts 1 and 2) achieved at least a PASI-75 or greater 2 weeks after the last dose of course 1, with 14 % in those receiving alefacept, versus 4 % in the placebo group. In course 1, 28 %, 56 %, and 23 % of the subjects achieved a PASI-50, PASI-75, and PGA of “clear” or “almost clear”, respectively, versus 8 %, 24 %, and 6 % in the placebo group, respectively. In course 2, 37 %, 64 %, and 30 % of subjects receiving alefacept achieved a PASI-75, PASI-50, and PGA of “clear” or “almost clear” respectively, versus 19 %, 49 %, and 30 % in the placebo group, respectively. The response to alefacept was long lasting. The subjects who received one course of alefacept therapy (cohort 2) and achieved a PASI-75 during or after treatment preserved a PASI-50 or greater for a median duration of more than 7 months. For subjects achieving two courses of alefacept (cohort 1), the median duration of response could not be determined since more than 50 % of these subjects maintained 50 % or greater improvement at the final end point, which was a year after the first dose of alefacept. An international, randomized, double-blind, placebo-controlled phase III trial of intramuscular alefacept in patients with chronic plaque psoriasis found similar

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efficacy to the intravenous dosing method. In this study, placebo, 10 mg, or 15 mg of alefacept was administered once weekly for 12 weeks followed by 12 weeks of observation. Mean reductions in PASI in the 15 mg alefacept, 10 mg alefacept, and placebo groups were 46 %, 41 %, and 25 %, respectively, 6 weeks after dosing. The percentage of subjects achieving at least a PASI-50 was 57 % and 53 % in the 15 mg and 10 mg alefacept groups, respectively, compared to 35 % in the placebo group. As the intravenous study showed, the response to alefacept was durable. Of those subjects receiving 15 mg of alefacept and who achieved a PASI-75, 74 % maintained at least a PASI-50 during the 12-week follow-up period. The overall response rates for the Physicians Global Assessment (PGA) of clear or almost clear were 24 %, 22 % and 8 % of patients in the 15 mg alefacept, 10 mg alefacept, and placebo groups, respectively. In a follow-up extension study of the intramuscular alefacept trial, patients who achieved 75 % or more reduction in PASI with the first course of intramuscular alefacept 15 mg maintained a PASI-50 for a median duration of 209 days. In addition, two courses of 15 mg intramuscular alefacept produced a PASI-50 overall response rate of 69 %, whereas only 57 % did so with a single course. 9.4.4 Safety The safety of alefacept has been evaluated in two placebo-controlled phase 3 clinical trials that enrolled over 1,000 patients. In course 1, 186 subjects received placebo, and 367 received 7.5 mg IV alefacept. In the second course, 142 subjects received placebo and 307 received alefacept. In the phase 3 intramuscular study, 173 subjects received 10 mg alefacept, 166 received 15 mg alefacept, and 169 received placebo. Both studies required weekly monitoring of CD4+ T-cell counts as alefacept acts by binding to CD2 receptors, therefore affecting the activity and quantity of cells expressing these receptors. In course 1, 38 patients (10 %) in cohorts 1 and 2 had at least one placebo substitution due to CD4+ lymphocyte counts below 250 cells/μl. There were seven permanent placebo substations due to four consecutive CD4+ T-cell counts below 250 cells/μl. In the second course, there was placebo substitution in 14 patients (9 %) in cohort 1 (2nd course 7.5 mg alefacept) and 8 patients (5 %) in cohort 3 (alefacept during 2nd course of therapy), while there was

only one permanent substitution in course 2. Throughout the study, no opportunistic infections and no associated infections were noted with CD4+ T-cell counts below 250 cells/μl. In the IM study, 4 % of patients had at least one placebo substitution for CD4+ T-cell counts below 250 cells/μl, and there were no permanent substitutions. At the end of treatment in both studies, T-cell counts recovered to normal levels. Also at the end of the 12-week observation period, 98 % of patients in both the IV and IM studies had total lymphocyte counts that were above the lower limit of normal. In all, alefacept therapy was well tolerated. For patients in the two course intravenous study, relative to the first course of therapy received in those patients in cohort 1, the incidence of each adverse event was similar or lower in the 2nd cohort. In the IV study, the only adverse event in course 1 that had a greater than or equal to 5 % higher incidence in the combined alefacept group (cohorts 1 and 2) versus placebo (cohort 3) was chills (10 % vs. 1 %). For subjects who received two courses of alefacept (cohort 1), this frequency substantially decreased in the 2nd course of therapy compared to the first (< 1 % vs. 13 %, respectively). Adverse events experienced in greater than 10 % of patients receiving a single course and two courses are shown in Table 9.1. Other commonly reported events were headache, pharyngitis, flu syndrome, and infection, which most frequently referred to the common cold. In the IM study, alefacept was also found to be well tolerated. Adverse events occurring at an incidence of 5 % or greater in the alefacept group compared to placebo were observed for pruritus, injection-site pain, and injection-site inflammation. The incidence of infections was monitored by CD4+ T-cell counts lower than 250 and at least 250 cells/μl. No relationship between the incidence of infections and decreased CD4+ T-cell counts was observed. In both studies, there was no single event that contributed significantly to drug discontinuation. In the IV study, 2 % of patients receiving alefacept, compared to 1 % in the placebo group, discontinued the study drug. Similarly in the IM study, 2 %, 2 %, and 1 % of patients in the placebo, 10 mg, and 15 mg alefacept groups, respectively, discontinued the drug. In addition, a study by Gottlieb and colleagues examined the effect of alefacept on T-cell-dependent humoral responses to a neoantigen and recall antigen (tetanus toxoid). The study found that alefacept did not alter primary or secondary antibody responses to a neoantigen or memory responses to a recall antigen.

9.6 Adalimumab

9.4.5 Off-Label Use Alefacept is currently approved for the treatment of adult patients with moderate to severe chronic plaque psoriasis who are candidates for systemic therapy or phototherapy. There have been numerous case reports and small case series regarding the use of alefacept for the management of diseases such as palmoplantar psoriasis, psoriasis affecting the nails, and erythema nodosum, lichen planus, pyoderma gangrenosum, and scalp psoriasis.

9.5 Infliximab Infliximab is a chimeric anti-TNF- [ monoclonal antibody that is given by intravenous infusion. It was the first anti-TNF therapy studied for the treatment of psoriasis, and is currently approved by the FDA for the treatment of moderate to highly active rheumatoid arthritis (in combination with methotrexate) and moderate to highly active Crohn’s disease or for those patients who have rheumatoid arthritis or Crohn’s disease and have failed previous conventional treatments, as well as psoriatic arthritis. A double-blind study by Chaudhari and colleagues showed that infliximab provides substantial clinical efficacy with high tolerability. Specifically, 82 % and 73 % of patients with psoriasis receiving 5 and 10 mg/kg of infliximab, respectively, achieved a PASI-75 at week 10. An open-label phase of the same study showed that at week 26, PASI response was maintained in 40 % and 73 % of patients receiving 5 and 10 mg/kg of infliximab, respectively. A double blind, placebo-controlled, phase 2 trial conducted by Gottlieb and colleagues found that at week 10 (intravenous infusions of placebo or infliximab at either 3 or 5 mg/kg given at weeks 0, 2, and 6), 72 % of patients treated with infliximab (3 mg/kg) and 88 % of patients treated with infliximab, 5 mg/kg, achieved a PASI-75 or greater, compared to only 6 % of those receiving placebo. These improvements were seen as early as 2 weeks. More recently a phase 3, double-blind study examined the utility of infliximab as an induction and maintenance therapy to moderate to severe psoriasis. Patients were randomized to receive infliximab 5 mg/kg or placebo at weeks 0, 2, and 6, and then every 8 weeks until week 46. Those patients receiving placebo were crossed

over to receive infliximab at week 24. At week 10, 80 % of patients treated with infliximab achieved a PASI-75 and 57 % achieved a PASI-90, compared to 3 % and 1 %, respectively, in the placebo group. PASI-75 scores were maintained at week 24 as well (82 % for infliximab compared to 4 % for placebo). At week 50, 61 % of the infliximab treated patients achieved a PASI-75 and 45 % achieved a PASI-90, showing a sustained effect. Overall, infliximab has been well tolerated in clinical trials. The most common adverse events reported in 10 % or more of infliximab treated patients include headache, nausea, and upper respiratory tract infection (Winterfield and Menter 2004). Similar safety issues are seen in patients receiving infliximab as are seen in etanercept treated patients including infections and reactivation of tuberculosis (with mandatory PPD testing), demyelinating conditions, congestive heart failure, malignancy, and autoimmunity. Infliximab is approved by the EMEA and is filed at the FDA for treatment of moderate to severe psoriasis. Its use in the treatment of many other dermatological conditions has been reported and includes the following: Beh¸cet’s, graft versus host disease, hidradenitis suppurativa, panniculitis, pyoderma gangrenosum, SAPHO (synovitis, acne, hyperostosis, and osteitis), sarcoidosis, subcorneal pustular dermatosis, Sweet’s syndrome, toxic epidermal necrolysis, and Wegener’s syndrome.

9.6 Adalimumab Adalimumab is a fully human anti-TNF- [ monoclonal antibody currently in phase 2 studies for the treatment of moderate to severe psoriasis. It is currently approved for the treatment of rheumatoid arthritis as a secondline agent, alone or in combination with a diseasemodifying anti-rheumatic drug (DMARD) and psoriatic arthritis. Adalimumab is given as a 40 mg subcutaneous injection every 2 weeks. A phase 2 trial by Chen and colleagues found that 53 % of study patients receiving 40 mg every other week achieved a PASI-75, and 80 % of patients receiving 40 mg weekly achieved a PASI-75. The percentages of patients achieving a PASI75 were statistically significantly greater than placebo as early as 4 weeks. As discussed with etanercept and infliximab, adalimumab has the same important side effects as the other TNF- [ inhibitors exhibit.

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Adalimumab is currently undergoing a phase 3 investigational study for the treatment of moderate to severe psoriasis. In addition to psoriasis, there have been case reports on the successful use of adalimumab in the treatment of recalcitrant acrodermatitis continua of Hallopeau (with acitretin), psoriatic onychopachydermo periostitis (POPP), pyoderma gangrenosum, and refractory rheumatoid arthritis-associated leg ulcerations.

References Chaudhari U, Romano P, Mulcahy L, Dooley L, Baker D, Gottlieb A (2001) Efficacy and safety of infliximab monotherapy for plaque type psoriasis: a randomized trial. Lancet 357: 1842 – 1847 Chen DM, Gordon K, Leonardi C, Menter A (2004) Adalimumab efficacy and safety in patients with moderate to severe chronic plaque psoriasis: preliminary findings from a 12 week dose-ranging trial. J Am Acad poster P2 Ellis CN, Kreuger GG (2001) Treatment of chronic plaque psoriasis by selective targeting of memory effector T lymphocytes. N Engl J Med 345(4):248 – 255 Gelfand JM, Berlin J, Van Voorhees A, Margolis DJ (2003) Lymphoma rates are low but increased in patients with psoriasis: results from a population-based cohort study in the United Kingdom. Arch Dermatol 139:1425 – 1429 Gordon KB, Papp KA, Hamilton TK, Walicke PA, Dummer W, Li N, Bresnahan BW, Menter A (2003) Efalizumab for patients with moderate to severe plaque psoriasis: a randomized controlled trial. JAMA 290(23):3073 – 3080

Gottlieb AB (2003) Immunobiologicals for psoriasis: Using targeted immunotherapies as pathogenic probes in psoriasis. In: Weinstein GD, Gottlieb AB (eds) Therapy of moderate to severe psoriasis. Marcel Dekker, New York, pp 239 – 260 Gottlieb AB (2005) Psoriasis: emerging therapeutic strategies. Nat Rev Drug Discov 4:19 – 34 Gottlieb AB, Chamian F, Masud S, Cardinale I, Abello VM, Lowes MA, Chen F, Magliocco M, Kreuger JG (2005) TNF inhibition rapidly down-regulates multiple proinflammatory pathways in psoriasis plaques. J Immunol 175:2721 – 2729 Lebwohl M, Tyring SK, Hamilton TK, Toth D, Glazer S, Tawfik NH, Walicke P, Dummer W, Wang X, Garovoy MR, Pariser D (2003) A novel targeted T-cell modulator, efalizumab, for plaque psoriasis. N Engl J Med 349:2004 – 2013 Leonardi CL, Papp KA, Gordon KB, Menter A, Feldman SR, Caro I, Walicke P, Compton P, Gottlieb AB (2005) Extended efalizumab therapy improves chronic plaque psoriasis: results from a randomized phase III trial. J Am Acad Dermatol 52(3):425 – 433 Mehlis SL, Gordon KB (2003) The immunology of psoriasis and biologic immunotherapy. J Am Acad Dermatol 49(2 Suppl):44 – 50 Menter A, Gordon KB, Carey W, Hamilton T, Glazer S, Caro I, Li N, Gulliver W (2005) Efficacy and safety observed during 24 weeks of efalizumab therapy in patients with moderate to severe psoriasis. Arch Dermatol 141:31 – 38 Papp KA, Tyring S, Lahfa M, Prinz J, Griffiths CEM, Nakanishi AM, Zitkik R, van de Kerkhof PCM (2005) A global phase III randomized controlled trial of etanercept in psoriasis: safety, efficacy, and effect of dose reduction. Br J Dermatol 152:1304 – 1312 Schön MP, Boehncke W-H (2005) Psoriasis. N Engl J Med 352:1899 – 1912 Winterfield L, Menter A (2004) Psoriasis and its treatment with infliximab-mediated tumor necrosis factor [ blockade. Dermatol Clin 22:437 – 447

Chapter 10

Biologic Agents in Psoriatic Arthritis P. Mease

10.1 Introduction Psoriatic arthritis (PsA) is a chronic, progressive form of inflammatory arthritis that occurs in individuals with psoriasis. It affects at least 0.3 % of the population, although estimates of its prevalence vary widely, and is generally considered an autoimmune disease with unknown antigenic determinants. There currently is no predictive marker indicating which psoriasis patients will develop arthritis (Mease 2004). PsA often is classified as a subtype of spondyloarthropathy, due to shared HLA associations among those with spinal involvement, and characteristic inflammatory clinical and immunopathologic features (Kruithof et al. 2005). Although it is heterogeneous in presentation, this disease often results in significant functional impairment and reduced quality of life (Husted et al. 2001).

10.2 Classification and Epidemiology The first classification criteria for PsA were proposed in 1973 by Moll and Wright (1973) defining an inflammatory arthropathy in patients with psoriasis, usually with negative rheumatoid factor, and five distinct clinical subsets: (1) oligoarticular (< 5 tender and swollen joints) asymmetric arthritis, (2) polyarticular arthritis, (3) distal interphalangeal joint (DIP) predominant, (4) spondylitis predominant, and (5) arthritis mutilans. Although several classification criteria for PsA have been proposed since the initial Moll and Wright criteria (Helliwell and Taylor 2005; Taylor et al. 2004), the Classification of Psoriatic Arthritis study group (CASPAR) has conducted an authoritative international study and has developed new classification criteria for PsA, in

order to improve classification sensitivity and specificity, the results of which are soon to be published (Taylor et al. 2005; Taylor 2006) (Table 10.2, p. 106). Partly because of relatively recent recognition as a discrete disease entity, as well as the heterogeneity of clinical presentation, diagnostic confusion with other diseases in the differential diagnosis, and chance of subclinical arthritis not yet symptomatic in a patient with psoriasis (Offidani et al. 1998), it is likely that the disease has historically not always been accurately identified. With these limitations in mind, it is known that psoriasis affects approximately 2 – 3 % of the general population and the prevalence of PsA in psoriasis patients is reported as being from 6 % to 39 % (Leonard et al. 1978; Shbeeb et al. 2000). Telephone surveys recently conducted in Europe and in the US, respectively, suggest a prevalence of 30 % (Salonen 2003) and 11 % (Gelfand et al. 2005). This wide range is related to the heterogeneity of the populations studied, as well as to differing methods of ascertainment. In any case, it is clear that there is a distinctive inflammatory arthritis (with varied patterns of presentation) associated with psoriasis (Gladman et al. 2005).

10.3 Genetic Epidemiology The relative risk for PsA amongst first degree relatives is second only to ankylosing spondylitis amongst rheumatic diseases; thus the genetic association for PsA is strong (Moll and Wright 1973; Rahman and Elder 2005). Current research evidence points to a multifactorial pattern of inheritance (Rahman and Elder 2005) with a possible parent-of-origin effect (paternal) (Rahman et al. 1999). The concordance of PsA in identical twins is 30 – 40 % (Sege-Peterson and Winchester 1999).

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PsA is associated with human leukocyte antigen (HLA) class 1 alleles. Linkage with the short arm of chromosome 6 has been shown, demonstrating associations with HLA-B13, B-17, B-27, B-38, B-39, HLACw6, and HLA-DRB1*07 (Gladman et al. 1986, 2003). Further research into regions outside the MHC region is underway.

10.4 Immunopathology Several key cell types participate in the inflammatory and destructive process of PsA, including T cells, macrophages, and dendritic cells in the joints and their analogues in the skin. Numerous pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF[ ), IL-1, and IL-6 also serve important roles in the disease. TNF- [ has gained much recent attention, since biologic agents that block its activity are now available (see below). Studies of these agents re-confirm the central role of TNF- [ in the inflammation of PsA and psoriasis. TNF- [ is produced by macrophages, keratinocytes, mast cells, monocytes, dendritic cells, and activated T-cells. It upregulates nuclear transcription factors, including NfkB, resulting in enhanced expression of many molecules central to the inflammatory response, including other cytokines (e.g., IL-1, IL-6) and chemokines. In the joints, TNF- [ mediates other biological processes that can result in cartilage and bone damage, including: expression of metalloproteinases by fibroblasts and chondrocytes, maturation and activation of osteoclasts from monocytic stem cells, and angiogenesis. In relation to both the joints and the skin, TNF- [ induces the expression of endothelial, keratinocyte and dendritic cell surface adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and E-selectin (CD62E). In addition to stimulating pro-inflammatory cells and cytokines in the skin, a key role played by TNF- [ is promotion of keratinocyte hyperproliferation and survival, which is important in the psoriatic lesion (Krueger and Bowcock 2005; Mease 2004a, b; Veale et al. 2005). Several groups have, in the last several years, further elucidated the features which set PsA apart from RA at an immunohistochemical level. These include features such as increased decreased synovial lining cellularity, increased vascularity, increased numbers of polymorphonuclear leukocytes (PMNs), CD163+ macrophages

as well as upregulation of Toll-like receptors 2 and 4, several of which are features consistent with activation of the innate immune system and a possible role for microbial antigens (Baeten et al. 2005; De Rycke et al. 2005; Kruithof et al. 2005; Veale et al. 2005). Comparative synovial sampling of patients with RA and three forms of SpA: AS, PsA, and undifferentiated spondyloarthropathy, demonstrate a high degree of similarity in cellular composition, immunohistochemistry, and vascularity amongst the SpA subtypes and clear distinction from RA, further supporting the common pathophysiology amongst the SpA subtypes (Kruithof et al. 2005). Recently, a Viennese group has developed an animal model for psoriasis and PsA. Inducible epidermal deletion of the gene JunB and its functional companion cJun in adult mice led to the histologic and immunohistochemical hallmarks of psoriasis and arthritis. In humans, JunB is a component of the activator protein 1 (AP-1) transcription factor, localized in the psoriasis susceptibility region PSORS6, and has diminished expression in human psoriatic skin lesions. They further showed that development of arthritis, but not psoriasis, required the presence of T and B cells and signaling through tumor necrosis factor 1 (TNFR1). Their conclusion was that deletion, or at least diminishment, of JunB/AP-1 in keratinocytes induces chemokine/ cytokine expression, which in turn recruits PMNs and macrophages to the epidermis, leading to both skin and joint lesions (Zenz et al. 2005). A comprehensive discussion of the pathophysiologic features of PsA can be found in recent reviews (Krueger and Bowcock 2005; Mease 2003, 2004a, b; Mease and Antoni 2005; Ritchlin 2005; Veale et al. 2005).

10.5 Clinical Features PsA is characterized by varied clinical patterns (Gladman and Rahman 2001) with varying degrees of involvement of joints (both peripheral and spinal), enthesium, and skin (Gladman and Rahman 2001; Gladman et al. 2005; Helliwell and Wright 2000). Psoriasis usually develops before joint involvement by many years, although occasionally arthritis may precede the psoriasis. Nail lesions, which occur in 87 % of patients with PsA (Gladman et al. 1986), help distinguish PsA from RA (Eastmond and Wright 1979).

10.5 Clinical Features

a

Fig. 10.1. Psoriasis plaque

Skin involvement usually occurs as plaque psoriasis, the most common form of psoriasis (Fig. 10.1). This is characterized by thickened, erythematous, hyperkeratotic skin lesions. These scaly patches usually occur over extensor surfaces, such as the elbow and/or knees, and may coalesce to cover large parts of the body. Other forms of psoriasis, such as guttate, pustular, and erythrodermic, are less common (Langley et al. 2005; Menter 2004). The severity of the psoriasis is not predictive of the severity of PsA, but recent studies show there may be a correlation between psoriasis severity and the occurrence of PsA (Gelfand et al. 2005; Gladman 1998). Joint involvement often is asymmetric, with frequent inflammation of distal interphalangeal (DIP) as well as other joints. Other characteristic features include enthesitis, dactylitis, and spine inflammation, particularly in the sacroiliac joints. About 40 % have spine involvement (Gladman et al. 2005). Enthesitis involves inflammation at sites where tendons, ligaments, and joint capsule fibers insert into bone, such as the insertions of the Achilles tendon and plantar fascia, as well as ligaments around the rib cage and pelvis. Dactylitis, swelling of a whole digit, includes both joint synovitis as well as enthesitis of tendon and ligament attachments in the digit. Another less frequent clinical feature is iritis (Gladman et al. 2005; Gladman and Rahman 2001; Helliwell and Wright 2000). Serum tests for rheumatoid factor (RF) usually are negative in PsA, but may occasionally be mildly elevated, which is also true for antibody to cyclic citrullinated protein (CCP) (Taylor et al. 2005). Elevations in levels of acute phase reactants, such as erythrocyte sedimen-

b

Fig. 10.2. a PIP and DIP synovitis. b DIP synovitis and psoriatic nail changes.

tation rate (ESR) or C reactive protein (CRP), are variable. Radiographic features include: (1) joint space narrowing and erosions; (2) lytic changes (such as the pencil-in-cup change) that reflects gross bone and cartilage lysis; and (3) evidence of new bone formation, such as complete ankylosis of joints and juxta-articular osteitis (Gladman and Rahman 2001; Helliwell and Wright 2000; Ory et al. 2005; van der Heijde et al. 2005). Several of these features help distinguish PsA from RA. A significant proportion of patients with PsA experience functional impairment and reduced quality of life (Gladman et al. 2005; Mease 2003; Mease et al. 2005b, Mease and Menter 2006, Husted et al. 2001, Gladman and Mease 2006).

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d

Fig. 10.2. c Dactylitis and psoriatic nail changes. d Iritis

Fig. 10.3a–d. Radiographic features. a X-ray of distal interphalangeal joint in PsA shows classic pencil-in-cup change due to severe erosion of bone on both sides of joint. b Joint shows joint space narrowing and erosions, whilst along the shaft of the bone in a juxta-articular location there is periostitis. c Pelvis X-ray shows asymmetric involvement of the sacroiliac joints wherein the right SI joint (viewer’s left) is spared and the left SI joint shows significant narrowing, erosion, and periarticular sclerosis.

a

b

c

10.7 Treatment Table 10.1. Measures of PsA outcome (Gladman et al. 2004; Mease 2005) ) ACRa Response Criteria: 20 %, 50 %, 70 % (validated in RA, not PsA) – Tender and swollen joint score (modified for PsA to include DIP and CMC joints) – 3/5: patient global, physician global, patient pain, HAQ,b acute phase reactant (sed. rate/CRP) ) Psoriatic Arthritis Response Criteria (PsARC) (not validated) – Improvement in at least two of four criteria, including: ) Physician global assessment (0 – 5) ) Patient global assessment (0 – 5) ) Tender joint score (> 30 %) ) Swollen joint score (> 30 %) – Improvement in at least 1 of 2 joint scores – No worsening in any criteria ) DASc (exploratory, adapted from RA) ) Skin assessment (PASI,d Target Lesion, Static Global) ) QoL/function/disability indices (HAQ, SF-36, DLQIe) ) Radiographic (Modified Sharp, Modified van der Heijde/ Sharp, Modified Steinbrocker, Wassenberg/Rau) a b c d e

Fig. 10.3. d Lateral view of the spine in PsA patient with intervertebral ligament calcification and exuberant syndesmophytes

10.6 Outcome Measures For the most part, outcome measures have been adapted from similar measures used in assessment of RA and psoriasis. These are used both in longitudinal studies of the natural history of PsA, as well as in clinical trials. These measures have been shown to effectively assess peripheral joint and skin inflammation, function, quality of life, fatigue, and structural damage determined by X-ray, and distinguish treatment from placebo (Table 10.1). Approaches to assessment of enthesitis, dactylitis, and spine involvement are still in development. Studies performed by members of the Group for Research and Assessment of Psoriasis and Psoriatic

American College Rheumatology Health Assessment Questionnaire Disease Activity Score Psoriatic Arthritis Skill Index Dermatology Life Quality Index

Arthritis (GRAPPA), an international research consortia of rheumatologists and dermatologists, have shown that for the most part, performance characteristics for these measures have been good (Fransen et al. 2006; Husted et al. 1997, 1998; Singh et al. 2005). A detailed review of these measures is given elsewhere (Gladman et al. 2004; Mease et al. 2005c; Mease and van der Heijde et al. 2006). Several studies have documented the effectiveness of ultrasound (D’Agostino et al. 2003; De Simone et al. 2003; Kane et al. 1999; Klauser et al. 2005; Ory et al. 2005; Wakefield et al. 2005) and MRI (Baraliakos and Braun 2006; McGonagle et al. 1998; Ory et al. 2005) in detecting inflammation in the joints and enthesium of SpA patients, as well as the extent of structural damage. As these tools become more refined, they also will enhance our ability to assess the effectiveness of new therapies on the progression of joint damage in PsA.

10.7 Treatment Treatment of PsA depends upon the severity of the condition and the features involved. The approach to treat-

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ment utilizes principles of both RA and psoriasis treatment. Given the heterogeneous presentation in PsA, there are specific challenges in determining which medications are most useful for each individual. For example, some medications preferentially impact psoriasis features, but not joint problems and vice versa. In addition, the potency of the medication must match the severity of the disease and the probability of progressive damage. In a large cohort of patients followed by Gladman, predictors of progressive disease included lack of response to nonsteroidal anti-inflammatory drugs (NSAIDs), elevated acute phase reactants, polyarticular disease, erosions on X-ray, and disability (Gladman et al. 1995). Treatment for PsA, therefore, may range from topical agents (such as vitamin D or corticosteroid cream) or light therapy for mild skin lesions, NSAIDs, intraarticular corticosteroid injections, and physical therapy approaches for mild joint involvement, to traditional disease-modifying anti-rheumatic drugs (DMARDs) and/or biologics for moderate to severe arthritis features. Corticosteroids must be used cautiously in patients with PsA, because withdrawal may trigger a flare of psoriasis. Beyond the scope of this chapter, a full review of topical and light therapy of psoriasis and traditional anti-inflammatory and DMARD therapy of arthritis can be found in a number of recent articles (Lebwohl et al. 2005; Mease 2004; Mease and Antoni 2005; Mease 2004; Mease et al. 2005; Nash and Clegg 2005; Winterfield et al. 2005). Biologic agents are used increasingly by rheumatologists and dermatologists to treat moderate to severely affected patients who do not respond adequately to traditional DMARDs. These agents, such as the anti-TNF[ medications, have contributed greatly to our ability to improve joint and skin disease, to inhibit progression of structural joint damage, and to improve patients’ functional ability and quality of life. Physical therapy also is an important component of treatment that helps to maintain and/or improve joint mobility, muscle strength and range of motion. Occupational therapists focus on procedures and devices to aid hand and wrist function. Orthopedic procedures, including joint modification or replacement, can help with more severely damaged joints.

10.8 Biologic Agents The biologic agents currently approved for treatment of PsA, i.e., those targeting the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF- [ ), have produced dramatic improvements in arthritis, enthesitis, dactylitis and psoriatic skin lesions, as well as function, quality of life, and inhibition of structural damage as measured radiographically (Antoni et al. 2005a, b; Mease 2003, 2004; Mease and Antoni 2005; Mease et al. 2000, 2004, 2005; Mease and van der Heijde et al. 2006). Current anti-TNF- [ agents include etanercept, infliximab, and adalimumab. Some of the observed histological and immunohistochemical effects of TNF[ inhibition include reduction of: synovial lining layer thickness, vascularity, endothelial expression of avb3 and vascular cell adhesion molecule (VCAM)-1, sublayer expression of intercellular adhesion molecule (ICAM-1) and E-selectin, T-cell and macrophage infiltration, vascular endothelial growth factor (VEGF) expression, synovial flk-1 expression and reduction in osteoclast differentiation and in the numbers of osteoclast precursors (Mease 2004b). Studies of patients with RA show that many of those who have inadequate efficacy with one anti-TNF- [ medication may switch to another and attain a good response (Hansen et al. 2004; Haraoui et al. 2004). Preliminary evidence suggests this is true in PsA as well (van den Bosch et al. 2006). 10.8.1 Etanercept Etanercept is a soluble receptor TNF- [ antagonist currently approved for treatment of RA, JA, PsA, AS and psoriasis. It typically is given as 25 mg subcutaneously twice a week or 50 mg once a week. Approval of etanercept in PsA is based on two placebo-controlled trials. In a single-center trial, PsA patients (n = 60) were randomized to etanercept or to placebo. Nearly one-half (47 %) of these patients were receiving a concomitant stable dose of MTX (Mease et al. 2000). Patients had on average a 20-year history of psoriasis and a 15-year history of inflammatory arthritis. In the 3-month placebocontrolled phase of the study, 87 % of patients receiving etanercept and 23 % of patients receiving placebo improved according to the Psoriatic Arthritis Response Criteria (PsARC) (p < 0.0001). In addition, 73 % of

10.8 Biologic Agents

patients receiving etanercept and 13 % of patients receiving placebo improved according to the composite ACR 20 response criteria, which are a standard for assessing treatment response in rheumatoid arthritis (p = 0.015). Skin lesions also improved significantly. Randomization in this trial was stratified according to use of MTX, utilized by nearly half of the patients. Concomitant use of MTX did not affect the results, suggesting that etanercept can be effective as monotherapy or in combination with a traditional DMARD. In a larger, multicenter study of similar design (n = 205), patients received either etanercept or placebo. Concomitant MTX was used by 42 % of patients (Mease et al. 2004b). Fifty-nine percent of etanercept patients and 15 % of placebo patients achieved an ACR 20 response at 3 months (Fig. 10.4a). PsARC response was observed in 72 % and 31 %, respectively. The treatment group showed significant changes in all individual measures of the composite criteria and significant improvements in measures of quality of life (SF-36, DLQI) and function (HAQ). At baseline 66 patients in the etanercept group and 62 in the placebo group had plaque psoriasis that involved at least 3 % of their body surface area, and thus were eligible for PASI scoring. At week 24, 23 % of the etanercept group achieved a PASI 75 response as compared to 3 % in the placebo group (p = 0.001) (Fig. 10.4b); 47 % and 18 % achieved a PASI 50 response respectively (p < 0.001). Radiographs showed inhibition of disease progression, the first demonstration of such an effect in PsA. After 48 weeks,

Fig. 10.4. Phase 3 Trial of Etanercept in PsA (Mease 2004)

there was no worsening of Total Sharp Score (TSS) in the active treatment group: a significant benefit compared to placebo, where worsening was observed (Mease et al. 2004, 2004) (Fig. 10.4c). As in the previous trial, background treatment with MTX did not affect outcome. Two year extension data from this trial ha been reported and shows persistent efficacy and safety (Mease et al. 2006b). 10.8.2 Infliximab Infliximab, a chimeric monoclonal antibody, is currently approved for RA, Crohn’s disease, PsA, AS, and ulcerative colitis. For PsA, it typically is administered as an intravenous infusion every 8 weeks. Two placebocontrolled trials in PsA have been conducted: the Infliximab Multinational Psoriatic Arthritis Controlled Trial (IMPACT, n = 104) (Antoni et al. 2005a) and a phase III study, IMPACT II (n = 200) (Antoni et al. 2005b). In both studies, the drug was administered 5 mg/kg intravenously and background DMARD use was allowed, but not required. In IMPACT II, MTX was the only DMARD allowed. In both studies, about half of enrolled patients received treatment with concomitant MTX. This did not have an impact on efficacy. Within IMPACT, 69 % of inflimab patients and 8 % of placebo patients showed an ACR 20 response at 16 weeks (p < 0.001), and 78 % and 18 % showed a PsARC response (p < 0.001), respectively. Patients with a good or moderate European League Against Rheu-

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matism (EULAR) response were 85 % and 23 %, respectively (p < 0.001) (Antoni et al. 2005). Enthesitis and dactylitis improved significantly. Thirty-nine patients had a baseline PASI of 2.5. Of these, 67 % had at least a PASI 75 response compared to 0.05 % in the placebo group (p = 0.001). In an open-label, 1-year follow-up of this group, patients originally taking placebo quickly achieved similar results with equivalent efficacy (Antoni et al. 2005). Radiographic data of hands and feet (including the DIPs of the hands) were analyzed according to the modified van der Heijde-Sharp method. These showed no disease progression in both groups over 50 weeks. Due to the short duration of placebo treatment (14 weeks), no difference in the treatment groups could be shown over 1 year. However, the calculated annual progression rate was reduced, indicating that even delayed treatment with infliximab after 14 weeks inhibited radiographic progression of PsA (Antoni et al. 2004). Results from IMPACT II showed an ACR 20 in 58 % and 11 % (p < 0.001) (Fig. 10.5a), PsARC response in 77 % of infliximab patients and 27 % of placebo patients (p < 0.001), and PASI 75 in 65 % and 2 %, respectively (p = 0.001) 14 weeks (Fig. 10.5b) (Antoni et al. 2005). Median PASI improvement in ACR 20 responders was 87 %, and 74 % in ACR 20 non-responders, suggesting that skin response could be achieved even when joint response was not demonstrated (Mease et al. 2004a). As in the etanercept trial, measures of physical function and quality of life showed significant improvement. Presence of dactylitis and enthesitis (palpation of

Achilles tendon and plantar fascia insertions) was assessed at baseline and at 6 months and showed significant improvement in the treatment group. Radiographic data showed no evidence of disease progression in infliximab patients as compared to progression noted in placebo patients at week 24 (van der Heidje et al. 2005). When PsA-specific radiographic features, including pencil-in-cup deformities and gross osteolysis, were examined, there was no difference between the treatment groups, a finding also found in radiographic results of the other anti-TNF- [ agents. 10.8.3 Adalimumab Adalimumab is a human anti-TNF- [ monoclonal antibody. It is administered subcutaneously, 40 mg every other week. It currently is approved for use in RA and in PsA. It was first shown effective for PsA in an open trial of 15 patients (Ritchlin et al. 2004). In a larger placebo controlled trial (n = 313) in PsA, in which 50 % of patients were on background MTX, 58 % of the treatment group and 14 % of the placebo group (p < 0.001) showed an ACR 20 response (Fig. 10.6a) at 3 months, the primary endpoint. Initial response in skin was rapid (as early as 2 weeks) and significant. As in the studies of the other TNF- [ inhibitors, concomitant use of MTX did not have an effect on efficacy. At 6 months, PASI 50/ 75/90 responses were 75 %, 59 %, and 42 % in the adalimumab group and 12 %, 1 %, and 0 % in the placebo group, respectively (p < 0.001) (Fig. 10.6b) (Mease et al.

Fig. 10.5. Phase 3 Trial of Infliximab in Ps/PsA (Antoni et al. 2005)

10.9 Other Biologic Agents

Fig. 10.6. Phase 3 Trial of Adalimumab in PsA (Mease 2005)

2005). Significant improvements in measures of physical function, quality of life and fatigue were demonstrated in the treatment group. Radiographic progression of disease was significantly inhibited by adalimumab, again showing no progression of structural damage in the treatment group (Mease et al. 2005) (Fig. 10.6c). A second smaller phase III study confirmed efficacy in multiple PsA domains (Genovese et al. 2005). Several other anti-TNF- [ agents are in various phases of development for PsA, including a human monoclonal antibody for subcutaneous administration (golimumab), a PEG-ylated Fab fragment of a monoclonal antibody (certolizumab pegol), and even oral agents that have TNF- [ inhibiting capability.

10.9 Other Biologic Agents Several agents being developed for use in RA or psoriasis eventually will be assessed for use in PsA and other SpA. The common feature of these agents is their ability to target different key cells (cytokines) of the immune response and inflammatory processes. Agents currently being tested for efficacy in PsA include drugs that inhibit T cells by blocking the ’’second signal’’ of T-cell activation. The ’’first’’ signal is provided by the cognate interaction of antigen appropriately presented in the context of major histocompatibility complex molecules on the surface of antigen-presenting cells, with specific

T-cell receptors on the surface of T cells. There are several pairs of receptor/counter-receptors capable of providing stimulatory ’’second’’ signals. Several of these agents, described below, are the targets of immunomodulatory therapies for autoimmune diseases. 10.9.1 Alefacept Alefacept is a human fusion protein that blocks the interaction between leukocyte function associated antigen-3 (LFA-3) on antigen-presenting cells and CD2 on T cells. It is given as a weekly (15 mg) intramuscular injection and is currently approved for use in psoriasis (Krueger et al. 2002; Lebwohl et al. 2003). Treatment leads to a depletion of T cells, preferentially memory T cells, via interactions through the molecule’s Fc piece. The drug is given as a regimen of 12 weeks on and 12 weeks off, partly to allow recovery of CD4 counts, which must be monitored during therapy. Despite the transient depletion of CD4 cells, there has been no increased risk of infection in psoriasis trials (Krueger et al. 2002; Lebwohl et al. 2003). In a small (n = 11) open-label trial of this compound in PsA, more than half of patients showed ACR 20 responses. Synovial biopsies showed a decrease in CD4, CD8, and CD68 (macrophage) cells in the synovial lining (Kraan et al. 2002). A recent RCT (n = 185) to evaluate the efficacy and safety of alefacept in combination with MTX for patients with PsA (Mease et al. 2005) found this combi-

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nation provided significant clinical improvement in PsA. Patients given a 12-week course of alefacept and MTX had significantly greater response rates in both PsA (ACR 20) and psoriasis (PASI 50) than patients given placebo-MTX. There was incremental improvement in ACR 20 response during the treatment-free phase, indicating that response to alefacept continues even when the drug is not being administered (Mease et al. 2006a). 10.9.2 Efalizumab Efalizumab is a humanized antibody to the CD11 subunit of LFA-1 that inhibits the interaction of LFA-1 with its counter-receptors, including ICAM-1. It can interfere with the activation of T lymphocytes, as well as migration of cells from the circulation to sites of inflammation. It has been approved for treatment of psoriasis (Lebwohl et al. 2003). In a 12-week trial of patients with PsA, in which efalizumab was administered subcutaneously once a week, 28 % of patients achieved an ACR 20 response versus 19 % in the placebo group (p = 0.2717) (Papp et al. 2004). Because there was not statistically significant differentiation between the treatment group and placebo, this agent cannot

currently be recommended to control arthritis in a psoriasis patient. 10.9.3 Abatacept Abatacept, previously known as CTLA4-Ig, is a recombinant human fusion protein comprising the extracellular domain of human CTLA4 along with the Fc piece of a human IgG1 molecule. CTLA4 is a naturally occurring inhibitor molecule that binds to CD80 and CD86 on antigen-presenting cells, thereby inhibiting the ability of CD28 to bind to these molecules and provide an activating signal. It is administered intravenously once per month and is approved for use in RA (Kremer et al. 2003). A Phase II trial for psoriasis has been conducted (Abrams et al. 1999). It is anticipated that further assessment of this drug will be conducted for PsA and for psoriasis.

10.10 Other Potential Treatments A number of other agents are being tested or could potentially be tested in PsA, including a number of

Established inflammatory musculoskeletal disease (joint, spine, or entheseal) With 3 or more of the following 1. Psoriasis

(a) Current†

Psoriatic skin or scalp disease present today as judged by a qualified health professional

(b) History

A history of psoriasis that may be obtained from patient, or qualified health professional

(c) Family history

A history of psoriasis in a first or second degree relative according to patient report

2. Nail changes

Typical psoriatic nail dystrophy including onycholysis, pitting and hyperkeratosis observed on current physical examination

3. A negative test for RF

By any method except latex but preferably by ELISA or nephlemetry, according to the local laboratory reference range

4. Dactylitis

(a) Current

Swelling of an entire digit

(b) History

A history of dactylitis recorded by a qualified health professional

5. Radiological evidence of juxtaarticular new bone formation

Ill-defined ossification near joint margins (but excluding osteophyte formation) on plain xrays of hand or foot

Table 10.2: Diagnostic criteria for PsA (CASPAR‡)*

‡Classification of Psoriatic Arthritis *The CASPAR criteria have specificity of 98.7% and sensitivity of 91.4%. †Current psoriasis is assigned a score of 2; all other features are assigned a score of 1.

10.12 Conclusion

cytokine inhibitors. A pilot trial of an IL-15 inhibitor in PsA has shown efficacy (McInnes and Gracie 2004). A recent trial to assess the efficacy and safety of an IL-1 antagonist, anakinra, in PsA did not show adequate efficacy (Gibb et al. 2006). A humanized antibody to the -subunit (CD25) of the IL-2 receptor that blocks IL-2 binding to the T-cell receptor has been tried in psoriasis, albeit with some loss of efficacy noted over time (Jung et al. 2005; Krueger et al. 2000). A monoclonal antibody to the IL-6 receptor (MRA) is in phase III development for the treatment of RA, having shown benefit in phase II and will likely be tested in PsA (Nishimoto et al. 2004). Several inhibitors of IL-12 are undergoing active evaluation in psoriasis, with good early success (Krueger et al. 2006), and will be likely assessed in PsA, as well. Similarly, it is anticipated that inhibitors of IL-18 will be studied. Conversely, some cytokines may have anti-inflammatory effects and thus their administration may be therapeutic. A recombinant IL-10 agent has been studied in psoriasis, with demonstration of preliminary benefit (Reich et al. 2001). However, a controlled study with human IL-10 in patients with PsA (McInnes et al. 2001) showed benefit in the skin but not in joints. Similarly, a recombinant human IL-11 has been utilized in psoriasis, with preliminary benefit noted clinically and histologically (Trepicchio et al. 1999). huOKT3 * 1, a monoclonal antibody to CD3, a component of the T-cell receptor complex, has demonstrated some benefit in PsA, although issues such as transient T-cell depletion and mild cytokine release symptoms have been noted (Utset et al. 2002). Pioglitazone is a ligand for PPAR * that is administered orally. It originally was developed to treat diabetes. It has been considered as a potential therapy for inflammatory autoimmune disease, such as PsA, due to observations that it led to a marked inhibition of angiogenesis and down-regulation of proinflammatory cytokines (Mease 2004). In an uncontrolled trial of pioglitazone treatment, 60 % of patients met the PsARC criteria and 50 % achieved an ACR 20 response after 12 weeks (Bongartz et al. 2005). This agent may be beneficial for treating PsA, but its efficacy must be evaluated in a well-controlled study.

10.11 Cost-Effectiveness Analysis Acquisition costs of biologic agents exceed that of older anti-rheumatic therapies. Therefore, appropriate pharmacoeconomic assessment must take into consideration the costs of the therapies themselves, but also the costs of the disease, in terms of work disability and the interference with the ability to perform activities of daily living. Highly effective therapies may be shown to be cost-effective if they are able to prevent some of the cost to the individual and society of a worsening of rheumatic disease. Several recent studies have demonstrated the potential cost-effectiveness of anti-TNF- [ agents in PsA (Bansback et al. 2004; Guh et al. 2005; Marra 2005).

10.12 Conclusion Numerous studies have increased our understanding of the basic pathophysiology of PsA, providing support for the clinical effects of targeted therapy, e.g., inhibition of TNF- [ . The consequent emerging treatments for PsA have demonstrated significant benefit for clinical signs and symptoms, inhibition of joint damage as assessed by radiographic progression, and improved quality of life and functional status. There are fewer studies documenting these benefits with traditional DMARDs, whose effectiveness does not seem as great as TNF- [ inhibitors. Targeted therapies that inhibit pro-inflammatory cytokines, such as the TNF- [ inhibitors, have proven highly effective in managing joint, enthesial and skin disease. Agents that block the cell– cell interactions required to activate T cells are effective in the skin and may benefit the joints, as well. Observation of the effectiveness of these agents has helped elucidate the pathogenesis of PsA and psoriasis which, in turn, may lead to more novel and effective interventions. Development of these targeted therapies has also increased interest in the accurate diagnosis and classification of PsA, which would facilitate the institution of appropriate therapy in a timely fashion. Significant efforts are underway to further develop and validate outcome measures that accurately assess the effect of therapies and determine the natural history of these diseases. This effort, along with the development of evi-

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dence-based treatment guidelines and general educational initiatives, is being led by international research consortia such GRAPPA and other groups. The benefits of biologic agents must be weighed against their cost: patient improvement and inhibition of disease progression on one hand, versus allocating limited resources on the other. Comprehensive health economic analyses are being developed to aid our ability to see the full impact of these more effective treatments on patient function, productivity, and quality of life in the context of society as a whole.

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function in patients with psoriatic arthritis. J Immunol 167:4075 – 4082 Mease PJ (2003) Psoriatic arthritis/psoriasis. In: Smolen J, Lipsky PE (eds) Targeted therapies in rheumatology. Martin Dunitz, London, pp 525 – 548 Mease PJ (2004a) Recent advances in the management of psoriatic arthritis. Curr Opin Rheumatol 16:366 – 370 Mease P (2004b) TNFalpha therapy in psoriatic arthritis and psoriasis. Ann Rheum Dis 63:755 – 758 Mease P (2004c) Targeting therapy in psoriatic arthritis. Drug Discovery Today 1:389 – 396 Mease P, Antoni C (2005) Psoriatic arthritis treatment: biological response modifiers. Ann Rheum Dis 64(Suppl 2):ii78–ii82 Mease PJ, Goffe BS (2005) Diagnosis and treatment of psoriatic arthritis. J Am Acad Dermatol 52:1 – 19 Mease P, van der Heidje D (2006) Joint damage in psoriatic arthritis: How is it assessed and can be prevented? International J Advances in Rheumatol 4:38 – 48 Mease PJ, Goffe BS, et al. (2000) Etanercept in the treatment of psoriatic arthritis and psoriasis: a randomised trial. Lancet 356:385 – 390 Mease P, Kavanaugh A, et al. (2004a) Infliximab improves psoriasis regardless of arthritis response in patients with active psoriatic arthritis: results from IMPACT 2 Trial [abstract]. Arthritis Rheum 50(Suppl 9):S616 Mease P, Kivitz A, et al. (2004b) Etanercept treatment of psoriatic arthritis: safety, efficacy, and effect on disease progression. Arthritis Rheum 50:2264 – 2272 Mease P, Ruderman EM, et al. (2004c) Etanercept in psoriatic arthritis: sustained improvement in joint and skin disease and inhibition of radiographic progression at 2 years [abstract]. Ann Rheum Dis 63(Suppl 1):99 Mease P, Antoni C, et al. (2005a) Psoriatic arthritis assessment tools in clinical trials. Ann Rheum Dis 64(Suppl 2):ii49–ii54 Mease P, Gladman D, et al. (2005b) Efficacy of alefacept in combination with methotrexate in the treatment of psoriatic arthritis [abstract]. Ann Rheum Dis 64(Suppl 3):324 Mease P, Gladman D, et al. (2005c) Alefacept in combination with methotrexate for the treatment of psoriatic arthritis. Arthritis Rheum 52 (Suppl 9):S280 Mease P, Gladman D, et al. (2005d) Adalimumab in the treatment of patients with moderately to severely active psoriatic arthritis: results of ADEPT. Arthritis Rheum 58:3279 – 3289 Mease P, Gladman D, et al. (2006a) Alefacept (Amevive®) in combination with methotrexate for the treatment of psoriatic arthritis: results for a randomized, double-blind, placebocontrolled study. Arth Rheum 54:1638 – 1645 Mease P, Kivitz AJ, et al. (2006b) Continued inhibition of radiographic progression in patients with psoriatic arthritis following 2 years of treatment with etanercept. J Rheumatol 33:712 – 721 Mease P and Menter A (2006c) Quality of life issues in psoriasis and psoriatic arthritis: Outcome measures and therapies from a dermatological perspective. Journal of American Academy of Dermatol 54:685 – 704 Menter A and Mease P (2004) The pathogenesis and treatment of psoriasis. In: Schiff M (eds) Rheumatology Educational Initiative (REDI): making a difference in rheumatology. Early and aggressive treatment = better outcomes for

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Singh A, Mease P, et al. (2005) Health Assessment Questionnaire has similar psychometric properties in psoriatic arthritis and rheumatoid arthritis. Arthritis Rheum 52 (Suppl 9):S402 Taylor W, Gladman D, et al. (2006) Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 54(8):2665 – 2673 Taylor W, Marchesoni A, et al. (2004) A comparison of the performance characteristics of classification criteria for the diagnosis of psoriatic arthritis. Semin Arthritis Rheum 34: 575 – 584 Taylor WS, Helliwell PS, Gladman DD, Mease P, Mielants M, Marchesoni A (2005) A validation of current classification criteria for the diagnosis of psoriatic arthritis – preliminary results of the CASPAR study. Ann Rheum Dis 64:107 Trepicchio WL, Ozawa M, et al. (1999) Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions. J Clin Invest 104:1527 – 1537 Utset TO, Auger JA, et al. (2002) Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial. J Rheumatol 29: 1907 – 1913 van den Bosch F, Reece R, et al. (2006) Adalimumab (Humira®) is effective and safe in treating psoriatic arthritis (PsA) in real-life clinical practice: Preliminary results of the STEREO trial. Arthritis Rheum 54(Suppl 9):S719 – S720 van der Heidje D, Gladman D, et al. (2005a) Infliximab inhibits progression of radiographic damage in patients with active psoriatic arthritis: 54 week results from IMPACT 2. Arthritis Rheum 52 (Suppl 9):S281 van der Heijde D, Kavanaugh A, et al. (2005b) Infliximab inhibits progression of radiographic damage in patients with active psoriatic damage in patients with active psoriatic arthritis: Results from IMPACT 2 trial. Ann Rheum Dis 64(Suppl 3):109 van der Heijde D, Sharp J, et al. (2005c) Psoriatic arthritis imaging: a review of scoring methods. Ann Rheum Dis 64(Suppl 2):ii61–ii64 Veale D, Ritchlin C, et al. (2005) Immunopathology of psoriasis and psoriatic arthritis. Ann Rheum Dis 65(Suppl 2):ii26– ii29 Wakefield RJ, Balint PV, et al. (2005) Musculoskeletal ultrasound including definitions for ultrasonographic pathology. J Rheumatol 32:2485 – 2487 Winterfield L, Menter A, et al. (2005) Psoriasis treatment: current and emerging directed therapies. Ann Rheum Dis 64(Suppl 64):ii87–ii90 Zenz R, Eferl R, et al. (2005) Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins. Nature 437:369 – 375

Chapter 11

Biologic Therapies for Rheumatoid Arthritis Targeting TNF-␣ and IL-1 P.C. Taylor

11.1 Introduction ’Biologics’ are protein-based drugs derived from living organisms and are designed to either inhibit or augment a specific component of the immune system. Examples include antibodies directed against very specific molecular components of the immune response, for example, pro-inflammatory cytokines, or naturally occurring cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra). The primary cause of rheumatoid arthritis remains unknown. Nonetheless, advances in molecular technology have facilitated identification of numerous novel therapeutic targets, including cytokines, cell subsets, and other molecules, such as those involved in signalling pathways, that contribute to the inflammatory and destructive components of rheumatoid arthritis. Concurrent advances in biotechnology made it possible to produce abundant high-quality chimerized mousehuman or even completely human monoclonal antibodies with specificity for relevant disease molecules. Other approaches to blocking pro-inflammatory molecules include the use of naturally occurring soluble receptors or inhibitory proteins.

11.2 Biologic Therapies Targeting TNF-␣ At the present time, biologics represent the only available class of specific TNF inhibitors available for clinical practice. Three drugs are currently approved. These are infliximab (Remicade), a chimeric monoclonal anti-TNF- [ antibody comprising a human IgG-1κ antibody with a mouse SV fragment of high affinity and neutralizing capacity; adalimumab (Humira), a mono-

clonal human antibody produced by phage display; and etanercept (Enbrel), an engineered p75 TNF receptor dimer with a fully human amino acid sequence linked to the Fc portion of human IgG-1. The monoclonal antibodies have specificity for TNF- [ . Binding assays using radioactively labelled TNF- [ demonstrate that antibodies such as infliximab bind both monomeric (inactive) and trimeric (biologically active) forms of soluble TNF- [ (Scallon et al. 2002). In contrast, the fusion protein etanercept acts as a competitive inhibitor of TNF- [ and can also bind lymphotoxin (TNF- q ). Etanercept forms relatively unstable complexes with TNF- [ , allowing dissociation and the potential to form a reservoir for binding TNF- [ (Scallon et al. 2002). Aside from the three currently approved biologic TNF inhibitors for rheumatoid arthritis, others are in development, including certolizumab pegol (formerly known as CDP-870, now Cimzia), a pegylated Fab fragment which can be produced in Escherichia coli (Hazleman et al. 2000). 11.2.1 Rationale for TNF Blockade in the Treatment of Rheumatoid Arthritis The predicted clinical success of anti-TNF therapy was based on several experimental observations. The first of these was the expression of TNF- [ and its receptors in rheumatoid arthritis synovial tissue (Maini and Taylor 2000). Secondly, evidence from in vitro experiments employing dissociated synovial cell cultures pointed to TNF- [ as a regulator of many other pro-inflammatory cytokines (Brennan et al. 1989; Butler et al. 1995; Haworth et al. 1991). Thirdly, a number of independent in vivo studies demonstrated that blockade of bioactive TNF in murine collagen-induced arthritis can ameliorate clinical symptoms and prevent joint destruction in

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11 Biologic Therapies for Rheumatoid Arthritis Targeting TNF-␣ and IL-1

established disease (Williams et al. 2000a). Finally, in a murine model, over-expression of a human TNF- [ transgene modified at its three prime ends to prevent degradation of its MRNA is associated with the development of a destructive form of polyarthritis 4 – 6 weeks after birth. This can be prevented by administration of a monoclonal antibody with specificity for human TNF (Keffer et al. 1991). An extensive range of pro-inflammatory cytokines can be detected at the protein level in human synovial samples, regardless of differences in donor disease duration, severity, or even drug therapy, and has been confirmed in studies from a number of laboratories. These findings imply that there is prolonged cytokine expression in the rheumatoid joint, contrasting with the transient production induced by mitogenic stimulation. This hypothesis was confirmed following the observation that pro-inflammatory cytokines are produced over several days in dissociated rheumatoid arthritis synovial membrane cell cultures in the absence of extrinsic stimulation (Buchan et al. 1988). This finding suggested the presence of one or more soluble factors regulating prolonged cytokine synthesis within the rheumatoid synovial membrane cultures. These dissociated cell cultures comprise a heterogeneous population of cells producing numerous cytokine and other non-cytokine molecular messengers. A key observation using this cell culture system was that addition of anti-TNF antibodies strikingly reduced the production of other pro-inflammatory cytokines including IL-1, GM-CSF, IL-6, and IL-8 (Brennan et al. 1989). Furthermore, using the same rheumatoid arthritis synovial cell culture system, blockade of IL-1 by means of the IL-1 receptor antagonists results in reduced IL-6 and IL-8 production but not that of TNF- [ (Butler et al. 1995). These observations led to the formulation of the hypothesis that TNF- [ occupies a dominant position at the apex of a pro-inflammatory cytokine network. At the time of these experimental findings, in the early 1990s, TNF- [ had also been described as a pleiotropic cytokine with biological properties that included enhanced synovial proliferation, production of prostaglandins and metalloproteinases (Dayer et al. 1985), as well as regulation of other pro-inflammatory cytokines. For all these reasons, TNF- [ was considered to represent a potential therapeutic target in rheumatoid arthritis. The first data in vivo to support the hypothesis that TNF- [ is a good therapeutic target for inflammatory

arthritis came from studies of murine collageninduced arthritis. Monoclonal anti-TNF antibodies or soluble TNF receptor-Fc fusion proteins, administered either during the induction phase of arthritis or, more importantly, in the established phase of disease after the onset of symptoms, were able to ameliorate clinical features and significantly inhibit joint destruction (Williams et al. 1992; Piguet et al. 1992). Further unequivocal validation of TNF- [ as a therapeutic target came following the administration of biologic agents to patients with rheumatoid arthritis. 11.2.2 Clinical Studies of Anti-TNF Therapy Data from numerous clinical trials with the anti-TNF agents infliximab, etanercept, and adalimumab have confirmed the validity of TNF- [ as a therapeutic target in rheumatoid arthritis. Proof of principle for TNF- [ blockade was initially established in an open-label study in which infliximab was administered intravenously in divided doses over 2 weeks (either 10 mg per kilogram a fortnight apart, or four doses of 5 mg per kilogram every 4 days). The results clearly demonstrated that these relatively large doses of antibody were tolerated without any immediate adverse reaction (Elliott et al. 1993). Although these early studies were not primarily designed to test efficacy, a remarkable reduction in pain, stiffness, swelling, and joint tenderness was observed within 24 h, with maximum benefit at around 2 – 4 weeks, and sustained for the entire 8-week duration of the study in the majority of patients. Relief of fatigue within hours of the infusion was consistently reported. Subsequent studies in both the early and established phases of disease confirm that long-term, repeated use of anti-TNF agents results in sustained improvement in symptoms and signs of disease in the majority of patients (Kremer et al. 2003; Maini et al. 2004; Lipsky et al. 2000; Klareskog et al. 2004; St Clair et al. 2004; Breedveld et al. 2004, 2006; Emery 2005; Bathon et al. 2000; Genovese et al. 2002). In studies of rheumatoid populations having failed to respond adequately after exposure to multiple DMARD therapies, with active disease despite ongoing methotrexate therapy, between 50 % and 70 % of patients are reported to achieve an ACR 20 response level at 6 months, as compared to between 20 % and 30 % of patients treated with methotrexate alone (Weinblatt et al. 1999; Maini et al. 1999; Keystone et al. 2004).

11.2 Biologic Therapies Targeting TNF-␣

However, it is important to note that caution should be applied when comparing differences between proportions of responders or magnitude of response between studies, as the nature of the study populations is very different with respect to baseline disease activity and rate of structural damage to joints. At the more stringent 50 % ACR response level, the difference between the proportion of patients with established disease responding on methotrexate alone and those responding on the combination of methotrexate and a TNF inhibitor is approximately 20 – 40 % at 1 year and, similarly, at the ACR 70 % response level, approximately 15 % of the study population (Weinblatt et al. 1999; Maini et al. 1999; Keystone et al. 2004). Most TNF inhibitors have been evaluated using a step-up clinical trial design in patients with active disease despite methotrexate therapy, who then received either the TNF inhibitor or placebo. In many of these trials, however, a biologic monotherapy arm was absent. Nonetheless, in a study of patients with an inadequate response to methotrexate, infliximab was administered at a dose of 1, 3, or 10 mg per kilogram, with or without a fixed low dose of methotrexate, while control groups were treated with placebo infusions and methotrexate (Maini et al. 1998). In this trial, the duration of response to repeated biologic administration was inversely related to the production of human antichimeric antibodies to the infliximab. Higher doses of infliximab were found to be less immunogenic than a low dose of 1 mg per kilogram, and concomitant methotrexate administration further reduced the occurrence of human anti-chimeric antibody responses. Although reduced immunogenicity of the biologic agent administered may be one mechanism whereby methotrexate has an additive or synergistic benefit when used in combination with TNF inhibitors, it is likely that there are other effects beyond this mediated through complementary mechanism of action, although these have yet to be fully elucidated. In the TEMPO study (Trial of Etanercept and Methotrexate with radiographic Patient Outcomes), the combination of methotrexate with etanercept was clearly superior to that of either methotrexate or etanercept alone (Klareskog et al. 2004). Similarly, in the PREMIER study (prospective, multi-centre, randomized, double-blind, active comparator-controlled, parallel groups study comparing the fully human monoclonal anti-TNF- [ antibody D2E7 given every 2nd week with methotrexate given weekly, and the combination of

D2E7 and methotrexate administered over 2 years in patients with early rheumatoid arthritis), the combination of adalimumab and methotrexate consistently demonstrated superior efficacy in every area over either methotrexate or adalimumab as monotherapies (Breedveld et al. 2006). In this study, at week 52, 43 % of patients on combination therapy achieved remission by DAS 28 criteria of less than 2.6 as compared with 23 % of patients receiving adalimumab alone and 21 % of patients receiving methotrexate alone. In the ASPIRE study of infliximab in early rheumatoid arthritis, 17 % of patients receiving 6 mg per kilogram of infliximab together with methotrexate achieved an ACR 70 response for six or more consecutive months and 12 % of patients receiving infliximab at 3 mg per kilogram together with methotrexate. This contrasted with under 8 % of patients on methotrexate alone (St Clair et al. 2004). Fifteen percent of patients on methotrexate alone achieved remission criteria by DAS 28 at week 54, whereas the addition of infliximab at 3 mg per kilogram enhanced this figure to 21 %, and further to 31 % at a dose of 6 mg per kilogram. One of the early studies to look at biomarkers after anti-TNF therapy demonstrated dose-dependent reductions in serum concentrations of pro-matrix metalloproteinase 1 and pro-matrix metalloproteinase 3 (Brennan et al. 1997). These reductions in mediators of cartilage degradation predicted that TNF blockade might have the benefit of significant inhibition of radiographic damage to joints. That this was indeed the case was confirmed in the 54-week analyses from the ATTRACT study (Lipsky et al. 2000). In this trial, infliximab, at doses of 3 mg per kilogram or 10 mg per kilogram, given every 4 or 8 weeks, when added to therapeutic doses of methotrexate was found to give sustained reduction in symptoms and signs of disease that were significantly greater than the reduction associated with methotrexate alone. In patients treated with methotrexate as a monotherapy, joint space narrowing and erosions progressed as anticipated. In contrast, therapy with infliximab plus methotrexate prevented the progressive joint damage characteristic of rheumatoid arthritis and even resulted in improvement in the radiographic score from baseline in a significant percentage of patients (Lipsky et al. 2000). Combination therapy halted progression of joint damage not only with limited radiographic destruction at baseline, but also in those with extensive damage. Similar findings have been reported for etanercept.

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When administered together with methotrexate, the combination was significantly better in inhibition of radiographic progression compared with methotrexate or etanercept alone, in a population of patients who were methotrexate-naive at study entry (Klareskog et al. 2004). Very similar findings have been reported for adalimumab given in combination with methotrexate in the established phase of disease (Keystone et al. 2004). There is also good evidence that antibodies targeting TNF significantly inhibit progression of structural damage in the early phase of disease (St Clair et al. 2004; Breedveld et al. 2006). These observations confirm the pathogenic role of TNF- [ in the early phase of rheumatoid arthritis and suggest that in the proportion of patients with high likelihood of radiographic progression, early intervention with a TNF inhibitor together with methotrexate may provide long-term benefits in terms of functional capability by preserving joint integrity (Breedveld et al. 2004). Surprisingly, significant inhibition of radiographic damage has even been reported in those patients failing to achieve a clinical response at the ACR 20 level (Smolen et al. 2005). This fascinating observation in effect defines a subpopulation in whom there is a dissociation between inflammatory and destructive pathways. Changes in functional status are assessed by means of the HAQ score. It has been determined that an improvement from baseline of 0.3 in HAQ score represents a clinically meaningful change. There is data for all three currently available anti-TNF inhibitors to show that repeated therapy over time, in particular when an anti-TNF agent is combined with methotrexate, leads to significant improvement in physical function and quality of life (Klareskog et al. 2004; Keystone et al. 2004; Maini et al. 2004). Preservation or improvement of function is a particularly important goal of therapy in the early phases of disease. In the ASPIRE study, 60 % of patients treated with methotrexate as a monotherapy achieved an improvement in HAQ exceeding 0.3, whereas 71 % of all patients receiving infliximab together with methotrexate achieved an improvement of this magnitude (St Clair et al. 2004). Similarly, in the PREMIER study, patients receiving adalimumab and methotrexate achieved a 1.1 improvement from baseline in HAQ score at the end of 1 year, as compared with a 0.8 improvement in patients treated with methotrexate alone. With adalimumab alone, the improvement was

0.9 (Breedveld et al. 2006). In the light of these findings, it is not a surprise, but nonetheless very welcome, to see emerging data indicating that in early disease, treatment with infliximab and methotrexate gives a higher probability of maintaining employment than treatment with methotrexate alone (Smolen et al. 2006). These data would predict a lowering of the indirect costs associated with rheumatoid arthritis, such as disability benefits, as well as reduced direct costs of patient care over time, such as those associated with joint arthroplasty; but as yet, there are few long-term data that can reliably offset the expense of anti-TNF therapies themselves against the anticipated long-term savings. 11.2.3 Safety of Biologic TNF Inhibitors In rheumatoid arthritis and many other chronic inflammatory diseases, many cellular and molecular processes contribute towards an immunological disequilibrium, in which normal homeostatic processes are unable to restore a healthy state and prevent the perpetuation of inflammatory processes. The success of TNF- [ blockade in therapy suggests that this molecule occupies such a critical position in the pathogenic process. However, blockade of TNF or any other key physiological molecules may have the downside of negating their beneficial role in generating protective immune responses. For TNF- [ antagonists, the key safety considerations include infection, both common and opportunistic, cytopenias, demyelinating disease, lupus-like syndromes, congestive heart failure and malignancies, particularly lymphomas. 11.2.4 Infectious Complications One of the most important and common safety concerns for the use of TNF inhibitors is the occurrence of infectious complications. In clinical trials, the rate of upper respiratory tract infections occurring in patients receiving TNF inhibitors, usually with concomitant methotrexate, is higher than that in patients receiving placebo injections or infusions together with methotrexate. However, the rate of serious infections has been consistently comparable between the groups receiving placebo and TNF inhibitor. Nonetheless, one of the most frequently occurring serious infections in the early days of anti-TNF treatment was Mycobacterium


tuberculosis, with extrapulmonary disease in about a third of cases. The incidence of complicating tuberculosis early in the history of infliximab therapy was approximately one in a thousand cases. However, the rate has substantially fallen since the introduction of screening programmes. Tuberculosis has been reported as a complication of all biologic anti-TNF agents but with a varying prevalence, depending on the country in which the drug was used. Occurrence of complicating tuberculosis is strongly influenced by age, low socio-economic status, and geography. The background rate in the population is important, as the majority of cases of tuberculosis occurring following exposure to anti-TNF agents are thought to represent reactivation of latent TB. This may be because TNF blockade is a particularly effective way of breaking down granuloma walls. Where tuberculosis has been reported, the median time of onset in patients receiving etanercept is approximately 11 months, whereas 97 % of patients treated with infliximab and having TB reported developed the complication within 7 months (Keane et al. 2001). Screening for and treating latent tuberculosis infection will prevent reactivation in most patients. Latent tuberculous infectious screening should include a careful history including history of BCG vaccination, tuberculin skin test, and a chest radiograph. Skin testing is problematic, however, because of the occurrence of anergy in rheumatoid arthritis and high rate of false negative tuberculin skin tests as a consequence. Furthermore, for those inexperienced in skin testing, it is not uncommon to place injections or read the results inappropriately and therefore training is required. Because of the difficulties in interpreting tuberculin skin testing, there is now much interest in a newer generation of ELISPOT tests, more sensitive, more specific, and more convenient than tuberculin skin tests. This test requires a single blood sample for the detection of interferon * -secreting T cells, with reactivity to peptides highly specific for latent Mycobacterium tuberculosis infection (Lalvani et al. 2001). However, the ELISPOT test is not yet widely available because it is costly and requires isolation of mononuclear cells, a procedure that is not performed in routine clinical laboratories. Where latent tuberculosis is diagnosed or strongly suspected, prophylactic treatment should be offered in accordance with local guidelines and advice; for example, isoniazid for 9 months (Keane et al. 2005). Although screening has greatly reduced the occurrence

of Mycobacterium tuberculosis with TNF antagonists, it has not completely eliminated it and there must always be a high degree of awareness for this and other granulomatous diseases. A number of opportunistic infections have been reported with TNF inhibitors, both in the context of clinical trials and in adverse events reporting after drug approval. Although such infections are relatively rare, the most frequently occurring include histoplasmosis, Pneumocystis carinii, listeriosis and aspergillosis. The occurrence of these infections varies according to geographical location. Because of the potential for infectious complications with TNF inhibitors, this class of drug is relatively contra-indicated in patients with chronic infectious states such as bronchiectasis or chronic sinusitis. 11.2.5 Congestive Cardiac Failure Anti-TNF inhibitors must be used with great caution or avoided altogether in patients with a history of congestive cardiac failure. Although there have been theoretical arguments to support the potential benefits of TNF inhibition in this condition, clinical studies with both etanercept and infliximab failed to demonstrate any benefit. Furthermore, in two clinical trials of etanercept in 2,000 patients with moderate to severe cardiac failure (New York Heart Classification functional class 2 – 4), the findings suggested the possibility of increased mortality in patients receiving etanercept, in particular at a dose regime of three times weekly. Infliximab has also been studied in a small group of patients with New York Heart Classification functional class 3 and 4 over a period of 1 year. In this study, there was an increased rate of hospitalization and mortality in patients receiving infliximab at a dose of 10 mg per kilogram at baseline, week 2, and week 6 (Keystone 2003). In a post-drug approval surveillance study, 47 patients treated with either etanercept or infliximab were identified as having congestive cardiac failure. Of these, 38 had new-onset disease and nine had exacerbations of prior disease (Kwon et al. 2003). There were no identifiable risk factors in half the patients with new onset heart failure, 29 of whom were treated with etanercept and 18 treated with infliximab. The onset of congestive cardiac failure ranged from 2 h after treatment administration to 2 years, with a median time of

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3½ months. Ten patients under the age of 50 developed new-onset congestive cardiac failure and three of these had pre-existing risk factors. The condition improved or resolved in nine of these ten patients after discontinuation of the TNF inhibitor and institution of therapy for congestive cardiac failure. 11.2.6 Solid Tumours and Lymphoma Just as any powerful immunosuppressive agent raises concerns regarding infectious complications, similarly there are concerns that it may diminish immunosurveillance of abnormal cells and thus raise the risk of malignancy. However, the available data for all antiTNF agents is reassuring and suggests that the observed occurrence of solid tumours in patients that have been exposed to TNF inhibitors is no different to that expected on the basis of frequencies reported in age-, sex-, and race-matched subjects from the surveillance, epidemiology, and end results database (SEER database of the National Cancer Institute of the USA). Lymphomas of both Hodgkin’s and non-Hodgkin’s type have been reported as occurring in patients after exposure to all of the available TNF inhibitors, but it is difficult to ascribe an unequivocal causal link to the therapy. This is because rheumatoid arthritis itself is a risk factor for lymphoma, with a risk that correlates with the activity and severity of disease (Baecklund et al. 1998). In one study, the odds ratio for development of lymphoma was 25.8 for rheumatoid patients with high inflammatory activity, compared with low inflammatory activity. The standardized incidence rates observed for lymphomas occurring with the use of TNF inhibitors in the context of clinical trials are within the range expected based on other publications for standardized incidence rates in rheumatoid arthritis itself. The contributions of other anti-rheumatic drugs to this risk, including azathioprine and methotrexate, are not clear. The majority of lymphomas observed with the use of TNF inhibitors are of non-Hodgkin’s type, with a mean time to onset of between 10 and 21 months. In conclusion, there is a higher rate of lymphoma occurrence in rheumatoid patients than there is in healthy age- and sex-matched controls. Lymphomas have been reported in patients treated with anti-TNF therapies, but it remains unclear whether the medication is a contributory factor or causal or simply indicative of a patient population that is likely to have had

more severe inflammatory disease activity prior to exposure to an anti-TNF drug (Keystone 2003). 11.2.7 Other Toxicity Issues New cases of central nervous system demyelination and exacerbations of pre-existing disease have been reported in patients with rheumatoid arthritis following exposure to TNF inhibitors. However, as in the case of lymphoma, any causal relationship remains controversial and unproven. In fact, TNF- [ has been implicated in the pathogenesis of multiple sclerosis, and because of this there have been studies of TNF inhibitors in this disease. In a Phase I open-label study, two patients with rapidly progressing multiple sclerosis refractory to high-dose intravenous corticosteroids were treated with intravenous infliximab. Both patients were found to have an increase in the number of gadolinium-enhancing lesions on MRI, together with increases in CSF leucocyte counts after each infusion. However, these changes were not accompanied by neurological deterioration (van Oosten et al. 1996). In a Phase II multicentre study, 168 patients with relapsingremitting and secondary progressive multiple sclerosis received monthly infusions of lenercept (a recombinant soluble TNF p55 receptor fusion protein) at three dose levels or placebo for up to 48 weeks. Although this treatment was not associated with an increase in the number of new lesions, on MRI scanning it was sufficiently associated with increased demyelination attack frequency but not duration or severity (Klinkert et al. 1997). Neurological events associated with demyelinating lesions on MRI have been reported in patients treated with anti-TNF agents (Mohan et al. 2001). However, the rate of new cases of multiple sclerosis reported in post-marketing surveillance for infliximab, etanercept, and adalimumab is at or below the number of expected cases based on the background rate for society as a whole, which is approximately six new cases per 100,000 per year (Noseworthy et al. 2000). Although there is not a proven relationship between TNF inhibition and onset of a demyelinating episode, it is wisest to avoid TNF inhibitors in patients with a history of optic neuritis, transverse myelitis, or other form of CNS demyelination. In clinical trial data, induction of anti-nuclear antibodies and antibodies to double-stranded DNA have been reported with higher frequency in patients receiv-


ing TNF inhibitors than in comparator groups not receiving these drugs. Although induction of antibodies to double-stranded DNA is relatively common, in the order of 10 – 15 % of patients, the occurrence of clinical features of SLE is much less common. Sulfasalazine, widely used in the treatment of rheumatoid arthritis, is also associated with a similar pattern of autoantibody induction. In reported cases of clinical lupus following exposure to TNF inhibitors, the onset of symptoms occurs between 1½ and 18 months after introduction of the anti-TNF agent, with a mean time of 4.4 months, and women are predominantly affected. The syndrome abates on cessation of the TNF inhibitor and introduction of corticosteroid therapy as appropriate (Mohan et al. 2002). 11.2.8 Injection Site Reactions or Infusion-Related Reactions Safety issues involving the anti-TNF agents as a class include the risk of injection site reactions or infusionrelated reactions. These are usually mild, however, and most often easily managed. For example, in the case of the self-administered subcutaneous delivery of etanercept, injection site reactions were reported in 37 % of etanercept-treated patients vs. 10 % of controls in placebo-controlled trials. These reactions are generally mild to moderate, occur sporadically in a minority of injections over time, and do not necessitate the discontinuation of the agent. Similarly, in the case of adalimumab, which is also given by self-administered subcutaneous injection, injection site reactions are the most commonly reported adverse event, occurring in 19.5 % of treated patients vs. 11.6 % of controls. Reactions may take the form of erythema, itching, haemorrhage, pain, or swelling at the injection site, although such events very rarely merit the discontinuation of the therapy. In the case of infliximab, infusion-related reactions may occur within 1 – 2 h of the infusion itself. In clinical trial data, 22 % of infliximab-treated patients experienced an infusion reaction compared with 9 % of controls. However, it is also the case from clinical trial data that less than 5 % of infliximab infusions are associated with infusion reactions, compared with 2 % of placebo infusions. Fewer than 1 % of infliximab-treated patients develop serious infusion reactions and it is rarely necessary to discontinue therapy. Most infusion-related reactions are mild and non-specific and may simply require slowing of the infusion rate and administration

of paracetamol and/or an antihistamine. Similarly, symptomatic injection site reactions with etanercept or adalimumab can be managed with local warm compresses and oral antihistamines if required. 11.2.9 Mechanism of Action of TNF Blockade A number of mechanisms of action of TNF inhibitors have been identified to date. These include de-activation of the pro-inflammatory cytokine cascade at the site of inflammation; reduction in mediators of joint destruction; diminished recruitment of inflammatory cells from the blood to the rheumatoid joint and diminished synovial vascularity. A role in modulation of apoptosis remains controversial. The first formal proof that TNF- [ regulates other pro-inflammatory cytokines in vivo was the observation that there is a rapid reduction in serum IL-6 concentrations, closely followed by falling serum CRP, following administration of infliximab (Elliott et al. 1994; Charles et al. 1999). Although IL-1 concentrations are often below the limit of detection in the peripheral blood of rheumatoid arthritis patients, where it is detectable, down-regulation has been reported in a proportion of patients (Lorenz et al. 1996). Similarly, in a small study of repeat synovial biopsies obtained before and 2 weeks after a single infusion of 10 mg per kilogram of infliximab, computerized image analysis of sections stained for cytokine-producing cells demonstrated a reduction in synovial IL-1 [ and IL-1 q in a subgroup (Ulfgren et al. 2000). It is clear that the benefits of anti-TNF therapy are not mediated by upregulation of endogenous pro-inflammatory cytokine inhibitors, since circulating IL-1ra and soluble TNF receptor levels fall after infliximab infusion (Charles et al. 1999). It is thought that a major mechanism of action of TNF inhibitors is likely to be mediated by modulation of inflammatory cell traffic. A dose-dependent rise in peripheral blood lymphocyte counts is observed following infliximab infusion, with a maximum rise within 24 h of treatment (Paleolog et al. 1996). This is mediated by modulation of both arms of the inflammatory cell recruitment cascade. Thus there is reduced histological expression of synovial cytokine-induced vascular adhesion molecules, such as E-selectin and VCAM1, following anti-TNF treatment (Tak et al. 1996) and a significant dose-dependent reduction in soluble serum E-selectin and ICAM-1 concentrations (Paleolog et al.

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1996), as well as significantly diminished immunohistological expression of the chemokines IL-8 and MCP1, with a trend to reduction in a number of other chemokines (Taylor et al. 2000). Further indirect evidence to suggest TNF blockade reduces inflammatory cell recruitment to the joint is based on the observation that infliximab therapy is associated with a reduction in numbers of synovial tissue macrophages and lymphocytes (Tak et al. 1996; Taylor et al. 2000). However, the definitive confirmation that TNF- [ blockade reduces leucocyte traffic to inflamed joints was obtained in an open-label clinical trial demonstrating a 40 – 50 % decrease in retention of autologous indium-111 labelled granulocytes in the hands, wrists, and knees 2 weeks after infliximab treatment (Taylor et al. 2000). There is a reduction in the marginating granulocyte pool after infliximab treatment, an observation that would normally be associated with a rise in peripheral blood granulocyte counts (Taylor et al. 1999). However, in contrast to peripheral blood lymphocyte counts, numbers of peripheral blood granulocytes decrease after infliximab with maximal changes within 24 h. The reason for this is that myeloid cell production is reduced secondary to downregulation of GM-CSF as a consequence of TNF blockade. Because of the short circulating half-life of the granulocyte, of approximately 8 h, a diminished rate of cell production dominates the peripheral blood picture. One factor contributing to the very rapid reduction in joint swelling observed by both patients and physicians after anti-TNF therapy is likely to be a reduction in tissue oedema and capillary leak, mediated by vascular endothelial growth factor (VEGF), a cytokine implicated in new blood vessel formation and found to be elevated in the serum of rheumatoid arthritis patients (Paleolog et al. 1998). Serum concentrations of VEGF show a dose-dependent reduction following infliximab infusions, but without normalization. There is also reduction in synovial vascular density and in particular a reduction in angiogenesis, as assessed by diminished expression of vessels expressed the [ V q 3 integrin (Taylor 2005). Further evidence for a reduction in synovial vascularity following TNF blockade is the observation that the vascular signal on quantitative power Doppler imaging is significantly reduced following infliximab therapy (Taylor et al. 2004, 2006). Relatively early in the history of clinical trials of TNF blockade, marked reductions in circulating concentra-

tions of the precursors of the matrix metalloproteinase enzymes MMP-1 and MMP-3 were reported (Brennan et al. 1997) as well as a significant reduction in synovial tissue expression of matrix metalloproteinases (Catrina et al. 2002). Similarly, serum levels of osteoprotegerin (OPG) and soluble receptor activator of nuclear factor κB ligands (sRANKL), both of which are elevated in rheumatoid compared to normal sera, are normalized following infliximab therapy without influencing the OPG:sRankL ratio (Ziolkowska et al. 2002). These observations predicted the disease-modifying capability of anti-TNF inhibitors, which is now established. One hypothesis for the failure of the p75 TNF receptor fusion protein etanercept to give clinical benefits in trials of Crohn’s disease, in contrast to the marked benefits demonstrated with monoclonal antibodies to TNF- [ , is that the antibodies may cause an increase in apoptosis of lamina propria and peripheral T cells through caspase-dependent mechanisms (Van den Brande et al. 2003). However, this topic remains controversial and the relevance of modulation of apoptosis as the mechanism of action of TNF inhibitors in rheumatoid arthritis is unclear. In one study, decreased synovial cellularity was reported as early as 48 h after infliximab administration, but with no corresponding evidence of apoptosis (Smeets et al. 2003). In another study, however, apoptosis in synovial macrophages was reported to be induced by both etanercept and infliximab, with a corresponding increase in active caspase-3 expression. No increase in lymphocyte apoptosis was observed, however (Catrina et al. 2005). The relevance of these interesting observations to the mode of action of TNF inhibitors in rheumatoid arthritis is not clear.

11.3 Targeting IL-1 Studies in animal models of arthritis have demonstrated the therapeutic potential of IL-1 blockade (van den Berg 2000). The dominance of IL-1 q over IL-1 [ in the pathogenesis of collagen-induced arthritis has been demonstrated in studies of cytokine blockade (Joosten et al. 1996; Williams et al. 2000b) and confirmed by the finding that IL-1 q gene knock-out mice show markedly reduced levels of inflammation following immunization with type II collagen. The use of genetically modified mice has also helped to confirm the physiological significance of IL-1ra as deletion of this gene in BALB/c

11.4 Combination Anti-cytokine Therapies

mice results in the spontaneous development of arthritis (Horai et al. 2000). Proof of principle for IL-1 blockade in rheumatoid arthritis has been established using once-daily, subcutaneously administered IL-1 receptor antagonist (IL1ra: anakinra), a naturally occurring inhibitor of IL-1 (Bresnihan et al. 1998). In a phase II placebo-controlled study, 472 patients received daily subcutaneous injections of placebo or one of three different doses of human IL-1ra; 30 mg, 75 mg, or 150 mg. Improvements were observed in all individual clinical parameters, including swollen and tender joint counts, pain score, duration of early morning stiffness, patient assessment of disease activity, and investigator assessment of disease activity, although no clear dose-response relationship was observed. At the end of the study period, significantly more patients on the higher dosage schedule achieved improvement at the ACR 20 % response level than placebo-treated patients. There were also significant reductions in ESR in all active treatment groups. However, the overall magnitude of clinical responses and changes in acute phase reactants were relatively modest, at 20 – 35 % from baseline, as compared with those reported for TNF- [ blockade. These observations do not necessarily imply that IL-1 is not a good target for therapy in rheumatoid arthritis, but may reflect pharmacokinetic challenges for IL-1ra as a means to achieve IL-1 blockade. For example, the kidneys excrete IL-1ra rapidly and therapeutic levels persist for a few hours only. Furthermore, IL-1 receptors are ubiquitously expressed and have a rapid turnover. Nonetheless, daily administration of human IL-1ra is reported to have the benefit of disease modification, with a reduction in the rate of radiographic damage in patients receiving active drug as compared with those on placebo. However, the reduction only reached statistical significance in patients receiving the two lower doses. The efficacy and safety of anakinra in combination with methotrexate has been tested in a multi-centre randomized double-blind placebo-controlled trial (Cohen et al. 2002). In this study, patients with moderate to severely active rheumatoid arthritis despite methotrexate therapy for six consecutive months, with stable doses for more than 3 months, were randomized to receive either single daily placebo injections or one of five different doses of anakinra. At week 12, the ACR 20 responses in the five active treatment plus methotrexate groups demonstrated a statistically significant

dose-response relationship over that in the placebo plus methotrexate group, and these responses were enduring through 24 weeks. The combination of anakinra and methotrexate was safe and well tolerated. Although IL-1 blockade using anakinra in combination with methotrexate has been shown to be clinically superior to methotrexate therapy alone, the disappointing magnitude of clinical efficacy as compared with that seen with TNF inhibitors has prompted the exploration of alternative strategies for exploring IL-1. These include the use of monoclonal antibodies with specificity for IL-1 q , and the IL-1 trap, an engineered protein comprising the two high-affinity signalling chains of the cell surface IL-1 receptor, linked by the Fc portion of IgG-1. Preliminary results presented at meetings demonstrate efficacy for the IL-1 trap at the higher dose tested.

11.4 Combination Anti-cytokine Therapies The widespread use of conventional DMARDs in combination with an apparent increase in efficacy without raising significant regards concerning toxicity or tolerability has prompted the investigation of combination anti-cytokine therapy (Genovese et al. 2004). The potential attractions of this approach include superior immunomodulation and hence enhanced efficacy. However, in a 24-week randomized controlled trial conducted in 242 patients with rheumatoid arthritis who had not previously been treated with biologic agents and were taking background methotrexate, the combination of etanercept 25 mg twice weekly together with anakinra 100 mg once daily resulted in an incidence of serious infection of 7 % and the occurrence of neutropenia in the combination group. The incidence of both infection and neutropenia was higher in the combination group than in the Enbrel alone group and higher than the rate observed in studies using anakinra alone. Furthermore, there was no therapeutic benefit of the combination treatment over etanercept alone. For this reason, the concomitant use of IL-1 blockade and TNF inhibitors is not recommended (Genovese et al. 2004).

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11.5 Conclusions The anti-TNF agents have been demonstrated to provide a marked improvement in outcomes in a proportion of patients with rheumatoid arthritis. Furthermore, they are well tolerated. Injection site reactions or infusion-related reactions are relatively common but easily managed and rarely lead to discontinuation of therapy. These drugs do lead to an increased incidence of upper respiratory tract infections and more rarely to serious opportunistic or tuberculous infections. With appropriate screening, some infections can be prevented and those that do occur generally respond to appropriate medical treatment. However, it is important to emphasize the value of screening, education, and monitoring of patients. Overall, the anti-TNF biologics have demonstrated a risk-benefit profile that markedly favours an overall benefit. IL-1 inhibitors have been less successful in the clinic but nonetheless clinical trials have validated IL-1 as a target for therapy in rheumatoid arthritis. New pharmacological approaches to IL-1 blockade are under investigation but current evidence strongly contraindicates dual blockade of IL-1 and TNF on the basis of no observed additional benefit and a considerable increase in risk of infective complications.

References Baecklund E, Ekbom A, Sparen P, Feltelius N, Klareskog L (1998) Disease activity and risk of lymphoma in patients with rheumatoid arthritis: nested case-control study. BMJ 317(7152):180 – 181 Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff MH, Keystone EC, Genovese MC, Wasko MC, Moreland LW, Weaver AL, Markenson J, Finck BK (2000) A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. N Engl J Med 343(22):1586 – 1593. Erratum in: N Engl J Med 2001 344(3):240. N Engl J Med 2001 344(1):76 Breedveld FC, Emery P, Keystone E, Patel K, Furst DE, Kalden JR, St Clair EW, Weisman M, Smolen J, Lipsky PE, Maini RN (2004) Infliximab in active early rheumatoid arthritis. Ann Rheum Dis 63:149 – 155 Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pavelka K, van Vollenhoven R, Sharp J, Perez JL, Spencer-Green GT (2006) The PREMIER study: A multicenter, randomized, double-blind clinical trial of combination therapy with adalimumab plus methotrexate versus methotrexate alone or adalimumab alone in patients with early, aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis Rheum 54(1):26 – 37

Brennan FM, Chantry D, Jackson A, Maini R, Feldmann M (1989) Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis. Lancet 2(8657):244 – 247 Brennan FM, Browne KA, Green PA, Jaspar JM, Maini RN, Feldmann M (1997) Reduction of serum matrix metalloproteinase 1 and matrix metalloproteinase 3 in rheumatoid arthritis patients following anti-tumour necrosis factoralpha (cA2) therapy. Br J Rheumatol 36(6):643 – 650 Bresnihan B, Alvaro-Gracia JM, Cobby M, Doherty M, Domljan Z, Emery P, Nuki G, Pavelka K, Rau R, Rozman B, Watt I, Williams B, Aitchison R, McCabe D, Musikic P (1998) Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum 41(12): 2196 – 2204 Buchan G, Barrett K, Turner M, Chantry D, Maini RN, Feldmann M (1988) Interleukin-1 and tumour necrosis factor mRNA expression in rheumatoid arthritis: prolonged production of IL-1 alpha. Clin Exp Immunol 73(3):449 – 455 Butler DM, Maini RN, Feldmann M, Brennan FM (1995) Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures. Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist. Eur Cytokine Netw 6(4):225 – 230 Catrina AI, Lampa J, Ernestam S, af Klint E, Bratt J, Klareskog L, Ulfgren AK (2002) Anti-tumour necrosis factor (TNF)alpha therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis. Rheumatology (Oxford) 41(5):484 – 489 Catrina AI, Trollmo C, af Klint E, Engstrom M, Lampa J, Hermansson Y, Klareskog L, Ulfgren AK (2005) Evidence that anti-tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages, but not lymphocytes, in rheumatoid arthritis joints: extended report. Arthritis Rheum 52(1):61 – 72 Charles P, Elliott MJ, Davis D, Potter A, Kalden JR, Antoni C, Breedveld FC, Smolen JS, Eberl G, deWoody K, Feldmann M, Maini RN (1999) Regulation of cytokines, cytokine inhibitors, and acute-phase proteins following anti-TNF-alpha therapy in rheumatoid arthritis. J Immunol 163(3):1521 – 1528 Cohen S, Hurd E, Cush J, Schiff M, Weinblatt ME, Moreland LW, Kremer J, Bear MB, Rich WJ, McCabe D (2002) Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial. Arthritis Rheum 46(3):614 – 624 Dayer JM, Beutler B, Cerami A (1985) Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 162(6):2163 – 2168 Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Katsikis P, Brennan FM, Walker J, Bijl H, Ghrayeb J, et al. (1993) Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha. Arthritis Rheum 36(12):1681 – 1690 Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Bijl H, Woody JN (1994) Repeated therapy with monoclonal

References antibody to tumour necrosis factor alpha (cA2) in patients with rheumatoid arthritis. Lancet 344(8930):1125 – 1127 Emery P (2005) Adalimumab therapy: clinical findings and implications for integration into clinical guidelines for rheumatoid arthritis. Drugs Today (Barc) 41(3):155 – 163 Genovese MC, Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff MH, Keystone EC, Wasko MC, Moreland LW, Weaver AL, Markenson J, Cannon GW, Spencer-Green G, Finck BK (2002) Etanercept versus methotrexate in patients with early rheumatoid arthritis: two-year radiographic and clinical outcomes. Arthritis Rheum 46(6):1443 – 1450 Genovese MC, Cohen S, Moreland L, Lium D, Robbins S, Newmark R, Bekker P (2004) Combination therapy with etanercept and anakinra in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully with methotrexate. Arthritis Rheum 50(5):1412 – 1419 Haworth C, Brennan FM, Chantry D, Turner M, Maini RN, Feldmann M (1991) Expression of granulocyte-macrophage colony-stimulating factor in rheumatoid arthritis: regulation by tumor necrosis factor-alpha. Eur J Immunol 21(10):2575 – 2579 Hazleman B, Smith M, Moss K, Lisi L, Scott D, Sopwith M, Choy E, Isenberg D (2000) Efficacy of a novel pegylated humanised anti-TNF fragment (CDP870) in patients with rheumatoid arthritis. Rheumatology 39 Abstracts Suppl 1 (abstract 158, p. 87) Horai R, Saijo S, Tanioka H, Nakae S, Sudo K, Okahara A, Ikuse T, Asano M, Iwakura Y (2000) Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J Exp Med 191(2):313 – 320 Joosten LA, Helsen MM, van de Loo FA, van den Berg WB (1996) Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF alpha, anti-IL-1 alpha/beta, and IL-1Ra. Arthritis Rheum 39(5):797 – 809 Keane J (2005) TNF-blocking agents and tuberculosis: new drugs illuminate an old topic. Rheumatology (Oxford) 44(6):714 – 720 Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, Schwieterman WD, Siegel JN, Braun MM (2001) Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 345(15):1098 – 1104 Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, Kollias G (1991) Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 10(13):4025 – 4031 Keystone EC (2003) Advances in targeted therapy: safety of biological agents. Ann Rheum Dis 62 Suppl 2:ii34 – 36 Keystone EC, Kavanaugh AF, Sharp JT, Tannenbaum H, Hua Y, Teoh LS, Fischkoff SA, Chartash EK (2004) Radiographic, clinical, and functional outcomes of treatment with adalimumab (a human anti-tumor necrosis factor monoclonal antibody) in patients with active rheumatoid arthritis receiving concomitant methotrexate therapy: a randomized, placebo-controlled, 52-week trial. Arthritis Rheum 50(5): 1400 – 1411 Klareskog L, van der Heijde D, de Jager JP, Gough A, Kalden J, Malaise M, Martin Mola E, Pavelka K, Sany J, Settas L, Waj-

dula J, Pedersen R, Fatenejad S, Sanda M; TEMPO (Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes) study investigators. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial. Lancet 363(9410): 675 – 681 Klinkert WE, Kojima K, Lesslauer W, Rinner W, Lassmann H, Wekerle H (1997) TNF-alpha receptor fusion protein prevents experimental auto-immune encephalomyelitis and demyelination in Lewis rats: an overview. J Neuroimmunol 72(2):163 – 168 Kremer JM, Weinblatt ME, Bankhurst AD, Bulpitt KJ, Fleischmann RM, Jackson CG, Atkins KM, Feng A, Burge DJ (2003) Etanercept added to background methotrexate therapy in patients with rheumatoid arthritis: continued observations. Arthritis Rheum 48(6):1493 – 1499 Kwon HJ, Cote TR, Cuffe MS, Kramer JM, Braun MM (2003) Case reports of heart failure after therapy with a tumor necrosis factor antagonist. Ann Intern Med 138(10):807 – 811 Lalvani A, Nagvenkar P, Udwadia Z, Pathan AA, Wilkinson KA, Shastri JS, Ewer K, Hill AV, Mehta A, Rodrigues C (2001) Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J Infect Dis 183(3):469 – 477 Lipsky PE, van der Heijde DM, St Clair EW, Furst DE, Breedveld FC, Kalden JR, Smolen JS, Weisman M, Emery P, Feldmann M, Harriman GR, Maini RN; Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group (2000) Infliximab and methotrexate in the treatment of rheumatoid arthritis. N Engl J Med 343(22): 1594 – 1602 Lorenz HM, Antoni C, Valerius T, Repp R, Grunke M, Schwerdtner N, Nusslein H, Woody J, Kalden JR, Manger B (1996) In vivo blockade of TNF-alpha by intravenous infusion of a chimeric monoclonal TNF-alpha antibody in patients with rheumatoid arthritis. Short term cellular and molecular effects. J Immunol 156(4):1646 – 1653 Maini RN, Taylor PC (2000) Anti-cytokine therapy in rheumatoid arthritis. Ann Rev Med 51:207 – 229 Maini RN, Breedveld FC, Kalden JR, Smolen JS, Davis D, Macfarlane JD, Antoni C, Leeb B, Elliott MJ, Woody JN, Schaible TF, Feldmann M (1998) Therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor alpha monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis. Arthritis Rheum 41(9):1552 – 1563 Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, Smolen J, Emery P, Harriman G, Feldmann M, Lipsky P (1999) Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. ATTRACT Study Group. Lancet 354(9194):1932 – 1939 Maini RN, Breedveld FC, Kalden JR, Smolen JS, Furst D, Weisman MH, St Clair EW, Keenan GF, van der Heijde D, Marsters PA, Lipsky PE; Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study

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11 Biologic Therapies for Rheumatoid Arthritis Targeting TNF-␣ and IL-1 Group (2004) Sustained improvement over two years in physical function, structural damage, and signs and symptoms among patients with rheumatoid arthritis treated with infliximab and methotrexate. Arthritis Rheum 50(4):1051 – 1065 Mohan N, Edwards ET, Cupps TR, Oliverio PJ, Sandberg G, Crayton H, Richert JR, Siegel JN (2001) Demyelination occurring during anti-tumor necrosis factor alpha therapy for inflammatory arthritides. Arthritis Rheum 44(12):2862 – 2869 Mohan AK, Edwards ET, Cote TR, Siegel JN, Braun MM (2002) Drug-induced systemic lupus erythematosus and TNFalpha blockers. Lancet 360(9333):646 Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG (2000) Multiple sclerosis. N Engl J Med 343(13):938 – 952 Paleolog EM, Hunt M, Elliott MJ, Feldmann M, Maini RN, Woody JN (1996) Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor alpha antibody in rheumatoid arthritis. Arthritis Rheum 39(7):1082 – 1091 Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M, Maini RN (1998) Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor alpha and interleukin-1 in rheumatoid arthritis. Arthritis Rheum 41(7):1258 – 1265 Piguet PF, Grau GE, Vesin C, Loetscher H, Gentz R, Lesslauer W (1992) Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor. Immunology 77(4):510 – 514 Scallon B, Cai A, Solowski N, Rosenberg A, Song XY, Shealy D, Wagner C (2002) Binding and functional comparisons of two types of tumor necrosis factor antagonists. J Pharmacol Exp Ther 301(2):418 – 426 Smeets TJ, Kraan MC, van Loon ME, Tak PP (2003) Tumor necrosis factor alpha blockade reduces the synovial cell infiltrate early after initiation of treatment, but apparently not by induction of apoptosis in synovial tissue. Arthritis Rheum 48(8):2155 – 2162 Smolen JS, Han C, Bala M, Maini RN, Kalden JR, van der Heijde D, Breedveld FC, Furst DE, Lipsky PE; ATTRACT Study Group (2005) Evidence of radiographic benefit of treatment with infliximab plus methotrexate in rheumatoid arthritis patients who had no clinical improvement: a detailed subanalysis of data from the anti-tumor necrosis factor trial in rheumatoid arthritis with concomitant therapy study. Arthritis Rheum 52(4):1020 – 1030 Smolen JS, Han C, van der Heijde D, Emery P, Bathon JM, Keystone E, Kalden JR, Schiff M, Bala M, Baker D, Han J, Maini RN, St Clair EW (2006) Infliximab treatment maintains employability in patients with early rheumatoid arthritis. Arthritis Rheum 54(3):716 – 722 St Clair EW, van der Heijde DM, Smolen JS, Maini RN, Bathon JM, Emery P, Keystone E, Schiff M, Kalden JR, Wang B, Dewoody K, Weiss R, Baker D; Active-Controlled Study of Patients Receiving Infliximab for the Treatment of Rheumatoid Arthritis of Early Onset Study Group (2004) Combination of infliximab and methotrexate therapy for early rheumatoid arthritis: a randomized, controlled trial. Arthritis Rheum 50(11):3432 – 3443

Tak PP, Taylor PC, Breedveld FC, Smeets TJ, Daha MR, Kluin PM, Meinders AE, Maini RN (1996) Decrease in cellularity and expression of adhesion molecules by anti-tumor necrosis factor alpha monoclonal antibody treatment in patients with rheumatoid arthritis. Arthritis Rheum 39(7):1077 – 1081 Taylor PC (2005) Serum vascular markers and vascular imaging in assessment of rheumatoid arthritis disease activity and response to therapy. Rheumatology (Oxford) 44(6): 721 – 728 Taylor PC, Peters AM, Glass DM, Maini RN (1999) Effects of treatment of rheumatoid arthritis patients with an antibody against tumour necrosis factor alpha on reticuloendothelial and intrapulmonary granulocyte traffic. Clin Sci (Lond) 97(1):85 – 89 Taylor PC, Peters AM, Paleolog E, Chapman PT, Elliott MJ, McCloskey R, Feldmann M, Maini RN (2000) Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis. Arthritis Rheum 43(1):38 – 47 Taylor PC, Steuer A, Gruber J, Cosgrove DO, Blomley MJ, Marsters PA, Wagner CL, McClinton C, Maini RN (2004) Comparison of ultrasonographic assessment of synovitis and joint vascularity with radiographic evaluation in a randomized, placebo-controlled study of infliximab therapy in early rheumatoid arthritis. Arthritis Rheum 50(4):1107 – 1116 Taylor PC, Steuer A, Gruber J, McClinton C, Cosgrove DO, Blomley MJ, Marsters PA, Wagner CL, Maini RN (2006) Ultrasonographic and radiographic results from a two-year controlled trial of immediate or one-year-delayed addition of infliximab to ongoing methotrexate therapy in patients with erosive early rheumatoid arthritis. Arthritis Rheum 54(1):47 – 53 Ulfgren AK, Andersson U, Engstrom M, Klareskog L, Maini RN, Taylor PC (2000) Systemic anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha synthesis. Arthritis Rheum 43(11):2391 – 2396 van den Berg WB (2000) Arguments for interleukin 1 as a target in chronic arthritis. Ann Rheum Dis 59 Suppl 1:i81 – 4. Review Van den Brande JM, Braat H, van den Brink GR, Versteeg HH, Bauer CA, Hoedemaeker I, van Montfrans C, Hommes DW, Peppelenbosch MP, van Deventer SJ (2003) Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease. Gastroenterology 124(7):1774 – 1785 van Oosten BW, Barkhof F, Truyen L, Boringa JB, Bertelsmann FW, von Blomberg BM, Woody JN, Hartung HP, Polman CH (1996) Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal antitumor necrosis factor antibody cA2. Neurology 47(6):1531 – 1534 Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleischmann RM, Fox RI, Jackson CG, Lange M, Burge DJ (1999) A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. N Engl J Med 340(4): 253 – 259

References Williams RO, Feldmann M, Maini RN (1992) Anti-tumor necrosis factor ameliorates joint disease in murine collageninduced arthritis. Proc Natl Acad Sci U S A 89(20):9784 – 9788 Williams RO, Feldmann M, Maini RN (2000a) Cartilage destruction and bone erosion in arthritis: the role of tumour necrosis factor alpha. Ann Rheum Dis 59 Suppl 1:i75 – 80 Williams RO, Marinova-Mutafchieva L, Feldmann M, Maini RN (2000b) Evaluation of TNF-alpha and IL-1 blockade in collagen-induced arthritis and comparison with combined

anti-TNF-alpha/anti-CD4 therapy. J Immunol 165(12): 7240 – 7245 Ziolkowska M, Kurowska M, Radzikowska A, Luszczykiewicz G, Wiland P, Dziewczopolski W, Filipowicz-Sosnowska A, Pazdur J, Szechinski J, Kowalczewski J, Rell-Bakalarska M, Maslinski W (2002) High levels of osteoprotegerin and soluble receptor activator of nuclear factor kappa B ligand in serum of rheumatoid arthritis patients and their normalization after anti-tumor necrosis factor alpha treatment. Arthritis Rheum 46(7):1744 – 1753

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Chapter 12

12 Biologics in Crohn’s Disease and Ulcerative Colitis: Focus on Tumor Necrosis Factor Antagonists J. Salfeld, P. Rutgeerts

Elucidation of the cellular and molecular mediators of tissue injury in Crohn’s disease (CD) and ulcerative colitis (UC) has expanded the potential management options for these diseases. The discovery of immunologic and inflammatory mediators, in particular, has paved the way for clinical research with biologic agents in this area. Although conventional treatments remain viable options for some patients, those who are intolerant to these agents and those with more serious or refractory disease may require newer biologic therapies.

12.1 Clinical Features of Crohn’s Disease Crohn’s disease is a disease of flares and remissions, although 10 – 20 % of patients have refractory, chronically active, or steroid-dependent disease (Rutgeerts 2002) (Fig. 12.1). It is characterized by chronic transmural inflammation that may affect any part of the gastrointestinal tract. In approximately one-third of patients, CD is confined to the small intestine, whereas 40 – 50 % of patients will have involvement of both the small intestine and the colon. Twenty % to 30 % of patients have only colonic involvement. When disease is restricted to the colon, it can be difficult to differentiate between CD and UC. This type of disease is referred to as indeterminate colitis and is seen in approximately 5 % of patients with CD. Symptoms include abdominal pain and tenderness, chronic and nocturnal diarrhea, rectal bleeding, weight loss, and fever (Hanauer and Present 2003). CD evolves over time from a primarily inflammatory disease into one of two clinical patterns – stricturing (obstructive) or penetrating (fistulizing) (Hanauer and Present 2003). Current treatment strategies for CD use a “step-up” approach. Oral 5-aminosa-

licylate agents, sulfasalazine, antibiotics, or budesonide are used as first-line therapies for mild to moderate disease. Moderate to severe disease is treated with systemic steroids, azathioprine, 6-mercaptopurine, methotrexate, or infliximab, the first biologic indicated for treatment of CD. Severe, fulminant CD requires intravenous steroids, infliximab, conventional immunosuppressant drugs (e.g., cyclosporine), or surgery (Fig. 12.2). The current treatment paradigm does not modify the natural course of CD. New treatment approaches are needed that will alter the natural history of CD and prevent the need for surgery. The advent of biologics is changing the goal of CD therapy. These agents have the ability to change the course of the disease and induce and maintain complete remission, thereby preventing complications (obstruction, fistulization), and reducing the need for surgery.

12.2 Pathogenesis of Crohn’s Disease Although significant advances in understanding the pathogenesis of CD have been made, there are many unknowns. CD is believed to be caused by a combination of genetic and environmental factors, affecting the mucosal immune system and culminating in an aberrant inflammatory response (Korzenik and Podolsky 2006) (Fig. 12.3). It is a polygenic disease with probable genetic heterogeneity. Some genes are associated with the disease itself, whereas others increase the risk of the disease or are associated with the location or behavior of the disease (Lakatos et al. 2006). Caspase recruitment domain family member 15 (CARD15, also known as IBD1 or NOD2) was the first specific gene associated with inflammatory bowel disease (IBD) and is believed to confer the critical mutation on chromosome 16

12.2 Pathogenesis of Crohn’s Disease

a

b

Fig. 12.1. a Anatomic distribution of Crohn’s disease. b Comparison of the appearance of normal and Crohn’s mucosa: gross (top); histologic (center); endoscopic (bottom). (Reprinted with permission from Bayless et al. 2006)

Fig. 12.2. Treatment algorithm of therapeutic options for Crohn’s disease. 5-ASA 5-aminosalicylate, IL interleukin, IV intravenous. (Reprinted with permission from Bayless et al. 2006)

(Hugot et al. 2001; Ogura et al. 2001). CARD15 is the intracellular receptor for peptidoglycan-derived muramyl dipeptide and is involved in cell activation via NFκB. CARD15 mutations are associated with diminished mucosal alpha-defensin expression, which could be the cause of decreased innate immune response to endogenous bacteria (Kobayashi et al. 2005; Wehkamp et al. 2004). Moreover, in healthy first-degree relatives of patients with CD, high mucosal permeability was associated with the presence of CARD15 mutation, indicating that genetic factors may be involved in the impairment of the intestinal barrier function in families with IBD (Buhner et al. 2006). Organic cation transporter 1 (also known as IBD5) also has been associated with CD, especially fistulizing disease. Mutations in this gene affect the ability of transporters to pump xenobi-

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12 Biologics in Crohn’s Disease and Ulcerative Colitis: Focus on Tumor Necrosis Factor Antagonists ¸

Fig. 12.4. Novel therapeutic targets in inflammatory bowel disease (IBD). Potential therapies in IBD encompass interventions targeting a variety of pathways in the inflammatory cascade. These include altering luminal factors, enhancing intestinal repair, augmenting the intestinal innate immune barrier function, inhibiting cell adhesion, and blocking cytokine activity. GM-CSF granulocyte-macrophage colonystimulating factor, ICAM-1 intercellular adhesion molecule 1, IFN interferon, IL interleukin, M ” macrophage cell, PMN peripheral mononuclear cell, SAM selective adhesion molecule, TNF tumor necrosis factor. (Reprinted with permission from Korzenik and Podolsky 2006) Fig. 12.3. Immunopathogenesis of Crohn’s disease. (Reprinted with permission from Bayless et al. 2006)

otics and amino acids across cell membranes (Peltekova et al. 2004). Drosophila Discs Large Homolog 5 (DLG5), located on chromosome 10q23, has been associated with IBD in the German population. This mutation is thought to impair the ability of DLG5 to function as a guanylate kinase and to maintain epithelial polarity, affecting epithelial permeability (Stoll et al. 2004). Other genes and loci are currently under investigation in CD (Noble et al. 2006). These investigations, in addition to aiding the understanding of the pathophysiology of the disease, may give way not only to potential screening and prevention programs targeted at specific genotypes, but also to future gene therapies for CD (Baert et al. 2004; Schreiber 2006). In patients with CD, the mucosal immune system may be more exposed to potentially harmful pathogens and foreign antigens, resulting in the production of inflammatory cytokines and T-cell differentiation. The mucosal inflammation of established CD is dominated by CD4-positive T lymphocytes with a type 1 helper phenotype, characterized by the production of interferon- * (IFN- * ) and tumor necrosis factor (TNF). Other components of the intestinal matrix, such as adhe-

sion molecules, matrix metalloproteinases, intestinal epithelial cells, fibroblasts, and granulocytes, interact in a complex network that balances inflammation and healing (Baert et al. 2004).

12.3 Biologics for Use in Crohn’s Disease There are a number of therapeutic targets for new biologics in CD (Fig. 12.4). Biologics aim to reduce mucosal inflammation by inhibiting the effects of proinflammatory cytokines [interleukin (IL)-1 q , TNF, IFN- * , IL12, IL-18], by inducing apoptosis of type 1 helper lymphocytes (anti–IL-6, TNF antagonists), or by boosting natural anti-inflammatory mechanisms (granulocytemacrophage colony-stimulating factor, transforming growth factor- q , IL-20) (van Deventer 2003). Table 12.1 provides a list of approved and potential future biologic therapies for CD and key clinical trial data are summarized in Table 12.2.

12.3 Biologics for Use in Crohn’s Disease

Fig. 12.4. Table 12.1. Evolving and approved biologics for Crohn’s disease and ulcerative colitis

a

Therapeutic approach

Agent

Target

Phase

Indication

Pro-inflammatory cytokines/ pathways

Adalimumab Infliximab Certolizumab Fontolizumab

TNF TNF TNF IFN

3 4a 3 2

CD CD/UC CD CD

Selective adhesion molecules

MLN-02 (LDP-02) Natalizumab

[4q7 [4

3 3

UC/CD CD

T-cell differentiation/subsets

ABT-874 Basiliximab CNTO 1275 Daclizumab Visilizumab MRA

2a 2 2 2 2–3 2

CD UC CD UC UC CD

IL-12/IL-23 IL-2R [ IL-12/IL-23 IL-2R [ CD3 IL-6R

Approved for use by the US Food and Drug Administration and the European Innate immune stimulation GM-CSF GM-CSF-R 3 CD Agency for the Evaluation Intestinal repair EGF EGF-R 3 UC of Medical Products for the Growth hormone hGH-R 2 CD treatment of Crohn’s disease and ulcerative colitis CD Crohn’s disease, EGF epidermal growth factor, EGF-R epidermal growth factor receptor, GM-CSF granulocyte-macrophage colony-stimulating factor, GM-CSF-R granulocyte-macrophage colony-stimulating factor receptor, hGH-R human growth hormone receptor, IFN interferon, IL interleukin, TNF tumor necrosis factor, UC ulcerative colitis

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12 Biologics in Crohn’s Disease and Ulcerative Colitis: Focus on Tumor Necrosis Factor Antagonists Table 12.2. Summary of clinical data for each biologic in Crohn’s disease Treatment

N

Study duration

Primary endpoint(s)

Adalimumab (160/80 mg, 80/40 mg, 40/20 mg) Placebo

76, 75, 74

4 weeks

Clinical remission (CDAI CDAI & 70: 59 %, 59 %, 54 % vs. 37 % CDAI & 100: 50 %, 40 %, 34 % vs. 25 % e 150) at Week 4: 36 %, 24 %, 18 % vs. 12 %

CLASSIC II (Sandborn et al. 2005)


221 in OL cohort; 55 in RCT arm

4 week OL segment then 52 week RCT or OL arm

Clinical remission (CDAI OL cohort: CDAI & 70: 69 % CDAI & 100: 61 % e 150) at Week 56: 43 % in OL cohort

CHARM (Colombel et al. submitted to DDW 2 006)

Induction: Adalimumab 80 mg at Week 0 and 40 mg at Week 2 Maintenance: Adalimumab 40 mg e.o.w., 260 257 40 mg weekly, 261 or placebo

56 weeks

Clinical remission (CDAI e 150) at Week 26: 40 %, 46 % vs. 17 % (p < 0.001) Clinical remission (CDAI e 150) at Week 56: 36 %, 41 % vs. 12 % (p < 0.001)

Clinical remission (CDAI e 150) + discontinuation of steroids: 29 % (p < 0.001), 23 % (p = 0.008) vs. 6 % Complete fistula closure: 36 %, 46 %, vs. 14 % (p < 0.027)

26 weeks

Clinical response (CDAI & 100) among CRP & 10 mg/l stratum: 62 % vs. 34 % (p < 0.001)

Clinical remission (CDAI e 150) among CRP & 10 mg/l stratum: 42 % vs. 26 % (p < 0.01) Clinical response among ITT: 63 % vs. 36 % (p < 0.001) Clinical remission (CDAI e 150) among ITT: 48 % vs. 29 % (p < 0.001)

26 weeks

Clinical response (CDAI & 100) at Week 6 among CRP & 10 mg/l stratum: 37 % vs. 26 % (p e 0.05) Clinical response (CDAI & 100) at Weeks 6 and 26 among CRP & 10 mg/l stratum: 22 % vs. 12 % (p e 0.05)

Clinical remission (CDAI e 150) at Week 6 among CRP & 10 mg/l stratum: 22 % vs. 17 % Clinical remission (CDAI e 150) at Weeks 6 and 26 among CRP & 10 mg/l stratum: 13 % vs. 8 % Clinical remission (CDAI e 150) at Week 6 among ITT: 22 % vs. 17 % Clinical remission (CDAI e 150) at Weeks 6 and 26 among ITT: 14 % vs. 10 %

54 weeks

Clinical remission (CDAI 54 weeks vs. 19 weeks

Clinical remission (CDAI 40 weeks vs. 14 weeks (p < 0.001)

No draining fistulas at Week 54: 365 vs. 19 % (p = 0.009)

Trial name Adalimumab CLASSIC I (Hanauer et al. 2006)

Certolizumab pegol Induction: PRECiSE-2 Certolizumab 400 mg at (Schreiber Weeks 0, 2, and 4 2005) Maintenance: Certolizumab 400 mg or placebo q4 weeks PRECiSE-1 (Sandborn et al. 2006)

Infliximab ACCENT I (Hanauer et al. 2002)

ACCENT II (Sands et al. 2004)

Induction: Certolizumab 400 mg or placebo at Weeks 0, 2, and 4 Maintenance: Certolizumab 400 mg or placebo q4 weeks

Induction with infliximab 5 mg/kg at Week 0 Group 1: Placebo at Weeks 2, 6 and q8 weeks Group 2: Infliximab 5 mg/kg at Weeks 2, 6, and q8 weeks Group 3: Infliximab 5 mg/kg at Weeks 2, 6, and 10 mg/kg q8 weeks Induction: Infliximab 5 mg/kg at Week 0, 2, and 6 Maintenance: Infliximab 5 mg/kg or placebo q8 weeks

Secondary endpoints

74

216 212

331 328

188 192 193

96 99

12.3 Biologics for Use in Crohn’s Disease Table 12.2. (Cont.) Trial name

Treatment

REACH (Cuccuiara et al. 2006; Hyams et al. 2006)

Induction: Infliximab 5 mg/kg at Weeks 0, 2, and 6 Maintenance: Infliximab q8 weeks or q12 weeks through Week 46

Natalizumab ENACT-1 (Sandborn et al. 2005c)

ENACT-2 (Sandborn et al. 2005c)

N

Study duration

Primary endpoint(s)

Secondary endpoints

10 weeks 54 weeks

Clinical response (PCDAI & 15 and PCDAI e 30) at Week 10: 88 %

Clinical response (PCDAI & 15 and PCDAI e 30) at Week 54: 63 % in the q8 week group versus 33 % in the q12 week group Clinical remission (PCDAI e 10) at Week 54: 56 % in the q8 week group versus 24 % in the q12 week group Median change in average daily corticosteroid dose at Weeks 10, 30, and 54: –0.2, –0.3, and –0.3 mg/kg/day

724 Induction: Natalizumab 300 mg or pla- 181 cebo at Weeks 0, 4, and 8

3 months

Clinical response (CDAI & 70) at Week 10: 56 % vs. 49 % Clinical remission (CDAI 90 % of B cells in non-Hodgkin’s lymphoma. CD20 expression is not detectable on haematopoietic stem cells, plasma cells, or non-haematopoietic tissues. The effect of rituximab is mediated by induction of apoptosis in target cells, antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. In the treatment of CBCL rituximab has been used as a systemic infusion as well as an intralesional injection.

14.3 Biologics in the Treatment of CTCL

In an applicational observation, Gellrich et al. reported on ten patients with CBCL (FCL, MZL) (Gellrich et al. 2005). The dosage was 375 mg/m2 body surface area once per week, up to 8 weeks. All patients received premedication with hydroxyhydrochloride and indomethacin. Complete remission was observed in seven out of the ten patients, while two of the ten had a partial remission. The mean duration of remission was 23 months. Observed side effects were infusionrelated fever, shivering and nausea. Laboratory analysis showed a nearly complete depletion of B cells, leaving other parameters, e.g. creatinine and alkaline phosphatase, unchanged. During the study two patients suffered from bacterial infections; albeit no severe adverse events were recorded. Intralesional administration of rituximab has also been shown to be effective in the treatment of CBCL (Piekarz et al. 2001). In a retrospective study, patients received 10 mg rituximab intralesionally in up to four lesions three times weekly. Cycles were repeated every 4 weeks for up to eight cycles. With this regimen a complete remission was observed in six out of seven patients. While in one patient the tumour recurred locally after 27 months, two patients had a recurrence at distant body sites after 12 and 14 months, respectively. Side effects were burning sensations at the injection site during and several hours after injections. Interestingly, B cells were depleted from the circulation, indicating systemic effects of locally applied rituximab. Intralesional injection of rituximab is especially suitable for solitary or a small number of lesions as systemic adverse events have not been observed and the amount of rituximab needed is less than for systemic administration, thus lowering treatment costs. Currently rituximab is recommended as second line treatment for patients with relapsing or refractory CBCL and is generally well tolerated. 14.3.4 90 Y-Ibritumomab Tiuxetan 90Y-ibritumomab tiuxetan (Zevalin) is a radioimmuno-

therapeutic agent composed of a monoclonal murine IgG antibody, which binds to the human CD20 molecule, and tiuxetan, a molecule which contains the radionuclide yttrium-90. Yttrium-90 is a q -radiator with a half-life of 64 h and a radiation distance within tissues of 5.3 mm. Binding of the antibody to cells expressing CD20 results in radiation-induced apopto-

sis of target cells and nearby cells (so called “collateral damage”). Whether other antibody-specific mechanisms contribute to the therapeutic effect of 90Y-ibritumomab tiuxetan is unclear today. Compared to the chimeric antibody rituximab described above, ibritumomab is cleared faster from the circulation. Therefore duration of exposure to radiation is limited. 90Y-Ibritumomab tiuxetan is approved in Europe for the treatment of B-cell non-Hodgkin’s lymphoma. CD20-positive cells are depleted from the circulation by administration of rituximab 1 week and several hours before the one-time infusion of YIT. The administered dosage is 0.4 mCi/kg. Preliminary results of a prospective study on treatment of CBCL showed complete response in eight of nine treated patients while one patient had progressive disease (Duvic et al. 2005). In the follow-up phase, one patient had a relapse after 6 months while seven out of nine patients remained in remission. Reported side effects were a decrease in ECOG status from 0 to 2 or 3 and a weight loss in 33 % of patients. Haematological toxicities (thrombopenia, lymphopenia and anaemia) resolved after 12 weeks. Adverse events, recorded in studies treating nonHodgkin’s lymphoma, were infusion-related side effects such as fever, nausea, chills and flu-like-symptoms. The myelosuppressive effects of 90Y-ibritumomab tiuxetan are observed 4 – 8 weeks after administration. Of observed grade 4 haematological toxicities, neutropenias were the most common, in 30 % of patients on average, with thrombocytopenia and anaemia occurring in 10 % and 3 %, respectively. It is noteworthy that neutropenia and thrombocytopenia required active therapy in 60 % of cases. However, most of the cytopenias are only short-lived. As the haematological side effects correlate with the pre-treatment platelet count, the dose has to be reduced if the pretreatment count is < 150×109 platelets/l. So far only preliminary data are available regarding treatment of CBCL with ibritumomab tiuxetan. Remissions have been achieved, but so far duration of remission remains unclear. Because of the generally benign prognosis of CBCL, 90Y-ibritumomab tiuxetan should be reserved for CBCL, which are refractory to other treatments or have systemic involvement.

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14.3.5 Histone Deacetylase Inhibitors The acetylation status of histones is involved in regulation of gene transcription. While histone acetylation favours DNA transcription, histone deacetylases keep the DNA in a transcriptional inactive state. Histone deacetylase inhibitors (HDACIs) increase the acetylation status of histones and thereby facilitate the transcription of genes involved in cell differentiation, cell cycle arrest and apoptosis. Currently two substances, namely depsipeptide (FR901228) and suberoylanilide hydroxamic acid (SAHA), from the group of HDACIs are being investigated for the treatment of CTCL. Depsipeptide has been given to three patients with CTCL refractory to previous topical or systemic therapies (7). The drug was administered intravenously on days 1 and 5 of a 21-day cycle. Improvement of skin erythema and oedema was reported for two patients with Sézary syndrome and nearly complete remission for a patient with tumour stage CTCL. Development of multiple subcutaneous abscesses in one patient was reported as an adverse event. Because of these promising results, a phase II trial was initiated. An oral formulation of SAHA has been tested in a phase II trial with 37 CTCL patients participating (8). The used dosage was 400 mg once daily. Reported results showed a partial remission in 27 % of patients. A decrease in lymphadenopathy and pruritus was seen in 58 % and 68 %, respectively. Observed adverse events were fatigue, symptoms, nausea, dry mouth, and decreased appetite. Thrombocytopenia (grade 3 or 4) occurred in 18 % of patients. In conclusion HDACIs showed clinical activity in CTCL patients, which were refractive to previous therapy. However, before they can be recommended for the treatment of CTCL, the existing results need to be confirmed by the ongoing studies.

References Duvic M, Talpur R, Zhang C, Goy A, Richon V, Frankel SR (2005) Phase II trial of oral suberoylanilide hydroxamic acid (SAHA) for cutaneous T-cell lymphoma (CTCL) unresponsive to conventional therapy. J Clin Oncol 23(16S):abstract 6571 Gellrich S, Muche JM, Wilks A, Jasch KC, Voit C, Fischer T, Audring H, Sterry W (2005) Systemic eight-cycle anti-CD20 monoclonal antibody (rituximab) therapy in primary cutaneous B-cell lymphomas – an applicational observation. Br J Dermatol 153(1):167 – 173 Lundin J, Hagberg H, Repp R, Cavallin-Stahl E, Freden S, Juliusson G, Rosenblad E, Tjonnfjord G, Wiklund T, Osterborg A (2003) Phase II study of alemtuzumab (Campath-1H) in patients with advanced Mycosis fungoides/Sezary syndrome. Blood 101:4267 Olsen E, Duvic M, Frankel A, Kim Y, Martin A, Vonderheid E, Jegasothy B, Wood G, Gordon M, Heald P, Oseroff A, PinterBrown L, Bowen G, Kuzel T, Fivenson D, Foss F, Glode M, Molina A, Knobler E, Stewart S, Cooper K, Stevens S, Craig F, Reuben J, Bacha P, Nichols J (2001) Pivotal phase III trial of two dose levels of denileukin diftitox for the treatment of cutaneous T-cell lymphoma. J Clin Oncol 19:376 – 388 Piekarz RL, Robey R, Sandor V, Bakke S, Wilson, Dahmoush L, Kingma DM, Turner ML, Altemus R, Bates SE (2001) Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report. Blood 98:2865 – 2868 Willemze R, Jaffe ES, Burg G, Cerroni L, Berti E, Swerdlow SH, Ralfkiaer E, Chimenti S, Diaz-Perez JL, Duncan LM, Grange F, Harris NL, Kempf W, Kerl H, Kurrer M, Knobler R, Pimpinelli N, Sander C, Santucci M, Sterry W, Vermeer MH, Wechsler J, Whittaker S, Meijer CJ (2005) WHO-EORTC classification for cutaneous lymphomas. Blood 105:3768 – 3785 Williams DP, Snider CE, Strom TB, Murphy JR (1990) Structure/function analysis of interleukin-2-toxin (DAB486-IL-2). Fragment B sequences required for the delivery of fragment A to the cytosol of target cells. J Biol Chem 265:11885 – 11889

Chapter 15

Biologics in Targeted Cancer Therapy D. Schrama, J.C. Becker

Treatment of cancer should be as potent as possible to completely destroy the tumor. However, precisely this aggressiveness often causes severe side effects. Indeed, due to the side effects, some promising therapeutics cannot be applied systemically. In addition, therapeutics like cytokines which physiologically function in a para- or autocrine fashion require a locally enhanced level to exert their effect appropriately. An elegant way to accumulate therapeutic agents in the tumor is by their conjugation/fusion to tumor-specific antibodies. This chapter presents an overview with preclinical and clinical data for different agents which were turned into targeted therapeutics.

15.1 Introduction Until the 19th century, when anesthesia, improved techniques and histological control made surgery more efficient, cancer was more or less regarded as incurable. Surgery was first complemented by radiation therapy. In this regard, the invention of the linear accelerator in the first half of the 20th century advanced radiotherapy from a palliative method to a cancer treatment with a curative intent. Both therapies, however, are in most cases not sufficient to control metastatic disease. Consequently, the introduction of nitrogen mustard in the early 1940s initiated chemotherapy, targeting proliferating cells in general (Papac 2001). Still, cancer remains one of the most life threatening diseases. Notably, despite intensive research on cancer and cancer therapy over the past 30 years the prognosis of metastatic cancer has not sufficiently improved as yet; however, many pathways and characteristics of different tumor entities have been unraveled. Based on this information, specific tumor therapies are being pursued either

by directly targeting the proteins involved in the neoplastic process, or by targeting toxic drugs to the tumor. Both strategies can be achieved using monoclonal antibodies (mAbs). In this regard, Paul Ehrlich already envisioned at the end of the 19th century antibodies as “magic bullets,” but not until 1975 when Köhler and Milstein described the generation of murine monoclonal antibodies did the necessary munition become available (Köhler and Milstein 1975). Among other problems, the patients’ immune response readily inactivating antibodies of non-human origin hampered the development of mAbs as therapeutic agents until these had been solved by technical advances in the process of antibody generation. To this end, the first therapeutic antibody for cancer therapy was approved by the US Food and Drug Administration (FDA) in 1997 and mAb based therapies became a major strategy in medicine (Grillo-Lopez et al. 2002). Unconjugated mAbs can exert their anti-tumor effect by inducing immune responses, blocking highly expressed and activated growth factor receptors on tumor cells or inhibiting angiogenesis. In addition, tumor specific antibodies also provide the means to target therapeutic measures to tumor cells. Indeed, conjugates of cytotoxic drugs, cytokines, toxins or radionuclides (see Chapter 6, this volume) and tumor-specific mAbs have been evaluated in preclinical and clinical settings with the aim of increasing the specificity of the therapeutic intervention and thereby minimizing the side effects while maximizing the desired effects. Here, we discuss recent preclinical and clinical data on immunconjugates of cytotoxic drugs and focus on components conjugated to antibodies which exert their therapeutic efficacy by utilizing biological processes.

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15 Biologics in Targeted Cancer Therapy

15.2 Chemoimmunoconjugates For most cancers, traditional chemotherapy is still the standard of care. The selectivity of cytotoxic agents, however, relies on the premise that rapidly proliferating cells are more prone to the cytotoxic effect of these drugs. As a consequence, toxicities against normal tissues characterized by enhanced proliferation rates are regularly observed side effects which increase the potency of the drug. To reduce this risk, anticancer chemotherapeutics are often given at suboptimal doses. A strategy to achieve tumor selectivity and thereby circumvent this problem is conjugating cytotoxic agents to a tumor specific antibody; thereby, the mAb serves as a delivery vehicle for the targeted application of cytotoxic drugs. Such chemoimmunoconjugates indeed display selectivity towards the cells which express the respective antigens (Johnson et al. 1981). After binding, the conjugate is internalized via receptor-mediated endocytosis and the parent drug is released from the lysosome into the cell to restore its cytotoxic activity (Fig. 15.1). Standard chemotherapeutic agents belonging to the antifolates, vinca alkaloids or anthracyclines have been conjugated to mAbs (Schrama et al. 2006). Although such immunoconjugates based on standard anti-cancer drugs demonstrated efficiency in preclinical models (Deguchi et al. 1986), they were not efficient in the clinical situation. This was due to the fact that therapeutic levels of the cytotoxic agents within the cells were not

achieved since these drugs possessed only moderate cytotoxic potency. A high cytotoxic potency is important as both the amount of cytotoxic drug which can be conjugated to the antibody and the expression of antigens on the tumor cell is limited (Allen 2002). From these initial studies, however, crucial information was gathered concerning the influence of the targeting device on the pharmacokinetics of the conjugates as well as the different chemical strategies of coupling cytotoxic agents to the antibody. For example, peptide linkers, which are stable in serum but can be readily degraded in intracellular compartments by specific enzymes, were found to be superior to hydrazone linkers. In this respect, disulfide linkers are suitable for conjugation of drug and mAbs since these linkers are cleaved by disulfide exchange with an intracellular thiol such as glutathione; this is of particular advantage as the gluthatione concentration in tumor cells is generally higher than in normal cells (Jaracz et al. 2005). In addition, progress in DNA engineering, enabling the production of chimeric, humanized and human mAbs, solved the major problem of early antibody-drug conjugates, i.e., the rather high clearance rate of the immunoconjugates due to the development of human antimouse antibody responses (HAMA) in the treated patients (Carter 2001). Two avenues of improving the cytotoxic potency of antibody-drug conjugates have been pursued: (1) Fig. 15.1. Internalization of antibody-drug conjugates. In order to regain their cytotoxic activity, the cytotoxic agent has to be cleaved from the chemoimmunoconjugate. Uptake of antibodies predominantly occurs via the clathrin mediated endocytosis pathway. After binding the respective antigen associated with coated pits, antibody-drug conjugates will readily be endocytosed, initiating their transit via several stages of transport and endosomal vesicles and finally ending up in a lysosome. Subsequently, linkers and antibody will be cleaved, releasing the cytotoxic agent which – after exit from the lysosomal compartment – exerts its cytotoxic effect

15.2 Chemoimmunoconjugates Table 15.1. Immunoconjugates in clinical developmenta (Schrama et al. 2006) Name

Targeting device

Immunoconjugates AVE9633 (huMy9 – Humanized anti-CD33 6-DM 4) mAb BB-10901 (huN901-DM 1) Humanized anti-CD56 mAb CMC-544

Humanized anti-CD22 mAb Gemtuzumab ozogamicin Humanized anti-CD33 mAb huC242-DM 4 MLN2704 SGN-15 with taxotere ADEPT A5CP + ZD2767P MFECP1 + ZD2767P

Immunotoxins BL22 Hum-195/rGel LMB-2 LMB-9 SS1(dsFv)-PE38

Immunocytokines EMD 273066 Bispecific double scFv BiTE MT103 rM28 a

Conjugate

Tumor

Phase

DM 4

AML

Phase Ib

DM 1

Recurrent or refractory lung cancer or other CD56+ solid tumors B-cell NHL

Phase I and II

Calicheamicin

Older patients with relapsed or untreated AML CanAg+ solid tumors

Phase II and III

Prostate cancer

Phase I and II

Prostate cancer

Phase II completed

Prodrug ZD2767P

Advanced CRC

Phase I

Prodrug ZD2767P

CEA expressing tumors

Phase I

Truncated Pseudomonas exotoxin A Recombinant gelonin

Leukemia and lymphoma Phase I and II

Calicheamicin

Humanized antiDM 4 CanAg mAb Humanized anti-PSMA DM 1 mAb Chimeric anti-Le(Y) Doxorubicin mAb Murine anti-CEA F(ab)2 fragment fused to CPG2 Murine anti-CEA scFv fragment fused to CPG2

Phase I

Murine anti-CD22 dsFv fragment Humanized anti-CD33 antibody Murine anti-CD25 scFv fragment Murine anti-Le(Y) dsFv fragment

Truncated Pseudomonas exotoxin A Truncated Pseudomonas exotoxin A

Murine anti-mesothelin dsFv fragment

Truncated Pseudomonas exotoxin

Humanized antiEpCAM mAb

IL-2

Rabbit anti-CD19 scFv fragment Murine anti-M-AP scFv fragment

scFv fragment of a B-cell tumors murine anti-CD3 mAb scFV fragment of a Metatstatic melanoma murine anti-CD28 mAb

Phase Ic

Advanced myeloid malig- Phase I nancies Leukemia and lymphoma Phase I and II Phase I completed Advanced pancreatic, esophageal, stomach cancer or CRC Phase I Mesothelin-expressing tumors like mesothelioma, ovarian and pancreatic adenocarcinoma Ovarian, prostate, CRC or Phase I NSCL cancers Phase Id Phase I and II; not yet recruiting

Information on ongoing trials was gathered from: http://utm-ext01a.mdacc.tmc.edu/dept/prot/clinicaltrialswp.nsf/Index/2004 – 0756 c http://www.idd.org/forms/PhaseI.pdf d http://www.micromet.de or from http://www.clinicaltrials.gov [all others]. Status quo October 2005 AML acute myelogeneous leukemia, CPG2 carboxypeptidase 2, CRC colorectal cancer, ds disulfide-stabilized, M-AP melanomaassociated proteoglycan, NHL non-Hodgkin’s lymphoma, NSCLC non-small cell lung cancer, PSMA prostate specific membrane antigen, ZD2767P bis-iodo phenol mustard b

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increasing the number of molecules delivered per targeting moiety by means of carriers like liposomes and polymers, and/or (2) the use of highly cytotoxic compounds. Indeed, targeting extremely cytotoxic agents, such as calicheamicin, the maytansine derivative DM 1 or monomethyl auristatin E to the tumor, results in a pronounced anti-tumor activity in vivo. Nevertheless, to date only one immunoconjugate containing a cytotoxic drug has been approved by the FDA for the treatment of cancer. This immunoconjugate (Mylotarg) consists of a humanized anti-CD33 mAb (gemtuzumab) linked to the cytotoxic antibiotic N-acetyl- * -calicheamicin. Mylotarg is approved for the treatment of elderly patients relapsed from CD33-positive acute myeloid leukemia (AML) (Bross et al. 2001). In a multicenter phase II trial the combined response rate was around 30 % with a median relapse-free survival of 7 months. A recent phase II trial demonstrated that Mylotarg can also be safely applied as first line therapy leading to objective responses in 27 % of the patients (Nabhan et al. 2005). Notably, preliminary studies suggest that Mylotarg treatment may be potentiated by concomitant intensive chemotherapy. Consequently, a phase III trial is currently testing Mylotarg’s impact on standard chemotherapy treatment in newly diagnosed AML patients (Tallman et al. 2005). Since Mylotarg demonstrated clinical efficiency in pediatric patients with advanced CD33-positive AML, further studies in combination with standard chemotherapy for this patient group are warranted. Other cytotoxic drugs currently being investigated for use in drug-antibody conjugates include doxorubicin, DM 1, CC-1065, second generation taxane, monomethylauristatin and geldanamycin E (Sanderson et al. 2005) (Table 15.1). For example, SGN-15, an antibodydoxorubicin conjugate in combination with docetaxel, is currently being tested in a phase II trial for patients with advanced non-small cell lung carcinoma (http://www.clinicaltrials.gov). The efficacy of therapeutic mAb may be significantly improved by chemical conjugation with cytotoxic drugs; e.g., the Herceptingeldanamycin conjugate demonstrated in a xenograft tumor model a much greater anti-tumor effect than the anti-HER2 mAb alone (Mandler et al. 2004). The therapeutic efficiency of chemoimmunoconjugates relies on their binding to and the subsequent internalization into the target cell. Thus, only tumor cells presenting the respective antigen are affected by this treatment. The antibody-directed enzyme prodrug

therapy (ADEPT) is a strategy to overcome this limitation by expanding the anti-tumor effect towards cells not expressing the respective ligand. ADEPT is a targeted therapy where an enzyme is directed to the tumor – by a tumor specific antibody – which converts a weakly toxic prodrug into a very toxic agent (Bagshawe 1987). In contrast to antibody-drug conjugates, the antibodyenzyme conjugate has to remain on the cell surface after binding the respective antigen. In addition, the enzyme-antibody conjugate has to be cleared rapidly from the circulation to prevent toxicity. The latter is achieved by mannose glycosylation of the targeting moiety (Chester et al. 2004). Three classes of enzyme have been used for ADEPT (Senter and Springer 2001): (1) enzymes of non-mammalian origin with no mammalian homologs, (2) enzymes of non-mammalian origin with a mammalian homolog, and (3) enzymes of mammalian origin. Each class has both advantages and disadvantages. For example, prodrugs cleaved by class I enzymes are not cleavable by endogenous enzymes, which avoids toxicity against normal cells. However, due to their foreign nature, class I enzymes evoke a strong immune response. In contrast, class III enzymes are generally not immunogenic, but endogenous enzymes may cleave prodrugs designed for class III enzymes at inappropriate sites. Preclinical studies have proven the feasibility of ADEPT (Sharma et al. 2005); clinical data, however, are limited and largely restricted to phase I trials. An example is the prodrug ZD2767P: The activated form of ZD2767P proved to be highly cytotoxic, which gives a very short half-life (Francis et al. 2002). Despite the fact that the clinical efficiency for ADEPT remains to be established, the preclinical data demonstrating the capacity of this approach to reach high concentrations of cytotoxic drugs in the tumor, which will kill antigen negative tumor cells without severe systemic toxicity, warrants further testing.

15.3 Immunotoxins Toxins, i.e., poisonous substances produced by living cells or organisms, constitute another class of highly cytotoxic agents suitable for mAb based tumor targeted therapy. Indeed, toxins or toxin subunits derived from plants, bacteria and fungi, such as diphtheria toxin, ricin, gelonin, saporin, pokeweed and Pseudomonas exotoxin A, have been conjugated to mAb and tested for

15.4 Antibody-Cytokine Fusion Proteins

anti-tumor therapy efficacy (Frankel 2005). Toxins are enzymes which exert their cytotoxic activity inside the cell and in most cases one single molecule is sufficient to kill the cell. In order to be used therapeutically, toxins have to be modified to remove their tissue binding sites (Frankel et al. 2000). In addition, deglycosylation of toxins avoids their rapid clearance by liver cells expressing mannose receptors. Toxins may be targeted to the tumor cells by conjugation to targeting moieties, which includes mAbs (immunotoxins) or growth factors, cytokines and peptide hormones (fusion protein toxins). For therapeutic efficacy the conjugates have to be internalized upon binding to susceptible cells. After endocytosis the released toxins cause cessation of protein synthesis, which results in subsequent cell death. Well defined biochemical properties determine the cytotoxic potency of immunotoxins. These properties include antigen-binding affinity, internalization rate, intracellular processing and the intrinsic potency of the toxin-domain (Hexham et al. 2001). It should be noted that since the uptake of the immunotoxins into the intracellular compartment seems rather inefficient, toxicities are not as high as would be expected from the toxicity of the toxin per se. Several clinical trials addressed or are currently addressing the efficiency of toxin conjugates for therapy of hematological and solid tumors (Table 15.1), demonstrating an impressive clinical efficiency with response rates greater than 30 %. Tested compounds include: (1) denileukin diftitox (Ontak) for the treatment of patients with therapy-refractory cutaneous Tcell lymphoma, which is approved by the FDA; (2) LMB-2 and (3) BL-22, both indicated for treatment of hairy cell leukemia; as well as (4) HN66000 for therapy of high grade glioma patients (Frankel et al. 2000). The FDA has approved denileukin diftitox, which consists of diphtheria toxin fragments fused to interleukin-2 (IL-2); the latter serves as a targeting device. Clinical cytotoxicity of targeted-toxin based therapies includes vascular leak syndrome and hepatocyte injury. Moreover, a major drawback of both immuno- and fusion protein toxins is their immunogenicity of the toxin; humoral immune responses to toxins can be observed as soon as after one treatment course (Posey et al. 2002). This immunological response not only reduces the serum half-life but also significantly inhibits the cytotoxic activity. These effects are particularly troublesome in cases where repeated treatment courses are necessary. Thus, several approaches have been pursued

to decrease the immunogenicity of toxins, namely the co-administration of immunosuppressive agents or modifications of the toxin. To date, however, these concepts were either not effective in patients or have not yet been tested in clinical trials (Frankel 2004). PEGylation of the toxin, i.e., its modification by conjugation with polyethylene glycol, and genetic engineering to generate humanized toxins, are promising approaches to overcoming this problem (Youn et al. 2005). The latter approach is exemplified by use of human RNase, which downregulates gene expression in the targeted cell; fusion proteins of RNase with humanized or fully human antibodies are expected to possess only limited immunogenicity. Two independent research groups generated fusion proteins consisting either of human pancreatic RNase and a fully human anti-ErbB-2 single chain Ab (De Lorenzo et al. 2004) or angiogenin and a humanized anti-CD22 single chain Ab (Arndt et al. 2005). Both constructs demonstrated specific binding to the cells expressing the respective antigen and exhibited cytotoxic/cytostatic activity towards these cells. For the anti-ErbB-2-human pancreatic RNase fusion protein, an anti-tumor effect could also be demonstrated in vivo, i.e., inhibition of tumor growth in a murine mammary carcinoma model (De Lorenzo et al. 2004). However, the expected low toxicity and any possible therapeutic effect awaits analysis in clinical trials.

15.4 Antibody-Cytokine Fusion Proteins Treatment of neoplastic disease was initially based on surgery, chemotherapy and radiation. However, since the identification of tumor specific or associated antigens, immunotherapy has evolved rapidly as an attractive alternative (Van Pel and Boon 1982). Notably, immunoregulatory cytokines have been demonstrated to improve anti-tumor immune responses. Indeed, systemic administrations of IL-2, granulocyte macrophage colony stimulating factor (GM-CSF) or IL-12 increase the immunogenicity of some tumors, thereby inducing or boosting immune response to a level that there is effective eradication of the tumor. Additional mechanisms of cytokines include direct effects on tumor or tumor stroma cells; for example, tumor necrosis factor- [ (TNF- [ ) directly damages the tumorassociated vasculature (Manusama et al. 1998). Physiologically, however, cytokines function as auto- or

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paracrine factors, with high concentrations only in close vicinity of the producing cell. Thus, systemic administration of cytokines neglects this idea, and doses which are needed in order to see a clinical effect are frequently accompanied by severe side-effects (Keilholz et al. 1997). To overcome this problem, cytokines were directly injected into solid tumors to achieve sufficient concentrations of the cytokines at the tumor site while reducing generalized toxicity (Mattijssen et al. 1994). Similarly, locally disseminated tumors can be amended by locoregional treatment, i.e., by isolated limb perfusion (Eggermont et al. 2003). However, advanced tumors in general are neither localized nor accessible, which are the prerequisites for either of these two approaches. An alternative means to achieve sufficient cytokine concentration at the tumor site which allows even micrometastases to be addressed is the genetic fusion of the cytokine to tumor specific antibodies. This strategy was published in 1991 by two independent research groups (Hoogenboom et al. 1991; Gillies et al. 1991). Importantly, both groups demonstrated that the fusion of cytokines to antibodies impaired neither the binding capacity of the mAb, nor – with the exception of a GM-CSF-antibody fusion protein – the biologic activity of the cytokine. Moreover, the clearance rate of these constructs in most cases lies between that of the mAbs and the cytokine, i.e., a marked prolongation of the serum half-life of the cytokine. The most elaborately studied antibody-cytokine fusion proteins are those containing IL-2. IL-2 was rec-

ognized early on to promote T-cell proliferation and to activate the cytotoxic capacity of T and NK cells. Moreover, it is FDA approved for therapy of advanced melanoma or renal carcinoma. An antibody-IL-2 fusion protein specific for the disialoganglioside GD2, an antigen commonly overexpressed on human melanoma and neuroblastoma cells, displayed anti-tumor efficacy in preclinical models in vitro and in vivo. To this end, the ch14.18-IL-2 fusion protein led to the eradication of established experimental and spontaneous metastases in xenogeneic and syngeneic tumor models (Becker et al. 1996). The therapeutic effect depended on the fusion of antibody and cytokine and could not be ascribed to the above mentioned increased in vivo half-life of antibody-IL-2 fusion proteins since neither the combined treatment of parental antibody and cytokine nor the application of an antibody-IL-2 fusion protein directed to an irrelevant antigen, demonstrated any therapeutic effects. Biodistribution analysis in the preclinical models demonstrated that the tumor specific fusion protein indeed accumulated within the tumor bearing organs (Becker et al. 1996). Interestingly, depending on the tumor model, the antibody-IL-2 fusion protein mediated its therapeutic effect either by innate or adaptive immunity, demonstrating IL-2’s broad spectrum of activity (Fig. 15.2). Referring to this, tumor eradication in the melanoma and colon carcinoma models was dependent on T cells, whereas in the neuroblastoma model the efficacy of the antibody-IL-2 fusion protein relied on NK cells. Indeed, in the melanoma model, the Fig. 15.2. Working mechanism of immunocytokines exemplified for tumor targeted IL-2. A mAb specific for a tumor-associated antigen allows the enrichment of cytokines in the tumor microenvironment. In the case of IL-2 it enhances antibody dependent cellular cytoxicity mediated by Fc-receptor positive effector cells like NK cells. In addition, tumor targeted IL-2 stimulates T cells to expand and attack the tumor. High concentrations of plasmin at the tumor site enable the cleavage of IL-2 from the fusion protein through the plasmin cutting site within the linker

15.4 Antibody-Cytokine Fusion Proteins

antibody-IL-2 fusion protein seemed to exert its therapeutic effect by boosting a pre-existing T-cell response and required CD4+ T-cell help. Consequently, in lymphotoxin- [ (LT- [ ) knock out mice, which are characterized by an impaired induction of immune responses – due to the absence of secondary lymphoid tissue – antibody-IL-2 fusion protein treatment is only effective if primed tumor specific T cells are adoptively transferred prior to IL-2 fusion protein therapy (own unpublished observations). The effect of antibody-IL-2 fusion proteins was also tested in adjuvant settings. For example, tumor targeted IL-2 clearly enhanced anti-tumor immune responses induced by DNA vaccination. Accordingly, therapeutic vaccination with tumor antigen loaded dendritic cells only induced a therapeutic effect in a murine melanoma model when vaccination was followed by IL-2 administration. This therapeutic effect could be clearly improved by the application of tumortargeted IL-2 instead of systemic IL-2 treatment (Schrama et al. 2004). Interestingly, tumor targeted IL-2 therapy influenced the development of a memory immune response: accordingly treated mice were protected against tumor rechallenge to the same organ, but not directed towards other organs. These findings are substantiated by a previous report demonstrating that high doses of antibody-IL-2 fusion proteins elicited protective immunity without memory in a murine B-cell lymphoma model (Penichet et al. 1998). Intriguingly, systemic IL-2 administration increases the activity of antibody-IL-2 fusion protein treatment in a murine neuroblastoma model (Neal et al. 2004). Antibody-IL-2 fusion proteins, however, not only boost vaccination induced immune response, but also increase the immunogenicity of antigens. Targeting IL-2 to a soluble, poorly immunogenic antigen triggered an immune response which led to significant tumor growth retardation (Dela Cruz et al. 2003). These encouraging preclinical data led to the clinical evaluation of antibody-IL-2 fusion proteins. The first phase I trials were conducted to test the efficacy of antibody-IL-2 fusion proteins specific for GD2 or EpCAM for the treatment of metastastic melanoma (King et al. 2004) or prostate cancer, respectively (Ko et al. 2004). The antibody-IL-2 fusion proteins were generally well tolerated; in very few patients did drug related toxicities equal to or larger than grade 3 occur. Translational studies revealed the biological activity of immunocytokines, i.e., an increase in total lymphocyte and NK-cell

counts as well as enhanced NK-cell and antibodydependent cellular cytotoxic activity. Although these trials were only designed to evaluate safety and the maximum tolerated dose, the clinical outcomes of the melanoma patients were reported. To this end, 58 % of the treated patients after the first course (three doses of fusion protein) and 28 % at the end of the second course of therapy – which was completed by 52 % of the patients – presented with stable disease. Consequently, a phase II trial investigating the therapeutic activity is currently in preparation (King et al. 2004). Other antibody-cytokine fusion proteins were generated with GM-CSF, IL-12, IFN- * , TNF- [ or LT- [ , but have not yet been as thoroughly investigated as the antibody-IL-2 fusion proteins. This may be at least in part due to the fact that their generation encountered some difficulties. For example, antibody-GM-CSF fusion proteins are cleared much faster from the plasma than the parental antibody (Dela Cruz et al. 2000). In addition, the biologic activity of TNF- [ , LT- [ and IL-12 within the fusion proteins is markedly decreased compared to the recombinant cytokines (Reisfeld et al. 1996). This is probably due to the nature of the cytokines: TNF- [ and LT- [ are active as trimeric structures and IL-12 as a heterodimer. Nevertheless, all these constructs possessed potent anti-tumor efficiency in preclinical models. Antibody-GM-CSF fusion proteins facilitate neutrophil antibody-dependent cellular cytotoxicity in vitro and elicit a strong anti-tumor immune response eradicating solid tumor in vivo (Dela Cruz et al. 2000). Interestingly, the therapeutic effect of an antibody-LT- [ fusion protein, originally designed to exert a direct apoptotic effect on tumor cells, crucially depended on the presence of immune competent cells. In a xenograft model, these were B and NK cells, whereas in a syngeneic melanoma model the anti-tumor effect was mediated by T cells (Schrama et al. 2001). The anti-tumor effect of LT- [ fusion protein in the syngeneic melanoma model was associated with an induction of tertiary lymphoid tissue next to the tumor, which actually may provide all the necessary requirements for T-cell priming. In this regard, these structures contained high endothelial venules which are essential for the emigration of na¨ıve T cells from the blood into lymphatic tissue and na¨ıve T and antigen presenting cells (Fig. 15.3). The observations that the Tcell infiltrate within the tumor was tumor-specific and the quantity and clonality of this infiltrate increased over the course of therapy, imply that this tertiary lym-

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Fig. 15.3. Induction of tertiary lymphoid tissue by antibody-LT- [ fusion proteins. Targeting LT- [ to the tumor induces peritumoral tertiary lymphoid tissue. Macroscopic (A), microscopic (B) and ultrastructural (C) appearance of tertiary lymphoid tissue. Double staining of kryosections with anti-CD8 antibodies (green) and TRP2/MHC class I tetramers (red) demonstrates the presence of tumor-specific cytotoxic T cells (yellow) in these tissues (B). Electronic microscopy reveals the induction of high endothelial venules, which allows the entry of na¨ıve T cells (C)

phoid structure enabled priming of tumor specific T cells (Schrama et al. 2001). In conclusion, the severe side effects of systemic administrations of high doses of cytokines can be prevented by targeting cytokines to the site of interest by an antibody and – most importantly – this approach demonstrated therapeutic efficiency in preclinical models. However, the clinical characterization of these molecules has just been started and it will be interesting to see if their preclinical therapeutic potential can be confirmed in clinical trials.

15.5 Evolving Approaches As the underlying mechanisms of oncogenic transformation are deciphered with increasing speed and the general knowledge of biological processes is steadily expanding, new approaches to fight cancer emerge. These attempts are based either on disrupting aberrant signaling in the tumor cell or enhancing processes enabling the eradication of tumors. In most cancers the mechanisms of apoptosis, i.e., programmed cell death, are dysregulated; many tumor

15.5 Evolving Approaches

cells have an altered threshold, but are still able to undergo apoptosis. Therefore, direct induction of programmed cell death is believed to be a powerful strategy to treat cancer. Apoptosis can be initiated by triggering of death receptors through extrinsic factors like FasL (CD95L), tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) or TNF. To this end, the feasibility of Fas ligation for cancer therapy has been demonstrated (Shimizu et al. 1996). However, systemic treatment with Fas agonists such as anti-Fas antibodies or multimeric recombinant FasL causes severe systemic toxicities (Timmer et al. 2002). To overcome this problem, Samel and colleagues targeted soluble FasL (sFasL) to the tumor using a tumor specific single chain antibody; upon binding to the respective cell surface molecule, these constructs aggregate, thereby mimicking the affect of membrane bound FasL (Samel et al. 2003). Treatment with this antibody-sFasL fusion protein did not cause any systemic toxicity, but prevented tumor growth in a xenogeneic tumor model. Analogously, a single chain antibody-sTRAIL fusion protein with specificities for either CD7 (Bremer et al. 2005a) or the EGFR (Bremer et al. 2005b) demonstrated preclinical efficacy. This approach offers the advantage that cancer cells are more sensitive to TRAIL-induced apoptosis than normal cells. Interestingly, targeting sTRAIL to antigen expressing tumor cells not only induced apoptosis in those, but also in antigen-negative bystander cells (Bremer et al. 2005b). Another method to directly harm the tumor cells is to interfere with the translation of messenger RNA and thereby to block the expression of proteins. This can be achieved by the use of antisense RNA, ribozymes, RNA interference or more broadly by RNases. RNA-based strategies hold the potential for exquisite selectivity; nuclease sensitivity, rapid plasma elimination and poor intracellular delivery, however, hamper their use as therapeutic agents. These problems have already been addressed by use of carriers and chemical modifications, thereby substantially prolonging their circulation lifetime. Selective delivery of these agents was achieved by their fusion to targeting devices such as mAbs and specific ligands. For instance, tumor targeting of chemically modified antisense oligonucleotides specific for c-myc (Ou et al. 2005) or antisense oligonucleotides specific for c-myb was increased by encapsulation within liposomes (Brignole et al. 2005). Short interfering (si)RNA – similar to antisense oligonucleotides – specifically silences gene expression. Since siR-

NA approaches are both efficient and robust, this technology has become a frequently used tool for specific gene silencing in vitro. Consequently, siRNA are now being seized upon as drug candidates but face the same problems as antisense oligonucleotides, i.e., poor stability, short elimination half-life and inefficient mechanisms for delivery of siRNA to respective cells. One way to deliver siRNA to tumor cells is by ligand-targeted nanoparticles (Schiffelers et al. 2004): such constructs demonstrated tumor selectivity as well as specific silencing of the targeted gene both in vitro and in vivo in preclinical models. Nevertheless, delivery of siRNA both with respect to specificity and efficacy remains to be improved prior to translation into clinical trials. Targeting either costimulatory or immunmodulatory molecules to the tumor microenvironment should enhance naturally occurring immune processes. In the early 1990s a large series of reports demonstrated that triggering of costimulatory pathways augments the initiation and/or efficacy of immune responses against the tumor; hence, it was conceived that aiming costimulatory molecules to the cell surface of tumors renders them immunogenic. Recombinant fusion proteins containing the costimulatory molecule B7 were able to mediate anti-tumor effects if directed to the tumor (Moro et al. 1999). Grosse-Hovest and colleagues improved this approach by generating a bispecific Ab which allowed the targeting of an anti-CD28 Ab to melanoma cells (Grosse-Hovest et al. 2003). This bispecific antibody mediated effective tumor eradication by activation of non-specific cytotoxic cells. The therapeutic potential of several other bispecific diabodies is currently being tested in vitro and in vivo in preclinical models. Diabodies specific for CD19 and CD3 or CD19 and CD16 mediated lysis of tumor cells by T cells or mononuclear cells, respectively (Bruenke et al. 2005) (Fig. 15.4). Interestingly, a synergistic anti-tumor effect of anti-CD19/CD3 and anti-CD19/CD16 diabodies was obvious in a murine non-Hodgkin’s lymphoma model (Kipriyanov et al. 2002). Furthermore, anti-p-glycoprotein/CD3 diabodies in combination with activated PBLs inhibited the growth of xenograft tumors expressing p-glycoprotein (Gao et al. 2004). The use of multivalent antibodies is likely to further improve this strategy; a trispecific Ab targeting simultaneously CD3 and CD28 and the tumor abolished the requirement of pre-activated T cells for tumor lysis (Wang et al. 2004).

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Fig. 15.4. Mode of action of immune modulating or apoptosis inducing antibody fusion proteins. Bispecific antibody fragments allow the targeting of immunomodulatory molecules to the tumor cell. As exemplified, anti-CD16/CD19 diabody or anti-CD19/CD3 bispecific scFv can enhance the NK-cell or T-cell response, respectively, to B-cell lymphomas. Triggering of death receptors by targeting extrinsic factors such as FasL or TRAIL to the cell surface of tumor cells can induce apoptosis in an autocrine as well as a paracrine way

15.6 Conclusions Monoclonal antibodies have emerged as important therapeutic agents for the treatment of malignant disease. Indeed, they have proved to be well tolerated and effective for the treatment of different cancers and have consequently been approved by the FDA. Besides their role as cancer therapeutics per se, their targeting potential earmarks them as compounds able to increase selectivity of different kinds of therapeutic measures. For some of these measures, such as toxins, fusion to such a targeting moiety is inevitable in order to use them therapeutically. However, for their broader use as a targeting device a few problems still have to be addressed, namely (1) immunogenicity, (2) selectivity

and (3) penetration into solid tumors. The generation of fully human antibodies will solve the problem of the inherited immunogenicity of earlier constructs. To increase the selectivity of antibodies and thus reduce unspecific toxicity of fusion proteins, new tumor antigens and the respective mAb have to be identified. This can be achieved by phage display technology, which not only enables the identification of differentially expressed antigens but can also be used to isolate respective Abs (Trepel et al. 2002). Notably, the behavior of the antigen upon antibody binding should be taken into account with respect to the delivered drug; those approaches aiming at the direct killing of the tumor cells require antigens which become internalized upon binding, whereas those modulating biological processes should stay on the surface of the tumor cells. Since lympho-hematopoetic malignancies are easier to access, the body of evidence for these diseases is much larger and most of the targeted therapies approved so far are for these indications. In order to affect solid tumors, the therapeutic agent must overcome several obstacles including the vascular endothelium, stromal and epithelial barriers and high interstitial pressure (Stohrer et al. 2000). In this regard, smaller recombinant mAb constructs like single chain antibodies should overcome some of these problems (Yokota et al. 1992), but have the disadvantage that they are more rapidly cleared from the plasma (Adams et al. 1998). An alternative approach for solid tumors is to target the tumor microenvironment in general and the endothelium of tumor blood vessels in particular (Hofmeister et al. 2005). The advantages of targeting the tumor vasculature are accessibility, the relative genetic stability (thereby avoiding antigen loss variants), and that many tumor cells, irrespective of their antigen expressing profile, are hit via this route. Recent years have demonstrated the wide variety of agents which can be targeted to tumors. In preclinical models, these tumor targeted therapies have revealed impressive results. The clinical value for targeted cancer therapy has to date only been confirmed for conjugates of cytotoxic drugs and radionucleotides. However, the success of tumor-specific mAbs per se is fueling optimism towards this attractive approach.

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Part III

Safety and Perspectives

III

Chapter 16

Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies C.K. Schneider, J. Löwer

16.1 Introduction Based on his observations from selective histological staining of bacteria, Paul Ehrlich in 1900 published his idea that certain compounds could be used as “magic bullets” to selectively target external pathogens or even tumours (Ehrlich 1900). With the description of the hybridoma technology by Köhler and Milstein in 1975, the production of targeted monoclonal antibodies (mAbs) became possible and Paul Ehrlich’s dream of magic bullets a reality. Currently, more than a dozen mAbs are licensed (Reichert et al. 2005), and more promising products are about to enter clinical development. While the first antibodies were of completely murine origin due to the underlying technology, scientists started an “evolution” of mAbs in order to reduce immunogenicity (Borchmann et al. 2001). Murine proteins are highly immunogenic, and continuous use in humans in most cases is not possible due to the occurrence of neutralizing antibodies, leading to loss of efficacy and/or safety problems like infusion reactions. Therefore, chimaeric antibodies such as infliximab (Remicade) were developed, reducing immunogenicity by replacement of the murine Fc part by the human counterpart. Still being considerably immunogenic, monoclonal antibodies were further “humanized” also by replacing large parts of the Fab part of the molecule by human sequences (for example, in trastuzumab, Herceptin). With the latest techniques, the production of fully human mAbs has also become possible, further reducing immunogenicity to a small but still existent extent. The only fully human mAb licensed so far in Europe is adalimumab (Humira), an anti-TNF- [ mAb (Salfeld and Kupper 2007). Despite these achievements in a more structural “evolutionary chain” of mAbs, mechanisms of action

are also evolving. While the “classical” mAbs have rather clear-cut hypotheses as regards the way they function, for example targeting tumour epitopes on the surface of tumour cells, newer mAbs appear to be becoming more specific, targeting distinct subepitopes of certain structures. A recent example is the anti-CD28 mAb TGN1412, which is directed against a certain substructure of the CD28 molecule, the C’’D loop (Luhder et al. 2003), exhibiting a distinct pharmacodynamic effect, representing a so-called “super-agonist”. This product has gained unfavourable publicity due to serious adverse events during testing in a first-in-man trial (Schneider et al. 2006; Suntharalingam et al. 2006). Cases like this demonstrate that therapeutic intervention with biologics can be harmful to a considerable extent, and that safety is a central aspect to be considered for these products. In this context it is important to note that possible adverse events might be deducible from the (putative) mechanism of action of a particular compound, for example potential toxicity to skin keratinocytes of mAbs directed against epidermal growth factor receptor-1 (EGFR-1), like cetuximab (Erbitux). However, experience clearly shows that such considerations might not be sufficient, and that other aspects need to be taken into account. mAbs might exhibit a certain fine specificity that discriminates them from others, although directed against the same antigen. As discussed above, TGN1412 might again serve as an example. Another important aspect influencing safety of mAbs is the design of the antibody with respect to the isotype. An IgG1 mAb can be expected to behave differently from, for example, an IgG4 mAb, although directed against the same epitope. This can be explained by the different effector mechanisms mediated by the Fc parts of the IgG isotypes. While IgG1 can fix and activate complement, thereby triggering complement-mediated cytotoxicity when directed against a

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cell-membrane bound antigen, IgG4 might be expected to only bind to and block the epitope, potentially without directly affecting the viability of the target cell, since IgG4 cannot activate complement. Monoclonal antibodies are under development with mutated Fc parts, modulating the interaction with Fc receptors. For such molecules, the possible effects cannot solely be imputed by considerations about blockage of the epitope. Chemical modification such as PEGylation can change pharmacokinetic behaviour, tissue penetration capacity, and immunogenicity, thereby considerably changing the pharmacodynamic behaviour of a mAb. Therapeutic intervention with mAbs can be achieved by several principles, for example, neutralization of soluble and/or membrane bound cytokines, blockage of membrane-bound molecules that mediate intercellular communication, and blockage of growth factor receptors with or without triggering of cell death. This chapter highlights recent examples of licensed mAbs representative for each of these mechanisms, discusses specific safety aspects concomitant with these interventions, and briefly describes the regulatory measures aiming at a reduction of these identified risks.

16.2 Intervention with Pleiotropic Cytokine Pathways One of the major breakthroughs in the development of efficacious medicines against various autoimmune diseases including rheumatoid arthritis and Crohn’s disease (CD) has been the introduction of the anti-TNF- [ principle (Taylor 2007; Salfeld and Kupper 2007). One of the key mediators of autoimmune inflammation is TNF- [ , a multipotent cytokine occurring both in a soluble and a non-soluble, membrane-bound form. Produced mainly by macrophages and T cells, TNF- [ induces a variety of further cytokines, resulting in a pleiotropic biological role in inflammation, host defence including mycobacteria, and cancer immunity (Beutler 1999). With the introduction of TNF- [ inhibitors, the therapeutic arsenal against RA and other autoimmune diseases was amended by a potent new mechanism of action. Three drugs are licensed, two monoclonal antibodies (infliximab, Remicade, and adalimumab, Humira) and one fusion protein consisting of the extracellular portion of the p75 TNF receptor fused to the Fc part of human IgG1 (etanercept, Enbrel). Remi-

cade was the first monoclonal antibody against TNF- [ to be licensed in Europe, and therefore a relatively large safety database exists for this product. Remicade was first licensed in the EU for the treatment of severe, active Crohn’s disease under “exceptional circumstances” (European Medicines Agency 2006a) due to promising efficacy in this frequently severe autoimmune disease. Remicade was licensed for treatment of patients who have not responded despite a full and adequate course of conventional treatment such as a corticosteroid and/or an immunosuppressant. However, after marketing authorization in this second line setting (i.e. requiring an immunosuppressor OR corticosteroids), the “efficacy” of Remicade became apparent also in a negative sense, since due to the strong inhibition of TNF- [ serious adverse events were also observed, such as reactivation of tuberculosis (TB), severe infections, and sepsis. Occurrence of serious infections is the key safety problem. Data suggest that TNF- [ plays a physiological role in the formation and maintenance of granulomas, a host defence mechanism against intracellular pathogens that cannot be killed by other mechanisms (e.g. mycobacteria) (Wallis and Ehlers 2005). Treatment with TNF- [ inhibitors apparently leads to delayed formation of these granulomas or the disruption of existing ones, resulting in reduced or even abolished host defence against certain pathogens like mycobacteria. Accordingly, cases of tuberculosis reactivation and other opportunistic infections have been reported in the post-marketing setting. Such reports have also been reported in Germany, as illustrated in the reports in 2004 of opportunistic infections to the competent national authority responsible for these kinds of products, the Paul-Ehrlich Institut (Table 16.1). These data from the reporting period of 2004 are representative results. A recent report in the literature describes a clinical case of a patient who developed widespread pulmonal TB during long-term treatment with infliximab for ankylosing spondylitis despite a 9-month isoniazide prophylaxis, showing a delayed response even to a five-drug anti-tuberculosis treatment (Vlachaki et al. 2005). Cases of patients like this, which are very difficult to treat, illustrate that the use of biologics like Remicade needs to be thoroughly considered on a case-by-case basis. Based on the observations on serious infections and also other safety issues in the time after marketing authorisation, an expert panel had convened at the European Agency for the Evaluation of Medicines (EMEA), and in early 2002

16.3 Intervention with Adhesion Molecules Table 16.1. Opportunistic infections with infliximab (Remicade) during February to August 2004 reported to the Paul-Ehrlich Institut, Germany Pathogen

No. of Deaths Crohn’s RA Other reports

Pneumocystosis 13 Atypical mycobacteria 8 TB 92 Histoplasmosis 8 Listeriosis 3 Aspergillosis 2 Coccidioidomycosis 15 CMV 14 Systemic candidiasis 9 Blastomycosis 2 Toxoplasmosis 1 Nocardia 5 Herpes 25 Total 200

2 0 11 3 0 0 2 6 4 1 0 0 1 50

0 1 13 1 1 1 0 3 6 1 0 2 6 35

11 5 55 7 1 1 14 7 1 0 1 1 18 122

2 2 24 0 1 0 1 4 2 1 0 2 1 39

the indication for the treatment of CD was restricted. This was implemented by the EMEA’s scientific committee, the Committee for Proprietary Medicinal Products (CPMP, now CHMP, see below), by an urgent safety restriction (European Medicines Agency 2006a). The resulting indication was a restriction on patients not responding to a full and adequate course of treatment with a corticosteroid AND an immunosuppressant (instead of “and/or”), resulting in a restriction to a third line indication. Narrowing the patient population to a more restricted subset is a strong regulatory measure, and one of the regulatory principles intended to ensure safe use of medicines only for patients where the benefits clearly outweigh the risks. To further enhance patient safety and also awareness both of patients and prescribers, a patient alert card was introduced in 2002, which has proven to represent a powerful tool for this purpose. Meamwhile, the indication for Remicade in the treatment of Crohn’s disease has been changed back to a second line indication, based on a more solid database that has emerged over the years.

16.3 Intervention with Adhesion Molecules Another promising principle of interference with the immune system is the inhibition of adhesion molecules on T lymphocytes, like integrins. Integrins are heterodimeric cell adhesion molecules, playing pleiotropic roles in fetal development, immune reactions, leuko-

cyte migration, haemostasis, and tumour biology (Hynes 2002). T cells in the blood circulation adhere to endothelium by interaction of alpha-beta integrins on their surface (Lobb and Hemler 1994). In 1993 it was established that T-cell migration into brain parenchymal tissue is mediated by a distinct subset of these integrins, the [ 4 q 1 integrin molecule (Baron et al. 1993), interacting with counter-receptors on endothelial cells. Observations like this lead to the idea of inhibiting Tcell migration in diseases where T cells are key mediators, like multiple sclerosis (MS), which is the most common inflammatory autoimmune disease of the central nervous system (Gold and Hohlfeld 2007). Antibodies against [ 4 integrin on T cells were shown to inhibit the MS animal model, experimental autoimmune encephalomyelitis (EAE) (Yednock et al. 1992). A humanized antibody was developed (natalizumab, Tysabri, formerly “Antegren”), which binds to the [ 4 subunit of human [ 4 q 1 and [ 4 q 7 integrin, for the treatment of patients with MS. First clinical data from patients dosed for 6 months revealed a nearly subtotal inhibition of inflammation, as measured by uptake of contrast medium in Magnetic Resonance Imaging (MRI) (Miller et al. 2003). These promising data also translated into clinical benefit, resulting in a significant reduction of relapse rate and delay in the progression of disability as measured by the Expanded Disability Status Score (EDSS) after 2 years of treatment (Polman et al. 2006). Based on 1-year data, which already showed a clinical benefit in terms of relapse rate reduction, the US Food and Drug Administration (FDA) granted priority review status for the regulatory assessment of the 1-year clinical data in June 2004, and granted an accelerated approval of Tysabri in November 2004. In mid February 2005, the marketing authorization holder published key 2-year efficacy data, but around 2 weeks later on 28 February 2005 announced a voluntary suspension of the marketing authorization due to the occurrence of a serious unexpected adverse event, progressive multifocal leukencephalopathy (PML). This event evolved in two patients in the long-term MS trials, both being concomitantly treated with beta-interferon (Kleinschmidt-DeMasters and Tyler 2005; Langer-Gould et al. 2005). One of the patients died, and one survived with severe disability. All clinical trials were immediately suspended, and no further drugs were administered to patients. PML is a rare non-inflammatory demyelination (Astrom 1958) mediated by reactivation of JC virus, a polyoma virus. PML is mainly seen

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in patients with severe immunosuppression, e.g. individuals with AIDS. The most probable explanation of how PML could have occurred in the two patients is that Tysabri potently suppresses the migration of any T cell, not only pathogenic autoreactive cells, but also T cells circulating for detection and elimination of viruses and other pathogens (T-cell surveillance). As a consequence, reactivation of viruses is thought to become insufficiently controlled by T-cell surveillance. Following these events, the marketing authorization holder evaluated most patients having been treated with Tysabri and found no other case of PML, despite a retrospective case of a patient who had died of PML in a clinical trial for Crohn’s disease (Van Assche et al. 2005). Based on the observed clinical efficacy and the medical need in the treatment of patients with MS, the Committee for Medicinal Products for Human Use (CHMP, until 2004: CPMP) at the European Medicines Agency EMEA (until 2004: European Agency for the Evaluation of Medicines) issued a positive opinion in April 2006, recommending a marketing authorization for Tysabri for the European Union to the European Commission. This was based on a clear-cut restriction of the patient population, firstly patients with high disease activity despite treatment with a beta-interferon, and secondly patients with rapidly evolving severe relapsing remitting multiple sclerosis. Together with a strict risk management system, educational programmes including algorithms for the detection of PML and other opportunistic infections, and a patient alert card, the CHMP considered the benefit-risk positive for the aforementioned patient population with more severe MS (European Medicines Agency 2006b). The European Commission granted a marketing authorization for Tysabri in the aforementioned setting at the end of June 2006. In June 2006, the US FDA also reintroduced Tysabri to the US market.

16.4 Intervention with Growth Factor Receptors Tumour cells frequently harbour growth factor receptors and/or structures that are rather restricted to these cells. The idea of using the immune system by specifically targeting these molecules with mAbs is one of the classical concepts in the therapeutic interventions with mAbs. Several antitumoural antibodies are licensed, e.g. cetuximab (Erbitux), rituximab (MabThera), alem-

tuzumab (MabCampath), and trastuzumab (Herceptin). The latter antibody, trastuzumab, is directed against the Her2/neu antigen (human epidermal growth factor receptor 2 protein) expressed on a subset of breast cancers (approximately 30 % of cases), and in Europe until recently was only licensed for the treatment of metastatic breast cancers which express a high level of this antigen, either as a “last line” monotherapy, or in combination with taxanes as first-line combination therapy. Her2/neu is a member of the epidermal growth factor receptor (EGFR) family and is encoded by c-erb B-2 (the human homologue of the rat neu oncogene), a proto-oncogene that codes for a transmembrane glycoprotein receptor tyrosine kinase (European Medicines Agency 2006c). Its physiological role is thought to be part of tissue homeostasis including lobuloalveolar differentiation and lactation in physiological breast tissue. Further, it participates in normal development and organogenesis of nervous and heart tissue (Olayioye et al. 2000; Yarden and Sliwkowski 2001). Adding Herceptin to chemotherapy exhibited a potent cytotoxic effect on breast cancers in clinical trials, leading to a market authorization of the mAb in 2000 in the European Union (European Medicines Agency 2006c). Soon it became clear that some patients treated with Herceptin showed signs and symptoms of cardiotoxicity, especially in combination or after treatment with anthracyclins. This unwanted effect on the adult organism cannot readily be explained by its physiological role in embryonic development. In preclinical studies during the clinical development programme before marketing authorization application, no tissue cross-reactivity with monkey or human heart tissue was observed, and no cardiotoxicity in a relevant animal surrogate model was noted (European Medicines Agency 2006c). Animal experiments performed subsequently show that selective Her2/neu deletion in left ventricular tissue in mice yields an apparently normal phenotype; however, mice with this deletion show signs of dilatative cardiomyopathy, and cardiomyocytes are more susceptible to anthracyclin-induced cardiotoxicity (Crone et al. 2002). Bioptic evaluations suggest that Herceptininduced cardiotoxicity could be distinct from that of anthracyclins (Ewer et al. 2005). While most cardiac events are usually asymptomatic and reversible, severe cardiac events have also been reported. Most events occur in combination with anthracyclins, which are themselves cardiotoxic compounds. As a result, the

References

warning statements in the European Summary of Product Characteristics (SmPC) were strengthened. Cardiac monitoring before and during therapy with Herceptin has become standard practice. Recently, the license for Herceptin has been extended to the treatment of patients with HER2 positive early breast cancer following surgery, chemotherapy (neoadjuvant or adjuvant) and radiotherapy.

16.5 Conclusion Biologics like monoclonal antibodies are innovative molecules that have in some cases revolutionized modern medicine due to unprecedented efficacy; however, experience shows that this efficacy also expands to unwanted side effects. Several principles of how mAbs can target human autoimmune diseases are known, ranging from intervention with pleiotropic cytokine pathways to inhibition of T-cell migration. Direct cytotoxicity is a classical principle of targeted therapies against tumours. All of these mechanisms are inherently associated with certain safety problems; however, regulatory measures can be implemented that nevertheless allow for a relatively safe administration of such drugs to patients. Such regulatory measures include a restriction of the indication to certain patients, usually those suffering from a more severe stage of the disease, and provision of educational information to prescribers and patients. Further research is needed to elucidate the exact mechanisms of action behind the typical safety problems. While most hypotheses appear convincing, still other unknown mechanisms could be responsible, and detailed knowledge could help further reduce the risks for patients.

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Borchmann P, Riethmuller G, Engert A (2001) [Monoclonal antibodies: development and clinical prospects] [in German]. Internist (Berl) 42(6):803 – 4, 807 – 814 Crone SA, Zhao YY, Fan L, Gu Y, Minamisawa S, Liu Y, Peterson KL, Chen J, Kahn R, Condorelli G, Ross J, Jr, Chien KR, Lee KF (2002) ErbB2 is essential in the prevention of dilated cardiomyopathy. Nat Med 8(5):459 – 465 Ehrlich P (1900) On immunity with special reference to cell life. Proc R Soc 66:424 – 448 European Medicines Agency (2006a) Remicade European Public Assessment Report. EMEA, London. http://www.emea.europa .eu/humandocs/Humans/EPAR/remicade/remicade.htm European Medicines Agency (2006b) Tysabri European Public Assessment Report. EMEA, London. http://www.emea. europa.eu/humandocs/Humans/EPAR/tysabri/tysabri.htm European Medicines Agency (2006c) Herceptin European Public Assessment Report. EMEA, London. http://www.emea.europa .eu/humandocs/Humans/EPAR/herceptin/herceptin.htm Ewer MS, Vooletich MT, Durand JB, Woods ML, Davis JR, Valero V, Lenihan DJ (2005) Reversibility of trastuzumab-related cardiotoxicity: new insights based on clinical course and response to medical treatment. J Clin Oncol 23(31):7820 – 7826 Gold R, Hohlfeld R (2007) Multiple sclerosis: new immunobiologics. In: Boehncke W-H, Radeke HH (eds) Biologics in general medicine, Chapter 13. Springer-Verlag, Heidelberg [this volume] Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110(6):673 – 687 Kleinschmidt-DeMasters BK, Tyler KL (2005) Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med 353(4):369 – 374 Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256 (5517):495 – 497 Langer-Gould A, Atlas SW, Green AJ, Bollen AW, Pelletier D (2005) Progressive multifocal leukoencephalopathy in a patient treated with natalizumab. N Engl J Med 353(4):375 – 381 Lobb RR, Hemler ME (1994) The pathophysiologic role of alpha 4 integrins in vivo. J Clin Invest 94(5):1722 – 1728 Luhder F, Huang Y, Dennehy KM, Guntermann C, Muller I, Winkler E, Kerkau T, Ikemizu S, Davis SJ, Hanke T, Hunig T (2003) Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists. J Exp Med 197(8):955 – 966 Miller DH, Khan OA, Sheremata WA, Blumhardt LD, Rice GP, Libonati MA, Willmer-Hulme AJ, Dalton CM, Miszkiel KA, O’Connor PW; International Natalizumab Multiple Sclerosis Trial Group (2003) A controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 348(1):15 – 23 Olayioye MA, Neve RM, Lane HA, Hynes NE (2000) The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 19:3159 – 3167 Polman CH, O’Connor PW, Havrdova E, Hutchinson M, Kappos L, Miller DH, Phillips JT, Lublin FD, Giovannoni G, Wajgt A, Toal M, Lynn F, Panzara MA, Sandrock AW; AFFIRM Investigators (2006) A randomized, placebo-con-

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16 Safety Aspects of Biologics: Lessons Learnt from Monoclonal Antibodies trolled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med 354(9):899 – 910 Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC (2005) Monoclonal antibody successes in the clinic. Nat Biotechnol 23(9):1073 – 1078 Salfeld J, Kupper H (2007) Adalimumab. In: Boehncke W-H, Radeke HH (eds) Biologics in general medicine, Chapter 3. Springer-Verlag, Heidelberg [this volume] Schneider CK, Kalinke U, Löwer J (2006) TGN1412 – a regulator’s perspective. Nat Biotechnol 24(5):493 – 496 Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, Panoskaltsis N (2006) Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med Sep 7; 355(10):1018 – 1028 Taylor PC (2007) Biologic therapies for rheumatoid arthritis targeting TNF- [ and IL-1. In: Boehncke W-H, Radeke HH (eds) Biologics in general medicine, Chapter 11. SpringerVerlag, Heidelberg [this volume]

Van Assche G, Van Ranst M, Sciot R, Dubois B, Vermeire S, Noman M, Verbeeck J, Geboes K, Robberecht W, Rutgeerts P (2005) Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn’s disease. N Engl J Med 353(4):362 – 368 Vlachaki E, Psathakis K, Tsintiris K, Iliopoulos A (2005) Delayed response to anti-tuberculosis treatment in a patient on infliximab. Respir Med 99(5):648 – 652 Wallis RS, Ehlers S (2005) Tumor necrosis factor and granuloma biology: explaining the differential infection risk of etanercept and infliximab. Semin Arthritis Rheum 34(5 Suppl 1):34 – 38 Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127 – 137 Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N (1992) Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature 356(6364):63 – 66

Chapter 17

New Biological Therapeutics in the Genome Age 17 T.N.C. Wells, S. Schnieper-Samec

17.1 Introduction The availability of the human genome sequence in the last decade has powered a new wave of protein therapeutics. Using a wide variety of bioinformatics technologies, it has been possible to identify a nearly complete list of all secreted proteins including important biotherapeutic classes of cytokines, growth factors and hormones. The challenge ahead is to identify the function of these secreted and cell surface proteins. A combination of classic low throughput and large scale industrialized biology approaches (functional genomics) promises to identify the roles of the newly discovered genes as well as to assign new roles for known proteins in both normal homeostasis and in the development and resolution of disease. Only a few proteins will be directly converted into protein therapeutics whereas others might become targets for monoclonal antibodies, the fastest growing segment of biotherapeutics. Key to the success of therapeutics is the validation of the target in human disease(s). Recent advances in genotyping technologies now allow us to scan for genetic associations on a whole genome basis. This technology together with target validation approaches applying molecular biology, cell biology and pharmacology defines a new paradigm that will increase our confidence in progressing molecules towards the clinic. As we learn more about the biological and therapeutic function of the first generation of protein therapeutics, new possibilities are emerging for second generations of modified proteins. Technologies are available that will increase the therapeutic efficacy of these parent products, by enhancing their potency and time of action, among other properties.

Fig. 17.1. The pyramid of new therapies from the human genome. The top level, finding new therapeutic hormones, is the most difficult – since most hormones have already been identified based on cellular or physiological activity. The second level, finding new activities for existing proteins, is arguably more fruitful. Many proteins are pleiotropic, they have many activities, and often the initial discoverers focused only on one indication. The third level is that of protein engineering or antibody generation – where we tailor the biological activities to the needs in disease pathology, rather than physiology. The fourth level is that of replacing the proteins with small molecules or other technologies to improve the convenience for our patients. The increase in width of the pyramid as we descend describes the increase in the number of possible solutions to the problem

Proteins – natural or engineered – and antibodies along with small molecules form the pyramid of our therapeutic arsenal (Fig. 17.1). Combining the access to the entire catalogue of secreted proteins with the ability to gain insights into the human disease through genetics, places us in a unique position to effectively find the protein therapeutic medicines of tomorrow.

17 New Biological Therapeutics in the Genome Age

17.2 Early Biotechnology Production of Human Cytokines and Hormones

early days finding the sequence was the difficult part. Now the challenge is to find the biological activities for orphan sequences that look like cytokines.

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Fig. 17.3. The market size for existing biotech products and projected markets over the next 3 years. a Most of the power of the biotechnology industry comes from classic products – molecules which were first identified by their biological activities, and then cloned and produced. b The second wave is the development of monoclonal antibodies, enabling us to block key responses, and leading to several blockbuster products. The commercialization of the genome era cytokines will follow on after this wave, with molecules such as TACI IG in the vanguard 19 80

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Over the past 20 years, the biotechnology industry has been extraordinarily successful in bringing a wide variety of new products to the market. It is often perceived that the biotechnology industry has been faster than the traditional pharma industry in bringing products to the market. However, the list (from insulin through growth hormones and gonadotrophins, to EPO) shows that the road in the early days was often long and difficult, and started from an ’in vivo’ demonstration of activity using crude extracts (Fig. 17.2). Indeed, the majority of the early products of biotechnology were proteins from natural sources (insulin, growth hormone) purified based on biological activity; subsequent protein sequencing was obtained, thus allowing cloning from cDNA libraries only when the recombinant DNA technology became available in the late 1970s. The triumph of the biotech industry has been in finding ways to produce the molecules on a large scale, and at a quality that allows their safe administration to humans (Fig. 17.3a). In the mid 1990s, large amounts of data from expressed sequence tag projects (Wells and Peitsch 1997; Yee and Conklin 1998) became publicly available, allowing us to rapidly identify homologous sequences or little brothers of the known cytokines. Now the scientific challenge had been turned on its head – in the 19 00

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Fig. 17.2. Timelines for the development of new proteins as therapeutics. Prior to 1995, all therapeutic proteins had been identified based on their biological activities and cloned. TACI represents a new generation of proteins, identified based on sequence with the biological activity determined afterwards. This has been a much faster route, although arguably with a much higher rate of attrition (there are still relatively few success stories). TREM-1 triggering receptor expressed on myeloid cells, BCSP-1 bone and cartilage stimulating peptide-1

17.4 Assembling the Complete Protein Collection: The Serono Secretome

17.3 Finding New Cytokine Orphans in the Human Genome: Early Excitement from Expressed Sequence Tags High throughput sequencing technologies were used to sequence the first 300 – 400 bases at the 5’ and 3’ ends of cDNA clones – a technology known as expressed sequence tags. No attempt was made to define the biology linked to these sequences, but they were deposited in their hundreds and thousands in public – particularly by the University of Washington in collaboration with Merck (Hillier et al. 1996; Aaronson et al. 1996; Marra et al. 1998) and private databases, with companies such as Human Genome Sciences building their own proprietary databases in parallel. The power of this technology is illustrated by the effect it had on chemokine search – a family of proteins playing a major role in cell migration. Chemokines are small proteins, around 80 amino acids, and so in many cases the entire coding sequence could be found on a single EST. Initially, searches based on sequence similarity at the level of DNA or protein were used, such as BLAST (Altschul et al. 1990). The result was that in a few months over ten new chemokines were found (for a review see Wells and Peitsch 1997). The challenge that has occupied the field since then has been to find the biological activity of the proteins: whereas it took only 10 weeks to identify a new wave of chemokines, in some cases it took up to 10 years to identify their function. In the majority of cases, it was clear that these proteins were pro-inflammatory, and therefore of potential interest as antibody targets. However, some chemokines such as MPIF-1 (Patel et al. 1997) and SDF-1 (Jankowski et al. 2003) have been shown to have activity on stem cells – making them potential therapeutic proteins in their own right. Unfortunately, the clinical results in this area have so far been disappointing – since neither MIP-1 [ (the original chemokine stem cell factor) nor MPIF (a more potent version) showed impressive effects clinically. Interestingly, ESTs collected using sequences of pathogens have led to the identification of several new chemokines in the genomes of viruses (Kledal et al. 1997; Wells and Schwartz 1997), and their characterization has given us an insight into the way viruses can control our physiology. These examples of chemokine identification have taught us that it is possible to find interesting new active cytokines and hormones using these new geno-

mic technologies. Nevertheless, we still need to gain a better understanding of how they intervene in human physiology, a topic we will address below under the scope of moving to a protein’s biological activity for targeting. Moreover, the question remained how we could apply this approach to assemble a complete collection of the targetable proteins from the genome, namely the secreted or transmembrane proteins as well as the decoy proteins – the so-called “secreted proteome” or “secretome”.

17.4 Assembling the Complete Protein Collection: The Serono Secretome Human genome sequencing opened up an even more complete view of the rich variety of human secreted proteins – the majority of which had not been studied in depth. The number of secreted proteins coded by the human genome has been a matter of extensive debate – with ranges of up to 8,000 (Kramer and Cohen 2004), many of which will not be druggable as defined by Hopkins (Hopkins and Groom 2002). We therefore set out to assemble a collection of 2,000 – 3,000 secreted proteins – our requirement being that any protein in our collection should be purified, and be characterized by SDS PAGE. The proteins are produced in transient transfections in human cells, which ensures that their glycosylation patterns resemble the natural pattern as much as possible. Typical expression levels of the most highly expressed proteins are 500 μg and are sufficient to determine an early biological activity in vitro. In order to assemble such a collection, we followed a variety of different approaches (see Fig. 17.4). 1. Signal sequence trapping. Early work to identify secreted proteins focused on the construction of cDNA libraries that could be enriched for secreted proteins using a technique called signal sequence trapping (Tashiro et al. 1993). The alternative approach to biological sequence tag selection was to use bioinformatics approaches to recognize signal sequences. This was the focus of the Genset Signal Tag collections first produced in 1997 (WO200037491). These sequences (almost 1,000 of them) have been expressed and purified, allowing us to then test these proteins in parallel in high content cell biology assays to find new biological activities.

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Fig. 17.4. The secretome. Our collection of secreted proteins comes from a variety of sources – from early database searching using signal sequences, through more and more complex bioinformatics sources. It is also clear that there are interesting proteins yet to be found, even more than10 years after the start of EST sequencing and 5 years after the completion of the human genome sequence

2. Multiple alignment searches in families of proteins. It is clear that the majority of cytokines have similar three-dimensional folds. The chemokines mentioned above have a three-stranded beta sheet and a carboxy terminal alpha helix. Most of the interleukins and growth factors are built from a bundle of four alpha helices. Doing a BLAST search with the simple sequence of the protein does not always find other members of the same structural class: interleukin (IL)-2 will not find IL-3, and IL-4 will not find IL-5 despite them all being four helix bundles (see Wells et al. 1994). Thus, more sensitive approaches have used multiple sequence alignment approaches, where profiles are built up of the average of the members of a family and these are used to search (programs such as PSI BLAST). Zymogenetics identified a whole range of post-genomic cytokines, including many new TNF family members and four helix bundles (Foster et al. 2004). These include IL20, IL-22, IL-24 and IL-31 and most importantly the B-cell modulator TACI (transmembrane activator and CAML inhibitor; Gross et al. 2000). A fusion protein, TACI-Fc, is now in clinical development for autoimmune diseases. 3. Using the three-dimensional structures of proteins – threading. More complex analysis of the proteins imbedded in the human genome was possible using threading. This is a technology where protein sequences are compared to all the known threedimensional folds for proteins, and therefore is looking for new proteins based on shape (Miller et al. 1996). We have been collaborating with Inphar-

matica since 2001 and have found almost 200 putative new open reading frames coding for new proteins and annotations. This technology requires an extremely large computing power, if the whole of the genome is used, and its development has been possible because of the availability of massive parallel computing systems. 4. Beyond the genome – new approaches to finding new secreted proteins Although the human genome sequence has been available now for 5 years, there are still new secreted proteins to be found. First, we know that splice variants exist in large numbers (Graveley 2001), and that some have interesting biological activities. Second, whole genome scans, where we are looking at differences in single nucleotide polymorphisms (SNPs) across large populations, are now relatively inexpensive. Using these approaches we can find SNPs linked with disease where no known protein is expressed – allowing the discovery of new disease-associated proteins. Approaches such as genomic tiling arrays (Bertone et al. 2006) can identify novel open reading frames in these areas – some of which have encoded previously unidentified secreted proteins amongst the non-coding RNAs (Washietl et al. 2005). Putting all this together, we have been able to assemble a collection of over 2,000 secreted proteins. This compares with an earlier collection of over a thousand genes, the Secreted Protein Discovery Initiative (Clark et al. 2003). In order to be part of our secretome collection, the cDNAs were inserted into a standard vector, and we purified protein on a highly parallel automated system. In comparison with earlier approaches, we have concentrated on the quality of the protein as an entry criterion into our collection. Work with combinatorial chemistry has taught us that approaches that fail to check if the protein is actually being produced generally result in a lot of lost time following up on false positives.

17.5 Moving from the Protein to the Biological Activity: The Post-Genome Era In the search for new therapeutic and/or biological functions, we first applied various biological tests to our protein collection. We thus work on high content rather than high throughput assays, meaning that rath-

17.5 Moving from the Protein to the Biological Activity: The Post-Genome Era

Fig. 17.5. Identification of osteopontin as a nerve remyelination factor (Selvaraju et al. 2004). Osteopontin was shown to be up-regulated in EAE samples in areas of active remyelination, and the protein was shown to be the most active cytokine from our collection in terms of nerve remyelination in coculture models. We have since been able to demonstrate that as well as reversing the pathology, osteopontin is able to restore function in rodent models of nerve crush

er than miniaturizing assays to increase throughput and reduce costs, we have transformed more and more complex biology to 96-well formats. For example, in our field of multiple sclerosis, we have pioneered a way to study remyelination of neurons in 96-well assays – a technology involving several cell types, a 21-day incubation period, and complex image analysis. It was this technology that led to the identification of several new proteins involved in neuronal repair – of which osteopontin is the forerunner (Fig. 17.5). In the immunology field we were the first to link the FACS cell sorter to our robotics system – and now routinely examine the cytokine production profile of cells (Besson et al. 2003). Whenever possible, we have used human cell or cellbased assays that reflect as closely as possible the mechanism of human physiology and pathology ’in vitro’. Out of our collection of 2,000 proteins and using approximately 10 assays, a large quantity of data points need to be collected. Proteins showing activity are moved in vivo using either of the three following approaches. First is the classical production and purification of the milligrams of protein for direct injection into animal models. Some of these models involve simple inflammatory stimuli such as LPS, concanavalin A or TNF to activate the immune system. Second, more rapid progress can be made with our technology for direct expression of the cDNA ’in vivo’ (see the example for IL18 bp in Mallat et al. 2001). This allows a rapid read-out of the biological activity, bypassing the need to express and purify the protein.

Third, in vivo data can also be obtained with a knock-out of the murine protein. Replacement of the gene of interest with LacZ allows us to understand the expression pattern of the protein, which can shed light on further disease models to be tested. The validity of this high throughput screening approach is underlined by the results obtained when known drug targets have been knocked out (Bolon and Galbreath 2002; Zambrovicz and Sands 2003, 2004). Given the amount of information produced by the above approaches, one of our key strategic advantages has been in investment in data processing, including treatment and analysis, so that we can easily distinguish real activities from false positives. Although we set out to find novel therapeutic proteins, we also identify in many cases new activities for known proteins. Often the first biological activity found has tended to bias a research activity. This has always been an issue – interferons for instance were originally identified based on their antiviral activity but interferon gamma is today used to treat lymphoma following autologous peripheral blood stem cell transplantation (Ohno et al. 2005), and interferon beta is an effective treatment for multiple sclerosis. From our protein pipeline, we have a number of projects now progressing through early preclinical studies, which shows the power of such approaches to identify new biological activities. Our next stage is to test these proteins in more and more diverse types of cellular biology, for which we are currently building a wide range of collaborations.

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17.6 Strategies for Blocking Responses Apart from identifying proteins that ameliorate pathological responses, we have also found proteins that exacerbate disease. A number of therapeutic interventions to address this problem have been developed over the last decade. 17.6.1 Monoclonal Antibodies A wide variety of platforms are now available to make monoclonal antibodies and they currently represent an important part of the therapeutics on the market (Fig. 17.3b). They have an advantage over other therapeutic proteins in that their physico-chemical and pharmacokinetic properties are generally similar – allowing rapid production of material and a relatively streamlined approach to preclinical and early clinical testing. For the genome era proteins, one example is the antiBAFF antibody [Lymphostat-B (belimumab)] used for the treatment of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Antibodies against other emerging targets (targets after 1995) are currently under clinical development in many companies – we are currently developing anti-IL-31 and anti-IL-22Ra antibodies for use in dermatological indications. 17.6.2 Receptor Fusion Proteins Fusing a protein to a stable portion of an antibody structure confers a longer circulating half-life to the protein, thus increasing its therapeutic benefits. The classic example is etanercept (Enbrel), a soluble p75-Fc dimer that blocks the action of TNF- [ , the fusion being between p75, the TNF- [ type II receptor (the TNF binding domain), and the Fc portion of human IgG (Mohler et al. 1993). More recently, we have collaborated with Zymogenetics on TACI-Ig. This is a fusion protein between the extracellular portion of the TACI receptor and the Fc portion of human immunoglobulin G. Since this protein inhibits Blys (B lymphocyte stimulating factor), APRIL (a proliferation-inducing ligand) and heterotrimers, it might have greater clinical potential than an antibody (Dillon et al. 2006). It is currently in clinical studies for SLE, RA and hematological malignancies.

17.6.3 Protein Antagonists In many cases, we have been able to engineer antagonists out of cytokines with relatively small changes to the primary sequence. The chemokine RANTES, for example, can be turned into an antagonist by the addition of an amino acid at the amino terminus (Proudfoot et al. 1996). Chemical modification of the amino terminus led to a molecule that was highly potent in blocking HIV infection: (AOP)-RANTES (Simmons et al. 1997). Amongst the four helix bundle cytokines, early work on IL-4 produced an antagonist R121D, Y124D (Pitrakinra) – which was taken into clinical development for allergy by Bayer (Tony et al. 1994). Work on another four helix bundle, IL-5, cytokine showed that potent antagonism is achieved by a single amino acid change E12 K (McKinnon et al. 1997). We can expect that examples of such antagonists based on other four helix bundle cytokines may enter clinical trials in the near future. 17.6.4 Small Molecules: The Convenience of an Oral Medicine The idea of antagonizing cytokines by blocking their receptors with a small molecule has been a long and difficult road. Early on (Labriola-Tompkins et al. 1991), IL-1 was shown to have a large surface area interacting with its receptor – highlighting why so many high throughput screens had failed to find a low molecular weight antagonist. Inhibitors have been found, but these are larger peptides, which can block the large surface area of the interaction (Akeson et al. 1996). More recently, examples based on rational design have been shown for TNF antagonists (He et al. 2005), based on allosteric binding modifying the structure and inhibiting function. Alternatively, binding may be designed for the ’hot spot’ that corresponds to protein interfaces particularly adept at binding to proteins, peptides and thus even small molecules (Arkin and Wells 2004).

17.7 Future Directions The availability of a large collection of secreted proteins has enabled us to look at physiology and pathology from a new viewpoint. However, our assays are always

References

Fig. 17.6. Combining the power of the human secretome and human genetics gives us a unique platform for drug discovery in the future. The secretome is a list of proteins that are by definition drug targets, since they can be administered to patients (not all proteins are druggable – see Hopkins et al. 2002). Genetics gives us the candidates which correlate best with disease. Where these two worlds intersect, then we have the best chance of finding a new therapeutic. Often the worlds do not intersect directly, and so it is important to understand the metabolic and protein-protein interaction pathways that link them

limited – either we are studying human cells in culture, or we are studying the in vivo activity in animal models. Although both give us a glimpse of the possible clinical use, neither gives us a complete picture. Before we get into clinical testing it is essential to know that our protein targets are actually linked with the human disease. Typically we look at this from three viewpoints. First, is the target actually expressed in the tissue during disease? Second, is there evidence that the protein will have an effect on cells from patients with the disease. This is typically only straightforward if the target is present on leukocytes, or if it is present in the skin or on tissue which is surgically resected as part of the treatment of advanced stage disease (such as arthritis). The third piece of data comes from the human genetics. We now live in the era of the whole genome scan (see Klein et al. 2005 for an example) – where it is possible to look at as many as 500,000 single nucleotide polymorphisms. Since we are studying the DNA of a large number of patients, we can try to detect whether SNPs in the gene of interest can correlate with the severity, speed of onset, or even subtype of disease. This is only a correlation, but it does enable us to take a look at molecules associated with the disease, and to see which of our proteins might play a role in the real human pathology. We have completed scans in the autoimmune diseases multiple sclerosis, psoriasis and SLE – allowing us to see which of our secreted proteins can be prioritized based on the genetic data. Combining these three viewpoints is a very powerful approach (Fig. 17.6) – protein discovery gives us a list of proteins – all tractable (easily made) – but with no a

priori correlation with disease. Whole genome scan analysis of human genetics gives us a list of the genes which link to disease – but with no a priori correlation as to whether they are tractable as targets (whether a drug could ever be made against them). Sometimes these two worlds will overlap directly – but in most cases they will not. To link the two worlds we have to make the most of pathway databases, which look at which proteins can and do interact with each other and to understand which tractable protein can be used to modulate the activity of a protein whose gene correlates with development of disease. The technologies of the genome era have enabled us to start to master the human secretome and to understand the biology of its members in much more depth. The bulk of the challenge still lies ahead of us – picking those proteins that will be of the greatest clinical benefit to our patients. This is a challenge which will keep us busy for many decades to come.

References Aaronson JS, Eckman B, Blevins RA, Borkowski JA, Myerson J, Imran S, et al. (1996) Toward the development of a gene index to the human genome: an assessment of the nature of highthroughput EST sequence data. Genome Res 6:829 – 845 Akeson AL, Woods CW, Hsieh LC, Bohnke RA, Ackermann BL, Chan KY, et al. (1996) AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1. J Biol Chem 271(48):30517 – 30523 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403 – 410

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17 New Biological Therapeutics in the Genome Age Arkin MR, Wells JA (2004) Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat Rev Drug Discov 3:301 – 317 Bertone P, Trifonov V, Rozowsky JS, Schubert F, Emanuelsson O, Karro J, et al. (2006) Design optimization methods for genomic DNA tiling arrays. Genome Res 16:271 – 281 Besson D, Yeow K, Lang P, Scheer A (2003) HTS and cellular biology at Serono. Curr Drug Discov 29 – 32 Bolon B, Galbreath E (2002) Use of genetically engineered mice drug discovery and development: wielding occam’s razor to prune the product portfolio. Int J Toxicol 21(1):55 – 64 Bougueleret L, Dumas J-B, Duclert A. Complementary DNA’s encoding proteins with signal peptides. Patent WO200037491 Clark HF, Gurney AL, Abaya E, Baker K, Baldwin D, Brush J, et al. (2003) The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. Genome Res 13:2265 – 2270 Dillon SR, Gross JA, Ansell SM, Novak AJ (2006) An APRIL to remember: novel TNF ligands as therapeutic targets. Nat Rev Drug Discov 5(3):235 – 246 Foster D, Parrish-Novak J, Fox B, Xu W (2004) Cytokine-receptor pairing: accelerating discovery of cytokine function. Nat Rev Drug Discov 3:160 – 170 Graveley BR (2001) Alternative splicing: increasing diversity in the proteomic world. Trends Genet 17(2):100 – 107 Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, et al. (2000) TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature 404:995 – 999 He MM, Smith AS, Oslob JD, Flanagan WM, Braisted AC, Whitty A, et al. (2005) Small-molecule inhibition of TNF- [ . Science 310:1022 – 1025 Hillier L, Lennon G, Becker M, Bonaldo MF, Chiapelli B, Chissoe S, et al. (1996) Generation and analysis of 280 000 human expressed sequence tags. Genome Res 6:807 – 828 Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727 – 730 Jankowski K, Kucia M, Wysoczynski M, Reca R, Zhao D, Trza E, et al. (2003) Both hepatocyte growth factor (HGF) and stromal-derived factor-1 regulate the metastatic behaviour of human rhabdomyosarcoma cells, but only HGF enhances their resistance to radiochemotherapy. Cancer Res 63: 7926 – 7935 Kledal TN, Rosenkilde MM, Coulin F, Simmons G, Johnsen AH, Alouani S, et al. (1997) A broad-spectrum chemokine antagonist encoded by Kaposi’s sarcoma-associated herpesvirus. Science 277:1656 – 1659 Klein RJ, Zeiss C, Chew EY, Tsai J-Y, Sackler RS, Haynes C, et al. (2005) Complement factor H polymorphism in age-related macular degeneration. Science 308:385 – 389 Kramer R, Cohen D (2004) Functional genomics to new drug targets. Nat Rev Drug Discov 3:965 – 972 Labriola-Tompkins E, Chandran C, Kaffka KL, Biondi D, Graves BJ, Hatada M, et al. (1991) Identification of the discontinuous binding site in human interleukin 1 q for the type I interleukin 1 receptor. Proc Natl Acad Sci U S A 88:11182 – 11186

Mallat Z, Corbaz A, Scoazec A, Graber P, Alouani S, Esposito B, et al. (2001) Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability. Circ Res 89:e41–e45 Marra MA, Hillier L, Waterston RH (1998) Expressed sequence tags – ESTablishing bridges between genomes. Trends Genetics 14(1):4 – 7 McKinnon M, Page K, Uings IJ, Banks M, Fattah D, Proudfoot AEI et al. (1997) An interleukin 5 mutant distinguishes between two functional responses in human eosinophils. J Exp Med 186(1):121 – 129 Miller RT, Jones DT, Thornton JM (1996) Protein fold recognition by sequence threading: tools and assessment techniques. FASEB J 10:171 – 178 Mohler KM, Torrance DS, Smith CA, Goodwin RG, Stremler KE, Fung VP, et al. (1993) Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. J Immunol 151:1548 – 1561 Patel VP, Kreider BL, Li Y, Li H, Leung K, Salcedo T, et al. (1997) Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors. J Exp Med 185(7):1163 – 1172 Ohno E, Ono K, Kikuchi H, Saburi Y, Utsunomiya A, Nasu M (2005) Prolonged remission of adult T-cell leukemia/lymphoma treated with interferon- * following autologous peripheral blood stem cell transplantation. Leuk Lymphoma 46(12):1843 – 1845 Proudfoot AEI, Power CA, Hoogewerf AJ, Montjovent M-O, Borlat F, Offord RE, et al. (1996) Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist. J Biol Chem 271(5): 2599 – 2603 Selvaraju R, Bernasconi L, Losberger C, Graber P, Kadi L, Avellana-Adalid V, et al. (2004) Osteopontin is upregulated during in vivo demyelination and remyelination and enhances myelin formation in vitro. Mol Cell Neurosci 25:707 – 721 Simmons G, Clapham PR, Picard L, Offord RE, Rosenkilde MM, Schwartz TW, et al. (1997) Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276:276 – 279 Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T (1993) Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261(5121): 600 – 603 Tony H-P, Shen B-J, Reusch P, Sebald W (1994) Design of human interleukin-4 antagonists inhibiting interleukin-4dependent and interleukin-13-dependent responses in Tcells and B-cells with high efficiency. Eur J Biochem 225: 659 – 665 Washietl S, Hofacker IL, Lukasser M, Hüttenhofer A, Stadler PF (2005) Mapping of conserved RNA secondary structures predicts thousands of functional noncoding RNAs in the human genome. Nat Biotechnol 23(11):1383 – 1390 Wells TNC, Peitsch MC (1997) The chemokine information source: identification and characterization of novel chemokines using the worldwideweb and expressed sequence tag databases. J Leukoc Biol 61:545 – 550 Wells TNC, Schwartz TW (1997) Plagiarism of the host

References immune system: lessons about chemokine immunology from viruses. Curr Opin Biotechnol 8(6):741 – 748 Wells TNC, Graber P, Proudfoot AEI, Arod CY, Jordan SR, Lambert MH, et al. (1994) The three-dimensional structure of human interleukin-5 at 2.4-angstroms resolution: implication for the structures of other cytokines. Ann N Y Acad Sci 725:118 – 127 Yee DP, Conklin D (1998) Automated clustering and assembly of large EST collections. Proc Int Conf Intell Syst Mol Biol 6:203 – 211

Zambrowicz BP, Sands AT (2003) Knockouts model the 100 best-selling drugs – will they model the next 100? Nat Rev Drug Discov 2:38 – 51 Zambrowicz BP, Sands AT (2004) Modeling drug action in the mouse with knockouts and RNA interference. Drug Discov Today 3(5):198 – 207

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Chapter 18

18 Evidence Based Medicine’s Perspective on Biologics B. Rzany, A. Nast

18.1 What is EBM? Since the first articles by Sackett in the 1990s (Sackett and Rosenberg 1995), evidence based medicine (EBM) has developed considerably. Defined as “the integration of the best research evidence with clinical expertise and patients’ values”, EBM is an attempt to improve the care of our patients. There are certain misconceptions about EBM. It is not a standard recipe and there are no standard patient and individual factors that have to be taken into account for definite decisions to be made. EBM is also not an exclusive concept, neither is it a sophisticated “l’art pour l’art” for specialists. If EBM were only practiced by specialists, it would be dead – EBM should thrive in daily practice. Last but not least, EBM is not a cost regulating instrument. Often, the best documented therapies – like biologics – are the most expensive ones.

18.2 EBM Steps to Treating an Individual Patient In order to clarify what EBM means in daily practice, it is important to bear in mind the steps of EBM in the treatment of an individual patient. First a structured and answerable set of questions based on the patient encounter needs to be generated. Next is the search for valid external evidence and then the critical appraisal of that evidence for its relevance and validity. At the end of this process the results of the appraisal of evidence should be applied to the patient, and last but not least one’s own performance should always be recorded and evaluated. Imagine therefore a 50-year-old patient with severe plaque psoriasis in your private practice. The past his-

tory reveals recurrent hospitalisation in the previous couple of years, intolerance of fumaric acid, unsatisfactory results with oral cyclosporin and an increase in liver enzymes while under treatment with methotrexate. Based on this case history, one question would be “What is the best and safest therapy for this patient?” As this question is quite broad, it is recommended to focus the question a bit more. Therefore if biologics are being considered as a treatment choice the question could be: “What is the best and safest biologic for this patient?” Based on either question the next step will be the search for evidence. Here there are several possibilities. One step is to search the primary literature, which means going to Pubmed or Medline and making the necessary searches to retrieve the relevant literature. As this is quite an exhausting way of finding the evidence, other possibilities, such as searching the secondary literature, should be considered. Depending on the question, systematic reviews from the Cochrane library, evidence based books such as Clinical Evidence (Naldi and Rzany 2005) or Evidence-Based Dermatology (Williams et al. 2003) as well as the evidence-based guidelines (Nast et al. 2006) might be helpful in gathering the relevant evidence. During searches of the evidence based literature for biologics, it has been quite clear that there are no systematic reviews for this group of new drugs in the Cochrane library. The Evidence-Based Dermatology book from BMJ/Blackwell does not discuss biologics either. There is a chapter in the most recent Clinical Evidence book from the BMJ group which discusses two of the four biologics used in the treatment of psoriasis. The EBM-based secondary literature might give recommendations for the use of biologics based on the evidence for efficacy and safety but it does not usually consider other aspects such as practicability and cost. These aspects might be covered by a set of guidelines.

18.4 EBM and Biologics

At the moment, two evidence based sets of guidelines for the treatment of psoriasis exist: a set of British guidelines (Smith et al. 2005), which consider only the use of the presently available biologics, and the German S3 guidelines (Nast et al. 2006), which focus on all the systemic and topical treatments of psoriasis including biologics.

18.3 German S3 Guidelines for the Treatment of Plaque Psoriasis As evidence-based guidelines, the German S3 guidelines follow a structured approach. The first step in the guideline process is to nominate the people who will contribute to the guidelines. This includes the guideline project team who coordinate the guidelines, the team of experts who review the literature and last but not least the extended committee who formulate and pass the proposed recommendations. The next step is to review the literature. As the hits based on the different literature databases are not very sensitive and specific, the relevant articles need to be pre-screened and selected for the review process. In the review process itself the papers are evaluated for inclusion/exclusion criteria, quality of the methods and presentation of the results. Finally, a grade of evidence is given to each paper. As several definitions for the “grade of evidence” exist, the grade of evidence itself needs to be defined. Usually, it is based on the recommendations from the Oxford Centre of Evidence Based Medicine. Using the Oxford classification as a basis, the German Guidelines Team developed an adapted version, ranging from systematic reviews with meta-analyses (the highest level) to expert opinions (the lowest level). As the grade of evidence is based on a single paper, a “level of evidence” was assigned to sum up the evidence Table 18.1. Efficacy of biologics: comparison of “Number Needed to Treat” according to biologic and dosage

of all reviewed papers for one intervention. Here again, the highest grade would be for an intervention that is based on systematic reviews or the consistent results of good clinical trials. The final “therapeutic recommendations” are formulated considering the evaluated evidence-based literature on efficacy, as well as other aspects such as safety, practicability and costs. These therapeutic recommendations should be real consensus statements and great efforts have to be taken to make sure that the majority of the guideline group agrees on the formulation.

18.4 EBM and Biologics Biologics are quite new drugs. The pre-marketing studies follow the high present standards of good clinical practice. Therefore, it is not surprising that the level of evidence is better for biologics than for many of the other older treatments of psoriasis, e.g. methotrexate. On the other hand, for most available biologics the clinical experience is limited and issues such as rare side effects and the results of long term treatment cannot be discussed conclusively. Comparison of the published trials on the efficacy of biologics will lead to the assumption that there is nothing like “THE” biologic. The biologics differ in efficacy and safety. Based on the recommended dosages, the highest efficacy can be found for infliximab, followed by etanercept and efalizumab. The Number Needed to Treat (NNT), an established EBM tool to clarify efficacy, gives numbers from 1.22 (1.10 – 1.37) for infliximab, 3.30 (2.62 – 4.44) for etanercept at the low dose and 2.18 (1.65 – 2.66) at the higher dose, to 4.49 (3.62 – 5.91) for efalizumab (Table 18.1). So what does it mean? For infliximab it means that if you treat approximately four patients with infliximab

Time of evaluation/dosing

Number of patients needed to treat to reach a 75 % PASI reduction

Source

Infliximab

10 weeks; 5 mg/kg in weeks 0, 2, 6

1.22 (1.1 – 1.37)

Gottlieb et al. 2004

Etanercept

12 weeks a) 2 × 25 mg/week b) 2 × 50 mg/week

a) 3.3 (2.62 – 4.44) b) 2.18 (1.65 – 2.66)

12 weeks; 1 mg/kg; 1/week

4.49 (3.62 – 5.91)

Efalizumab

Leonardi et al. 2003

Menter et al. 2005

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three will achieve a reduction of the PASI of 75 % or more. This means that only one patient will fail the benchmark of a reduction of the PASI of 75 %. However, it should be taken into account that efficacy is not everything. Effectiveness that means “How well does the drug do in clinical practice” is very important. The next most important factor is safety. You want to have a drug that is safe for this chronic inflammatory disease. Other factors to consider are the practicability for patients and doctors as well as costs. When talking about costs it is important to remember that it is just not the price of the drug but also the cost of laboratory investigations, other investigations such as X-rays and in addition the days not lost to hospitalisation. We would like to finish this chapter with some thoughts on safety. It is important to remember that safety cannot be measured effectively by clinical trials. Only a limited number of patients are included in clinical trials. If 3,000 subjects are exposed prior to drug marketing, one can only be 95 % certain that any event which does not occur in this population will occur in no more than 3 in 3,000 subjects or has an incidence rate of less than 0.001. Therefore, post-marketing surveillance is very important to discover rare and also delayed reactions, especially in biologics, which apart from a few exceptions have not been on the market for long.

18.5 Where Do Biologics Stand Among Other Systemic Treatments of Psoriasis? In order to answer this question, all the aspects have to be considered: efficacy, effectiveness, quality of life, safety, practicability, and pharmacoeconomics. As previously mentioned, biologics are a quite heterogeneous group and each biologic needs to be evaluated separately.

Since biologics are a new group of drugs and clinical experience is still limited, it is too early to make final recommendations on the use of biologics. Over time, guided by EBM, biologics will find their proper place in the treatment of patients with psoriasis.

References Follmann M, Sterry W, Rzany B für die Psoriasis-Leitlinienkerngruppe (2005) Development of the evidence-based guidelines for psoriasis – a project of the German Dermatological Society (DDG). J Dtsch Dermatol Ges 3(9):678 – 689 Gottlieb AB, Evans R, Li S, Dooley LT, Guzzo CA, Baker D, Bala M, Marano CW, Menter A (2004) Infliximab induction therapy for patients with severe plaque-type psoriasis: a randomized, double-blind, placebo-controlled trial. J Am Acad Dermatol 51(4):534 – 542 Leonardi CL, Powers JL, Matheson RT, Goffe BS, Zitnik R, Wang A, Gottlieb AB (2003) Etanercept as monotherapy in patients with psoriasis. N Engl J Med 349(21):2014 – 2022 Menter A, Gordon K, Carey W, Hamilton T, Glazer S, Caro I, Li N, Gulliver W (2005) Efficacy and safety observed during 24 weeks of efalizumab therapy in patients with moderate to severe plaque psoriasis. Arch Dermatol 141(1):31 – 38 Naldi L, Rzany B (2005) Chronic plaque psoriasis. Clin Evid 13:2070 – 2098 Nast A, Kopp I, Augustin M, Banditt KB, Boehncke WH, Follmann M, Friedrich M, Huber M, Kahl C, Klaus J, Koza J, Kreiselmaier I, Mohr J, Mrowietz U, Ockenfels HM, Orzechowski HD, Prinz J, Reich K, Rosenbach T, Rosumeck S, Schlaeger M, Schmid-Ott G, Sebastian M, Streit V, Weberschock T, Rzany B (2006) S3 Leitlinie zur Therapie der Psoriasis vulgaris. J Dtsch Dermatol Ges 4 (Suppl 2):S1 – 126 Sackett DL, Rosenberg WM (1995) The need for evidencebased medicine. J R Soc Med 88 (11):620 – 624 Smith CH, Anstey AV, Barker JNWN, Burden AD, Chalmers RJG, Chandlers D, Finlay AY, Griffiths CEM, Jackson K, McHugh NJ, McKenna KE, Reynolds NJ, Ormerod AD (2005) British Association of Dermatologists guidelines for use of biological interventions in psoriasis. Br J Dermatol 153:468 – 497 Williams H, Naldi L, Bigby M, Herxheimer A, Diepgen T, Rzany B (2003) Evidence-based dermatology. BMJ Bookshop, BMA House, London

Chapter

Subject Index

abatacept 105 ABT-874 135 ACCENT trial 130 acetaminophen 91 acrodermatitis continua of Hallopeau 84 acropustulosis 84 ACT studies 137 acute myeloid leukemia (AML) 50 acyclovir 78, 150 adalimumab 14–28, 74, 77, 79, 82, 86, 95, 96, 102, 104, 105, 111–117, 127, 128, 131–134, 168, 169 – ankylosing spondylitis 25 – Crohn’s disease 17, 25, 128, 131 – psoriasis 17, 25 – rheumatoid arthritis 17, 18, 20, 26 – safety profile 26 ADCC, see antibody-dependent cellular cytotoxicity ADEPT, see antibody-directed enzyme prodrug therapy alefacept 79, 81, 82, 86, 92–95, 104, 105 alemtuzumab 60, 144, 147, 150, 171 alicaforsen 137 allergenic asthma 60 allo-reactive plasma cell 74 ammonia 61 anakinra 106, 119 anemia 60 ankylosing spondylitis 25, 169 – adalimumab 25 – etanercept 33, 37 – infliximab 7 anthracycline 154 antibody IL-2 fusion protein 159 antibody-dependent cellular cytotoxicity (ADCC) 17, 56, 72, 134 antibody-directed enzyme prodrug therapy (ADEPT) 156 – ADEPT trial 23, 24 antibody-LT-alpha fusion protein 159 anti-Fas antibody 161 antifolate 154

antigen-presenting cell (APC) 45, 86, 89 – antigen complex 45 anti-IL-12 antibody 129 APC, see antigen-presenting cell apoptosis 5, 161 ARMADA trial 19 arthritis 92 aspergillosis 115 ASPIRE study 114 ATLAS trial 25 ATTRACT study 10, 113 Auger – electron 52, 54 – emitter 54 autoimmune bullous skin disorder 69 – rituximab 72 azathioprine 71, 75 B cell 75 baby hamster kidney (BHK) 61 bacterial artificial chromosome (BAC) 72 basiliximab 127 Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) 25 BCA, see bifunctional chelating agent belimumab 179 Bexxar 50, 53 BHK, see baby hamster kidney bifunctional chelating agent (BCA) 54 bismuth radioisotope 53 BLAST 176, 177 blister formation 69 Borrelia burgdorferi 149 brain parenchyma 143 bronchitis 27 Brunsting-Perry pemphigoid 71 bullous pemphigoid 71 calicheamicin 156 Campath-1H 144 CARD15, see caspase recruitment domain family member

carrier substance 1 CASPAR study 97 caspase recruitment domain family member 15 (CARD15) 124 CBCL, see cutaneous B-cell lymphoma CCP, see cyclic citrullinated protein CD4+ cells 37 – T cell count 94 CDC, see complement-dependent cytotoxicity cell culture process 63 c-erb B-2 171 certolizumab 127, 128, 133, 134 – pegol 105, 111, 128, 133 – – Crohn’s disease 128 cetuximab 168, 171 CHARM 132 chemokine 176 Chinese hamster ovary (CHO) 61 CHMP, see Committee for Medicinal Products for Human Use CHO, see Chinese hamster ovary CLA, see cutaneous lymphocyte antigen CLASSIC study 25, 131, 132 clobetasol propionate 71 c-myb 161 c-myc 161 CNTO 1275 136 colchicine 75 colectomy 137 Committee for Medicinal Products for Human Use (CHMP) 171 complement-dependent cytotoxicity (CDC) 56 congestive heart failure (CHF) 27, 89, 115 cotrimoxazole 150 C-reactive protein 16, 19, 37, 99, 133 Crohn’ disease 118, 124, 128, 169 – adalimumab 17, 25 – certolizumab pegol – infliximab 6, 7 – – pharmacokinetics 9 – pediatric 9

188

Subject Index Crohn’s Disease Activity Index (CDAI) 130 CTCL, see cutaneous T-cell lymphoma cutaneous – B-cell lymphoma (CBCL) 147, 149 – lymphocyte antigen (CLA) 46, 86 – T-cell lymphoma (CTCL) 147 cyclic citrullinated protein (CCP) 99 cyclosporine 39, 79 cynomolgus monkey 11 cytokine 158, 175 – inhibitor 105 cytotoxic agent 154 daclizumab 127, 144, 145 dactylitis 99, 100 dapsone 71 decoy protein 176 dendritic cell 45, 98, 148 denileukin diftitox 157 depsipeptide 152 dermatitis herpetiformis 70 – Duhring 71 Dermatology Life Quality Index (DLQI) 90 desmoglein (Dsg) 69, 74 diffuse large B-cell lymphoma (DLBCL) 149 digoxin 35 [ 4 q 7-dimer 135 disease-modifying antirheumatic drug (DMARD) 18, 22, 39, 95, 102, 119 distal interphalangeal 99 DLBCL, see diffuse large B-cell lymphoma DMARD, see disease-modifying antirheumatic drug DNA vaccination 159 docetaxel 156 DOTA 55 doxorubicin 56 Drosophila Discs Large Homolog 5 (DLG5) 126 EAE, see experimental autoimmune encephalomyelitis EBA, see epidermolysis bullosa acquisita EBM, see evidence-based medicine EDSS, see Expanded Disability Status Score efalizumab 42–48, 81, 82, 89–92, 106, 184 – safety 91 EGFR, see epidermal growth factor receptor ELISA, see enzyme-linked immunosorbent assay ELISPOT test 115

emitter – [ 53 – Auger 54 – q 52 ENACT-1 135 endocytosis 48 enthesitis 99, 101, 104 enthesopathy 38 enzyme 59 enzyme-linked immunosorbent assay (ELISA) 10 epidermal growth factor receptor (EGFR) 161, 168, 171 epidermolysis bullosa acquisita (EBA) 70, 71 – mechanobullous 71 – rituximab 75 Eprex 65 erythrocyte sedimentation rate (ESR) 37, 99 erythrodermic psoriasis 82 erythropoietin 65 Escherichia coli 44, 61, 111 E-selectin 46, 77, 98, 117 etanercept 1, 6, 16–18, 32–40, 60, 77–79, 81, 82, 86–89, 95, 102–104, 111–119, 134, 169, 179, 184 – ankylosing spondylitis 33 – elderly patients 34 – infection rates 36 – pediatric patients 34 – psoriasis 33 – rheumatoid arthritis 33 European League Against Rheumatism (EULAR) 22 evidence-based medicine (EBM) 183 Expanded Disability Status Score (EDSS) 170 experimental – arthritis 145 – autoimmune encephalomyelitis (EAE) 142, 170 extracorporal photopheresis 148 Fab-1 43 FasL 161 FCL, see follicle centre cell lymphoma fibrinogen 16 FISH, see fluorescent in situ hybridization fistula 131 flexural psoriasis 84 fluorescent in situ hybridization (FISH) 56 follicle centre cell lymphoma (FCL) 149 fontolizumab 127, 136 fusion protein toxin 157 gamma heavy chain 43

gentuzumab 60, 156 – ozogamicin 60, 155 German S3 guidelines 184 glycosylation 61 GM-CSF, see granulocyte-macrophage colony-stimulating factor golimumab 105 gonadotropin 175 good manufacturing practice (GMP) 63 granulocyte macrophage colony-stimulating factor (GM-CSF) 15, 157 granuloma annulare 92 GRAPPA 101 gro-a 137 growth hormone 175 guttate psoriasis 81 HACA, see human anti-chimeric antibody HAMA, see human anti-murin immunoglobulin antibody HAQ, see Health Assessment Questionnaire Harvey-Bradshaw Index (HBI) 133 HDACI, see histone deacetylase inhibitor Health Assessment Questionnaire (HAQ) 19 – score 114 HEK, see human embryonic kidney hematological toxicity 76 hemodialysis 35 hepatic insufficiency 34 – etanercept 34 hepatocyte injury 157 HER2, see human epidermal growth factor receptor 2 Herceptin 56, 171 histone deacetylase inhibitor (HDACI) 152 histoplasmosis 115 HIV infection 143 HLA, see human leukocyte antigen Hodgkin lymphoma 116 human – anti-chimeric antibody (HACA) 10, 11, 76 – anti-murin immunoglobulin antibody (HAMA) 50, 154 – embryonic kidney (HEK) 61 – epidermal growth factor receptor 2 (HER2) 60 – genome sequence 174 – leukocyte antigen (HLA) 98 – retina 61 hybridoma 50, 168 hydroxyhydrochloride 151 hyperkeratosis 44

Subject Index hyperproliferation

46

IBD, see inflammatory bowel disease IBDQ, see Inflammatory Bowel Disease Questionnaire 90 Y-ibritumomab tiuxetan 151 ICAM, see intercellular adhesion molecule IgG 1 κ immunoglobulin 5 IHC, see immunohistochemistry IMID, see immune-mediated inflammatory disease immune – cell migration 141 – system 1 immune-mediated inflammatory disease (IMID) 1 immunoconjugate 155 immunohistochemistry (IHC) 56 immunosuppressive therapy 79 immunotoxin 157 IMPACT trial 103 indomethacin 151 inducible nitric oxide synthase (iNOS) 87 infectious disease 60 inflammatory – arthritis 102 – bowel disease (IBD) 124 Inflammatory Bowel Disease Questionnaire (IBDQ) 139 infliximab 5–12, 14, 16, 17, 74, 77, 78, 82, 86, 89, 95, 103, 104, 111–117, 124, 127, 128, 130, 131, 133, 134, 137, 168–170, 184 – ankylosing spondylitis 7 – antibody 10 – Crohn’s disease 6, 7, 128, 130 – failures 132 – pharmacokinetics 8 – plaque psoriasis 7 – psoriasis 7 – psoriatic arthritis 7 – rheumatoid arthritis 6, 7, 10, 11 – ulcerative colitis 6, 7 intercellular adhesion molecule (ICAM) 42 – ICAM-1 45, 46, 77, 86, 89, 98 – – keratinocyte level 47 [ 4 q 1 integrin molecule 170 interferon – IFN- [ 59, 65 – IFN- q 144 – IFN- * 42, 85, 126 interleukin – IL-1 15, 37, 112 – – blockade 118, 119 – IL-2 45, 85 – – DAB389 149

– IL-6 15, 37, 86, 112, 136 – IL-8 112 – IL-9 137 – IL-12 135 – IL-23 135 iodine isotope 53 JunB 98 Jurkat cell 5, 44 keratinocyte 98 Koebner phenomenon 81 lactate level 61 LacZ 178 Langerhans cell 45 lenercept 88, 116, 142 LET, see linear energy transfer LFA-1, see lymphocyte function-associated antigen linear energy transfer (LET) 52 listeriosis 115 lymphocyte – count 48 – function-associated antigen (LFA-1) 42, 45, 46, 86, 89 lymphoma 27, 88, 116 lymphotoxin 78 – LT- [ 15, 159 macrophage 98 MAK195 14 manufacturing 62 marginal zone lymphoma (MZL) 149 Master Cell Bank (MCB) 62 matrix metalloproteinase (MMP) 16, 37, 86, 118 MCB, see Master Cell Bank melanoma 158 memory effector cell 92 messenger RNA 161 metastatic melanoma 159 methotrexate (MTX) 7, 11, 19, 35, 39, 95, 112, 113, 119, 131 MHM24 43 mixed lymphocyte response assay (MLR) 44 MLN-02 135 MLR, see mixed lymphocyte response assay monomeric FAB fragment 5 monomethyl auristatin E 156 mouse myeloma 61 MPIF-1 176 MTX, see methotrexate multiple sclerosis (MS) 88, 116, 141, 178 murine collagen-induced arthritis 111

Mycobacterium – pneumonia 150 – tuberculosis 115 mycophenolate mofetil 71, 78, 79 mycosis fungoides 148 mylolarg 50, 156 MZL, see marginal zone lymphoma natalizumab 127, 129, 135, 143, 170 neuritis 145 neuroblastoma 158 neuromyelitis optica 145 NF-κB 87, 98 NHL, see non-Hodgkin’s lymphoma NNT, see number needed to treat non-Hodgkin’s lymphoma (NHL) 50, 73, 116, 148, 151 nonsteroidal anti-inflammatory drug (NSAID) 102 number needed to treat (NNT) 184 OLE study 22, 24 onercept 134 onycholysis 84 OPG, see osteoprotegerin osteoclast 38 osteoprotegerin (OPG) 118 paclitaxel 56 palmoplantar – psoriasis 84 – pustulosis 84 paracetamol 150 parakeratosis 44 PASI, see Psoriasis Area and Severity Index Pautrier microabscess 148 PBMC, see peripheral blood mononuclear cell pemphigoid 70 pemphigus 69 – foliaceus 69 – rituximab 73 – vulgaris 69, 74 – – TNF- [ 78 peptide hormone 59 peripheral blood mononuclear cell (PBMC) 44 p-glycoprotein 161 phage display technology 14 Physician’s Global Assessment (PGA) 93, 94 Pichia pastoris 61 pioglitazone 106 plaque psoriasis 44, 81, 87, 93 – infliximab 7 Pneumocystis carinii 75, 115 polyarthritis 112 polymorphonuclear leukocyte (PMN) 98

189

190

Subject Index PRECISE 133 prednisolone 75 prednisone 79 PREMIER study 22, 28, 113, 114 primary cutaneous lymphoma (PCL) 147 progressive multifocal leukoencephalopathy (PML) 143, 170 pro-matrix metalloproteinase 113 protein vehicle 56 pruritus 71, 94 PSA, see Psoriasis Symptom Assessment PsA, see psoriatic arthritis Pseudomonas 156 psoralen 39 psoriasis 38, 81, 92 – adalimumab 17, 25 – clinical features 83 – efalizumab 44 – etanercept 33, 38, 86 – flare 92 – infliximab 7 – – pharmacokinetics 9 – vulgaris 44 Psoriasis Area and Severity Index (PASI) 24, 48, 91, 84, 87, 90, 93 Psoriasis Symptom Assessment (PSA) psoriatic – arthritis (PsA) 23, 38, 95, 97 – – etanercept 38 – – infliximab 7 – nail changes 100 Psoriatic Arthritis Response Criteria (PsARC) 102 purified protein derivative (PPD) 88 pustular psoriasis 84 quality – assurance (QA) 64 – control (QC) 64 radioimmunotherapy (RIT) 53 radioisotope 51 radionuclide 51, 52 RANTES 179 Raptiva, see efalizumab REACH 131 recombinant – DNA molecule 59 – therapy 1 renal insufficiency 34

– etanercept 34 respiratory tract infection 27, 114 rheumatoid arthritis 111, 116, 119, 169, 179 – adalimumab 17, 18, 20, 26 – etanercept 33, 37, 86 – infliximab 6, 7, 10, 11, 73 rheumatoid factor 99 rhinitis 27 ribozyme 161 RIT, see radioimmunotherapy Rituxan, see rituximab rituximab 50, 56, 72–77, 78, 144, 145, 147, 150, 171 – autoimmune bullous skin disorder 72 – epidermolysis bullosa acquisita 75 – pemphigus 73 – rheumatoid arthritis 73 – toxicity 75

TAA, see tumor-associated antigen targeted monoclonal antibody 168 T-cell receptor (TCR) 42 TCR, see T-cell receptor TEMPO study 113 tetanus toxoid 94 thrombocytopenia 92, 152 tissue plasminogen activator 59 Total Sharp Score (TSS) 19 toxin 157 trastuzumab 56, 60, 168, 171 TSS, see Total Sharp Score tuberculosis 27, 94, 115, 169 tumor necrosis factor – TNF- [ 5, 32, 37, 77, 98, 111, 126, 142 – – blockade 118, 119 – – inhibition 88 – – pemphigus vulgaris 78 – TNF- * 65 tumor-associated antigen (TAA) 50

SAHA, see suberoylanilide hydroxamic acid sargramostim 129, 137 secretome 176, 180 Sézary syndrome 148, 152 Short Form-36 (SF-36) 19 signal sequence trapping 176 single nucleotide polymorphism (SNP) 177, 180 SLE, see systemic lupus erythematosus soluble receptor activator of nuclear factor κB ligands (sRANKL) 118 somatropin 137 SONIC study 131 SPECT 53 spine inflammation 99 spondyloarthropathy 37 – etanercept 37 STAR trial 22 STAT-1 87 static Physician’s Global Assessment (sPGA) 90 suberoylanilide hydroxamic acid (SAHA) 152 sulfasalazine 35 systemic lupus erythematosus (SLE) 179

ulcerative colitis 124, 137 – infliximab 6, 7 ultraviolet A phototherapy 39 urinary tract infection 27

T cell 44, 92, 105 – proliferation 47 – surveillance 171

Yttrium-90

varicella zoster 78 vascular – cell adhesion molecule (VCAM) 86 – – VCAM-1 117 – endothelial growth factor (VEGF) 118 – leak syndrome 157 VCAM, see vascular cell adhesion molecule vinca alkaloid 154 visilizumab 127, 137 Visual Analogue Score (VAS) 90 VLA-4 143 von Zumbusch pustular psoriasis 84 warfarin 35 WCB, see working cell bank white blood cell (WBC) count 47 WHO-EORTC classification 147 working cell bank (WCB) 62 xenograft tumor 151

Zevalin 50, 53

161

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