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ADVANCES IN PROTEIN CHEMISTRY Volume 49 Antigen Binding Molecules: Antibodies and T-cell Receptors

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ADVANCES IN PROTEIN CHEMISTRY EDITED BY FREDERIC M. RICHARDS

DAVID E. EISENBERG

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California

PETER S. KIM Department of Biology Massachusetts Institute of Technology Whiteheadlnstitute for Biomedical Research Howard Hughes Medical lnstitute Research Laboratories Cambridge, Massachusetts

VOLUME 49

Antigen Binding Molecules: Antibodies and T-cell Receptors EDITED BY EDGARHABER Department of Biological Sciences Harvard School of Public Health Harvard Medical School Boston, Massachusetts

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1996 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:lfwww.apnet.com Academic Press Limited 24-28 Oval Road, London NWl 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Serial Number: 0065-3233 International Standard Book Number: 0-12-034249-9 PRINTED IN THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 0 1 B C 9 8 7 6 5

4

3 2 1

To the memory of Christian B. Anfinsen,

without whose insights into the structure of protein molecules much of what is written here could not have been accomplished.


CONTENTS

PREFACE

.

.

.

.

.

.

.

.

.

.

.

.

.

xi

Antigen-Specific T-cell Receptors and Their Reactions with Natural Complexes Formed by Peptides with Major Histocompatibility Complex Proteins HERMAN N . EISEN, YURISYKULEV, AND THEODORE J. TSOMIDES Overview . . . . . . . . . . History and Background . . . . . . . T-cell Receptor Genes . . . . . . . T-cell Receptor Proteins . . . . . . . T-cell Receptor Ligands: Peptide-MHC Complexes T-cell Responses to PepMHC . . . . . T-cell Receptor Accessory Proteins . . . . Altered Peptide Ligands: Partial Agonists and Antagonists . . . . . . . . . . IX. MHC Restriction by Self and Nonself MHC: Paradox of Alloaggression . . . . . . . . . X. Concluding Remarks . . . . . . . References . . . . . . . . . .

I. 11. 111. IV. V. VI. VII. VIII.

. . . . . . .

1 3 8 11 15 22 39

.

41

. . .

43 47 48

. .

57 68 74 79

X-Ray Crystallography of Antibodies EDUARDO A. PADUN

Introduction . . . . . . . . . X-Ray Crystallography of Whole Antibodies . . X-Ray Crystallography of Fc . . . . . . X-Ray Crystallography of Antigen Binding Fragments X-Ray Crystallography of Complexes of Antibodies with Specific Ligand . . . . . . . . . VI. Conclusions . . . . . . . . . . References . . . . . . . . . .

I. 11. 111. IV. V.

. . .

. .

87 125 128

...

CONTENTS

Vlll

Insight into Antibody Combining Sites Using Nuclear Magnetic Resonance and Spin Label Haptens

HARDEN M. MCCONNELL AND MARIAMARTINEZ-YAMOUT I. Introduction . . . . . . . . 11. Antibodies ANOn . . . . . . . 111. Difference Spectra . . . . . . . IV. Selective Deuteration . . . . . . V. NMRSignalAssignments . . . . . VI. Diamagnetic Haptens . . . . . . VII. Distance Titrations . . . . . . . VIII. Combining Site Dynamics and Reaction Kinetics IX. Crystal Structure ofAN02 . . . . . X. Conformational Heterogeneity . . . . XI. Synopsis and Outlook . . . . . . . . . . . . . . References .

.

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*

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135 136 138 138 139 141 143 143 144 145 147 148

Computational Biochemistry of Antibodies and T-cell Receptors JIRI

NOVOTNY AND JURCEN BAJORATH

I. Background . . . . . . . . . . 11. Tools of Computer Analysis . . . . . . 111. Structures, Sequences, and Superfamilies . . . IV. Molecular Anatomy of Antibody Binding Site . . V. In Search of Effector Sites . . . . . . VI. Protocols for Three-Dimensional Modeling of Binding Sites . . . . . . . . . . . VII. Binding Affinity and Specificity . . . . . VIII. Molecular Basis of Protein Antigenicity . . . IX. Antibody Engineering . . . . . . . X. T-cell Receptor Modeling and Engineering . . References . . . . . . . . . .

150 154 162 168 173 177 194 213 220 232 236

Catalytic Antibodies

EDWARD M. DRICCERS AND PETER G. SCHULTZ

. . . . . I. Introduction 11. Immunological Evolution of Catalysis 111. Structural Studies . . . .

. . .

. . .

. . .

. . .

261 262 266

ix

CONTENTS

IV. V. VI. VII. VIII.

Evolving Functions Not Yet Found in Nature Unnatural Cofactors . . . . . . Difficult ChemicalTransformations . . Future Directions . . . . . . Conclusion . . . . . . . . References . . . . . . . .

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270 273 275 280 283 284

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289 290 298 300 303

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The Nature of the Antigen MICHAEL SELA AND ISRAEL PECHT

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI.

. . . . . . . Introduction Antigens, Antigenicity, Immunogenicity . Molecular Criteria forhtigenicity . . Role ofConformation inhtigenicity . . Antibody-Antigenic Epitope Interactions . Conformational Transitions Induced by Hapten Binding . . . . . . . . . T-Cell-Antigenic Epitope Interactions . . Thymus-Independent Antigens . . . Superantigens . . . . . . . Tumor Antigens . . . . . . . Concluding Remarks . . . . . References . . . . . . . .

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306 31 1 316 319 32 1 323 323

Antibody Binding Sites S. HUSTON, MICHAELN. MARGOLIES, AND EDGAR HABER I. Overview . . . . . . . . . . . 11. Protein Chemistry of Antibody Fragments and Antigen Binding Regions . . . . . . . . . . . . . . . 111. Engineered Antibody Binding Sites IV. Antibody Combining Site Structure: Antiarsonate and . . . . . . . . Antidigoxin Antibodies V. Enhancing Enzyme Selectivitywith Substrate-Selective Antibodies . . . . . . . . . . . References . . . . . . . . . . . JAMES

330 330 347 380

427 439

CONTENTS

X

Maturation of the Immune Response CESAR

MILSTEINAND MICHAEL S. NEUBERGER

. . . . . . . . . I. Introduction 11. Genetic and Structural Diversity of Primary Repertoire 111. Available Repertoire and Onset of Immune Response IV. Hypermutation . . . . . . . . . V. Antigenic Selection . . . . . . . . . . . VI. Structural and Evolutionary Implications References . . . . . . . . . . AUTHOR INDEX SUBJECT INDEX

. .

. .

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. .

. .

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45 1 454 46 1 464 470 477 48 1 487

525

PREFACE

Immunology began at the end of the nineteenth century when Paul Ehrlich became fascinated by precipitation reactions in the serum of immunized animals. His work was followed by a systematic analysis by Karl Landsteiner of the exquisite specificity of what we now know to be the antigen-antibody interaction. Yet the precipitin reaction remained in the realm of phenomenology until the 1950s, during which Rodney Porter and Gerald Edelman defined the modular structure of the antibody molecule and demonstrated that antigen binding was the property of only one domain of this multifunctional molecule. Renaturation experiments from my laboratory and those of Michael Sela and Charles Tanford soon implied that there had to be a great many different antibody molecules to account for the wide range of antigen binding specificities, since the amino acid sequence of an antibody determined its specificity. After the earliest amino acid sequences of myeloma proteins (homogeneous surrogates for the very heterogeneous serum antibodies) became available in 1965 from Norbert Hilschmann and Lyman Craig, it was apparent that a hypervariable region accounted for the antigen binding domain. The ability to clone examples of real antibodies by the hybridoma method of George Kohler and Cksar Milstein then led to a surge of activity in the 1970s that defined many aspects of the structure and function of the antigen binding site. Soon it was clear that the same principles of binding applied not only to humoral antibodies but to T-cell receptors and many members of what we now call the immunoglobulin superfamily. Interest in antigen binding waned for a decade while the detailed mechanisms of the cellular immune response were explored and defined. Now in the 1990s we are witnessing a revival of interest in antigen bindings as its broad relevance to many immunologic processes has become apparent and a variety of new analytic tools have allowed for deeper insights into the nature of antigen binding. Advances in molecular engineering permit the recapitulation of clonal selection and affinity maturation of antibodies in vitro. It is possible to produce antibodies to selected antigens without the mediation of an animal host, and it is even possible to turn an antigen binding site into a highly selective enzyme. This volume brings the reader up to date on the covalent and threedimensional structures of the antibody molecule’s antigen binding domain and the synthesis and use of this domain as a separate small molecule. The reader will find a full account of antibody three-dimensional structure (as xi

xii

PREFACE

revealed by x-ray crystallography and computational biochemistry) as well as an analysis of how antigens bind. The nature and the structure of an antigen are defined, and the affinity maturation of antibodies is examined in relation to gene structure and diversification. The exciting field of catalytic antibodies has advanced to include a range of enzymatic functions not even contemplated when the first examples were described a few years ago. The T-cell receptor, which has some elements in common with the antibody molecule, is far more complex in its antigen recognition function. The T-cell receptor is analyzed here in the context of its binding to antigen and to the essential major histocompatibility complex. The role of the T-cell receptor’s accessory proteins in binding and activation is also defined. Not only immunologists but also biologists and chemists should profit from reading this volume: it reveals a mature yet evolving field of broad interest to other areas of science. EDGAR HABER

ANTIGEN-SPECIFIC T-CELL RECEPTORS AND THEIR REACTIONS WITH COMPLEXES FORMED BY PEPTIDES WITH MAJOR HISTOCOMPATIBILITYCOMPLEX PROTEINS By HERMAN N. EISEN, YURl SYKULEV, and THEODORE J. TSOMIDES Center for Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts02139

I. 11. 111. IV. V. VI.

VII.

VIII. IX. X.

Overview ............................................... History a ound . . . . . . . . . . T-cell Receptor Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T-cell Receptor Proteins T-cell Receptor Ligands: Peptide-MHC Complexes. . . . . . . . . . . . . . . . . . . . T-cell Responses to PepMHC A. Affinity: Intrinsic Equilibri B. Kinetics . . . . . . . . . . . . . . . . . C. Time Required to Approach .......................... D. Epitope (PepMHC) Density E. TCR-PepMHC Engagement: .................... F. Specificity, Degenerac T-cell Receptor Accessory Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. C D 3 a n d c . . . . . . . . . . . B. C D 4 a n d C D 8 . . . . . . . . Altered Peptide Ligands: Partial Agonists and Antagonist MHC Restriction by Self and Nonself MHC: The Paradox Concluding Remarks. . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 8 11 15

22 23 26 29 30 32 35 39 39 40 41 43 47 48

I. OVERVIEW

The most distinctive feature of the vertebrate immune system is its ability to recognize an enormous number of organic molecules and molecular complexes, termed antigens, distinguishing broadly between those that are foreign to the responding animal (nonself) and those that are indigenous (self). This property is due to antigen-specific receptors on lymphocytes, small cells that comprise -5% of all cells in the body (estimated at 101'-lO1z out of about loL3 cells in an adult human). The receptors on the two major classes of lymphocytes, B and T cells, are similar structurally but profoundly different functionally. On B cells the receptors ADVANCES I N PROTEIN CHEMISTRY W1. 49

1

Copyright 63 1996 by Academic Press, Inc. All rights of reproduction in any form reselved.

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HERMAN N . ElSEN ET AL.

are immunoglobulins (Ig) embedded in the cell surface as integral membrane proteins; in response to recognition of antigens, B cells produce large amounts of the receptors and secrete them as soluble antibody molecules. The antigen-specific receptors on T cells (T-cell receptors or TCR) are also Ig-like cell surface integral membrane proteins; their recognition of antigens triggers T cells to exercise a great variety of functions but not to secrete the receptors. Both Ig and TCR molecules are heterodimers, each subunit consisting of two or more domains, with each domain having a characteristic threedimensional shape called the Ig fold. The N-terminal domains (termed variable or V domains) differ in amino acid sequence from one lymphocyte clone to another. The variable domains of each heterodimer pair to form a single antigen binding site that determines the unique ability of each clone to recognize and respond to only very few of the millions of different antigens to which an individual animal can respond. The enormous diversity of B- and T-cell receptors arises from the many germline gene segments that encode them; as each lymphocyte matures, different combinations of these segments are joined (combinatorial diversity) and additional variations in sequence are introduced at the junctures (junctional diversity), leading to an immense number of variable domain sequences. Despite extensive similarities in amino acid sequence between Ig and TCR and in the organization and recombination of the gene segments that encode them, these receptors differ remarkably in the universe of antigens they recognize. The antigens recognized by antibodies (or their membranebound form on B cells) vary enormously: Physically they may be soluble, colloidal, particulate, or parts of virions or microbial or eukaryotic cells, and chemically they may be proteins, peptides, carbohydrates, lipids, nucleic acids, or any of a limitless number of diverse small organic molecules. In contrast, the TCR reviewed here normally recognize and respond only to complexes formed between small peptides and a specialized set of proteins encoded by the major histocompatibility complex (MHC). Because MHC molecules (sometimes called histocompatibilityantigens) are integral membrane proteins, these complexes (termed pepMHC) are confined to cell surfaces, and the TCR of a T cell is therefore normally able to recognize antigenic complexes only on the surfaces of other cells, called antigen presenting cells or target cells. Inasmuch as both TCR and their natural pepMHC ligands are embedded in cell surface membranes, analysis of their interaction at the molecular level poses a major challenge. Later in this review we focus on how this challenge is being met and emphasize recent results that illuminate the way in which TCR react with (or “recognize”) their natural pepMHC ligands, particularly the TCR on those T cells (called cytotoxic T lymphocytes or CTL) that destroy other cells (termed target cells).

ANTIGEN-SPECIFIC T-CELL RECEPTORS

3

11. HISTORY AND BACKGROUND The immune system has been under study for about 100 years, but only in the past 30 years have T and B cells been distinguished and only in the past 12 years have TCR molecules and the genes encoding them been identified. Nevertheless, for decades before T cells and TCR emerged as recognized entities, their existence was foreshadowed by certain antigenspecific inflammatory responses produced by injecting antigens into the skin of individuals with previous exposure to such antigens, either through natural infection of deliberate inoculation (immunization). Around 1890, Robert Koch showed that the injection of tubercle bacilli (or a mixture of proteins called “tuberculin” from supernatants of tubercle bacilli cultures) elicited an intense inflammatory response in guinea pigs if they had been previously infected with these microorganisms. The responses appeared 1 2 4 8 hr following antigen injection, and similar delayed-type hypersensitivity (DTH) responses, always specific for the original inciting antigen, were subsequently demonstrated with crude protein mixtures from many other microbes (bacteria, fungi, and later, viruses). Responses sometimes occurred following deliberate immunization with purified proteins (e.g., ovalbumin) or even with small organic molecules applied to the skin, providing they reacted in situ to form covalent derivatives of skin proteins (as with 2,4-dinitrochlorobenzene,or a catechol in the case of poison ivy). In contrast to the late appearance of DTH responses, many other antigen-specificskin responses appear almost immediately, e.g., within 1 min or sometimes 2-3 hr. Because the transfer of serum antibodies from an immunized to a nonimmunized (naive) individual confers on the recipient the same prompt antigen-specific responses, it was clear that these rapid or immediate-type responses were mediated by antibodies. But serum failed to transfer the delayed-type responses (e.g., to tuberculin). Although efforts were made to reconcile these failures with a role for special antibodies, it came to be widely believed that antigen-specific cells rather than soluble antibody molecules were the direct mediators of DTH responses. This belief was supported by the finding that DTH responses could be transferred to naive recipients with inflammatory cells from immunized donors, although the transferred cells were complex mixtures of leukocytes that probably included some antibody-forming cells. The resolution of all doubt came after T cells were distinguished from B cells, largely through studies involving extirpation of the thymus from newborn mice. These studies led to the establishment of a clear dichotomy between those lymphocytes that develop from immature precursors in the thymus (T cells) and those that develop to maturity in the bone marrow (B cells). B cells were shown to be the source of Ig and antibodies, and T cells were

4

HERMAN N . EISEN ET AL

shown to be required for the optimal production of antibodies. In addition, highly purified populations of T cells could transfer DTH responses, suggesting that T cells bear receptors that recognize antigens. Early studies on the nature of the antigen-recognizing receptors on T cells were marked by intense disagreements arising largely, it appears in retrospect, because the T-cell populations studied were often contaminated by small numbers of B cells and because reliance was placed almost exclusively on serological analyses using anti-Ig antisera. Antigen-specific molecules on T cells were variously suggested to be Ig molecules firmly attached to T cells, to be some other (non-Ig) type of cell-associated protein, or to be some other type of nonprotein informational macromolecule. The debate was resolved by the development of monoclonal antibody (MAb) technology and the ability to grow T-cell clones in culture. Using MAb raised against T-cell clones, one a malignant lymphoma and the others normal T cells, three independent studies succeeded in immunoprecipitating a T-cell surface protein that was unique to cells of the immunizing clone (Allison et al., 1982; Meuer et al., 1983; Haskins et al., 1983). The clonally specific (or clonotypic) protein was in each case a disulfidelinked heterodimer (-90 kDa) consisting of a relatively acidic membranebound a chain and a more basic membrane-bound p chain. The idea that these clone-specific heterodimers were antigen-specific receptors was strengthened by two findings. First, antibodies against them could block antigen-driven responses of the corresponding T-cell clones (Lancki et al., 1983). Second, amino acid sequences of proteolytic fragments from isolated a and p subunits suggested that, like Ig heavy and light chains, they had some regions where amino acid sequences varied from clone to clone and others where these sequences were invariant (Kappler et al., 1983; Meuer et al., 1984; McIntyre and Allison, 1983). The isolation and sequencing of cDNA clones for the b subunit (Yanagi et al., 1984; Hedrick et al., 1984; Saito et al., 1984a) and then for the a subunit (Saito et al., 1984b; Chien et al., 1984) finally demonstrated unambiguously that the clonally diverse heterodimers greatly resembled Ig and had all the characteristics expected of cell surface integral membrane proteins serving as antigen-specific receptors. Moreover, introduction of genes for both the a and /3 subunits into a T-cell clone having unrelated specificity transferred the antigen-specific responsiveness of the donor cell to the recipient cell (Dembic et al., 1986; Saito et al., 1987). In the course of searching a T-cell cDNA library for clones for the a and p subunits, cDNA for a third related subunit was found (Saito et al., 1984a). Termed y, the third gene turned out to encode an Ig-like chain that paired with the Ig-like product of a fourth gene, 6, to form a y6 heterodimer (Chien et al., 1984). Closely similar to the ab TCR, y6 heterodimers are


5

another type of antigen-specific receptor found on a subset of T cells located primarily in epithelia. yd cells constitute about 1-5% of peripheral T cells in mice and humans; these TCR will not be considered further here, as their natural ligands are not as well-defined as those recognized by aB TCR and the hnction of yd T cells is still not clear. Ten years before the molecular identity of the ab TCR began to take shape, several observations pointed to the special character of its natural ligands. By transferring T cells to athymic (nude) mice, Kindred and Shreffler (1972) saw that these cells reacted only when the T cells and the recipients had the same MHC type. The ineffectiveness of MHC-dispartate T cells could not be attributed to their immune elimination in the recipient because athymic mice do not reject allografts (see Alloaggression, Section IX). It was therefore concluded that the MHC “must play an active role in ensuring cooperation between B and T cells.” Shortly thereafter, in vitro studies of T-cell enhancement of the production of antibodies by B cells (Katz et al., 1973) and of T-cell responses to antigens presented by macrophages (Rosenthal and Shevach, 1973) clearly indicated that successful T-cell responses required the responding T and B cells, or T cells and macrophages, to have the same MHC type. Then, in a seminal publication, Zinkernagel and Doherty (1974) reported that virusinfected target cells were lysed by T cells from a mouse infected by that virus only if the target cells expressed the same MHC products as the infected animal. Work with different antigens revealed similarly that antigenspecific lysis of antigen-bearing target cells by cytotoxic T cells depended on target cell expression of a proper MHC protein (Shearer, 1974; Bevan, 1975; Gordon et al., 1975).This dual requirement of antigen recognition, referred to as “MHC restriction” (Zinkernagel and Doherty, 1979), has since been found to be characteristic of all a/?TCR-mediated reactions. To explain MHC restriction, two models were proposed. In the tworeceptor model, T cells had one receptor for an MHC product and another for antigen, and both had to be occupied for a successful T-cell response (Cohn and Epstein, 1978). According to the alternative onereceptor model, each T cell had a single type of receptor that recognized an antigen-MHC complex. The one-receptor model was shown to be correct by various approaches. In one, a hybridoma resulting from the fusion of two cells recognizing antigen A and MHC X or antigen B and MHC Y responded specifically to cells expressing either A + X or B + Y but not to those expressing A + Y or B X, indicating that the T cell did not see the antigen and the MHC product separately (Kappler et al., 1981). The antigeneMHC complex was sometimes referred to as “altered self’ because in this context the restricting MHC is indigenous (self) with respect to the responding T cells and is therefore nonimmunogenic, while the

+

+

6

HERMAN N. EISEN E T AL

antigen is foreign (nonself) and somehow alters the character of self MHC, imposing “foreignness” and ability to elicit a reaction. Mature T cells bearing ap TCR fall into two groups, each marked by one of two cell surface glycoproteins. Termed CD4 and CD8 (formerly called Lyt-1 and Lyt-2,3 in the mouse), both are present on,immature (double positive) T cells in the thymus, and one of them is lost by what appears to be a stochastic process (Corbella et al., 1994) to yield single positive (CD4+ or CD8+) mature T cells. Some mature CD4+ T cells, termed helper (Th2) cells, are required for optimal B-cell responses, while other helper cells (Thl) produce cytokines that cause inflammation, as in DTH responses. In contrast, CD8+T cells, which also produce some cytokines, behave primarily as CTL, lysing target cells that bear appropriate antigenic MHC complexes. The CD4/CD8 T-cell dichotomy also extends to the types of MHC protein that restrict antigen recognition. Class I MHC proteins (MHC-I) restrict antigen recognition by CD8+ T cells, and class I1 MHC proteins (MHC-11) restrict antigen recognition by CD4+ T cells (Table I). Virtually all cells express MHC-I and can present peptides to CD8+ cells, whereas only specialized cells (principally macrophages, dendritic cells, and B cells) express MHC-I1 for interactions with CD4+ cells. In common usage, cells that present pepMHC-I1 complexes to CD4+ cells are called antigen presenting cells (APC), whereas those that present pepMHC-I complexes to CD8+ CTL are termed target cells. Once it became established that a single heterodimeric receptor on T cells reacts with an antigen displayed on the surface of another cell and TABLE I

Peptide Binding to MHC-I and MHC-II Molecules Parameter Distribution Domain structure Accessory molecule Typical T-cell response Origin of most bound peptides Length of bound peptides Pockets in MHC groove Peptide N and C termini Many critical contacts Equilibrium constant, Ka

MHC-I

All nucleated cells az, a3

+ Pzm

CD8 Cytolytic activity Cytosolic (endogenous) Usually 8-9 residues Yes Buried in groove Peptide side chains -104-109 M-I measured

MHC-I1 Specialized APC al, a2 + P I ,PZ CD4 B-cell help, DTH Extracellular and membranous proteins Variable, 12-25 residues Yes May extend outside groove Peptide backbone 106-1 OH h.T’measured

-

-


7

that the antigen includes both a self (restricting) MHC protein and a bona fide foreign element (such as a virus-encoded protein), it was reasonable to assume that the MHC protein and the foreign protein combine to form an antigenic complex in the plasma membrane of the target cell (e.g., Cohen and Eisen, 1977). However, the subsequent finding that T cells responding to viral infections are paradoxically specific for intracellular proteins not found at the cell surface (e.g., influenza virus nucleoprotein) (Townsend and McMichael, 1985; Yewdell et al., 1985) led Townsend and colleagues to the discovery that short peptides (about 8-25 amino acids in length) derived from internal viral proteins can sensitize target cells bearing appropriate MHC molecules for lysis by CD8+ T cells (Townsend et al., 1986). It has since become clear that (1) the natural ligands for TCR are cell surface complexes each consisting of a short peptide and an MHC protein (a pepMHC complex); (2) under normal conditions the peptides are produced intracellularly by limited proteolysis of intracellular proteins (processing) and are then transported as pepMHC complexes to the cell surface (reviewed by Heemels and Ploegh, 1995); and (3) this physiological pathway can be circumvented by adding, to whole cells, synthetic peptides that bind directly to a subset of cell surface MHC molecules that are free of naturally processed peptides or bind them weakly and lose them readily by dissociation. The idea that fragmented protein antigens, i.e., peptides, can be recognized by T cells actually emerged from the earlier studies of Unanue and Allen (1987) on antigen presentation to CD4+ T cells by MHC-I1 on APC. Indeed the first unequivocal and quantitative demonstration of the binding of a defined peptide to an MHC molecule was obtained for MHC-I1 (Babbitt et al., 1985). In mice and humans, MHC-I proteins are encoded by genes at three linked loci (termed K, D, and L in mice, and A, B, and C in humans), and MHC-I1 proteins by several genes at two or three linked loci (termed IA and IE in mice, and DP, DQ, and DR in humans). Most of these genes are extremely polymorphic, some having as many as 50 (or more) allelic variants. Any particular individual inherits alleles for only a small number of MHC proteins, at most six MHC-I and six MHC-I1 in humans, because of heterozygosity at each gene locus. Some inbred mouse strains have only two MHC-I proteins and a single MHC-I1 protein. Remarkably, however, these few proteins can effectively present an enormous number of different peptides to a vast number of TCR, each MHC protein having the capacity to bind to thousands of different peptides with equimolar (1 :1) stoichiometry. Although some peptides can be bound by more than one MHC protein, each MHC protein binds to distinctly different sets of peptides. How each of these protein molecules is able to bind to so many different peptides, and yet retain a significant degree of selectivity, has

8

HERMAN N. EISEN E T AL.

become clear as a result of striking advances over the last 5 years, particularly in the solution of several three-dimensional structures of crystallized pepMHC complexes (see Section V).

111. T-CELLRECEPTOR GENES

The genes that encode TCR a and chains are assembled in developing T cells in the thymus by the juxtaposition of variable (V), diversity (D), and joining (J) gene segments to form VJ or VDJ or VDDJ exons, separated by a short intron from an exon for the constant (C) domain. The great similarity in organization of TCR and Ig gene segments is striking (Davis, 1990) and includes the canonical heptamer and nonamer signal sequences adjacent to V, D, and J segments; these signal sequences are almost the same for TCR and Ig genes and indeed are interchangeable (Yancopoulos et al., 1986). TCRP gene segments are spaced over 700-900 kb of human (or mouse) DNA, with an array of multiple VP segments separated from two tandemly duplicated sequences each including one DP, six JP, and one CP segment. All these segments have the same transcriptional orientation except for one downstream VP segment (Fig. 1). TCR a gene segments are also distributed in a long, linear array, but these were found surprisingly to be interrupted by J and C sequences of the TCR 6 chain (Chien et al., 1984). The pairing of a 6 with a y chain forms the yd heterodimeric receptor on the small subset of T cells (yd+) referred to previously. Because a single locus contains gene segments for both a and y chains, the rearrangements and expression of a and y genes are regulated by an intricate process (Winoto and Baltimore, 1989; Diaz et al., 1994). Evidently, a V gene from the same upstream array in this locus can join either to Jd (or Dd and Jd

5 4

4 “VVY JyCyl

“y

Jut*

- - -

VY Cfl(‘r)

-C)-..e...++t...+...+...i)c - -

TcRy

FIG 1. Arrangements of V (variable), D (diversity), J (junction), and C (constant) TCR gene segments in three loci in the mouse genome. Transcriptional orientation is indicated by arrow. (From Davis, 1990.)


9

segments) to form a complete VJSCS (or VDSJSCS) sequence for a Q chain, or to one of many Ja segments to form a complete VJaCa sequence for an a chain. In T cells that express an a/3TCR, the joining of a V gene to a Ja eliminates 6 gene sequences and thus precludes expression of a 6 chain. In T cells that express yS receptors, the joining of a V segment to a DS and/or JS takes place first and evidently suppresses subsequent rearrangements involving a gene segments. Despite many similarities in the organization of Ig and TCR genes and their extensive amino acid sequence homology, they differ in the following important respects. 1. Localization of hyperuariable sequences in TCR V domains. In Ig there are three especially variable (hypervariable) regions in V domains of light and heavy chains, and X-ray crystallographic studies show that these form the boundaries of antigen binding sites of antibodies (Davies et al., 1990). Because they determine the complementary fit of antibody to antigen, they are referred to as complementarily determining regions or CDR (i.e., CDRl, CDR2, and CDR3). Comparisons of the amino acid sequences of a and /3 variable domains from many TCR have shown that, in contrast to those of Ig, the CDRl sequences of a/3 TCR vary very little and the CDR2 sequences vary only slightly more, but that the CDR3 sequences vary considerably in length and sequence. The great variation in CDR3 reflects the very large number of TCR J gene segments as compared with those for Ig genes (e.g., about 50 Ja and 12 J/3 segments in contrast to 4J H and 5 J K segments for murine Ig). As discussed later, the extensive variation in CDR3 and J sequences suggests that the most diverse element of the pepMHC ligand that binds to a T-cell receptor, the peptide moiety, might make contact primarily with the CDR3 region of the receptors, whereas the less variable flanking a-helical regions of the MHC protein might make contact primarily with CDRl and CDR2. 2. Alternative RNA splicing. Alternative splicing of RNA transcripts of Ig genes plays a critical role in modifying the 3' ends of mRNA in order to generate proteins that are secreted as antibodies or retained as cell surface antigen-specific B-cell receptors. In contrast, RNA splicing is not involved in the expression of TCR genes, and TCR molecules are not secreted; they invariably remain as integral membrane proteins on T-cell surfaces. 3. Isotype switching. Gene sequences for the V domains that determine the specificity of antibodies for antigens can be spliced to one of several different immunoglobulin C regions to yield various forms of heavy chains called isotypes. Different Ig isotypes have different functional roles: For example, certain isotypes bind to receptors on B cells and macrophages, certain others can be transported across the placenta, and still others bind

10

HERMAN N . EISEN ET AL

to receptors on mast cells to mediate anaphylactic reactions. During the course of normal B-cell differentiation, switching of isotypes can occur (with no change in antigen specificity). For TCR, there is no comparable switching because there are no forms homologous to Ig isotypes. 4. Somatic mutation. A distinctive feature of the antibodies produced in response to antigenic stimulation is the progressive increase over time in the intrinsic affinities of the antibodies made against an antigenic epitope. These changes are the result of extensive somatic mutation that takes place in the V, D, and J regions of rearranged Ig genes during prolonged or repeated antigenic stimulation, together with selection by the antigen of B cells producing high-affinity antibodies (for a recent minireview see Foote and Eisen, 1995). Comparisons among the sequences of several hundred V regions from TCR a and /? mRNA or cDNA failed to reveal systematic evidence for somatic mutation, as the sequences nearly always matched one or another of the germline sequences (Davis, 1990).The important implication is that progressive increases or maturation in the intrinsic affinities of TCR does not occur, and thus TCR affinities for their ligands generally reflect the distribution of affinities arising from germline sequences and variations introduced by joining V, D, and J gene segments. However, Zheng et al. (1994), in a careful analysis of lymphocytes in lymph mode germinal centers (where B cells undergo somatic mutation), found evidence for somatic mutation of TCR V regions. Whether antigen selection of the mutated T cells takes place, as it does for B cells, is not yet clear. 5. Allelic exclusion. In a newly developing, immature thymocyte, productive (in-frame) rearrangement of a V/? gene segment in one chromosome prevents rearrangement of additional V/? gene segments in the other chromosome (Van Meerwijk et al., 1991; Malissen et al., 1992). However, Va segments on both chromosomes rearrange at the same time and can continue to rearrange until a complete ap TCR is formed and the cell is stimulated (by positive selection in the thymus) to mature into a single positive T cell (CD4+ or CD8+). As a result, almost one-third of mature T cells in the periphery carry two productively rearranged a genes and have on their surface two TCR species, differing in the a chain but having the same/? chain (Padovan et al., 1993; Heath and Miller, 1993; Mason, 1994). Because antibodies can bind via their constant (Fc) region to certain receptors (called FcR) on target cells, some antibodies to a TCR can mimic the natural ligand of the TCR and trigger “redirected lysis” of an FcR+ target cell by CTL expressing that TCR (Kranz et al., 1984b), even though the target cell lacks a pepMHC complex that is recognized by the TCR. Thus, a target cell expressing FcR was shown to be lysed by a CTL having two TCR, call them alp1and a$,, in the presence of either antibody to al


11

or antibody to az,indicating that each TCR of such a two-receptor T cell can, when ligated via its Va domain, trigger T-cell activation (Padovan et al., 1993). This circumstance raises an interesting possibility, not yet critically tested, that a mature two-receptor T cell that is stimulated to proliferate via one TCR will result in an increased number of cells expressing both TCR molecules, perhaps increasing the possibility of reactions with different pepMHC. Because the two receptors on such a T cell would be expected to have different specificities, they might increase opportunities for reactions against autoantigens or MHC-disparate cells (see Section IX). Experiments with mice transgenic for a TCR p subunit suggest, however, that in antigenically stimulated T cells there may be unknown mechanisms that hinder expression of a nonutilized TCR (Hardardottir et al., 1995), as in the case of Ig receptors on B cells transfected with multiple K chain genes (Lozano et al., 1993).

IV. T-CELLRECEITOR PROTEINS Once the primary structures of several T-cell receptors became known through the sequences of their V, D, J, and C genes, it became possible to look for clues regarding their tertiary structures. It was obvious on the basis of sequence homologies that TCR belong to the so-called immunoglobulin superfamily. That is, the domains of a heterodimeric TCR WaVp and CaCP) were expected to have the same chain topology and secondary structure elements as the canonical Ig fold (reviewed elsewhere in this volume). However, this observation did not lead to an understanding of how TCR recognize an antigenic universe consisting of relatively few MHC proteins combined with a great many different peptides (thousands for each MHC protein). Further analyses of sequence homologies between Ig and TCR (roughly 25-30% identity between the two groups) led to more detailed structural predictions. Following the first complete cDNA sequence of the TCR, Novotny et al. (1986) aligned the sequences of TCR a, p, and y chains with those of Ig of known three-dimensional structure and concluded that a TCR molecule was likely to possess a single antigen binding site that is essentially no different from that of an Ig. Later, Chothia et al. (1988) identified 40 amino acid residues critical for the conserved structure of Ig variable domains, either because they serve as framework residues or because they are crucial for interdomain contacts (VL-V, or VH-CHl), and compared them with the corresponding residues in -200 TCR Va and Vp sequences. They found a high percentage of the same or very similar residues at these positions, often 90+% identity, and they also found im-

12


portant similarities in the distribution of hypervariable regions between Ig (three such regions per VL or VH chain) and TCR (Va and Vp chains). As mentioned earlier, these noncontiguous hypervariable regions combine in the three-dimensional folded structure of an Ig to form the antigen binding site and are termed complementarity determining regions (CDR). Unlike Ig, however, the first and second predicted CDR of a TCR are much less variable than the third, called CDR3 (approximately residues 95-105 from the N terminus). Building on the discovery that peptides are an integral part of the antigenic structures recognized by T-cell receptors, Davis and Bjorkman (1988) proposed a model for T-cell recognition in which two CDR3 regions (from Va and Vp) interact primarily with peptide, while CDRl and CDR2 (each from both V a and Vp) interact with the two a helices flanking the peptide binding site (see Figs. 2 and 3). These authors also emphasized the greater diversity in TCR CDRS regions than in CDRl and CDR2; the latter are fully encoded by germline V genes, of which there are relatively few, whereas CDR3 is formed by the joining of V and J genes (in TCR a and y) or V, D, and J genes (in TCR /3 and 6 ) . Because of prevalent N-region additions at these junctions (due presumably to terminal deoxynucleotidyltransferase), as well as other mechanisms for diversification, TCR variability is highly concentrated in the CDRS region (Davis and Bjorkman, 1988). Because the MHC sites in contact with a TCR are generally less variable than the peptide residues recognized by a TCR, the preceding model provided a rationale for the greater sequence diversity of CDRS than of CDRl or CDR2; it also accommodated functional data from studies in which variations in only the CDR3 residues within a set of T-cell clones affected peptide recognition but not MHC specificity (Fink et al., 1986; Winoto et al., 1986). Furthermore, this model, independently suggested by Chothia et al. (1988) and by Claverie et al. (1989), was consistent with the dimensions observed in the first crystal structure of an MHC protein (Bjorkman et al., 1987a): The peptide binding groove is -10 A wide and 25 A long (Fig. 2), while the spacing between the predicted CDR3 segments in the TCR is 10-15 A. The two CDRS regions were projected

-

FIG.2. Ribbon diagram of MHC-I and MHC-I1 proteins. Shown for the MHC-I heterodimer are three domains of a or h e p y chain (the polymorphic a1 and a 2 domains, which together form the peptide binding groove, and the conserved a3 domain) and the 82microglobulin or light chain. Shown for the MHC-I1 heterodimer are the two domains for each subunit, i.e., the polymorphic a2 and82 domains. In this view the a helices flanking the peptide binding groove are clearly evident. Although there is virtually no sequence identity between MHC-I and MHC-I1 proteins, their three-dimensional structures are remarkably similar (Brown et al., 1988).(Courtesy of L. J . Stern and D. C. Wiley.)

Class I

Class II

14


A

B

FIG. 3. Ribbon diagram of a top view of the peptide binding sites of (A) MHC-I and (B) MHC-I1 proteins (HLA-A2 and HLA-DR1, respectively). Note that the peptide adduct is longer (10-20 amino acids) in MHC-I1 than in MHC-I (8-10 amino acids) proteins. (Courtesy of L. J. Stern.)


15

to interact with about five residues of a bound peptide (Claverie et al., 1989; see also Section IX). In a revealing study, Jorgensen et al. (1992) immunized mice transgenic for either the a or the /? chain of a particular TCR with a series of variant peptides. Analysis of the T-cell responses in these mice indicated that some peptide variants elicited T cells having complementary charges in the CDR3 regions of their TCR; furthermore, the contact sites for two peptide side chains could be assigned to either the TCR V a or V/?chain. To date, no three-dimensional structure of a complete TCR molecule has been reported. Such a structure is eagerly awaited, not only to illuminate the structure of the TCR itself but hopehlly to explain in molecular terms how the TCR universe of antigens is limited to pepMHC complexes while that of antibodies is virtually limitless. The recently reported structure of a TCR /? chain revealed Ig-like domains and three CDR loops plus an additional hypervariable loop whose significance is not yet clear (Bentley et al., 1995). V . T-CELLRECEPTOR LIGANDS: PEPTIDE-MHC COMPLEXES

Beginning with Townsend and colleagues (Townsend and Bodmer, 1989), a common method for studying the ligands recognized by T cells on various target cells (e.g., virus-infected, tumor, or allogeneic cells) has been to add synthetic peptides to different target cells that express an appropriate MHC protein, forming pepMHC complexes that can elicit a measurable T-cell response. This approach has been successfd in part because the added synthetic peptide need not precisely match the natural ligand (e.g., a processed viral peptide); it can be substantially longer, for example, and then undergo limited trimming by proteases in the assay medium or on cell surfaces to reach the optimal length for binding to an MHC protein. However, in some instances, it is important to know the exact identity of a naturally processed peptide, in order, for instance, to determine the number or density of specific pepMHC complexes on the surfaces of target cells (reviewed in Tsomides and Eisen, 1993a). An understanding of the relationship between the synthetic peptides that can be used to elicit T-cell responses and the naturally processed peptides that are generated within cells has rested on two principal lines of investigation: ( 1) X-ray crystallographic determination of MHC structures; and (2) direct biochemical isolation and sequencing of endogenous peptides associated with MHC molecules. The nature of the MHC and its ligand was greatly illuminated when the first crystal structure of an MHC protein, the human MHC-I protein called HLA-AP, was determined by Bjorkman

16

HERMAN N . EISEN

Er

AL

et al. (1987a). The molecule consisted of four domains of -90 amino acids each, three derived from an a chain (a,,a2,and a3)and one comprising P2-rnicroglobulin V2m). While the membrane proximal a3 and P2m domains resembled Ig domains, a l and az paired to form a novel structure: an eight-stranded antiparallel fi sheet underlying two long a helices (Figs. 2 and 3). Between the two a helices was a continuous region of unassigned electron density later shown to represent a complex mixture of peptides that copurified with HLA-A2; since A2 purification takes several days at least, this observation proved to be the first clue that some naturally processed peptides can form extremely stable complexes with MHC-I proteins, often having dissociation half-lives in the tens of hours (Cerundolo et al., 1991; Tsomides et al., 1991; Olsen et al., 1994).The crystal structure of HLA-A2 furthermore suggested that the unknown peptides harbored between the two a helices might be 8-20 amino acids long, based on the approximate dimensions of the binding site (25 x 10 x 11 A) (Bjorkman et al., 1987b). More precise definition of some natural ligands was achieved through an acid elution technique used to separate peptides from MHC molecules (Buus et al., 1988; Rotzschke et al., 1990a), followed by characterization of the isolated peptides. The first naturally processed peptides to be identified were viral products obtained from infected cells (van Bleek and Nathenson, 1990; Rotzschke et al., 1990b) and were eight or nine residues long. Other MHC-I-associated peptides present at relatively high levels were resolved by high-performance liquid chromatography (HPLC) and sequenced by Edman degradation Uardetzky et al., 1991; Corr et al., 1992) or by the mass spectrometric approach pioneered by Hunt and colleagues (1992a); these peptides proved to arise from a variety of intracellular proteins and were often eight or nine residues in length, though sometimes longer (Henderson et al., 1992; Wei and Cresswell, 1992). Rammensee and colleagues then performed a critical experiment in which they subjected the total pool of peptides eluted from MHC-I molecules to sequencing by Edman degradation (Falk et al., 1991a).While they found each amino acid at virtually every cycle during the Edman procedure, they discovered several key facts about naturally processed peptides: (1) For a given MHC-I protein (such as HLA-A2), the eluted peptides contained a predominance of one or two amino acids at certain key “anchor” positions (e.g., leucine or methionine at position 2, and valine or leucine at position 9); (2) amino acid yields dropped precipitously after nine cycles, suggesting that most MHC-I bound peptides are nonamers (octamers for certain MHC-I molecules); and (3) the “motifs” characterizing peptide length and anchor positions were distinctive for each MHC-I protein. These motifs have been highly useful in the rapid identification of candidate peptides recognized by T cells when the parent protein sequence is


17

known (Rotzschke et al., 1991a; Pamer et al., 1991). However, this approach is not infallible for several reasons: (1) Some MHC-binding peptides do not conform to the expected motif and therefore will be missed (e.g., Udaka et al., 1992); (2) synthetic peptides found to be active need not always correspond to naturally processed peptides, even when optimized for activity, since heteroclitic reactions can occur for T-cell receptors (Bodmer et al., 1988) as well as for antibodies; and (3) nonphysiological crossreactions can be observed when target cells are sensitized with high doses of synthetic peptides because of the resulting artificially high pepMHC densities (Milligan et al., 1990; Schild et al., 1990; Dutz et al., 1994; Tsomides et al., 1994). Despite these caveats, several naturally processed peptides recognized by T cells have turned out to match precisely the peptides predicted from motifs (Rotzschke et al., 1990b, 1991a; van Bleek and Nathenson, 1990; Pamer et al., 1991; Tsomides et al., 1994) (Table 11), leading to the reasonable assumption that this is often the case. All the biochemical information about naturally processed peptides bound to MHC-I molecules fits neatly with the emerging crystallographic data. Monopeptidic pepMHC complexes were obtained and crystallized, revealing important structural similarities and differences among peptides binding to the same MHC-I molecule (Fremont et al., 1992; Zhang et al., 1992; Madden et al., 1993).Pockets within the MHC binding site were shown to accommodate peptide side chains at the positions described as anchors (Garrett et al., 1989; Matsumura et al., 1992a; Young et al., 1994). Peptides longer than nine residues could fit by bulging out in the middle, with both peptide termini substantially buried in the binding site (Guo et al., 1992). Within a short time, a coherent picture emerged of how a single MHC molecule can bind selectively to an enormously diverse variety of peptides. Comparable findings (peptide motifs, MHC pockets) were later made for MHC-I1 molecules, although in this case the situation proved more complex. Naturally processed peptides binding to MHC-I1 molecules turned out to include sets of nested peptides sharing a core sequence but having different N and C termini (Demotz et al., 1989; Nelson et al., 1992; Chicz et al., 1992). These peptides tended to be longer than those eluted from MHC-I proteins, ranging from about 12 to 25 residues (Rudensky et al., 1991; Hunt et al., 1992b). Again, this information correlated well with the results of crystallography. The structure of an MHC-I1 molecule complexed with endogenous peptides (Brown et al., 1993) or with a single viral peptide (Stern et al., 1994) revealed two major differences from MHC-I: (1) The MHC-I1 binding groove allows bound peptides to extend out at both ends rather than having their termini tucked into the binding site, thereby explaining the length heterogeneity among naturally occurring MHC-I1 associated peptides (see Fig. 3B); and (2) many of the peptide-

TABLE 11

Naturally Processed Peptides Associated with MHC Molecules" Class and MHC protein Class I H-2Kd H-2Db H-2Kh H-2Kb H-2Kd H-219 H-2Ld HLA-A2.1 HLA-A2.1 Class I1 IAb

IEb

IAk

HLA-DR1

Peptide origin Influenza NP 147-155 Influenza NP 366-374 Vesicular stromatitis virus NP52-59 Ovalbumin 257-264 Listeria monocytogenes listeriolysin 91-99 Mouse a-ketoglutarate dehydrogenase Mouse a-ketoglutarate dehydrogenase HIV-1 reverse transcriptase 476-484 HIV-I gag 77-85 Murine leukemia virus envelope 145-1 57 145-158 I-E a chain 56-73 Invariant 39-53 Murine leukemia virus envelope 454-467 454-468 454-469 Bovine serum albumin 141-154 Hen egg lysozyme 48-60 48-6 1 48-62 52-64 Invariant chain 105-118 105-1 19 96-1 18' 96-1 19' 96-120' 97-1 18' 97-1 19' 97-120' 98-1 18' 98-1 lgL 99-1 18'

Peptide sequence'

Refs.

SIINFEKL GYKDGNEYI

Rotzschke et al. (1990b) Rotzschke et al. (1990b) Van Bleek and Nathenson (1990) Rotzschke et al. (1991a) Pamer etal. (1991)

LSPFPFDL

Udaka et al. (1992)

VAITRIEQLSPFPFDL

Udaka et al. (1993)

ILKEPVHGV

Tsomides et al. (1 994)

SLYNTVATL

Tsomides et al. (1994)

HNEGFWCPGPHR HNEGFYVCPGPHRP ASFEAQGALANIAVDKA KPVSQMRMATPLLMR

Rudensky el al. (1991)

SPSYVYHQFERRAK SPSYVYHQFERRAKY SPSYVYHQFERKAKYK

Rudensky et al. (1991)

TYQRTRALV ASNENMETM RGYVYQGL

GKYLYEIARKHPYF DGSTDYGILQINS DGSTDYGILQINSR DGSTDYGILQINSRW DYGILQINSRWWC

Nelson et al. (1993)

KMRMATPLLMQALP KMRMATPLLMQALPM LPKPPKPVSK ...P LPKPPKPVSK...PM LPKPPKPVSK...PMG PKPPKPVSK...P PKPPKPVSK...PM PKPPKPVSK...PMG KPPKPVSK...P KPPKPVSK...PM PPKPVSK...P

Chicz el al. (1992)

' Adapted from Tsomides and Eisen (1993a).

'

Only MHC-I binding natural peptides that are recognized by established T-cell clones are shown; MHC-I binding sequences identified on the basis of their relatively high abundance are not included. ' For brevity, invariant chain residues 106-1 17 are represented by ellipses (...).


19

MHC-I1 contacts involve the peptide backbone rather than specific peptide side chains, implying different mechanisms for degenerate peptide binding by MHC-I and MHC-I1 proteins (Table I). Once it was appreciated that purified MHC-I molecules are normally occupied by stably bound endogenous peptides, the failure of early attempts to demonstrate significant peptide binding to purified MHC-I molecules (Chen and Parham, 1989; Tsomides and Eisen, 1990) became understandable. Soon techniques based on whole-cell binding assays (Christinck et al., 1991), MHC-I immunoprecipitation from cell lysates (Cerundolo et al., 1991), or in vitro binding to empty MHC-I molecules purified from transfected Drosophila cells (Matsumura et al., 199213; Saito et al., 1993) or other cells (Boyd et al., 1992; Ojcius et al., 1993; Fahnestock et al., 1994) allowed the measurement of equilibrium binding constants for a wide variety of peptide-MHC-I reactions. Parker et al. (1992, 1994) used ppm dissociation as a surrogate indicator for the stability of pepMHC-I complexes, and Olsen et al. (1994) found that peptides were able to bind well to purified MHC-I molecules at reduced temperatures. Each of these experimental systems suffers from certain limitations, a shared one being that the binding of synthetic peptides to fully formed MHC-I molecules may not accurately mirror events in the ER, where this reaction ordinarily takes place. Nevertheless, these equilibrium values seem to reflect the specificities otherwise observed for peptide-MHC-I reactions, and similar measurements have proved useful as indicators of potential peptide immunogenicity (Feltkamp et al., 1993; Celis et al., 1994; Sette et al., 1994). Given the ability to measure equilibrium constants for the peptideMHC reaction, the number of pepMHC complexes per target cell required to trigger the activity of a given T-cell clone can be estimated in assays where T-cell activity depends on the concentration of synthetic peptides (added to the extracellular medium). The estimates are based on the Karush (1970) form of the law of mass action (Day, 1990):

r = Kcnl(1 + Kc)

(1)

In Eq. ( l ) , r is the number of pepMHC complexes per target cell, K is the equilibrium association constant for a peptide-MHC reaction, c is the free concentration of peptide that sensitizes target cells for a particular level of lysis (e.g., half-maximal) by a given CTL, and n is the total number of MHC binding sites per target cell accessible to extracellular peptide. For several reasons, the calculated values of r should be regarded as only reasonable approximations, e.g., because the peptide may be subject to proteolytic degradation during the assays. More importantly, the number of accessible sites ( n )changes over time as newly synthesized MHC molecules migrate to the target cell surface and some (unknown) proportion have accessible peptide binding sites, either because they are empty or because

20

HERMAN N . EISEN E?’ AL.

they are occupied by rapidly dissociating natural peptides that exchange readily with peptides in the extracellular medium. Nevertheless, Eq. (1) yields reasonable approximations for those peptides whose reactions with MHC (on intact cells) reach a steady state within a short time (e.g., 2-3 hr). As discussed later, and in contrast to values of a few hundred reported in earlier studies (Harding and Unanue, 1990; Demotz et al., 1990; Vitiello et al., 1990; Christinck et al., 1991), the minimum number of activating complexes per target cell was found to vary over several orders of magnitude depending on the particular T cell, MHC-I molecule, and peptide, from several thousand per target cell to fewer than 10 in optimal combinations (Kageyama et al., 1995) (see Section V1,D). How do pepMHC complexes recognized by T cells arise? CD8+ T cells react with pepMHC-I complexes that form within the ER of a cell as newly synthesized MHC-I molecules assemble (reviewed by Monaco, 1992; Yewdell and Bennink, 1992; Germain and Margulies, 1993; Heemels and Ploegh, 1995). The peptides are generated by limited proteolysis in the cytosol and translocated into the ER by MHC-encoded peptide transporters (transporters associated with antigen processing, TAP) (Spies et al., 1991), or in some cases via TAP-independent pathways (Anderson et al., 1991; Henderson et al., 1992; Zweerinket al., 1993; Hammond et al., 1993; Zhou et al., 1993). Once inside the ER, peptides may or may not be subjected to further proteolysis (Falk et al., 1990; Yewdell et al., 1994) before binding to nascent MHC-I molecules, which are then exported to the cell surface as mature pepMHC complexes. Given several thousand intracellular proteins potentially available for degradation in any given nucleated cell, and manyfold higher numbers of peptides theoretically available for transport into the ER and for subsequent binding to MHC-I molecules, it is apparent that competition among peptides must be a significant feature of antigen presentation. It may be that only tightly binding peptides compete effectively for MHC-I binding sites, explaining the slow dissociation rates that have been measured. Certainly peptide selectivity exists at the levels of MHC binding (Falk et al., 1991a; Schumacher et al., 1991) and TAP-mediated peptide translocation into the ER (Shepherd et al., 1993; Neefjes et al., 1993; Schumacher et al., 1994), and perhaps also at the level of proteolysis (Goldberg and Rock, 1992), but much remains to be clarified about the generation of MHC-Ibinding peptides in vivo. It is clear that the pepMHC-I complexes ultimately arriving at the surface of a cell represent a sampling of the contents of that cell, with some peptides present in relatively high copy numbers, e.g., several hundred to perhaps several thousand identical pepMHC-I complexes per cell, and a greater number of peptides relatively scarce, e.g., between 1 and 100 com-


21

plexes per cell (van Bleek and Nathenson, 1990; Falk et al., 1991b; Hunt et al., 1992a; Udaka et al., 1992; Tsomides et al., 1994). Most natural MHCI-binding peptides arise from the normal self proteins of an individual and are not recognized efficiently by the mature T cells of that individual, which are purged of most self-reactive cells as they develop in the thymus (negative selection) or rendered unresponsive (anergic)in the periphery (Schwartz, 1989). However, peptides from a foreign protein, e.g., originating from a virus or other intracellular microbe (Townsend and Bodmer, 1989), or an anomalous self protein, such as the mutated protein of a tumor cell (Lurquin et al., 1989; Mandelboim et al., 1994), can (in association with a self MHC-I protein) elicit and react with CD8+ T cells. Peptides from certain normal (nonmutated) self proteins in some tumors can also be recognized, in association with MHC-I, by T cells that react against the tumor (Boon, 1994); for example, CD8+ T cells that can be isolated from excised human melanomas (Kawakami et al., 1994a,b; Coulie et al., 1994; Castelli et al., 1995; Bakker et al., 1994; Tsomides et al., 1996) or lymph nodes (Cox et al., 1994) have recently been shown to recognize peptides from nonmutated melanocyte-specific proteins in association with HLA-A2. The underlying requirement for all these responses to be productive is that the abundance of the naturally processed pepMHC complex be sufficient and the TCR affinity and kinetics be favorable (see Section VI). CD4-t T cells, in contrast to CD8+ cells, react with pepMHC-I1 complexes on specialized APC (e.g., macrophages, dendritic cells, B cells). Peptides that bind to MHC-I1 proteins generally arise from integral membrane proteins or from endocytosed proteins, either soluble or membraneassociated (Tables I and 11). These peptides are produced in special endosomal organelles by a set of cellular proteases that differ from those that generate MHC-I-binding peptides. Newly synthesized MHC-I1 molecules, complexed with a nonpolymorphic invariant chain, traffic into this endocytic pathway (Neefjes et al., 1990) and come into contact with the available peptides. There the invariant chain is partially degraded, allowing some of the peptides to bind to MHC-I1 molecules under the acidic conditions that prevail in these organelles (pH optimum -5.0) and leading to egress of pepMHC-I1 complexes to the cell surface. Studies aimed at quantitating the binding between synthetic peptides and MHC-I1 molecules succeeded before those involving peptide-MHC-I reactions (Babbitt et al., 1985; Buus et al., 1986; Jardetzky et al., 1990; Roche and Cresswell, 1990; Roof et al., 1990; Rothbard and Gefter, 1991). As in the case of MHC-I-binding peptides, immunodominance describes the phenomenon whereby only one or a few peptides from a given protein are bound ef€iciently by a given MHC-I1 molecule and presented to T cells, accounting for the bulk of a polyclonal T-cell response to that protein.

22

HERMAN N . EISEN El' AL.

Of considerable interest are the kinetics of the reactions between peptides and MHC proteins. The first kinetic data, obtained for MHC-11, indicated unusually slow on- and off-rates for the peptide-MHC reaction [e.g., tl,2(off) = 5-10 hr at 37"C, Buus et al., 19861. Sadegh-Nasseri and McConnell (1989) subsequently found evidence for rapid formation of a quickly dissociating complex that slowly converted to a stable form [tl,2(off) > 30 hr]; the long half-life for peptide dissociation was shown to limit the association rate for an added peptide (Tamp6 and McConnell, 1991). These kinetically distinguishable pepMHC-I1 complexes correlated with different behaviors in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: the short-lived complex dissociated into MHC-I1 a and p subunits, whereas the more stable complex migrated as an intact heterotrimer (a,p, peptide) (Sadegh-Nasseri and Germain, 1991, 1994; Stern and Wiley, 1992). Nelson et al. (1994) found that the dissociation half-times of pepMHC-I1 complexes vary widely, with selective persistence of the most stable complexes on APC helping to account for the striking immunodominance of certain peptides. Conversely, while a rapidly dissociating pepMHC-I1 complex is expected to be relatively ineffective at eliciting T-cell responses, it might also be ineffective at eliciting negative selection in the thymus and thus allow maturation of the corresponding autoreactive T cells. Thus, Fairchild et al. (1993) and Mason and McConnell (1994) suggested that, for this reason, a self peptide from myelin basic protein might fail to induce T-cell tolerance toward this peptide. The failure would leave potentially autoreactive T cells that could eventually cause autoimmune encephalomyelitis if activated by cross-reacting viral peptides (see Oldstone, 1987; Wucherpfennig and Strominger, 1995; Section V1,F). It remains to be seen whether such a mechanism also applies to peptides interacting with MHC-I proteins. A related possible mechanism for evading tolerance could be extrathymic posttranslational modification of a self peptide, leading to inadequate negative selection of T cells specific for the modified peptide (Wu et al., 1995). VI. T-CELL RESPONSES TO PEPMHC

The intensity of mature T-cell responses to pepMHC complexes on other cells is greatly affected by the cell surface abundance of these complexes and by small changes in peptide sequence, as illustrated in Fig. 4. When the same T cells and target cells (or APC) are used in the representative assays shown, synthetic peptides with closely related sequences can differ as much as several millionfold in the concentrations required for half-maximal intensity of the responses they elicit (SD5o values, Tsomides

23

AN'IIGEN-SPECIFIC T-CELL RECEPTORS

-+- LSPFPFM p X a

--+-- ANERADLIAYLKQATK

MCC ANERADLIAYLEOATK MCC(99E) --t ANERADLIAYLKQASK MCC(t02S) --&- ANERADLIAYLKQTAK PCC

-+- QLSPFPFDL

QL9 LSPFPFDLL LL9

U

1000

B

- 1 4 - 1 3 - 1 2 - 1 1 - 1 0 - 9 -8 -7 -6 -5

-9

-8

-7

-6

-5

-4

-3

Log peptide concentration, M FIG. 4. Responses of T cells to target cells sensitized with various concentrations of cognate peptides or their structural analogs. (A) Cytotoxic responses by a CD8+ CTL clone (2C) to a naturally occurring octapeptide p2Ca from a-ketogluterate dehydrogenase and three contiguous overlapping nonapeptides from this protein (QL9, SL9, and LL9) presented by the MHC-I protein Ld on I?-transfected target cells (T2-Ld) shown as percentspecific lysis of the target cells (Sykulev et al., 1994h). (B) IL-2 production by a CD4+ T-cell hyhridoma (2B4) in response to peptides from cytochrome c of mouse (MCC) or pigeon (PCC)presented by the MHC-I1 protein IEk on IEk-transfected CHO cells (Matsui et al., 1994).

et al., 1991). In attempts to account for these enormous differences, some T cell-target cell systems have been analyzed extensively in terms of (1) the affinity of T-cell TCR for particular pepMHC complexes; and (2) the abundance of these complexes on target cells (epitope density). As discussed in Section VI,C, TCR reactions with pepMHC complexes approach equilibrium rapidly, indicating that intrinsic equilibrium constants for these reactions (TCR affinities) may be relevant to understanding the responses illustrated in Fig. 4. A. Afinity: Intrinsic Equilibrium Constants of TCR-PePMHC Reactions

Several approaches have been used to determine the equilibrium binding constants of TCR-pepMHC reactions. They include (1) competition assays in which soluble pepMHC complexes compete with lZ5I-labeledantibodies (or Fab' fragments) for binding to TCR on intact T cells; (2)direct binding of soluble '*51-labeledpepMHC complexes to TCR on intact cells; or (3) binding of unlabeled soluble pepMHC complexes to TCR molecules produced in soluble form by genetic engineering, immobilized on a solid

24

HEKMAN N . EISEN

Er

AL.

support, and the reaction evaluated by surface plasmon resonance (SPR, below). All these approaches have been sufficiently consistent (Table 111) to suggest the fimctional equivalence of the genetically engineered soluble analogs of TCR and MHC proteins to their native membrane-associated forms. The first measurement of an intrinsic TCR-pepMHC equilibrium constant involved competition for the TCR on intact CD4+ T cells between a soluble pepMHC-I1 complex and 1z51-labeledFab fragments of an antibody to the TCR VP domain (Matsui et al., 1991). The free pepMHC concentration that inhibited binding of the lZ5I-labeledFab by 50% was 5 x 10-5M . This value (Kd) and the other equilibrium constants discussed here are all expressed in the following discussion as association constants (K, = 1/&) (Table 111).A slightly higher K , was found in another study that used another soluble TCR to inhibit the response of an intact CD4+ T cell (measured by T-cell cytokine production) to an intact APC. By comparing the inhibitory effect of the soluble TCR with the inhibitory effect of an anti-MHC Fab' fragment, it was estimated that the intrinsic affinity of the TCR for the pepMHC-I1 complex was lo3M-I (Weber et al., 1992). This result should be viewed with the understanding that it was indirect and based not on a measured physical interaction between TCR and its ligand but on the production of interleukin-2 (IL-2). The equilibrium constants determined by Matsui et al. (1991) and Weber et al. (1992) (2 x 104M-' and -lo5 M-I, respectively)were consistent with the often expressed expectation that TCR affinities would generally fall in the low range exhibited by antibodies having germline V domain (i.e., not somatically mutated) sequences (e.g., Eisen, 1986; Davis, 1990). However, TCR with substantially higher intrinsic affinities were subsequently found by Sykulev et al. (1994a): 1-2 x lo6 M-l in a binding assay involving the Iz5I-labeledFab' fragment of an anticlonotypic antibody to the TCR on a CD8+ T-cell clone (2C) in competition with a soluble pepMHC complex [formed by the MHC-I protein Ld and an octapeptide from a-ketoglutarate dehydrogenase, LSPFPFDL, termed p2Ca (Udaka et al., 1992, 1993)] (Table 111). Later, the same TCR was found to have a 10-fold higher affinity (1-2 x lo7M-') for a related pep.Ld complex in which the peptide differed from p2Ca only by an additional glutamine residue at the N terminus (QLSPFPFDL, QL9) (Sykulev et al., 199413). Given this high intrinsic affinity, it proved possible to measure the direct binding of QL9.Ld complexes (trace-labeled with Iz5I)to TCR on intact 2C cells, obviating the need for indirect measurement by competition with an antireceptor antibody. The values obtained by direct and indirect approaches were in close agreement, indicating the feasibility of measuring TCR affinities on diverse T-cell clones without requiring rare (e.g., clonotypic) anti-TCR antibodies.

-

TABLE I11

Equilibrium Association Constantsfor Binding of PeptUie-MHC Complexes to TCR T-cell clone (CD4 or CD8)

Peptidea Name

MHC

Sequence

Class

Allele

Equilibrium constant, P (M-I)

Method6

Refs.'

5C.C7 (CD4) 228.5 (CD4)

MCC MCC(99E)

ANERADLIAYLKQATK ANERADLIAYLEQATK

I1 I1

IEk I Ek

2.0 x 104 1.9 x 104

Competition Competition

(1) (11

14.3d (CD4)

m110-120

CFERFEIFPKE

I1

IEd

2.0 x 105

Competition

(2)

QL9 QL9 p2Ca SL9 p2Ca-A3 p2Ca-A5 p2Ca-A8 p2Ca pvsv pOV8 p2Ca p2Ca

QLSPFPFDL QLSPFPFDL LSPFPFDL SPFPFDLLL LSAFPFDL LSPFAFDL LSPFPFDA LSPFPFDL RGYVYQGL SIINFEKL LSPFPFDL LSPFPFDL

1

Ld Ld Ld Ld Ld Ld Ld

LO x 107 2.0 x 107 2.0 x 106 1.4 x 104 2.0 x 104 1.6 x 104 1.7 x lofi 3.0 x 103 Rl are rolled over the surface and define the accessible surfaces as (continuous) lines traced by their centers. Their contact surfaces are the (discontinuous) lines of contact with the individual atoms. Their reentrant surfaces are the (discontinuous) lines that fail to make contact with the probe. Together, the contact and reentrant surfaces define the (continuous) molecular surface. Note that, as the probe radius goes to inifinity, so does the accessible surface while the contact surface converges to a small value. (Reproduced with permission from Richards, 1977. Annu. Rev. Biophys. Bioeng. Vol. 6. 0 1977 by Annual Reviews, Inc.)

surfaces of the same protein, effectively defined by differently sized probes, may vary and may have somewhat different properties. Large probes ( r = 10 A) that can sample, by direct contact, only the most protruding parts of a protein surface, “see” a surface with an atomic compositio? rather different from that seen by a smaller, water-sized probe (r = 1.4 A) (Table I). The concept of solvent accessibility inspired a large body of work. The studies by Chothia (1974) and others established proportionality between the Lee and Richards solvent-accessible surfaces and the magnitude of the hydrophobic effect of the solute. The Lee and Richards (1971) algorithm also became the basis of various programs for the display of molecular surfaces (Fig. 3), most notably the dot surface diagrams due to Connolly (1983) and the “plaster” surface approximations of the program GRASP

159

COMPUTATIONAL BIOCHEMISI'RY OF ANTIBODIES

TABLE I

Accessibility ofAmino Acid Residues to Probes ofDzfferent Radii" Probe radius = 1.4 8,

Probe radius = 11.4 8,

Accessibility

Accessibility

Amino acid

(A")

Amino Acid

(AZ)

'4% LYS Gln Glu Asn Tyr ASP Pro Thr Ser His Trp Ala Gly Val Leu Met Ile Phe cys

101 103 79

Lys k g Gln Asn Glu ASP Pro Ser TYr Thr His Ala Gly Val Leu Met Ile TrP CYS Phe

223 219 152 1 I5 115 104 92 79 77 74 40 36 33 22 20

66 65 61 59 53 46 43 41 32 28 25 25 22 22 20 18 13

14

14 14 5 5

Adapted from Novotny et al. (1987). The values represent averages of calculations performed on 11 single-domain proteins.

(Nicholls, 1992). Richmond (1984) presented an analytical surface representation employing differential geometry and allowing for some measure of surface minimization. More recently, Pascual-Ahuir and Silla (1990), in their program GEPOL, gave us a tool for the generation of a molecular surface (see Fig. 2) and proposed that it is a more direct correlate of hydrophobicity than the solvent-accessible surface is (Tufion et al., 1992). Richards (1974) and Finney (1975) described computer algorithms that partitioned the space occupied by a protein into atomic volumes based on Voronoi polyhedra (Fig. 4). This allowed the local packing density of proteins to be determined as -0.75, a very high density indeed, approaching that of closely packed ideal spheres. Volume (Connolly, 1985, Stouch and Jurs, 1986) and surface partitioning is an important component of various computerized docking procedures (Kuntz and Crippen, 1979; Connolly, 1986; Gregoret and Cohen, 1990, 1991; Jiang and Kim, 1991; Cherfils

160

A

JIRI NOVOTNY AND JURGEN BAJORATH

.

B

FIG.4. Volumes occupied by protein atoms (lioronoi polyhedra). (A) A two-dimensional sketch of the Voronoi algorithm. Points show centers of atoms. Vectors are drawn from an atom to all its neighbors within a sphere of 8 A radius. Planes perpendicular to the vectors, and normal to the van der Waals radii of the contacting atoms, are constructed, defining the smallest polygon (a polyhedron in three dimensions, the Voronoi polyhedron) that encloses the central atom. The ratio of the atomic van der Waals volume to that of the Voronoi polyhedron is the packing density of the atom. (B) The Voronoi polyhedron of the Oy atom in a serine residue. Atoms are shown as spheres with radii one-quarter of their van der Waals radii. [Reprinted with permission from Harpaz et al. (1994).

et al., 1991) that determine the best shape complementarity in protein-

ligand pairs. The importance of inter- and intraprotein packing to protein function and stability has been hotly debated, with the focus on the following issue: Is tight packing of complementary side chains the decisive determinant of protein folds [i.e., the “jigsaw puzzle” or “watchmaker” paradigm of Harpaz et al. (1994)], or could tight packing be readily achieved with any nonspecific assemblage of side chains [e.g., the “nuts-and-bolts-in-a-bag” paradigm of Bromberg and Dill (1 994)]? Evolutionary evidence seems to favor the latter view. Side-chain-side-chain contacts in proteins of similar threedimensional structure show high variability (Russel and Barton, 1994), often with as few as 12%common contacts and virtually no conservation of energetically favorable side-chain-side-chain interactions. Gerstein et al. (1994) analyzed volume changes in side chains occurring in protein evolution and found that only about half of the protein cores strongly conserved their volume (to within 10% variation). The rest of the positions showed various degrees of variation that, in some core sites and in nearly all surface sites, approached random variation (28% and more). An inspection of interfaces in protein-protein (Krystek et al., 1993) and, particularly, in antibody-

COMPUTATIONAL BIOCHEMISTRY OF ANTIBODIES

161

antigen (Lawrence and Colman, 1993) complexes revealed relatively loose complementarity and enough empty space to suggest that, on the whole, the main role of (approximate) shape complementarity may be expulsion of water from hydrophobic interfaces and/or modulation of interprotein interactions by surface-prebound water molecules (see Section VII,C,3), rather than precisely engineered atomic interactions and steric repulsions.

C. Structural Superpositions Statistical analysis of the available Brookhaven Protein Databank entries has indicated that there may be no more than about 1000 distinct proteinfold families (Chothia, 1992), and that the many millions of protein amino acid sequences may merely repeat the same folding motifs over and over again. Although the three-dimensional dissimilarity of two proteins parallels their sequence diversity (Chothia and Lesk, 1986), proteins with no sequence similarity may still share the same fold. Three-dimensional similarities that persist in proteins despite sequence differences accumulated in evolution can be quantitatively analyzed by pairwise structural superpositions. The measure of overall corespondence of the two compared sets of atoms is the root-mean-square (rms) deviation (A):

McLachlan (1972), Diamond (1976), Kabsch (1976), and Rossmann and Argos (1975,1976) introduced formalisms for finding the best rotation to fit a given set of atoms to a target set of coordinates. Structural motifs (sets of backbone and/or side-chain atoms) are first identified in the two structures being compared, for example, residues participating in heme binding in globins and in cytochromes. These are then used to carry out the best rigid-body rotation and translation that matches the Cartesian coordinates of one atomic set (the source) to those of the other atomic set (the target) such that the (weighted) sum of squared deviations is minimized. In the McLachlan (1972) formulation, given the two sets of N coordinate vectors hba(a = 1, ... N)we seek an orthogonal rotation matrix R and a translation t that convert the coordinates aza(i = 1,2,3) to

and minimize the residual

162

JIKI NOVOTNY AND JUKGEN BAJORAlH

where w, is an arbitrary weight. Note that the formula implies equivalence of predefined pairs of atoms in the two structures. Several powerful algorithms were developed that find the best fit for two sets of atoms (structures) through systematic application of rotations and translations in the three-dimensional Cartesian space (Rossman and Argos, 1975; Diamond, 1976; Lesk, 1991). It is important to realize that there is no a przorz correct way of equivalencing protein structures and parts thereof. In current practice, the selection and definition of groups of superposed atoms implicitly contain a hypothesis of the cause of structural similarity; at the same time, the hypothesis is being tested by the superposition and the ensuing analysis of the results. For example, in order to measure differences in relative orientation of the Vr*and VH domains in different antibodies, it is possible to equivalence Ca atoms of one domain (e.g., V,) by least-squares superpositions, and to calculate the rotational-translational matrix required to best superpose the other domain pair (e.g., VH). However, given the existence of the invariant motifs at the VL-VH interface (Section IV,A), we may argue that a more meaningful way of equivalencing pairs of dimeric VL.-VH modules would be to least-squares superpose the six aromatic rings of the conserved “herringbone” cluster and the pair of Gln residues forming interdomain side-chain hydrogen bonds, rather than the complete domain backbones (Novotny and Sharp, 1992; Bajorath et al., 1995). It is reasonable to assume that the solvent-exposed /3 sheets are unimportant in formation of the interface and that by their excessive sequence (mass) variations, they may obscure the best alignment of the key interface-forming segments.

111. SmucruKEs, SEQUENCES, AND SUPERFAMILIES The relationship between amino acid sequence and three-dimensional structure is often described as a stereochemical code that transposes primary protein structures into tertiary protein structures (Epstein, 1964, 1966). The code is degenerate: Although the chemical structure (amino acid sequence) of the protein is known to determine the three-dimensional structure (Anfinsen and Haber, 1961; White, 1961), many different sequences can adopt the same fold. From the point of view of the stereochemical code, structural variability of immunoglobulin polypeptide chains offers an interesting, well-studied example of structural degeneracy exploited by nature for generation of the immune repertoire. It is generally accepted that each immunoglobulin sequence represents a unique antigenic specificity. Indeed, by refolding a denatured immune Fab and

COMPUTAI’IONAL BIOCHEMISI‘KY OF ANTIBODIES

163

demonstrating that it had regained its original specificity, Haber (1964) showed for the first time that the information of immune specificity resides in the amino acid (and, implicitly in the DNA) sequence alone. In this section, we will consider sequence similarity from the general perspective of polymer solubility and from the point of view of an immunoglobulin superfamily of structures.

A . Gross Immunoglobulin Structure It is of functional significance that immunoglobulins have a modular design (Hill et al., 1966; Singer and Doolittle, 1966; Edelman, 1970; Fig. 5). Both the light (25,000 Da) and the heavy chains (50,000 Da) are composed of several domains, each of which consists of approximately 110 amino acid residues. The antigen binding function is concentrated in the amino terminal, the so-called variable domains of both the light and heavy chains (Hilschmann and Craig, 1965; Titani et al., 1965). It has been demonstrated experimentally that the isolated variable domains can refold without changing the rest of the polypeptide chain (Hochman et al., 1973). Antibody modules share the same architectural motif as noncovalently formed domain dimers. The domains themselves are formed by two antiparallel P sheets closely packed face to face (Richardson, 1981; Lesk and Chothia, 1982; Novotny et al., 1983; see Section II1,C). The basic building blocks of immunoglobulin domains are therefore P-strand segments and reverse loops connecting these strands. The domains are linked together by rather extended “hinge” or “switch” peptides long enough to permit the movement of domains with respect to each other. Comparison of currently available X-ray structures of Fab fragments shows clearly that the “elbow” angle between the long axes of the VL-VH and CL-CHl dimeric modules can vary from essentially extended (180”) to quite sharp (100”). Flexibility of the hinge between the Fabn and the Fc is well documented, e.g., by the electron microscopic studies of Valentine and Green (1967) and the hydrodynamic studies of Tanford and co-workers (Noelken et al., 1965; Fig. 5). Some immunoglobulin subclasses (e.g., the mouse IgG2.J are more flexible than others (e.g., the relatively rigid mouse IgGl) and the polypeptide segments crucial to flexibility have been mapped by domain swap experiments (see Section IX,A) to the hinge and the CHI loop 131139 (Schneider et al., 1988). Contrast-matching, small-angle scattering of neutrons determined the mean antigen-antigen distance between the and IgGnb molecules as 11.7-12.4 nm, with a bivalent mouse IgC,, IgCZA, large variance (-4 nm, Sosnick et al., 1992). For all three subclasses, the scattering data could be fitted only with a distribution of distances rather

Fv

Fab

-45m-

FIG.5. Various models of a complete I@ molecule. (hj) a sketch of covalent structure showing the light (L) and heavy (H) chains, interchain disulfide bonds (SS), and approximate dimensions of the Fab and Fc fragments. Variable domains are hatched. (Middle)Model derived from hydrodynamic studies by Noelken et al. (1965).A and B chains refer to the H and L chains, respectively; fragments I and I1 are the Fab fragments, and fragment I11 is the Fc fragment. (Rzght)a-Carbon tracing of a complete I g C molecule (Harris et al., 1992). Light line, L chain; heavy line, H chain.


165

than with a single distance. Thus, antibody molecules in solution sample a large selection of hinge and elbow angles at any given moment, a structural trait undoubtedly important for efficient antigen engagement. B. Polymer Solubility and Amino Acid Sequence Variability

The conformational states of a polymer in solution (random coil or folded, compact or extended) depend on a relative balance of polymerpolymer and polymer-solute interactions (Flory, 1969). The fact that in proteins many different sequences adopt the same compact fold strongly suggests that there is one gross (solubility)property of natural amino acids that overwhelmingly determines the structure of the folded state (Dill, 1990). Polypeptide chains that may have only 1 or 2 amino acids out of 10 in common, but conserve the sequence distribution of hydrophobic and hydrophilic residues, retain the same fold (Bashford et al., 1987), suggesting that the hydrophobicity pattern of an amino acid sequence is the most likely determinant of protein structure. The computer experiments of Chan and Dill (1990), enumerating the complete conformational spaces of two-dimensional polymers composed of two types of residues (black and white, hydrophobic and hydrophilic), reproduced many of the features of folded proteins, in particular the existence of unique compact structures attainable by different sequences, the families of related sequences, and the secondary structure patterns. It is often asked whether the native protein structure is the one of global free energy minimum, and whether kinetic aspects of protein folding are not equally important (Baker and Agard, 1994). Although no definitive answer is currently available, some recent results emphasize the importance of a thermodynamic minimum in the determination of native folds. Sippl (1990), Bryant and Lawrence (1991), Bowie et al. (1991), and others developed pseudo potentials that capture, in numerical form, important features of the known protein structures, e.g., the averages and distributions of pairwise residue-residue distances, solvent exposures of all 20 amino acids, distances (and implicitly, electrostatic interactions) of formally charged residues, and the character of the immediate neighborhood of side chains in various folds. Native amino acid sequences examined with these pseudo potentials virtually always show a distinct energy minimum associated with only one, the native, fold. Energies obtained with misfolded structures (i.e., fits of the sequence into alternative folds of other proteins) are distinctly less stable. Likewise, the computer folding experiments of Sali et al. (1994), with proteinlike simplified polymers on a threedimensional lattice, seemed to imply that the sole criterion for a fast folding step toward compact, nativelike structures (molten globules) was the existence of a deep global minimum in the free energy landscape.

166

JIKI NOV0I”Y

AND J U R G E N UAjORATH

C. Immunoglobulin Superfamily

Lesk and Chothia (1982) were the first to define the core of immunoglobulin domains (VK,VA, Vy, CZ, Cyl, Cy2, Cy3) on the basis of leastsquares comparisons of available X-ray structures. Although the core of each of the domains consisted of about 35-36 homologous residues, forming two p sheets packed face to face, only 3 residues were common to all the domains: 2 cysteines that formed a disulfide bridge between the p sheets, and a tryptophan that packed against them (Fig. 6). The other interior residues tended to retain hydrophobic character but varied greatly in size. Williams and Barclay (1988), Harpaz and Chothia (1994), and others defined groups of amino acid sequences designated the immunoglobulin superfamily (IgSF). In the superfamily, sequence similarity ranges from a clear homology, virtually guaranteeing an identical fold (-4040% identical residues), to weak similarities (10-1 5%), and a putative three-dimensional structure for the more controversial members of the family was debated for some time. For example, a folding motif radically different from that of immunoglobulins was proposed for the CD2 antigen: the (ap fold of this molecule (Clayton et al., 1987) was seemingly favored by circular dichroism measurements showing a significant proportion of a helix in the CD2 extracellular domain(s), whereas Williams et al. (1987) argued CD2 similarity with immunglobulins on the basis of sparse, conserved sequence motifs. X-ray (Jones et al., 1992) and NMK studies (Driscoll et al., 1991, Withka et al., 1993) confirmed the presence of an immunoglobulin fold in the CD2 leukocyte antigen. Given the marginal sequence similarity of CD2 to immunoglobulins, elucidation of this structure constituted strong support for the concept of an immunoglobulin superfamily. It appears that, within the immunoglobulin family, the number of constituent p strands may vary but the general folding topology, i.e., the Greek key motif (Richardson, 1981), remains conserved. According to Harpaz and Chothia (1994), the current superfamily tree can be subdivided into sets, i.e., clusters of domains that are structurally more similar to each other than to a member of any other set. Sets are distinguished by the number ofp strands in the two individual sheets and their length (Fig. 6). An antiparallel p sandwich is one of the commonest protein folds observed, and Bork et al. (1994) used the computer program DALI to carry out a complete three-dimensional classification of the known P-sheeted structures. The common structural core of four p strands, B, C, E, and F, was found to be shared by nine distinct families. Subclass distinctions arose as additional /3 strands were appended to the core. Disulfide bridges were not necessarily invariant in number and location within the subclasses. The four major topological subtypes were described as (1) the c-type, i.e., classical seven-stranded, Ig constant-domain topology; (2) the s-type, a

FIG 6. Folding motifs of the immunoglobulin superfamily (Williams and Barclay, 1988; Harpaz and Chothia, 1994). (Top left) V domain fold (the VH domain of the McPC 603 Fab fragment) with two four-stranded /3 sheets. (Top rzght) C1 domain fold (the CI, domain of the McPC 603 Fab fragment), a p sandwich with a four-stranded and a three-stranded p sheet with the strand topology A-B-D-E and C-F-G. The core of the immunoglobulin domain (Lesk and Chothia, 1982), i.e., the intradomain disulfide and a tryptophan side chain packed against it, is also shown. (Bottom) C2 fold (the fourth extracellular domain of CD4) is essentially the C 1 fold with the D strand missing. See Bork el al. (1994) for a more encompassing definition of the immunoglobulin fold. [Graphics by Molscript (Kraulis, 1991).I

168


seven-stranded “switched” type with strand topology E-B-A and G-F-C-C’; (3) the h-type, a hybrid between (1) and (2) where the strand C’/D is kinked and hydrogen-bonds to the strand C; and (4) the v-type, a nine-stranded type typified by variable domains. In this broad definition, the immunoglobulin fold comprised proteins as diverse as the growth hormone receptor domain 2, fibronectin, neuroglian domains 1 and 2, fungal galactose oxidase domain 3, the PapD protein domain 1, and cyclodextrin glycosyltransferase domain D. In all these folds, the core motif of four antiparallel 4, strands conserved its tight packing, strand curvature, and sheet-sheet angle.

ANATOMY OF ANTIBODY BINDING SITE IV. MOLECULAR Primary structures of different light and heavy chain variable domains and V H ) contain segments conspicuously variable both in length and amino acid composition (Wu and Kabat, 1970). There are three such segments in both VL and V H , and their positions within the domain sequences are homologous. It has been hypothesized that these hypervariable loops, designated L1, L2, L3, H1, H2, and H3, are responsible for antibody specificity and form the surface of the antigen combining site. Accordingly, they are also known as complementarity-determining regions (CDRs). X-ray crystallographic structures of many Fab fragments indeed confirmed that, by noncovalent association of the VL and VH domains, all six loops come into close contact and form a contiguous area on the surface of the VL-VIj dimer from which the binding site (the paratope in serological parlance) is constructed (Fig. 7). (VL

A . VL-VH Interface /3 Barrel Domains from different chains associate by noncovalent forces to form domain heterodimers. Each domain can be viewed as having two /3-sheet surfaces, and in the complete immunoglobulin molecule one of the /3 sheets faces the solvent, whereas the other mediates contact with the other domain of the pair. The principal difference between a VL-VH type of dimerization and constant-constant domain pairs lies in the fact that the /3 sheets involved in the domain interface are different in the two domain types (Edmundson et al., 1975). It has been noticed that /3 sheets are not flat, as originally suggested by Pauling and Corey (1951), but twisted (Chothia, 1973). The VL-VH contact /3 sheets are not only twisted but also strongly curved, giving an impression of being wrapped counterclockwise around an elliptical-hyperboloidal surface (Novotny and Haber, 1985). Thus, the VL-VH interface seems to


169

give rise to a third p barrel formed by walls of the two interacting p barrels of the VL-VH domains. Structural fingerprints of this unique fold are (1) the two /3 bulges in the edge p strands G of the VL and VH domains, with sequences Phe-Gly-Gly-Gly and Trp-Gly-X-Gly, respectively; (2) an aromatic ring cluster of at least six Tyr/Phe residues forming a second layer of side-chain interactions in the VL-V, interface; and ( 3 ) a pair of Gln residues forming a pair of hydrogen bonds across the VL-VH interface (see Fig. 7; Novotny and Haber, 1985). The conserved features of antibody binding sites are worth recounting in greater detail. The VL-VH interface forms a close-packed, twisted p barrel characterized by cross-sectional dimensions 1.04 x 0.66 nm (10.4 x 6.6 A) and a top-to-bottom twist angle of 212". The geometry of the interface is preserved via the invariance of about 12 side chains, both inside the domain and on their surface. Buried polar residues form a conserved hydrogen-bonded network that has a similar topological connectivity in the two domain types. The two hydrogen bonds contributed by invariant Gln side chains extend across the interface and anchor the p sheets in their relative orientation. Invariant aromatic residues close-pack at the bottom of the binding site p barrel with their ring planes oriented perpendicularly in the characteristic herringbone packing mode. About 18 nm2 of protein surface is buried between the domains and about 3040% of this contact surface is contributed by the hypervariable regions (Novotny and Haber, 1985). The p sheets that form the interface have edge strands that are strongly coiled by /3 bulges. As a result, the edge strands fold back over their ownp sheet at two diagonally opposite corners. In the VL-VH dimer, residues from these edge strands form the central part of the interface, resulting in what we call three-layer packing; i.e., there is a third layer composed of side chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing (Chothia et al., 1986) is different from the common aligned or orthogonal /3-sheet packing found in other P-sheeted proteins (Chothia and Janin, 1981, 1982). Conservation of the geometry of the VL-VH interface strongly contrasts with the variability of the CDR loops that provide connections between the /3 strands of the interface barrel. A schematic diagram of the binding region is shown in Fig. 7. It is interesting to note that one of the p strands in each of the domains corresponds to the J gene segment and is encoded separately from the rest of the variable-region gene (Early et al., 1980; Bernard and Gough, 1980; Newel1 et al., 1980). The region of the V geneJ gene junction maps into the L3 and H3 hypervariable loops, with additional amino acid sequence variablility generated by frame shifting the splice point between the two gene segments. In the heavy chain segments,

170

JIRI NOVOTNY AND JURGEN BAJJORATH

there is a third short gene segment, D, that becomes inserted in the V-J junction (Early et al., 1980; Siebenlist et al., 1981); the D segment corresponds exactly to the H3 loop. The overall picture that emerged from structural analysis is that of a conserved structural scaffold or framework (the Vl,-VH interface p barrel) to which the various hypervariable regions can be attached. This structural concept has been supported by the loop swap experiment of Jones et al. (1986) whereby a transfer of antigenic specificities, via gene splicing and protein expression, was accomplished between two different antibody V region scaffolds (see Section IX,D). It is obvious that amino acid sequence variations in the hypervariable loops change the shape of the outer parts of the binding site. However, many examples of antibody specificity modulation can be mapped to amino acids participating in formation of the bottom of the binding site and inaccessible to solvent or antigen (Stevens et al., 1980; Horne et al., 1982). Side chains of residues Leu L96, Glu H35, and Asp H101, known to be important to the specificity of phosphorylcholine-binding myelomas (Rudikoffet al., 1981; Cook et al., 1982, Rudikoffet al., 1982; Chothia et al., 1992), are either totally or partially buried in the V,-VH interface. Other hypervariable residues, such as C-terminal parts of L1 and H1 and Nterminal parts of J gene segments, also form part of the VL-VH contact surface. The calculated residue free energy contributions to the stability of antibody-antigen complexes (AGrc,,due;see Section VI1,C) also emphasized the importance of the bottom part of the binding site. For example, Tulip et al. (1994) and Rauffer et al. (1994) found that, as a rule, the highly destabilizing mutations of the N9 neuraminidase-NC4 1 antibody complex occurred at rigid residues, just as protein folding is more destabilized by mutations in the core than by mutations at mobile residues (Alber et al., 1987). B. Binding Potential of Surface Cavities In virtually all known protein structures, binding sites have the shape of cavities or grooves. In antibodies, the antibody binding site is located at the generally concave interface between the light and heavy chain variable domains. In one case, a small hapten (phosphorylcholine) enters deeply into the interdomain pocket (Satow et al., 1986), in another case the principal residue of a protein antigen (glutamine-121 of hen egg white lysozyme) is buried in the interface while the rest of the antigen-antibody contact area is rather flat, if irregularly undulated ( h i t et al., 1986). It is natural for the curvature of binding site cavities to match that of the antigens: Binding sites that accommodate small ligands have high curvatures and appear as pockets; those directed toward large protein antigens


171

have a low curvature, being more akin to valleys and grooves than to deep crevices (de la Paz et al., 1986). Why should the concave surface be favored as a specific combining site? Although no definitive answer is available at present, at least four good reasons can be cited. First, diffusion away from cavities is significantly slower than from flat surfaces or through the solvent. Primitive binding properties of simple concave organic molecules such as crown ethers and cavitands demonstrate this very clearly (Cram, 1983). Second, electrostatic fields are enhanced or focused in cavities, even though solvent quenches the field at other, flat or convex parts of the protein surface (Zauhar and Morgan, 1985; Klapper et al., 1986). Third, the hydrophobicity of a surface is a function of its curvature relative to the size and curvature of a water molecule, and concave surfaces are more hydrophobic than flat or convex surfaces (Sharp et al., 1991; Nicholls et al., 1991). Fourth, a hydrodynamic drag that develops at concave sites as a ligand molecule approaches may give rise to a a steering torque that forces the ligand into the pocket. The magnitude of this torque force is estimated to be significant, even larger than the electrostatic torque (Brune and Kim, 1994). The dielectric enhancement of an electrostatic field at cavities is illustrated in Fig. 3A. There the negative field generated by a pair of carboxyl groups embedded in a low-dielectric ( E =2) protein (with no other charges present) is compared to that generated by the same groups in water (i.e., a dielectric constant continuum of 80). The low dielectric of the protein not only allows the field to extend into a larger region of space but also modifies the field according to the shape of the dielectric boundary. The field enhancement inside the binding site cavity is striking (Novotny and Sharp, 1992). Computer simulations of antigen diffusion toward the antibody HyHEL-5 (Kozack and Subramaniam, 1993) suggested that the electrostatic field generated by charged amino acids at the binding site increases reaction rates, and effectively steers the antigen molecule into the site. The precise matching of charged groups with charged groups of the opposite sign is an example of chemical complementarity between antibody and antigen which, in addition to shape complementarity, determines the specificity of interaction. Complex specificity imposed by this charge distribution can, however, be achieved only by paying a price in free energy of desolvation which decreases complex stability (see Section VI1,B). C. Side-Chain Compositional Bias

It has been noted that antigen combining sites of antibodies have an unusual amino acid composition (Padlan, 1990). There is a statistically significant preference for aromatic rings and dipolar side chains, in partic-

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JIRI NOVOTNY AND JURCEN BAJORATH

ular Tyr and Asn. A similar side-chain bias emerges from empirical free energy calculations that make it possible to rank, in a semiquantitative manner, the combining site residues according to their binding signifiNovotny et al. 1989). cance (AGres,due, Is the side-chain bias seen in antibody combining sites shared by recognition sites of other proteins? A comparison with enzymes (Krystek et al., 1993) reveals substrate binding sites of neutral proteases populated by amino acids very different from those of antibody binding sites: small, rigid side chains that incur no conformational-entropic penalty on complex formation (Pro, Ala, Cys) or Gly, which has no side chain. Hence enzymes and their inhibitors on the one hand, and antibodies and their antigens on the other hand, employ different strategies for harnessing the free energy of binding in their respective complexes. Neutral proteases bind uncharged substrate molecules, and the nonentropic side chains in their binding sites (1) serve a better purpose than charged and dipolar amino acids, and (2) contribute favorably to binding energies by minimizing the stiff conformational entropy penalty. By analogy, the aromaticdipolar residue bias in antibody combining sites is likely to reflect the nature of antigenic surfaces. The majority of antigenic epitopes consist of loops where charged and dipolar amino acids are very common (Section VII). It seems that in the antibody combining sites the best binding surfaces are constructed with formally charged side chains carrying charges opposite those displayed by the antigens. Indeed, charge matching is frequently seen in antigen-antibody complexes, but it may be difficult to generate a perfect charge-charge match to sufficiently stabilize the complex. Further, (free) energy needed for charge desolvation on complex formation represents a large energetic penalty, so large that it may actually destabilize the complex (Section IV,D). From these points of view, aromatic rings stabilizing buried charges via “aromatic hydrogen bonds,” i.e., charge-ring n electron interactions (Levitt and Perutz, 1988), may represent a good alternative to charge neutralization and burial. Dipolar (amide) side chains represent, next to the formally charged and aromatic residues, another possible tool of charge neutralization. D. Electrostatic Perspective

Novotny and Sharp (1992) analyzed electrostatic fields generated by antibodies and their antigens, and discussed their importance in binding. For convenience, their conclusions are briefly summarized here. (1) The calculated field contours corresponded closely to the distribution of formally charged side chains on the surface. (2) By and large, the sign of the field at the binding site was found to be opposite that of the hapten. For dipolar (zwitterionic) molecules, the field dipole helped to


173

orient the ligand. Binding sites to neutral molecules may have measurable fields (menadione, galactan), or may be neutral (digoxin). The fields did not extend very far (i.e., beyond -4A) into the solvent. Outside the binding site region, the electrostatic potentials were complex and seemingly uncorrelated with the antigen binding. (3) Although there were local regions of electrostatic complementarity for large antigens, absolute complementarity was not a prerequisite for complex formation (e.g., D1.3lysozyme). (4) Fields at the 4-4-20 binding site specific for fluorescein suggested an electrostatically guided, lateral, two-dimensional diffusion of the ligand along the antibody surface into the site. ( 5 ) The HyHEL-5 and HyHEL- 10 antibodies against lysozyme had large negative fields complementing large positive fields of the antigen. In HyHEL-10, side chains that did not contact the antigen acted through space to augment the field and increase antibody affinity for the antigen. V. INSEARCH OF EFFECTOR SITES An antibody is a dual-purpose molecule: The antigen binding site allows for recognition of a virtually unlimited range of antigenic structures, and the constant domains mediate interactions with molecules belonging to the effector systems participating in antigen elimination. The y chain CH2 domain in particular plays an important role by engaging the Clq component of complement. The triggering event in the classical complement pathway is the binding of the first component of complement, C 1, to the Fc region of immunoglobulins aggregated in immune complexes. The structure of Clq, proposed by Reid and Porter (1975), has the appearance of a bunch of tulips: 18 chains, each about 200 amino acid residues in length, are linked in threes in the base and stalk regions to form collagen-like triple helices. Each of the six superhelices terminates with a globular head region thought to contain the Fc binding site. Apparently, the single Fc binding sites are of relatively low affinity ( K s 100-1000 M P ) and only the aggregation of several Fc fragments on immune complexes affects tight C l q binding due to multivalency mediated by the six C l q heads. Identification of the C l q binding site on IgG antibodies represents an early example of successful computer-aided structural analysis.

-

A. C l q Banding Site on Fc Fragment Burton et al. (1980) argued that the residues of the CH2 domain involved in interaction with C l q should fulfil two criteria. First, they should be accessible for C l q binding, that is, not buried in the interior of the

174


domain. Second, they should be highly conserved in those immunoglobulin molecules that bind Clq; this followed from the observation of crossspecies reactivity of C l q and IgG antibodies. Starting with the crystallographic structure of the Fc fragment (Deisenhofer, 1981), residues of the CH2 domain in contact with solvent were identified using the Lee and Richards (197 1) algorithm and a water-sized probe (radius 1.4 A). Of the 104 residues of the cH2 domain, 72 were found to be accessible to water. An inspection of contiguous, solventexposed hydrophobic patches identified 27 residues clustered into four large patches (> 100 Az),and 8 other residues found in two small patches or as isolated side chains. The location of three of the large patches made it unlikely that they were involved in C 1 q binding. Analysis of interspecies residue conservation then helped to eliminate all the patches but one. The sequence conservation analysis employed human and mouse myeloma proteins, and rabbit and guinea pig heavy chains, some of which did and some of which did not activate complement. Some invariant positions could be readily eliminated from being involved in Clq binding by being buried, covered with the carbohydrate, or affecting sugar attachment. Of the remaining positions, the invariance of some could be understood on structural grounds without invoking functional significance. All these considerations led to emergence of the continuous residues on the C-F-G /?-sheet face as a potential binding region composed of exposed, highly conserved, and mostly charged (five Lys, two Glu) residues. These residues were proposed to form the C l q binding site (Fig. 8). In 1988, Duncan and Winter systematically altered surface residues in the mouse IgG2, isotype and localized the binding site for C l q to essentially three side chains, Glu-318, Lys-320, and Lys-322, contained within the originally proposed seven residues of the CH2 C-F-G/?-sheet face. B. Fc Receptor Binding Sites and Rheumatoid Factor-Reactive Sates

The solvent accessibility-sequence conservation argument previously outlined was also used by Woof et al. (1986) to propose location of the Fcy receptor binding site to the hinge-link region of the CH2 domain (Woof FIL 8. Effector sites of the Fc fragment. (To$ lefl) Fc fragment (Deisenhofer, 1981). p Strands are shown as ribbons, and the oligosaccharide at the c H 2 / c H 2 interface as ball-andstick. T h e p hairpin predicted by Burton et nl. (1980) to be the C l q binding site is shown in heavy lines. (To$ right) Detail of the C"2 domain. Residues mutated in the F /3 strand by Duncan and Winter (1988). are also shown. (Lower left) Fc fragment with the residue Pro-238 highlighted. The Fc receptor binding site was localized to the polypeptide N-proximal to residue 238. This peptide, however, is not visible in the crystallographic structure of the human Fc fragment. (Lower rzght) Detail of the CH2 domain with the residue Pro-238 highlighted. [Graphics by Molscript (Kraulis, 1991).]

-

\/

\\

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et al., 1986; Burton, 1985). Duncan et al. (1988) engineered a single amino acid change in a mouse IgGeb antibody, E235L, which enabled the antibody to bind the high-affinity FcyRI receptor with a 100-fold improvement in affinity. Finally, Chappel et al. (1991), through the use of recombinant IgG1/IgGphybrids and site-directed mutagenesis, localized the essential receptor binding activity to the CH2 sequence Glu-Leu-Leu-Gly-Gly-Pro (residues 233-238) (Fig. 8). A rather complex effector system is that of immunoglobulin E and its various cellular receptors: the high-affinity FceRI, the low-affinity FceRII (also known as CD23), FceRIII, and complement receptor 2 (CR2, also known as CD21; see Sutton and Gould, 1993). The three-dimensional structure of IgE is unknown, but the amino acid sequence of the E chain suggests an overall structure similar to that of other classes of antibodies, in particular, a p chain with an additional constant domain in place of the hinge region. The two C-terminal domains of IgE were computermodeled on the basis of the crystallographic structure of the human IgG Fc fragment (Padlan and Davies, 1986). Highlights of the IgE model were (1) a disulfide bridge linking CLto CE1, and (2) two SS bridges linking the two Ce2 domains of the pseudo symmetry-related heavy chains. The predicted fold of the C Eand ~ C Edomains ~ assumed Asn-394-linked oligosaccharide chains to lie in between the Ce3 domains. With the use of recombinant peptides and domain exchange techniques, a segment of E chain sequence at the N terminus of the Ce3 domain (residues 330-335 of the E sequence) has been implicated in F e R I receptor binding. In order to localize rheumatoid factor (RF)-reactivesites on human IgG molecules, Peterson et al. (1995) used overlapping heptapeptides derived from the C H domain ~ sequence as competitors in RF-binding assays. To design the heptapeptides, they relied on the calculated, large-probe accessible “antigenic epitopes” postulated in the IgG CH3domain by Novotny et al. (1986a; see Section VIILA). About 10 residues were identified as critical for rheumatoid factor binding, clustered in the two segments between amino acids 343-354 and 401-439 of the human H chain sequence. C. Conserved Elbow Joint in Fab Fragments

Although not an effector site per se, the elbow joint described by Lesk and Chothia (1988) is an intriguing molecular device that is localized in a small region of the antibody molecule and mediates segmental domain flexibility (see Section 111,A). Based on computer graphics inspection of several Fab fragments with different elbow angles, Lesk and Chothia


177

(1988) concluded that movement of the VL-VH dimer relative to the CLCHI dimer involved interactions of three VH and two CHI residues that formed the molecular equivalent of a ball-and-socket joint, plus a few additional contacts that varied from structure to structure. Residues 11, 110, and 112 in VH packed against residues 149 and 150 in CHI, and the three VH residues belonging to the P-sheet framework contacted the two adjacent, turn-located CHI residues. The extent of the shift between members of any pair of Fab structures was estimated by superposing the CHI residues in different Fab fragments and calculating the mean position differences of the VHresidues. In one case, the VHresidues were displaced by a translation of 4.4 A and a rotation of 36". Inspection of space-filling models showed that the relative movement of the residues resembled that occurring in a ball-and-socketjoint, with the CHI residues forming the ball and the VHP-sheetresidues forming the socket (Fig. 9). VI. PROTOCOLS FOR THREE-DIMENSIONAL MODELING OF BINDING SITES Basic information about the nature and dimensions of antigen combining sites was derived in the precomputer age by solution chemistry and serology. Combining sites were probed by series of chemically similar haptens in the early works of Landsteiner (1962), Pressman and Grossberg (1968), Karush (1962), Kabat (1970), Haber (Haber et al., 1967, 1976), and Schechter (1971), to name but a few. The approximate dimensions of binding sites were such as to accommodate about six monosaccharide units or about three or four peptide units. In favorable cases, ingenious resonance transfer and chemical modification studies provided more detailed atom-atom distance information (e.g., Rosenstein and Richards, 1976). A. N M R Spectroscopy Combined with Computer Model Building In chemistry, it is common to derive structural formulas of compounds from spectroscopic methods such as nuclear magnetic resonance, spin resonance, circular dichroism, and infrared. The same methodology has been applied to proteins, and particularly to antibodies. If an atomic detail-level model is sought, the complexity of the task also calls for the use of computational methods. Some of the first atomic details of antibodyligand interactions emerged from a combination of NMR spectroscopy and computer-aided model building in the work of Dwek's group (Dwek et al., 1975, 1976; Sutton et al., 1977; Willan et al., 1977; Dower et al., 1977; Wain-Hobson et al., 1977) These studies paved the way for similar analyses

178

J I K I NOVO'I'NY A N D JURCEN BAIOKA'I'H

FIG.9. Ball-and-socketjoint at the elbow of the Fah fragment McPC 605. VH domain is at the upper left, arid CFI1 domain is at the lower right. The VH side chains 1 1, 110, and 1 12 are shown in sp;ice-filling representation (heavy lines), and the C k ~ lspace-filling side chains 149 and 150 are shown as dashed lines. These side chains form a mechanical equivalent of a ball-and-socketjoint, facilitating Fah elbow movements. [Graphics by Molscript (Kraulis, 1991).]

on anti-spin label monoclonal antibodies (Anglister et al., 1984, 1987; Levy et al., 1989; Zilber et al., 1990) and on the anti-2-phenyloxazolone Fv fragments (McManus and Riechmann 1991). Dwek and co-workers used then state-of-the-art magnets (270 MHz) and various spin-labeled haptens to deduce that the combining site of the MoPC 315 myeloma protein for dinitrophenyl was a (hydrophobic) cleft with overall dimensions 11 x 9 x 6 A. The use of lanthanide ions allowed

COMPUlA’I’IONAI. BIOCHEMISTRY O F ANTIBODIES

179

the exact equilibrium binding constants for dinitrophenyl haptens to be measured (- 1 p M ) . Dinitrophenyl was buried in the site to about 11 A depth, and interacted closely with the A r g side-chain VL 95 and an “aromatic box” of residues Trp L93, Phe H34, and Tyr L34. Asn L36 contributed hydrogen bonds to the nitro groups of the hapten. Reference to the computer-built model of Padlan et al. (1977) allowed the assignment of three nearby histidine residues, H 102, L97, and L44. Eventually, inclusion of chemical modification data (Dwek et al., 1977) and NMR spectroscopy on selectively nitrated tyrosine residues (Leatherbarrow et al., 1982) led to a refined model that accounted for the binding experimental data from a large range of cross-reacting haptens (Fig. 10). B. General Stratagems

Computer-aided analyses of antibody combining sites (Section IV,A) formulated the concept of a conserved framework with hypervariable loops implanted on it. Since 1986, it has become possible, via gene cloning (Jones et al., 1986), to create chimeric proteins with CDR loops grafted onto foreign frameworks that carry antigen-binding capacity essentially indistinguishable from that of the parent antibody. If indeed it is possible to manipulate antigenic specificities at will, then accurate modeling of structures of arbitrary loops will become an important practical goal. To derive the structure of an antigen binding site from its amino acid sequence, one is faced with the problems of (1) finding the most appropriate three-dimensional Fv framework (the template) among the existing X-ray crystallographic structures, and (2) replacing the six CDR loops of the crystal structure with loops in conformations corresponding to those of the amino acid sequences at hand. A successful loop swap experiment requires (a) a modeling protocol generating natural loop conformations from amino acid sequences, (b) an exact definition of the boundaries between the framework and the N and C termini of each loop, (c) knowledge of the best possible succession in which the loops are built back onto the framework, and (d) identification of any framework residue that influences CDR conformations. These critical residues must be carried over from the template to the framework of the newly constructed antibody. Different approaches have been developed to accomplish these tasks. C. Knowledge-Based Methods

Knowledge-based (homology) modeling methods (see, e.g., Greer, 1991; Bajorath et al., 1993; Bajorath and Aruffo, 1994; and Sali, 1995, for concise overviews) allow one to construct an approximate three-dimensional

180

JIRI NOVOTNY AND JURCEN BAJOKATH

H!

c

66

L3 . n

FIG. 10. Three-dimensional structure of the MOPC 315 antigen binding site, as derived by NMR spectroscopy and computer modeling (see Section VI,A for details). (Reproduced with permission from Dwek et al., 1977. Nature 266, 31-37. 0 1977 Macmillan Magazines Ltd.)

model of a protein from its amino acid sequence, and atomic coordinates of a similar protein structure. Jones and Thirup (1986), in their analysis of protein structures from the Brookhaven Protein Data Bank, pointed out that loops with similar N- and C-terminal end points and identical length

(~OMPU'I'A'I'IONAL BIOCHEMISTRY OF ANTIBODIES

181

often had similar conformations. The conjecture that CDR loops with identical length and different sequences may adopt similar conformations formed the basis of early antibody modeling protocols (Kabat and Wu, 1972; Padlan et al., 1977; Feldmann et al., 1984; de la Paz et al., 1986; Roberts et al., 1987; Smith-Gill et al., 1987). Independently, structural classification of loops in proteins (Sibanda and Thornton, 1985; Ring et al., 1992) also facilitated modeling, particularly of the P-hairpin loops L2, L3, H2, and H3. This type of structure-based modeling was automated by Levitt (1992) with his segment matching algorithm: Given a loop sequence, the Protein Data Bank can be searched for short, homologous backbone fragments (e.g., tripeptides) which are then assembled and computationally refined into a new combining site model. A milestone event in knowledge-based modeling was introduction of the canonical CDR structure concept by Chothia, Lesk, and colleagues (Chothia and Lesk, 1987; Chothia et al., 1989). They found that five of the six CDR loops (all except the H3) adopted only a limited repertoire of backbone conformations that were readily predictable from the sequence. These canonical conformations were determined by specific packing, hydrogen bonding interactions, and stereochemical constraints of only a few key residues (structural determinants). Examples of canonical motifs are presented in Fig. 11. In its most general form, the canonical structure concept assumes that (1) sequence variation at other than canonical positions is irrelevant for loop conformations, (2) canonical loop conformations are essentially independent of loop-loop interactions, and (3) only a limited number of canonical motifs exist and these are well represented in the database of currently known antibody crystal structures. Every one of these assumptions may fail in isolated practical instances. In their definition of the five canonical CDR loops, Chothia and Lesk (1987) and Chothia et al. (1989) described the CDR loops as overlapping, but not identical to, the hypervariable regions of Kabat et al. (1977).When a large number of antibodies were scanned for the presence of canonical sequence motifs in their CDR loops and frameworks, 50%-95% (depending on the particular loop) of murine and human sequences were found to contain one. Chothia et al. (1992) also showed that the vast majority of VH domains in antibodies display one of the seven different canonical H 1 and H2 conformations. The canonical repertoire of CDR loop Ll was enriched by Wu and Cygler (1993) who analyzed the crystal structure of a murine antibody with a 1 light chain (Cygler et al., 1991). Canonical features are also thought to determine conformations of loops in proteins other than immunoglobulins (Tramontano et al., 1989; Tramontano and Lesk, 1992). Structural determinants of canonical CDR loops include residues in the framework regions. The side chain of the heavy chain residue 71, in the

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JIRI NOVOTNY AND J U R C E N BAJORATH

@-strandE, determines the conformation of the CDR loop H2 (Tramontano et al., 1990). A critical role for other framework residues in preserving productive antibody-antigen interactions was demonstrated in the mutagenesis experiments of Foote and Winter (1992). 1. Results: Canonical Loop Modeling of Binding Sites The canonical loop concept has met with success in structure prediction of several antibody binding sites. Chothia and colleagues predicted all six CDR loop conformations in the lysozyme-binding antibody D1.3 (Chothia et al., 1986) and five canonical loop conformations in four other antibodies (HyHEL-5, HyHEL-10, NC41, and NC10) before their crystal structures were known (Chothia et al., 1989). In the early D1.3 prediction conformations of four CDR loops (Ll, L2, H2, and H3) were accurately described with backbone rms deviations (including @ carbons) 0.5-0.9 A from the X-ray structure. Subsequently, backbones of 14 out of 19 canonical loops were predicted with an accuracy of better than 1.0 A rms (but in no case worse than 1.4 A rms). A level of accuracy of -0.7 A was achieved by Eigenbrot et al., (1993) and Essen and Skerra (1994) in single canonical loop predictions. Several papers reported the construction of models for which crystallographic structures do not exist and whose accuracy cannot be evaluated yet. The study of Roberts et al. (1994), on model building of the catalytic (esterolytic-amidolytic) NPN43C9 antibody included a careful selection of the framework based on an inspection of various VL-V, interfaces. The model correctly predicted previously unknown binding and catalytic hnctions of the Arg residue L96, and suggested a mechanism by which the antibody stabilized high-energy transition states during catalysis. Chothia et al. (1989) observed that even correctly predicted CDR loops may be spatially misplaced (rigid-body shifts) by up to 3 A relative to their crystallographic counterparts. Such effects are critical for accurate modeling of antibody binding sites, as they may substantially alter the shapes of binding sites. In this sense, assessment of the accuracy achieved in individual CDR loop predictions may be misleading. The often reported backbone, or even all-atom rms values obtained by direct least-squares fit of isolated individual loops, do not take into account the rigid-body misplacements of whole loops, and the actual accuracy of the complete binding site model may be lower (Bajorath et al., 1995). As the database of high quality crystal structures has grown, some canonical motifs have become well established. In other cases (e.g., the H2 loop), the ensemble of observed and classified canonical conformations may still be incomplete. Unusually long L1 loops, whose tip portions are not very well defined conformationally and probably sample several different backbone configurations, also may not be accurately predictable. Fur-


183

thermore, an assignment of sequence motifs to canonical conformations may remain ambiguous in a few instances, e.g., when conformationally “odd” amino acids, such as multiple prolines or glycines, are found in antibody sequences. In general, however, the canonical structure concept provided the model building of combining sites with an excellent tool. For the many newly determined antibody sequences it is highly likely to find, in the Brookhaven Protein Data Bank, templates (frameworks) with very high sequence similarity; more than 80% identity is not unusual. Such templates often include one or more CDR loops of the same canonical class as the loops to be modeled. In this case loop splicing is not necessary and the template loop can be directly copied into the model. At the current rate of Brookhaven Data Bank growth (close to a new antibody X-ray structure every month), and the number of antibody Fab structures currently available (more than 50), knowledge-based modeling will only become easier and even more reliable. Automatic loop search procedures, such as the distance matrix-based methods of Jones and Thirup (1986)will help to identify loops with sequences identical, or nearly identical, to those of loops with unknown conformations. Fast fetching algorithms for structure comparison (Holm and Sander, 1994) and relational databases (Bryant, 1989) will allow efficient handling of the rapidly growing numbers of antibody structures and sequences. 2. Framework-Loop Relationship

Having determined canonical backbone motifs of selected CDR loops, one is faced with remaining problems such as (1) how to model conformations of noncanonical loops, (2) how to place side chains on CDR loop backbones, and (3) how to combine CDR loops with the best framework model. Routinely, it is assumed that side-chain conformations of homologous residues are similar (Summers et al., 1987). Thus, the side-chain conformations most frequently observed at the corresponding positions in other CDR loops of the same canonical conformation are selected in the model. Libraries of preferred side-chain rotamer conformations assembled from databases can also be consulted (Ponder and Richards, 1987). The generality of these procedures, however, has frequently been questioned (see, e.g., Schrauber et al., 1993). As the best way of splicing loops onto the framework, Tramontano and Lesk (1992) suggested placing loop backbones on the end points of the framework after a weighted, least-squares superposition of four framework residues N- and C-terminal to the loop. This suggestion, however, does not eliminate differences in framework structures, and in the resulting rigid-body shifts in relative loop positions. An example will illustrate this point. Seven crystal structures of antibody combining sites, refined to

184


better than 2.0 A resolution, were superimposed with average rms shifts of 0.4-0.6 A on the backbones, a very good agreement indeed (see Fig. 12 and Table I1 for details). In order to assess how well the individual loops of the same canonical class agreed in the context of the frameworks, two different rms deviations were calculated (Bajorath et al., 1995). The conventional value (rms) was calculated after direct least-squares superposition of only the loop backbone atoms and directly reflects the differences in the backbone conformation of the loops. The other value, called here spatial rms (s-rms), was calculated for the loops afier superposition ofthe conserued Fv framework segments as described in Table 11. The s-rms values reflected not only the conformational differences in various loops, but also their different orientations with respect to the conserved framework (the “takeoff’ angles resulting in rigid-body shifts). A comparison of two CDR loops that have a similar conformation but are spatially displaced via rigid-body shifts would therefore result in a low rms value and a high s-rms value. As an example, we limited our comparison to the H1 and L2 loops (Fig. 12), all of which are of the same respective canonical structure type. The average backbone rms deviation for the pairwise H 1 loop comparisons (seven residues long; see Table 11) was 0.5 A, however, the s-rms deviations were much greater, 1.6 A. For the L2 loop, the average backbone rms shift of the seven L2 loops (three residues long) was 0.2 A, but their average s-rms deviation was 1.2 A. These observations are generally valid for all canonical CDR loops (Novotny and Bajorath, unpublished observations). That is, isolated loop conformations always superpose much better than loops in the context of their individual frameworks. For example, three of the seven L3 loops shared canonical structure type 3, and the backbone conformations of these loops were remarkably similar. In contrast, their relative spatial positions were quite different (see Fig. 12). Examination of the superimposed p strands supporting the CDR loops shows that the positions of the framework termini differ and that their average painvise difference is typically larger than 1.0 A. The central strands of the p sheets essentially diverge at the very ends but edge strands (such as C”) show greater structural differences throughout. To a large extent, CDR loop displacements correspond to differences at the CDRframeworkjunctions. The definition of the framework-loop junctions is, to some extent, subjective and arbitrary and a judicious selection of loop splice points may have a major impact on the quality of the final model. For example, the CDR loop H2 of the 5539 Fab fragment, when implanted on the HIL framework, would result in an s-rms deviation of about 2.5 A, despite the fact that the H2 loop conformations in 5539 and HIL are essentially identical (0.3 A rms).

-


'TABLE 11

Painvise ms Comparisons of Seven H I CDR Loops of Same Canonical Type Root-mean-square deviation Fv Fragments superimposed" 4-4-20 to KOL H52 to KOL D1.3 to KOL D1.3 to 5539 D1.3 to 4-4-20 H52 to 4-4-20 H52 to Dl .3 H52 to 5539 KOL toJ539 Se155-4 to 4-4-20 HIL to D1.3 5539 to 4-4-20 HIL to KOL HIL to 5539 Sel55-4 to KOL Se155-4 to HIL 4-4-20 to HIL H52 to HIL Se155-4 to D1.3 Se155-4 to H52 Se155-4 to 5539

rmsb

s-rms'

0.6 0.4 0.9 0.9 1.2 0.6 0.8 0.5 0.2 0.7 0.8 0.6 0.2 0.3 0.4 0.3 0.6 0.3 0.6 0.3 0.5

0.9

(4

(4

0.8

1.4 1.4 1.7 1.1 1.4 1.2 1.2 1.9 2.0 1.8 1.4 1.6 1.8 1.a

2.1 1.8 2.3 2.1 2.7

' 4-4-20, Mouse monoclonal antifluorescein Fah (Herron et al., 1989), PDB code 4FAB; KOL, human myeloma Fah (Marquart et al. 1980), PDB code 21G2; D1.3, mouse mnooclonal antilysozyme Fah (Fischmann et al., 1991), PDB code 1 FDL; 5539, mouse monoclonal antigalactan Fah (Suh etal., 1986), PDB code 2FBJ; H52, humanized mouse myeloma antiCD18 Fv (Eigenbrot et al., 1994), PDB code IFGV; HIL, human Fab (Saul and Poljak, to be published) PDB code SFAB; Se155-4, mouse monoclonal antioligosaccharide Fah (Cygler et al., 1991), PDB code 1MFE. To obtain the rms values listed, all the atoms of the two H I loop backbones (residues H26-H32) were least-squares superimposed. ' To obtain the s r m s values, backbone atoms of the conserved VL-VH interface-forming residues (H36-H40, H94-H96, L35-38, and L86-88; see Novotny and Sharp, 1992) in each of the two Fv fragments were least-squares superimposed. The operation brought the complete antigen combining site regions (i.e., parts of the inner /3 barrel and all the CDR loops) of the two Fv fragments into equivalent positions. Thus superimposed, the root-mean-square deviation between the pair of the H1 loop backbones was calculated as the s-rms value.

185

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JIKI NOVOTNY AND J U R C E N BAJORATH

Changes in the spatial relation of CDR loops may provide, in addition to sequence and length variability, a means of increasing the repertoire of shapes available for antigen recognition. From this point of view, antibody sequences must include determinants not only of CDR loop conformation but also of CDR loop position. The canonical CDR loop library consists of an overlapping and, in general, smaller set of residues than that for hypervariable regions (Kabat et al., 1977). Backbone segments, which on the basis of canonical loop definitions are classified as frameworks, include sequence variability which may modulate interactions among adjacent loops and may account for some or all of the effectsjust described. One way of addressing this framework bias in practical modeling may be to select frameworks not only on the basis of overall sequence similarity (currently the most common procedure) but also on the basis of conserved patterns of residues in the regions adjacent to the CDR loops (see Section IX,D). D. Conformational Searches and Monte Carlo Methods

Protein conformation is determined by values of backbone and sidechain torsional angles @ (C-N-Ca-C),11, (N-Ca-C-N), and x (N-Cu-C/?-Cy, Ca-C/?-Cy-CG,and so on). Three-dimensional modeling involves finding specific values for all these torsional degrees of freedom. A particularly promising approach is therefore to use automatic computer algorithms that uniformly sample the complete conformational space of a polypeptide chain segment (Bruccoleri and Karplus, 1987; Shih et al., 1985; Snow and Amzel, 1986; Moult and James, 1986; Fine et al., 1986; Amzel, 1992). Ideally, all the backbone and side-chain conformations compatible with the rest of the protein structure can be generated. The lowest energy conformation should correspond to the naturally occurring one. In practice, technical problems associated with an exhaustive sampling of conformational space restrict searches to short polypeptide segments only. The CONGEN program (Bruccoleri and Karplus, 1987) represents an efficient realization of this modeling stratagem. Given an accurate Gibbs function and a short loop sequence, reliable structural prediction using CONGEN appears to be remarkably easy: Generate all the stereochemically acceptable structures of the loop, calculate their energies, and take the one with the lowest energy. In practice, however, two major problems must be overcome first: Loops are not always short, and accurate energy functions do not yet exist. In the case of antibodies, most loops are sufficiently short that a complete search is feasible given modern computer workstations. The second problem presents a more fundamental hurdle (see Section VI1,C).

COMPUTATIONAL BIOCHEMISIKY OF ANTIBODIES

187

In its most general form, a conformational search is a set of nested iterations of the degrees of freedom (e.g., torsional angle values on a preselected grid, say, 30") in the system. In CONGEN, the generation of backbone coordinates depends heavily on the Go and Scheraga (1970) chain closure algorithm as modified by Bruccoleri and Karplus (1986). Given stereochemical parameters for construction of the polymer and six adjustable torsion angles between the two fixed end points, the algorithm calculates values for the six torsional angles needed to perfectly connect the (rigid-angle) polymer from one end point to another. T o generate conformations of loops with more than three residues, the backbone torsion angles of all but three residues are systematically sampled, and the Go and Scheraga procedure is used to close the backbone. Bruccoleri and Karplus (1986) allowed for small variations in the peptide bond angles which greatly improved the efficiency of chain closure. CONGEN sampling of backbone torsion angles is done with the aid of a conformational energy map. A set of maps giving energies as a function of discrete values of q5, q, and o corresponding to grids of 60" down to 5" has been precalculated. Typically, a 30" sampling is sufficent to include the natural torsion and to guarantee good results. The backbone can be searched either forward from the N terminus or backward from the C terminus, and both cis and trans peptide bond angles can be sampled. After the backbone has been constructed, side chains can be placed by a variety of methods, each allowing complete freedom in sampling the complete side-chain conformational space. In 1987 a seven- to eight-residue loop was often too long to be searched exhaustively, particularly if it contained amino acids with many torsional degrees of freedom (glycine backbones, lysine side chains). With present-day computers, and their increasing speed of computation, it has become possible to search loops with 10 or more amino acids exhaustively, and the limit is steadily increasing (Table 111). Random searches of backbone conformations is an alternative way of producing a library of possible CDR loop structures. The random tweak method of Fine et al. (1986) consists of Monte Carlo simulation in loop torsion angles followed by energetic minimization of the randomly generated starting conformations. Random torsion angles are forced to satisfy geometric constraints of the framework and splice into the remainder of the molecule. The constraints are applied as a set of Lagrange multipliers in a computationally fast iteration scheme [one inversion of a 4 x 4 matrix is required per iteration (Bajorath and Fine, 1992)l. The tweak method has been implemented in two programs, Levinthal's PAKGRAF and the HOMOLOGY/INSIGHT program commercially available from Molecular Simulations Inc. (San Diego, CA). Higo et al., 1992 (see also Gibrat et al.,

188

JIRI NOVUTNY AND J U R G E N BAJORATH

TABLE 111

Computer Workstation Speed in CONGEN Conformational Searches CPU speed Year

Computer

1987 1990 1994 1994 1995

MicroVaxII" IRIS 4D/706 ONYX, 1 processor6 ONYX, 10 processors" CHALLENGE, 10 processors"

'I

MHz 1.5 16 150 -1300 -2600

Search time per residue'

nsec

(set)

667 62 6.7 -0.8 -0.4

200 10 1 0.1 0.05

Digital Equipment Corporation, Maynard, MA.

' Silicon Graphics, Inc., Mountain View, CA.

' Averages obtained from constructions of six CKD loops of various lengths and amino acid sequences; see e.g., Bruccoleri et al. (1988), Novotny et al. 1991), Bassolino-Klimas et al. 1992).

1992) described a different Monte Carlo protocol for CDR loop modeling. It combines the Metropolis et al. (1953) search algorithm, and weights applied to potential energy terms (forcing potentials), in a simulated annealing scheme (Kirkpatrick et al., 1953) to generate loops that satisfy the framework constraints. In order to identify the native conformation in the (often vast) number of all those that are stereochemically possible (including side-chain rotamers), one has to rely on a free energy criterion. Indeed, it is the free energy of the complete system, rather than its potential energy in vacuo, that determines the native fold. In the past, the calculated in vacua potential energies (Brooks et al., 1983) were unable to distinguish between correctly and incorrectly folded protein structures, whereas modified potentials with nonbonded interactions including solvent-exposed (surfacedependent) terms could discriminate between the two types of structures (Novotny et al., 1984). An approximate representation of solvent effects was incorporated into the loop selection algorithm by Bruccoleri et al. (1988). The generated loops were first ranked by the in vacuo potential, and then solvent-accessible surfaces of the loops in the lowest potential energy window of 1 kcal (4.2 kJ) were calculated; the loop with the least accessible surface was then selected. Alternative approximations to free energy developed and reported over time were (1) a complete exclusion of intramolecular van der Waals interactions from the potential (Martin et al., 1989), and (2) ranking on the basis of the electrostatic energy obtained from a finite difference solution of the Poisson-Boltzmann equation (including the solvation term), and

-


189

atomic solvation parameters (Smith and Honig, 1994). None has been completely satisfactory. If indeed loop-loop contacts are important for correct binding site structure prediction, then both the knowledge-based methods and automatic computer algorithms will eventually need a robust free energy functional to gauge the nativeness of the completed assemblies of all six loops. In CONGEN construction, the lowest free energy partial solutions may continuously be selected as the buildup of the loops progresses. In knowledge-based procedures, once the correct backbones are found, the Gibbs function may be used to guide side-chain construction. 1. Results Obtained with Conformational SearcheslMonte Carlo Methods

The first use of the CONGEN program involved an automated protocol that constructed two antibody binding sites, one for the small hapten phosphorylcholine, McPC 603, and the other for the protein antigen (antilysozyme HyHEL-5). Reasonable accuracy was achieved in both cases (1.4-1.6 A s-rms for all CDR backbones, 2.4-2.6 total), with the best results obtained for the McPC 603 L3 loop (six residues, 0.8 A backbone rms, 1.4 A total) and the McPC 603 H1 loop (five residues, 0.7 A backbone rms, 1.7 A total) and the HyHEL-5 L2 loop (six residues, 0.8 A backbone rms, 1.7 A total). Significant side-chain misplacements occurred on isolated aromatic rings and formally charged residues in the L3 and H3 of HyHEL-5 (total rms 14.1 A and 2.7 A, respectively; backbone rms 1.1 A in both cases) and the H3 in McPC 603 (total rms 2.9 A, 1.1 A on the backbone). The exceptionally long L1 loop in McPC 603 had to be built in several successive runs, each constructing only part of the 100~1,and also showed a large rms deviation from the X-ray structure (2.6 A on the backbone, 3.0 A total). However, the tip of this long loop showed relatively high backbone B factors in the crystal and its electron density was difficult to interpret unequivocally (Satow et al., 1986; see also comments in Brookhaven Protein Data Bank entry 1MCP). Several CONGEN computer modeling experiments were carried out using antibody sequences whose three-dimensional structure became available only later. Nell et al. (1992) reported construction of the binding site of an anti-insulin antibody 123, based on its homology with HyHEL-5 antilysozyme antibody. In the case of the antidinitrophenyl antibody AN02 (Bassolino-Klimas et al., 1992), the backbone rms shift of the six loops (43 amino acid residues) was 2.6 A. The shortest loop was 6 residues long, and the longest 9 residues long. Due to the length of the loops, several side-chain misplacements occurred (total rms 3.9 A). The antidigoxin antibody 26-10 (Bruccoleri and Novotny, 1992)was modeled from the McPC 603 framework and only five loops were built by conformational searches. The H1 loop backbone was copied directly from the crystal-

190

JIRI N O V O T N Y A N D J U R C E N BAJORATH

lographic template because the only sequence difference in the definition of the loop was Glu H35 to Asn. This turned out to be a mistake: When the X-ray structure of the 26-10 antibody became known (Jeffrey et al., 1993), it was found that the H1 loop conformation was substantially different from the template for residues 28-30, outside the range of the loop as initially defined for the CONGEN run. As a result of this, and of the sequential construction of the loops, the H3 and H2 loops were constructed incorrectly. The fact that polypeptide segments with essentially identical sequences may differ substantially in conformation (Kabsch and Sander, 1984) presents a major challenge to all protein modeling protocols. In the first application of the random tweak method, Fine et al. (1986) generated conformations for four loops, H1, H3, L2, and L1, from the binding site of McPC 603. Starting from random structures, a large number of conformations for the loop backbones were generated, followed by either minimization or molecular dynamics to find minimum energy conformations for both the backbone and loop side chains. The same method was used to construct the entire antigen combining site of the CEA antibody specific for a carcinoembryonic antigen, a known colon cancer cell marker (Mas et al., 1992). Gibrat et al. (1992) reported backbone rms agreements in the range 0.9-2.6 A on reconstruction of the complete HyHEL-5 binding site with the Metropolis-simulated annealing Monte Carlo method. 2. Single-Residue Mutants, Indirect Effects on Binding CONGEN calculations were also used to supply a structural rationale for experimentally produced single-residue mutations and their effects on antigen binding. In the antidigoxin antibody 40-150 (Novotny et al., 1990; Ping et al., 1993), a spontaneous mutant 40-150 A2.4 carried a replacement Ser-+Argin its heavy chain (H94) and had altered specificity.A secondorder mutant, 40-150 A2.4P.10, lacked two residues at the N terminus of its H chain and had a specificity profile approaching that of 40-150 antibody. The N terminus and the position H94 were distant from the antigen binding site of the antibody, and the structural basis of the specificity changes was not immediately apparent. Approximate structures of the 40-150 antibody and its mutants were constructed by the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. The torsional spaces of the polypeptide backbone and side chains around position H94 were uniformly sampled and the lowest energy conformations were analyzed in detail. The results indicated that, when Arg H94 is substituted for Ser, Arg H94 can hydrogen-bond to side chains of Asp H101, Arg L46, and Asp L55 (Fig. 13).This resulted in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N-terminal residues increased solvent accessibility of Arg H94.


191

The solvation may have caused a hydrogen bond between Arg H94 and Asp HlOl to be lost, restoring the structure to one similar to that of 40-150. The preceding structural hypothesis was tested by Ping et al. (1993) who prepared genes coding for N-terminally deleted heavy chains with both the Ser H94 and Arg 94 side chains, respectively, in the position H94. When antibody activity of the engineered and reconstituted antibodies was measured, N-terminal deletion had little effect on binding of the Ser H94 structure, whereas two-residue truncations increased affinity approximately 40-fold for the Arg H94 mutant, consistent with the hypothesis. Chien et al. (1989) described computational analysis of a single residue mutation similar to that in 40- 150. In their work Asp H 101, when replaced with Ala, totally abolished the phosphorylcholine-binding capability of the antibody S107. Computer-aided analysis of this effect highlighted the importance of Arg H94, and the salt bridge between the residues Arg H94 and Asp H101, in maintaining proper conformation of the H3 loop. RuffJamison and Glenney (1993) used the CONGEN program to build a model of another phosphorylcholine binding Fv fragment, Py20, and to simulate binding site mutants on a computer. In the course of their work, they discovered two mutations (V, Tyr-105-tAla and VH Tyr-1064Ala) with a moderately increased affinity for the antigen. In mutagenesis experiments on the antidigoxin antibody 26-10, CONGEN computer modeling of mutated side chains helped to rationalize diverse mutagenesis data, e.g., at hapten-contacting residues Asn H35 and Tyr-50 (Schildbach et al., 1991, 1993a, 1994). Modulation of antibody affinity by side chains in the hapten-distal position H52 was also analyzed by Schildbach and co-workers (1993b). Several mutant side chains were modeled using the crystal structure as a starting point. The results suggested that diverse residues could be accommodated within the antibody without substantial structural rearrangement, and that none of the substituted side chains were able to contact hapten. At the same time, the modeled H52 mutant conformations suggested plausible ways in which noncontact residues could modulate affinity indirectly through their impact on the orientation of the hapten-contacting side chains. E. Issues, Combined Protocols, Future Improvements

Several relatively accurate methods are currently available for modeling loop structure from its sequence. An inherent accuracy limit of the canonical concept is a fraction of an angstrom if backbones are considered in isolation, and -1.5 A for all atoms. Automatic loop construction via uniform conformational sampling or random tweaking essentially always contains the native loop structure in the set of the generated loops but, as mentioned

192

JIRl NOVDTNY AND JURGEN BAJORAlH

earlier, the key problem here is to have a reliable Gibbs function to identify the native loop correctly. In many cases, the calculated free energy of the loop will be correct only if all the (potentially manifold) interloop interactions are correctly modeled, too. This raises the question of accurate modeling of the complete binding site, i.e., an ensemble of six interacting loops. Even if knowledge-based methods successfully find every single loop, one will still need a reliable procedure for correctly assembling the backbones in the context of the site, and adding side chains in their correct native conformations. Reflection on all these complexities of construction seems to favor a hybrid knowledge-based-conformationalsearch protocol, perhaps in the form of an automatic construction and database selection of every single loop, and an iterative loop combination-side-chain placement algorithm that would then completely assemble the site. Such a general protocol has not been developed yet, although the Martin et al. (1989) procedure (now commercially available from Oxford Molecular, Ltd.) represents the best attempt so far to combine CONGEN conformational searches with database loop selection methods. Pedersen et al. (1992) described an assembly of the complete D1.3 antilysozyme binding site using a combination of database searches for the corresponding canonical templates for five loops, side-chain construction by CONGEN, and CONGEN construction of the complete H3 loop, for which no canonical template was available. Very good rms values were reported for all the individual loops. One of the successful applications of a combined, knowledge-basedconformational search protocol is construction of the binding site of the anticancer antibody BR96 (Bajorath, 1994). In this work, framework regions were combined from VL and Vw domains of two different X-ray structures, 4-4-20 and 17/9 (Rini et al., 1992). This allowed the conformations of CDR loops L2, L3, and H1 to be directly included in the model. The splicing of the remaining CDR loop backbones on the framework was accomplished by superposing five residues N- and C-terminal to the loop and the framework end points, respectively. The unusually long CDR loop L1 in BR96 (12 residues) shared length and canonical determinants, but only limited sequence similarity, with the corresponding loop in 4-4-20 and was modeled based on the 4-4-20 structure. CDR loop H2 in BR96 represented another ambiguous canonical motif. Its sequence was consistent with the H2 canonical structure type 3 characterized by a glycine and exceptional torsion angles at position 54, but it had two glycine residues at positions 53 and 54, making it difficult to predict its conformation. The canonical conformation, with the glycine at position 53 in regular torsions, and the glycine at position 54 in exceptional torsions, was retained in the model as one possibility, and an alternative conformation was generated using CONGEN conformational searches. The best CONGEN-generated conformation, in fact, contained exceptional backbone torsions at position

193


53 and the glycine at position 54 with @ and 1/, angles in the allowed region of the torsional space. To model the H3 loop, the most common conformation at the base of the loop was assumed, and the residual portion of the loop was obtained from complete CONGEN conformational searches in the presence of the other loops. Crystal structure of the BR96 Fab fragment, determined and refined to 2.5 A resolution, was reported by Jeffrey et al. (1995). Table IV summarizes the rms and s-rms deviations between the BR96 X-ray structure and the model, as originally reported by Bajorath and Sheriff (1995). Overall, a good agreement exists for the backbone rms of all the loops (see Table IV). The largest rms was obtained for the L1 loop which also shows rather high B factors in the crystal. Also, as in other models, the s-rms values for all the loops were higher than the rms values. In the future, as computers become faster (approximately doubling the speed of the central processing unit every 10 months), computer time will become less of an issue in modeling, and all the stereochemically acceptable conformations of long loops can be fully sampled. In automatic modeling methods (CONGEN, random tweak), it is important to avoid situations where a small imprecision in, say, positioning a side chain of one loop, is amplified into a gross error in modeling the neighboring loop (see examples in Section IV,D,l). For a complete combining site buildup, Bruccoleri et al. (1988) developed a sequence of loop constructions based on the relative positions of the loops in the local frame of reference provided by the /3 sheets of the VL-VH domain interface. The three shorter loops, L2, L3, and HI, are placed “low” (closer to the midpoint of the P-barrel interface) than the others (Fig. 7B). They do not interact with each other and provide a natural basis for construction of the remaining “high” loops, H2, H3, and L1. Thus, the low loops are built first, e.g., in the order L2, H1, L3, followed by H2, H3, and L1. TABLL IV

CDR Loop Cornpanson: BR96 Model ~

~~

~

rms deviation

~~

(A)

71s

BR96 X-ray‘ ~~

s-rms deviation (8,)

Loop

Backbone

All atoms

Backbone

All atoms

L1

1.6 0.2 0.3 0.3 0.3 1.5

2.9 0.4 0.9 0.6 0.9 3.3

2.0 0.3 1.0 0.9 1.4 2.9

3.2 0.5 1.:3 1.0 1.8

L2 L3 H1 H2 H3

At 2.5 8, resolution.

3.8

194

JlRI NOVOTNY AND JURGEN BAJORAlH

VII. BINDING AFFINITY

AND

SPECIFICITY

The question of antibody specificity is central to molecular immunology. Even problems seemingly unrelated to specificity, such as the immune repertoire and maturation of the antigenic response require, for their full understanding, a reference to antigen binding and affinity changes in the process of clonal selection. What are the main physical components of binding and specificity? Do antibody and antigen structures change on complexation, and, if so, by how much? What is the atomic origin of affinity and, implicitly, of specificity? Only recently, with the emergence of dozens of X-ray structures of antigen-antibody complexes including both low- (- 1pM) and highaf€inity ( 1 nM-10 pM) ones, have we had an opportunity to explore these questions in precise atomic terms. Our explorations have been met with a stiff challenge. In the structures of the complexes, there are hundreds of atoms in intimate interactions: van der Waals, hydrogen bonding, salt bridging. It is not a simple task to relate this molecular jungle to experimental observables (equilibrium binding constants, KM). The most basic biophysical concepts necessary for formulation of any binding theory are still being debated, and there are those who doubt whether atomic origins of binding specificity will ever be fully understood (Mark and van Gunsteren, 1994; Janin, 1995). Rigorous calculations of binding constants may currently be unattainable, however, the situation is not so hopeless that any meaningful insight into the attribution of binding energies would be denied us. As we hope to demonstrate in this section, qualitative and semiquantitative estimates of binding energy attribution are possible in many cases, and can be directly compared to experimental perturbations of binding (AAG values obtained from site-directed mutants of antibodies and/or antigens). Gradually, a conceptual framework for the rationalization of binding data is emerging and will be refined, with time, into a truly quantitative tool. The current situation may remind us of the empirical and semiempirical quantummechanical calculations used in chemistry. They, too, provide a valuable insight into structures of compounds by aproximating, rather than rigorously solving, the Schrodinger equation for many electron molecules.

A. Thermodynamics of Binding Concepts such as binding specificity and complex stability (affinity) have their origin in the thermodynamics of bimolecular reactions. For example, in the reaction Antigen

+ Antibody = Complex


195

molar concentrations of the three molecular species are measured at equilibrium, and the strength (specificity) of the complex is estimated from its relative concentration, i.e., the ratio KAS = [complex]/[antibody][antigen]. The experimentally measured KAs relates to the Gibbs free energy of complex formation, AG, as AG = RT log KAS (where R, the gas constant, is the product of the Boltzmann constant, k, and the Avogadro constant, L, and is equal to 8.314 kJ mol-' K-', and T is the temperature in kelvins). Changes in Gibbs free energy, a thermodynamical quantity, can in principle be obtained from atomic structures of all the molecules involved in the reaction, provided all the physical forces responsible for the complex formation are accurately known. The specificity of protein-ligand interactions comes from a large difference between binding constants characterizing the binding of specific and nonspecific ligands. Thermodynamically, the higher binding constants for specific ligands arise from stronger overall forces (Gibbs free energy differences) between the antibody and the antigen. Most of the forces responsible for binding are distinctly short-range: Van der Waals-London dispersion forces (London, 1930), hydrophobic force [i.e., a difference in surface solvation] (Kauzmann, 1959), and hydrogen bonds (Pauling, 1960) have vanishingly small values at distances greater than 3 4 A, a local distance even on an atomic scale. While the first two types of forces can be said to be isotropic (spherically symmetric), hydrogen bonds are strictly directional and require orientation of the participating groups. Electrostatic forces act at a distance but the high dielectric constant of water effectively limits their reach. Thus, the burial of charged atoms at a protein-protein interface distinctly increases their effective electrostatic field, but only at the expense of the energetically unfavorable desolvation of charged groups. A consequence of all this is that the two molecular surfaces, the antibody paratope (i.e., the binding site) and the antigenic epitope, will enter into a stable complex only if they have complementary molecular shapes over a large area, and their surface charge distribution is such that the interaction of opposite charges on the epitope and the paratope provides sufficient Coulombic attraction to stabilize the complex (Novotny et al., 1987). These attractive contributions are counterbalanced by conformational entropy losses of side chains immobilized at the antigen-antibody interface. In the omnipresent thermal motion at room temperature, torsions of free side chains tend to rotate randomly and distribute themselves equally in the three lowest energy conformational states, trans (180") and f gauche (k60"). On complex formation, only one of these states becomes locked in the tightly packed interface. Enforcement of this torsional preference over the natural equalizing tendency of the Brownian motion costs energy, and some of the Gibbs free energy of complex formation must be expended on it.

196

JIRI N O V O T N Y AND JUKGEN UAJORATH

Although the quantitative aspects of these biophysical components of binding are still debated, estimates have been summarized as follows (Novotny and Sharp 1992). (1) Van der Waals or dispersion-repulsion (London) interactions probably constitute most of what is termed shape complementarity in binding, in that they penalize intermolecular contacts that provide either overlap of atoms, or cavities, either directly or through induced strain. Well-packed contact regions at antigen-antibody interfaces, though, are probably not much more favorable than the antibodysolvent and antigen-solvent contacts experienced by the free molecule in the complex. Surface tension data from organic liquid-water systems show that the work of adhesion between hydrocarbon and water interfaces is essentially the same as that between two hydrocarbon interfaces (Nicholls et al., 1991, Adamson, 1976). Furthermore, dispersion forces are relatively small in magnitude, and their possible differences amount to even smaller magnitudes. ( 2 ) The hydrophobic effect is the major stabilization factor of complex formation contributing, according to various estimates, between 25 and 7 2 cal (6 and 17 J) of Gibbs free energy stabilization per 1 of protein-protein contact area (Chothia, 1974; Chothia and Janin, 1975; Sharp et al., 1991b). (3) Electrostatic effects accompanying complex formation involve the following. (a) Creation of new clusters of charged atoms in the low dielectric constant ( E = 2) environment at the protein-protein interface of the complex; these charge-charge interactions can stabilize the complex through Coulombic attraction, the net effect being proportional to the atomic charges. (b) Desolvation of charged groups; this is proportional to the square of the atomic charge, and so the desolvation process is always unfavorable, and often stronger, than the net Coulombic attraction (see Section VI1,A). (4) On complex formation, immobilization of a freely rotatable torsional degree of freedom (a side chain exposed to solvent) carrying a free energy penalty of about 0.6-0.7 kcal ( 2 . 5 kJ; Privalov, 1979; Novotny et al., 1989; Nicholls et al., 1991; Pickett and Sternberg, 1993).

x2

B . Lock and Key or Induced Fit Do antigens andlor antibodies change structure on binding [induced fit hypothesis, first explicitly proposed for antibodies by Pauling (1940)l or is the binding essentially an association of two rigid bodies [the lock-and-key paradigm of Fischer (1894)l. The X-ray crystallographic structures, in fact, reveal both binding modes. Examples of lock-and-key complexes, currently much more numerous, include the McPC GO3 antiphosphorylcholine myeloma (Satow et al., 1986); D1.3 antilysozyme (Amit et al., 1986; Bhat et al., 1994); NC41 and NClO antineuraminidase (Tulip et al., 1994;

COMPUI’AI‘IONAI. BIOCHEMISI‘KY OF ANTIBODIES

197

Malby et al., 1994); 4-4-20 antifluorescein (Herron et al., 1989); and 26-10 antidigoxin ueffrey et al., 1993). The best examples of large induced fits on complexation are the antipeptide antibodies 17/9 and 50.1 where the whole H3 loop pivots as if on two anchor points (Stanfield et al., 1990, 1993; Rini et al., 1992; Schulze-Gamen et al., 1993). On complex formation, either lock-and-key or induced fit, one of the possible surface side-chain conformers is always selected and “rigidified” at the antigen-antibody interface. Because of this generality, and because its energetic cost can be well estimated, this shift from a preformed conformational equilibrium need not be considered an induced fit. The simplest case of a backbone-induced fit may also involve stabilization of a disordered loop segment in one of the thermally available conformational states, introducing a shift in the preexisiting equilibrium (Fersht, 1984). Given the relatively high cost of conformational entropy (-0.6 kcal per aliphatic side-chain torsion or 0.4 kcal per backbone torsion; see Section VII,A), it may well be that those antigen-antibody systems where tight binding is required evolved toward rigid (lock-and-key)binding. Can the induced fits be operational in cross-reactivity?The only structurally well-documented case of antibody cross-reactivity, that of the highaffinity (- 1 &) mouse monoclonal antiprogesterone DB3 which binds four different progesterone-like steroids (Arevalo et al., 1993, 1994), involves no structural changes in the antibody on engaging the various ligands. Cross-reactivity was accompanied by two distinct modes of hapten orientation (i.e., the steroidp ring with two methyl groups) with respect to the Trp side chain H50, one of the major hapten-contacting residues. Incomplete binding surface complementarity between the haptens and the antibody seemed to be the key structural feature promoting cross-reactivity. Bruccoleri and Karplus (1990), Hoffren et al. (1992), and de la Cruz et al. ( 1 994) reported molecular dynamics simulations of hypervariable loops that may provide us some idea of the shape variations the binding site undergoes with thermal fluctuations. The Bruccoleri and Karplus (1990) simulations were carried out at different temperatures in vacuo, and with the goal of sampling the complete conformational space of each loop. Comparison with the results of CONGEN searches revealed that molecular dynamics sampled the loop conformational space less completely, and less efficiently, than the CONGEN uniform searches. De la Cruz et al. (1994) simulated dynamics of a free and a complexed antibody binding site (Fv fragment of the antirhinovirus serotype 2 heptadecapeptide) at room temperature and with an explicit solvent. They reported average rms deviations of up to 1.4 for the free hypervariable loops of the Fv fragment, and less so for those complexed with antigen. A systematic drift from the X-ray positions was also observed that, e.g., for the

198

JIRI NOVOTNY AND JURGEN BAIORATH

CDR H2 loop approached 3 A after a 100-psec dynamics run (in the presence of an explicit solvent). This may have been an artificial by-product of the particular technical conditions of the simulations (the GROMOS potential, nonbonded interactions evaluated with a cutoff of 8 A, and a longer cutoff of 15 applied every fifth step). By comparison, other work (Kitson et al., 1993) showed that, under different conditions (class I1 DISCOVER potential, cutoff 25 with no switching function or employing Ewald summation),2 the average structure simulated by molecular dynamics did not differ from the X-ray structure by more than a fraction of an angstrom. This is indeed to be expected if the solvated protein structure represents a natural system at Boltzmann equilibrium with itself.

C. Empirical Gibbs Functions As early as 1975, Chothia and Janin used the rule of proportionality between hydrophobic effect and solvent-accessible surface area (Chothia, 1974) to estimate hydrophobic stabilizations of bovine pancreatic trypsin inhibitor (BPTI), hemoglobin a/3 dimer, and insulin dimer. After a correction for conformational entropy loss on complex formation, a good correlation between the solvent-accessible protein-protein contact areas and the measured K M was achieved. Nevertheless, the concept of accessible surface area as the only measure of the Gibbs free energy change in complex formation was an oversimplification. For example, the areas of contact between trypsin and BPTI (Huber et al., 1974) and between the immunoglobulin Fc fragment and the fragment B of staphylococcal protein A (Deisenhofer, 1981) are identical (13.9 nm2), while the affinities of these two complexes differ by at least six orders of magnitude (0.1 pM in the case of the trypsin inhibitor and -1pM or less in the case of Fc fragmentfragment B). By now, approximations of binding constants have been refined by including empirical estimates of all the atomic thermodynamic contributions deemed to be important in the attribution of binding energy (Section VI1,A). Empirical Gibbs (free energy) functionals, in the form of simple

'

Evaluation of nonhonded (van der Waals and electrostatic) interactions is timeconsuming during molecular dynamics simulations; often the painvise interactions are neglected beyond an arbitrary cutoff distance. Switching functions are sometimes added to the potential around the cutoff distance to smooth the abrupt truncation of interactions, hut this practice was shown to generate large artificial forces and unwanted perturbations to the system. Ewald (1921) summation allows calculation of all the electrostatic interactions of a set of charges to inifinity faster than simple summation of all the painvise interactions can.


199

formulas, were proposed by Novotny et al. (1989), Williams et al. (1991), Nicholls et al. (1991), Wilson et al. (1991), Horton and Lewis (1992), Murphy et al. (1993), and Abagyan and Totrov (1994). The many simplifications necessarily adopted in these estimates (see Section VII,C, 1) require that the results not be interpreted too finely, and various improvements are being explored (Pickett and Sternberg, 1993; Jackson and Sternberg, 1994; Vajda et al., 1994). Nevertheless, the present forms yielded reasonable estimates in many cases and showed predictive power where sitedirected mutagenesis results distinguished between residues important and unimportant for binding. Research now focuses on (1) elimination of technical errors in electrostatic calculations such as, e.g., poor treatment of charge desolvation effects on complex formation (see Bruccoleri et al., 1996), (2) refinement of surface scaling constants (Novotny, Bruccoleri, Davis, Sharp, manuscript in preparation), and, where applicable, ( 3 ) a proper account of interface-bound water and its impact on the energetics of complexation (see Section VII,D). 1. Functional Forms and Limits of Approximation

The treatments of Novotny et al. (1989), Murphy et al. (1993), and Abagyan and Totrov (1994) are the only ones systematically applied to antigen-antibody interactions and are discussed here in detail. In the Novotny et al. (1989) method, atomic coordinates of an antibody-antigen complex are the only data on which the calculation is based. By using the Gibbs functional as detailed in the following discussion, the atomic free energies are calculated for each molecule, first for the uncomplexed form and then for the complex. The difference between these two values gives the atomic Gibbs free energy differences on complex formation, AG,,,,,, values. These are the sums of the individual contributions due to hydrophobic, electrostatic, and side-chain conformational entropy effects. From the atomic values, the AGresidurcontributions and the complete AG of the reaction are obtained by further summations. In this empirical scheme, and assuming rigid macromolecules, (1) the hydrophobic effect, AGHB, is directly proportional to the contact solvent-accessible area (in square angstroms) (Lee and Richards, 1971)between the two molecules (Chothia, 1974):

AGHB = (contact area) x 25 cal

(10)

(1 cal = 4.2 J), and (2) electrostatic (Coulombic) interaction between the two molecules, AGEL,is empirically “screened” by an effective dielectric constant:

200

JIRI N O V O T N Y A N D J U R G E N BA J O M I ' H

where QJ is the partial atomic charge, r is the distance between the ath and jth atoms, and E is the effective dielectric constant. Hydrogen bonding is treated as an electrostatic phenomenon, included in Eq. (1 1). Equation (11) approximately reproduces the differential strength of protein-protein hydrogen bonds compared to protein-solvent hydrogen bonds, as estimated by Fersht et al. (1985) in site-directed mutagenesis experiments. These usually attractive interactions are counteracted by a loss of conformational entropy of surface side chains immobilized at the contact surface (Privalov, 1979; Rashin, 1984): -TAS( = NRT log 3 = 0.6N kcal

(12)

where R is the gas constant, T , the temperature, equals 300 K, and N is the number of side-chain torsional degrees of freedom lost. Equation (12) implies that each torsion has approximately three equienergetic states available in free solution (i.e., trans and 2 gauche) but becomes locked in one conformation on the formation of a complex (hence the log 113 = -log 3 term. Finally, for the absolute AG value calculations, estimates are made for the cratic and translational-rotational entropy changes (AS(R and A S T R , respectively) that accompany complex formation (see Novotny et al., 1989): TASCR= 2 kcal

(13)

TASIK= 9 kcal

(14)

Murphy et al. (1994) and Finkelstein and Janin (1989) offered somewhat different treatments and values for both the cratic and translationalrotational entropy terms. Assumptions implicit in Eq. (9-14) are that (1) the solute-solute van der Waals interactions are essentially of the same magnitude as the solutesolvent van der Waals interactions and effectively cancel out (see Section VII,A), and (2) that changes in vibrational entropy between the two free proteins and the protein-protein complex are likewise unimportant. Indeed, the largest changes are expected to occur in low-frequency, collective modes of vibrations [e.g., lobe opening and closing, as suggested by, e.g., Tidor and Karplus (1994), which are known to be effectively damped in aqueous solutions (Cusack et al., 1988). 2. Culculutaons on Indavaduul Antagen-Antabody Complexes

Calculations carried out to date include those for about 10 antibodyantigen complexes and are summarized in Tables V-VII. Overall, the absolute AG are in the range of experimentally observed values, with two exceptions (HyHEL-5 and NC41) due to a poor electrostatics approximation [Eq. (1l)]. Experimentally determined AAG values characterizing the effects of single-residue mutations on binding are available for the NC 10

20 1

COMPUTATIONAL BIOCHEMISTRY OF ANTIBODIES TABLE V

Empirical Free Energy Calculationsfor X-ray Structures of Antibody-Antigen Complexes ~A

A G (kcdl)

Complex

~ G ~ (kcal)

D1 .3-lysozymer Hy HEL-5-lysozyme HyHEL-10-lysozyme NC4 1-N9 neuraminidase NC 10-N9 neuraminidase McPC603-phosphorylcholine

-11.4 -14.2 -13.0 -9.7 -10.8 -6.6

-9 -3 2 -13 -22 -13

36.7 1-phenyl arsonate 4-4-20-fluorescein

-4 -12.3

-7 -16

' The experimentally

~~

~

~ Refs.

~

~

Novotny et al. (1989) Novotny et al. ( 1 989) Novotny (1991) Tulip et ul. (1994) Tulip et al. ( 1994) Novotny et al. (1989); Novotny (1991) See Table VI See Table VII

4

determined AG value.

* The calculated AG value.

' The original calculation reported by Novotny et al. (1989) was carried out on the Amit et al. (1986) X-ray coordinates. Crystal structure has since been refined and several water molecules were found at the antigen-antibody interface, however, empirical AG calculations have not yet been repeated on the refined coordinates.

TABLE VI Phenyl Arsonate-36-71 Fab Complex: Calculated &&p,dUp ~

~

~~

Contact surface

and Experimental AAG Valuesa ~~

Residue

(A2)

AGH$

AGEL'

N, torsion

Asn H35 Ser H99 Trp H47 Tyr H50 Ala H59 Phe H 108 Tyr H106 Arg L96 Leu L94

0.8 0.8 0.6 32.4 3.4 1.6 25.9 17.0 15.9

0 0 0 -0.8 -0.1 0 -0.6 -0.4 -0.4

-0.7 -0.7 -0.4 -0.2 0 0 -0.1 -4.1 0

0 0 0 1.5 0 0 2.0 1 1

- T A S L F ~ Total

0 0 0 0.9 0 0 1.2 0.6 0.6

-0.7 -0.7 -0.4 -0.4 -0.1 0 +0.2 -3.9 +0.2

AAGEx~ >-3.5 -3.4 -2.3 -3.5

-3.1

' Values in kcalimol. The experimental A A G E x ~values (Sompuram and Sharon, 1993) represent the measured differences between the AG of the wild type and that of the Ala mutant at the same position. Phenyl arsonate partial atomic charges were derived from STO-3G Gaussian90 calculations (T. Stouch and J. Novotny, 1991, unpublished results); the arsonate group was assumed to be monoionic (Pressman and Grossberg, 1968). Residue numbers are consecutive through the polypeptide chain. h The hydrophobic term; see Eq. (9). The electrostatic term; see Eq. (10). The TASC:F(conformational entropy) term; see Eq. (1 1).

202


TABLE VII

Fluorescein-4-4-20 Fab Complex: Calculated AGrcsidueand Experimental AAG Valuesa Contact surface Residue

(A*)

Gly H104 Trp H33 Arg H52 Arg H74

6.5 48.1 0 0 29.6 0 0 39.9 16.2 5.5

TyrH103 Lys H54 Lys H67 Tyr H56 Tyr H102 Ser HlOl Asp H31 Arg L39 Lys H55 His L 31 Gln L33 Ser L96 Ser L94 Trp LlOl Phe L103 Tyr L37

5.4

4.2 0 32.1 6.9 8.6 5.0 17.9 5.7 36.5

N, A G E L ~ torsion

-0.2 -1.2 0 0 -0.7 0 0 -1.0 -0.4 -0.1 -0.1 -0.1 0 -0.8 -0.2 -0.2 -0.1 -0.4 -0.1 -0.9

-0.8 -0.1 -0.7 0 -0.9 0 0 0 -0.2 -0.2 0.8 -3.3

0

-2.2

0 1 0 2 0

0 0 -1.2 0 -0.1 0 0.5

1 0 0

2 0

0 2 2

2 1 0

1 1

2

-TASCF~ Total 0-1.0 0.6 0

0 1.2 0 0 1.2 1.2 1.2 0.6 0 0 0.6 0 1.2 0 0.6 0.6 1.2

-0.7 -0.7 -0.5 -0.4 -0.3 -0.2 +0.2 +0.6 +0.9 f1.3 -3.4 -2.2 -0.2 -0.2 -0.2 -0.1 +0.1 +0.5 +0.8

AAGEXP

-2.2 0

-2.4

a Values in kcal/mol. The experimental AAGEXP values (Denzin et al., 1993) represent the measured differences between the AG of the wild type and that of the Ala mutant at the same position. Fluorescein partial atomic charges were derived from STO-3G Gaussian90 calculations (T. Stouch and J . Novotny, 1991, unpublished results). Residue numbers are consecutive through the polypeptide chain. In this crystal structure of the 4-4-20 complex, one molecule of the solvent 2-methylpentane-2.4-diolis found in a cavity inside the Fv fragment beneath fluorescein but makes no contact with the hapten. The KO of complex formation in methylpentanediol is different (lower) from that in physiological solution (Herron et al., 1989). The mcthylpentanediol molecule was present in the empirical AG calculation. The hydrophobic term; see Eq. (9). The electrostatic term; see Eq. (10). The TASCF(conformational entropy) term; see Eq. (11).

and NC4 1 antineuraminidase complexes, antiphenylarsonate 36-7 1 (Table VI), antiphosphorylcholine McPC 603, and the antifluorescein 4-4-20 (Table VII) complexes. Often, the relative ranking of residues agrees in the experiment and the calculations but the absolute values differ, sometimes by as much as 3 kcal (e.g., Table VI). The calculated AGresidueof Tyr H106 in the 36-71 antibody, Tyr-H33 in McPC 603, and Trp LlOl in 4-4-20 are all in error due to an overestimation of the side-chain con-


203

formational entropy term. These side chains were probably constrained to a limited set ofXl torsions even prior to complex formation. On the other hand, some agreements of the calculated residue contributions with experimental AAG data are notable. Kam-Morgan et al. (1993), working on the HyHEL- 10 complex, measured AAG values for several mutants in the Arg-2 1 and Asp- 101 positions and compared them with the AGresiduecontributions calculated by Novotny (199 1). Mutagenesis of Asp101 was well rationalized by the calculated AGHB,AGELand TASCFterms. The Arg-2 1 AGresiduccontribution, originally calculated as -11 kcal/mol, was measured to be only about -2 kcal/mol. Tulip et al. (1994) compared binding energy attributions in complexes of the influenza N9 neuraminidase with those of two different antibodies, NC41 and NC10. In this work, mutations at the energetically important positions (i.e., side-chain substitutions at sites for which the calculated AGresiduec -1 kcal/mol) did not bind the antibody, while neutral mutations (at sites where AGr,,idue > -1 kcal/mol) had no effect on binding, as tested against the binding data reported by Nuss et al. (1993) and Webster et al. (1987). The trend was valid for NC41 in 19 out of 27 cases at 13 neuraminidase sites, or 24 out of 27 if steric clashes and backbone hydrogen bonds, immutable by sidechain replacements, are taken into account. It was valid for NClO in five out of seven neuraminidase sites [corrected experimental data of Gruen et al. (1994) as opposed to the seven out of seven correlation originally reported by Tulip et al. (1994)l. Describing the effect of mutations made in the D1.3 antilysozyme combining site, Hawkins et al. (1993) noted that “much of the energetics of interaction seems to be driven by contacts from . . . the segment G117 to Q121 of lysozyme,” and that, in the antibody, VH residues T30, Y32, R99 and VL residues Y50, T53, and S93 were less important. These rankings are in a good overall accord with those given in Novotny et al. (1989). In the most recent coordinates of the D1.3 complex (Bhat et al., 1994) water molecules were found at the interface that were not considered in the earlier calculations. The question of water-mediated binding is discussed separately in Section VII,D. Hawkins et al. (1993) also remarked that “the number of contacts appears to be at least as reliable a guide to predicting the energetics of the interaction of the D1.3 antibody and lysozyme as semi-empirical calculations.” However, in the Tulip et al. (1994) calculations, retention of binding in the I368R mutant, at the spatial center of the epitope, was successfully predicted while, e.g., the K432N mutation at the edge of the interface markedly reduced binding, an effect expected from the calculations. Perhaps the most important effects suggested by the calculations and subsequently borne out by experimental evidence were those involving (1)

204

JIRI NOVOI‘NY AND JUKGEN BAJOKATH

the existence of an “energetic epitope” (Novotny et al., 1989; Jin et al., 1992), more fully discussed in Section VIII, and (2) the very different attribution of binding affinities in the two overlapping neuraminidase epitopes (also see Section VIII). Later calculations employ a significantly improved formula for electrostatics (Bruccoleri et al., 1996), a hydrophobicity term based on scaling contact molecular surfaces and on conformational entropy estimates enumerated by uniform conformational sampling of all the side chain torsional degrees of freedom in CONGEN. A blind test of the method attempting to reproduce AAG values measured on 10 lysozyme singlechain mutants that affected binding of the HyHEL-10 antibody Kirsch, University of California, Berkeley, personal communication; Novotny, Bruccoleri, Davis, and Sharp, manuscript in preparation) yielded encouraging results, as shown in Fig. 14.

u.

3. Binding Energies from Calorimetric Data

Murphy et al. (1993) carried out a calorimetric study of complex formation between the Fab fragment of the antibody 13. l and its antigen, angiotensin 11. Association of the two molecules was accompanied by an enthalpy change (AH) of -8.9 2 0.7 kcal mol-’ and a heat capacity change (AC,) of -240 2 20 cal K-I. From these values, the free energy change of the reaction, AG, at 30°C was estimated as -1 1 kcal mol-’ with a AS component of 6.9 cal K-’ mol-’ (TAS 22.3 kcal). Thus, complex formation was favored both enthalpically and entropically. Structural interpretations of AC, and AH changes invoked proportionality between accessible polar (AApo1) and apolar (AA,,)contact surface areas calculated from the structure of the complex, and the AC, and AH values (Murphy and Freire, 1992; Privalov and Makhatadze, 1990; Spolar et al., 1992). Thus,

-

AC, = 0.45AAap- O.26AAp,I

(15)

The enthalpy change was related to the temperature at which the apolar contribution was assumed to be zero, T*H - 100°C:

AH

= AH*

+ (Acap+ h c , , ~ )( T - T*H)

(16)

AH* = 35AAp,,

(17)

Here,

To derive KM from the X-ray structure of the complex, no structural changes were assumed in the antibody binding site, and an arbitrary extended conformation of angiotensin was used to estimate the intramolecular surface changes accompanying the assumed change in angiotensin conformation on complex formation.

205


I

I

I

I

I

I

I

I

/I

I / /

OKWD

12.0

/ /

-

/

/

10.0

-

/ / /

- correl. cosi

/

8.0

R21E

0

8.0

-

/ /

'OKWG

,,

without K98M: correl. coef. 0.78

-

/

4.0

0.68

/

(ave. error

2.8 kcal)

/

/

-

2.0 0.0 /'

-2.0

,ti: 0

-

OKSEM

-

ODlOlK

/

-4.0

w62y

/

(9

-

R21K

W83Y

/

-6.0

I

I

I

I

I

I

I

I

I

Experimental AAG values [kcal]

FIG.14. Empirical Gibbs free energy estimates on 10 single-residue hen egg lysozyme mutants complexed with the HyHEL-10 antibody. (The experimental data are courtesy of J. Kirsch, University of California Berkeley.) The calculated AAG values (mutant-wild type) are based on CONGEN-generated coordinates of the respective mutants starting from the X-ray crystallographic coordinates of the wild-type lysozyme-HyHEL- 10 complex [e.g., in the W63Y mutant the lysozyme Trp-63 was replaced by Tyr, etc. (Novotny, Bruccoleri, Davis, and Sharp, manuscript in preparation)]. The calculations employed a scaled molecular surface ([contact area]*70). as the hydrophobic term, a finite difference Poisson-Boltzmann algorithm with dielectric boundary and charge smoothing-antialiasing as the electrostatic term, and conformational entropy estimates carried out by the exhaustive CONGEN enumeration of the trans and gauche torsional degrees of freedom. The correlation coefficient for all the data points is -0.6; it is -0.8 if the K96M mutant is ignored (the lysozyme wild type Lys-96 is exceptional in that it participates in buried intramolecular hydrogen bonds and its substitution for Met is likely to lead to global structural changes). The average error for the latter nine AAG values, comparing the experiment and the calculation, is *2.8 kcal.

Based on Eq. (15), a AC, value of -250 cal K-' mob' was obtained, in close agreement with the experimental results, and the A H estimate, with the use of Eq. (15), yielded -8.4 kcal mol-I, compared to the experimentally determined AH of -8.9 kcal mol-I, a remarkable result considering that the angiotensin conformation in free solution was not well known and its total surface areas, A, and LP,had to be approximated.

206


The entropy change in the complexation was assumed to be related to the heat capacity change as

AS = AS*

+ (AC,., + Ap,pol)In (T/T*s)

(18)

where T*s, the temperature at which the apolar contribution to the entropy change is zero, equals 112”C,and AS*, the residual entropy change, is interpreted as consisting of configurational and “other statistical” contributions to AS:

AS* = AS,,,

+ ASsc + AS,,

(19)

where AS,,, is the change in backbone torsional degrees of freedom, ASSC is the change in side-chain torsional degrees of freedom, and AS,,, is the change in the number of particles in solution. The values of the entropy estimates, i.e., the terms of Eq. (19), were substituted from literature data and the overall AS estimate fell in the range 7-9 cal K-’ mol-’, in good agreement with the experimental value of 6.9 cal K-’ mol-’. These results were interpreted as showing both the loss of configurational entropy and a larger entropy gain from solvent release due to the hydrophobic effect on complexation. Enthalpically, binding was also favored by hydrogen bonding. It can be said that the main focus of the Murphy et al. (1993) work was on finding structural correlates of the extensive thermodynamic functions of state rather than approximating the absolute AG of the reaction from its atomic components. Structural interpretations of protein thermodynamics (Kauzmann, 1959) have had a long and fruitful tradition (Privalov, 1979), however, some of its important issues are still unresolved (Sturtevant, 1994; Naghibi et al., 1995). For example, the hydrophobic effect is considered to be a manifestation of water entropy changes induced by solutes but, depending on the reference state with respect to which the effect is measured (aliphatic alcohol, vacuum) and the theoretical framework used (classical or Flory-Huggins theory; Sharp et al., 1991; Sitkoff et al., 1994; Chan and Dill, 1994), differences in solute-solvent interactions vary in magnitude, contain varying amounts of enthalpic and entropic (mixing volume) contributions, and may enter into the free energy balance with different significance. Electrostatic interactions, on the other hand, are mostly considered to be of enthalpic origin, yet they involve desolvation of charges on attainment of compact solute states (folded protein, proteinprotein complex). Solvation-desolvation events are accompanied by changes in the entropy of water (electrostriction) so large that they almost certainly overshadow the hydrophobic effect encountered at nonpolar surfaces. Efforts trying to relate any macroscopic theory (thermodynamics) to the microscopic, atomic description of matter are essential for an understand-


207

ing of biological specificity, however, the phenomenological gap that must be overcome is very wide indeed. Thus, the development of empirical atomic rules of specificity may have a better chance of success when focusing on the interpretation of just one-the major- experimental observable (AG and the binding constant, KD) rather than dissecting this observable into its individual macroscopic parts (calorimetric enthalpy and entropy). This is because every thermodynamic quantity of state represents an equally bewildering mix of microscopic events, each of them as complex in its atomic origins as the parent one and each involving mixtures of solute and solvent effects. Thus, by dividing the problem, one may paradoxically make it more and more complex, in proportion to the number of functions of state being considered. All the (unobservable) microscopic states and their transitions manifest themselves to us only indirectly, as macro observables. The interfacial tension, for example, is not only a direct measure of hydrophobicity, reflecting, in ensemble, all the events happening on solute-solvent mixing, but also a quantity proportional to the solute molecular surface. Molecular surface is a straightforward attribute of structure and, as such, is readily amenable to measurement and analysis. The advantage of simple empirical rules such as the correlation between the hydrophobic effect and solute surface is in circumventing the microscopic complexity of the solution and substituting an aspect of solute structure instead. In this way, quantitation of hydrophobicity can be easily carried out based on the structure of the solute alone. 4. Antigen-Antibody Docking

One way of probing intermolecular interactions is to carry out antigenantibody docking simulations in a computer. A successful computer docking experiment requires (1) a rapid and ef€icient generator of the many sterochemically acceptable contact orientations of the two molecules, and (2) a robust Gibbs functional that can correctly identify the native complex among the many antigen-antibody pairs generated in the course of orientational sampling. Because of the important role of the Gibbs functional, the docking problem is similar in spirit to the theories of binding energy attribution discussed previously. However, the importance of shape complementarity has been highlighted by the work of Norel et al. (1994) who reported successful docking on the basis of surface shape matching alone. Building on the earlier work of Connolly (1986), they represented molecular surfaces by “critical points” describing prominent holes and knobs as minima and maxima of a shape function. Their automatic algorithm considered the entire molecular surfaces of 16 protein pairs from the known

208


complexes, but no additional information about the structure of the binding sites. Fifteen complexes were successfully docked, including the antibody-lysozyme complexes HyHEL-5 and HyHEL-10. The most ?ccurate ab initio prediction of antibody-antigen association, to within 1.6 A of the X-ray structure, was reported by Totrov and Abagyan (1994) for lysozyme and the HyHEL-5 antibody. Their docking algorithm used an original, “biased probability,” Monte Carlo procedure. The Metropolis et al. (1953) Monte Carlo algorithm is a succession of random steps in the generalized coordinate space followed by energy evaluation of the new state, E , and its acceptance in proportion to the Boltzmann factor,

AE kT In the biased method, the random step and its acceptance are modified by a probability function that favors the energetically most preferred regions of the configurational (coordinate) space. The biased algorithm does not waste much time sampling the forbidden regions of the energetic landscape, the main problem associated with Metropolis Monte Carlo searches. Another important innovation introduced by Abagyan et al. (1993) was consistent use of the internal coordinate space, rather than the Cartesian coordinate space, for configurational sampling. The Gibbs functional used to evaluate the calculated antigen-antibody configurations was that of Abagyan et al. (1993) and Abagayan and Totrov (1994). It consisted of three terms: (1) surface energy, (2) electrostatic polarization free energy, and (3) side chain entropy. Although conceptually similar to the Gibbs functional described in Section VII,C,l [Eqs. (10)(12)], its formal implementation was different. Thus, surface terms employed the Eisenberg and McLachlan (1986) solvation parameters to scale the solvent exposed and/or contact surfaces. To this term a precomputed conformational entropy term was added that approximated side-chain conformational entropy changes in the individual side-chain types. Sidechain entropy estimates were made on the basis of preferred conformational zones, essentially rotamer libraries:

u=l

where P is the probability of the vth state and R is the gas constant, corrected if necessary for an additional number of states: Sadd

= -R 1%

(Nadd)

(21)

where N is the number of additional states. The electrostatic term, the

COMPUTA?’IONAL BIOCHEMISTRY OF ANTIBODIES

209

modified image electrostatics, makes use of (1) a rigorous analytical solution to dielectric boundary effects for an ideal spherical body (Kirkwood, 1934; Friedman, 1975), and (2) a fast approximation (a surface projection) of this solution to the irregular shape of a protein. The final electrostatic equations were of the Coulombic type and contained, in addition to the partial atomic charges, Qi, the fictitious image charges, Qi””,created at the dielectric boundary:

where E,,, and E~ are solvent and protein dielectric constants, R is the spherical protein radius, and xi is the distance from the ith atom to the center of the sphere). Cherfils et al. (1991), in their docking of the lysozyme-HyHEL-5 complex, employed simplified protein models with one sphere per residue and simulated annealing algorithms driven by a pseudo energy function proportional to the protein interface area. Docked complexes were subjected to conformational energy refinement with full atomic detail. Although a near-native complex configuration was generated and identified as a low-energy one, some other nonnative complexes could not be rejected based on the criteria used. Jiang and Kim (1991) developed a “soft” docking algorithm utilizing a cubical grid and a full molecular mechanics potential. When applied to the lysozyme-HyHEL-5 antibody complex, the correct docking solution was found to be among the top 500 configurations out of about 20,000 generated. Independently, Walls and Sternberg (1992) developed a soft algorithm that allowed for structural changes during docking. Docked structures were evaluated quantitatively based on protein surface complementarity and a simple electrostatic model that screened out unfeasible interactions. When applied to the HyHEL- 10, D1.3, and HyHEL- 10 antibody-lysozyme complexes, the method identified between 15 and 40 possible docking orientations with the native structures being ranked 3rd, 5th, and 30th. Pellegrini and Doniach (1993) reported on computer docking experiments involving lysozyme complexes with D1.3, HyHEL-5, and HyHEL10 that employed rigid structures and a two-step approach. First, a coarsegrained painvise atomic potential of the Sippl (1990) type was used to bring the two molecules together. The configurations obtained were then refined with use of the all-atom OPLS potential of Jorgensen and TiradoRives (1988) and a distance-dependent dielectric function. The native configuration was consistently found to be the preferred solution for all three complexes. Friedman et al. (1994) docked epitopic fragments (heptapep-

210


tides) to the binding site of the antipeptide antibody B1312 (both the free and complexed X-ray structures), using the Metropolis Monte Carlo docking program of Goodsell and Olson (1990). The peptides Pro-His and Val-Pro-His, which contain residues experimentally identified as important for binding, docked correctly to both antibody structures, but all larger peptides docked correctly only to the complexed Fab, even when torsional flexibility was allowed in the ligand. 5. Hypothesis of Functional (Energetic) Epitopes

Immunochemists often observed that a small portion of an antigenic determinant was of crucial importance in defining its specificity. The term immunodominant (Sela, 1969, quoting Heidelberger) has commonly been used to describe this phenomenon. Does immunodominance have an identifiable molecular basis? The empirical Gibbs free energy calculations for antibody-antigen (Novotny et al., 1989; Novotny, 1991; Tulip et al., 1994) and enzyme-inhibitor (Krystek et al., 1993) complexes consistently indicated that only a small number of amino acids, approximately 30% of the total contact surface area, contributed actively to binding energetics. In the antibodies, the bottom part of the antigen binding cavity often dominated the energetics of binding, whereas in lysozyme, the energetically most important residues defined small (2.5 to 3 nm2) energetic epitopes. Thus, a concept of protein antigenicity emerged that invoked the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area (see also Section VIII). The concept offered resolution of an apparent paradox: on the one hand, a multitude of side-chain-side-chain interactions at the interface [-16 side chains in the antibody as well as in the antigen; see, e.g., Amit et al. (1986)J and, on the other hand, the experimentally derived size of the binding site as four to six amino acids (Haber et al., 1967; Sela, 1969; Schechter, 1971; Kabat, 1970). A number of experimental data consistent with the hypothesis of an energetic epitope have accumulated, including those obtained on antibodies to human growth hormone (Jin et al., 1992; Cunningham and Wells, 1993),3human placental lactogen (Lowman et al., 1991), the A repressor (Breyer and Sauer, 1989), the antilysozymes HyHEL-10 (Lavoie et al., 1992) and D1.3 (Hawkins et al., 1993), the anti-N9 neuraminidase NC41 (Nuss et al., 1993), anticytochrome c (Mylvaganam et al., 199l), and anticyclosporin (Rauffer et al., 1994) antibodies. Smythe and von Itzstein (1994) accepted the concept of a functional epitope as a starting point for their synthesis of

Jin et al. (1992) suggested the termfinctional eptiope instead of energetic epitope.


21 1

a biologically active, constrained cyclic peptide that mimicked the NC4 1 antineuraminidase antibody. The size of the functional epitope varies somewhat according to the criterion used for its definition. Jin et al. (1992) found that, per epitope, the number of alanine substitutions causing a >2- or >20-fold effect on binding affinity was on average eight and three, respectively, which is more in accordance with Nuss et al. (1993) than with the CONGEN calculations (Tulip et al., 1994; Novotny et al., 1989). Part of the discrepancy may arise because the large negative AGreslduc values of some residues in the CONGEN functional epitopes are due to favorable contacts made by main-chain atoms only (Arg-327 in N9-NC41) and can be “invisible” to mutagenesis. In the utmost limit of low molecular weight haptens with no formal charges and only one or two functional groups (e.g., digoxin; see Near et al., 1993), the functional epitope concept may not be applicable (Webster et al., 1994). In the calculations reported by Novotny et al. (1989) and Tulip et al. (1994), charged residues were high on the list of energetic residues. Jin et al. (1992), Kelley and O’Connell (1993), and Cunningham and Wells (1993) also reported that charged residues played a prominent role in most functional epitopes and that out of the average number of 8 residues causing a >2-fold reduction in affinity, on the average 2.8 are charged. The functional epitopes are mostly discontinuous (Jin et al., 1992; Tulip et al., 1994).As for predictions that some of the contact residues act in a repulsive manner and destabilize the complex, further experimental data are required to confirm that this is the case. The alanine scan of Jin et al. (1992) sometimes identified side chains that hindered antibody binding. Getzoff et al. (1988) reviewed the evidence indicating that, in heteroclitic antibodies, the contributions of some residues to binding affinity can be increased. D. Water-Mediated Binding

The complex of D1.3 antibody with hen egg white lysozyme is currently one of the best resolved antigen-antibody complexes, at 1.8 A (Bhat et al., 1994). At this resolution, about 50 water molecules were reported at or near the interface, with few of them actually trapped at the interface. The structure of the lysozyme complexed with the D1.3 mutant W92D (VL domain) was also solved (Ysern et al., 1994). Titration calorimetry of W92D mutant complex formation showed that the AAG of the reaction (--4 kcal, wild type-mutant) could be attributed to a smaller negative binding enthalpy (3.8 kcal) with few net changes in binding entropy. In the structure of the mutant, two water molecules occupied the space created by the smaller size of the Asp side chain compared to Trp.

212

FIR1 NOVUI‘NY AND ,JLiRC,EN BAJORATH

The phenomenon of water-mediated binding has been most extensively discussed for the trp receptor-operator system (e.g., Shakked et al., 1994). There, the specificity of DNA-protein interaction could be explained not only by direct hydrogen bonding but also by water-mediated hydrogen bonds. Comparison of the X-ray structures of the free and bound states of the DNA operator regulatory sequence made it clear that “the three hydration sites used to mediate protein contacts to the three critical bases of the operator half-site sequence are already fully occupied in the free DNA. The water molecules can thus be regarded as non-covalent extensions of the DNA bases which may be used as stereospecific recognition elements of the DNA target sequence” (Shakked et al., 1994). A full account of water-mediated interactions in antigen-antibody complexes requires knowledge of whether any given water molecule was bound to a protein prior to complex formation, or whether it became passively trapped at the interface. In the first instance, a prebound water molecule can be looked at as another protein side chain, and its interactions in the complex can be evaluated in a straightforward manner based on its protein contact and its partial atomic charge (see Section VI1,C). In the second instance, the protein-water-protein complex becomes a ternary complex of a “solute” water molecule and two protein molecules. In this case, it is necessary to estimate correctly the entropy decrease of the system due to imprisonment of the water molecule at the interface. A water molecule bound to protein may acquire new, productive waterprotein interactions (hydrogen bonds, van der Waals contacts) at the expense of those existing in bulk water prior to complex formation. One of the four possible water-protein hydrogen bonds would stabilize the complex by a varying amount, depending on the quality of the bond and the partial charge of the participating protein atom. In bulk water at room temperature, there are -3.5 hydrogen bonds per molecule (Lemberg and Stillinger, 1975) and the average hydrogen bond energy of liquid water is probably in the range 2-3 kcal/mol (Eisenberg and Kauzmann, 1969). Thus, to recover the bulk interaction energy, a protein-entrapped water molecule should gain at least -8 kcal in hydrogen bonding to protein groups, i.e., nearly -3 kcal per H bond if, as reported by Williams et al. (1994), three H bonds per molecule is the most common form of waterprotein interaction. The entropic cost of the transfer and complete immobilization of a water molecule from the liquid to the protein has been estimated to be -2 kcal/mol (Dunitz, 1994).In most situations, the free energy associated with entrapment of water in the compIex is expected to carry a AG close to zero at best, and would probably be unfavorable in many cases. This may be the reason why, in tight protein-protein complexes, intersurface-bound water is a relatively rare phenomenon.


213

VIII. MOLECULAR BASISOF P R O T E I N ANTIGENICITY

The early serological experiments of Landsteiner (1962) and others showed that virtually any chemical structure attached to a protein molecule can elicit an antigenic response. The early era of hapten research has given way to the current age of antigenicity research on peptides and proteins. The comparison of properties of antibodies elicited with protein antigens, on the one hand, and their mimics, short peptides, on the other hand, has formed most of our views (Sela, 1969; Benjamin et al., 1984; Tainer et al., 1985; Novotny et al., 1987; Getzoff et al., 1988; Colman, 1988) on the molecular origin of antigenicity. In discussing the molecular basis of antigenic response, it is important to distingiush between the terms antigenicity and immunogenicity (Benjamin et al., 1984). Antigenicity refers to the ability of a protein surface region to be potentially antigenic, while immunogenicity refers to the ability of any antigenic site to elicit such a response under particular experimental conditions such as the immunization protocol, and the genetic constellation of the organism [an “immunopotent” determinant according to Sela (1969)]. However, bona fide antigenic sites may not be immunogenic in certain experimental situations (“immunosilent,” Sela, 1969). Identification of antigenic epitopes can be based only on indirect experimental procedures, such as methods involving the comparative strength of binding of the same specific antibody to homologous proteins with a small number of amino acid replacements (Benjamin et al., 1984); NMR hydrogendeuterium exchange experiments (Paterson et al., 1990); and X-ray crystallography of antibody-antigen complexes ( h i t et al., 1986). Antiprotein antibodies sometimes specifically recognize short peptides (tetra- to hexapeptides), and such antibodies can be elicited by synthetic peptide antigens. Often a single native conformation of a peptide is recognized, such as the disulfide-bonded loop peptide of lysozyme: antiloop antibodies do not react with peptides in which the disulfide bond has been reduced (Amon et al., 1971). The majority of antigenic sites in proteins, however, seem to consist of amino acids that are not contiguous in the amino acid sequence (composite or discontinuous epitopes). This is simply a consequence of the large contact area between antibodies and antigens (- 800 A*), and the low probability that such a large surface would be contributed by a contiguous polypeptide segment (Barlow et al., 1986). Based on a long history of experimental work, some researchers concluded that several discrete antigenic sites exist on protein surface (e.g., Atassi, 1975, 1978), implying that certain surface regions are more antigenic than others. Other researchers have argued that many more mutually overlapping epitopes exist on protein surfaces and that the whole protein surface is antigenic (Benjamin et al., 1984).

214

J l K I N O V U I N Y AND JUKGEN BAJOWI'H

A. Segmental Flexibility and Surjace Exposure Westhof et ul. (1984) and Tainer et al. (1984) noticed a correlation between the average backbone crystallographic B factors and locations of antigenic sites and proposed that segmental flexibility (assumed to be associated with the cause of high B factor values) is an important component of antigenicity (Tainer et al., 1984, 1985). Implicit in this proposal was the notion that most antigen-antibody interactions are accompanied by an induced fit in the antigen, and that antigenic epitopes frequently rearrange their conformations to maximize productive noncovalent (electrostatic, van der Waals) interactions with binding sites (Geysen et al., 1987, Getzoff et al., 1987). An alternative antigenic theory was suggested by Padlan (1985) who proposed that the antigenic potential of a polypeptide segment was a simple additive function of atomic properties such as surface exposure and polarity. The segmental flexibility theory of antigenicity was challenged by several groups (Novotny et al., 1986b; Fanning et al., 1986; Thornton et al., 1986) on the grounds that ( 1 ) B factors represented parameters combining the effects of thermal mobility and static crystalline disorder into one measure, often making it dificult to correlate them unequivocally with either the static or the dynamic aspects of the structure; and that (2) molecular properties other than segmental flexibility (in particular, surface protrusion) were also correlated with protein antigenic sites, and the average backbone B factors. For example, the prominent antigenic epitopes may simply be the most protruding parts of the surface, easily accessible to the large antibody molecules. In fact, correlation among surface protrusion (static accessibility), segmental flexibility, and antigenicity was so strong that it was difficult to design experiments that would isolate the relative importance of these various properties. In this context, analysis of flexibility and antigenicity properties in scorpion neurotoxins, small molecules of 46 amino acids containing four disulfide bridges (Fig. 15), was particularly illuminating. The experimental work oFEl Ayeb et al. (1983, 1984) and Bahraoui et al. ( 1986) established four antigenic epitopes in the Androctonus australis neurotoxin and localized them in the amino acid sequence. Novotny and Haber (1986) calculated large-probe (r = 10 A, comparable in size to antibody domains) accessibility profiles of the Centruroides sculpturatus neurotoxin, a molecule closely similar to the of A. australis neurotoxin, using the X-ray coordinates of Almassy et al. (1983). Six prominently exposed regions were identified, clustered in four surface patches that were identical to, or overlapped with, the experimental antigenic epitopes. Next, Novotny and Haber (1986) carried out molecular dynamics simulations on the C. sculpturatus structure, computed average backbone B fac-


215

FIG 15. Ribbon diagram ofthe scorpion neurotoxin fold. Disulfide bonds are shown as light sticks and large probe-exposed loops (antigenic regions). are highlighted in a dark color. The N terminus of the polypeptide chain is approximately in the middle of the figure and the C;-terminal Cys is at the lower left. Two turns of an a helix are visible at the upper right, and beneath the a helix is a three-stranded /3 sheet.

tors from the simulation, and compared results with the X-ray-derived B values. Most of the neurotoxin structure and, in particular, three out of the four antigenic sites, were found inflexible, asjudged both by the computed and the crystallographic B factors (Fig. 16). The remaining flexible epitope was associated with only marginal above-average maxjma of backbone B values, corresponding to rms displacements of 0.5 A. It thus appeared that, at least in this molecule, antigenicity was determined by an exceptional surface exposure of relatively short loop segments, and that segmental flexibility was not an essential component of antigenicity. These conclusions were supported by a later study (Granier et al., 1989) based on the crystal structure of the A. australis toxin determined at 1.8 resolution (Fontecilla-Campset a,l., 1988).On refinement to 1.3 A resolution (Housset et al., 1994), the average backbone B factor of the A . australis toxin structure remained exceptionally low: 10 A2,with a maximum at 16.2 A2 and a minimum at 7.2 .k2.

216


7.

8.

1.

0.

10

20

30

40

60

e0 .

Sequence number

FIG. 16. Large-probe accessibility contact surface and crystallographic B factor profiles for the A. australis scorpion neurotoxin molecule. Heavy line represents the smoothed accessible contact surface calculated with a spherical probe 10 %, in radius, light line represents B factors. Antigenic peptides (see Section VIII,A) are delineated by small squares at the top of the figure.

It seems significant that the calculated energetic epitopes cluster along the most exposed regions in the two proteins whose antigenicity has been studied most thoroughly: hen egg white lysozyme and influenza neuraminidase (Fig. 17). Similarly, the complete antigenic analysis of human growth hormone (Jin et al., 1992) showed the epitopes to correlate well with the most protruding regions of the molecule. It is interesting that both the experimental data of Jin et al. (1992), and the calculated frequency of side chains occurring in protein surfaces accessible to large spherical probes (Novotny et al., 1987), show a prominence of long, formally charged or dipolar side chains (Arg, Lys, Glu, Gln, Asp, Asn; see Table VI). Antibodies themselves can become antigens of other antibodies and Novotny et al. ( 1986a) investigated a correlation between immunoglobulin antigenic epitopes (i.e., locations of the idiotypic, allotypic, and isotypic serological markers) and large-probe accessibility profiles of selected antibodies. The experimental epitopes always corresponded to convex parts of an antibody surface made by reverse turns. The computed protruding surfaces occurred in homologous positions in all the immunoglobulin chains for which the computations were carried out, and most of the Bsheet surfaces of the domains were found to be poorly antigenic. The C H ~

217


A 7.

D1.3. Hy-10

Hy-5

Hy-5. Hy-10

Hy-10

D1.3

e.

c (

H

5.

X

"8

4.

5 ti

3.

8

2.

s 1.

0.

LO

20

30

60

50

40

90

80

70

110

100

120

13C

Sequence number

B I

I

m

4.

-

0

0

2

3.

X

"!

5 f;

2.

sI .Id

0

1.

0.

l!L 320

I

I

o

ym

I

I 0

rn

I 0

I

I

NClO epitope

m

:II 380

400

I

I

420

Sequence number

FIG. 17. Large-probe accessibility contact surface profiles for (A) lysozyme and (B) influenza neuraminidase. Positions of the energetically most important residues (the functional epitopes) are highlighted by vertical bars and squares and circles at the top of the figure.

218

JIRI N O V O T N Y AND J U R G E N BAJORATH

and CH3domains had many more calculated antigenic sites than the Fab fragment. Variable-domain epitopes (idiotopes) involved both hypervariable and framework residues, and only about 25% of the hypervariable residues were strongly antigenic. Pedersen et al. (1994) have carried out a statistical analysis of all the surface-accessible residues in human and murine Fv domains. They found that precise patterns of exposed residues were different in the two species, and that most surface positions had strong preferences for a small number of residue types. These observations have practical implications for the humanization of murine antibodies (see also Section IX,E). A large body of indirect evidence seems to indicate that surface protrusion is an important characteristic of antigenic sites in proteins. Side-chain polarity and higher-than-average backbone B factors also correlate well with antigenicity, but a causal link between these properties and antigenicity is less straightforward than that between protrusion and antigenicity. The conjecture that antigenicity is mostly determined by surface protrusion provides a natural link between the two extreme antigenicity theories (“distinct antigenic epitopes exist” vs “the whole surface is antigenic”) by introducing the concept of antigenic probability which varies along the surface. B. What Is a Protein Epitope?

“An antigenic epitope” is an operational definition whose factual content differs depending on whether we emphasize energetics of complex formation, complementarity of antigen-antibody surfaces, or other phenomena. According to the method used to define the epitope, one may thus arrive at different conclusions about antigenicity (Geysen et al., 1987; Laver et al., 1990; Greenspan, 1992). In addition to crystallographic epitopes and functional (energetic) epitopes, we also have NMR epitopes defined by the extent to which bound antibody prevents deuteriumhydrogen exchange on the backbone segments of the antigen (Paterson et al., 1990; Benjamin et al., 1992) and mutational epitopes deduced from the effects of single-residue substitutions (either synthetic or natural) on the strength of binding (Smith-Gill et al., 1987; Smith and Benjamin, 1991; Smith et al., 1991; Prasad et al., 1993). The mutual relations of these various definitions are only now beginning to be delineated. Sheriff et al., (1987), Padlan et al. (1989), and Prasad et al. (1993) compared the X-ray structures and epitope mutational data on the HyHEL-5, HyHEL-10, and phosphocarrier protein HPr-Jell42 antibody complexes. By and large, the crystallographic and mutational epitopes overlapped well. The limitations of the mutagenesis approach became apparent when, of the 14 amino acid

COMPUTA?’IONAL BIOCHEMISTRY OF AN‘I’IBODIES

219

residues of the HPr protein found in contact with the antibody binding site, 9 were correctly assigned by the mutagenesis studies, 1 could not be altered by mutations, 2 appeared to be critical for the protein fold, and 2 other peripheral side chains had a minimal effect on antibody binding. Interestingly, 4 amino acids adjacent to the epitopic residues were incorrectly assigned to the Jell42 epitope. The concept of a functional (energetic) epitope (see Section VI1,D) provides an additional perspective on antigenicity. The calculated energetic epitopes (Novotny et al., 1989; Novotny, 1991; Tulip et al., 1994) clustered along the most exposed regions in the two proteins whose antigenicity has been studied the most thoroughly, i.e., hen egg white lysozyme and influenza neuraminidase (Fig. 17). The complete antigenic analysis of human growth hormome gin et al., 1992) also showed the functional epitopes to correlate best with the most protruding regions of the molecule. Both the experimental data of Jin et al. (1992) and the calculated frequency of side chains occurring on protein surfaces accessible to large spherical probes (Novotny et al., 1987) showed a prominence of long, formally charged or dipolar side chains (Arg, Lys, Glu, Gln, Asp, Asn; see Table I) in the functional epitopes. At the same time, however, the size of the crystallographic epitope indicates that a surface area larger than the functional epitope must be complementary to the antibody surface. C. Cross-Reactivity in Proteins: Influenza Neuraminidase How cross-reactive are individual protein epitopes? How degenerate are proteins as antigens? The fact that a delimited patch of protein surface can support several overlapping, different epitopes has by now been well established (Darsley and Rees, 1985; Malby et al., 1994; Lescar et al., 1995; Bottger et al., 1995). In the two cases where cross-reactive epitopes were studied in atomic detail, degenerate binding of the same antigenic motif (Malby et al., 1994), or two different antigenic motifs by the same antibody (Lescar et al., 1995), did not require any chemical similarities between the different epitopes or different binding sites. In the N9 neuraminidase complexes, -80% of the NC41 and NClO antibody epitopes overlap (Malby et al., 1994), and one might expect about two-thirds to threequarters of the energetic neuraminidase residues to be identical, and to contribute comparable binding energies, in the two complexe~.~ This was clearly not the case, however, as established both by experiment (Malby This was the situation invariably found with enzyme-inhibitor complexes such as, e.g., the eglin inhibitor in complex with chymotrypsin and with subtilisin. See Krystek et al. (1993) for more details.

220


et al., 1994; Nuss et al., 1993; Webster et al., 1987) and by calculations (Tulip et al., 1994). The two antibodies, NC41 and NC10, engaged different side chains of the same neuraminidase surface to create stable complexes (Table VIII). Is an area of protein surface a single antigenic epitope, or is it a multitude of different overlapping epitopes? The term epitope being an operational one, the answer to this question may also be formulated operationally. About 50-60% of protein surfaces is made of polar atoms including those that are formally charged. The charged atoms occur mostly at prominent surface convexities where they are most efficiently solvated. The Asp, Glu, Lys, and Arg side chains are not only preferentially located in loops but are themselves the longest side chains. Naturally, protrusions and their polar atoms constitute the best anchor points for a firm attachment of antibodies but, depending on the topography of the larger, adjacent surface area and relative dispositions of the multitude of surrounding polar atoms (both capable of making, and when in the complex required to make, hydrogen bonds) many alternative solutions exist for approximately complementary binding site surfaces. Thus, the antigenic code, akin to the stereochemical code of protein structures (Section III,B), may be degenerate (Malby et al., 1994) in the sense that many different surface shapes can complement an antigenic determinant. The degeneracy of both the stereochemical and antigenic codes may not be accidental. Protein-protein interactions follow the same physical rules as protein folding events, and approximately similar phenomenology can be expected in both types of interactions. IX.

ANTIBODY

ENGINEERING

The modular three-dimensional architecture of immunoglobulins (Section II1,A) and T-cell receptors lends itself well to protein engineering schemes that shuffle, transpose, and reconnect the domains into chimeric proteins with hybrid structures and novel properties. The rapid development of antibody engineering has been stimulated by three important technological advances: ( 1) Rapid development of gene cloning technologies and the advent of the polymerase chain reaction (PCR), allowing subcloning of eukaryotic genes into bacterial plasmids (Boss et al., 1984; Cabilly et al., 1984). Nevertheless, the expression of chimeric immunoglobulins in transformed lymphoid cells such as myeloma or hybridoma (Rice and Baltimore, 1982; Oi et al., 1983; Ochi et al., 1983; Rusconi and Kohler, 1985) has remained a powerful experimental tool. (2)Progress in the controlled expression of proteins from plasmid-inserted genes in bacteria and other organisms, i.e., yeast (Wood et al., 1985) and baculovirus


22 1

TABLE VIII

AGresduem Calculated for N 9 Neuraminidme Epitopes (-80% Overlapping) in NC41 and NCI 0 Antibody Complexesa NC41 Antibody complex Residue Attractive LYS-463 Lys-432 Ala-369 Asn-400 Thr 401 Arg 327 Ile 368 Pro 43 1 Ser 367 Lys 435 Neutral Asn 329 Pro 328 Leu 399 Pro 326 Gly 343 Trp 403 Ser 370 Asn 345 Man 200D Asn 344 Man 200F Ile 149 Asn 347 Ile 366 Ser 372 Asp 434 Repulsive Asp 402 Glu 433

NClO Antibody complex Residue

-4.8 -4.0 -3.4 -2.6 -2.4 -1.9 -1.8 -1.4 -1 .o -1 .o -0.8 -0.7 -0.6 -0.5 -0.5 -0.5 -0.3 0.0 0.0 0.1 0.2 0.3 0.3 0.5 0.7 0.7

1.5 2.0

AGresidue

Attractive Lys.432 Asn-329 Man 200F Ala-369 Thr 401 Pro 328 Gly 343 Ser 370

-3.6 -2.2 -2.0 -1.6 -1.5 -1.3 -1.0 -1 .o

Neutral Pro 331 Pro 342 Trp 403 Man 200E Man 200D Asn 400 Ile 368 Thr 332 LYS-336 Ile 366 Val 333 Ser 367 Asp 330 Asn 344

-0.6 -0.6 -0.6 -0.5 -0.3 -0.2 0.0 0.0 0.0 0.2 0.2 0.3 0.9

Repulsive Tyr 341 Ser 372

1.1 1.1

a Data in kcal/mol; see Tulip et al. (1994) for details. Amino acids set in boldface type are those mutated by Nuss et al. (1993) and Webster et al. (1987).

and plant cells (Hiatt et al. (1989). Proteins were obtained either as secreted, refolded, soluble species or as insoluble, denatured intracellular aggregates, inclusion bodies, that were easy to isolate but had to be solubilized and renatured before a functional protein was obtained. (3) Computer-

222


aided structural design techniques, supported by the numerous X-ray crystallographic coordinates of diverse antibodies. In this section, we briefly discuss chimeric antibodies and T-cell receptors, covalent Fv fragments (single-chain or disulfide-bonded), heterospecific bifunctional constructs, and various domain humanization schemes (Neuberger et al., 1984; Riechmann et al., 1988) with a focus on computeraided structural design. A . Chimeric Antibodies via Domain Interchange Some of the first synthetic chimeras were mouse antigen binding domains and V,) implanted on human constant-region domains (Boulianne et al., 1984; Morrison et al., 1984; Takeda et al., 1985);VH domains spliced onto CLdomains giving rise to functional, antigen-specific, VH-CJVI.-CL L chainlike dimers (Sharon et al., 1984); recombinant mouse antibodies with novel effector functions engineered via H chain constant domain swaps (Neuberger et al., 1985; Schneider et al., 1988); and domain deletions or insertions to produce shortened antibody-like molecules (Igarashi et al., 1990) and antibodies with altered oligomerization states. As a rule, the structural design of these hybrids was straightforward (i.e., genes coding for complete domains were swapped, by subcloning, between molecules). More-ambitious examples of antibody engineering included the replacement of selected immunolgobulin domains with foreign proteins [e.g., enzymatically active bacterial P-lactamase (Goshorn et al., 1993; De Sutter and Fiers, 1994)l. Highlights of these constructions include (1) CD4 and CTLA4 immunoadhesins (proteins composed of IgG constant domains, the Fv modules being replaced with two extracellular domains of the CD4 receptor (Capon et al., 1989) or the CTLA4 marker (Linsley et al., 1991) for use in acquired immunodeficiency syndrome (AIDS)therapy; (2) immunoligands such as the interleukin-2 molecule fused to IgG constant regions (Landolfi, 1991); (3) an exogenous peptide epitope implanted in lieu of the H chain third hypervariable loop (Sollazzo et al., 1990); (4) metal coordination sites engineered into an antibody binding pocket (Roberts et al., 1990); and divalent molecules combining the complete class I major histocompatibility complex (MHC) molecule with an immunoglobulin heavy chain (Dal Porto et al., 1993). Finally, various chimeric constructs consisting of T-cell receptor (TCR) C domains and antibody V domains, or TCR-antibody polypeptide chain aP or yd heterodimers, were also assembled and shown to carry functional traits characteristic of both parent molecules (Gascoigne et al., 1987, Gross et al., 1989, Mariuzza and Winter, 1989; Becker et al., 1989; Goverman et al., 1990; Schearman et al., 1991; Gregoire et al., 1991; Eilat et al., 1992). (VL


223

B. Single-Chain Fv Fragments, “Diabodies, Fv-Toxin Congugates ”

The production of large quantities of stable Fv fragments in bacteria was seriously hindered by the fact that two polypeptide chain segments, VL and VH, had to be separately refolded and reformed into a noncovalent dimer. The problem has been circumvented by the design and successful construction of a single-chain Fv where the C terminus of a VH (alternatively, V,) domain was connected to the N terminus of the VL (VH)domain by a polypeptidic linker. The following issues had to be considered in the design of the linker: (1) connection polarity, i.e., the question of a possible preference for leading the linker from the C terminus of the Vr. domain to the N terminus of the VH domain, or vice versa; ( 2 ) the minimal, and possibly the maximal, length of the linker; (3) the nature, i.e., the detailed amino acid sequence, of the linker, with special reference to protein folding and proteolysis. Two different single-chain Fv designs were described in 1988, both involving the use of computer modeling to design the molecule but approaching the preceding questions in different ways. In the work of the HarvardJ Creative Biomolecules group (Huston et al., 1988), the order of domain connection was deemed unimportant in view of the pseudo symmetry of the Fv fragment, and the polarity chosen was VH+VL. In the work of the Genex group (Bird et al., 1988) the polarity was VL+VH. The success of both designs immediately proved the functional equivalence of both solutions. The HarvardKreative Biomolecules group obtained the distance to be spanned by the linker from Fab crystal stryctures as -3.5 nm which, considering the length of a peptide unit (3.8 A), would require at least 11 amino acid residues. To interfere minimally with refolding of the two immunoglobulin domains, and also to maximize the flexibility of the linker, a sequence rich in glycines, (Gly-Gly-Gly-Gly-Ser)3,was chosen. Such a sequence should also be rather resistant to proteases. The design philosophy of the Genex group relied on searches through the Brookhaven Protein Data Bank for peptides of proper molecular dimensions to bridge the interdomain distance and to introduce correct peptide bond angles at the N and C termini of the prospective linkers. Alternatively, linkers were designed by an incremental addition of short peptides from the C terminus of the VL to the N terminus of the VH. Some linkers were designed to minimize interactions with the Fv, whereas others were designed to fit into a groove on the back of the Fv structure primarily with the use of alternating glycine and serine residues and Glu and Lys included to enhance solubility. Thus, one of the successhl linkers had the sequence EGKSSGSGSESKST. Since the original studies, dozens of papers have been published describing the use of single-chain Fv fragments for various diagnostic and

224


therapeutic purposes, and in investigating various aspects of the design (see, e.g., Huston et al., 1991, and Pluckthun, 1992, for reviews). It seems that many amino acid sequences can satisfy the purpose of the linker, although some were claimed to be superior from the point of view of bacterial secretion (Takkinen et al., 1991). Argos (1990) published a detailed survey of oligopeptide linkers in natural multidomain proteins and recommended candidates for general gene fusion work. He concluded that, for the linker to be optimally extended, it should contain small (Gly) and polar (Ser, Thr) amino acids. Thus, e.g., the improved linker described by Adams et al. (1993) and Hilyard et al. (1994) had the sequence (Ser-Ser-Ser-Ser-Gly)3,while that used by Whitlow et al. (1993) had the sequence GSTSGSGKPGSGEGSTKG. For single-chain Fv fragments, the critical length requirement for the linker seems to be 12-13 residues, as 10-residue linkers did not sterically allow the VL-VH domain dimer formation. This observation was cleverly exploited by Holliger et al. (1993) and Hudson et al. (1994) in the design of “diabodies,” i.e., small bivalent and bispecific antibody fragments. By using a linker too short to allow pairing between the two domains of the same chain (either 5 or 10 residues of linker length), the VL and VH domains were forced to pair with the complementary domains of another chain to create two different antigen binding sites. An efficient domaindomain packing at the two contacting ends of the dimeric Fv modules was investigated by computer graphics (Holliger et al., 1993), and it was found that it might be possible to join the C terminus of the VH domain directly to the N terminus of the VL domain and dispense with the linker polypeptide. Indeed, fragments with no linker proved to be dimeric and bispecific when expressed in bacteria and the X-ray structure of the diabody L5MK16, specific for phosphatidylinositol, turned out to be very similar to that predicted by modeling (Perisic et al., 1994). According to Desplancq et al. (1994) and Whitlow et al. (1994), single-chain Fv fragments have a natural tendency to form heterobivalent dimers and perhaps even higher oligomers. Thus, heterodimers of antifluorescein and antitumor scFv fragments 4-4-30 and CC49, respectively, formed with a linker as long as 12 residues (Whitlow et al., 1994). Bivalent dimers of the antitumor antibody B72.3 were obtained even with a linker 30 residues long, and better activity was observed with the domain arrangement VL-linker-VH compared to VH-linker-VL (Desplancq et al., 1994). NMR data from the McPC 603 single-chain Fv fragment using the (GGGGS)s linker (Freund et al., 1993) indicated relative independence of the linker from the rest of the structure, and confirmed its high flexibility. Two X-ray crystallographic structures have been reported for single-chain Fv fragments (Kortt et al., 1994; Zdanov et al., 1994). In both, including the


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1.7 A resolution structure of the carbohydrate-binding antibody Se155-4 (Zdanov et al., 1994) single-chain Fv complexed with the antigen, the linker was largely invisible due to crystalline disorder. Antibody binding sites, with their unique specificities, can be used to target proteins (enzymes, toxins) to cells and tissues. An example of this approach to human therapy is antibody-targeted dissolution of infarctionrelated blood clots by proteolytic enzymes (Fab urokinase, Haber et al., 1989). The single-chain Fv technology has been particularly successful in supporting bacterial production of effective immunotoxins. Thus, singlechain Fv of antitumor specificity with a modified Pseudomonas exotoxin (PE40) attached to its C terminus is a powerful and specific anticancer agent (Chaudhary et al., 1989). An extensive literature, exploring various aspects of targeting, toxin structure, optimal expression, and other aspects of this topic exists by now, and is not reviewed here in detail. One practically relevant bifimctional construction was the antidigoxin single-chain Fv fragment with the -40-residue fragment B of the staphyloccocal protein A attached to its N terminus (Tai et al., 1990). The fragment B effector domain, through its affinity for the immunoglobulin Fc fragment, simplified purification (via affinity chromatography) of the Fv construct. The single-chain technology has also been applied successfully to T-cell receptors and MHC proteins. Fv-like, soluble single-chain T-cell receptor V P a fragments were expressed in bacteria and shown to possess essentially the same antigen specificity as the parent receptor (Novotny et al., 1991; Ward, 1992; So0 Hoo et al., 1992; Schodin and Kranz, 1993; Kurucz et al., 1993). Functional, soluble MHC-like proteins were prepared by tethering the three extracellular domains (al,a2and a3)of the mouse H-2 class I a chain to the p2 domain (Mottez et al., 1991, Mage et al., 1992). Finally, Eshhar et al. (1993) designed and constructed chimeric genes composed of a single-chain Fv domain linked with y or 5 chains, the common transmembrane, signal-transducing subunits of the immunoglobulin, and T-cell receptors. The chimeric genes were expressed as functional cell surface receptors in a cytolytic T-cell hybridoma, and they triggered interleukin-2 secretion from the cells on encountering antigen to which the single-chain Fv specificity was directed (the hapten trinitrophenyl). Such chimeric receptors can provide T cells and other lymphocytes with antibody-type recognition coupled directly to cellular activation. A diagrammatic summary of Fv fragment-based single-chain constructs is given in Fig. 18.

C. Disuljide-Bonded Fv Fragments It is known that Fv fragments of different specificities, and different amino acid sequences, vary widely in stability (Padlan, 1994).Although the

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FIG 18. Protein engineering of antibody binding sites: a schematic diagram. VH and VI. domains are shown as open circular sections, peptide linkers as thick lines, leucine zippers (a helical dimers) as filled rectangles, and cell toxins, enzymes, etc., as filled circles. (Tol,) From left to right, a single-chain Fv fragment (scFv), a disulfide-bonded Fv fragment (SSFv), a bivalent diabody, and a bivalent, disulfide-bonded scFv dimer. (Bottom) From left to right, an scFv attached to an effector molecule (e.g., Pseudomonas PE40 cellular toxin), a bivalent scPv-leucine zipper construct, and a bivalent scFv chimera with three scFv fragments chained together by a flexible linker. Both homo- and heterodimeric leucine zipper sequences are known, allowing in principle a noncovalent assembly of homobivalent and heterobivalent antibody-like chimeras. Similarly, homo- and heterobivalent, linker-chained scFv constructs are possible. Bivalent scFv chimeras based on four-helix bundles (the ROP dimer of the a helix-turn- helix motif). were also described (see Section IX,F) but are not shown. Note also that antigen combining sites (i.e., the three hypervariable loops in the VH and V1, domains) can be transferred from an Fv framework to another uones et al., 1986), and different pairs of VH and V1- domains can be recombined to give novel antigen binding sites.

critical interdomain side-chain contacts accomplishing the “three-layer” P-sheet-P-sheet packing are conserved in the variable domains of antibodies and T-cell receptors, about 40-50% of the domain-domain interface is contributed by hypervariable loop residues, suggesting that the strength of domain-domain contacts may be modulated considerably. Quantitative information is scarce, but one of the most stable VL-VH

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dimers may be that of the antidigoxin antibody 26-10 (Anthony et al., 1992), approaching the V r P H association constant of 1 nM. At the other extreme, the McPC 603 Fv fragment was found to be only marginally stable (Glockshuber et al., 1990), with an estimated domain-domain association constant of less than 1 p M . According to Glockshuber et al. (1990), three different strategies, namely, (1) chemical cross-linking, (2) introduction of disulfide bonds, and (3) generation of a single-chain protein, all stabilized the Fv fragment in a comparable way. To introduce disulfide bonds into the Fv fragment, Glockshuber et al. (1990) used the computer program of Pabo and Suchanek (1986) that systematically scans intra- or intermolecular residue pairs and selects those with the Cp-Cp distance close enough to support a SS bridge (-4-5 A). The two disulfide bridges Glockshuber et al. (1990) introduced into the McPC 603 VH and Vr domains were relatively close to the third CDR loop: V1.55 and VH108 (L chain and H chain, respectively), and G56C and T106C (L chain and H chain, respectively). Jung et al. (1994) used molecular graphics and computer model building tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment, distal from the antigen combining site (Fig. 19). Of the two sites identified, i.e., VH44-vL105 and vH111VL48, the former was tested by constructing a chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of the monoclonal anticancer antibody B3 (Brinkmann et al., 1991; see also Webber et al., 1995). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Reiter et al. (1994a) then showed that the latter disulfide site, VH11 l-Vl~48, could also be used to generate a functional disulfide-bonded B3 Fv fragment. Reiter et nl. (1994b) extended the disulfide-bonded constructs to two more Fv fragment-Pseudomonas toxin chimeras, generating cytotoxic proteins with full activity, improved stability, and a good yield in bacterial expression. Thus, SS-bridged Fv fragments may be more useful than single-chain Fv immunotoxins as therapeutic and diagnostic agents where inexpensive production and large quantities of refolded material are required, The single-chain Fv fragments may retain their usefulness in applications such as recombinant membrane-bound Fv receptors (Eshhar et al., 1993), phage surface display of complete binding sites, and heterospecific bifunctional miniantibodies.

D. Humanization of Mouse Monoclonals Monoclonal antibody therapy has become an attractive alternative in the cure of several disease states, such as allergy (Kolbinger et al., 1993) and cancer. Immunoglobulin-doxorubicin conjugates (Trail et al., 1993)

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FIG.19. Disulfide bond engineered into the Fv fragment. The Molscript ribbon diagram shows the VL (heavy lines) and the VH (light lines) domains. Carbons of side chains H44 and L100, which were replaced with cysteines by Jung et al. (1994), are shown as van der Waals radii spheres.

and single-chain Fv-Pseudomonas toxin chimeras are currently in human clinical trials. One of the major potential drawbacks of prolonged administration of mouse hybridomas, or fragments thereof, is onset of an immune response against the mouse framework antigenic determinants. To diminish this problem, murine monoclonals can be humanized by protein engineering. In therapy based on complete antibodies or Fab fragments,


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most foreign determinants can be eliminated by grafting the mouse VLand VH domains onto the human constant domains (Sahagan et al., 1986, Sun et al., 1987). More fundamentally, the six murine hybridoma hypervariable loops forming the antigen combining surface can be implanted onto a human VdV, framework, producing functional chimeric Fvs that retain the original antigenic specificity (Jones et al., 1986; Riechmann et al., 1988; Verhoyen et al., 1988).This fine molecular surgery is not always successful. As discussed in Section VI,C,2, the relative orientations of the CDR loops and their conformations are supported by several framework residues. Experience has shown that these side chains must be transplanted together with the loops in order to retain the full antigenic activity of the chimera. Computer-aided structural analysis of framework-CDR loop interactions helped to rationalize humanization protocols. Aspects of this analysis have been discussed in Section VI, but a few additional comments are given here. Difficulties encountered in CDR loop grafting are illustrated, e.g., by the work of Kao and Sharon (1993) on a hybrid antibody consisting of the anti-p-azophenyl arsonate framework (hybridoma 36-65) and antidextran CDR loops (hybridoma 26.4.1). Without the use of structural analysis or modeling, and with the Kabat et al. (1977) definition of CDR loops, all attempts failed to produce a functional chimera with VH and VL 36-65 frameworks and 26.4.1 CDR loops, although a partial chimera, constructed from a hybrid H chain and the native antidextran L chain, was fully active. The humanized anti-Tac antibody (with antiinterleukin-2 receptor activity) was prepared by Queen et al. (1989) with the use of human frameworks that maximized homology with the anti-Tac framework sequences. In addition, a computer model of the anti-Tac antibody, built with the ENCAD program of Levitt (1983), was used to identi5 several framework positions (H chain 27, 30, 48, 67-68, 98, and 106, L chain 47 and 59) which were likely to interact with the CDR loops or antigen. The humanized antibody retained about one-third of its affinity for the antigen, compared with the wild-type antibody. In the trial-and-error approach of Gorman et al. (199 1) a chimeric form of an anti-CD4 antibody, based on the framework of the human myeloma KOL, possessed essentially native antigen affinity,while a chimera based on the NEW antibody framework showed only a poor affinity. The most successful humanization CDR loop grafting protocols documented in the literature employed computer-built models of the chimeric Fv fragments. Typically, these studies report a judicious choice of frameworks, transfer of the critical loop-supporting residues from the wild-type antibody, and the canonical loop structure approach to model generation. In one case (Nakatani et al., 1994) the CDR loops were grafted on, and

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QUANTNCHARMm homology models were built for, as many as nine different frameworks. Each construct was assessed for biological affinity. One of the humanized anti-IL-2 (B-B10) receptor antibody variants, M5, showed nearly the same activity as the mouse wild type. Some of the best documented examples of the humanization of a group of antibodies were reported by the Genentech group (Carter et al., 1992; Kelley et al., 1992; Presta et al., 1993) who also reported crystal structures of three humanized fragments, one Fv and two Fab (Eigenbrot et al., 1993, 1994). The crystallographic structures represented different variants of the 4D5 antibody against a proto-oncogene HER2 gene product p185. The X-ray structures attested to an excellent accuracy of model building: The average rms deviation of the computer-built models from the X-ray structures was within the range of those observed among the X-ray structures themselves. The modeling protocol of the Genentech group relied on the generation of consensus coordinates based on the crystal structures of seven different Fab fragments. The consensus structure is believed to have eliminated inappropriate structural idiosyncrasies that may be present in a single structure. Derivation of the consensus framework, with use of the program INSIGHT (Molecular Simulations, Inc.), involved ( 1) independent definition of the p-sheeted segments; (2) least-squares superposition of the consensus p strands from all the structures onto the same template structure; (3) redefinition (filtering) of consensus segments based on a Ca-Ca distance criterion (i.e., only a carbons closer than 1 A to the template were retained in the template; generally, p strands passed this test, whereas many loops did not); (4) calculation of average Cartesian coordinates for all the consensus backbone atoms; (5) addition of conserved side chains to the consensus structure; and, finally, (6) addition of the modeled CDK loops to the consensus structure, based on the Chothia et al. (1989) classification of canonical loops. Often, no antibody template could be found for the H3 loop. In that case, loops of the same length were imported from nonimmunoglobulin structures and the resulting models energy-minimized in the DISCOVER (Molecular Simulations, Inc.) or CHARMm (Molecular Simulations, Inc.) program. The humanization procedure reported by Hsiao et al. (1994) likewise relied on the comparison of several immunoglobulin frameworks. The most homologous sequences were selected as structural templates and the consensus framework was generated by superposition of invariant residues at the VL-VH interface (Novotny and Haber, 1985; Novotny and Sharp, 1992). Where possible, the Chothia et al. (1989) canonical loops were utilized; alternatively, the loop selection procedure of Jones and Thirup (1986) was employed to extract approximate loop conformations from the

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Brookhaven Protein Data Bank. Then, with the use of computer graphics, side chains were compared residue by residue to identify framework positions potentially critical for the structural integrity of the combining site. For such positions, the murine residues were retained in the final model. In an attempt to develop a general Fv humanization algorithm, Studnicka et al. (1994) classified each amino acid position in the variable region according to the benefit of achieving a more humanlike antibody vs the risk of decreasing or abolishing specific binding affinity. With use of the Chothia et al. (1987,1989) definitions of CDR loop end points, knowledge of Fv solvent accessibility (Novotny et al., 1986a; Padlan, 1991), VL-VH interface conservation (Novotny and Haber, 1985, Chothia et al. 1986), and the conservedP-sheeted motifs (Lesk and Chothia, 1982), a consensus table was developed to identify, in a semiquantitative manner, low-risk positions (exposed to solvent but not contributing to binding or antibody structure), moderate- and high-risk positions (directly involved in antigen binding, CDR stabilization, or internal packing). The consensus table was tested experimentally by humanizing the anti-CD5 antibody H65 whose binding activity was greatly reduced by two previous “blind” attempts at CDR grafting. The new humanized H65 antibody, with 20 low-risk human consensus substitutions, retained the full binding avidity of the wild type. Another engineered antibody with 14 more moderate-risk substitutions had unexpectedly three- to seven-fold-enhanced avidity. The Studnicka et al. (1994) “position-risk scheme” is similar in spirit to the solvent accessibility analysis reported by Pedersen et al. (1994) (see Section VIII).

E. Heterospecijic Polyvalent Constructs, “Miniantibodies” The conceptually simplest bivalent, bispecific single-chain construct is the one where two single-chain Fv fragments of different specificities were connected by a C-terminal, disulfide bond-forming Gly4Cys or Cys5Hisj extension (Adams et al., 1993; Kipriyanov et al., 1994), or by means of designed Gly- and Ser-rich linkers (Mallender and Voss, 1994; Mallender et al., 1994; Hayden et al., 1994; Mack et al., 1995; Kurucz et al., 1995). A more complicated design of heterofunctional “miniantibodies” consisting of a single-chain Fv fragment, a flexible IgG3 hinge and an amphiphilic a-helical segment (leucine zipper), was reported by Pack and Pluckthun (1992) (Fig. 18; see also Pack et al., 1993). The dimer-forming propensity of the a helices drives spontaneous generation of a noncovalent, bifunctional, heterospecific chimera. When expressed in Escherichia coli, the bivalent fragments associated readily and were able to bind to surface-bound antigen under conditions in which bivalent but not monovalent antibody fragments bind. Packet al. (1993) reported that two single-

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chain Fv fragments with a C-terminal hinge followed by a helix-turn-helix motif formed bivalent noncovalent dimers in vivo with significantly higher avidity than those based on the leucine zipper-containing constructs. The improved avidity may have resulted from the greater stability of the fourhelix bundle formed on association of the helix-loop-helix motifs, from the antiparallel orientation of the Fv binding sites, or from both. Better still, tetravalent miniantibodies were assembled on a parallel, four-bundle a-helical scaffold (Pack et al., 1995; Fairman et al., 1996). A similar dimerization motif, i.e., the Fos and Jun leucine zipper genes fused to VH and CH1domain genes, was used to bring together Fab‘ fragments with two different specificities (Kostelny et al., 1992) in a mammalian expression system. Initially, bivalent, monospecific Fab’ fragments were expressed individually in the myeloma cell line Sp2/0 from plasmids containing genes for the hybrid H chains and those encoding the complete L chains. When these homodimers were reduced at the hinge region and their mixture reoxidized, mostly Fab’-zipper heterodimers formed and could be readily isolated. One of the exciting developments of antibody engineering has been the advent of combinatorial libraries of immunoglobulin polypeptides, and their expression on the surface of filamentous phage vectors (see, e.g., Winter and Milstein, 1991, or Marks et al., 1992, for review). One of the components of this design is use of the single-chain Fv fragment for phage surface expression. In principle, the method may supersede hybridoma technology and facilitate a mass production of antibodies with desired specificities. In practice, the recovery of rare “original” L and H chain pairs constituting a potent antibody depends on an efficient screening of very large (> lo8) combinatorial libraries. Screening of V gene repertoires becomes very efficient when single-chain combinations of VL and VH genes are cloned into a filamentous fd phage vector in its gene I11 or gene VI proteins. Modified phages display functional single-chain Fv modules on their surfaces and readily bind to antigen in a specific manner. Phage variants as rare as lo-‘ can be isolated in a single affinity chromatography step. X. T-CELLRECEPTOR MODELING AND ENGINEERING Before March 1995, when the X-ray crystallographic structure of the T-cell receptor chain was published by Bentley et al., all our structural knowledge of thie receptor had been derived indirectly, from analyses of T-cell receptor amino acid sequences, homology modeling, and sitedirected mutagenesis of T-cell receptor binding sites.


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A . Outline Structure Based on Sequence The first nucleotide sequences of T-cell receptor a and P chain genes (Chien et al., 1984; Hedrick et al., 1984; Saito et al., 1984a,b; Yanagi et al., 1984) indicated that these polypeptides corresponded in size to immunoglobulin L chains and were distantly related to immunoglobulin chains. The initial comparisons of small numbers of P chain variable domains (Patten et al., 1984) emphasized either similarities or differences between immunoglobulins and led, accordingly, to different conclusions about their functions. For example, Patten et al. (1984) hypothesized, purely on the basis of a sequence variability index, that VP segments had more hypervariable regions than the three CDR segments of immunoglobulins. The three additional nonimmunoglobulin hypervariable segments might be involved in interactions of T-cell receptors with polymorphic MHC determinants. On the other hand, Arden et al. (1985) suggested, on the basis of sequence analysis of 19 Va genes, that variable domains of T-cell receptors and immunoglobulins are similar in structure, and that it is unnecessary to postulate any special sites, apart from the classical antigen binding site, for binding properties of T-cell MHC-restricted antigen receptors. Amid these conflicting claims, Novotny et al. (1986~) analyzed sequence similarity of immunoglobulins and T-cell receptors from the point of view of structural fingerprints known to be conserved in antibody domains and in the antigen combining site (Lesk and Chothia, 1982; Novotny ef al., 1983; Novotny and Haber, 1985; Chothia et al., 1986; see Section IV). Based on these conserved sequence patterns, the T-cell receptor a, P, y, and CD8 chains were postulated to fold into immunoglobulin-like domains consisting of multistranded antiparallel P-sheet bilayers. Since the invariant side-chain motifs mediating domain-domain interactions were also found to be conserved T-cell receptor chain^,^ it appeared that the binding site of the T-cell receptor was fundamentally no different than the conventional binding site of an antibody. Thus, a/?receptors and immunoglobulins were likely to accommodate, in their respective binding sites, antigens in the same size range. If there was a single T-cell receptor binding site (as opposed to separate sites, one for the antigen and the other for the presenting MHC molecule), and if the binding sites of T-cell receptors and antibodies were fundamentally no different, what was the structural basis for the difference in antigen recognition between B and T cells? The answer to this question was likely The crystallographic structure of the CD8 extracellular domain reported by Leahy et al. (1992) confirmed that CD8 associated into a Fv-like homodimer.

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to be found in the structure of the antigen recognized, rather than in the binding sites themselves. The epitopes recognized by binding sites of antibodies were derived entirely from antigen, while those recognized by T-cell receptors may have been derived in part from the nominal antigen and in part from the restricting MHC element. Fundamentally a restatement of the “altered self’ hypothesis (Zinkernagel and Doherty 1974), the preceding conjecture accommodated the experimental evidence for complex formation between an immunogenic peptide and an MHC class I1 molecule (Babbitt et al., 1985). In the Ca and C/3 T-cell receptor domains, the segments corresponding to A-B-D-E /3 sheets showed a higher degree of conservation than their putative solvent-facing C-F-G /3-sheets, consistent with a dimeric Va/V/3 (VJVH-like) and CalCp (CLJCHl-like) domain modules. However, the interface of the C domains in the T-cell receptors resembled most the corresponding interface of the antibody CH3 domains which is relatively rich in electrically charged residues. Stable P-sheet-P-sheet contacts involving buried charges required formation of neutralizing ion pairs. The net charges of the putative Ca/C/3 domain dimers suggested favorable domaindomain electrostatic interactions, whereas some other domain-domain pairs were electrostatically less favorable (Novotny et al., 1986~). This observation further justified the search for a missing chain compatible with the Cy domain. Indeed, the 6 chain was identified by Brenner et al. (1986) and Bank et al. ( 1 986). Chothia et al. (1988) developed an outline structure of the T-cell a/3 receptor, based on a large set of amino acid sequences and assuming that the VaVp dimer has a framework structure very close to that of immunoglobulins. The loops that formed the antigen binding site were found to be similar in size to those commonly found in immunoglobulins, although perhaps with different conformations, and only limited sequence variability was found in the a I and PI hypervariable loops, suggesting that they mainly interacted with the constant parts of the MHC proteins. Claverie et al. (1989) built a complete model of the VaVp dimeric module, with use of the FRODO program and the Fab backbone as the starting point. The model was optimized by energy minimization with CHARMm and CONGEN, and was used to investigate various alternative arrangements of the receptor and the MHC molecules in putative antigenic complexes. The main conclusions were that the a and /3 chains were functionally equivalent, and that the third hypervariable loops of both chains may mainly interact with the antigen. The first and second regions were in positions favorable for making contacts with residues pointing up from the two a helices of the MHC structure. Similar suggestions about the relative MHC-peptide-receptor orientations were put forward by Davis and


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Bjorkman (1988). It was comforting to see many of the previously mentioned predictions (e.g., the immunoglobulin fold and the character of the Cp interdomain surface) to be confirmed by the X-ray structure of the p chain (Bentley et al., 1995). An interesting observation was made by Jores et al. (1990) in their studies on sequence variability in T-cell receptor /3 chains. In addition to the three hypervariable loops homologous to those of antibodies, the /3 chains possessed a distinct fourth hypervariable loop between residues 70 and 74, intermediate to the p strands D and E. In the antibody-like model of the T-cell receptor, the four hypervariable regions formed a contiguous surface area available for contacts with putative antigens. B. Engineering and Mutagenesis of T-cell Receptor Binding Site

If indeed the T-cell receptor structure is very similar to that of antibody, equivalent engineering designs, such as construction of a soluble Fv-like fragment, should be possible with the receptor. A strategy for the production of such small, soluble, single-chain T-cell receptor fragments was reported by Novotny et al. (1991). A gene encoding the RFL3.8 receptor, specific for the hapten fluorescein in the context of major histocompatibility complex class I1 and composed of the Va and Vp domains joined by a flexible peptide linker, was assembled in an E. coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated Va-Vp model showed several surface-exposed hydrophobic residues. When these were replaced by polar side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact receptor of the RFL3.8 T-cell clone (see Section IX,B for complete references on single-chain VaVp constructs). The computer-generated model of the RFL3.8 antifluorescein receptor served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues (Ganju et al., 1992). To localize the potential antigen-contacting residues in the model, advantage was taken of the fact that the crystallographic structure of an antifluorescein antibody, the murine monoclonal 4-4-20, has been solved (Herron et al., 1989). Using atomic coordinates of the 4-4-20 antibody, the most conserved parts of its Vr-VH interface were superimposed on the corresponding parts of the RFL3.8 model. On this superposition, the fluorescein molecule bound to the 4-4-20 antibody was found close to a conspicuous cavity on the surface of the RFL3.8 model. The cavity was surrounded by V a and Vp hypervariable loops and was therefore a promising candidate for the RFL3.8

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antigen binding site (see Fig. 3B). Strikingly, side chains chemically similar to those comprising the most important hapten-contacting residues of the 4-4-20 antibody (Trp H33, Tyr L37, Ser L94, Arg L39) were found at the surface of the putative RFL3.8 fluorescein-contacting cavity (Tyr-31, Tyr166, Ser-227, Arg-94) despite the fact that the amino acid sequences of the 4-4-20 and RFL3.8 hypervariable loops showed little similarity to each other. This chemical similarity extended to the layer of aromatic residues directly under the bottom of the binding site cavity. All these observations suggested that similar chemical motifs may be used by antibodies and T-cell receptors to engage the same antigen. Altogether, six potential amino acid contacts with the antigen were selected for alanine-scanning mutagenesis. The mutated single-chain T-cell receptors were expressed in E. coli, purified, refolded, and assayed for fluorescein binding (Ganju et al., 1992). Five out of six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues were distributed among the p3, al and a:! CDR loops. Given that fluorescein is one of the smallest T-cell antigens available, it seems reasonable to expect that the majority of receptors use multiple CDR loops to contact antigen in combination with the MHC proteins.

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CATALYTIC ANTIBODIES By EDWARD M. DRIGGERS and PETER G. SCHULTZ Howard Hughes Medical Institute Department of Chemistry University of California Berkeley, California 94720

I. Introduction. . 11.

..............

111. IV. V. VI . VII. VIII. Conclusion . . . References . . .

............

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26 1 262 266 270 273 275 280 283 284

I. INTRODUCTION

The humoral immune system is perhaps the best single example in which large, diverse libraries of molecules are generated and screened for a specific biological function. One of the first examples in which the chemical potential of large libraries of this sort was exploited was the use of transition state theory to select catalysts from among the large population of antibody molecules (1). Since these early experiments, which involved relatively simple transformations, the reactions catalyzed by antibodies have increased in complexity and degree of difficulty (2). At the same time strategies for generating antibody catalysts have become increasingly sophisticated. Structural and mechanistic studies show that the chemical notions used to generate catalytic antibodies are indeed reflected in their active site structures (3-5). Today, there is interest in screening libraries not only of antibodies but also of Fab fragments (6), RNAs (7), peptides (S), synthetic organic molecules (9),and even solid state materials (lo), for interesting new properties and functions. This article surveys advances in the field and lessons that have been learned from the study of antibody catalysis.

AD5’ANCE.S IN PROTEIN CHEMISTRI’, Vld. 49

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Copyright 0 1996 by Academic Press, Inc.

MI rights of reproduction in any form reserved.

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11. IMMUNOLOGICAL EVOLUTION OF CATALYSIS In contrast to the situation for enzymes, the binding affinity and selectivity of antibodies evolve rapidly, through a process involving genetic recombination of variable (V),joining and diversity (D) genes followed by affinity maturation events. The net result is that tremendous molecular diversity can be screened in a period of weeks for a desired specificity ( 1 1 ) . Although immunological and natural selection both afford selective, highaffinity binding sites, enzymes generally evolve to maximize catalytic efficiency and are therefore selected based on affinity for high-energy transition states (12),whereas antibodies evolve to maximize affinity for molecules in their ground state. Consequently, in order to tap the catalytic potential of antibodies, the immune system must be provided with information relating to the rate-limiting transition state of a particular reaction. One strategy employed successfully to generate catalytic antibodies is the use of a stable analog of the transition state (TST) as the immunogen (1). This effectively directs immunological evolution along the same pathway as enzymatic evolution; the end result is an antibody with catalytic activity. To what degree can one reproduce the natural evolution of enzymes using these ideas? Consider, for example, the enzyme ferrochelatase, which catalyzes the insertion of Fez+ into protoporphyrin as part of the heme biosynthetic pathway (13). A potent inhibitor of this enzyme is the bent porphyrin N-methylprotoporphyrin (14),which is thought to resemble the transition state for porphyrin metallation in which distortion of the macrocyclic ring system forces the nitrogen lone pairs into a geometry better able to bind the incoming metal ion. If this hypothesis is indeed correct, then antibodies generated against a bent N-alkylmesoporphyrin should act as a ferrochelatase. In order to test this notion, monoclonal antibodies were generated against the mesoporphyrin derivative 1 (Fig. 1) (15). In the case of antibody G1-A12, catalysis was observed with a variety of transition metals including Zn(II), Co(II), Cu(II), and Mn(I1). The catalytic properties of this antibody are similar to those of the enzyme ferrochelatase. The reported Michaelis constant, K,, values for the enzyme ferrochelatase range from 10 to 70 p M , compared to 50 p M determined for the antibody (Cu"), and both enzyme and antibody have comparable affinities for the N-methylporphyrin (13-15). The enzyme and antibody insert a variety of divalent transition metals into porphyrins, with similar turnover numbers (L). A calculated value of k,,, for ferrochelatase with Zn(I1) is 800 hr-', and the experimentally determined value of k,,, for the antibody is 80 hr-' (15). Clearly in this case, immunological evolution against a transition state analog produced a catalytic antibody with properties quite close to those of

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CATALYTIC ANTIBODIES

263

1

FIG.1. Antibody-catalyzed porphyrin metallation reaction.

the corresponding enzyme (13-1 5). Moreover, the similarity between the antibody and the enzyme strongly support the hypothesis that the enzyme has evolved to bind a distorted heme structure. Among the most ubiquitous and well-characterized enzyme-catalyzed reactions are the acyl transfer reactions. A number of novel mechanisms have evolved for these enzyme-catalyzed reactions, including the formation of acyl-enzyme intermediates, direct acyl transfer between two substrates, and metal-dependent acyl transfer (16).These reactions have been an attractive target for antibody catalysis and many examples have been documented in the literature, including stereo- and regioselective ester hydrolysis (17) and amide bond hydrolysis (18). One can again ask, To what degree can immunological evolution recapitulate the properties of enzymes for this important class of biochemical reactions? Consider, for example, the antibody-catalyzed transesterification reactions illustrated in Fig. 2 (structures 2-9). Antibodies raised against phosphonates 2 or 3 were expected to bind the corresponding substrates in a reactive orientation and thereby catalyze the bimolecular reaction by acting as “entropy traps” (19,20). In addition, the dipole of the P=O bond reflects the developing negative charge on the carbonyl oxygen in the transition state. The fact that the antibodies are complementary to tetrahedral phosphonate esters should be reflected in lower affinities for the trigonal reaction products.

264

EDWARD M. DRIGGERS AND PEI'ER G. SCHULTZ F)

NCCHzO+

n

YJo

0

6

AO-Ph O

H 5

I

+

FIG.2 . Antibody-catalyzed transacylation reactions.

Antibodies generated to each hapten were found to be remarkably efficient catalysts for the corresponding transesterification reactions. The effective molarity for the antibody-catalyzed transesterification of thymidine 4 with the alanyl ester 5 is approximately 5 x lo4M (20). The transesterification reaction of the vinyl ester 7 with racemic 2-phenethyl alcohol 8 is estimated to have an effective molarity on the order of 105-106 M (19). These values, which represent very large rate enhancements for these bimolecular reactions, underscore the importance of entropic factors in biological catalysis. The efficiency and selectivity of these reactions are reflected by the fact that neither antibody catalyzes acyl transfer to water to


265

any appreciable extent, even though water is present in both cases at a concentration approximately 1O5 times that of the more hindered secondary alcohol. This ability to exclude water from participating in reactions is characteristic of many enzyme-catalyzed reactions. The two antibodies were found to function via different mechanisms. In the case of thymidine aminoacylation (20), the differential binding affinity of the antibody to the phosphonate diester relative to substrates appears to account for a large fraction of the catalytic advantage. This result is consistent with the classic notion of transition state complementarity in enzymatic catalysis put forth by Haldane and Pauling (12). In contrast, the second transesterification reaction was found to proceed through a PingPong mechanism involving a covalent acyl-antibody intermediate ( 19). Moreover, formation of the acyl intermediate appears to depend on an induced fit phenomenon. The preceding results suggest that these two catalytic antibodies can in fact be viewed as primitive enzymes following two distinct evolutionary pathways. The ideas used to generate these catalytic antibodies have recently been extended to the generation of antibodies that catalyze peptide bond formation (21,22). One such antibody has been shown to catalyze this bimolecular reaction with an effective molarity of roughly 1000 M , without catalyzing the hydrolysis of the peptide product or racemization of the ester substrate (Fig. 3) (22). These results raise the intriguing possibility of using antibodies as tools for polypeptide synthesis.

u 0. -0

FIG.3. Antibody-catalyzed peptide bond formation.

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EDWARD M. DKICGERS AND PETER G. SCHULTZ

111. STRUCTURAL STUDIES

Structural studies support the notion that catalytic antibody active sites evolve in response to the mechanistic information contained in the structure of the immunogen (3-5). Consider, for example, the antibody 17E8 which was raised to phosphonate 10 (Fig. 4,structures 10-12) (4).This antibody catalyzes hydrolysis of the corresponding formyl norleucine phenyl ester 11 with a k,,, of 223 min-' and a rate acceleration lIcaJlcuncatof 2.2 x lo4. The X-ray crystal structures of the antibody-transition state analog complex and the Michaelis complex (in which an amide analog of the substrate is bound) (4,23) reveal a recognition pocket in the active site for specific binding of the side chain of the amino acid substrate as well as a light chain lysine residue (Lys-97L)which likely functions to stabilize the negatively charged transition state, much like the oxyanion hole found in serine proteases. A comparison of the structures of the Michaelis complex and the transition state analog complex clearly shows the increased binding interactions that occur as the reaction progresses toward the transition state configuration. The antibody active site has a Ser-His catalytic dyad proximal to the phosphorus of the bound hapten, resembling the Ser-HisAsp catalytic triad of the serine proteases. The positions of the antibody and enzyme side chains relative to the acyl group undergoing nucleophilic addition are virtually superimposable (Fig. 5), suggesting that the antibody resembles in many respects a primitive serine protease. Steady-state

FIG.4. Antibody-catalyzed ester hydrolysis.


267

FIG. 5 . The active site of antibody 17E8 superimposed on the active site of trypsin complexed with BPT14.

hydroxylamine partitioning experiments lend support to a catalytic mechanism involving rate-limiting formation of a covalent acyl-antibody intermediate. By introducing additional catalytic groups, such as an aspartate or glutamate in close proximity to the histidine, one might be able to further enhance the activity of this catalytic antibody. For example, it has been shown previously that mutagenesis of T ~ r - 3 to 3 ~histidine in the phosphorylcholine binding antibody S 107, which catalyzes the hydrolysis of choline esters, results in a 10-fold increase in the esterolytic activity of this antibody (24,25). Another comparison between enzyme- and antibody-catalyzed reactions can be made in the case of the conversion of chorismic acid 13 to prephenic acid 14 which is catalyzed by the enzyme chorismate mutase. This 3,3-sigmatropic rearrangement, which is a key step in the biosynthesis of

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EDWARD M. DKIGGERS AND PEI'EK G . SCHULTZ

aromatic amino acids in plants and bacteria, is known to proceed through an asymmetric chairlike transition state (26, 27). One might expect that an antibody combining site complementary to the conformationally restricted transition state would accelerate this rearrangement, again by acting as an entropy trap. Antibodies 1F7 and 2E11, elicited to derivatives of a bicyclic transition state inhibitor of chorismate mutase, have been shown to increase the rate of the rearrangement 10'-fold (hapten 15) (28) and lo4fold (hapten 16) (29), respectively, over that of the uncatalyzed reaction (Fig. 6, structures 13-16), compared to the approximately 106-foldacceleration (k,,Jk,,,,,) of the enzyme chorismate mutase. Both antibodies, like the enzyme, display high specificity for the (-)-isomer of chorismate. Antibody 2E11 (29) functions primarily by increasing the entropy of activation for the reaction (AS++= -1.2 eu, AH++ = 18.3 kcal/mol), antibody 1F7 (28) primarily decreases the enthalpy of activation (AS++= -22 eu, AH++= 15 kcal/mol), and the enzyme affects both (AS++= 0.0 eu, AH++= 15.9 kcal/mol) (27). The X-ray crystal structures of the enzyme and antibody 1F7 have been solved (3). Structural data suggest that the enzyme and the antibody stabilize the same conformationally restricted chairlike transition state (Fig. 7). The overall shape and charge complementarity between the combining sites and the transition state analog result in binding of the correct enantiomer of chorismate in the conformation required for reaction. Differences in the number and nature of specific interactions available for restricting conformational entropy and stabilizing the highly polarized transition state may account for the observed lo4 times lower activity of the antibody 1F7 relative to that of the natural enzymes.

OH

OH 13

14

FIG.6. Antibody-catalyzed Claisen rearrangement of chorismate to prephenate.

269


\

0 ' 'U

A

0'

h H 3 3

Cys75J

FIG.7 . Schematic diagram comparing the hydrogen bonding and electrostatic interactions of the transition state analog with relevant side chains of 1F7 (A) and Bacillus subtilis chorismatr mutase (B). Dashed lines indicate hydrogen bonds. (3)

The X-ray structures just described show that the properties of catalytic antibodies do reflect the mechanistic information contained in the hapten structure, and other mechanistic studies also bear this out. For example, it has been shown that by raising antibodies to positively charged hapten 19, one can generate an antibody (43D4-30D3) with a complementary negatively charged residue in the combining site (Fig. 8, structures 17-22) (30). This residue, which has been identified as Glu-43" by affinity labeling and mapping techniques, is positioned to abstract the proton a to the carbonyl group in substrate 17, leading to p-elimination of HF with a rate acceleration 105-foldover the second order rate constant for the acetate-catalyzed reaction (31). Hilvert and co-workers have exploited charge complementarity to generate very efficient catalytic antibodies that also operate via general base catalysis (32).An antibody specific for hapten 22 was found to catalyze the decomposition of substituted benzisoxazole 20 to give 2cyanophenol 21 with greater than 108-foldrate accelerations. In addition to demonstrating the large catalytic advantages that can be realized with antibodies, this experiment also began to define the degree to which optimally positioned general bases and acids can contribute to enzymatic catalysis. Although in each example described here the catalytic antibody evolved in response to a synthetic immunogen, some reports suggest that the immune system can produce catalytic antibodies in the absence of such immunogens (33,34). For example, autoimmune antibodies isolated from patients with systemic lupus erythematosus were found to hydrolyze DNA with a /@' value of 14 min-' and a K, value of 43 nM (33). These kinetic constants, which were obtained using highly purified Fab fragments, are remarkably close to those of restriction enzymes such as EcoRI. Unraveling the mechanism by which these antibodies are generated during the im-

270

EDWARD M. DRIGGERS AND P E E R G. SCHULTZ

Jyp_,,-C^"a 17

+

HF

18

FIG.8. Antibody-catalyzed elimination reactions.

mune response could prove extremely useful in the development of other effective antibody catalysts for biological and biomedical applications.

Iv. EVOLVING FUNCTIONS N O T Y E 1 FOUND IN NATURE Because the immune system responds rapidly to a given set of chemical instructions, one can attempt to evolve antibodies that catalyze reactions for which enzymes have yet to be found. Concerted pericyclic reactions, which include cycloaddition reactions, sigmatropic rearrangements, and electrocyclic ring closure reactions, represent one such opportunity. These reactions have not only received a greal deal of theoretical and mechanistic attention from chemists, but have also found many applications in organic synthesis (35).There are few examples of pericyclic reactions catalyzed by enzymes (27). Consequently, pericyclic reactions represent an ideal target for testing the degree to which catalytic antibodies can be generated for reactions that are rare or do not occur in nature.

27 1


The first such reaction to be considered was the Diels-Alder reaction, which in its simplest form consists of the reaction between butadiene and ethylene to yield cyclohexene (36). The transition state involves a highly ordered cyclic array of interacting orbitals in which carbon-carbon bonds are broken and formed in a single concerted step (Fig. 9). As a result of the stringent alignment of orbitals in the bimolecular transition state, an unfavorable entropy of activation (AS$) on the order of -30 to 4 0 eu is generally observed. The design of a hapten for generating catalytic antibodies for this reaction must address two issues: (1) an entropy sink must be provided such that the two substrate molecules are oriented in a reactive conformation on binding, and (2) a mechanism must be included for avoiding product inhibition because the product of a condensation reaction might be expected to bind more strongly than either substrate. Hilvert and co-workers were successful in designing a bicyclo[2.2.1]hapten, 23, that satisfies both criteria (37). The Diels-Alder reaction they chose to investigate was the addition of tetrachlorothiophene dioxide 24 to N-ethylmaleimide 25 which gives rise to an initial bicyclic Diels-Alder adduct (Fig. 10, structures 23-30). This adduct spontaneously extrudes sulfur dioxide, resulting in the dihydrophthalimide product 26 which has significantly less structural similarity to the hapten than the initial DielsAlder adduct, thereby minimizing product inhibition. An antibody raised to hapten 23 that resembles the transition state for the addition reaction was an efficient catalyst with an effective molarity of 100 M . At the same time, a second, more general strategy for catalyzing the Diels-Alder reaction was developed (38). In this case the reaction involved cycloaddition of the acyclic diene 27 to N-phenylmaleimide 28 to yield the cyclohexene product 29 (Fig. 10). Hapten 30 is based on a bicyclo[2.2.2]octene skeleton in which the ethano bridge locks the cyclohexene ring into a conformation resembling the Diels-Alder transition state. Because the

R

7 Diene

+

'R'

R' Dienophile

Product Transition state FIG.9. Diels-Alder reaction of a diene and a dienophile to yield a cyclohexene product.

272

EDWARD M. DRIGGERS AND PETEK G. SCHULTZ

24

P

0

FIG 10. Antibody-catalyzed Diels-Alder reactions.

reaction product does not contain this hydrophobic bridge and has a conformation distinct from that of the hapten, it was expected to bind less tightly in the antibody combining site. In fact, an antibody generated to hapten 30 catalyzed the formation of the Diels-Alder adduct with a k,,K,, value of 900 M-' sec-' (diene); product was a bound with KO of 10 p M , which can be compared to a K O of 126 nM for the hapten. Another well-studied pericyclic reaction for which enzymatic catalysis has yet to be demonstrated is the Cope rearrangement (39). In order to generate catalysts for this reaction, antibodies were raised against the diphenyl-substituted cyclohexane derivative 3 1. This hapten was expected to mimic the six-membered ring transition state for this concerted rear-


273

rangement (Fig. 11, structures 31-33) (40). Four antibodies were isolated that catalyze the oxy-Cope rearrangement of diene 32 to 33, one with a kcaJkuncatvalue of 5300. NMR analysis of this antibody by transferred NOE studies in accord with the hapten design indicates that it does in fact constrain the substrate in a cyclic conformation (unpublished results). These studies on the Diels-Alder and oxy-Cope reactions underscore the ability of the immune system, when guided by carefully defined mechanistic criteria, to evolve catalysts for a wide range of chemical transformations, some of which have heretofore been unknown in nature.

v.

UNNATURAL COFACTORS

Many enzymatic reactions depend on a set of small nonpeptidyl cofactors such as hemes, flavin, pyridoxal, and nicotinamide. Chemists have developed their own set of chemical auxillaries including metal hydrides, transition metals, and Lewis acids, which perform similar functions. The large number and utility of the cofactors available to the chemist raise the question whether antibodies can be used to extend biological catalysis to reactions not normally considered within the purview of nature. Specifically, can antibodies be developed that use chemical “cofactors” in addition to the limited set of enzymatic cofactors (41)? One such example is the antibody-catalyzed oxygenation reaction of a sulfide to the corresponding sulfoxide (42). The monooxygenase enzymes responsible for this transformation use flavin or heme cofactors that typically require NADPH for cofactor regeneration (43). Hsieh and co-workers asked whether the inexpensive oxidant, NaI04, could be used to carry out this reaction. This eliminates the need for cofactor recycling, a significant

0

32

33

FIG.11. Antibody-catalyzed oxy-Cope rearrangement.

274

EDWARD M. DRIGGEKS AND PETER G. SCHULTZ

barrier to the use of many enzymes in synthesis (Fig. 12, structures 34-36). Hammett u-p studies, solvent isotope effects, and pH-dependent studies on sulfide oxidation by periodate (44) suggest that the transition state for this reaction resembles that depicted in Fig. 12. Consequently, aminophosphonic acid 34 was designed to generate antibodies that catalyze the conversion of sulfide 35 to sulfoxide 36. Because hapten 34 contains an amine that is protonated at physiological pH, antibodies specific for 34 were expected to stabilize the incipient positive charge on sulfur present in the transition state. A phosphonic acid moiety was introduced into the hapten to provide a binding site for the periodate ion. Antibody 28B4.2, which catalyzes the NaI04-dependent oxidation of sulfide 35 with a k,,, of 8.2 sec-l, was isolated, and no inactivation due to antibody oxidation was observed. The turnover number and catalytic efficiency of this antibody are comparable to those of the corresponding enzymes (43). A second example of antibody catalysts that use unnatural cofactors is the antibody-catalyzed reduction of ketones to the corresponding alcohols (45,46). The enzymatic reduction of carbonyl groups to alcohols usually requires the cofactor NADH or NADPH. Chemical reductions, on the other hand, are usually carried out with inexpensive and versatile metal hydrides, such as NaBH4 or LAlH4. In an effort to generate antibodies that catalyze the metal hydride dependent reduction of ketone 37,antibodies were generated against N-oxide 39 (Fig. 13).Antibodies raised against this hapten were expected to stabilize the tetrahedral transition state arising from nucleophilic attack of hydride on the carbonyl group, as well as to provide a site to accommodate the reductant. The chiral active site of an antibody

L

J

36

FIL. 12. Antibody-catalyzed, periodate-dependent oxygenation reaction.

275


NaCNBH,, Ig

QN

QN

37

38

FIG. 13. Antibody-catalyzed, borohydride-dependent ketone reduction. Ig, Immunoglobulin.

specific for one of the two enantiomers of hapten 39 should also discriminate between the enantiotopic faces of a prochiral substrate, affording high stereoselectivity.Antibody 378.39.3 was found to catalyze the reduction of substrate 37 to the corresponding alcohol 38 with a k,,JK, value of 1.9 x 103M-' min-' (R=C2H5)(46). As expected, the antibody stabilized one of two possible enantiomeric transition states to give the S-alcohol in 96%enantiomeric excess. The reduction of ketones containing branched and aryl substituents, including the highly symmetric l-nitrophenyl-3phenyl-2-propanone, also showed high enantioselectivity. This straightforward strategy may find general applicability in the regio- and stereoselective reduction of a broad range of compounds and may be useful for reactions not amenable to existing biological or chemical approaches. Other examples involving antibodies that use novel cofactors include the peroxycarboximidic acid dependent oxidation of unfunctionalized alkenes to yield epoxides (47). The enantioselectivity for this reaction was greater than 98%, and exceeds that which can be achieved with the hemedependent enzyme chloroperoxidase. VI. DIFFICULT CHEMICAL TRANSFORMATIONS The field of catalytic antibodies has begun to focus on catalytic transformations that are difficult to carry out using existing chemical methods. These include reactions that have been termed disfavored, i.e., kinetically controlled reactions in which the products arise not from the lowest energy transition state (favored) but from a higher energy transition state (disfavored). In practice it has proven difficult to discriminate chemically and to control the relative energies of these two transition states. Consider, for

276

EDWARD M . DRIGGERS AND PETEK G. SCHULFZ

example, the Diels-Alder reaction discussed earlier. In the case of a monosubstituted diene and dieneophile, eight products are possible. Four products correspond to the regio- and stereoisomers resulting from endo attack and four arise from exo attack (Fig. 14). For reactions under kinetic control the endo pathway is typically favored over the exo pathway as a result of secondary orbital interactions in the transition state. Gouverneur and co-workers asked whether the binding energy of antibodies could be programmed to selectively catalyze formation of the disfavored exo pathway (48). The reaction chosen for study was the cycloaddition between trans-lN-acylamino-1,3-butadiene 40 and N,N-dimethylacrylamide 41 (Fig. 15, structures 40-45). In the absence of a catalyst the reaction proceeds under aqueous conditions to give an 85: 15 mixture of the endo 42 to exo adduct 43. A bicyclo[2.2.2]octeneframework was again used to mimic the boatlike transition state for the pericyclic reaction. Hapten 44 mimics the endo approach in the transition state, because the amide group of the dienophile is oriented toward the n orbitals of the diene. Conversely, in the exo transition state, the dienophile substituent is oriented away from the n system. This geometry is mimicked by hapten 45, which should generate

endo approach

Re face

Ri

< + ' ! R2

Ri

em approach

Si face

ex0

approach Re face L

FIG. 14. Geometric features of the four transition states that control the endo/exo enantioselectivity of the Diels-Alder transformation.

277


’CONMe2 0

Y

NH

b

R

b

40

42

43

0

R

A

&

N&CONhle, H

CONMe2

NH

oAR 44

0 J--A 45

FIG.15. Antibody catalysis of an exo Diels-Alder reaction.

antibodies whose binding sites stabilize the exo transition state and catalyze formation of the corresponding trans adduct, 43. Antibody 7D4 (specific for hapten 44) and antibody 22C8 (specific for hapten 45) catalyze exclusive formation of the cis (endo) and trans (exo) adducts, respectively (48). The effective molarities for the antibody-catalyzed reactions were 4.8 M (7D4) and 18 M (2268). In each case the degree of enantioselectivity was greater than 98%. Another disfavored reaction that has been successfully catalyzed by antibodies is the 6-endo-tet cyclization of the epoxy alcohol illustrated in Fig. 16 (structures 46-49) (49). This antibody-catalyzed reaction is formally a violation of Baldwin’s ‘‘rules’’for ring closure reactions (50), which state that the preferred product arising from the 180”transition state geometry of an intramolecular nucleophilic substitution reaction is the 5-exo-tet product. In order to catalyze 6-endo-tet cyclization, it was necessary to generate an antibody that not only lowers the energy barrier for epoxide ring opening but also overcomes the entropic barrier and strain necessary to bring the hydroxyl group into a geometry that favors a six-membered (disfavored) vs a five-membered (favored) ring transition state geometry. It was anticipated that hapten 46 would generate a combining site that would stabilize both the developing charge in the breaking C-0 bond and the six-membered ring geometry (49). The difference in dipole between the hapten and the reaction product was expected to minimize product inhibition. Two antibodies generated against N-oxide 46 were found to catalyze the regioselective ring opening of epoxide 47 to form the six-membered ring

278

EDWARD M. DRIGGERS AND PETER G . SCHULTZ

~

FK. 16. Antibody catalysis of an anti-Baldwin cyclization reaction.

product 49. Comparison of the Ltvalue with that of the uncatalyzed reaction was not possible, because only the five-membered Baldwin ring closure product was formed in the absence of antibody. In addition, only the S,Sepoxide was a substrate for antibody-catalyzedpyran ring formation. Thus, the antibody controls both the regio- and the stereochemistry of this reaction. Another challenge in chemistry is the rational design of catalysts capable of complete regio- and stereoselective control over reaction products. For example, it would be difficult using known chemical methods to selectively reduce diketone 50 to afford only one of eight possible reaction products because all eight products arise from transition states that are expected to be similar in energy (Fig. 17, structures 50-53). However, it was shown that one antibody (37B39.3) generated against N-oxide 39 (see

OH H 02

E

'

H

3

NaCNBH,

- - .

52

50

OH

, 02Nd

/

I =H

3

53

FIG. 17. Regio- and stereoselective, antibody-catalyzed diketone reduction.

279


later discussion) catalyzed the reduction regioselectively with greater than 75:1 selectivity for one of the two nearly equivalent ketone moieties (46). Moreover, the reaction was highly stereoselective, affording the S enantiomer of hydroxy ketone 51 in 96% enantiomeric excess. In contrast, the nitrobenzyl carbonyl group was reduced more slowly than the methoxybenzyl carbonyl group in the uncatalyzed reaction (V,.el= 0.74).The overall yield of hydroxy ketone (S)-51 was 94%, which is significant in light of the fact that the background reaction produces all eight products. Another example in which antibodies were able to selectively stabilize one of a number of nearly equivalent transition states is a diasteroselective esterolytic reaction (51). Antibodies were generated against each of four diastereomeric phosphonate analogs of the transition states for the hydrolysis of the corresponding l-(benzyloxy)-2-fluoro-2-methyl-3-hydroxybutane esters 54. Each of the four esters was hydrolyzed in 2 97% enantiomeric excess and in greater than 23% overall conversion (theoretical yield, 25%) with the corresponding antibody (Fig. 18,structures 54-56). Given that at present no general chemical methods exist for generating catalysts of this sort, such antibodies might find applications in the chiral resolution of synthetic intermediates containing either acid or alcohol functionality. Regioselective esterolytic antibodies have also been reported that are able to selectively deprotect an acylated carbohydrate (52). Many other examples are appearing in which antibodies provide the chemist with a high degree of control over the outcome of chemical reactions. Antibodies can effectively exclude solvent from participating in reactions. Keinan and co-workers showed that an antibody generated against the quaternary ammonium ion hapten 57 was able to catalyze the cyclization of hydroxyethyl enol ether 58 to the corresponding ketal59 in water with high enantiomeric purity (Fig. 19,structures 57-60) (53).Ordinarily,

54

55

-

H 0 2 C m P < o + Me

I

NHAc

56

FIG.18. Diastereoselective antibody-catalyzedester hydrolysis reaction.

280

EDWARD M . DRIGGERS AND PETER G. SCHULTZ

50

Ar

,

59

FIG. 19. Exclusion ofwater from an energetic intermediate in an antibody active site

ketal formation does not occur under aqueous conditions; the oxocarbonium ion intermediate is rapidly trapped by water to yield a hemiketal and ultimately ketone 60. Antibodies have also been reported that control the syn/unti ratio of the oxime product that results from condensation of hydroxylamine with a ketone (54) as well as the formation of 3-hydroxyoxepane rings from simple hydroxy epoxides (55). Finally, antibodies generated against a quaternary ammonium hapten have been shown to catalyze the stereoselective hydrolysis of an enol ether under aqueous conditions to give the corresponding aldehyde with greater than 98% enantioselectivity (56).

VII. FUTURE DIRECTIONS

Antibody catalysis will undoubtedly be extended to many other interesting classes of reactions. For example, antibodies can catalyze a cationic cyclization reaction (57). Antibody 4C6, which was generated against hapten 61, was found to catalyze the cationic rearrangement of 62 to cyclohexene 63 (2%) and trans-2-dimethylphenylsilylcyclohexanol 64 (98%) (Fig. 20, structures 61-64). Such a narrow distribution of products is surprising because cationic cyclization reactions conducted under solvolysis condi-

28 1


0

I

63

64

62

-0

bNU H

OH

61

F I G20. Antibody-catalyzed cationic cyclization reaction.

tions usually yield a plethora of products. The almost singular production of the cyclized product, 64, was attributed to the ability of an antibody to both enforce a pseudocyclic transition state geometry and trigger the reaction under conditions so mild that there is no detectable background reaction. These studies will undoubtedly lead to efforts aimed at larger multiring cyclization reactions. We will also continue to see the development of new methods for generating catalytic antibodies. One promising approach involves the use of mechanism-based screens in which covalent modification by a hapten is used to identify antibodies from a phage display library with an appropriately positioned nucleophile group in the active site. For example, a semisynthetic combinatorial antibody library was successfully screened for antibodies containing a cysteine residue in the complementarity determining regions (58). Libraries were panned with a-phenethylpyridyl disulfide 65 attached to a solid support. This reagent undergoes efficient disulfide exchange with an appropriately placed thiol in an antibody combining site (Fig. 21, structures 65, 66). Out of 10 randomly picked clones, two antibodies contained an unpaired cysteine in the combining site. One of these was found to accelerate the hydrolysis of thioester 66, in which the electro-

282

EDWARD M. DRIGGERS AND PETER G. SCHULTZ

FIG 2 1. Mechanism-based selection for active site nucleophiles.

philic carbonyl occupied the same position as the reactive sulfur atom in 65 during selection. As expected, the reaction involves formation of an acylantibody intermediate. This result suggests that iterative mechanismbased selection procedures may prove quite useful in screening for antibodies with specific functional groups in the combining site. In addition to strategies involving synthetic immunogens, more biologically oriented approaches are being developed for generating catalytic antibodies. For example, the hydrolytic antibody 48G7 (see earlier discussion) has been efficiently expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity (59). Conditions were found that permit the secretion of active recombinant antibody into the periplasm of an E. coli strain deficient in biotin biosynthetic genes (Abio-gal).A number of substrates were synthesized that, upon hydrolysis by the antibody, yielded free biotin, a required nutrient for cell growth. These substrates and selections are being used to identifji mutants of the antibody with altered activities. This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds. Similar approaches are being developed for selecting antibodies with enhanced activity that catalyze biosynthetic reactions, such as the conversion of chorismic acid to prephenic acid (60), and the decarboxylation of orotate (61).For both reactions, antibodies have been found that complement auxotrophs lacking the corresponding biosynthetic enzymes. In the latter case, this complementation was used as the basis for screening a library of antibodies generated from a mouse immunized with a synthetic immunogen. Other interesting strategies have also appeared for generating catalytic antibodies, including the development of rapid plate assays for screening large numbers of antibodies directly for catalysis (62,63). For example, Green and co-workers used an antibody specific for the product of a reaction to screen hybridoma supernatants for catalysts (63).A second strategy involves the generation of transgenic mice that produce antibodies with a high percentage of metal ion-binding light chains (64). This approach

CATALYI'IC ANTIBODIES

283

may facilitate the generation of catalytic antibodies that utilize metal ions and other cofactors. Catalytic antibodies with cholinesterase activity have also been generated by immunizing mice with a monoclonal antibody directed against the active site of acetylcholinesterase (65). The catalytic efficiencyof this antibody (kLaJkuncar= 4 x lo8)is quite high, suggesting that this approach may prove useful for generating structural and functional variants of enzymes. It is likely that antibody catalysis will soon be applied to practical problems in chemistry and medicine. Already there have been reports in the literature of antibody-catalyzed cocaine hydrolysis (66) and antibodycatalyzed prodrug activation (67,68). In the latter case, the use of a catalytic antibody makes prodrug activation possible by reactions not catalyzed by endogenous enzymes. In addition, efforts are focusing on methods for efficiently generating polyclonal catalytic antibodies in the hope that someday researchers may be able to actively immunize and produce therapeutic catalytic antibodies (69). A demonstration that enantioselective conversions can be carried out by antibodies on a multigram scale (70), as well as efforts aimed at extending antibody catalysis to organic solvents (71), should facilitate the use of antibodies in organic chemistry. A report describes the use of a catalytic antibody to carry out the key synthetic step, the enantioselective protonolysis of an enol ether, in the total synthesis of (-)-a-multistriatin, the aggregation pheromone of the European elm bark beetle (72). This reaction, which was carried out in greater than 99% enantiomeric excess and 98% chemical yield, was followed by 10 chemical steps with all four asymmetric centers originating from the chirality achieved using the catalytic antibody. Finally, many of the ideas that form the basis for the work described in this article are being applied elsewhere (6-1 0). For example, the planar phenanthrene transition state analog 67 has been used to screen nucleic acid libraries for RNAs that catalyze isomerization of the substituted biphenyl 68 (Fig. 22, structures 67-69) (73). In addition, mechanism-based selections have been used to identify RNAs that undergo efficient selfalkylation reactions (74).Such experiments are extending RNA catalysis from phosphoryl transfer reactions to new classes of chemical transformations.

VIII. CONCLUSION Although tremendous progress has been made in the field of catalytic antibodies, major challenges still exist. Methods have to be developed to further improve the catalytic efficiencies of antibodies. Many classes of reactions also remain to be surveyed, and effective haptens must be de-

284

EDWARD M. DRIGGERS AND PETER G. S C H U L T Z

FIG.22. RNA-catalyzed isomerization reaction.

signed to catalyze such reactions. Improved methods for expressing and screening antibodies need to be developed, and many practical problems associated with the use of antibodies in bioreactors must be solved. However, the progress made in the field in recent years suggests that these challenges will be met. Moreover, the success of chemistry in exploiting the molecular diversity of the immune system to generate selective catalysts points to the tremendous potential of combinatorial libraries. Indeed the lessons learned from the catalytic antibodies have recently found their way to the physics community with the report that libraries encompassing virtually the entire periodic table can now be screened for interesting new physical properties such as superconductivity and magnetism (10).

ACKNOWLEDGMENTS We are grateful for financial support for this work from the National Institutes of Health, the Ofice of Naval Research, and the US. Department of Energy under Contract No. DEAC03-76SF00098. PGS is a Howard Hughes Medical Institute Investigator.

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THE NATURE OF THE ANTIGEN By MICHAEL SELA and ISRAEL PECHT Department of Immunology Weizmann lnstltute of Science Rehovot 76100, Israel

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A. Definitions

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Antigen-binding molecules include antibodies and T-cell receptors (TCR), as well as class I and class I1 antigens encoded by the major histocompatibility complex (MHC). This article will define and illustrate the notion of antigens. Macromolecules, such as enzymes and hormones, have well-defined biological activities and are uniquely specific. The same is largely true for the previously mentioned biological macromolecules with the exception of antigens. Antigens may be small or large, may have other defined biological activities, or may be confined to their ability to react with antigen-binding molecules. Thus, defining and surveying antigens is difficult, as it could cover almost all facets of immunology. We will provide a concise message, and will refer primarily to reviews, key articles, and recently published papers, and we will use certain results as examples. We also had to make some arbitrary decisions: we will briefly describe tumor ADVANCES IN PROTEIN CHEMISTRY, Vol. 49

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antigens but will not discuss antigens used for vaccination against infectious diseases or those involved in autoimmune disease and allergy. We will address several more quantitative aspects of the interactions between antibodies and their respective epitopes. The remarkable recent increase in our knowledge of the three-dimensional structure of antibodies has led to an appreciation of conformational transitions that are induced on antigen binding. Earlier spectroscopic and kinetic evidence for the operation of an induced fit mechanism in antibodies has received detailed support from structural studies. Moreover, X-ray crystallography has provided crucial insights into the mechanisms by which antigenic epitopes are presented to T cells: Structures of both class I and class I1 MHC-encoded molecules in their peptide-bound states were combined with the results of an immense volume of immunological and biochemical studies to construct a detailed model of MHC-epitope interactions. Thermodynamic and kinetic studies have started to provide input to this relatively static structural outline. Most importantly, several investigations of the mechanism of binding interactions between recombinant soluble TCR and its ligand-MHC-peptide complexes have been carried out. These studies have begun to yield more quantitative information about this crucial process for selection of the T-cell repertoire and its activation. Undoubtedly, we are entering an exciting and promising phase in immunology in which a detailed molecular understanding of central cellular processes is emerging. 11. ANTIGENS, ANTIGENICITY,

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IMMUNOGENICITY

A . Definitions Antigen (Ag) is any substance (molecule) that provokes the production of a specific antibody or immunocyte (immune cell), or that interacts specifically with these products of the immune response, when penetrating the body of a vertebrate (Paul, 1993). The first part of this definition, namely, the capacity to provoke an immune response, is also called immunogenicity and the substance provoking such a response is called an immunogen (Sela, 1969). The second part, the specific interaction, is called antigenic specificity. A chemical or physical change in a molecule that results in an increased immune response enhances its immunogenicity, even though it may or may not change its antigenic specificity. It is the antigenic determinant (also called an epitope) that is responsible for the specificity. The notion of antigen also includes the capacity to induce specific immunological tolerance (e.g., Swat et d., 1994), or anergy, defined as

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a state of T-lymphocyte unresponsiveness characterized by the absence of proliferation (human T-cell clonal anergy, Gimmi et al., 1993). Immunogenicity is the capacity to provoke an immune response. When one refers to an immunogenic molecule, this means that the molecule is capable of triggering an immune response by itself, not when conjugated to another macromolecule. Thus, the term immunogenic peptide, for example, refers to a peptide that is immunogenic by itself, whereas a peptide that serves as an important epitope while attached to a macromolecule should be described as immunopotent or, if it is more immunopotent than related peptides, as immunodominant. The ability to mount an immune response ultimately depends on the interplay between the chemistry of the antigen and the physiological state of the host. Thus, immunogenicity is operationally dependent on the experimental conditions of the system, including parameters such as the antigen, the mode of immunization, the organism being immunized, and its genetic background, as well as the sensitivity of the methods used to detect a response. For thymus-dependent antigens, an eficient immune response necessitates the collaboration of T- and B-cell epitopes (reviewed by Milich, 1989; see also Sharma et al., 1993).A normal antibody response to T-cell-dependent antigens requires physical contact between antigenspecific B and T cells. It has been shown that the germinal centers in lymph nodes are an in vivo site where antigen-specific T and B cells interact (Fuller et al., 1993). It is worth stressing that we use the word antigen sometimes for a molecule, sometimes for a virus or a bacterium, and sometimes for an organ or a tissue, and that antibodies have a combining site (also defined as a paratope), with all its distinctive features, of a more or less similar size and cavity. These combining sites are not complementary to a complete bacterium or to a complete heart, but they are always complementary to a unique antigenic determinant which is of limited molecular size. Among the immunocytes we recognize B cells (the precursors of antibodyproducing cells) and T cells. The combining sites on B cells are essentially the same as those of the antibodies that their progeny will produce. On the other hand, the combining sites of T-cell receptors are distinct from those of antibodies. What is characteristic of T-cell receptors is that they recognize the antigen after it has been processed within a cell and presented to the T-cell receptor in conjunction with either class I or class I1 antigens. These are antigens defined by immune response genes that are part of the MHC. Most antigens are thymus-dependent, which means that they need to be recognized in this manner. A minority of antigens, called thymus-independent, do not need recognition by T cells and may lead to efficient antibody formation after interacting exclusively with B cells.

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The antigenic determinants may be parts of proteins, nucleic acids, polysaccharides, lipids or glycolipids, or other biological macromolecules. Very often they have unique steric conformations and are part of a native structure. We thus have to distinguish between sequential determinants (epitopes) and conformation-dependent (or conformational) determinants (epitopes) (Sela et al., 1967). The operational definition holds that if antibodies against a protein, for example, react well with a tetra-, penta-, or hexapeptide derived from that protein, then the antibody is against a sequential determinant. The antibody, on the other hand, is against a conformation-dependent determinant if it is made against a juxtaposition of atoms in space that results from a unique conformation of the macromolecule, and any peptide derived from such a protein cannot, after denaturation, react with the antibody. It is of interest that for most globular proteins and native nucleic acids, almost all the antigenic determinants are conformation-dependent, whereas for most polysaccharides, fibrilar proteins such as silk fibroin, and single-stranded nucleic acids the determinants are sequential. The use of homopolymers of amino acids or sugars and of peptidyl proteins as antigens has established that the determinant is composed of four to six amino acid or sugar residues which contribute unequally to binding with the antibody combining site. Not all antigenic determinants express themselves all the time: Some are more immunopotent than others. Some do not express themselves at all under a certain set of conditions and are called immunosilent, even though under other conditions they may provoke an efficient immune response. Thus a determinant may be immunosilent within a complete macromolecule but quite immunopotent when a segment of the macromolecule on which it is present is used for immunization. Situations are also known in which a determinant is immunosilent but becomes immunopotent in animals made tolerant to other parts of the immunogenic macromolecule of which the particular determinant is a part. We may thus define immunopotency as the capacity of a region of an antigen molecule to serve as an antigenic determinant and induce the formation of a specific immune response. The term hapten, in its strictest sense, designates any substance, large or small, that does not elicit an immune response by itself but can be shown to react with an antibody provoked by immunization with a complete immunogen of which the hapten forms a part. In practice, most investigated haptens are small chemical substances-smaller than a complete antigenic determinant. When attached to a protein, a hapten such as dinitrophenyl or penicilloyl might be defined as an immunodominant part of an antigenic determinant (an epitope). In studies on determinants of a polysaccharide or polypeptide nature, it is of interest to establish the immunodominant portion.

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Generally, an organism distinguishes between self (material that is its own) and nonself (any foreign material). The immune system of an organism reacts against any foreign compound (antigen) and is tolerant (unable to react) toward its own body components, which may be good immunogens in other organisms. This self-tolerance is acquired during fetal or neonatal development, and the immune system can be made tolerant to even foreign material or tissue introduced during this period. Such material, which can induce immunological tolerance or unresponsiveness, is called a tolerogen. Another phenomenon that should be mentioned here is antigenic competition (Taussig et al., 1973). This may occur between different antigens (intermolecular) or between different specificity determinants on the same antigen (intramolecular), in which case we define it as competition between antigenic determinants. This phenomenon may account for some determinants being immunosilent under certain circumstances. Antigenic competition may be defined as inhibition of the immune response to one antigen or determinant by the administration of another antigen or determinant. B. Molecules as Antigens

The two types of natural macromolecules most investigated as antigens are proteins and polysaccharides (Sela, 1973-1 987). These also include glycoproteins, nucleoproteins, lipoproteins, etc., as well as peptidoglycans, glycolipids, and other conjugates. Nucleic acids are also antigenic. Lipids are poor immunogens, but antibodies against them can be obtained, and liposomes play a role here, as they do in enhancing the immunogenicity of various other antigens. Synthetic antigens, especially synthetic polypeptides, have played an important role in elucidation of the molecular basis of antigenicity and many other immunological phenomena, and they will be discussed later in more detail. Other synthetic polymers have also been shown to be immunogenic (e.g., polyvinylpyrrolidone). All proteins are probably immunogenic, although individual proteins differ markedly in immunogenicity. Denatured proteins are often less immunogenic than the corresponding native proteins. Self-aggregation of a protein is usually associated with a negligible change in its antigenic specificity but with a considerable increase in immunogenicity. Like other antigens, proteins possess a continuous spectrum of antigenic determinants that correspond to discrete portions of the surface structure and are preferentially located in regions most exposed to the external environment. The relationship between structure and antigenicity is, however, more complex for globular proteins than for other antigens in that it depends to a very large extent on the overall conformation of the molecule. The exploration of antigenic regions on the surface of proteins has become easier

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with the advent of monoclonal antibodies and rapid methods of peptide synthesis.The antigenic sites may be described as surface domains composed of amino acid side chains that may be distant in sequence but close in space (conformation-dependent determinants). Such domains are probably overlapping and cover most of the protein surface. On the other hand, fibrous proteins possess sequential determinants, whose size may be determined with synthetic peptides to be in the range of three to six amino acid residues. Special attention has been given by immunologists to proteins that possess distinct and easy-to-measure biological properties. These include enzymes and enzyme inhibitors, protein hormones, toxins, and antibodies (i.e., immunoglobulins as antigens), as well as proteins composing viral coats. Immunological studies have also been most successful in following the evolution of proteins. In the case of enzymes, antibodies directed against them-depending on the site of the particular epitope against which they are derived-may lead to total or partial inhibition of the enzyme, form a complex with the enzyme that is fully active, cause enhancement of enzymatic activity, and in some cases even stabilize enzymatic activity at elevated temperatures (Amon, 1973). Polysaccharides are the other class of antigenic substances. Although complex, they nevertheless provide antigens of relatively simple structure through which many of the detailed structural aspects of antigenic determinants and antibody combining sites have been worked out. Microbial polysaccharides are located on the cell surface and are therefore of importance in recognition and immune responses of higher organisms to microbial infection. The simplest polysaccharide antigens are dextran, (a polymer composed entirely of glucose) and levan, composed entirely of fructose. Another important group consists of the capsular polysaccharides of pneumococci. Complex lipopolysaccharide antigens, endotoxins, are found in a large variety of microorganisms, notably in gram-negative Enterobacteriaceae such as Salmonella and Shagella. The polysaccharide determinants are predominantly sequential, consisting usually of four to six sugars, but some antibodies may have combining sites that are smaller. Blood group antigens are the other important category of polysaccharide antigens. They are gene-dependent structures expressing the individuality of cell surfaces, body fluids, and secretions. Chemical characterization of blood group structures has been fully developed. The chemical structures of ABH and Lewis antigens have been elucidated using watersoluble blood group substances isolated from secretions (ovarian cyst mucin, gastric mucin). These structures, when on erythrocytes and other cells, are part of more complete glycolipid or glycoprotein antigens. Antigenic functions of nucleic acids were recognized much later than those of proteins or polysaccharides. No immunogen has yet been pre-

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pared that is capable of inducing antibodies to double-stranded DNA in experimental animals. Such antibodies are, nevertheless, present in humans suffering from systemic lupus erythematosus, as well as in mice and dogs with similar diseases. Their sera usually contain antibodies to single-stranded, denatured DNA, as well as to RNA, double-stranded RNA, histone, and nucleoprotein. It is possible to induce antibodies to single-stranded RNA or DNA either by immunizing with them, complexed with a macromolecule of opposite electric charge, or by preparing conjugates of nucleosides, nucleotides, or oligonucleotides with proteins or synthetic polypeptide antigens and using them as immunogens. Complex formation of methylated bovine serum albumin with synthetic polynucleotides, including some that were double-stranded and triple-stranded, yielded a mosaic of antibodies, some of which definitely recognized the higher-order structure of these macromolecules. Progress in the field of lipid immunology is more recent, largely because of advances in lipid and membrane chemistry. This has led to a heightened awareness of lipids as important cellular antigens. Among the lipids, phosphatides (such as sphingomyelin and cephalin) and glycosphingolipids (such as galactocerebroside) are the most important immunologically. Antibodies against lipoidal extracts of various tissues appear during the course of syphilitic infection, which has led to a standard serological test for syphilis. Another lipid antigen is the Forssman antigen, which is mainly responsible for hemolysis of sheep erythrocytes in the presence of antiserum and complement. The greatest barriers to advancement in the field of lipid immunology have always been the chemical and physical properties of the lipids themselves, particularly water insolubility. The problem of reactivity of soluble antibodies with lipid antigens was partially overcome by including auxiliary lipids such as lecithin and cholesterol in the antigen suspension. Mixtures of lipids in the form of liposomes, consisting of concentric spherules of lipid bilayer membranes, can mimic precisely many of the immunological aspects of intact cell membranes. Liposome availability has permitted study of the immunogenicity of membrane-associated lipids. C. Immunoglobulins, Major Histocompability Complex Products, T-cell Receptors

Immunoglobulins are mentioned here exclusively in terms of their antigenicity. Immunological data usually preceded structural information, and they were crucial, e.g., in the discovery of allotypy and idiotypy. Today a detailed antigenic analysis of immunoglobulins (Ig) of every class and type is possible because of the availability of monoclonal antibodies (MAb).

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The epitopes within one immunoglobulin molecule vary greatly in their relative immunopotencies. Thus, some determinants on the Fc fragment of an IgG are immunodominant compared with determinants on Fab, but when Fab is injected, in the absence of Fc, either by itself or as the dimer (Fab)2, antibodies are produced efficiently against epitopes on the Fab fragment of IgG. Antigenic analysis helped not only to distinguish among the various classes (IgG, IgM, IgA, IgE, IgD) and subclasses of immunoglobulins but also to define their phylogeny. Allotypes are immunologically detectable genetic differences in particular constant regions, whereas idiotypes are the unique antigenic determinants found on the variable regions of antigenbinding receptors; i.e., they represent paratopes of different specificities (idiotypes) in terms of their antigenic variation (Greenspan and Bona, 1993). Regulation of the immune system through idiotype-anti-idiotype interactions can be achieved without the presence of the antigen and thus is particularly well suited to the maintenance of steady states in lymphoid populations after the antigenic stimulus has been removed. Indeed, idiotypeanti-idiotype relationships have the potential to link diverse members of the immune system into a network, and so the activation of one set of clones within the system may have far-reaching and quite unanticipated effects elsewhere within the immunological network. All plant and animal cells possess antigens that can express themselves in a foreign host. Many animal and human antigens can trigger autoimmune phenomena. Some antigens may be organ-specific, whereas others are present essentially on all cells (e.g., histocompatibility antigens). The central role of the MHC in immune processes has been recognized and its antigenicity has been described (e.g., Klein, 1986; Rotzschke and Falk, 1991; Bradley et al., 1992). The MHC, a cluster of diverse genes that mediate and regulate a variety of immune mechanisms, appears to exist in all higher vertebrate species (HLA in humans; H-2 in mice). Genetic, structural, and functional studies on the multiple MHC products have defined three broad classes of genes and molecules. Class I products are glycoproteins expressed on the membranes of all nucleated cells (histocompatibility or transplantation antigens). These are the main targets of the graft rejection reaction, and they mediate the recognition and destruction of virus-infected or neoplastic cells. Class I1 products, defined by immune response (IT)genes, are expressed principally on the membranes of antigen presenting cells (e.g., macrophages, dendritic cells, B lymphocytes) and mediate the regulation, through so-called helper and suppressor effects, of a variety of humoral and cellular immune responses. When incompatible, these Ir gene products also play a potentiating role in trans-


297

plant rejections. Class I11 genes determine the structures of several discrete components of the complement system that cause destruction and elimination of bacteria and other foreign cells. All the products of these genes are antigenic, and the antibodies they provoke have been investigated. Antibodies recognizing TCR or their component chains have been used extensively in their study. Some of the early investigations have been summarized by Marrack and Kappler (1986).

D. Complex Antigens Antigens of importance in practical immunology are generally cellular or multicellular structures, not dispersed molecules. Of course, the specificity is ultimately definable in molecular terms in all cases. Viruses, sometimes even crystallizable, are among the simplest of such structures, with most of the antigenic specificity residing in their coats which are sometimes purely protein but often include lipids or polysaccharides. In some cases, inner core proteins are also efficient antigens capable of providing a protective immune response. More has been learned about immunology from studies on bacteria than on any other group of natural antigens, and their antigenic determinants have been elucidated in great detail in many cases, most of them polysaccharide in nature but also involving proteins and teichoic acids. Lipids often participate in these specificities as well. Progress has been more limited in the investigation of mycoplasmas and chlamydiae. On the other hand, there has been tremendous progress in the immunology of parasite diseases, due to both helminths and protozoa, including the great antigenic variations characteristic of some agents. Fungi are another group of antigens that express an enormous number of antigenically different entities about whose molecular nature relatively little is known. Allergens are antigens that cause allergic reactions of either the immediate or delayed type. They may be of widely different origins such as dust, fungi, hair, pollen, bacterial proteins, food, or drugs. The immediate-type allergy is induced mainly through a mechanism triggered by IgE class antibodies, whereas the delayed-type allergy is T cell-mediated. No listing of natural antigens would be complete without mention of all the cellular markers, receptors, and tissue antigens. There is a growing number of immunologically and chemically defined antigens characteristic of various types of T cells, B cells, and macrophages. There are also the water-soluble cytokines, molecules produced in cells and spilled into body fluids, such as interleukins, interferons, and thymic and tumor factors. Tumor antigens, superantigens, and T-independent antigens will be discussed later in this article.

298


111. MOLECULAR CRITERIA FOR h T I C E N I C I 7 Y The availability of synthetic antigens permitted a systematic approach to elucidating the role of various molecular aspects in antigenicity and immunogenicity (Sela, 1969; Novotny et al., 1987). Landsteiner first showed that small molecules (e.g., 2,4-dinitrophenol), when injected into animals, do not elicit antibody formation. However, dinitrophenyl proteins, in which dinitrophenyl is attached to a macromolecular carrier, elicit the formation of antibodies reacting specifically with the dinitrophenyl group. Such small molecules were termed haptens. Thus, a new antigenic specificity may be grafted on to an antigen. On the other hand, a limited enrichment of gelatin with tyrosine increased its immunogenicity without significantly changing its specificity. Thus, it is possible to change both immunogenicity and antigenic specificity by chemical modification. An increase in immunogenicity by means of appropriate adjuvants has developed significantly due to the availability of synthetic adjuvants which may also be attached covalently to the antigen. Synthetic polypeptides (polymers of amino acids), linear and branched, have been used extensively in immunological research, as their structures are both simple and well known. This permits the construction of hundreds of antigens for the purpose of elucidating the molecular basis of antigenicity, and later the molecular basis of manifold immunological phenomena. Knowing the chemistry of the copolymers made it possible to arrive at conclusions about the role of various structural features in their antigenic function. It was determined that the immunogenically important area of the molecule must be readily accessible and not hidden in the interior of the molecule. By chemical modification antigenic materials may be converted into nonantigens [e.g., by attachment of polyethylene glycol or by poly(o~-alanylation)],whereas nonantigenic materials may become immunogenic. Recent examples of a decrease in immunogenicity are described by Fuertges and Abuchowski (1990) and by Sasaki et al. (1993). An interesting example of increasing immunogenicity involves the insertion of a peptide from interleukin- 1 into poorly immunogenic recombinant proteins (Beckers et d., 1993). The presence of electric charges on a macromolecule is not a minimum requirement for it to be immunogenic. The overall shape of the molecule does not seem to be a crucial factor in immunogenicity, whereas the size seems to be important: Very few molecules of less than 2 kDa are immunogenic, and immunogenicity increases steadily with molecular size. The presence of aromatic amino acids increases the chance of a small molecule’s becoming immunogenic by itself. Generally, in our current understanding, such a small molecule will be

THE NATURE O F THE ANTIGEN

299

immunogenic if it can serve both as a B-cell epitope and a T-cell epitope at the same time. Appropriately constructed polymers of D-amino acids may be immunogenic in a similar way to polymers of natural L-amino acids. To detect this antigenicity, however, it is necessary to immunize animals with very low doses, as in the dose range required to prove the immunogenicity of Lamino acid polymers, the D-polymers may induce immunological tolerance, also called paralysis. This is probably because they are metabolized very slowly, if at all. Through studies of structurally related immunogens, it was possible to establish that antigens, such as pneumococcal polysaccharides, Escherichia coli lipopolysaccharides and D-amino acid polymers, which possess repeating antigenic determinants and are slowly metabolized, are T-independent, i.e., they do not need the cooperation of helper T cells and B cells, whose progeny produce antibodies, for an efficient immune response. In contrast, most antigens, including L-amino acid copolymers, are T-dependent. The purely cellular immune response is limited to the T-cell population, of which many subpopulations are now known, and it probably involves the cooperation of helper T cells and effector T cells. Antigens leading to an immune response of essentially any desired specificity can be prepared synthetically, including the production of antibodies against peptides, oligosaccharides, oligonucleotides, tRNA, and lipids, as well as against haptens such as penicillin, prostaglandin, dinitrophenol, pyridoxal, and ferrocene. Similarly, antibodies have been obtained against many biologically active peptides such as angiotensin, bradykinin, and vasopressin. Moreover, totally synthetic immunogens, including peptide segments of viral coat proteins, e.g., MS2 bacteriophage, hepatitis virus, and bacterial toxins (diphtheria, cholera) have been produced, and they have led to the production of antibodies capable of neutralizing the virus or inducing protection against diphtheria and cholera in experimental animals, thus opening the conceptual door to the production of synthetic vaccines (for a review, see Milich, 1989). To give just one of the many examples where synthetic antigens were of crucial importance in detecting or elucidating a defined immunological phenomenon, the genetic control of immune response should be mentioned. The capacity to respond well or poorly to a certain antigenic stimulus is under strict genetic control. This observation has been made possible largely because of synthetic antigens (chemically simple) and inbred strains of animals (genetically simple). Using defined branched synthetic polypeptides, differing only in a limited manner in their antigenic determinants, it was possible to prove conclusively that genetic control of the

300


immune response is determinant-specific (McDevitt and Sela, 1965, 1967). McDevitt showed later that the immune response to these synthetic antigens was linked to the major histocompatibility locus of the species (McDevitt and Chinitz, 1969). The Zr genes and their products (Ia) have been extremely important in enabling us to understand the phenomena of immunity and resistance to diseases.

N . ROLEOF CONFORMATION IN ANTIGENICITY

Spatial folding of proteins plays an important role in determining their antigenic specificity, as is apparent from the poor reaction-or total lack of cross-reaction-between denatured proteins and antibodies to the same proteins in their native form (Sela, 1969; Todd et al., 1982; Novotny et al., 1987; Laver et al., 1990; Roberts et al., 1993; Ota et al., 1993). For example, antibodies to native bovine pancreatic ribonuclease do not react at all with the ribonuclease oxidized by performic acid, which is a randomly coiled chain devoid of disulfide bridges. Nor do antibodies to ribonuclease oxidized by performic acid react with the native enzyme (Brown et al., 1959). Similarly, antibodies prepared in goats against rabbit IgG do not react with the rabbit IgG after all its disulfide bridges have been opened by reduction (Freedman and Sela, 1966).This is undoubtedly due to changes in the conformation of the protein molecule, resulting in loss of the original antigenic determinants. Conformation here designates a particular arrangement of atomic positions in a molecule that can be achieved without the reorganization of chemical bonds. An antigen can provoke antibodies against many different determinants present in its molecule, and some of these antibodies may be overlapping. Thus, antisera against a protein antigen usually contain a population of antibodies of differing specificity, having as a common denominator the capacity to react immunospecifically with the antigen. There are many reported examples in which antibodies have been used for the detection of different conformations in proteins: Metmyoglobin forms a reddish-brown precipitate with antiserum to metmyoglobin. Antisera to apomyoglobin give white precipitates with apomyoglobin but also with metmyoglobin. The release of ferriheme from metmyoglobin must have been due in this case to a change in the conformation of the crossreacting antigen on reaction with antibodies to myoglobin (Crumpton, 1966). With synthetic models it has been possible to build, with the same peptides, immunogens that possess either exclusively sequential or conformationdependent epitopes. Thus, antibodies to the helical polymer (Tyr-AlaGlu), do not react with the peptide Tyr-Ala-Glu, nor do they react with


301

larger peptides in which the sequence Tyr-Ala-Glu is repeated several times. On the other hand, antibodies to the Tyr-Ala-Glu peptide do not cross-react with the helical polymer (Schechter et al., 1971). A not yet helical peptide, in which Tyr-Ala-Glu was repeated 11 times, was found to cross-react with antibodies to the helical polymer, and-when followed by circular dichroism-was shown to become helical on reacting with the Fab derived from antibodies to the helical polymer (Schechter et al., 1971). This may have been the first reported case where two biologically active macromolecules reacted with each other via their active sites and transconformation occurred in one of the molecules in order to permit the interaction. Antibodies to a polymer of Pro-Gly-Pro, which has the characteristic triple helix of collagen, cross-reacted with fish, rat, and guinea pig collagen (Borek et al., 1969). This was the first instance in which antibodies to a synthetic antigen reacted significantly with a natural protein, and this cross-reaction was clearly due to their higher-order structure. Similarly, a peptide analogous to a stretch of the hen egg white lysozyme sequence can be synthesized, closed into a ‘‘l00p” by a disulfide bridge, and attached to a synthetic branched polymer. The resulting macromolecule leads to production of antibodies that cross-react efficiently with a unique conformationdependent region of a native protein (Arnon et al., 1971). Antibodies to a peptide will cross-react with the parent protein either if they have a free-solution conformation similar to the one within the native protein or if they can easily transform into that conformation. Thus, on a priori grounds, if the protein segment is more flexible, the chance of crossreaction is higher, even though there are cases where a small peptide is already capable of having a relatively rigid conformation similar to the one it possesses within a native protein. It is obvious for these reasons that it is not realistic to expect that antibodies to any peptide corresponding to a sequence segment within a native protein will necessarily cross-react with that protein. In a study on the evaluation of surface areas on proteins that would be accessible to contacts with large ( 1 nm radius) spherical probes, Novotny et al. (1986) concluded that the primary reason why certain polypeptide chain segments are antigenic is their exceptional surface exposure, making them readily available for contact with antigen combining sites. Exposure of these segments frequently results in high mobility and, consequently, in the reported correlation between antigenicity and segmental flexibility. From the structures determined by X-ray diffraction of five complexes of a monoclonal antibody Fab fragment with its antigen, the epitopes all occupy large areas composed of 15-22 amino acid residues on several surface loops (Laver et al., 1990). The antigenicity of these epitopes is absolutely dependent on the conformation of the native proteins. Each epitope

302

MICHAEL SELA AND ISRAEL PECHl

has a buried surface area on the antigen of 650-900 square antigens. There are 75-120 bonds between the antibody and antigen, as well as salt links and hydrophobic interactions. Energetic calculations suggest that a smaller subset of 5-6 of the 15-22 residues contributes most of the binding energy, with the surrounding residues merely indulging in complementarity. When a native protein is used for immunization, it must be taken into consideration that, besides an immune response to the intact antigen, there will also be an immune reaction (antibodies and specific T cells) against a whole variety of partial degradation products. In other words, some of the cross-reactive antibodies may be a result of the immune response to such results of degradation. Illustrations of such a possibility have been described by Leder et al. (1994). Immunological cross-reactions between totally different antigens are sometimes reported, and may even lead to autoimmune phenomena (e.g., in rheumatic fever) because of the antigenic similarity between epitopes on cardiac tissue and those on streptococci (M protein andlor polysaccharides). A discussion of the molecular mimicry between microbial, viral, and host antigens is given by Shoenfeld and Isenberg (1989). Laine and Esser ( 1 989) have shown that polyclonal rabbit antibodies against melittin, the soluble hemolytic peptide present in bee venom, recognize human C protein C9, the terminal component of the membrane attack complex, and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence that together match a continuous segment in the melittin sequence. For thymus-dependent antigens it is logical to assume that even though the antibodies are made against conformation-dependent epitopes, T-cell help is triggered by sequential epitopes derived from the same antigen, and the same should be true for the triggering of cytotoxic T cells. Indeed, most of the T-cell clones described to date cannot discriminate between the native and the denatured form of the antigen (Benjamin et al., 1984). Lysozyme-specific T-cell clones have been described that discriminate between native and denatured antigen (Manca et al., 1994). While our current knowledge about the detailed interaction between peptides and the grooves of class I and class I1 MHC antigens is very extensive, nothing is known about the interactions of such grooves with epitopes of a nonpeptide nature, such as oligosaccharides, oligonucleotides, or small organic molecules acting as haptens. In a study on glycopeptides, Unanue and colleagues (Harding et al., 1993) proposed that recognition by T cells does not involve specific interaction between the carbohydrate moiety and the T-cell receptor. It seems, nevertheless, that oligosaccharides and other nonpeptide epitopes should be recognized by MHC class I and class I1 antigens. Otherwise, they would all have to be thymus-independent or to have help from minute amounts of peptides within the antigen.


303

V. ANTIBODY-ANTIGENIC EPITOPE INTERACTIONS

The pioneering kinetic studies on hapten binding to specific polyclonal antibodies (Froese et al., 1962; Day et al., 1963) suggested the operation of a single step binding mechanism:

where Ab, H, and Ab.H are the antibody, the hapten, and the complex, respectively. The specific rate of association is k,, and of dissociation, k,,E. This simple mechanism has been modified with the advent of the very first homogeneous antibody preparations secreted by induced myeloma cells. These allowed a more detailed and rigorous kinetic analysis of the association step. A systematic comparison of the kinetic behavior of a large family of related haptens interacting with an antigen binding site on the IgAMOPC315 indicated that the association process is even more complex (Haselkorn et al., 1974). These results fit a two-step association mechanism which is essentially applicable to all macromolecule-ligand association processes in solution (Eigen, 1974): Ab + H

[Ab.H] k-i

8

Ab.H

where [Ab.H] is an encounter complex in which the antibody and hapten form part of the solvation sphere of each other and Ab.H is the final specific complex. In the [Ab.H] some nonspecific interactions, e.g., electrostatic or hydrophobic, may be formed because of the mere process of the H becoming part of the Ab solvation sphere, in which case one should take Ab.H to be an outer sphere encounter complex. The preceding reaction scheme [Eq. (2)] was employed in a refined kinetic examination of a large number of haptens interacting with a given Ab site. It allowed the resolution of the encounter step and the following step, where establishment of the specific elementary interactions between the contact residues of the sites and the hapten occurs. Most of the reactions listed in Table I were discussed in detail earlier. [For the original detailed treatment, see Pecht and Lancet (1977).] The availability of monoclonal antibodies which allowed many new structures to be resolved led to a disappointingly small number of new kinetic studies on the interactions between specifically raised MAbs and their ligands. Hence current insights are still mainly based on earlier work employing chemical relaxation measurements. For the case of a diffusioncontrolled process, all the parameters of the overall forward reaction are expected to be similar to those of the encounter step formation. Identifica-

304


tion of those binding steps that are diffusion-controlled reactions requires knowledge of several parameters: (1) AH,, in the range 4-5 kcal/mol; and (2) for hapten binding, k,, larger than about 3 x lo7 M’ sec-’.There may still be reactions where these two conditions hold, yet k2 >> k-1 does not. This happens when AH2 is in the range of 4 kcaVmo1 and -TA& is not very large (12.5 kcal/niol). In such cases it may be difficult to determine whether the particular reaction is diffusion-controlled. However, the previously mentioned study on a series of different haptens with the same binding site (Haselkorn et al., 1974) was helpful in this respect: A value of k , , practically independent of the chemical nature of the hapten would then imply diffusion control. The k,,, values determined for the reactions listed in Table I suggest that many may indeed be difbsion controlled. Still, the AH,,, value, together with the variation in k,, for different haptens, seem to indicate that the condition kz >> kl applies strictly for none of these, and that some of them may be at best “almost diffusion-controlled” (k2 k.1). Haptens that are simple aromatic compounds have relatively high rates of association with the antibody because of their rigidity and the hydrophobic nature of the interactions involved. The transition from diffusion control to difbsional preequilibrium is effected by a decrease in kn. This may be the result of a higher AHz, or a higher -TASz, or both. One possible reason for an increase in A H 2 is hydration of the reactants which has to be overcome before binding can occur. Other effects that may lower k2 are ( 1 ) nonspecific electrostatic interactions that have to be broken before binding; and (2) a conformational equilibrium between different forms of the hapten (this may be the case for oligopeptides or oligosaccharides that do not have a preferred conformation in solution). In such cases -TAS2 will be higher, yet no distinct conformational change will necessarily be resolved; and (3) conformational changes in the protein. A good illustration of this case has been reported (Maeda et al., 1977) for the reaction of homogeneous antipneumococcal polysaccharides with di- and tetrasaccharide haptens (Table I). As stated above, the number of more recent kinetic studies on antibodyepitope interactions is relatively small. Results of some representative ones are also presented in Table I. Unfortunately the very different experimental methods employed in these studies allow only a qualitative consideration and limited comparison among them. In the studies on fluorescein binding to specific MAbs, some of which have rather high affinity, a pattern similar to that of other hapten-binding MAb was observed (Kranz et al., 1982): k,, values close to lo7 M-’ sec-’, i.e., approaching diffusion control. Dissociation rates were thus the main cause for affinity variation. The induction of conformational transition in the MAb on hapten binding was assumed for a few cases, though not clearly resolved by the methods

-

TABLE I Kinetics $Antibody-Antzgen Interactionsa Immunoglobulin or antibody MOPC 3 15 Anti-NP MOPC 3 15 MOPC 460a MOPC 3 15 Antiflorescein HOPC 8 MOPC 3 15 Antidigitoxin Antidigitoxin Antipolyalanine Antilactose IgMc Antilactose IgMd Antidextran Anti-Pn. pdysaccharide MAb 4-4-20 MAb 20-4-4 MAb 2B5 MAb 2F8 MAb E225 (antiidiotype) MAb E5.2 (antiidiotype)

kon

Hapten or antigen

(M-I 8')

DNP-NH-CH3 DHNDS-NP a-N-DNP-glycine &-N-DNP-lysine &-N-DNP-lysine Fluorescein Phosphorylcholine DNP-(1ysine)g Digitoxin Ouabain Ala-NH-(CH)zNH-DNS Lactose-dye Lactose-dye IM4-NPFI Hexasaccharide

5.2 x 1.8 x 1.9 x 1.3 x 1.3 x 6.0 4.1 3.3 1.4 1.3 4.0 x 3.9 x 1.1 x 4.4 1.1 x

Fluorescein Fluorescein Cytochrome c Cytochrome c Antilysozyme idiotope Antilysozyme idiotope

6x 4.7-35 x 6.5 1.5 x 1x

lo8 108 108

10' lo8 107 107 107 107

107 lo6 106 106

105 106 lo6 106 105 lo6 103

2.3 x lo5

koff (s-9

540 760 1300 580 53 g X 10-5 110

32 2.4 lo4 1.4 x 10-2 2 29 5 3.9 12 3.7-3.4 x 9-12 x -8 1.0 3.6 x

lo4 10-2 10-5

lo4 lo4

5.2 x lo4

konboff

M-'

9.6 x lo5 2.4 x lo5 1.5 x 105 2.2 x 105 2.4 x lo6 6.6 x 1011 3.3 x 105 1.0 x 106 5.8 x 1010 9.3 x 108 2 x 106 1.3 x 105 2.2 105 1.1 105 g x lo4 1.7-1.8 x 5-27 -gX 1.5 x 1.5 x

AHon

-T&n

(kcaVmol)

(kcallmol)

6.4'

-0.86

7.1

-0.28

6.1 5.1

2.3 4.1

6.0

10" 107 109

lo1' 105

4.3 x 108

5.5 3.8

3.8 5.2

Temp. ("C)

Ref.

21 25 25 23 25 18.5 25 21 22 22 25 25 25 25 25

Haselkorn (1975) Froese and Sehon (1965) Pecht et al. (1972) Lancet and Pecht (1976) Pecht et al. (1972) Levison et al. (1975) Hartmann et al. (unpubl.) Haselkorn (1975) Smith and Skubitz (1975) Smith and Skubitz (1975) Licht (unpubl.) Blatt and Pecht (1976) Blatt and Pecht (1976) Lancet et al. (unpubl.) Maeda et al. (1977)

2 2 20 20 20.4

Kranz et al. (1982) Kranz et al. (1982) Raman et al. (1 992) Raman et al. (1992) Tello et al. (1994)

25

Tello et al. (1994)

a DNP, 2,4-Dinitrophenyl; NP, 4-nitrophenyl; AC, aminocaproate; DNS, 4-dimethylaminonaphthalene-1-sulfonyl-NH-; IMm-NPFI, (isomaltose)n-1-(m-nitropheny1)flavazole; lactose-dye,N-(a-N-acetyl-~-N-DNP-L-lysyl)~-aminophenyl-~-lactoside. Average value.

'

306


employed. Interesting insights will hopefully emerge from kinetic studies on MAbs binding to protein epitopes. This is illustrated by the study on MAbs raised to cytochrome c both with an affinity in excess of lo9M-' and specific rates of association of about lo6M-' sec-' which, for the two macromolecular reactants, is also at the diffusion control limit. Once more, the affinity is therefore reflected by rather slow dissociation rates (Raman et al., 1992; Kelley et al., 1992). Interestingly, differences in reactivity were resolved toward distinct conformers of the antigen, yet none in the MAbs themselves. The structural analysis of anti-idiotypic MAbs (Tello et al., 1994), notably those to antilysozyme MAbs opens up another exciting area for kinetic and thermodynamic studies (Bhat et al., 1994; Braden and Poljak, 1995). In addition, application of advanced forms of nuclear magnetic resonance (NMR) spectroscopy is also starting to provide information about the solution structure of Fv domains (Freund et al., 1994). Detailed analysis of the elementary steps of hapten binding to the IgA secreted by MOPC-315 is both of broader importance for other protein binding site-ligand recognition processes that take place in different parts of the immune system. Moreover, it may also provide a basis for deeper insight into the mechanisms that underlie the maturation of antibody response (Foote and Milstein, 1994). Thus, the original simplistic assumption that binding site-epitope binding could be represented essentially by the single-step equilibrium [Eq. (l)]resulted in the interpretation of dissociation rate constants as indicators of binding affinity. However, in the mid- 1970s kinetic studies had already resolved conformational transitions to be induced on epitope binding and hence to markedly affect Ab-epitope affinity (Lancet and Pecht, 1976). Significantly, an increasing number of homogeneous antibodies in which distinct conformational changes are induced on epitope binding have been resolved in recent years by X-ray crystallography studies (Rini et al., 1992; Padlan, 1994; Braden and Poljak, 1995). Thus, the view that an induced fit process characterizes antibodyepitope interactions, which first emerged from kinetic studies, is now being firmly substantiated and elaborated on by structural studies and will be briefly reviewed below.

VI. CONFOKM,~'I'IONAI. TRANSITIONS INDUCED BY

HAITEN

BINDING

The extent and significance of structural changes induced in an antibody molecule on binding of its specific hapten or antigen has been a problem addressed by a variety of physicochemical methods including the very early X-ray crystallographic studies (Davies et al., 1988; Padlan, 1994; Wilson and Stanfield, 1994). This was mainly because of interest in the

THE NA'I'UKE

O F THE ANTIGEN

307

mechanism by which immunoglobulin effector functions are initiated. However, it is only recently that direct evidence for the induction of such changes has emerged from structural studies. In fact, a surge in such reports, has taken place during the last few years and has yielded ample evidence for conformational transitions and underscored their relatively wide occurrence and unexpected large extent (Wilson and Stanfield, 1994). Establishing the functional significance of these transitions obviously depends, among other things, on resolving where, i.e., in which Ig domains, they take place. However, it is noteworthy that a range of different experimental methods had earlier shown that antigenic epitope binding induces conformational changes in antibodies of diverse specificities (Pecht, 1976). The time-resolved analysis of hapten-induced conformational transitions, though suffering at that stage from limited knowledge of their extent and functional significance, is becoming part of the clearest and better-established supporting evidence (Zidovetzki et al., 1980). Binding of a hapten (or an antigen) to the immunoglobulin may cause changes in the spatial arrangement of residues in the binding site. These changes may be limited to the contact residues or involve a larger part of the protein. In the latter case, other loci on the immunoglobulin may be affected; i.e., allosteric phenomena will arise (Monod et al., 1965).An allosteric mechanism for the induction of physiological activities of antibodies by antigen binding thus requires evidence for the involvement of longrange conformational changes. Such a model, proposed by Huber et al. ( 1976) based on X-ray crystallographic studies, raised the possibility that these changes include those in noncovalent interactions among the immunoglobulin domains, as well as in the hinge peptides and in the position of the Fab regions relative to the Fc. Spectroscopic and other static methods have also supported the induction of conformational changes, yet their nature and significance have not been unambiguously determined and are still a topic of current debate and experimentation (Schlessinger et al., 1975; Guddat et al., 1995). As in other physicochemical approaches to the study of immunoglobulins, the heterogeneity of normally induced antibodies was a major obstacle to achieving an unambiguous analysis of the kinetics of interaction with haptens. From the results reviewed here later, it is now quite clear that epitope binding-induced conformational transitions are a general characteristic of antibodies. Following the first kinetic study describing the hapten binding-induced conformational transition €or the nitroaromatic hapten-specific murine IgA MOPC460 (Lancet and Pecht, 1976), several other antibodies were studied in great detail and were all observed to conform to the same general pattern. The homogeneous myeloma immunoglobulin MOPC460 was found to display two distinct relaxation times in

308


its reaction with e-dinitrophenyl-L-lysine, the first in the range 0.25-1 .O msec and the slower in the range 10-18 msec (Lancet and Pecht, 1976). The well-resolved relaxation spectrum of protein MOPC460 with its haptens, as monitored via quenching of the protein tryptophan fluorescence, led to the proposal that this antibody exists in two conformational states and that the equilibrium between them is shifted by hapten binding. This systematic kinetic study of MOPC460 was later extended to a number of other monoclonal antibodies of different specificities and belonging to classes other than IgA. A common, most probably general, mechanism was consistently found to govern these reactions. The homogeneous murine IgM-MOPC 104E is specific for a(1+3)dextran (Schepers et al., 1978). The kinetics of interaction of a series of a-~-glucopyranosyl-(l~3) oligomers of different size and structure with this antibody were extensively investigated. In all cases the observed chemical relaxation spectrum exhibited two well-resolved relaxation times. Thus far data have been analyzed only partially (Schepers et al., 1978; Blatt et al., 1979): For the interaction with the tetrasaccharide both relaxation times and amplitudes fit the following mechanism: Ab+H

r:

Ab.H

R, kR

Ab*.H

ko k-o

Ab*+H

The kinetics of interactions between three galactan-binding homogeneous IgA molecules, XRPC24,5539, and TEPC601, and their oligogalactose haptens was another system studied extensively by the T-jump method. By monitoring the intrinsic fluorescence of the proteins, the chemical relaxation spectra of all three proteins reacting with (Gal)3were found to be composed of two relaxation processes: the faster related to the hapten binding, and the slower to a conformational transition of the proteins (Vuk-Pavlovicet al., 1978; Zidovetzki et al., 1980). A detailed analysis of the concentration dependence of the relaxation times and amplitudes has also shown that this system behaves according to the preceding general mechanism. Both the intact immunoglobulins and their Fab fragments exhibited identical kinetic behavior, indicating that conformational changes are not affected by the Fc and therefore probably do not extend beyond the Fab. To further examine the generality of this mechanism, the kinetics of hapten binding to a series of heterologous recombinants of heavy and light chains prepared from the latter galactan-specific antibodies (X24, 5539,

T H E NATURE OF T H E ANTICXN

309

and T601) was investigated (Zidovetzki et al., 1980). This group of hybrid molecules had earlier been shown to maintain an affinity for theP-D-(1-*6)oligogalactose haptens comparable to that of their parent molecules (Manjula et al., 1976). These antibody-hapten systems also exhibited two relaxation times. The kinetic and amplitude data for the hybrids were found to fit the same general mechanism as that followed by their parent proteins: They exist in two conformational states and the equilibrium shifts to the higher affinity state on hapten binding. Furthermore, some of the specific rates and the thermodynamic parameters of these different steps were found to have values very close to those of their parent molecules. The kinetic and thermodynamic parameters obtained for this group of related antibody molecules were compared in the context of the differences in their amino acid sequences. Particularly interesting is a comparison between a hybrid and its parent proteins. A significant correlation with the parent light chain donor is found only for the rates of conformational transition of the hapten-bound state (kl and k I )of the hybrids. This observation is instructive, as the light chains of T60 1 and X24 have identical sequences except for an alanine exchanged for a serine at position 100. 5539 differs from the former at five positions, and from the latter at six positions (Rao et al., 1979; Rudikoff et al., 1980). The VH regions of X24 and T601 differ at six positions, three of which are clustered in the J segment. VHof 5539 differs from that of the two previous ones in a larger number of positions, both in the D and J segments, as well as in the rest of this domain (Rao et al., 1979). Examinations of the positions that the substitutions in the chains constituting these hybrids occupy in the threedimensional structure of their domains (R. Feldman, personal communication, 1980) are rather informative: They clearly reveal that several of the more important exchanges (e.g., Ser to Ala at L-100) are in the VL-VH contact areas. This is in line with the J segment having a decisive role in the light chain folding and, most probably, also in the heavy-light chain association. Thus, the recombinational events joining Vr. with JI and VH with D and JH, apart from affecting the nature and morphology of the combining site directly, modulate it because of their presence at the VL-VH interface. The rates of structural transitions of the hapten-bound protein exhibit the widest variation, ranging over four orders of magnitude. The hapten association rates are similar to those found for other saccharide-binding proteins (Table I; Clegg et al., 1977; Pecht, 1976) and are two orders of magnitude slower than association rates observed for nitroaromatic-binding immunoglobulins (Pecht and Lancet, 1977). Even slower bimolecular rate constants were found for the binding of saccharides to lectins (Clegg et al., 1977; Loontiens et al., 1977). As suggested earlier (Pecht and Lancet, 1977; Vuk-Pavlovic et al., 1978), these slow rates

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MICHAEL SELA AND ISRAEL PECHI

of saccharide (and peptide) binding to proteins may be a result of the flexibility of these ligands and/or the need to disrupt and form several hydrogen bonds on association. The operation of a common reaction mechanism, shared by heterologous chain recombinants, constitutes strong evidence for the hapteninduced conformational transition being an inherent property of the tertiary domain structure of the immunoglobulin molecules. The variations found in the reaction rates constants most probably reflect modulation effected by the structural variance. For the few heterogeneous antibody preparations that were examined (e.g., equine antilactose antibodies, Blatt and Pecht, 1976), there is also evidence that the preceding general mechanism is operative. The well-characterized group of phosphorylcholinebinding homogeneous antibodies (S 107, T15, H8) has also been studied kinetically. Though only a single relaxation has been resolved, a detailed analysis of the concentration dependence indicates that a more complex mechanism is operative (Oratore et al., 1981). Two questions arise from these observations: (1) What are the structural corrollaries of these kinetically observed conformational transitions? (2) What is their functional significance? The increasing number of reports describing X-ray crystallographically determined antibody structures that undergo conformational transitions provides at least a partial answer to the first question (Rini et al., 1992; Wilson and Stanfield, 1994). It is gratifying that the earlier mechanistic conclusions are now supported by structural studies. Thus, from the activation parameters of the conformational transitions, as well as from the fact that these are monitored via changes in the intrinsic extinction and emission properties of the proteins, one is led to conclude that at least the whole variable module is involved in these transitions. An interesting model of the possible transmission of the structural transitions into the second (constant) domains of immunoglobulin has been suggested: namely, that the hapten binding induces longitudinal interaction spanning the whole length of the light chain dimer of protein 315 (Zidovetzki et al., 1979). Studies on Fd’ interactions with L1, CL1and VL (Alexandru et al., 1980) provided another illustration of the feasibility of longitudinal interactions within the Fab. These interactions could be attained through changes in the lateral contacts between the domains (Abola et al., 1980) and are illustrated in considerable detail by several structural studies (Lascombe et al., 1992; Guddat et al., 1995; Braden and Poljak, 1995; Stanfield et al., 1993). Though conformational transition induced by antigen binding is now becoming a common, generally accepted mechanism, the functional implications of this mechanism remain to be established. The specific rates and activation parameters observed for conformational transition of the range of different systems clearly support the


31 1

occurrence of substantial rearrangements. A minimal hypothesis accounting for the observed reaction patterns would be that it reflects readjustment of the proteins to a better binding conformation, i.e., an induced fit mechanism. Significantly, in their studies on humoral immune diversity and its maturation, Foote and Milstein (1991, 1994) have examined the kinetics of hapten (2-phenyl-5-oxazolone) binding to a family of (40) different specific MAbs, and several of these reactions were found to exhibit multistep binding equilibria. They concluded that maturation of the humoral immune response may in fact attain increased affinities of epitope binding via a given preferential conformer and that this may also be the basis for an increase in the diversity of the repertoire.

VII. T CELL-ANTIGENIC

EPITOPEINTERACTIONS

The recognition of antigens by the T-cell compartment of the immune system is a multistep process culminating in the formation of a ternary complex between the TCR for antigen and the binary complex of class I or class I1 MHC-encoded molecules, with short peptides derived from the antigen. Thus, the specificity of the TCR is for a neo-antigenic determinant composed of both the genetically restricting MHC molecule and the bound peptide (Bevan et al., 1994; Rammensee et al., 1993; Rothbard and Gefter, 1991). The recognition process may therefore be considered to consist of two distinct phases, the first in which peptides are produced and bound to the MHC-encoded proteins, and the second in which the TCR binds this binary complex. Since MHC-encoded molecules may bind peptides derived from both self and nonself proteins, the fundamental implication of this is the need for a selection process where cells with TCR that bind self peptides are eliminated during the development process in the thymus, yielding a repertoire of mature T cells that recognize MHC complexes with nonself peptides. Understanding the molecular basis of this selection and maturation process constitutes a major challenge to contemporary immunology. It has been proposed that this process is based on the affinity (and resultant cell surface avidity) between the TCR and a given MHC-peptide complex. Specifically, low avidity would lead to a positive selection, whereas high avidity would result in a negative one. Quantitative analyses of the binding interactions leading to this ternary complex are only beginning (e.g., Kageyama et al., 1995). High resolution three-dimensional structures of several human and murine class I molecules have been complemented by that of the human class I1 molecule (Bjorkman et al., 1987; Garrett et al., 1989; Brown et al., 1993). Both classes of molecules are heterodimeric proteins. Class I is

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MICHAEL SELA AND I S M E L P E C H I

composed of a membrane-anchored heavy chain noncovalently associated with the P2-microglobulin.The peptide binding groove formed by the two membrane-distal heavy chain domains has been resolved: Two a helices form the walls of the groove, while its floor is provided by a @-pleated sheet. Class I1 molecules are membrane-anchored a and p glycoproteins forming a peptide binding groove, analogous to that in class I molecules. The polymorphism of both these proteins is primarily located in the grooves, producing allele-specific morphology. As a consequence of distinct intracellular processing pathways, class I molecules primarily present peptides derived from endogeneously synthesized (e.g., viral) proteins, whereas class I1 molecules present peptides of exogenous proteins that were taken up endocytotically by the antigen presenting cell (Rammensee et al., 1993; Rothbard and Gefter, 1991). The structure of complexes of defined antigenic peptides with human and murine class I molecules has also been resolved by X-ray crystallography (Madden et al., 1991; Fremont et al., 1992; Zhang et al., 1992). These structures have provided detailed insights into the way in which the groove accommodates amino acid residues of the fitting peptides and “locks” its C and N termini. Examination of the nature and position of the former so-called anchoring residues rationalized the formation of allelespecific peptide “motifs.” These structural insights were preceded and complemented by a large body of information derived from indirect, mainly immunological, studies. This work has yielded some general structural requirements for peptide binding and the resultant exposure of peptide residues to the recognizing TCR. The early demonstration that synthetic peptides representative of viral epitopes can be added to cells carrying appropriate class I molecules, and are therefore recognized efficiently by specific cytotoxic T lymphocytes (CTL), initiated a major effort employing this approach aimed at defining antigenic epitopes. Still, this approach was obviously fraught with uncertainties caused by possible intra- and extracellular proteolysis as well as by the binding mechanism of the added peptides competing with those already bound. It was the demanding challenge of isolating and characterizing the peptides naturally associated with the MHC molecules that provided the required chemical definitions of the different binding motifs (Rammensee et al., 1993). The limited number of different antigen presenting MHC molecules expressed by a given individual implies promiscuity; i.e., that a relatively large number of different molecules bind to each of the expressed MHC molecules in order to provide the required comprehensive response to a dynamically changing repertoire of pathogen-derived peptides. Indeed, estimates were made that more than lo4 different peptides can be bound


313

to a given MHC molecule. How this feature is accommodated with the binding affinity and the eventual specificity of the interactions with the TCR are problems that so far have only partial solutions. As stated earlier, X-ray crystallography has yielded high-resolution structures. Detailed analysis of both the structure of the groove formed by the a I and a pdomains of class I molecules and the analogous domains in class I1 together with those of the bound peptides have shown how these ligands interact by a combination of two or three van der Waals contacts between the peptide side chains residing in pockets or anchoring sites as well as by an array of hydrogen bonds. The shape and loci of the pockets were shown to be characteristic for a given MHC molecule and hence to determine the nature of the peptides that can bind to it. Moreover, specific sets of hydrogen bonding lock the C and N termini of peptides bound to class I molecules, thus setting constraints on their length. These binding patterns may in turn affect the conformation of the bound peptides, and vice versa. For example, the MHC structure may be influenced by the bound peptide. Thus, while a few class I molecules would bind octapeptides, most would prefer nonapeptides. In some cases, however, longer peptides would also bind, yet bulge out of the groove with different degrees of loss of binding energy. Still, peptides longer than 11 residues probably would not be bound because of the decline in binding energy. In addition, the question arises as to what extent there is interference in interactions between such bulging binary peptide-MHC complexes and the TCR or whether such structures are recognized at all. The more general issue of the extent to which binding interactions are formed between the TCR and the MHC itself as compared with those of antigenic peptides will be further discussed later. The conformation of bound peptides of equal length has been examined in complexes of the HLA-A2 molecule with four distinct epitopes (Madden et al., 1993). It was shown that as a result of “stapling” of the peptide at both ends via the anchor residues, as well as C and/or N termini, the central parts vary considerably in both chain and side-chain conformation. Thus, the conformation of bound peptides may be a function of both their sequence and their mode of interaction with the groove. Consequently, other residues and possible main-chain elements may be solvent- exposed and hence become TCR contact points. This may provide the rationale for the exquisite sensitivity to minor changes in peptide structure whereby a single residue change transforms a peptide into an antagonist in the T cell-induced response (Madrenas et al., 1995). Thus, while considerable constraints may operate in determining whether a peptide is bound because of its length and the nature of the anchor residues, great variability can exist in the exposed residues. The preceding considerations did not take into account the possible changes induced in

3 14


the conformation of the MHC molecule itself on peptide binding; since the TCR is assumed to recognize different composite structures constituted of contacts with both MHC and bound peptide elements, the induction of distinct MHC conformers on binding different peptides would provide an effective enlargement of the repertoire limited by the number of MHC alleles. While only side-chain conformation was observed to be peptide structure-dependent in the HLA-A2 molecule (Madden et al., 1993), the a - a helix of the H-2K" molecule was found to vary between two different peptide complexes (Fremont et al., 1992). This observation may perhaps indicate the wide range of potential structural cases to be expected. Pioneering studies aimed at quantitative understanding of peptideMHC interactions have already yielded insights into the thermodynamic and mechanistic aspects. Both soluble forms and cell surface-bound MHC molecules were employed in these studies, and it was assumed that the same complexes are formed with certain extraneously added peptides as with those produced by intracellular processing of antigen. Moreover, no knowledge of the actual state of the peptide binding sites was available in most cases, and another assumption was either that they are empty or that bound peptides are displaced. Still, monitoring in different ways the results of adding peptides is a widely accepted practice, and it has also been employed in efforts to quantitate affinity and the number of complexes required for CTL activation. Indeed, while earlier studies did not distinguish between intracellular and surface bound peptides, Kageyama et al. (1995) developed a more rigorous protocol for that purpose. Binding affinities were observed in a range similar to that reported by earlier studies (105-10xMI). The critical number of peptide MHC complexes required to become a CTL target ranged from several thousands to less than 10 per cell. This once more implies a relatively wide range of response and hence flexibility exhibited by this branch of the immune response. The marked similarity of the three-dimensional structure of class I1 MHC molecules implied on the basis of biochemical properties, domain organization, and genomic structure was clearly established by crystallographic analysis of the HLA-DR1 molecule. The similarity also extends to the overall architecture of the peptide binding site. However, striking differences are observed in the mode of peptide binding and hence in the nature of the peptides bound to the two classes. This is first noted in the length of the peptides bound; in contrast to the limited length of those bound to class I, apparently no such limit applies to those binding to class I1 (Chicz and Strominger, 1992; Hunt et al., 1992).The structural basis for these differences lies in the relatively small differences between the sites and the positioning of key residues forming H bonds with the peptide: In the structure of one defined peptide bound to the DR1 molecule (Stern et al., 1994) both ends of the 13-residue-long ligand extend outward and

THE NATURE OF T H E ANTIGEN

315

no hydrogen bonding is observed at the end of the groove. Probably because of the lack of clear relation between the end of the peptide and the anchor positions it is more difficult to characterize binding motifs for class I1 alleles. From random screening of libraries and binding measurements of systematically varied peptides, it became apparent that the motifs include more anchor residues than those of class I motifs and are less specific (Sette and Grey, 1993). The distinct modes of peptide binding to the groove of these two classes of MHC-encoded proteins also lead to marked differences in the pattern of peptide exposure. The class I-bound peptides are exposed mainly in the center of the groove, while those bound to class I1 present potential contact points distributed all along the groove (though approximately similar surfaces are produced in both cases). Results indicate that minor differences in the detailed structure of the composite peptide-MHC surface available for engagement with the TCR may provide distinct signaling (Madrenas et d., 1995). One of the parameters that may determine the distinct signals is the orientation of the engaged and clustered TCRs with respect to the above surface (Ortega et al., 1988). While the rigor of quantitative studies of MHC-peptide interactions on intact cells is somewhat limited experimentally, other problems have confronted investigations using soluble class I or I1 products. One is the relatively limited stability of these molecules in the “empty,” i.e., peptide-free, state and another involves the use of detergents. Still, the availability of recombinant-soluble class I molecules like the H2-Kd molecule, which exhibits considerable stability in the empty state, complemented studies done on intact cells and substantiated their validity (Fahnestock et al., 1994). Furthermore, extensive studies on peptide-class 11 binding kinetics have been carried out by McConnell and associates (Beeson and McConnell, 1994; Witt and McConnell, 1994; de Kroon and McConnell, 1994). This work showed that both peptide association to and dissociation from class I1 sites are slow (5 hr < t l / z < 100 hr). While the slow rate of binding is attributed to the required dissociation of the site-occupying peptide, the dissociation rate was found to be relatively constant and insensitive to the structure of the bound peptide (Witt and McConnell, 1992). The mechanism of binding is thus complex and involves both short- and long-lived peptide-protein complexes. The short-lived complex is probably an intermediate transformed into the long-lived species in a monomolecular step. Significantly it is the latter state of the complex that is assumed to be recognized immunologically. It was hrther proposed that formation of these intermediary species is a general feature of peptide-class I1 binding mechanism and that some of these species are due to two peptides being transiently associated with one protein molecule. Properties of the peptide-MHC class I complexes have also been probed by studying their intrinsic fluorescence as well as by monitoring

316

MICHAEL SELA A N D ISRAEL PECHT

fluorescence resonance energy transfer (Gakamsky et al., 1995; Catipovic et nl., 1994). While an expanded groove conformation was proposed for the empty state, it becomes more compact on peptide binding. An intriguing issue that emerges in considering the mode of peptide binding to class I and particularly class I1 MHC molecules is whether their grooves can accommodate molecules other than peptides. This question gathers considerably more significance in view of observations that T-cell response can be dramatically modulated even by minor changes in the sequence of bound peptides. The ability of class I1 sites to accommodate relatively long peptides has been shown to extend even to peptides with constrained conformations such as the loop peptide (residues 64-82 of hen egg white lysozyme), which is disulfide-bonded in addition to containing two prolyl residues (Visser et al., unpublished, 1990). Some evidence for the binding of ligands not derived from polypeptides emerged from studies on I-A extracts (Demotz et al., 1989; Rudensky et al., 1991). Moreover, helper T-cell reactivity toward streptococcal carbohydrates (Jackson et al., 1984) and evidence for direct binding of DNA to class I1 molecules (Mozes et al., 1996)clearly raise the interesting possibility of rather distinct molecular entities being bound and presented, at least on the latter MHC gene products. A different though important type of antigenic epitopes being presented by both class I and I1 molecules have structural elements that protrude from the bound peptide. Thus, chemically modified peptides bound to MHC molecules were shown to be the epitopes recognized by some specific T cells. In addition to the fundamental importance of this finding, there are obvious bearings on a wide range of drug and chemically induced allergic and autoimmune diseases. It also provides a rationale for T-cell reactivity toward transition metal ions coordinated to peptides (Von Bonin et nl., 1992; Martin et al., 1992). Moreover, the complexity of T-cell recognition of antigen has recently been illustrated by data showing that tails of MHC-bound peptides can also interact with the T-cell coreceptor CD4, increasing the affinity of these multipoint interactions (Vignolli and Strominger, 1994).

ANTIGENS VIII. THYMUS-INDEPENDENT

Most antigens are thymus-dependent; i.e., they require the cooperation of T and B cells for efficient antibody formation. Thus, soluble protein antigens are classified as T-dependent antigens because they do not elicit antibody responses in the absence of T-lymphocyte help, which may involve the presence of either T lymphocytes or T lymphocyte-derived factors (Mosier and Subbarao, 1982; Fitch et al., 1993).This is most probably

THE NATURE OF 'I'HE ANTIGEN

317

correct for cytotoxic T cells also, which may equally need helper T cells for an efficient, purely cellular response. On the other hand, T-independent antigens are those that can activate B lymphocytes in the absence of T lymphocytes (more precisely after depletion of T lymphocytes by the best methods available at the time of the experiment). There are two types of T-independent antigens. Bacterial lipopolysaccharides have intrinsic mitogenic activity and can cause polyclonal activation of murine B lymphocytes; they have been classified as type 1 T-independent antigens. Responses to this type of antigen probably require cytokines either provided by macrophages or produced by B lymphocytes in an autocrine fashion. T-independent antigens of type 2 are large polymeric molecules that contain multiple repeating antigenic epitopes (Feldmann and Basten, 1971) and are metabolized either not at all or only very slowly (Sela et al., 1972). This category includes many polysaccharides (pneumococcal polysaccharides, dextran, Ficoll, levan), polymerized flagellin, polymers of o-amino acids, and polyvinylpyrrolidone. Type 2 T-independent antigens may not be entirely independent of T-cell help since small numbers of T lymphocytes might greatly augment their response (Mond et al., 1980). The suggestion that repeating antigenic subunits are required for an immunogen to be T-independent (Feldman and Basten, 1971) may be a necessary but insufficient requirement for thymus independence since several multichain synthetic polypeptide immunogens, all possessing repeating antigenic determinants, need both thymus- and bone marrowderived cells to elicit efficient humoral immune responses (e.g., Shearer et al., 1972). The response to a multichain amino acid copolymer composed mostly of proline was T-dependent when the copolymer was composed exclusively of L-amino acids but totally T-independent when it was built entirely from D-amino acids. When a branched polymer was investigated in which peptides of L-phenylalanine and L-glutamic acid were attached to multichain poly-wproline, the response to the determinants with only Lamino acids on the outside was T-dependent, whereas the response to the inside region with only D-amino acids was T-independent (Sela et al., 1972). An important feature of T-independent antigens is a slow rate of metabolism, which results in the persistence of detectable amounts of antigens in macrophages of the spleen and lymph modes several weeks after immunization (Janeway and Humphrey, 1968; Medlin et al., 1970; Van den Eertwegh et al., 1992). The synthetic ordered polymer of the tripeptide L-prolylglycyl-L-proline, designated (Pro-Gly-Pro)", resembles collagen in its three-dimensional structure both in solution (Engel et al., 1966) and in the solid state (Traub and Yonath, 1966). Antibodies to this collagen-like polymer cross-react with several collagens (Maoz et al., 1973) but not with a random copolymer of proline and glycine similar to the

318

MICHAEL SELA AND ISR4EL PECHT

ordered polymer in its amino acid composition but not collagen-like. Fuchs et al. (1974) have found that the collagen-like (Pro-Gly-Pro) is T-independent, whereas the random polymer is T-dependent. Similarly, the native collagen was found to be T-independent, whereas in order to elicit a response to its denatured product, gelatin, thymus cells were required. Can one establish T-cell hybridomas specific to T-independent antigens? Zisman et al. (1993) described the establishment and characterization of T-cell hybridomas specific to the T-independent branched polymer composed exclusively of D-amino acids and compared them to T-cell hybridomas specific to the polymer of the same composition and structure built exclusively of L-amino acids and thus T-dependent. The T-independent wpolymer is presented to T cells by Ia molecules on antigen presenting cells, and the affinity of interaction with Ia molecules is similar to that of the T-dependent L polymer. Incubation with the D-polymer induces increased expression of membranal Ia molecules similar to the response reported for other T-independent antigens, namely, trinitrophenylated Brucella abortus (Vitetta et al., 1987) and antibody-conjugated dextran (Brunswick et al., 1989). The D-polymer has a faster kinetics of presentation than the L-polymer. This faster kinetics may explain the antigenic competition between stretches of D- and L-peptides within poly(DL-peptidyl) proteins, which elicited predominantly antibodies toward D isomers following immunization with poly(n1.-alanyl, tyrosyl, or phenylalanyl) proteins (Schechter and Sela, 1967). Rigorous T-cell depletion may completely abrogate the antibody response to T-independent antigens of type 2, indicating that T cells may be necessary for a bona fide response (Mond et al., 1980, 1983; Endres et al., 1983; Van den Eertwegh et al., 1993). The ligand for CD40 is gp39, and it is essential for T helper cell-dependent B-cell activation. Immunization with the T-independent trinitrophenyl-Ficoll led to a high frequency of gp39+ T cells (Van den Eertwegh et al., 1993). In the case of the Tindependent response of type 2 the process of T-cell activation could be explained by activated B cells being responsible for the activation of T cells. These results are similar to those described earlier from the work of Zisman et al. (1993) who showed that T-independent type 2 antigens were able to activate T cells. Snapper et al. (1994) recently suggested an in uitro model for T-independent induction of humoral immunity based on the requirement for NK cells. In this model in vitro-activated NK cells stimulated cells to secrete antibodies in the absence of any added cytokine. Polyvinylpyrrolidone is a typical T-independent type 2 antigen that is immunogenic in both enthymic and athymic nude mice (Anderson and Blomgren, 1971; Lake and Reed, 1976). Helper T cells activated by polyvinylpyrrolidone could be prepared, and they are similar in cell surface


319

phenotype to classic TH1 cells (Van Buskirk and Braley-Mullen, 1987). However, by several criteria they are not identical. Activation of polyvinylpyrrolidone-specific helper T cells requires that B cells be present during the first few weeks after the birth of the mice (Braley-Mullen et al., 1994). The examples of specific T cells against type 2 T-independent antigens just given lead to many questions which may be resolved only on additional experimentation.

Ix.

SUPERANTIGENS

No discussion of antigens would be complete without mentioning superantigens. The term superantigen was introduced to describe a group of microbial antigens that differ in several respects from conventional protein or peptide antigens (White et d.,1989). Most importantly, the recognition of superantigens by TCR appears to depend almost entirely on the variable domain of the TCR chain (Va),with little regard for the other diversity components (Kotzin et al., 1993). Because the relative number of Va genes is limited, a given superantigen is capable of interacting with a large fraction (5-30%) of the T-cell repertoire, whereas the corresponding response to a conventional antigen is usually much less than 1 in 1000. Like peptide antigens, superantigens are presented by class I1 MHC molecules, but they do not engage the peptide groove. Instead the intact, unprocessed superantigen interacts with conserved amino acid residues on the outside of the peptide-binding cleft. Polymorphic differences in MHC that affect peptide binding do not usually affect superantigen binding or presentation to TCR Va, and recognition of superantigens is not normally MHC-restricted (Kotzin et al., 1993). Two types of superantigens have been studied in detail. One type is the so-called minor lymphocyte-stimulating (Mls) antigens (to differentiate them from MHC antigens) first described by Festenstein (1973). These superantigens are encoded by endogenous retroviral genes [in the case of the MISfirst described, it was the product of mouse mammary tumor virus Uaneway, 1991)]. The other type of superantigen includes certain bacterial, mycoplasmal, and viral proteins, of which staphylococcal and streptococcal enterotoxins have been the most studied. Other members of this class include streptococcal M protein and mycoplasma arthritidis mitogen (Kotzin et al., 1993). T-cell receptor-MHC class I1 interaction is required for the T-cell response to bacterial superantigens (Labrecque et al., 1994). Both bacterial and retroviral superantigens share a common binding region on class I1 MHC antigens (Torres et al., 1993). Avery et al. (1994) have described a

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MICHAEL SELA AND ISRAEL PECH7

novel MHC-independent T-cell activation pathway for some bacterial superantigens, such as staphylococcal enterotoxins C and E, that leads to both clonal expansion and expression of CTL effector function, in class IInegative mice. Staphylococcal enterotoxin superantigens, as mentioned earlier, bind class I1 MHC molecules on antigen-presenting cells and on cell-to-cell contact stimulate proliferation of T cells expressing appropriate Vcl gene products. In addition, they can also deliver negative signals to antigen-specific T cells, resulting in a state of unresponsiveness or a loss of viability (Miethke et al., 1993). In some cases the staphylococcal enterotoxin superantigen induces programmed death (apoptosis) in a majority of antigen-specific CD4+ T cells accompanied by genomic DNA fragmentation (Damle et al., 1993). A better knowledge of superantigens has been obtained through elucidation of the crystal structure of staphylococcal enterotoxin B (Swaminathan et a,l., 1992). The unusual main-chain fold containing two domains may represent a general motif adopted by all staphylococcal enterotoxins. The TCR binding site encompasses a shallow cavity formed by both domains, and the MHC class I1 molecule binds to an adjacent site. The crystal structure of another superantigen, toxic-shock syndrome toxin- 1 (TSST-1), has also been reported (Acharya et al., 1994). Despite low sequence conservation, the TSST-1 topology is similar to the structure of the staphylococcal enterotoxin B mentioned earlier, but TSST- 1 lacks some structural features considered central to superantigen activity in the staphylococcal enterotoxin B. A more advanced study, on the three-dimensional structure of a human class I1 molecule complexed with staphylococcal enterotoxin B, has been reported (Jardetzky et al., 1994). The enterotoxin binds to t h e p l domain of class I1 molecules, positioning a TCR binding site above and to the side of the MHC peptide binding site. Antigenic peptides are not inhibitors of superantigen stimulation, and the structure demonstrates that the peptides and the enterotoxin occupy two distinct regions of the class I1 MHC molecule. Jardetzky and co-workers infer that the interaction of related superantigens with class I1 molecules may differ from that reported by them, as different superantigens do not all cross-compete in binding studies. Indeed, a subsequent study (Kim et al., 1994) showed the crystal structure of TSST-1 complexed with a human class I1 MHC molecule and found that the two binding modes differed. Superantigens play a crucial role in certain human diseases such as toxic shock syndrome, toxicity induced by bacterial toxins, and possibly even some autoimmune diseases such as Kawasaki syndrome, rheumatoid arthritis (Drake and Kotzin, 1992), and even insulin-dependent diabetis mellitus (Conrad et al., 1994). The role of superantigens in human diseases is not yet understood, and in many cases probably not even realized, but as


32 1

it is clear that superantigens have a dramatic effect on the immune system, it can be predicted that they may also have immunomodulatory effects that

might be beneficial for human health.

X. TUMOR ANTIGENS

A promising way to combat cancer-besides surgery, radiotherapy, and chemotherapy-is immunotherapy . This treatment includes efforts to increase immunity in a nonspecific way as well as specific immunotherapy, in which one uses either antibodies or T cells with a specificity toward antigens on tumor cells, or conjugates of such antibodies with drugs, toxins, or radioactive molecules for immunotargeting (Vogel, 1987; Pietersz et al., 1994), thus combining immunotherapy with radiotherapy or chemotherapy. The main problem in this approach is the search for truly tumorspecific antigens. One has to distinguish them from organ-specific antigens and histocompatibility antigens. In most cases studied, the antibodies used were against antigens present in much higher density on tumor cells as compared with normal cells, but they were never totally absent from the latter cells. Such antigens would therefore be more correctly defined as tumor-selective antigens. Tumor antigens are classified according to the origin of the tumor: experimentally induced (by chemical, physical, or viral carcinogens) or spontaneous. The category of oncodevelopmental antigens is of special interest. These antigens, exemplified by a-fetoprotein and by the carcinoembryonic antigen of the colon, are present in normal individuals before birth but disappear thereafter (or stay at an extremely low level) and reappear in the body fluids in the adult only concurrently with specific cancer diseases. Their quantification may therefore be of diagnostic value. Before discussing the few tumor antigens, described only in the last few years, that seem indeed to be tumor-specific, a few examples will be given from our own work on the successful use of antibodies against some tumorselective antigens in experimental in vivo studies. This may serve as an illustration of several similar studies reported in recent years. One example is the epidermal growth factor receptor, overexpressed in various types of human cancers such as epidermoid and squamous cell carcinomas and gliomas. A monoclonal antibody against the receptor was efficient against KB carcinoma in vitro and in nude mice (Aboud-Pirak et al., 1988). Another example is the Neu/ErbB-2 receptor tyrosine kinase (Stancovski et al., 1994). The monoclonal antibodies prepared against it induced either positive or negative growth effects on tumor growth in athymic mice (Stancovski et al., 199 1). A correlation between the growth-stimulating ef-

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fect of MAb N28 in viuo and activation of the tyrosine kinase function of the receptor was found. The MAbs that inhibited tumor growth had little effect on Neu phosphorylation but specifically induced phenotypic cellular differentiation that included markedly altered cytoplasm and nuclear morphology, synthesis and secretion of milk components (casein and lipids), and translocation of the Neu/ErbB-2 protein to cytoplasmic and perinuclear sites (Bacus et al., 1992). While these antibodies were demonstrated to have a tumor-inhibiting capacity, the inefficient accessibility of antibodies to solid tumors limits their clinical use. To redirect effector lymphocytes to tumor cells, we have constructed and functionally expressed in T cells chimeric single-chain receptor genes incorporating both the antigen binding domain of anti-NeuIErbB-2 antibodies and a transducing subunit of TClUCD3 complex. The resulting T cells, equipped with anti-NedErbB-2 specificity as the ligand binding domain of the chimeric receptor, respond specifically to NedErbB-2-bearing target cells (Stancovski et al., 1993). Several truly tumor-specific antigens have been described-proteins that seem to appear on tumor cells but not on healthy ones (Boon et al., 1994; Tsomides and Eisen, 1994).Attempts to identify the target antigens by biochemical fractionation of tumor cells have failed thus far, with the important exception of the identification of underglycosylated mucins present on breast and pancreatic carcinomas. Gene transfection approaches have proved more successful. A gene family named MAGE codes for antigens recognized by autologous CTL on a melanoma tumor (van der Bruggen et al., 1991; Traversari et al., 1992; Wolfel et al., 1993). These genes are not expressed in normal tissues except the testes. One gene, MAGE- 1, has been isolated (van der Bruggen et al., 199I), and the protein MAGE- 1 has been found to be located in the cytosol of human melanoma cells (Amar-Costesec et al., 1994). A peptide encoded by human gene MAGE-3 has been shown to induce CTLs that recognize tumor cells expressing MAGE-3 (van der Bruggen et al., 1994).When immunized with a melanoma cell vaccine, melanoma patients produced antibody responses to recombinant MAGE-1 antigen (Hoon et al., 1995). Using similar techniques, Kawakami et al. (1994) have reported on the gene (MART-1) for a shared melanoma antigen recognized by tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. MART-1 RNA was detected in melanocytes and melanomas, but not in other cell types, with the exception of retina cells. Identification of the gene for an antigen expressed by most melanomas and recognized by TILs in association with A2 represents another important advance in understanding and perhaps manipulating antitumor immune responses. Thus, hopes are raised again that we are close to the moment when the armamentarium of antigens for combating cancer will be sufficiently developed to undertake specific

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immunotherapy of this disease with vaccines (Bystryn et al., 1993). Roth etal. (1994) have prepared a review article on the immune response against tumors.

XI, CONCLUDING REMAKKS

The progress of immunology in the last few years has been enormous. This sentence could have been written anytime in the last 30 years and always be true. What is changing are the main subjects of this progressthey have moved from antigens to antibodies to immunocytes. The tremendous amount of knowledge acquired about T cells moves now to similar goals concerning B and NK cells. After achieving exciting results concerned with the nature of the signals triggering these cells, our sophistication aims at understanding in detail the actual steps taking place within the activated cells. Notwithstanding all this progress, within the context of this article it is worth stressing that antigenic stimulus is the key factor in any immune response and that the role and nature of the antigen will always play a central role in immunology.

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Progression” (R. Dickson and M. Lippman, eds.), pp. 161-191. Kluwer Academic, Boston. Stanfield, R. L., Takimoto-Kamimura, M., Rini, J. M., Profy, A. T., and Wilson, I. A. ( I 993). Structure 1, 83-93. Stern, L. J., Brown, J. H. Jardetzky, T. S. Gorga, J. C. Urban, R. G., Strominger, J. I.., and Wiley, D. C. (1994). Nature 368,215-221. Swaminathan, S., Furey, W., Pletcher, J., and Sax, M. (1992).Nature 359, 801-805. Swat, W., von Boehmer, H., and Kisielow, P. (1994). Eur.J. Immzmol. 24, 485487. Taussig, M. J., Mozes, E., Shearer, G. N., and Sela, M. (1973). Cd1. Immunol. 8, 299-3 10. _ . lello, D., Eisenstein, E., Schwarz, F. P., Goldbaum, F. A., Fields, B. A,, Mariuzza, R. A,, and Poljak, R. J. (1994).J. Mol. Recogn. 7, 57-62. Todd, P. E. E., East, I. J., and Leach, S. J. (1982). TIES 7, 212-216. Traub, W., and Yonath, A. (1966).J. Mol. Biol. 16, 404-414. Traversari, C., van der Bruggen, P., Van den Eynde, B., Hainaut, P., Lemoine, C., Ohta, N., Old, L., and Boon, T. (1992). Immunogenetics 35, 145-152. Tsomides, T. J., and Eisen, H. N. (1994). Proc. Natl. Acad. Sci. U.S.A. 91, 3487-3489. Van Buskirk, A. M., and Braley-Mullen, H. (1987).J. Immunol. 138, 1031-1037. Van den Eertwegh, A. J. M., Laman, J. D., Schellekens, M. M., Boersma, W. J. A,, and Claassen, E. (1992). Eur.J. Immunol. 22, 719-726. Van den Eertwegh, A. J. M., Noelle, R. J., Roy, M., Shepherd, D. M., Aruffo, A,, Ledbetter, J. A., Boersma, W.J. A., and Claasen, E. (1993).J. Exp. Med. 178, 1555-1565. van der Bruggen, P., Bastin, J., Gdjewski, T., Coulie, P., Boel, P., De Smet, C.,lraversari, C., Townsend, A,, and Boon, T. (1994). Eur.1. Immunol. 24, 3038-3043. van der Bnrggen, P., Traversari, C., Chomerz, P., Lurguin, C., De Plaen, E., Van den Eynde, B., Knuth, A,, and Boon, T. (1991). Science 254, 1643-1647. Vignolli, D. A. A., and Strominger, J. L. (1994).J. Exp. Med. 179, 1945-1956. Vitetta, E. S., Bossie, A., Fermandez-Botran, R., Myers, C. D., Oliver, K. G., Sanders, V. M., and Stevens, 7‘.L. (1987). Immunol. Rev. 99, 193-239. Vogel, C.-W. (ed.) (1987). “lmmunoconjugates.” Oxford University Press, Oxford. Von Bonin, A., Ortmann, B., Martin, S., and Weltzien, H. U. (1992) Int. Iirimunol. 4, 869. Vuk-Pavlovic, S., Blatt, Y., Glaudemans, C. P. J., Lancet, D., and Pecht, I. (1978). Riop/~ys.J. 24, 161-174. White, J., Herman, A., Pullen, A. M., Kubo, R., Kappler, J . W., and Marrack, P. (1989).Cell 56,27-35. Wilson, I. A,, and Stanfield, R. L. (1994). C u m Opin. Stmet. Riol. 4, 857-867. Witt, S. N., and McConnell, H. M. (1992)./. Am. Chem. Sac. 114, 3506-3511. Witt, S. N., and McConnell, H. M. (1994). Biochemzctry 33, 1861-1868. Wolfel, T., Hauer, M., Klehmann, E., Brichard, V., Ackermann, B., Knuth, A., Boon, ‘I.., and Meyer m m Buschenfelde, K.-H. (1993). Znt.J. Cancer 55, 237-244. Zhang, W., Young, A. C. M., Imaral, M., Nathenson, S. G., and Sacchetini, J. C. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 8403. Zidovetzki, R., Blatt, Y . , Glaudemans, C. P. .J., Manjula, B. N., and Pecht, 1. (1980). Biochemi.ytq 19, 2790-2795. Zisman, E., Dayan, M., Sela, M., and Mozes, E. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 994998.

ANTIBODY BINDING SITES By JAMES S. HUSTON,’ MICHAEL N. MARGOLIES,t and EDGAR HABER* ‘Creative BioMolecules, Inc. Hopkinton, Massachusetts01748 tDepartment of Surgery MassachusettsGeneral Hospital and Harvard Medical School Boston, Massachusetts02114 *Cardiovascular Biology Laboratory Harvard School of Public Health, and Department of Medicine, Harvard Medical School Boston, Massachusetts02115

I. Overview . . . . . 11. Protein Chemist A. Antibody Fragmentation and Chain Separation. . . . . . B. Refolding and Reassembly of Combining Site , . . . . . . . . . . . . . . . . . . . . . . . C. Antibody Paradigms and Analysis of Primary Structure . . . . . . . . . . . . . . . . . D. Immunoglobulin Shape and Domain Structure E. Immunogenetics of Antibody Formation 111. Engineered Antibody Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Architecture of Fv and Design of sFv Analog . . . . . . . . . . . . . . . . . . . . . . . . . B. Studies of Fv and sFv Proteins ..... ..................... C. Targeting in Vivo by Antibody Sites. D. Binding Equilibria and Linkage in Antibody IV. Antibody Combining Site Structure: Antiarsonate and Antidigoxin A. Antiarsonate Response in Inbred Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Crystal Structure of Combining Site of Fab 36-71 ..... C. Site-Directed Mutagenesis Studies of Antiarsonat Contact Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Enhancement of Antiarsonate Antibody Affinity by Mutation of Noncontact Residues ... .................. E. Certain Mutations of Antiarsonate Antibodies Engineered ex Vho that Confer Increased Affinity Are Not Observed in Vivo. . . . F. Engineering Changes in Antiarsonate Antibody Specifici G. Structure of Digoxin Hapten and Analogs, and Utility as Model System. . . . H. Diversity among Antidigoxin Antibodie I. Variants of Antidigoxin Antibody 40 J. Structure and Mutagenesis of Antidi K. X-Ray Crystal Structure and Binding Specificity of Antidigoxin Antibody 40-50: Comparison of Structures and Binding Modes of Two Antidigoxin Antibodies, 40-50 and 26-10 V. Enhancing Enzyme Selectivity with Substrate-Selec A. Chemically Cross-linked Antibody-Enzyme Conjugates. . . . . . . . . . . . . B. Bispecific Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Antibody-Enzyme Fusion Proteins D. Targeted Prodrug Activation. . . . . E. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ADVANCES IN PROTEIN CHEMISTRI’, Vol. 4 9

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330 330 331 333 34 1 343 346 347 349 358 373 377 380 380 381 383 386 390 392 397 398 400 403

42 1 427 428 430 43 1 437 439 439

Copy)-ight0 1996 by Academic Press, Inc. All righta uf~eproductionin any form reserved.

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I. OVERVIEW Recombinant antibodies and their engineered fragments offer remarkable opportunities for theoretical study and practical development (Haber, 1992). Many facets of protein chemistry are integral to antibody engineering, as they have been to immunology (Porter, 1970; Nezlin, 1991). This chapter assesses advances in the understanding and manipulation of antibody binding sites and variable regions. In certain cases, current research is viewed in the context of early discoveries about antibodies to emphasize the continuity of protein chemistry in the field of immunology. This perspective can reveal important generalizations that transcend the limits of studies based solely on polyclonal, monoclonal, or engineered antibodies.

11. PROTEIN CHEMISTRY OF ANTIBODY FRAGMENTS AND ANHGEN BINDING REGIONS Our understanding of antibody structure has grown progressively more refined with advances in protein chemistry, molecular biology, and cellular immunology. The complexities of antibodies have been deciphered by applying a reductionist approach through which the immunoglobulin structure is simplified in stages to reveal its molecular organization. Antibody engineering has reached the vanguard of immunology by combining insights about the three-dimensional structure of antibody fragments with the ability to manipulate genes encoding subregions of antibodies and express them in forms that yield native protein. In 1940 Linus Pauling made one of the earliest efforts at developing a comprehensive theory of antibody structure and formation. It was based on a minimum of tenets, including the correct assumption that serum globulin of the 157,000 molecular weight class had a binding valency of 2 (Pauling, 1940). However, use of a simple model for antibody diversity led him to the incorrect proposition that “all antibody molecules contain the same polypeptide chains as normal globulin, and differ from normal globulin only in the configuration of the chain; that is, in the way that the chain is coiled in the molecule.” Subsequent experiments from his laboratory supported this hypothesis, apparently because specific and nonspecific interactions between antigen and immunoglobulin were indistinguishable (Pauling and Campbell, 1942). The mechanism of antibody diversity remained undetermined for several decades more as protein chemistry and molecular immunology developed the sophistication necessary to analyze antibody structure-fimction relationships. Dissection of the antibody molecule was central to solving

ANTIBODY BINDING SITES

33 1

the mystery of its capacity for binding diversity, as were concurrent breakthroughs in understanding the structure and renaturation properties of proteins. Key concepts that emerged included an appreciation for the hydrophobic effect in protein folding (Kauzmann, 1959) and an understanding of the procedures involved in refolding ribonuclease (Anfinsen et al., 1961; Anfinsen and Haber, 1961; Haber and Anfinsen, 1961, 1962).The antibody folding problem was framed by denaturation and renaturation studies of immunoglobulin proteins under conditions that kept disulfide bonds intact (Buckley et al., 1963; Noelken and Tanford, 1964).The more demanding but critical experiments were subsequently performed, demonstrating the recovery of antibody binding activity from fully reduced and denatured antibody fragments (Haber, 1964; Whitney and Tanford, 1965a,b). The latter work contributed to our understanding of the basis of antibody diversity (see Section 11,B).Polyclonal antibodies were used in these early investigations. The homogeneous immunoglobulin proteins that became available during this period, as accidents of human disease or as nonspecific products of experimentally induced mouse plasmacytomas, proved to be more uniform substrates for antibody binding experiments. These reagents predated, by decades, the tailored antibody binding sites now available from hybridomas, transfectomas, and combinatorial libraries. A. Antibody Fragmentation and Chain Separation The preferential susceptibility to proteolysis exhibited by the antibody heavy (H) chain hinge region is a fortuitous aspect of immunoglobulin structure that has had an enormous impact on our ability to study antibody structure. Work on chain separation dates back to the investigations of Petermann and Pappenheimer on the crude enzymatic digestion of native antibodies (Petermann and Pappenheimer, 1941; Petermann, 1942, 1946), which led to the first study by Rodney Porter on the papain hydrolysis of rabbit antibodies to ovalbumin (Porter, 1950). The enzyme preparations of that era were so impure and separation methods so cumbersome as to preclude accurate measurement of antibody digestion products. Nevertheless, Petermann showed that crude papain could digest the antibody into fragments of roughly one-quarter of its original size (Petermann, 1946), while Porter (1950) found that the products of antiovalbumin antibody fragmentation inhibited ovalbumin aggregation mediated by intact antiovalbumin antibody. In the years that followed Porter studied various aspects of antigen-antibody interactions (Porter, 1957), improved his techniques for fractionating rabbit y-globulin and antiovalbumin (Porter, 1955), and improved his enzyme cleavage experiments. Better separation methods and pure crystalline papain finally allowed him to carry his early

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JAMES S. H U S I O N ET AL.

study (Porter, 1950) to a decisive conclusion. In 1958 Porter published the first proof of the tripartite structure of immunoglobulin G (IgG), on the basis of his successful fractionation of its papain digestion products (Fig. l), which he described as follows (Porter, 1958): These results suggest that the gamma-globulin molecule may be made up of three parts: one of these (my crystallinefraction 111) is identical in all gamma-

globulin molecules and is responsible for the common antigenic specificity of gamma-globulins observed by Treffers and Heidelberger (see Treffers, 1944).The other two parts [fractions I and 111presumably contain the antibodycombining centres, and are also responsible for the physical heterogeneity of gamma-globulins; these parts may be expected to vary from molecule to molecule. This suggested structure of an identical section in all molecules of gamma-globulins and two variable sections which contain the antibodycombining sites is very similar to the picture drawn by Pauling in 1940.

The complete details of this investigation were published several months later (Porter, 1959). However, some critical aspects of antibody structure remained unclear, in part because N-terminal sequence data were consistent with only a single type of polypeptide chain. This confusion arose from a now well-recognized phenomenon that the light (L) chain N-terminus is typically amenable to end group analysis and sequencing but the H chain

0

160 320 480 640 800 960 1120 1280 Volume of eluate (ml)

FIG.I. Fractionation of a papain digest of rabbit y-globulin. Separation of Fab (fragments I and 11) and Fc (fragment 111) by gradient elution of digest adsorbed on a carboxymethylcellulose column. Sodium acetate, pH 5.5, gradient 0.01-0.9 M . (Reproduced with permission from Porter, 1958. Nature 182, 670-671. 0 1958 Macmillan Magazines Ltd.)

333


N-terminus is not, as it is usually blocked and unreactive in the Sanger or Edman sequencing procedure. At this time, Edelman discovered that the molecular weight of human IgG in denaturant was dramatically lowered on the addition of a reducing agent (Table I), suggesting “that human y-globulin contains subunits linked at least in part by disulfide bonds” (Edelman, 1959).Edelman and Poulik (1961) then isolated two distinct polypeptide chains from reduced and denatured protein after chromatography on carboxymethyl cellulose in 6 M urea, and the Porter group completed analysis of the polypeptide chain organization of rabbit IgG (Fleischman et al., 1963; reviewed by Porter, 1970). Pepsin was shown to attack the hinge region differently than did papain, cleaving residues C-terminal to the hinge region disulfide bonds to yield a bivalent fragment of 100,000 molecular weight (Nisonoff et al., 1960). Fragments I and I1 (described by Porter as fractions I and I1 in 1958) proved to be functionally similar mixtures of polyclonal antigen binding fragments. After 1965 fragments I and I1 were generally called Fab fragments. Fragment I11 (fraction I11 in Porter) became known as the Fc fragment, being crystallizable and complement binding. B. Refolding and Reassembly of Combining Site

Denaturants were used not only to separate immunoglobulin chains but were also applied in protein denaturation and renaturation studies to clarify the relation between the structure and antigen-binding properties of antibodies. In the early 1960s it was still debated whether binding sites T.4BLE I

Effect of Vurious Reagents on M,pp (1 - ii#) Vulues of Human y-Globulin‘ M a p p (1 - Vp) k standard deviation

Solventb

0.2 M KC1 6 M urea + 0.2 M KC1 0.1 M MEA + 0.2 M KC1 0.1 M M U + 6 M urea + 0.2 M KCI Reduced in 8 M urea 0.1 M M U , next dialyzed against 6 A4 urea + 0.02 M iodoacetamide, then 6 M urea 0.2 M KCI

+

(4.8 f 0.1) x (3.0 f 0.3) x (3.5 f 0.1) x (0.93 k 0.07) x (0.92 0.05) x

*

lo4 lo4 lo4 lo4 lo4

MaPP 192,000 158,000 140,000 48,000 48,000

+

‘ This sample of Cohn fraction I1 (Lederle) contained a small amount of heavy material sedimenting faster than the main 7s component. Reproduced with permission from Edelman, 1959.J. Am. Chem. Sac. 81, 3155-3156. 0 1959 American Chemical Society. b MEA, P-mercaptoethylamine hydrochloride.

334

JAMES S . HUSTON E T AL.

differed strictly through variation in H and L chain sequences or through induction of antigen-specific conformations during folding. The proposition was resisted that every binding site to a distinct antigen differed in polypeptide sequence because it required the existence of an incredibly large number of proteins for a normal repertoire of antibodies to form in an individual. This proposition would have appeared more reasonable to immunochemists had they fully accepted the implications of the successful refolding of ribonuclease, which proved that primary sequence was sufficient for recovery of its unique native conformation and enzymatic activity. The fact that ribonuclease was a small, thermostable, globular protein of about 13 kDa, however, led many skeptics to postulate that its refolding did not reflect a general principle of protein chemistry but only a phenomenon peculiar to a subset of proteins. The wide acceptance of the Karush hypothesis (1958),which stated that antigen combining site diversity depended on permutations of disulfide bonding, indicated how little serious consideration was given to the proposition that each antibody refolded uniquely, with each distinct binding site the product of a different polypeptide sequence. In the 1960s the availability of monovalent antibody fragments was coupled with advances in physicochemical methods for monitoring protein structure and improved techniques for measuring antigen-antibody interactions. This climate produced a marked decline in the use of classical serological techniques for analyzing immunoglobulin structure and allowed rigorous studies of antibody renaturation that began from fully reduced and denatured monovalent antibody fragments (Haber, 1964; Whitney and Tanford, 1965a). This work is discussed in the following sections on refolding Fab fragments derived from antibodies to the small enzyme ribonuclease or to the dinitrophenyl (DNP) hapten. 1. Refolding of Antiribonuclease Fab

The insights from definitive research on ribonuclease structure and folding made it an attractive reagent for use in studies on antigen-antibody interaction. The association of fluorescein-labeled antigens with antibody could be monitored, and differences in fluorescence polarization could be used to distinguish aggregates from free antigen (Haber and Bennett, 1962). Spectrofluorimetry in conjunction with chromatography was also used to monitor the distribution of free and complexed antigen (Bennett and Haber, 1963). This research showed that antiribonuclease antibody could be purified from its precipitating ribonuclease antigen by size-exclusion chromatography under conditions that denatured the ribonuclease but left the antibody in its native state. This work also showed that any residual fluorescein-ribonuclease that might remain bound after immunoprecipitation could be detected with great sensitivity. For these studies, polyclonal y-globulin directed against ribonuclease was raised in rabbits. Papain

335


cleavage of the antiribonuclease antibody and ion exchange separation of its digestion products yielded a monovalent Fab population (fragment I or F- 1) specific for ribonuclease. Procedures were established that allowed sensitive measurement of Fab binding activity for 'z51-labeledribonuclease. Experiments were conducted to decide whether the primary sequences of the polypeptides in the antiribonuclease Fab were intrinsically capable of refolding into their native conformations with recovery of their original antigen-binding properties. Denaturation of the antiribonuclease Fab (F-1) was studied under various conditions to find those that guaranteed complete unfolding of the reduced polypeptide chains. As shown in the optical rotatory dispersion analysis in Fig. 2, 6 M guanidine hydrochloride (GuHCl) abolished all evidence of residual structure. The existence of

'

** 3000

-

.

*2wo

-

+loo0

-

n

[II

0.

U

-1000

-ZOO0

-3000

-

I ' 2 10

I

1

230

I

1

250

I

1

270

FIG. 2. Ultraviolet optical rotatory dispersion (ORD) of refolded antiribonuclease Fab fragments. Progressive loss of ordered protein structure was monitored by ORD of' antibody Fab fragments (fragment I, F-I) in native solution or under denaturing conditions, before or after reduction and carboxymethylation of free sulfhydryls, A, Anti-RNase F-I in 0.1 M NaC1; A,anti-RNase F-I in 8 M urea; 0,anti-RNase F-I reduced and carboxymethylated in 8 M urea; 0, anti-RNase F-I reduced and carboxymethylated in 6 M guanidinium chloride. [From Haber (1 964).]

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IAMES S. HUSTON E T AL

residual structure would undermine any proof of the hypothesis in question, as would the presence of antigen. The extent of cysteine reduction under a variety of denaturant conditions was monitored by alkylation followed by S-carboxymethylcysteineanalysis. The values obtained were compared with the moles of cysteic acid per mole of F-1,as determined by amino acid analysis of performic acid-oxidized F- 1 protein. This analysis indicated that 6 M GuHC1, and probably 10 M urea, fully exposed the F-1 sulfhydryls to reduction and alkylation. Data from the refolding and reoxidation of antiribonuclease Fab (F-1) under various conditions of denaturation and reduction are summarized in Table 11. Reoxidation was performed at a low protein concentration of 10 pg/ml, which recovered levels of binding activity that were several times higher than those obtained at higher concentrations of 45-90 pglml. The aggregation state of the renatured protein was also monitored on a sucrose density gradient, which indicated the presence of native Fab with a sedimentation coefficient of 3.2s.Although 56% of the expected binding activity was recovered for the protein that had been denatured in 8 M urea, there was 20% recovery for the antiribonuclease Fab that had been fully denatured and reduced in 6 M GuHCl containing 0.1 M /3-mercaptoethanol,

TABLE I1

Antigen Binding ofAnti-RNme F-I Reduced and then Oxidized with AiP Protein remaining in solutiond

Binding activity after oxidation'

Conditions of reduction6

Conditions of oxidation'

(a)

(%I

8 M Urea, pH 7.5

1 mM Mercaptoethanol, pH 8.0 Carboxymethylated 0.1 M Acetic Acid 1 mM Mercaptoethanol, pH 8.0 Carboxymethylated 0.1 M Acetic Acid 1 mM Mercaptoethanol, pH 8.0 1 mM Mercaptoethanol, pH 8.0

30

56

45 52 62

0 0 22

65 46 53

0

0 27

58

20

8 M Urea, pH 7.5 8 M Urea, pH 7.5

10 M Urea, pH 7.5 10 M Urea, pH 7.5 10 M Urea, pH 7.5

6 M Guanidinium chloride, pH 8.0 6 M Guanidinium chloride, pH 8.5

'"Reproduced with permission from Haber (1964).

* All reductions were done in 0.1 M mercaptoethanol.

' All oxidations were done at a protein concentration of 10 pgiml. Determined by radioactive counting. Expressed as a percentage of activity of an equal concentration of [acetyl-I4C]anti-RNase F-I.

ANIIUODY BINDING SITES

337

conditions under which the antiribonuclease Fab appeared to have lost all residual secondary structure (Fig. 2). This yield of activity was at least 400-fold higher than what would be predicted from a random reformation of disulfide bonds in the protein, leading to the following conclusion (Haber, 1964): “These findings make it highly likely that the amino acid sequence alone determines the conformation of the binding site, and that the specificity of an antibody must be determined at some time prior to protein synthesis.” This recovery of binding activity not only demonstrated renaturation of the component chains but also demonstrated their reassociation into functional binding sites, thus indicating that both tertiary and quaternary structure are derived from primary sequence. 2. Refolding of Antidinitrophenyl Fab Fragments

Refolding studies on monovalent antibody fragments were also being pursued in Charles Tanford’s laboratory. This group had first shown that Fab fragments recovered their antigen-binding activity if their original disulfide bonds were kept intact (Buckley et al., 1963); Fab (against bovine serum albumin) renatured, in the absence of antigen, from concentrated GuHCl under oxidizing conditions. For their primary model system, Noelken and Tanford (1964) purified rabbit Fab (F-1) from rabbit antidinitrophenyl hapten antibodies and likewise recovered DNP-binding activity after refolding of denatured but unreduced anti-DNP Fab. These experiments disproved Pauling’s hypothesis that antibody specificity arose from noncovalent bonds formed within the antibody in the presence of antigen. Fully reduced and denatured anti-DNP Fab was refolded, and significant binding activity was recovered, but with a much lower refolding yield than for the unreduced protein (Whitney and Tanford, 1965a). The physical properties of similarly renatured Fab fragments from nonspecific IgG were also characterized (Whitney and Tanford, 1965b). Renatured anti-DNP Fab preparations were titrated with DNP-lysine to assess binding properties by fluorescence quenching titrations (Fig. 3) (Velick et al., 1960; Eisen and Siskind, 1964). Refolding experiments were carried out on anti-DNP fragment I1 that had been both reduced and denatured before refolding, either in the absence (Fig. 3A) or presence (Fig. 3B) of hapten. The renatured anti-DNP fragment I, refolded from protein with intact disulfide bonds, showed strong quenching of fluorescence (Fig. 3C), with a steep linear profile at low hapten concentrations indicating the presence of high-affinity binding sites (Noelken and Tanford, 1964). In contrast with the profile in Fig. 3C, the shallow curvature of the fluorescence quenching in Fig. 3A suggests a loss of high-affinity binding sites when the original chain pairing was lost, which may have also contributed to the lower magnitude of quenching. When the denatured and re-

JAMES

70 -

s. HUSI'ON

E-r AL

Reoxidized nonspecific fragment I n

n

n

n

Y

0

65

751

I

0

0.05

I

I

I

0.10 0.15 DNP-lysine (ml lop4 M)

0.25

0.20

~

0

0.05

DNP-lysine (ml

0.15

0.10

M)

FIG.3. Fluorescence quenching titrations of refolded polyclonal Fab fragments against dinitrophenyl (DNP) hapten with DNP-lysine. (A) Duplicate DNP-lysine titrations of refolded and reoxidized nonspecific or antibody Fab (fragment 11). The upper curve represents a control experiment using nonspecific rabbit Fab (fragment I) that had gone through the same cycle of unfolding and refolding reactions as the antibody Fab fragment. (B) Titration of nonspecific Fab (fragment I ) and antibody Fab (fragment 11) after reoxidation in the presence of a 100-fold excess of DNP-lysine. The hapten was removed from the reoxidized

339


70 I I

11 Renatured Non-Specific 50 80

ff

I

I

I

I

I

I

F 70

E

d

1

60

Y

50 40

30

01

Renatured Anti-DNP

0.02 0.04 0.06 0.08 0.10 0.12 0.14 DNP-lysine (ml lop4 M)

protein before the titration was carried out. (C) DNP- lysine titration of y-globulin Fab (fragment I) refolded from the denatured state with disulfides kept intact. The top curve is for nonspecific Fab (fragment I) at a concentration of 0.017 g/lOO ml. The lower curve is for renatured anti-DNP Fab at a concentration of 0.018 g/lOO ml. [(A and B) from Whitney and Tanford (1965a); (C) from Noelken and Tanford (1964).]

duced chains of the anti-DNP fragment I1 were refolded in the presence of a 100-fold excess of DNP-lysine (Fig. 3B), the apparent affinity of the binding sites for hapten increased, presumably by enhancing productive chain pairing during refolding of the polyclonal mixture of anti-DNP L and Fd (the N-terminal half of the H chain). In related experiments, Cathou and Haber (1967) observed hapten stabilization of anti-DNP Fab exposed to 4 M GuHC1. They concluded that noncontiguous parts of the antibody molecule were involved in the binding of antigen, implying that discontinuous parts of each chain contribute to the combining site. 3. Refolding of Intact IgG

Although the renaturation of fully reduced and denatured IgG was a logical extension of Fab refolding studies, it represented a formidable tech-

340

IAMES S. HUSTON E'I' AL.

nical problem because of the high molecular weight and heterogeneity of the H and L polypeptides in polyclonal IgG preparations. Renaturation of reduced and denatured whole antibody was first attempted by Freedman and Sela (1966a,b) in studies on IgG modified by the addition to lysine &-amino groups of poly-DL-alanyl side chains. Complete reduction and denaturation of polyalanylated IgG in 8 M GuHCl showed improved solubility of the denatured H and L chains. When these derivatized polypeptide chains were renatured, antigenic determinants characteristic of nonspecific IgG reappeared (Freedman and Sela, 1966a).Also, specific antigenbinding activity was recovered from renatured anti-bovine serum albumin IgG (Freedman and Sela, 196613). The isolation of H and L chains followed by their recombination to form IgG under native conditions represented another approach to the problem of chain association. Bjork and Tanford (1971a,b) examined rabbit IgG in which the interchain disulfide bonds had been reduced and alkylated but the intrachain disulfide bonds had been left intact. The unmodified H and L chains were isolated from nonspecific rabbit IgG and characterized in isolation, demonstrating that the H chain existed as a stable dimer, whereas the L chain comprised a stable population of monomers and dimers. The reassembly of these H and L chain preparations under native conditions resulted in full recovery of IgG physical properties (Bjork and Tanford, 1971~). The study of chain recombination was next applied to H and L chain populations isolated from rabbit anti-DNP IgG. The DNP binding sites were analyzed in the parent antibody and in its isolated chains (Painter et al., 1972a), as well as in reconstituted IgG (Painter et al., 1972b). As suggested by previous experiments on the binding properties of renatured anti-DNP Fab (Whitney and Tanford, 1965a), when chain pairing was randomized during reconstitution the average affinity for DNP hapten dropped by a factor of about 1000. Another closely related experiment with polyclonal chains involved comparing homologous and heterologous chain recombinants, where the pairing of the anti-DNP H chain population with nonspecific L chains resulted in lower affinity than when an anti-DNP L chain population had been paired (Haber and Richards, 1966; Painter et al., 1972a). Calculation of binding constants from molal concentrations of reaction components yielded the average unitary free energy change on binding, which simplified interpretation of the recombination experiments (Painter et al., 197213). On the basis of this analysis, chain recombination experiments indicated that the sum of the interactions between DNP and the separated H and L chains equaled that between DNP and the original anti-DNP IgG preparation. However, the mispairing of chains present in reconstituted anti-DNP IgG resulted in a drastic reduction in average


34 1

binding affinity in comparison with that of unrandomized chain combinations.' These recombination studies further substantiated that both chains contributed directly to the antibody binding site and that recombination between different H and L chains could provide for a vastly increased binding site repertoire. These refolding studies first proved the hypothesis that antibody binding sites derive their specificity and affinity for antigen strictly from the sequences and pairing of their component polypeptide chains. This insight provided an important foundation for our understanding of antibody diversity at the protein level. Subsequent progress on the structurefunction relationships of antibodies showed the Fab to possess a highly conserved architecture capable of incorporating enormous diversity within its combining sites. The heterogeneity of antibodies was gradually proven to be directly associated with the regulated biosynthesis of a vast number of immunoglobulin proteins by the immune system. C. Antibody Paradigms and Analysis of Primary Structure

Sequence analysis provided the first indication of domain organization in immunoglobulins. Chemical studies of primary structure became practical with the availability of discrete H and L chain populations, in conjunction with antigen or complement binding fragments (Fab or Fc, respectively) and their component polypeptides. Initially, manual protein sequencing methods were applied to antibodies, and they spawned considerable interest in the comparative study of immunoglobulins. Advances in sequencing technology began with automated instruments (Edman and Begg, 1967; Waterfield et al., 1970) and continued with the development of DNA sequencing techniques (Sanger et al., 1977; Maxam and Gilbert, 1977) coupled with widespread application of the polymerase chain reaction (PCR) to gene cloning (Marks et al., 199 1 a). General features of the complex relation between antibody structure and immunogenetics began to emerge slowly. In 1965 Dreyer and Bennet suggested the daring theory that each H or L chain polypeptide was derived from a combination of two types of genes, variable (V) and constant (C). This hypothesis of antibody biosynthesis incorporated many contemporaneous concepts from protein chemistry. At the same time the studies of Hilschmann suggested the existence of a discrete pattern of variability in the N-terminal half of the L chain and, by implication, in the corresponding segment of the H chain (the Fd) (Hilschmann and Craig, 1965; Hilschmann, 1967). As a simplified model for natural antibodies, investiThis randomization effect has also been discussed in reference to the construction of combinatorial libraries, where original pairings tend to be lost (Gherardi and Milstein, 1992).

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JAMES S. H U S l O N E T AL

gators made use of Bence Jones proteins, which consist of the homogeneous L chains secreted by patients with multiple myeloma. This malignancy is derived from proliferation of a single plasmacyte, resulting in massive production in the circulation of intact monoclonal immunoglobulin (myeloma protein) and, in the urine, hundreds of milligrams per day of homogeneous L chain (termed Bence Jones proteins). Myeloma protein can also be accumulated by plasmaphoresis in quantities as high as hundreds of grams from a single patient. These homogeneous immunoglobulins, contributed by many patients, became the cornerstones on which immunochemistry developed in the 1960s and 1970s. This development culminated in determination of the three-dimensional structure of an Fab by X-ray crystallography (Poljak et al., 1973). Before the structure was published, Wu and Kabat (1970, 1971) analyzed homologies between available L chain sequences from Bence Jones and myeloma proteins. They identified positions constituting three hypervariable segments in the light chain (V,) and, in conjunction with the corresponding segments of the heavy chain (VH),proposed that this region formed the antibody combining site. Homogeneous immunoglobulins directed against specific antigens or haptens-i.e., true antibodies of known specificity-were pursued by several routes. Highly repetitive antigens were sometimes found to elicit an antibody of highly restricted homogeneity, as in the case of rabbit antipneumococcal or antistreptococcal cell wall antisera, which represented an immune response so limited that it was possible to obtain the sequences of hypervariable segments (summarized by Haber, 1970, 1971; Krause, 1970). During this period Sela and co-workers began their long-term studies on antibodies raised against repetitive peptide antigens, which also produced very restricted responses in some cases. The induction of mouse plasmacytomas provided another source of homogeneous antibodies which were tested against panels of various haptens to find those that bound most tightly, i.e., those that were putative antigen analogs. In this manner McPC 603 was found to bind to phosphorylcholine, and the antigen-antibody complex was solved crystallographically (Segal et al., 1974), as was the complex of Fab’ NEW with the several vitamin K analogs to which it bound (Amzel et al., 1974). The MOPC 315 IgA mouse myeloma protein represented another important system, wherein the putative hapten was the 2,4-dinitrophenyl group (Eisen and Siskind, 1964). In 1966 the entire polyclonal rabbit Fc was sequenced by R. L. Hill and colleagues (1966a,b). The group found that the Fc was homogeneous, as Porter had surmised (1958, 1959), and the sequence data revealed that two homologous units constituted the rabbit Fc sequence. The Hill group proposed that the H and L chains evolved from a common ancestral gene that had undergone a series of gene duplications. While this work on the


343

C-terminal half of the rabbit H chain did not directly address the nature of combining site diversity, it led to the sequence analysis of the N-terminal half of the rabbit polyclonal H chain by the Porter laboratory (Fruchter et al., 1970; Mole et al., 1971). This region, the Fd, represents the H chain component of the Fab. Porter’s group was able to obtain unique sequence throughout the Fd, except in the V H at allotypic sites showing a mixture of alternative residues and within each of the three hypervariable segments (now referred to as complementarity-determining regions or CDRs). Because the rabbit Fd was polyclonal, Edman degradation of Fd positions within hypervariable segments resulted in an indecipherable mixture of phenylthiohydantoin products. The final sequence of rabbit Fd thus showed blanks over the three hypervariable segments, in contrast to the sequence determined for the remaining three-fourths of V region framework and the entire first H chain constant region (CH1) structure. The immortalization of B lymphocytes secreting specific antibodies (Kohler and Milstein, 1975) finally provided access to true monoclonal antibodies raised by immunization with a specific antigen. This fostered increasingly sophisticated methods in molecular immunology, leading to analysis of the genetic basis for antibody diversity (Tonegawa, 1983) and maturation of the immune response (reviewed in this volume by Milstein and Neuberger).

D. Immunoglobulin Shape and Domain Structure Although it was clear from the work of Porter (1958, 1959) that the bivalency of IgG resided in a pair of antigen binding regions connected to a crystallizable complement binding region, it was not clear how the two Fab and the Fc were disposed spatially in solution. The Y-shape model of IgG was first supported experimentally by the analysis of hydrodynamic data collected on intact IgG and its papain fragments (Noelken et al., 1965) (Fig. 4A), and then substantiated by electron microscopic pictures of discrete antigen-antibody complexes (Valentine and Green, 1967) (Fig. 4B and C). The first three-dimensional structures of immunoglobulin fragments were solved for a human myeloma Fab (NEW; Poljak et al., 1973) and Bence Jones L chain (Schiffer et al., 1973), which ushered in a new era of atomic resolution in our appreciation of antibody structure. Poljak and colleagues recognized the immunoglobulin fold as the common structural element of domain folding in both the V and C regions. Increasingly refined interpretations of variable region fragment (Fv) structure evolved in the years that followed (see chapters in this volume by Padlan and by Novotny and Bajorath).

344

A)

JAMES S. HUSTON ET AL.

B chain

I or II

Fragment Ill

FIG.4. The experimental demonstration of the Y shape of IgG. The gross conformation of IgG as predicted in (A) from hydrodynamic analysis of its fragments and intact structure. In (B) and (C) this shape was further substantiated by analysis of electron micrographs of intact anti-DNP IgG antibody complexes with bivalent DNP ligand (shown above). (A) Schematic representation of a possible model for immunoglobulin G . The short heavy line represents the single disulfide bond between the two A chains. Other disulfide bonds are not shown. [From Noelken et al. (1965).](B) Scale diagram of a hapten-linked trimer of IgG molecules, based on patterns visualized by electron microscopy [note (C)]. The distance between the extremes of the Fc fragments (C) was 225 rt 3 8, (mean and standard deviation of the mean). The length of two Fab fragments linked by the hapten was 120 f 3 A. The difference between these figures gave the estimate for the length of Fc (allowance being made for orientation). The widths of Fab and Fc measured on the electron micrographs varied between a maximum of f10 %, from the mean. (C) A high-magnification electron micrograph of anti-DNP IgG complexes with bivalent DNP ligand showing the projection at each corner of the polygonal shapes. The interpretation of the structure of these complexes is given in panel (B). Magnification: X 500,000. [From Valentine and Green (1967).]


345

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JAMES S. HUS'I'ON ET AL

E. Immunogenetics of Antibody Formation

The discovery of hybridoma technology by Kohler and Milstein (1975) provided a source of antigen-specific monoclonal antibodies, which were of pivotal importance in the development of immunology. This approach has given critical access to homogeneous antibodies of preselected specificity and provided sources of genes encoding their specific chains. This technology catalyzed advances in cellular and molecular immunology and permitted delineation of the major features of antibody biosynthesis. Late in the 1980s the advent of PCR technology allowed the large-scale generation of VHand VL gene repertoires, which have been assembled randomly into antibody combining regions that appear to function as well as those derived by immunization of mice, rabbits, or humans. This combinatorial approach had been anticipated in early efforts to partially explain the extraordinary diversity of the natural antibody repertoire by the random combination of H and L chains (Cathou and Haber, 1967; Tanford, 1968). Remarkable progress has been made in understanding antibody immunogenetics in relation to binding site structure (reviewed by Cook and Tomlinson, 1995; Milstein and Neuberger, this volume). It has been established that the VH gene is assembled in two stages from its component germline genes, with combination of the D andJHgene segments followed by joining to the VH gene segment to yield VH(D)JH.Likewise the final VL gene comprises a germline VL gene segment fused to a J L where both V and J segments are K or il.The assembly of these genes introduces numerous sources of sequence variation, and the resulting somatic mutations contribute to the diversity of the natural immune repertoire. Rigorous analysis of the human genome and compilation of all data on immunoglobulin loci have revealed the surprising insight that very limited polymorphism exists in human Ig germline genes. A complete map of the human V H locus on chromosome 14 has been constructed. It is 1100 kb in length and has 5 1 functional VH segments and 44 nonfunctional segments (pseudo genes). There are an additional 24 functional VH segments on chromosomes 15 and 16 (Cook and Tomlinson, 1995).Thus, a total of 119 VH germline gene segments have been identified, which can be divided among seven gene families in three clans. In addition, a restricted set of germline genes is involved in assembly of the C-terminal region of VH encoding the H3 loop (D segment) and adjacent FR4 framework region (JH segment), with approximately 30 D 6 J H segments. The human Vr. loci also comprise about 100 germline V gene segments, divided between K and ilfamilies. The presumably complete set of human germline genes for the VH,V,, and V,I segments, as well as the H chain diversity segment (DH) and joining regions (JH, J, JK, and J,I segments) have been identified and


347

compiled for distribution through the World Wide Web (VBASE by I. M. Tomlinson, S. C. Williams, S. J. Corbett, J. P. I. Cox, and G. Winter; http://www.mrc-cpe.cam.ac.uk/; Cook and Tomlinson, 1995). Limited polymorphism of the VH loci is graphically analyzed in Fig. 5, which illustrates that, even prior to the V(D)J joining that adds H3 and FR4 to these sequences, the 51 functional VH segments show diversity over their whole length, including the FR1-3 and the first two CDRs. The H2 loop is clearly the region of greatest germline diversity, but several positions in FRs are comparable to H1 in their levels of diversity. Germline polymorphism or somatic mutations in these CDRs and FRs can be critical to the specificity and affinity of a particular combining site, as evidenced by the results of detailed structure-function studies on the binding sites discussed in Section IV. VBASE was used by Tomlinson and co-workers to decipher the general pattern of somatic mutation during maturation of the immune response: sequence diversity spreads outward from the center to the periphery of the binding site (Tomlinson et al., 1996). Molecular modeling of the complete set of germline H 1 and H2 loop conformations consisted of only 83 canonical structures (Chothia et al., 1992). The major sites of diversity in the H chain are in the H3 loop because of permutations and combinations of VH, D, and J H genes, as well as variable splicing of these genes. The 50 functional VH segments have been used to construct combinatorial libraries in which the H3 loop and FR4 were added synthetically to optimize the diversity of the resulting single-chain Fv (sFv) library (Nissim et al., 1994). These findings also provide a genetic basis for the observed success of molecular modeling in the prediction of CDR loop conformations. The experimental observation of a set of restricted canonical CDR loop structures (Chothia et al., 1989) is consistent with the combination of highly conserved framework architecture and a limited set of germline genes encoding the CDRl and CDR2 loops of each chain. This restricted polymorphism in germline antibody genes probably contributes significantly to making the modeling of antibody binding sites a tractable problem (Bruccoleri and Karplus, 1987; Bruccoleri et al., 1988). By comparison, the overall protein folding problem remains beyond our grasp. 111. ENGINEERED ANTIBODY BINDING SITES In 1972 the antigen binding region of an antibody was reduced to a 25-kDa minimum comprising a noncovalent heterodimer of VH and VL domains (Inbar et al., 1972). Discovery of this fragment, the Fv, marked the end of an era when protein chemistry was the principal source of structural

CDR1

10

20

30

CDR2

40

50

60

70

80

90

Amino acid position FIG.5. Germline variability of the 51 hnctional VH segments. Germline sequence variability was calculated for each residue in the VH domain using the formula of Kabat et al. (1991) (number of different amino acids at a particular position divided by frequency of the most common amino acid at that position). Numbering of amino acids and complementarity-determining regions (CDRs) are according to Kabat et al. (1991); designations of the H1 and H2 loops (lighter tint) are according to Chothia et al. (1992). [From Cook and Tomlinson (1995).]


349

information about immunoglobulins, as the first high-resolution crystal structures were described in 1973 for an Fab (Poljak et al., 1973)and an L chain dimer (Schiffer et al., 1973).Since then, our understanding of antibody architecture has depended increasingly on X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular modeling. This activity has produced a profusion of antibody combining sites in novel forms, derived from biosynthetic sources ranging from Escherichia coli to plants. The development of engineered antibodies gained significant impetus from transfectoma expression methods, which allowed all or part of the L or H chain to be fused to an effector protein (Neuberger et al., 1984).For example, Fab fusion proteins were constructed to enhance the activity of recombinant thrombolytic agents through targeted delivery at sites of arterial occlusion (Schnee et al., 1987;see Section V,C). The first reports of recombinant antibodies synthesized in bacteria were published in 1984, involving expression in E. coli of an entire IgG antibody against carcinoembryonic antigen (Cabilly et al., 1984) and of an IgM antibody against 4-hydroxy-3-nitrophenylacetylhapten in E. coli (Boss et al., 1984)and in yeast (Wood et al., 1985). Although the yields in these systems were modest, proof of the synthetic principle was obtained in both cases. Concern about the antigenicity of murine monoclonal antibodies led to the engineering of humanized forms of V regions by the method of CDR grafting, in which murine CDRs are spliced between human FRs to form a chimeric Fv region (Jones et al., 1986).In their first report Jones et al. described the expression of a humanized VH region within an otherwise murine IgE, where it was fused to H chain C regions, placing the original murine VHCDRs of the binding site into a human framework.

A. Architecture of Fv and Design of sFv Analog The minimal antibody combining region, the Fv, was first isolated as a discrete entity in experiments on the MOPC 315 antibody (Inbar et al., 1972;reviewed in Givol, 1991).Because the MOPC 315 Fv heterodimer was intrinsically stable, it suggested that the antibody binding site was a discrete unit of structure, despite its two-chain composition, and that Fv integrity might be sufficient for targeting applications. However, efforts to prepare similar fragments by proteolysis from other homogeneous antibodies were met with the greatest difficulty. The following 15 years of research on Fv proved frustrating, as only a few additional papers appeared in the scientific literature. Finally, in 1988,general strategies for making the Fv were proven to be workable through recombinant DNA methods, suggesting new dimensions for future research in this field. Native Fv were expressed in myeloma cells (Riechmann et al., 1988)as well

350

~ A M E S s. HUSTON

Er

AL.

as in E. coli (Skerra and Pliickthun, 1988; Field et al., 1988), and the first sFv species were likewise produced by refolding of bacterially expressed protein (Huston et al., 1988a; Bird et al., 1988). A comparison of IgG and its proteolytic fragments with the sFv and model sFv fusion proteins is given in Fig. 6. Some of the engineered antibody binding regions of current interest are drawn schematically in Fig. 7, which emphasizes the central position of the Fv in these studies. I . Linker Considerationsfor Bridging V Domains

There are several reasons for covalently linking the V domains that associate to form a functional Fv region. The most significant is that the strength of the interaction between VHand VL domains can vary over many orders of magnitude, and dissociation of an Fv region reduces or eliminates its population of active binding sites. Linking V domains in a way that is conducive to normal binding site function is thus a very useful device. The most commonly utilized method for connecting V regions is to join them at the gene level, which allows a given sFv to be conveniently utilized as a targeting vehicle for any ancillary peptide or effector protein. Gene fusion also has the distinct advantage that genotype and phenotype may be linked in selection methods that rely on phage antibodies or other combinatorial methods (Winter et al., 1994). The choice of linker has FIG.6. Schematic comparison of immunoglobulin G and its Fab and Fv fragments, and single-chain Fv and its fusion proteins. (A) IgG antibody: four chains connected by disulfides, 150,000 molecular weight. Fab fragment: two chains connected by a disulfide, 50,000 molecular weight. Fv fragment: two chains, 25,000 molecular weight. sFv protein: one chain, about 26,000 molecular weight; the sFv is shown in its two possible permutations, VL-VH and VH-VL, with the dotted linker on the back face and the solid linker on the front, for the given orientation of V regions. (B) Single-chain Fv polypeptide chain. The typical sFv protein consists of about 250 amino acids and has a molecular weight of approximately 26,00027,000, with the specific value depending on the actual sequences of the V regions and linker segment. (C) Polypeptide of an sFv fusion protein with a protein effector fused to the C terminus of the sFv. (D) Polypeptide of an sFv fusion protein with a protein effector fused to the N terminus of the sFv. (E) Schematic drawing of an effector-sFv fusion protein in its native conformation [corresponding to the N-terminal fusion protein in (D)]. (F) Schematic drawing of an sFv-effector fusion protein in its native conformation [corresponding to the C-terminal fusion protein in (C)]. Numbers indicate parts of the proteins in (E) and (F) as follows: (1) first residue of the effector domain, which is the N terminus of the fusion protein; (2) N-terminal effector protein domain; (between 2 and 3) spacer sequence that facilitates dual function of effector and sFv; (3) first amino acid residue of VH; (4)last amino acid residue of VH; (between 4 and 5) sFv linker segment; (5) first amino acid residue of VL; (6) last amino acid residue ofV1.; (between 6 and 7) spacer sequence that facilitates dual function of effector and sFv; (7) C-terminal protein effector domain; (8) C terminus of the fusion protein. Light chain and VL, white; heavy chain and VH, gray; sFv linker, black; effector protein, such as a toxin or growth factor, hatched or black. Antigen binding site is part of the Fv region and is indicated by a V-shaped docking site. (Reproduced with permission from Huston et al., 1993b. Int. Rev. Immunol. 10, 195-217. 01993 by Gordon and Breach F’ubl.)

35 1


@$J VH-linker-VL

V,-lin ker -V,

Linker

I

sFv

II

I

1 VH

Linker

VL

F) Antigen Binding Site

Antigen Binding Site

Amino Terminus Carboxyl Terminus Effector-sFv

sfv-effector

352

JAMES S . HUSTON ET AL.

DAbs

Fv

sFv'

sFv

Single-chain bispecific SFVI-SFV~!

Miniantibody (sFv-amph. helix);,

J .... ................... .... ......... .......... ......... )= ......... ......... ............... ........., ,... .... ..... .. ......... ........... ....... .... .... ....... . *

FIG.7. Schematic representations of minimal forms of monovalent and divalent combining site species. (A) VH domain binding site (12 kDa), also termed DAbs (Ward et al., 1989; Davies and Riechmann, 1995). (B) Fv heterodimer, comprising noncovalently associated VH and VL domains (Inbar et al., 1972). (C) Single-chain Fv (26-27 kDa) with linker peptide spanning the distance in the native conformation between the C terminus of the first domain and the N terminus of the second. (D) sFv', fusion of an sFv with a C-terminal cysteinyl peptide. (E) Fab fragment (50 kDa) comprising the L chain and Fd (amino-terminal half of H chain). (F) (sFv'):! dimer (54 kDa) consisting of two disulfide-linked sFv' binding regions (Adams et al., 1993; Kipriyanov et al., 1994; McCartney et al., 1995; Tai et al., 1995). (G) Single-chainbispecific (Huston et al., 1991), sFv2, also termed CRAbs (for chelating recombinant antibodies) (Neri et al., 1995). A 30 kDa dual binding site form of the sFv has been described (Keck and Huston, 1996), termed xBABS (for chimeric bispecific antibody binding site), in which the second binding site is grafted onto the bottom of the sFv to make a single-chain bispecific species. (H) Miniantibody form of sFv-amphiphilic helix fusion proteins (Pack and Pluckthun, 1992). (I) Bis Fv-Cys dimer (Cumber et al., 1992).u) Diabody form of tandem Fv dimers (Holliger et al., 1993; Whitlow et al., 1994). [Modified from Huston et al. (1994).]


353

gradually become relatively routine because of the widely proven effectiveness of the original 15-residue linker (Gly4-Ser)3,used to make the 26-10 sFv (Huston et al., 1988a) and its fusion with the B domain of staphylococcal protein A (FB), FB-sFv (Tai et al., 1990). Other linkers have also proven useful, particularly the series designed by the Genex and Enzon group (Bird et al., 1988; Whitlow et al., 1993), but their properties are more complex than those of the 26-10 linker. Alternative methods for cross-linking the V domains of a given Fv were first described by Glockshuber et al. (1990), including genetic modification to introduce a pair of opposing cysteinyl residues that can form an interchain disulfide bond between the domains. In this approach no linker is used, and these disulfide-stabilized Fv (dsFv) species have enhanced resistance to denaturation, relative to the corresponding Fv or SFV.The disadvantages are that it requires considerable additional protein engineering and does not lend itself easily to phage library selection; furthermore, in those Fv regions that undergo conformational changes or domain translation on antigen binding, as is the case for D1.3, the interchain disulfide may preclude the normal fluctuations of structure with a corresponding impact on the ability to bind antigen. Nonetheless, this approach has proven to be of particular interest in constructing Fv-immunotoxins in cases where sFv fusion proteins have been difficult to prepare. Beyond the absolute requirement to be noninteracting and allow normal V domain refolding, the most important consideration in linker design is to choose a linker of sufficient length to bridge the distance between the C terminus of the first V domain and the N terminus of the second V domain. As noted in Table 111, compilation of these bridging distances has been calculated for a number of proteins with crystallographically determined structures. These numbers are given in terms of the linear or Euclidean distance between these points and thus are always less than the distance required operationally to produce a stable sFv. Thus, although the peptide unit length is 3.8 A, a 38-A linker of 10 residues is not sufficient to generate a stable sFv but rather leads to the formation of interchain Fv dimers termed diabodies (Fig. 7, panel J), as first described by Holliger et al. (1993). The effect of linker length is quite dramatic, as shown in Fig. 8 for a series of 26-10 sFv analogs with linkers comprising one to five units of (Gly4-Ser),such that 5- and 10-residue linkers result in 3.5s dimers, whereas 15- and 25-residue linkers yield stable sFv monomers with a 2.5s sedimentation coefficient. Longer linker lengths have been reported to result in less tendency to aggregate (Whitlow et al., 1993; Desplancq et al., 1994), which can possibly result from a variety of effects. In cases where a given linker is strained within the sFv structure, a longer linker always lessens or eliminates such strain in the native conformation; for example, Table I11 indicates that a VL-VH orientation of domains in an

354

JAMES S. H U S T O N ET AL

TABLE 111

Comparison of Interdomain Bridging Distances in Fu Regions of Known Structure" Euclidean distance between linker ends

(At

Structure

PDB

VH-VL

VL-VH

26-10' McPC 603 KOL 4-4-20 D1.3

ligi 2mcp 2fb4 4fab 1fdl 2hfl 3hfm 6fab 2tbj lfl9 1nca 3fab

35.92 34.55 33.06 34.27 35.44 35.28 3 1.93 36.09 34.72 34.74 36.66 29.30

-

-

39.10 39.18 39.50 43.27 42.97 36.55 41.34 4 1.44 43.04 37.20 39.80

4.55 6.12 5.23 7.83 7.69 4.62 5.25 6.72 8.30 0.54 10.50

Hy HEL-5 HyHEL-10 36-7 1 5539 R1Y.9 NC4 1 NEW

(VL-VH)-(VH-VI.)

' Reprinted with modifications from Huston et al. (1993a). With use of the Biosym Insight I1 program and the Protein Data Bank (PDB) coordinates noted, the Euclidean (linear) distance between linker ends was estimated by the following method. The algorithm was used to calculate the distance between the C-terminal a-carboxyl carbon of the first V region and the Nterminal a-amino nitrogen of the second V region; the linker was peptide bonded to each of these positions in their assumed sFv configurations. For the VH-linkerVL configurations, these end points are VH position 128 to VL position 1 according to the structural position numbers in Huston et nl. (1993a). For the VI_linkerVH orientation, these termini are VL position 117 and VH position 1. For all but the 26-10 structure, the differences in Euclidean bridging distances were determined for the VL-VH and the VH-VL constructs, yielding (VL-VH) - (VH-VI.); this distance was in all cases longer for the VL-VH isomer. The 26-10 crystal structure (Jeffrey et al., 1993) did not define the position of VH residue 1, and therefore the PDB data set for 26-10 Fab (ligi was used here) did not permit calculation of the bridging distance for a VL-VH isomer of the 26-10 SFV.

sFv always requires a longer bridging distance than a VH-VL. It has been shown by nuclear magnetic resonance (NMR) relaxation studies (Freund et al., 1993) that the (Gly4-Ser)slinker, within the context of the McPC 603 sFv, is fully hydrated, fluctuates rapidly in configuration, and is devoid of specific interactions with the V domain surfaces except at its ends. Therefore, another explanation for the impact of longer linkers depends on the volume excluded increasing with length of a highly mobile linker. Since self-association of sFv species appears to sometimes be mediated by con-

355


1 .o h

P 0.8

a, Q

U

a,

$

0.6

v)

0,

.-P

g

0.4

0.0 0.0

1.0

2.0 s*

3.0 4.0 (Svedbergs)

5.0

6.0

FIG.8. Sedimentation profiles Ig(s*) versus s*] for 26-10 sFv species with different linker lengths. The (Gly4-Ser), linker was used, with n = 1, 2, 3, 5, resulting in linker lengths ranging from 5 to 25 residues; each 26-10 protein was refolded and affinity-purified as described previously (Tai et al., 1990; Huston et al., 1995). Consistent with the results of Huston et al. (1988a), the 2.5s monomer was formed spontaneously by sFv with 15 or 25 residue linkers, while abnormally short linkers of 5 or 10 residues resulted in noncovalent dimeric species of 3.5S, termed diabodies by Holliger et al. (1993). In this experiment, the diabodies consisted of tandem Fv dimers having the V F and ~ VL on one sFv polypeptide chain associated with the VI. and VH, respectively, on another sFv polypeptide chain (Fig. 75).The short 5 or 10 residue linkers resulted in an inability of the adjacent variable domains to form the intramolecular contacts of the normal unstrained sFv with 15 or 25 residues. These diabodies, or related multivalent Fv species described by Whitlow et al. (1993), thus offer alternative conformations that maintain variable domains in an Fv configuration. [From Tai et al. (1996).]

tacts with the bottom of the Fv, it is thus possible that longer linkers can act to inhibit self-association through steric hindrance of the contact surface on the bottom of the Fv region (McCartney et al., 1995; P. C. Keck and J. S. Huston, unpublished results). The (Gly4-Ser)Jlinker has probably been advantageous because it is devoid of charged or hydrophobic residues that might form strong interactions with V domain surfaces, which may occur with linkers such as the 212 version of the Genex linkers (Whitlow et al., 1993). This may explain why some groups have observed an improvement in antigen-binding affinity of as much as 10-fold on switching from a charged linker to the uncharged (Gly4-Ser)3(Newton et al., 1994; Rybak et al., 1995). During in vivo studies of sFv targeting, an alternative linker (Ser4-Gly), and its truncations also were tested (Adams et al., 1993; McCartney et al., 1995; Tai et al., 1995), but in fact this work has shown an increased tendency of sFv to dimerize in comparison with sFv species with

356

JAMES S . HUSTON E T AL.

the standard linker. This may be due to the potential for additional hydrogen bond formation provided by the additional serines or may relate to the restriction of linker motion caused by adding bulkier side chains along the linker. 2. Combinatorial Libraries of Antibody Binding Sites Genetic selection methods have been critical to the emergence of antibody engineering. Whereas proteolysis of IgG and IgA offered no general route to the Fv, PCR cloning methods have provided ready access to murine and human V genes, so that Fv regions may in theory be reproduced from the paired V H and VL genes of any given hybridoma cell line. The extension of this strategy to the use of multiple primers for cloning mixtures of VH and VL genes in wholesale fashion has allowed the generation of V genes that can be assembled into Fab or sFv libraries. The antigen-driven selection of sFv binding sites from these combinatorial libraries of randomly paired VH and VL genes can be accomplished, for example, by expressing them as sFv fusions with a phage coat protein (Winter et al., 1994); methods in use also allow the screening of Fab libraries (Barbas et al., 1991; Burton et al., 1991). The preparation of phage libraries comprising human V region genes (Marks et al., 1991a) has allowed the selection of sFv binding sites composed of authentic human VH and VL domains (Marks et al., 1991b) and offers an efficient and elegant alternative to CDR grafting (Marks et al., 1992, 1993; Schier et al., 1995). Although individual recombinant antibody binding sites may also be derived from monoclonal antibodies and may be prepared either by secretion or by refolding of expressed protein, the combinatorial library is preferable, as its selection process yields sFv species that will necessarily be secreted by E. coli. Studies on the anti-carcinoembryonic antigen (CEA) MFE-23 sFv emphasize the advantages of an sFv carefully selected from a phage library. The sFv-phage library was derived from V genes of CEAimmunized mice, yielding MFE-23 sFv with a CEA affinity of 5 x 10' M-', secretion levels of 20 mg/l (Chester et al., 1994), and superb targeting of tumor metastases in the first sFv clinical trial (Begent et al., 1996).

3. Specificity and Afinity Measurements of Combining Sites Underlying advances in recombinant DNA and PCR methodology, carehl affinity and specificity measurements of antibodies continue to be critically important, as they have throughout the course of immunochemistry. These properties define the fimctional attributes of a combining site of interest, whether derived from a phage library, a monoclonal antibody, or a polyclonal antibody fragment. As this field moves increasingly toward the derivation of combining sites from phage display libraries, it is important to emphasize the


357

cardinal rule of immunology, that the ultimate usehlness of a given combining site is generally governed by its antigen affinity and specificity. The properties of particular antibody binding sites can be defined only within the limits of available assays. Thus, even nearly ideal model systems are subject to experimental limitations. Under the best circumstances, one has the purified antigen and sensitive in vitro assays available, while in the most difficult situations one may have only an in vivo target antigen accessible in animal models. Given the former system, one might need only to compare several high-affinity combining sites selected against the same antigen from a phage library or hybridoma cell lines. Beyond obvious differences in affinity or specificity, such a comparative analysis may uncover subtle yet important differences, for example, in stability, solubility, self-aggregation, or production yield. In addition to equilibrium binding affinity the kinetic rate constants, for antigen association with an antibody binding site and their dissociation, are of particular interest. They are important to current discussions about what would be the most effective properties for targeted delivery of therapeutic agents (Schier et al., 1996) and what may be the natural limitations of the immune system (Foote and Eisen, 1995). This discussion has been fueled by a wealth of new data derived from widely used biosensor instrumentation that functions by surface plasmon resonance or resonant mirror techniques (Van Regenmortel, 1995). The use of stopped flow techniques may still be preferred for the physicochemical measurement of association and dissociation rate constants in the millisecond range. However, this classical approach is more technically demanding than the biosensor methods, which measure the increase or decrease in refractive index within a microscopic binding layer above the biosensor surface. By immobilizing an antigen of interest to the cross-linked dextran coating on the biosensor surface, both the binding and dissociation reactions with soluble antibody species may be monitored. These methods facilitate the comparative assessment of large numbers of analog molecules for binding site optimization or for quantitative assessment of species selected from combinatorial libraries (Schier et al., 1996). 4 . Structural Homology between Fv Regions

Despite the statistically enormous variation in binding properties of the Fv region, its architecture is conserved to a remarkable degree. A number of invariant structural features have been noted within each V domain (Novotny and Haber, 1985). These include the positions of the intradomain disulfide bridge and the adjacent buried tryptophan residue. In the process of expanding the database for this analysis, a scheme was devised for structural homology modeling that allows rapid alignment of a new sequence with a set of proteins having crystallographically determined

358

IAMES S. HUSTON ET AL.

three-dimensional structures. Thus, the conserved architecture of the Fv region is displayed with a schematic diagram of the D1.3 Fv peptide backbone (Fig. 9), wherein the beta strands have been numbered in consecutive order as outer strands (0s)if they are exposed to solvent, or as inner strands (IS) if buried at the interface between V regions. The corresponding structural alignment of this and other V region sequences is reviewed by Huston et al. (1993a). B . Studies of Fv and SFVProteins 1. Limited Proteolysis in Domain Isolation

Factors important in the application of limited proteolysis to immunoglobulins for the production of intact, compactly folded regions have included (1) the presence of specific protease recognition sequences at appropriate positions between the Fab and the Fc or in the switch regions that connect V and C domains; (2) the combination of accessible target sequence and backbone flexibility to promote reaction conditions that favor preferential cleavage at restricted positions; (3) the rigidity of the native domain structure, which helps to protect additional recognition sites from limited cleavage; and (4) the possibility of favoring cleavage between domains by appropriately chosen solvent conditions that enhance domain stability without protecting the hinge or switch regions. The resistance of native V and C domains to proteolysis contributed to the success of attempts to preferentially hydrolyze interdomain sequences. In IgG and IgA, this structural integrity typically served to restrict cleavage to the hinge region and spare the regions between the V and C domains. This protection of the switch region was enhanced by interactions between the Fd and L chains of polyclonal rabbit Fab, which proved resistant to the gentle conditions of papain hydrolysis that had cleaved the isolated Fd chains into 11.5-kDa globular regions (Huston et al., 1972). VI. and CI, domains were also cleaved from Bence Jones L chains (Solomon and McLaughlin, 1969) and served as the basis for characterizing their discrete refolding properties (Bjork et al., 1971). In the case of IgM, the acidic conditions required for peptic cleavage were able to dissociate L and Fd chains, which enhanced preferential cleavage of the interdomain switch regions. Preparation of Fv from IgM proteins by cold pepsin cleavage proved to be the only potentially general procedure for obtaining these fragments (Lin and Putnam, 1978), which involved reaction at pH 4 and 4°C for 24 hr. However, since the IgG and IgA classes of immunoglobulins have not typically yielded Fv under these conditions, and because of the low antigen-binding affinities of individual IgM binding sites, this approach has seen limited use.

VL

N IN-t

C-t FIG 9. Structural conservation of the immunoglobulin fold. (A) Tertiary folds of the Fv region drawn to emphasize conserved elements of Fv structure, based on the D1.3 VH and VL domains within the D1.3 Fab structure solved by Fischmann et nl. (1991) using the coordinates in PDB file lfdl from the Brookhaven National Laboratory (Bernstein et nl., 1977).Variable region amino termini (N-t) and carboxyl termini (C-t) are noted for VH and VI~,which were separated by horizontal translation from their heterodimeric state in the D1.3 Fab structure. Drawings are based on molecular graphics models constructed using the Biosym Insight I1 Program (Biosym Technologies, San Diego, CA). Conserved elements of V regions are designated by nomenclature related to that of Amzel and Poljak (1979). The 0-sheet contacts between the VH and VI- domains are formed by inner 0 strands (IS), indicated as shaded ribbons. The solvent-exposed outer0 sheets are made up of outer0 strands (OS), indicated as white ribbons. Loops in this figure are denoted as top loops (TL) and bottom loops (BL), which correspond to front and back loops, respectively, as defined by Amzel and Poljak (1 979). The CDRs are part of top loops that are drawn as ropelike connections between 0-strand ribbons; bottom loops are represented by linear connections between a carbons. The 0 strands drawn as ribbons in this diagram correspond to the definition of structurally conserved regions (Greer, 1991), being common to all aligned structures. [Reproduced with modification from Huston et nl. (1993a).]

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JAMES S. HUSTON E T AL.

2. Derivation of MOPC 315 Fv by Limited Proteolysis and Recombinant

DNA Methods The antidinitrophenol MOPC 3 15 murine IgA myeloma protein was an early paradigm for homogeneous antibodies (Eisen et al., 1968), from which the first Fv was generated. Peptic cleavage of the 315 Fab’ (Inbar et al., 1972) or IgA (Hochman et al., 1973) resulted in cleavage of both the L chain and Fd region to yield the intact Fv heterodimer. Peptic digests of 315 Fab’, conducted at pH 3.7 and 37°C for 4 hr, were chromatographed on DNPlysine Sepharose to isolate 48% of the protein as hapten-binding species, 90% of which were Fv fragments. As it turned out, this was a particularly fortuitous example of Fv production (Sharon and Givol, 1976; Givol, 1991). The protein chemistry of 3 15 Fv was explored in a series of papers from the Givol group and later exploited by others as well. In their first two papers (Inbar et al., 1972; Hochman et al., 1973), the classic solution properties of the protein were defined and compared with data for intact 315 IgA derived by Eisen et al. (1968). The Fv was shown to be a heterodimer of VH and VL domains, which associated to form the complete antigen binding site which was indistinguishable from the original DNP binding site of 3 15 IgA or Fab’. These were the first experiments to unambiguously prove that the V domains of an antibody can associate strongly enough to maintain a competent antigen binding site in the absence of an interchain disulfide or C domain interactions. In addition to this characterization, the refolding properties of 3 15 Fv were characterized by Hochman et al. (1973, 1976). These results were consistent with previous studies on the recovery of L chain domain structure after refolding (Bjork et al., 1971; Rowe and Tanford, 1973) insofar as the VH and VL domains could be refolded alone and reassociated or could be refolded together provided that the intrachain disulfide bond was left intact or oxidized prior to renaturation. Formation of the intrachain disulfide bond was an obligatory step in the refolding of 3 15 protein whether it was an isolated V region or an equimolar mixture of both VH and VL. Recombinant forms of the 315 Fv and sFv have been investigated by two groups (McCartney et al., 1991; Cheadle et al., 1992). The simplicity of the classical protein chemistry used to generate 315 Fv contrasts with the highly technical process of engineering the 3 15 Fv and SFV.However, the conventional methods of protein chemistry that yielded 3 15 Fv have never proven general, whereas the recombinant approaches offer broadly applicable strategies for making Fv and sFv proteins. The natural 3 15 Fv refolding properties have been found to be characteristic of the recombinant 3 15 Fv and 3 15 sFv proteins. Furthermore, the disulfide-restricted refolding pathway required by 3 15 Fv is representative


36 1

of other V domains and Fv regions, such as the RllDlO sFv (Nedelman et al., 1993) and 520C9 sFv (McCartney, Oppermann, and Huston, unpublished results, 1991). Refolding of isolated V regions by this approach is a high-yield process because only a single intrachain disulfide can form in vitro. However, in the sFv such oxidation results in one native disulfide cross-linked form and two nonnative fully oxidized forms (Fig. 10) or nine nonnative partially and fully oxidized forms (including the fully reduced form). These multiple oxidized states make such a refolding strategy an inherently low-yield process. McCartney et al. (1991) obtained only about a 4% yield of active 3 15 sFv for this reason. 3. Antidigoxin 26-1 0 sFv

The 26-10 antidigoxin monoclonal antibody has been the subject of comprehensive studies dating from its initial description (Mudgett-Hunter et al., 1982). These investigations continue with the detailed studies of its combining site and three-dimensional structure described in Section IV. The present discussion emphasizes its use as a paradigm in antibody engineering, particularly in the exploration of sFv (Table IV) and sFv fusion proteins. The 26-10 sFv has served as a valuable model system over many years (Huston et al., 1988a) and most recently aided in the development of genetically fined sFv-peptide chelates that provide for the facile coordinaT~ et al., 1995). tion of radiometals such as g g m (George a. sFv Species Based on Anti-Digoxin 26-10 IgG. The 26-10 monoclonal antibody was chosen for development of the sFv (Huston et al., 1988a) both for its potential clinical relevance and for its practical advantages: (1) The V region sequences of the 26-10 IgG had been determined by both protein and DNA sequencing (Novotny and Margolies, 1983; Huston et al., 1988a), giving confidence that, once expressed, synthetic VH and VL genes based on these known sequences would faithfully reproduce the authentic 26- 10 V region polypeptides; (2) the binding sites of the 26- 10 IgG and Fab were of high affinity (K, = 2.4 x log A 4 - l ) ; (3) assays of great sensitivity were available for monitoring the recovery of antigen-binding activity by recombinant 26- 10 species; (4)ouabain-amine-Sepharose affinity chromatography could be used for purification, with elution possible under native conditions on addition of 20 mM ouabain, a highly soluble analog of digoxin; (5) quantitative methods for affinity and specificity analysis allowed the detection of very small changes in the binding site contacts with antigen. b. Expression and Renaturation of 26-10 sFv Proteins. Early 26-10 antibody engineering predated common use of PCR methods, and consequently the 26- 10 V regions were constructed synthetically from overlapping synthetic oligonucleotides (Huston et al., 1988a). The sFv species were expressed as fusion proteins with leaders that enhanced the production of cytoplasmic

362

A)

JAMES S. HUSTON E T AL

Dilution refolding

-

-

Dilution refolding

Oxidation

B) Redox refolding

-

Redox-refolding __.__c

C) Disulfide restricted refolding

,(&, SH

N

formation Disulfide

SH

c

-€d

cl-

N

C - N

~

Nonnative

-

Renatur ation

FIG..10. Refolding schemes for sFv proteins. Structures on left-hand side represent the fully reduced and denatured sFv polypeptide, with the crenelated line indicating the linker segment. The intermediate state of the refolding process is indicated by the middle diagrams, and the right-hand schematics depict the native configuration of the sFv and its disulfide bonds. These disulfides bridge the innerp sheets (hatched walls) and outerp sheets (shaded walls) of each variable domain. (A) Dilution refolding involves a two-stage refolding process, with renaturation of the reduced protein followed by oxidation to form disulfides. (B) The redox-refolding process does not involve isolated intermediates since refolding and reoxidation occur concurrently in solution. (C) Disulfide-restricted refolding leads to multiple intermediates, of which only the correctly oxidized configuration (bottom intermediate) is capable of refolding to form native sFv. There are three possible intermediates if the sFv has been fully oxidized (indicated schematically by the intermediates) and seven other possibilities if the mixture also contains partially and fully reduced forms. [From Huston et al. (1991).]

363


TABLE IV Single-Chain Fv Organization of 26-10 Constructions sFv or sFv’ 26- 10 (acid cleavage) 26-10 (direct expression) 26-loin (direct expression) 26- 1OiS 26- 10-1 26-10-2

Leader

VI

Linker

Vp

Tail

P A A A A A

VH VH VH

GGGGSGGGGSGGGGS GGGGSGGGGSGGGGS (GGGGS),, n = 1-5 SGSSSSGSSSSGS SGSSSSGSSSSGS SGSSSSGSSSSGS

VL VL

SGGGGC

VH VH VH

VL

VL VL VL

sc

inclusion bodies. The polypeptide leaders were removed by extended treatment of the fusion protein in denaturant at pH 2.5, which cleaved the protein at an acid-labile Asp-Pro peptide bond between the leader and sFv polypeptides. After removal of partial cleavage products by Whatman DE 52 anion-exchange chromatography in 6 M urea buffer, acid-cleaved sFv protein was renatured from a reduced, denatured state by dilution in 0.01 M sodium acetate, pH 5.5, followed by exhaustive dialysis to remove denaturant and foster reoxidation of intrachain disulfides. The renatured 26- 10 sFv was purified by ouabain-amine-Sepharose affinity chromatography, which eliminated inactive material and, on elution with 20 mM ouabain, concentrated the active sFv fractions in a relatively small volume. Characterization of the renatured 26- 10 sFv showed significant recovery of binding activity, with retention of most hallmarks of the 26- 10 specificity profile. However, the solubility properties of this early form of the 26-10 sFv were very poor in 0.15 M NaCl + 0.05 M potassium phosphate, pH 7.0, + 0.03% NaN3 (PBSA).Thus, all measurements of binding affinity were conducted in 0.01 M sodium acetate buffer, pH 5.5, containing 0.25 M urea to minimize aggregation. Furthermore, affinity measurements relied on immunoprecipitation with a polyclonal second antibody against V region framework determinants, resulting in perturbation of the Fv region under the conditions of this assay. The digoxin affinities (K,) measured in these experiments gave an association constant for the 26-10 sFv of (3.2 f 0.9) x 1O’M-l and for the 26-10 Fab control of (1.9 f 0.2) x lo8M-’, which were both lower than the normal value of 2 x lo9M-’ obtained for 26-10 IgG in PBSA at pH 7. Once immunoprecipitation was replaced with ultrafiltration, the need for direct binding by a second antibody was eliminated, and the free and bound 3H-labeled digoxin concentrations at equilibrium with each 26-10 species were used to define binding isotherms (Tai et al.,

364

JAMES S . HUSTON ET AL.

1990; Huston et al., 1991). Within experimental error, the 26-10 sFv, FBsFv, Fab, and IgG had identical binding affinities for digoxin of 2.4 x lo9M-l. Refolding of directly expressed 26-10 sFv protein by the glutathioneurea redox buffer procedure yielded 26-10 sFv with excellent solution properties and native 26-10 antigen-binding properties, as given in Table V (Tai et al., 1990; Huston et al., 1995). The inclusion bodies and refolded sFv proteins were prepared without recourse to acid treatment, which had been used in earlier studies to remove the leader (Huston et al., 1988a, 1991). These experiments proved unequivocally that 26-10 sFv can recover a native binding site that faithhlly reproduces the specificity and affinity properties of the natural 26-10 combining site. These results gave credence to the principle that these molecular designs are fundamentally sound and can result in sFv proteins that can recover competent antibody combining sites in general. This prompted our engineering of sFv proteins for immunotargeting to tumor antigens, which are often difficult to characterize in vitro. Direct expression of the 26-10 sFv and other sFv proteins has provided a routine source of inclusion bodies suitable for refolding (Huston et al., 1995; McCartney et al., 1995). With current procedures, at least 30%of the TABLE V

Relative Dissociation Constantsfor 26-10 Binding Sites and Cardiac Glycosidesa

Cardiac glycoside ~~~~~

sFv

FB-SFV

Fab

IgG

4.5 3.6 5.9 5 11.7 50 176

4.2 3.8 4.6 4.2 4.6 37 105

~

Digoxin Digoxigenin Digitoxin Digitoxigenin Acetylstrophanthidin Gitoxin Ouabain

4.2 5.5 10.1 7.6 11.8 50 143

4 4.8 4.4 4.4 6.8 52 160

a Kd,app values are given for 26-l0/digoxin complexes, measured by ultrafiltration to be 4.2 x lo-'' M, within experimental error. Results for other cardiac glycoside complexes were calculated from specificity assays, wherein affinity purified goat anti-mouse Fab was adsorbed to microtiter plates, followed by the 26-10 species of interest. values were derived from the product of the corresponding digoxin Kd,app value and the normalized concentration of glycoside that inhibited up to 50% of maximal 1251-digoxinbinding. For each type of 26-10 species, normalization involved dividing the concentration of each glycoside at 50% inhibition by the concentration of digoxin at 50%inhibition. Reproduced with permission from Tai et al., 1990.J. Biochemistry 29, 8024-8030. 0 1990 American Chemical Society.


365

26-10 sFv protein renatures properly, with yields as high as 830 mg of product per liter of fermented cells when using controlled fermentation to obtain very high expression levels. However, renaturation is the most limiting step, as it typically must be conducted at protein concentrations of 0.1 mg/ml or lower, which in the case of 26-10 sFv yields about 30 mg of affinity-purified 26-10 sFv per liter, and thus demands rather large volumes for refolding gram quantities of protein. In this respect, the use of secretion methods can be competitive, as levels of 5-20 mg/liter can be obtained from the fermentation media or periplasm of cells using the standard secretion vectors for sFv produced from combinatorial libraries (Chester et al., 1994; Schier et al., 1995). Secretion can also be advantageous if an affinity purification step is unavailable, because it usually produces fully active protein. Refolding yields a mixture of active and inactive sFv that must be separated by chromatographic or affinity methods. If the secreted sFv has been engineered to have hexahistidine (His6) at its C terminus, it will complex with an immobilized metal affinity chromatography resin that is properly charged with a divalent cation; this step can both concentrate and purify the sFv that has been secreted. c. Folding Properties of sFv Proteins. The production of biosynthetic antigen binding regions has frequently relied on protein folding, in concert with secretion as an alternative that yields folded protein directly. The interpretation of protein folding reactions is of much current interest, and at the level of applied research, the producibility and stability of engineered antibody proteins is a critical factor in whether they can be clinically useful. The 26-10 sFv refolds spontaneously either by dilution refolding or redox refolding (Fig. 10).This contrasts with the refolding pathways followed by MOPC 3 15 sFv and other sFv molecules that utilize a disulfiderestricted refolding process. The transition curves for the antidigoxin 261O/S sFv in urea or guanidine hydrochloride (Fig. 11) substantiate that refolding is reversible as long as disulfide bonds remain intact during unfolding and refolding. d. N- Terminal Polypeptide Fusions to Form a Bafinctional FB-sFv Fusion Protein. The 26-10 sFv was also tested in the context of a bifunctional sFv fusion protein to investigate if an sFv was capable of refolding while in immediate proximity to an effector domain such as another globular protein. The B domain of staphylococcal protein A was a convenient hsion partner in this experiment, as it served in the dual capacity of enhancing FB-sFv expression while folding into a functional FB domain. In fact, measurements of digoxin and other cardiac glycoside affinities proved that the sFv binding site, with or without FB fusion, was the same as the parent 26-10 IgG combining site (Table V), and that the FB moiety was simultaneously capable of specific association with the Fc region of IgG (Huston and

366

JAMES S. HUSTON E T AL

0

2 4 6 8 Denaturant concentration (M)

FIG. 11. Transition curves for the 26-10/S sFv in guanidine hydrochloride (GuHCI) and urea. The coincidence of denaturation (X or +) and renaturation points (solid squares or triangles) indicates reversibility of the folding process when the disulfide bonds of the variable regions remain intact. The fraction denatured was based on the tryptophan fluorescence of the 26-10 V regions. The 3 hl urea conditions used for refolding 26-10 sFv are thus seen to correspond to mostly native species at equilibrium. Conditions of experiments were the following: a Perkin-Elmer LS-5 spectrofluorometer was used with excitation at 295 nm and emission at 350 nm; samples were incubated at a constant temperature in all experiments between 22.4" and 22.7"C; tryptophan fluorescence was enhanced by unfolding and quenched by refolding. All stock protein solutions were in PBSA and the 26-10 sFv concentration for measurements was 0.75 pA4. The linker used in this molecule was Ser-Gly-(Ser4-Gly)2-Ser.[From Huston et al. (1995).]

Oppermann, 1988; Huston et al., 198813, 1989; Tai et al., 1990). These experiments also proved that an effector domain could be fused to the N terminus of the sFv, which was an important facet of these novel molecular designs, since the N termini of V domains are always free in immunoglobulins and other members of the Ig superfamily. In fact, the sFv itself provided the first demonstration of fusion to the N terminus of a V region, but having a constrained linker fused at both ends to rigidV domains of an sFv is topologically quite different from having a mobile domain fused at the N or C terminus of the V domains (Huston et al., 1991). Access to the

367


combining site of the FB-sFv may have been facilitated by the known flexibility of FB residues 48-58. The apparent advantage of keeping a flexible spacer between the effector and sFv regions has been important in the design of toxin fusions to the sFv N terminus (Nicholls et al., 1993). e. C-Terminal Peptade Fusions to Form Minimal Chelation Sites for y9mTc. During the development of sFv species for targeted immunotherapy, sFv’ proteins were designed with C-terminal cysteinyl peptides (Fig. 12). This C-terminal cysteine was incorporated to facilitate both site-specific labeling and the formation of disulfide-bonded (sFv’)~ dimers (Adams et al., 1993; McCartney et al., 1995). The specific biotinylation of sFv’ species has been demonstrated by Kipryanov et al. (1994), who likewise used cysteinyl peptides as a basis for covalent dimerization. An important medical application would be to design the SFV’for optimal chelation of radiometals. In

sFv Linker

sFv‘ Linker

Tail

Refolding wifh glutathione redox couple

sFv‘-GI

...... ......... ... .... ...... ...,.... ....

:v::

WC?:

:v -S-[Qiulalhi~nyl]

FIG. 12. The ( s F v ’ ) ~engineering scheme, showing the progressive stages of sFv’ condimers. struction and expression, sFv’ renaturation, and oxidation to disulfide-linked (sFv’)~ The sFv and sFv’ polypeptides are each depicted in the VH-VL domain orientation. The sFv‘ has been drawn schematically to emphasize the unpaired -SH at the C terminus. The sFv’ was constructed by fusing the sFv gene to an oligonucleotide encoding the appropriate cysteinyl peptide. The sFv‘ was refolded in a urea-glutathione redox buffer, resulting in a native sFv’ in a C-terminal mixed disulfide with the glutathionyl peptide (sFv’-GI). The protected sulfhydryl group was deblocked by gentle reduction and oxidation to yield disulfide-bonded (sFv‘)~. From McCartney et al., 1995. Protein Eng. 8, 301-314. Reproduced by permission of Oxford University Press.

368


the past, antibody fragments were chemically conjugated with chelates for binding radiometals such as 99mTc,the preferred isotope for gamma camera imaging. Toward this goal, a chelation site was made by genetically incorporating Gly4-Cysat the sFv C terminus to form 26-10-1 sFv’ (George et al., 1995; McCartney et al., 1995). This 26-10-1 sFv‘ construction was based on 26-10/S sFv (Table IV), which has the same V regions as the original 26-10 sFv (Huston et al., 1988a),but direct expression resulted in N-terminal alanine, and a serinerich linker of 13 residues [S(G&)BGS]was utilized in place of the generally preferred 15-residue linker of - 10 sFv [(GGGGS)s]. These serine-rich linkers evolved to test the possibili of enhancing solubility with extra hydroxyl groups. The 26-10/S sFv and lated sFv‘ analogs were made as controls for studies with 741F8 sFv, whi targets the c-erbB-2 tumorassociated antigen (Adams et al., 1993; McCa ey et al., 1995). However, the additional serine in these linkers enhance the tendency for selfassociation in some forms of these sFv proteins (Tai, Stafford, and J. S. H., unpublished observations, 1992). Thus, (Ser4Gly)3or its variations should not be substituted for the (GlySer)~linker in routine sFv constructions (Huston et al., 1988a, 1995) but may be useful in enhancing dimerization, when desirable, or in improving the solubility of very marginally soluble Fv regions, as was the case for a T-cell receptor sFv (Hilyard et al., 1994). Ouabain binding by the uncomplexed 26-10-1 sFv’ was measured to determine if hsion of the peptide had an adverse impact on the combining site (George et al., 1995). When a resonant mirror biosensor was used to measure the rate constants for binding and dissociation, the 26-10-1 sFv‘ protein was shown to bind ouabain-labeled bovine serum albumin with a K d of (2.3 k 0.5) x lo-’ M , which was indistinguishable from the value of (2.1 k 0.3) x lO-’M for the 26-10/S sFv. These data confirmed that the presence of the C-terminal fusion peptide did not perturb the Fv in a way that compromised the combining site. The labeling studies presented in Fig. 13 defined optimal conditions for 99mTcchelation under which the 26-10-1 SFV’was able to coordinate 0.5-50 mCi of ggmTcin a high-affinity complex with its C-terminal cysteinyl peptide. Standard labeling conditions involved exposure of 5Opg of 26-10-1 sFv’ (0.5 mg/ml) to 18.5 MBq ggmTcO+in 0.22 mM SnFP at a final pH of about 9.5 for 60 min, which resulted in >97% incorporation of the radiometal. The control 26-10/S sFv was subjected to the same procedure with 6.4 f 2.7% incorporation, which verified that the C-terminal cysteinyl peptide was the principal coordination site in, 26- 10-1 sFv’. The coordination complex was also shown to be very stable when challenged with serum, saline solutions, or DTPA to attempt transchelation (Table VI). In addition, the ggmTc-sFv displayed much better retention of antigen-binding activity after 24 hr in vivo than protein radioiodinated by the chloramine-T method, showing

\

369


2

-

-

20

1 m

-

A

TABLE VI Stability of 99mTc-[26-10-1sFv'j Complexa Sample Control (60 min) Control (24 hr) DTPA PBS Saline Serum

ITLC

TCA precipitation

97.6 f 0.7 98.2 k 2.1 97.0 f 3.6 96.4 +- 3.6 97.1 f 0.5 ND

92.2 f 2.0 ND ND ND ND 92.9 f 4.8

a 9gmTc-[26-10-1sFv'] was prepared and incubated for 24 hr either without dilution or following dilution in DTPA (diethylenetriamine entaacetic acid), PBS, saline, or serum. The radiopharmaceutical purity of the 'gmTc-sFv' was then determined, using instant thin-layer chromatography (ITLC) or trichloroacetic acid (TCA) precipitation, as indicated. In each case, the fraction of total counts that remain complexed by the sFv' is expressed as the mean percentage k standard deviation of three or more experiments. ND, Not done. Reproduced with permission from George et al. (1995).

that 74.2 f 3.9% (n = 8) of the g g m T ~ ~ -complex F~' was able to bind ouabain-Sepharose, whereas only 53.8 k 6.6% of the lz51-labeled26-10-1 SFV'bound to the immunoadsorbent. The 9 9 m T ~ - ~was F ~also ' more stable in vivo than '251-sFv' as measured by trichloroacetic acid precipitation.

370

JAMES S. HUSTON ET A1

4 . Anti-c-erbB-2 741F8 sFv and SFV' Proteins

To extend sFv studies to immunotargeting, the V regions were isolated by PCR cloning from the 741F8 monoclonal antibody directed against the c-erbB-2 tumor-associated antigen (also known as HER-2/neu or p 185H"K2). This oncogene protein has been shown to be abnormally amplified on the surfaces of tumor cells in approximately 25% of cases of breast cancer (Slamon et al., 1987) and in a significant percentage of other adenocarcinomas. The c-erbB-2 extracellular domain (ECD) had been previously cloned and expressed in mammalian cells (Hudziak et al., 1987; Hudziak and Ullrich, 1991), and subsequently a high-expressing Chinese hamster ovary (CHO) cell line was constructed to produce an ECD with a His6 C-terminal extension (McCartney et al., 1995) to facilitate purification by immobilized metal affinity chromatography (Porath, 1992). The purified 90-kDa ECD glycoprotein was used to characterize the 741F8 sFv' and 741F8 (sFv')e dimers (Adams et al., 1993; McCartney et al., 1995). The ECD was found to be a well-behaved monomer in solution, which simplified studies on its interactions with the self-associating 741F8 sFv species. Dimerization of the 26-10 and 741F8 sFv' proteins may be assumed to involve bottom surfaces diametrically opposite to their combining sites (note schematic diagrams in Fig. 14) (McCartney et al., 1995).The discussion here also includes studies on the 26-10 SFV'and (sFv')~proteins, which were used as reference proteins throughout these studies. a.Features of sFv and sFv' Constructions. The V region genes of the 741F8 IgG were cloned from the parent hybridoma cell line using general PCR primers that introduced some mutant residues into the N-terminal FR1 sequence of VH. Refolding of the sFv comprising this VH sequence (version I1 in Table VII) yielded no detectable antigen-binding activity. To correct these errors, refolding properties were compared for two other analogs of the V H in the context of 741F8 sFv. Refolding of the sFv incorporating the version I sequence gave five times the yield of the version I11 analog. Ultimately the anti-c-erbB-2 741F8 Fv region (Dorai et al., 1994) was found to be closely related to the antineuraminidase NC41 Fv (Colman et al., 1987). Excluding the CDR3 loops, their Fv sequences are about 94% identical, and the FRl sequences of the NC41 and version I-741F8 VH regions are identical apart from their N-terminal residues, being Glu in 741F8 and Gln in NC41. Such homologies can be expected to provide a basis for correcting PCR errors in other VH sequences, if necessary. These results suggest that, in isolated cases, primer-induced errors within the FR1 sequence may have a pronounced effect on refolding. In attempting to reproduce a given monoclonal antibody as a recombinant sFv protein, cloning details may thus require close attention in the event that folding


37 1

+ X

X

FIG 14. Models for the reversible self-association of sFv’ subunits and conformational isomerization of (sFv‘):,dimers. (A) This reaction depicts the tendency of the blocked sFv’ species to self-associate through contact surfaces in the vicinity of the sFv C terminus. The true symmetry ofthe dimer cannot be predicted. The protecting group, X, is attached to the C-terminal tail peptide and has been sandwiched at the ( s F v ’ )interface ~ in the noncovalently associated form of the right-hand side. (B) This representation of (sFv‘)~ in solution emphasizes an equilibrium between two extreme conformations, each theoretically capable of bivalent antigen binding. In the compact species on the right-hand side, the sFv’ subunits interact noncovalently as well as through a disulfide bond. On the left-hand side of the reaction scheme, the sFv‘ subunits are linked by an interchain disulfide in an extended conformation, leaving contact surfaces exposed to solution. From McCartney et al., 1995. Protein Eng. 8, 301-314. Reproduced by permission of Oxford University Press.

yields are problematical. Combinatorial libraries are preferred for binding site isolation, as they incorporate selection methods and assays for sFv or Fab that detect only properly refolded molecules, as phage antibodies and as secreted sFv or Fab. 6. Preparation and Analysis of sFv’ and ( S F V ’ )Proteins. ~ The practicality of folding sFv’ proteins has been demonstrated (Adams et al., 1993; Kipriyanov et al., 1994; McCartney et al., 1995), as reasonable yields were obtained by redox-refolding procedures that should be applicable to any

372


TABLE VII

VHAmino-Terminal Sequences Used for 741F8 sFvIa

Source

Sequence

Heavy chain’

E V Q L Q Q S G A E L V K P G A

Version I‘

E I * Q L V * Q S G P * E L K * K P G E * T V K

Version I I ~

E V Q L Q E * S G P * E L K * K P G E * T I K I

Version IIIe

E V Q L Q Q S G P*E L V K P G E*T I K I

I

a Residues in versions 1-111 that vary from the 741F8 heavy chain N-terminal sequence are indicated by an asterisk. The 741F8 sFv molecules were constructed with three different VH amino-terminal sequences. From McCartney et al., 1995. Protein Eng. 8, 301-314. Reproduced by permission of Oxford University Press. 741F8 VH sequence obtained by Edman sequencing of the 741F8 heavy chain. VH sequence of the 520C9 anti-c-erbB-2 IgG. VH sequence of 741F8 from cDNA sequence (variant residues introduced by FR1 PCR primer). VH sequence of the 26-10 antidigoxin IgG.

SFV’analogs that refold spontaneously from the reduced random coil (McCartney et al., 1995). The yields for 741F8 SFV’species vary, depending on their particular sequence, but are similar to those for unmodified 741F8 sFv (McCartney et al., 1995). The sFv’-G1proteins were stable at 4°C on extended storage, and their propensity for self-association was minimized substantially in the (sFv’)~ forms. The sFv’-G1 species were easily deblocked by mild reduction with 2 mM dithiothreitol at pH 8.5, and dialysis against the same buffer without dithiothreitol (0.05 M Tris-HC1, 0.15 M NaC1, pH 8.5) allowed oxidation to (~Fv’):! homodimers (McCartney et al., 1995; Tai et al., 1995), as indicated schematically in Fig. 12. The main 741F8 sFv’ constructions and variants used in these studies are listed in Table VIII. All proteins were generated by direct expression, typically using the PET-3d expression vector in the BL21-DE strain of E. coli (McCartney et al., 1995). The 26-10 antidigoxin sFv’ (Table IV)and the 741F8 anti-c-erbB-2sFv’ were refolded in urea-glutathione redox buffers (Tai et al., 1995; McCartney et al., 1995), which resulted in protection of the C-terminal cysteine, presumably as a mixed disulfide with the glutathionyl peptide (Gl). These species have been studied in animal tumor models (Adams et al., 1993; Weiner et al., 1995), and they can be targeted effectively by single or repetitive bolus injections. Continuous infusion maintains a high serum concentration but results in loss of targeting specificity (Weiner et al., 1995), which may nonetheless prove useful in certain sFv scavenging applications for the reversal of drug overdoses.

373


TABLE VIII Single-Chain Fv Organization of 741F8 Constructions" sFv'

74 1F8 74 1F8- 1 741F8-2 74 1F8-3 74 1F8-4C

Leader

VI

Linker

Vph

Tail

A A ADNKFNKDP A A

vu

SSSSGSSSSGSSSS SSSSGSSSSGSSSS SSSSGSSSSGSSSS SSSSGSSSSGSSSS GSSGGGGSGGGGSMA

VL VL VL VL VH

GGGGC GGGGC SC GGGGC

VH VH VH

VL

-

" From McCartney et al., 1995. Protein Eng. 8, 301-314. Reproduced by permission of Oxford University Press. 11 The 741F8 VL domain terminated with the first three residues (RAD) of the switch region. Switch region residues represent part of the linker (Huston et al., 1991), and hence the 741F8-4 linker is formally 18 residues in length, consisting of RADGSSGGGGSGGGGSMA.

C. Targeting in Vivo bj Antibody Binding Sites

Radioimmunotargeting with engineered sFv antibody binding sites is attractive because the rapid sFv clearance yields high target-to-background ratios, both for radioiodinated species (Fig. 15) and specifically labeled Y Y m T ~ -species ~ F ~ ' (Fig. 16). After initial investigations of the 26-10 sFv and its fusion with the FB fragment, the 26-10 sFv became an important reference molecule in the development of other sFv proteins suitable for tumor targeting. This section on therapeutic applications focuses partly on an antitumor model, the anti-c-erbB-2 741F8 sFv, but controls involved various 26-10 sFv analogs studied in parallel experiments. The special merits of g g " l T ~ - ~complexes F~' in targeting tumors (George et al., 1995) suggest that they will be suitable for in vivo diagnostic imaging. Similar coordination chemistry applies to the incorporation of '"Re and '"Re for use in radioimmunotherapy. In virtually any practical application of engineered or natural antibodies, analysis of antigen binding to the combining site is of hndamental importance. The quality of binding in terms of specificity and afinity is very often a decisive factor in whether a given antibody binding site will prove clinically useful. In dealing with in vivo targeting, theoretical and practical issues can be very complex, particularly for multivalent antibody species binding to cells with multiple target epitopes. However, consideration of a simple model for such cellular targeting can avoid many of these complexities and be enlightening. The binding of monovalent antibody species to monomeric surface antigens on a cell represents a situation of

374

JAMES S. HUSI'ON E T AL

2

-s

1.5

v

P

E

l 0.5

0 Tumor

Blood Lungs Organ

Liver Kidneys

FIG. 15. Comparison of pooled 24-hr biodistribution data for 741F8 sFv, sFv'-CAM, Fab, and (sFv')z. The results of eight studies performed with radioiodinated 741F8-2 (sFv')~ (black), three studies performed with 741 F8-3 sFv'-CAM (hatched), and seven studies performed with 741F8 sFv (VH-VL; gray) are compared with 741F8 Fab (white) (Adams et al., 1993). Each separate study was performed with 20-100 pg of radiopharmaceutical administered intravenously in 100 pl to scid mice bearing 100- to 200-mg subcutaneous SK-OV-3 tumors known to express c-erbB-2. Groups of three to eight mice were sacrificed at each time point. Error bars were left out if the standard error of the mean was less than 0.02% IDlg. From McCartney et al., 1995. Protein Eng. 8, 301-314. Reproduced by permission of Oxford University Press.

considerable practical importance. The targeting of both monovalent and divalent species to antigenic sites on a cell surface were analyzed by Reynolds (1979) in terms of general thermodynamic equations and relevant cellular parameters, such as the diameter and curvature of the cell. The binding equilibrium between a monovalent protein and a monomeric ligand is governed by the law of mass action. The same equation applies when antibody binding site and antigen are in solution or when one is cell-bound and the other is in solution (Reynolds, 1979). For example, the targeting of an sFv monomer to the extracellular domain of a cell surface receptor is described as follows, where square brackets refer to the molar concentration of the given species in solution:

sFV

+ ECD

sFv~ECD

and the association constant is K=

[sFv~ECD] [sFv][ECD]


375

FIG 16. Imaging of human ovarian tumor xenografts in scid mice by 9 9 m T741F8-1 ~ anti-c-erbB-2 sFv'. Gamma camera images were obtained at 1, 6, and 24 hr after the intravenous administration of 99mTc741F8-1 sFv' to scid mice bearing SK-0%'-3tumor xenografts. The 20,000-count anterior images from a representative mouse with a 478-mg tumor show radioactivity mainly in the tumor (t) and bladder (b). Kidneys are visible as two patches above the tumor. The moderate bladder and kidney activities detectable at 1 and 6 h r were aided by stimulation of urinary output, which would likewise be affected in a clinical setting. The 24-hr image also shows higher relative activity in the kidneys and bowel; in this imaging experiment the animal was not perfectly flat on the stage, as in the other images, resulting in the asymmetric tumor image. [Modified from George et nl. (1995).]

Including the equations for conservation of mass, one obtains several useful relationships. The solution concentration of the noncovalent sFv.ECD complex may be expressed by either of the following ratios, wherein SFVT and ECDT are the sum of the free and complexed sFv protein or ECD ligand, respectively: [sFv*ECD]- K[ECD] { 1 + K[ECD]} [SFVT] or with equal validity, the fractional saturation of ECD,

376


The interaction of monovalent sFv and s sites of monovalent ECD on the cell surface can be treated by essentially the same equation, modified to give the average number of bound sFv molecules per cell, Nay(where N,, = sN): sK[sFv] Nav

= +-j

Graphical representation of the fractional saturation (N) of ECD on the cell offers some useful insights into immunotargeting. As shown in Fig. 17, the fractional saturation graphed as a function of the variables K[sFv] is a hyperbolic function. This function emphasizes that the fractional saturation may be raised by increasing the sFv binding afinity for ECD or by increasing the concentration of sFv in solution. There is a steep dependence of fractional saturation on the product of K and [sFv], which suggests that one can significantly improve target localization by increasing this product, especially in the early part of the curve, by increasing the target affinity of the sFv (Schier et al., 1996), its systemic concentration, or both. Data from Schlom’s group have shown in model radioimmunotherapy studies that a distinct therapeutic advantage was seen with only a 10-fold increase in binding affinity, from 2.5 x 10’ M-’ to 2.8 x 1O’O M-’ (Schlom et al., 1992).

c

0 ._ Y

F 3

a

Y

-

2 .Y

0

F

0.2

2O*4m0.0 O 0

10

20

30

40

50

K[sFv] FIG. 17. A simple model for sFv targeting. The effect of targeting a cell surface antigen (receptor) with soluble antireceptor sFv monomers is to associate with some portion of the available antigenic sites, interpreted as the fractional saturation, N . The value of N exhibits a hyperbolic dependence on the product of association constant, K, and free sFv concentration, [sFv], based on the equations of Reynolds (1979). For example, the quantity K[sFv] needs to reach only 9 for the fractional saturation of target sites to reach 90%. [From Huston et al., 1996.1


377

Reynolds also solved equations for a divalent antibody species binding to a cell-bound ligand, envisioned as the combination of two steps: (1) collision between one binding site of the divalent antibody [or (SFV’)~] in solution and the cell-bound receptor, and (2) reaction between the remaining free combining site of the divalent antibody and additional cell-bound receptors, which takes place entirely on the surface of the cell where effective concentrations of antibody species differ from those in the bulk solution. In this case, the binding reaction depends not only on bulk concentrations and intrinsic binding constants but also on the cell radius or surface area. There are also other treatments of this targeting problem (Hogg et al., 1987) and related issues (Dower et al., 1981a,b). Theoretical studies on solid tumors have been proposed to present a complication described by Weinstein as a “barrier effect” that permits less penetration as higheraffinity antibody is used to target tumors or micrometastases (Weinstein et al., 1986, 1987; Saga et al., 1995). This is disputed for smaller species, the size of Fab and below, by immunotargeting studies on multicell spheroids (Langmuir et al., 1991) and tumor xenografts; Weinstein has also calculated that the dependence of the effect on molecular weight is IgG > F(ab’)2> Fab, and presumably sFv or Fv is still less susceptible to the effect. D. Binding Equilibria and Linkage in Antibody Binding Site Proteins 1. Linked Functions and Reciprocal Effects

For a rigorous appreciation of the antibody binding site, it is useful to apply a unifying viewpoint to the interactions, dynamic changes, and functional attributes of antibodies. The Wyman theory of linked hnctions (Wyman and Gill, 1990) provides such a theoretical framework for relating the disparate properties of antibodies. Within a coherent theory, the Wyman relations permit interpretation of all the thermodynamic equilibria for a given protein that are linked because it is a common reactant, even if it exists in different functional, conformational, or associated states. This theoretical approach has been used as the basis for a general thermodynamic interpretation of allosteric proteins, such as hemoglobin (Wyman, 1948, 1964). It has also been applied to diverse aspects of protein chemistry, from the interactions of tubulin in solution (Shearwin and Timasheff, 1992, 1994; Shearwin et al., 1994) to the role of water in protein folding (Tanford, 1969) and the effects of cosolvents on protein structure (Arakawa et al., 1990). The application of Wyman theory to antibodies can quantitatively relate changes in antibody structure and function. Thus, binding of antigen to an antibody Fv region may lead to meaningful changes at the bottom surface diametrically opposite the binding site. In the D 1.3 Fv this

378


antigen-induced conformational change involved the relative translational movement of the VH and VL domains. Concomitant with these changes, distribution of the surface electrostatic potential may change, for example, and thereby alter pK, values of ionizable residues on the surface that could modulate other interactions. 2. Linked Functions in Antigen-Antibody Interactions

Until recently, the application of linked function theory to antibodies might have been premature, but current research has begun to measure the interactions that form the basis for such an analysis. Potentially, a wide range of experimental data may be quantitatively interpreted by these linked reactions and corresponding linkage hnctions. The details of these reactions are becoming available through efforts in X-ray crystallography, NMR spectroscopy, binding and dissociation kinetics, analytical ultracentrifugation (Stafford, 1994a,b), and other physicochemical methods. For the present discussion, a hypothetical case is described based on the Fv region of an antibody. Some of the equilibria that could take place are presented in the following hypothetical series of linked reactions. a. This model Fv contains a CDR loop which changes conformation from a relatively flexible state (F) to a rigid conformation that favors ligand binding (R), and thus even in the absence of antigen the following equilibrium takes place: K, c o d FvF

FvR

b. The differential binding affinity of antigen (Ag) in these states is thus described in the following reaction scheme, where KZaSSac

N

?T ? T

I

I

l

I

I

I

I

I

I

I

-T

I

I

I

I

I

I

I

I

V ) 4

+I N

' 0

o A N

4

m

V

& d

C

U

U

I

&


antibody-effector molecules in which the effector protein.

VH

403

N terminus is linked to an

J . Structure and Mutagenesis of Antidigoxin Antibody 26-1 0 1. Spontaneous Variants and Site-Directed Mutants

a. Spontaneous Variants. Because antibody 26- 10 had been crystallized (Rose et al., 1983), it was studied intensively. Spontaneous variants displaying altered binding were isolated using two-color, fluorescence-activated cell sorting, which permitted the selection of variants with subtle binding differences. Eleven variants were isolated, cloned, and sequenced, and their binding characterized. Among these, 10 different, unique V region amino acid sequence mutations were identified, all of which were in CDRs-9 in V H and 1 in VL. Eight variants had single amino acid replacements, while three contained multiple clustered VH region mutations, suggesting that they were due to gene conversion or recombination. Among nine VH mutants, two contained single mutations at position 35, and the three variants with multiple mutations all included position-35 mutations. Other mutations occurred at VH 50, 52, 58, 100B, and VL 92 (see Fig. 24). b. Mutations in Antibody HCDRB: Mutagenesis Studies on Contact and Noncontact Residues. The spontaneous variant 26-1OLL2 contains a single mutation, HTyr50His, which results in a 40-fold reduction in affinity for digoxin (Schildbach et al., 1991). To define the pattern of hapten recognition, the binding of 33 digoxin analogs was compared to that of digoxin in competition assays. As shown in Table XIV, antibody 26-10 does not recognize the C-3-attached sugar moieties, although there is a slight decrease in binding if the 3-hydroxyl is a rather than p. Antibody 26-10 is also indifferent to the presence (digoxin) or absence (digitoxin) of the 12hydroxyl. However, the mutation HTyr50His results in a combining site with altered specificity. This mutant is sensitive to the absence of the 12hydroxyl, as the relative affinities for digitoxin, digitoxigenin, evomonoside, and neriifolin are lower than for digoxin. Specificity changes for a second HCDR2 mutant, 26- 10LB4 (HSer52Phe), are less marked (Schildbach et al., 1993b). Both antibody 26-10 and in particular the mutant Tyr50His FIG. 23. Engineered 40-150 mutant antibodies and cleavage sites of signal peptidase and corresponding affinities for digoxin. Amino acid sequences are shown in the one-letter code for the 40-150 H chain signal peptide C-region, N-terminal region of the mature H chain, and residues 92-100 in antibody 40-150 and engineered mutants. Mutations resulting in amino acid replacements are in boldface. A deleted (delta) amino acid is indicated by a box. The vertical arrows indicate cleavage sites by signal peptidase, based on the results of amino acid sequences of expressed H chains (Ping et nl., 1993). Dashes indicate identity with the topmost sequence. FW,Framework. [Modified from Ping et al. (1993).]

-

CDR 1

HEAVYCHAINS

20

10

CDR2

t

40

2 0 52 a

30 t t 26-10 E V Q L Q Q S G P E L V K P G A S V R M S C K S S G Y I F T D F Y M N W V R Q S H G K S L D Y I G Y I S P Y S G V T G Y 4 0 - 5 0 < ~ - H - K E - - - G - - A - S O - L S I T - T V - - F S L - T Y G V H - F - - P P - - G - E W L - L - W ' A G - N - D 60

26-10 40-50

I

70

90

80 82 a b c

LIGWT'CH~S

$

10

26-10 40-50

100s b c d e

. * *

..

20 2 7 a b c d e 30 40 D V V M T Q T P L S L P V S L G D Q A S I S C R S S Q S L V H S N G N T Y L N W Y L Q K A G Q S P K L L I Y - I - L - - S - A - - A - - - - Q R - T - - - - A - R - V S T f S - Y S H I H - - Q - - p - - p - - - - - -

*

CDRZ 26-10 40-50

f

b

N ~ K F K G K A T L T V D K S S S T A ~ M E L R ~ L ~ ~ E D ~ A ~ ~ ~ ~ A G ~ ~ G N K - S A L M S R L S I N K - N - K - Q V F L K M ~ - - Q A D - ~ - M - - - - R F R F A ~ ~ ~ D Y A V - - - - Q - ~ - - - - - 4

Lp

OR3

100 50 60 70 80 90. K V S N R F S G V P D R F S G S G S G T D F T L K I S R V E A E D L G I Y F C S Q T T H V P P T F G G G T K L E I K R

..

L A - I L E - - - - A - - - - - - - - - - - - - N - H p - - E - - A A T - y - Q H ~ R E y - L - - - A - - E - - L - t

FIG.24. Amino acid sequences of the VH and VL regions of antidigoxin antibodies 2 S 1 0 and 40-150. Identities with the topmost sequences are indicated by dashes.

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