`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`CHARLES A JANEWAY«»«PAUL TRAVERS
`
`i
`
`1FrWeSha
`
`Ps
`
`7 - a * " " a b t
`
`Lassen - Exhibit 1038, p. 1
`
`

`

`
`Immuno
`iologye
`
`THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Charles A. Janeway, Jr.
`
`Yale University School of Medicine
`G
`Paul Travers
`
`Anthony Nolan Research Institute, London
`&
`Mark Walport
`
`Imperial College School of Medicine, London
`on)
`Mark J. Shlomchik
`
`Yale University School of Medicine
`
`Sots
`
`4<o
`
`
`
`a*
`
`
`<o
`Op
`a
`& Franci®
`
`Lassen - Exhibit 1038, p. 2
`
`Lassen - Exhibit 1038, p. 2
`
`

`

`Vice President:
`Text Editors:
`Managing Editor:
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`Penelope Austin, Eleanor Lawrence
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`Blink Studio, London
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`
`© 2001 by Garland Publishing.
`All rights reserved. No part of this publication may be reproduced,stored in a retrieval
`system or transmitted in any form or by any means—electronic, mechanical, photocopying,
`recording, or otherwise—withoutthe prior written permission of the copyright holder.
`
`Distributors:
`Inside North America: Garland Publishing, 29 West 35th Street,
`New York, NY 10001-2299.
`Inside Japam Nankodo Co.Ltd., 42-6, Hongo 3-Chrome, Bunkyo-ku,
`Tokyo, 113-8410, Japan.
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`
`ISBN 0 8153 3642 X (paperback) Garland
`ISBN 0 4430 7098 9 (paperback) Churchill Livingstone
`ISBN 0 4430 7099 7 (paperback)International Student Edition
`
`Library of Congress Cataloging-in-Publication Data
`Immunobiolegy : the immune system in health and disease / Charles A, Janeway, dr....
`[et al.].-- 5th ed.
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-8153-3642-X (pbk.)
`1. Immunology. 2. Immunity. |. Janeway, Charles. ||. Title.
`
`QRi81 1454 2001
`616.07'9--de21
`
`2001016039
`
`This book was produced using QuarkXpress 4.11 and Adobe Illustrator 9.0
`
`Published by Gariand Publishing, a memberof the Taylor & Francis Group,
`29 West 35th Street, New York, NY 10001-2299.
`
`Printed in the United States of America.
`15 1413 1211109876543 2
`
`Lassen - Exhibit 1038, p. 3
`
`Lassen - Exhibit 1038, p. 3
`
`

`

`
`
`
`
`Oo2©}Oo©=SSSso>©»©57oUtmoOo==os=oBo0o®2oa5==afwmp
`
`Basic Concepts in Immunology
`
`Innate Immunity
`
`Antigen Recognition by B-cell and T-cell Receptors
`
`The Generation of Lymphocyte Antigen Receptors
`Antigen Presentation to T Lymphocytes
`
`Index
`
`Afterword Evolution of the Immune System:Past, Present, and Future,
`by Charles A. Janeway, dr.
`
`PART!|AN INTRODUCTION TO IMMUNOBIOLOGY AND INNATE IMMUNITY
`
`PART Il|THE RECOGNITION OF ANTIGEN
`
`PARTfll|THE DEVELOPMENT OF MATURE LYMPHOCYTE RECEPTOR
`REPERTOIRES
`
`Chapter 6—Signaling Through Immune System Receptors
`QO=DosSsa a N
`
`The Development and Survival of Lymphocytes
`
`PART IV|THE ADAPTIVE IMMUNE RESPONSE
`
`Chapter8
`
`—T Cell-Mediated Immunity
`
`Chapter9
`
`The Humoral Immune Response
`
`Chapter 10 Adaptive Immunity to Infection
`
`PART V|THE IMMUNE SYSTEM IN HEALTH AND DISEASE
`
`Chapter 11
`
`Failures of Host Defense Mechanisms
`
`Chapter 12 Allergy and Hypersensitivity
`
`Chapter 13 Autoimmunity and Transplantation
`
`Chapter 14 Manipulation of the Immune Response
`
`Appendix!
`
`Immunologists’ Toolbox
`
`Appendix Il CD Antigens
`
`AppendixIll Cytokines and their Receptors
`
`Appendix IV Chemokines andtheir Receptors
`
`Appendix V_
`
`Immunological Constants
`
`Biographies
`
`Glossary
`
`Lassen - Exhibit 1038, p. 4
`
`Lassen - Exhibit 1038, p. 4
`
`

`

`THE RECOGNITION
`
`OF ANTIGEN
`
`Lassen - Exhibit 1038, p. 5
`
` |
`
`Lassen - Exhibit 1038, p. 5
`
`

`

`“=
`
`Antigen Recognition by B-cell
`and T-cell Receptors
`
`
`
`
`
`Wehave learned in Chapter 2 that the body is defended by innate immune
`responses, but these will only work to control pathogens that have certain
`molecular patterns or that induce interferons and other secreted yet non-
`specific defenses. Mostcrucially, they do not allow memory to form as they
`operate by receptors that are coded in the genome. Thus,innate immunity is
`good for preventing pathogens from growingfreely in the body, but it does
`not lead to the most importantfeature of adaptive immunity, which is long-
`lasting memory ofspecific pathogen.
`To recognize and fight
`the wide range of pathogens an individual will
`encounter, the lymphocytes of the adaptive immunesystem have evolved to
`recognize a great variety of different antigens from bacteria, viruses, and
`other disease-causing organisms. The antigen-recognition molecules of B
`cells are the immunoglobulins,or Ig. These proteins are produced by B cells
`in a vast range of antigen specificities, each B cell producing immunoglobulin
`of a single specificity (see Sections 1-8 to 1-10). Membrane-bound
`immunoglobulin on the B-cell surface serves as the cell’s receptor for anti-
`gen, and is known asthe B-cell receptor (BCR). Immunoglobulin of the same
`antigen specificity is secreted as antibody by terminally differentiated B
`cells—the plasmacells. The secretion of antibodies, which bind pathogensor
`their toxic products in the extracellular spacesof the body, is the main effector
`functionofB cells in adaptive immunity.
`
`Antibodies werethefirst molecules involved in specific immunerecognition to
`be characterized and arestill the best understood. The antibody molecule has
`two separate functions: one is to bind specifically to molecules from the
`pathogen that elicited the immune response; the otheris to recruit other cells
`and molecules to destroy the pathogen once the antibody is boundtoit. For
`example, binding by antibody neutralizes viruses and marks pathogens for
`destruction by phagocytes and complement, as described in Section 1-14.
`These functions are structurally separated in the antibody molecule, one part
`of which specifically recognizes and binds to the pathogen or antigen whereas
`the other engages different effector mechanisms. The antigen-binding region
`varies extensively between antibody molecules and is thus known as the
`variable region or V region, The variability of antibody molecules allows each
`antibody to bind a different specific antigen, and the total repertoire of anti-
`bodies madeby a single individual is large enough to ensure that virtually any
`structure can be recognized. The region ofthe antibody molecule that engages
`theeffector functions of the immunesystem does notvary in the same way and
`is thus knownas the constant regionor C region.It comes in five main forms,
`which are each specialized for activating different effector mechanisms. The
`membrane-bound B-cell receptor does not have these effector functions, as
`the C region remains inserted in the membraneoftheBcell. Its function is as
`a receptor that recognizes and binds antigen by the V regions exposed on
`the surface of the cell, thus transmitting a signal that causes B-cell activation
`leading to clonal expansion andspecific antibody production.
`
`Lassen - Exhibit 1038, p. 6
`
`Lassen - Exhibit 1038, p. 6
`
`

`

`
`
`94
`
`Chapter 3; Antigen Recognition by 8-cell and T-cell Receptors
`
`
`
`
`
`The antigen-recognition molecules of T cells are made solely as membrane-
`bound proteins and only function to signal T cells for activation. These
`T-cell receptors (TCRs) are related to immunoglobulins bothin their protein
`structure—having both V and C regions—andin the genetic mechanism that
`produces their great variability (see Section 1-10 and Chapter 4). However,
`the T-cell receptor differs from the B-cell receptor in an important way:
`it does not recognize and bind antigen directly, but instead recognizes short
`peptide fragments of pathogen protein antigens, which are bound to MHC
`molecules on the surfaces of other cells.
`
`The MHC molecules are glycoproteins encodedin the large cluster of genes
`known as the major histocompatibility complex (MHC)(see Sections 1-16
`and 1-17). Their most striking structural feature is a cleft running across
`their outermost surface, in which a variety of peptides can be bound. As we
`shall discuss further in Chapter 5, MHC molecules showgreat genetic varia-
`tion in the population, and each individual carries up to 12 of the possible
`variants, which increases the range of pathogen-derived peptides that can
`be bound. T-cell receptors recognize features both of the peptide antigen
`and of the MHC molecule to which it is bound. This introduces an extra
`dimension to antigen recognition by T cells, known as MHCrestriction,
`because any given T-cell receptoris specific not simply for a foreign peptide
`antigen, but for a unique combination of a peptide and a particular MHC
`molecule. The ability of T-cell receptors to recognize MHC molecules, and
`their selection during T-cell developmentfor the ability to recognize the
`particular MHC molecules expressed by an individual, are topics we shall
`return to in Chapters 5 and 7.
`
`In this chapter we focus on the structure and antigen-binding properties of
`immunoglobulins and T-cell receptors. Although B cells and T cells recognize
`foreign molecules in two distinct fashions, the receptor molecules they use
`for this task are very similar in structure. Wewill see howthis basic structure
`can accommodate great variability in antigen specificity, and how it enables
`immunoglobulins and T-cell receptors to carry out their functions as the
`antigen-recognition moleculesof the adaptive immuneresponse.
`
`Thestructure of a typicalantibodymolecule.
`
`Antibodies are the secreted form ofthe B-cell receptor. An antibodyis identical
`to the B-cell receptorof the cell that secretes it except for a small portion of
`the C-terminus of the heavy-chain constant region, In the case of the B-cell
`receptor the C-terminus is a hydrophobic membrane-anchoring sequence,
`andin the case of antibodyit is a hydrophilic sequence that allows secretion.
`Since they are soluble, and secreted in large quantities, antibodies are easily
`obtainable and easily studied. For this reason, most of what we know about
`the B-cell receptor comes from the study of antibodies.
`
`Antibody molecules are roughly Y-shaped molecules consisting of three
`equal-sized portions, loosely connected bya flexible tether. Three schematic
`representations of antibody structure, which has been determined by X-ray
`crystallography, are shown in Fig. 3.1. The aim ofthis part of the chapteris to
`explain howthis structure is formed and howit allows antibody molecules to
`carry out their dual tasks—binding on the one handto a widevariety of anti-
`gens, and on the other handto a limited number of effector molecules and
`cells. As we will see, each of these tasks is carried out by separable parts of the
`molecule. The two armsofthe Y end in regions that vary between different
`
`Lassen - Exhibit 1038, p. 7
`
`N terminus
`
`Variable
`
`region
`
`
`
`Constant
`fegion
`
`
`
`C terminus
`
`
`Fig. 3.1 Structure of an antibody
`molecule. Panela Illustrates a
`ribbon diagram based on the X-ray
`crystallographic structure of an IgG
`antibody, showing the course of the
`backbones of the polypeptide chains.
`Three globular regions form a Y. The
`two antigen-binding sites are at thetips
`of the arms, which are tethered to the
`trunk of the Y by a flexible hinge region.
`A schematic representation of the
`structure in a is given in panel b,
`illustrating the four-chain composition
`and the separate domains comprising
`each chain. Panel c shows a simplified
`schematic representation of an antibody
`molecule that will be used throughout
`this book. Photograph courtesy of
`A. McPherson and L. Harris.
`
`r
`
`Lassen - Exhibit 1038, p. 7
`
`

`

`The structure of a typical antibody molecule
`
`antibody molecules, the V regions. These are involved in antigen binding,
`whereas the stem ofthe Y,or the C region,is far less variable andis the part
`that interacts with effector cells and molecules.
`
`All antibodies are constructed in the same way from paired heavy andlight
`polypeptide chains, and the generic term immunoglobulinis used forall such
`proteins. Within this general category, however, five different classes of
`immunoglobulins—IgM, IgD, IgG, IgA, and IgE—can be distinguished by
`their C regions, which will be described more fully in Chapter 4. More subtle
`differences confined to the V region accountfor the specificity of antigen
`binding. Wewill use the IgG antibody molecule as an example to describe the
`general structural features of immunoglobulins.
`
`3-1
`
`IgG antibodies consistof four polypeptide chains.
`
`IgG antibodies are large molecules, having a molecular weight of approximately
`150 kDa, composed of two different kinds of polypeptide chain. One, of
`approximately 50 kDa, is termed the heavy or H chain, and the other, of 25
`kDa, is termed thelight or L chain (Fig. 3.2). Each IgG molecule consists of
`two heavy chains and twolight chains, The two heavy chains are linked to
`each other by disulfide bonds and each heavy chainis linked to a light chain
`by a disulfide bond. In any given immunoglobulin molecule, the two heavy
`chains and the twolight chains are identical, giving an antibody molecule
`two identical antigen-bindingsites (see Fig. 3.1), and thus theability to bind
`simultaneously to twoidentical structures.
`Two types of light chain, termed lambda(A) and kappa(x), are foundin anti-
`bodies. A given immunoglobulin either has « chains or A chains, never one of
`each. No functional difference has been found between antibodies having i
`or « light chains, and eithertypeoflight chain may be found in antibodies of
`any of the five major classes. The ratio of the two types oflight chain varies
`from species to species. In mice, the average x to A ratio is 20:1, whereas in
`humans it
`is 2:1 and in cattle it is 1:20, The reason for this variation
`is unknown. Distortions of this ratio can sometimes be used to detect
`the abnormalproliferation of a clone of B cells. These would all express the
`identical light chain, and thus an excess of ) light chains in a person might
`indicate the presenceofa B-cell tumor producing A chains.
`
`;
`|
`
`1
`|
`
`|
`
`By contrast,the class, and thusthe effector function, ofan antibody,is defined
`by the structure ofits heavy chain. There are five main heavy-chain classes or
`isotypes, some of which have several subtypes, and these determine the func-
`tional activity ofan antibody molecule.Thefive major classes ofimmunoglob-
`ulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin
`G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). Their heavy
`chains are denoted by the corresponding lower-case Greekletter (p, 6, y, @,
`and €, respectively). lgG is by far the most abundant immunoglobulin and has
`several subclasses (IgGl, 2, 3, and 4 in humans). Theirdistinctive functional
`Properties are conferred by the carboxy-terminal part of the heavy chain,
`whereit is not associated with the light chain. We will describe the structure
`and functionsof the different heavy-chain isotypes in Chapter4, The general
`Structural featuresofall the isotypes are similar and wewill consider IgG,the
`Most abundantisotypein plasma, as a typical antibody molecule.
`
`UP of two heavy chains (green) and two
`
`Fig. 3.2 immunoglobulin molecules
`are composed of two types of protein
`Chain: heavy chains and light chains.
`'mmunoglobulin molecule is made
`
`light chains (yellow) joined by disulfide
`bonds so that each heavy chain is linked
`to a light chain and the two heavy chains
`are linked together.
`
`Lassen - Exhibit 1038, p. 8
`
`Lassen - Exhibit 1038, p. 8
`
`

`

`
`
`
`
`Chapter 3: Antigen Recognition by B-cell and T-cell Receptors
`
`
`3-2.
`
`Immunoglobulin heavy and light chains are composed of constant
`and variable regions.
`
`The amino acid sequences of many immunoglobulin heavy andlight chains
`have been determined and reveal two important features of antibody mole-
`cules. First, each chain consists of a series of similar, although not identical,
`sequences, each about 110 amino acids long. Each of these repeats corre-
`spondsto a discrete, compactly folded region of protein structure known as
`a protein domain. Thelight chain is made up of two such immunoglobulin
`domains, whereasthe heavy chain ofthe IgG antibody containsfour(see Fig,
`3.1a). This suggests that the immunoglobulin chains have evolved by repeated
`duplication of an ancestral gene correspondingto a single domain.
`
`The second important feature revealed by comparisons of amino acid
`sequencesis that the amino-terminal sequences of both the heavy andlight
`chains vary greatly between different antibodies. The variability in sequence
`is limited to approximately the first 110 amino acids, corresponding to the
`first domain, whereas the remaining domains are constant between
`immunoglobulin chains of the same isotype. The amino-terminal variable or
`V domainsof the heavy andlight chains (Vq and V,, respectively) together
`make up the V region of the antibody and confer onit the ability to bind
`specific antigen, while the constant domains (C domains) of the heavy and
`light chains (Cy and C;, respectively) make up the C region (see Fig. 3.1b, c).
`The multiple heavy-chain C domains are numbered from the amino-terminal
`end to the carboxy terminus, for example Cj;1, Cy2, and so on.
`
`3-3.
`
`The antibody molecule can readily be cleaved into functionally
`distinct fragments.
`
`The protein domains described above associate to form larger globular
`domains, Thus, when fully folded and assembled, an antibody molecule
`comprises three equal-sized globular portions joined by a flexible stretch of
`polypeptide chain known asthe hingeregion (see Fig. 3.1b). Each arm ofthis
`Y-shaped structure is formed by the association of a light chain with the
`amino-terminal half of a heavy chain, whereasthe trunk ofthe Y is formed by
`the pairing of the carboxy-terminal halves of the two heavy chains. The asso-
`ciation of the heavy andlight chains is such that the Vj; and V,, domains are
`paired, as are the Cy1 and C;, domains. The Cy3 domainspair with each other
`but the Cy2 domains do notinteract; carbohydrate side chains attached to
`the Cy2 domainslie between the two heavy chains. The two antigen-binding
`sites are formed by the paired Vy and V;, domains atthe ends of the two arms
`of the Y (see Fig. 3.1b).
`
`Proteolytic enzymes (proteases) that cleave polypeptide sequences have been
`used to dissect the structure of antibody molecules and to determine which
`parts of the molecule are responsible forits various functions. Limiteddigestion
`with the protease papain cleaves antibody molecules into three fragments
`(Fig. 3.3). Two fragments are identical and contain the antigen-binding activity.
`These are termed the Fab fragments, for Fragment antigen binding, The Fab
`fragments correspond to the two identical arms of the antibody molecule,
`which contain the completelight chains paired with the Vj, and C,1 domains
`of the heavy chains. The other fragment contains no antigen-bindingactivity
`but was originally observed to crystallize readily, and for this reason was
`named the Fc fragment, for Fragment crystallizable. This fragment corre-
`spondsto the paired ©);2 and C3 domainsandis the part of the antibody
`molecule that interacts with effector molecules and cells. The functional
`differences between heavy-chain isotypes lie mainly in the Fc fragment.
`
`
`
`Lassen - Exhibit 1038, p. 9
`
`Lassen - Exhibit 1038, p. 9
`
`

`

`
`
`The structure of a typical antibody molecule
`
`
`
`Fig. 3.3 The Y-shaped immunoglobu-
`lin molecule can be dissected by
`partial digestion with proteases.
`
`Papain cleaves the immunoglobulin
`
`molecule into three pieces, two Fab
`
`fragments and one Fc fragment (upper
`
`panels). The Fab fragment contains the
`
`V regions and binds antigen. The Fc
`
`fragmentis crystallizable and contains C
`
`regions, Pepsin cleaves immunoglobulin
`
`to yield one F(ab’)s fragment and many
`
`small piecesof the Fe fragment, the
`
`largest of whichis called the pFc’ frag-
`
`ment (lower panels). F(ab’)is written
`
`with a prime becauseit contains a few
`
`more amino acids than Fab, including
`
`the cysteines that form the disulfide
`bonds.
`
`
`
`
`!
`
`|
`|
`i
`
`
`
`
`
`The protein fragments obtained afterproteolysis are determined bywhere the
`protease cuts the antibody moleculein relationto the disulfide bonds thatlink
`the two heavy chains. These lie in the hinge region between the Cy] and Cq2
`
`domains and,as illustrated in Fig. 3.3, papain cleaves the antibody molecule
`on the amino-terminal sideof the disulfide bonds. This releases the two arms
`
`of the antibody as separate Fab fragments, whereas in the Fe fragment the
`
`carboxy-terminal halves of the heavy chains remain linked.
`
`
`Another protease, pepsin, cuts in the same general region of the antibody
`molecule as papain but on the carboxy-terminal side of the disulfide bonds
`
`(see Fig. 3.3). This produces a fragment, the F(ab’)2 fragment,in which the
`
`two antigen-binding arms of the antibody molecule remain linked. In this
`
`case the remainingpart ofthe heavy chainis cut into several small fragments.
`
`The F(ab’), fragment has exactly the same antigen-binding characteristics as
`
`the original antibody but is unable to interact with any effector molecule.It is
`
`thus of potential value in therapeutic applications of antibodies as well as in
`
`Tesearch into the functional role ofthe Fc portion.
`
`Genetic engineering techniques also now permit the construction of many
`
`different antibody-related molecules. One importanttypeis a truncated Fab
`
`Comprising only the V domain of a heavy chain linked by a stretch of
`
`synthetic peptide to a V domain ofalight chain. This is called single-chain Fv,
`
`named from Fragmentvariable. Fv molecules may becomevaluable thera-
`
`Peutic agents because of their small size, which allows them to penetrate
`
`tissues readily, They can be coupled to protein toxins to yield immunotoxins
`with potential application, for example, in tumor therapy in the case of a Fy
`Specific for a tumor antigen (see Chapter14).
`
`
`
`
`
`Lassen - Exhibit 1038, p. 10
`
`Lassen - Exhibit 1038, p. 10
`
`

`

`
`
`Chapter 3; Antigen Recognition by B-cell and T-cell Receptors
`
`
`
`3-4 = molecule is flexible, especially atthe hinge
`
`n
`
`The hinge region that links the Fe and Fab portions of the antibody molecule
`is in reality a flexible tether, allowing independent movementof the two Fab
`arms, rather than a rigid hinge. This has been demonstrated by electron
`microscopy of antibodies bound to haptens. These are small molecules of
`various sorts, typically about the size of a tyrosine side chain. They can be
`recognized by antibody but are only able to stimulate production of anti-
`hapten antibodies when linked to a larger protein carrier (see Appendix |,
`Section A-1). An antigen madeof two identical hapten molecules joined by a
`short flexible region can link two or more anti-hapten antibodies, forming
`dimers, trimers, tetramers, and so on, which can be seen by electron
`microscopy (Fig. 3.4), The shapes formed by these complexes demonstrate
`that antibody molecules are flexible at the hinge region, Someflexibility is
`also found at the junction between the V and C domains, allowing bending
`and rotation of the V domain relative to the C domain. For example, in the
`antibody molecule shown in Fig. 3.1a, not only are the two hinge regions
`clearly bent differently, but the angle between the V and C domainsin each of
`the two Fab arms is also different. This range of motion has led to the junction
`between the V and C domains being referred to as a ‘molecular ball-
`and-socketjoint.’ Flexibility at both the hinge and V—C junction enables the
`binding of both arms of an antibody molecule to sites that are various
`distances apart, for example, sites on bacterial cell-wall polysaccharides.
`Flexibility at the hinge also enables the antibodies to interact with the
`antibody-binding proteins that mediate immuneeffector mechanisms.
`
` Angle between arms is 0° Angle between arms is 90°
`
`'
`
`Fig. 3.4 Antibody arms are joined by
`a flexible hinge. An antigen consisting
`of two hapten molecules (red balls in
`diagrams) that can cross-link two
`antigen-binding sites is used to create
`antigen:antibody complexes, which can
`be seen in the electron micrograph.
`Linear, triangular, and square forms are
`seen, with short projections or spikes.
`Limited pepsin digestion removes these
`spikes (not shown in the figure), which
`therefore correspond to the Fc portion of
`the antibody; the F(ab‘)z pieces remain
`cross-linked by antigen. The interpre-
`tation of the complexes is shown in the
`diagrams. The angle between the arms
`of the antibody molecules varies, from
`0°in the antibody dimers, through 60°
`in the triangular forms, to 90° in the
`square forms, showing that the
`connections between the arms are
`flexible. Photograph (x 300,000)
`courtesy of N.M. Green.
`
`
`
`Lassen - Exhibit 1038, p. 11
`
`Lassen - Exhibit 1038, p. 11
`
`

`

`Thestructureofatypicalantibody molecule [
`
`
`4.5
`The domains of an immunoglobulin molecule have similar structures.
`
`‘As we saw in Section 3-2, immunoglobulin heavy and light chains are
`~omposed ofa series of discrete protein domains. These protein domainsall
`nave a similar folded structure. Within this basic three-dimensional struc-
`
`ture, there are distinct differences between V and C domains.The structural
`gimilarities and differences can be seen in the diagram ofa light chain in Fig.
`
`35. Fach domain is constructed from two B sheets, which are elements of
`protein structure made up of strands of the polypeptide chain (f strands)
`
`wacked together; the sheetsare linked by a disulfide bridge and together form
`aroughly barrel-shaped structure, known as a B barrel. The distinctive folded
`
`eructure of the immunoglobulin protein domain is known as the
`immunoglobulin fold.
`
`Both the essential similarity of V and C domains andthecritical difference
`‘between them are most clearly seen in the bottom panels of Fig. 3.5, where
`‘the cylindrical domains are opened outto reveal how the polypeptide chain
`
`folds to create each of the B sheets and howit forms flexible loops asit
`changes direction. The main difference between the V and C domains is that
`flexible loops of the V domains form the antigen-binding site of the
`the V domain is larger, with an extra loop. Wewill see in Section 3-6 that the
`
`immunoglobulin molecule.
`
`
`
`|
`
`|
`|
`
`
`
`Fig. 3.5 The structure of immuno-
`globulin constant and variable
`domains. The upper panels show
`schematically the folding pattern of the
`constant (C) and variable (V) domains
`of an immunoglobulin light chain. Each
`domain is a barrel-shaped structure in
`which strands of polypeptide chain
`(B strands) running in opposite directions
`(antiparallel) pack together to form two
`f sheets (shown in yellow and green in
`the diagram of the C domain), which are
`held together by a disulfide bond. The
`way the polypeptide chain folds to give
`the final structure can be seen more
`clearly when the sheets are opened
`out, as shownin the lower panels. The
`B strandsare lettered sequentially with
`respect to the order of their occurrence
`in the amino acid sequence of the
`domains; the order in each B sheet is
`characteristic of immunoglobulin
`domains. The B strands C’ and C”that
`are found in the V domains but not in
`the C domains are indicated by a blue
`shaded background. The characteristic
`four-strand plus three-strand (C-region
`type domain)or four-strand plusfive-
`strand (V-region type domain) arrange-
`ments are typical immunoglobulin
`superfamily domain building blocks,
`found in a whole range of other
`proteins as well as antibodies and
`T-cell receptors.
`
`Lassen - Exhibit 1038, p. 12
`
`Lassen - Exhibit 1038, p. 12
`
`

`

`
`
`
`Chapter 3: Antigen Recognition by B-cell and T-cell Receptors
`
`
`
`Manyofthe amino acids that are common to C and V domains of immuno.
`globulin chains lie in the core of the immunoglobulin fold and are critical to
`its stability. For that reason, other proteins having sequences similar to those
`of immunoglobulins are believed to form domains ofsimilar structure, and in
`many cases this has been demonstrated by crystallography. These
`immunoglobulin-like domains are present in many other proteins of the
`immune system, and in proteins involved in cell-cell recognition in the
`nervous system and other tissues. Together with the immunoglobulins and
`the T-cell receptors, they make up the extensive immunoglobulin superfamily,
`
`Summary.
`
`The IgG antibody molecule is made up of four polypeptide chains, comprising
`twoidentical light chains and twoidentical heavy chains, and can be thought
`of as forming a flexible Y-shaped structure. Each of the four chains has a
`variable (V) region at its amino terminus, which contributes to the antigen-
`binding site, and a constant (C) region, which determines the isotype. The
`isotype of the heavy chain determines the functional properties of the
`antibody, The light chains are bound to the heavy chains by many non-
`covalent interactions and by disulfide bonds, and the V regions of the heavy
`and light chains pair in each arm ofthe Y to generate two identical antigen-
`bindingsites, which lie at the tips of the arms of the Y. The possession of two
`antigen-bindingsites allows antibody molecules to cross-link antigens and to
`bind them much morestably. The trunk of the Y, or Fe fragment, is composed
`of the carboxy-terminal domainsof the heavy chains. Joining the armsof the
`Y to the trunk are the flexible hinge regions. The Fe fragment and hinge
`regions differ in antibodies of different isotypes,
`thus determining their
`functional properties. However, the overall organization of the domainsis
`similar in all isotypes.
`
`Theinteractionoftheantibodymoleculewith
`specificantigen.
`
`We have described the structure of the antibody molecule and how the V
`regions of the heavy and light chains fold and pair to formthe antigen-binding
`site. In this part of the chapterwewill look at the antigen-binding site in more
`detail. We will discuss the different ways in which antigens can bind to anti-
`body and address the question of how variation in the sequences of the
`antibody V domains determines the specificity for antigen.
`
`3-6
`
`Localized regions of hypervariable sequence form the antigen-
`bindingsite.
`
`The V regions of any given antibody moleculediffer from thoseof every other.
`Sequence variability is not, however, distributed evenly throughout the V
`regions but is concentrated in certain segmentsofthe V region. The distribution
`of variable aminoacids can be seen clearly in whatis termed a variability plot
`(Fig. 3.6), in which the amino acid sequences of many different antibody
`V regions are compared. Three segments of particular variability can be
`identified in both the V,; and V,, domains. They are designated hypervariable
`regions and are denoted HV1, HV2, and HV3.In the light chains these are
`roughly from residues 28 to 35, from 49 to 59, and from 92 to 103,respectively.
`
`
`
`Lassen - Exhibit 1038, p. 13
`
`Lassen - Exhibit 1038, p. 13
`
`

`

`The interaction of the antibody molecule with specific antigen
`
`
`
`
`
`
`Fig. 3.6 There are discrete regions
`of hypervariability in V domains. A
`variability plot derived from comparison
`of the amino acid sequences of several
`dozen heavy-chain and light-chain V
`domains. At each amino acid position
`the degree of variability is the ratio of
`the numberof different amino acids
`seenin all of the sequences together
`to the frequency of the most common
`amino acid. Three hypervariable regions
`(HV1, HV2, and HV3) are indicated
`in red and are also knownas the
`complementarity-determining regions,
`CDR1, CDR2, and CDR3. They are
`flanked by less variable framework
`regions (FR1, FR2, FR3, and FR4,
`shownin blue or yellow).
`
`Lassen - Exhibit 1038, p. 14
`
`The most variable part of the domain is in the HV3 region. The regions
`between the hypervariable regions, which comprise the rest of the V domain,
`show less variability and are termed the framework regions. There are four
`such regions in each V domain, designated FR1, FR2, FR3, and FR4.
`
`The framework regions form the f sheets that provide the structural frame-
`work of the domain, whereas the hypervariable sequences correspond to
`three loops at the outer edge ofthe f barrel, which are juxtaposedin the folded
`domain (Fig. 3.7). Thus, not only is sequence diversity concentrated in
`particular parts of the V domainbutit is localized to a particular region on the
`surface of the molecule. When the Vj; and V;, domains are paired in the anti-
`body molecule, the hypervariable loops from each domain are brought
`together, creating a single hypervariable site at the tip of each arm of the
`molecule. This is the binding site for antigen, the antigen-binding site or
`antibody combiningsite. The three hypervariable loops determine antigen
`specificity by forming a surface complementary to the antigen, and are more
`commonly termed the comple