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`PFIZER EX. 1058
`Page 2
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`

`

`()
`M T;[r ·r
`
`ANNUAL REVtEW OF
`IMMUNOLOGY
`
`Volume 1, 1983
`
`WILLIAM E. PAUL, Editor
`National lnstitutes of Health, Bethesda, Maryland
`
`C. GARRISON FATHMAN, Associate Editor
`Stanford University, Stanford, California
`
`HENRY METZGER, Associate Editor
`National lnstitutes of Health, Bethesda, Maryland
`
`ANNU A L REV IEWS IN C.
`
`- ~ - - - - - - - - - - - - -
`PALO ALTO. CA LIFORNIA 94306 USA
`4139 EL CA MINO WAY
`
`PFIZER EX. 1058
`Page 3
`
`

`

`; I
`
`ANNUAL REVIEWS INC.
`Palo Alto, California, USA
`
`COPYRIGHT@ 1983 DY ANNUAL REVIEWS INC., PALO ALTO, CALIFORNIA, USA. ALL
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`conditions, of articles published in any Annual Review serial before January 1,
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`to make a single copy of an article without charge for use in research or
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`PRINTED AND BOUND IN THE UNITED STATES OF AMERICA
`
`PFIZER EX. 1058
`Page 4
`
`

`

`Annual Review of Immunology
`Volume 1, 1983
`
`CONTENTS
`
`GETTING STARTED 50 YEARS AGo--ExPERIENCEs,
`PERSPECTIVES, AND PROBLEMS OF THE FIRST 21
`YEARS, Elvin A. Kabut
`CELLULAR MECHANISMS OF IMMUNOLOGIC TOLERANCE,
`G. J. V. Nossal
`
`T-CELL AND B-CELL RESPONSES TO VIRAL ANTIGENS AT
`THE CLONAL LEVEL, L. A. Sherman, A. Vitiello, and
`N. R. Klinman
`STRUCTURAL BAsis OF ANTIBODY FuNcTION, David R.
`Davies and Henry Metzger
`
`GENETICS OF THE MAJOR HISTOCOMPATIBILITY
`CoMPLEX: THE FINAL ACT, J. Klein, F. Figueroa,
`and Z. A. Nagy
`!MMUNODIOLOGY OF TISSUE TRANSPLANTATION: A
`RETURN TO THE PASSENGER LEUKOCYTE CONCEPT,
`Kevin J. Lafferty, Stephen J. Prowse, Charmaine
`J. Simeonovic, and Hilary S. Warren
`AuTOIMMUNITY-A PERSPECTIVE, Howard R. Smith and
`Alfred D. Steinberg
`MECHANISMS OFT CELL-B CELL INTERACTION, Alfred
`Singer and Richard J. Hodes
`
`CoMPLEMENT LIGAND-REcEPTOR INTERACTIONS THAT
`MEDIATE BIOLOGICAL RESPONSES, Douglas T. Fearon
`and Winnie W. Wong
`CYTOLYTIC T LYMPHOCYTES, M. Nabholz and Jl R.
`MacDonald
`
`REGULATION OF B-CELL GROWTH AND DIFFERENTIATION
`BY SoLUBLE FACTORS, Maureen Howard and William
`E. Paul
`MEDIATORS OF INFLAMMATION, Gary L. Larsen and Peter
`M. Henson
`
`THE RoLE oF CELL-MEDIATED IMMUNE REsPoNsEs IN
`RESISTANCE TO MALARIA, WITH SPECIAL REFERENCE
`TO OxiDANT STRESS, Anthony C. Allison and Elsie M.
`Eugui
`
`33
`
`63
`
`87
`
`119
`
`143
`
`175
`
`211
`
`243
`
`273
`
`307
`
`335
`
`361
`
`(continued)
`
`PFIZER EX. 1058
`Page 5
`
`

`

`CONTENTS (continued)
`
`BIOSYNTHESIS AND REGULATION OF IMMUNOGLOBULINS,
`Randolph Wall and Michael Kuehl
`THE BIOCHEMISTRY OF ANTIGEN-SPECIFIC T-CELL
`FACTORS, D. R. Webb, Judith A. Kapp, and Carl
`W. Pierce
`IMMUNOREGULATORY T-CELL PATHWAYS, Douglas R.
`Green, Patrick M Flood, and Richard K. Gershon
`THE COMPLEXITY OF STRUCTURES INVOLVED IN T-CELL
`Acnv ATION, Joel W. Goodman and Eli E. Sercarz
`IMMUNOGLOBULIN GENES, Tasuku Honjo
`GENES OF THE MAJOR HisTocoMPATIBILITY CoMPLEX oF
`THE MousE, Leroy Hood, Michael Steinmetz, and
`Bernard Malissen
`
`GENETICS, EXPRESSION, AND FUNCHON OF IDIOTYPES,
`Klaus Rajewsky and Toshitada Takemori
`EPITOPE-SPECIFIC REGULATION Leonore A. Herzenberg,
`Takeshi Tokuhisa, and Kyoko Hayakawa
`T-LYMPHOCYTE CLONES, C. Garrison Fathman, and John G.
`Frelinger
`Sumner INDEX
`
`393
`
`423
`
`439
`
`465
`499
`
`529
`
`569
`
`609
`
`633
`657
`
`Special Announcement: New From Annual Reviews
`
`Some Historical and Modern Aspects of Amino Acids, Fermentations
`and Nucleic Acids, Proceedings of a Symposium held in St. Louis,
`Missouri, June 3, 1981, edited by Esmond E. Snell. Published
`October, 1982. 141 pp.; softcover; $10.00 USA/$12.00 elsewhere,
`postpaid per copy
`
`PFIZER EX. 1058
`Page 6
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`

`

`Ann. Rev. lmmunol. 1983. 1:87-117
`Copyright 'iJ 1983 by Annual Reviews Inc. All rights reserved
`
`STRUCTURAL BASIS
`OF ANTIBODY FUNCTION
`
`David R. Davies and Henry Metzger
`
`National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases,
`National Institutes of Health, Bethesda, Maryland 20205
`
`INTRODUCTION
`
`It is less than 20 years since the general architecture of antibodies was
`elucidated and even less time since the explicit molecular basis of antibody
`specificity first became clear. In the subsequent explosion of information
`about the immune system, two basic principles have emerged: (a) Antibod(cid:173)
`ies remain the only known structures whose diversity is sufficient to explain
`the fine specificity exhibited by the immune response; and, (b) antibody
`function is mediated by a molecule whose structure consists of two distinct
`regions-one that carries a recognition site for antigenic determinants, and
`a second by which the antibody reacts with receptors of a variety of effector
`systems.
`In this review we examine the current information on the structure of
`antibodies. We do not describe again the basic four-chain structure of
`immunoglobulins nor the division into variable and constant regions, which
`arc by now well known (e.g. 4, 90, 118). We instead concentrate on the
`higher resolution data, much of which is still in the course of refinement.
`We discuss the Fabs, in particular with reference to the combining site and
`the specificity of binding to hapten; the Fe region with reference to the
`binding site of protein A of Staphylococcus aureus and of C1q; and the
`structure of the hinge with reference to its possible role in separating Fab
`and Fe.
`Whereas the characterization of the structures of individual proteins and
`of their interactions with small molecules can now be carried out with some
`87
`
`0732-0582/83/0410-0087$02.00
`
`PFIZER EX. 1058
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`

`88
`
`DAVIES & METZGER
`
`sophistication, the interaction of two or more macromolecules still presents
`considerable difficulties. It is not surprising therefore that our under(cid:173)
`standing of how antibodies interact with the macromolecular receptors on
`effector systems (e.g. C1q in the complement pathway, Fe receptors on cell
`membranes) is much less advanced. Our review of this aspect of antibody
`structure and function therefore involves more questions than answers.
`
`IMMUNOGLOBULIN STRUCTURE
`
`General Comments
`Our knowledge of the three-dimensional structure of antibodies at atomic
`resolution rests mainly on X-ray diffraction investigations of fragments.
`Intact proteins for which X-ray analyses have been carried out consist of
`Kol (106), an IgG1(A) human myeloma protein, the protein Dob (161), an
`IgG 1(K) human cryoglobulin, and recently the human myeloma IgG 1(A)
`protein, Meg (129). In Kol, and also apparently in another immunoglobu(cid:173)
`lin, Zie (58a), the crystal structure contains an unusual feature: The Fe
`occupies a number of different positions in the crystal that are not crystallo(cid:173)
`graphically related, with the result that no significant electron density oc(cid:173)
`curs in this region of the crystal, there being an abrupt drop in density at
`the end of the hinge. In both Dob and Meg there is a 15-amino-acid-residue
`deletion in the hinge (63, 169), thus, presumably, reducing the flexibility of
`the molecule and enabling the Fe to be located in the electron density,
`although in the case of Dob the crystals are disordered and do not diffract
`to high resolution.
`The structures of three Fabs have been published, Newm (148), Kol
`(106), and McPC603 (152), as well as the structures of a number of VL
`dimers and L chain dimers (4). The structure of human IgG Fe has been
`determined, and also its complex with protein A of Staphylococcus aureus
`(43). These structures have been reviewed most recently by Amzel & Poljak
`(4) and are not covered comprehensively in this review.
`Because of the deficiencies in the crystals of the intact molecules, our
`knowledge of the whole antibody molecule has to be a sum of its parts. The
`flexibility of the molecule, in particular in the region between the Fab and
`the Fe, may preclude for some time visualizing directly by X-ray diffraction
`an intact molecule with intact hinge at atomic resolution. However, the
`checks that can be made on this composite three-dimensional picture of the
`antibody molecule are reassuring. Thus, the Kol Fab in isolated form in the
`crystal is quite similar to the Fab of the whole molecule in its crystal form.
`Also, the Fe in Dob has, within the experimental error of the comparison,
`the same overall structure as does the Fe in the isolated Fe crystals.
`
`PFIZER EX. 1058
`Page 8
`
`

`

`ANTIBODY FUNCTION
`
`89
`
`Fab Structure and the Antibody Combining Site
`McPC603 AND THE PHOSPHOCHOLINE BINDING SITE The structure
`of McPC603 Fab, a mouse myeloma lgA (K) with phosphocholine (PC)
`binding capability, has been determined at 3.1-A resolution ( 41, 42, 125,
`152) and is being refined to 2.7 A (Y. Satow, D. R. Davies, manuscript in
`preparation). The overall three-dimensional structure of the Fab is illus(cid:173)
`trated in Figure 1, which demonstrates the strong lateral association be(cid:173)
`tween domains of the light (L) and heavy (H) chains, together with the
`relatively weak longitudinal interactions along each chain. Figure 1 also
`shows the clustering of six of the seven hypervariab1e regions at the tip of
`the Fab, forming the complementarity-determining surface (89, 90). The
`variable domains have a very similar three-dimensional structure for both
`the Land H chains and across species (4, 42, 123). The constant domains
`CL and C111 are also very similar. Both the variable and the constant pairs
`of domains are related by approximately twofold (rotation about the V axis
`of 180° will superpose V L on V u) and the angle between the two axes has
`been referred to as the elbow bend of the Fab and has been observed to vary
`G 52C
`N 28
`N 28
`
`Figure 1 The a-carbon backbone of McPC603 Fab. The heavy chain is represented by the
`thick line. The two variable domains arc at the top and the constant domains are at the b?ttom
`of the figure. The complementarity-determining residues (CDR) are shown as filled ctrcles.
`Two residues in each CDR loop have been labeled.
`
`PFIZER EX. 1058
`Page 9
`
`

`

`90
`
`DAVIES & METZGER
`
`between approximately 137° for Fab Newm (4), 135° for McPC603 (152),
`147° for Dob (161), and approximately 170° for Kol (106).
`Figure 2 shows the combining site of McPC603 with ~C ?ound .. (~~
`Satow, E. A. Padlan, G. H. Cohen, D. R. Davies, manuscnpt tn pr~pc1r~
`tion). The choline is attached at the bottom of a pocket located p~mctpal Y
`between the hypervariable regions I-13 and L3. The phosphate ts on the
`surface and contacts residues from the heavy chain. It is apparent that PC
`is a small molecule and that the greater part of the hypervariable surface
`is not directly in contact with it. At the front of the pocket there are two
`hydrogen bond donors positioned within reasonable hydrogen bonding dis(cid:173)
`tance of the phosphate oxygens; these are the hydroxyl group of Ty~ 3~H
`and the guanidinium group of Arg 52H (152). The residues lining the mstde
`of the pocket are Tyr 94L on the right side, Asp 91 L on the left, Leu 96L
`at the back, (125, 146), and the side chain of Trp lOOaH at the top left. In
`addition, the backbone of residues 92-94L form the lower rim of the front
`of the pocket.
`
`CONFORMATIONAL CHANGE One of the mechanisms proposed for
`effector function activation involves an allosteric change upon antigen bind(cid:173)
`ing (110). Since crystals of immunoglobulins have large solvent channe!s
`and can bind to haptens soaked in through these channels, crystallographic
`investigation offers a direct way for observing conformational changes,
`when they occur.
`When PC binds to McPC603 in the crystal, no significant conformational
`change occurs in the protein. There is a small movement of Trp 1 04a away
`
`Figure 2 Stereo drawing of the combining site ofMcPC603 with phosphocholine bound. The
`lower residues (91-96 and F32) are from the light chain. The remaining residues belong to
`the three complementarity-determining regions of the heavy chain. The phosphocholine has
`the phosphate group in front with the choline moiety buried in a pocket.
`
`PFIZER EX. 1058
`Page 10
`
`

`

`ANTIBODY FUNCTION
`
`91
`
`from the pocket, but no other change of any significance. However, there
`are several reasons why it cannot be concluded from this observation that
`antigen-antibody interaction results in no conformation change:
`1. The crystals are grown in a concentrated ammonium sulfate solution,
`and it has been observed that in the absence of PC there is a peak at the
`phosphate binding site interpreted to be a sulfate ion (126). A conforma(cid:173)
`tional change might have been triggered by the presence of this sulfate ion,
`so that no additional change would be observed upon PC binding. However,
`in this respect it should be noted that in Fab Newm, (148) no conforma(cid:173)
`tional change occurs upon binding of a neutral vitamin K 1 derivative.
`2. PC is small and the association constant ('·---105 M-1) with McPC603
`might be insufficient to trigger a conformational change that could be
`induced by a larger, more tightly binding antigen. The same consideration
`applies to Fab Newm.
`3. The only two structures at atomic resolution of immunoglobulins with
`known binding specificity are of Fabs. Although improbable, one cannot
`rule out the possibility that changes that occur with the intact molecule
`might not occur with fragments (81 ).
`Thus, although there is no support from X-ray diffraction for a conforma(cid:173)
`tional change associated with antigen binding, such a change cannot be
`rigorously excluded.
`
`McPC603: THE CONTACTING RESIDUES AND THE EFFECT OF
`CHANGES IN TilE COMDINING SITE The PC molecule is in direct con(cid:173)
`tact with only a limited number of residues. They include side chains from
`all three heavy chain hypervariable regions and from one (L3) light chain
`region. The next most distant range of contacts contain many residues that
`play a role in positioning the directly contacting residues and changes in
`these might be expected to influence PC binding. An example of such a side
`chain is Glu 3511, a residue in the interface between Vu and VL that makes
`a hydrogen bond with the hydroxyl of Tyr 94L, which is in turn a major
`contacting residue with hapten. A mutant of S107, a PC-binding myeloma
`protein, has been observed that has lost the ability to bind PC and also that
`fails to agglutinate PC-SRBC (144). Amino acid analysis showed that the
`mutation results in substitution of an alanine for glutamic acid in position
`3511. Although a change of this magnitude is likely to produce a significant
`rearrangement of side chains in its vicinity simply because of the difference
`in volume of the two side chains, the loss of contact with Tyr 94L reduces
`an important constraint on a residue in direct contact with hapten.
`Another mutant observed by Cook et al (38) is more puzzling. The
`mutant still bound PC, but it bound less well than S107 to PC coupled to
`
`PFIZER EX. 1058
`Page 11
`
`

`

`92
`
`DAVIES & METZGER
`
`different carriers, and showed a decrease in affinity for a variety of PC(cid:173)
`carrier conjugates. The only amino acid change observed was that ~f Asp
`~ Ala in the fifth position of the heavy chain J region. Changes tn the
`carboxy terminal half of VL were not entirely ruled out, but they ~id ~ot
`appear in a tryptic peptide analysis. The strange aspect of this mutatiOn stte
`is that it is spatially well removed from the PC pocket so that it might not
`have been expected to be involved in antigen binding. Another curious
`feature is that diverse carriers coupled to PC were all affected, which implies
`they all have some contact with this residue, or with a region influenced by
`it.
`
`SEQUENCE COMPARISON OF PC-BINDING ANTIBODIES Sequences
`and binding data are available for a variety of PC-binding myeloma proteins
`and monoclonal antibodies. They have recently been reviewed by Rudikoff
`(145) and are only discussed here in relation to the three-dimensional
`structure of McPC603.
`
`The heavy chain The sequences of 19 heavy chains of BALB/c immuno(cid:173)
`globulins that bind phosphocholine have been analyzed (74, 145). Ten of
`these are identical and employ the Tl5 sequence. The remaining nine differ
`by one to eight residues from the T15 sequence. Gene isolation and analysis
`have revealed that all 19 of the V u regions must have arisen from the single
`germline Tl5 Vu gene segment (39). M167 is the most divergent protein
`with eight V u substitutions. In both M 167 and HPCG 13 the same change
`(Thr at position 40) occurs, but all the other substitutions are unique,
`occurring in only one protein.
`The PC-contacting residues Tyr33 and Arg52, together with Glu35, are
`present in all of these sequences. Similarily, all of the BALB/c sequences
`with the single exception of M167 contain a tryptophan at 100b. There is
`considerable variation in the D region for these proteins, accompanied by
`only relatively small changes in PC affinity, consistent with the fact that,
`with the exception of TrplOOb, CDR3 of the heavy chain does not play a
`major role in defining the PC pocket.
`
`The light chains The light chains of PC-binding antibodies can be repre(cid:173)
`sented by the three BALB/c myeloma proteins, Tl5, M603, and M167 (34,
`35). These light chains differ considerably in sequence, despite the similarity
`of their corresponding heavy chain sequences. However, in the PC-binding
`region, in particular with the contacting residues Tyr94, Pro95, and Leu96,
`their sequences are the same. They also all employ the same h sequence
`(J5).
`
`PFIZER EX. 1058
`Page 12
`
`

`

`ANTIBODY FUNCTION
`
`93
`
`The invariant residues in these light and heavy chains provide strong
`support for the suggestion that these PC-binding antibodies all have the
`same overall combining site for PC (125), with differences in binding speci(cid:173)
`ficity being contributed by the amino acid substitutions.
`
`f'ab Ncwm The structure of the Fab of Newm, a human IgG (A.) myeloma
`protein, has been refined to a nominal 2-A resolution (4, 148). The elbow
`bend is 137° and the L chain has a seven-residue deletion that includes
`residues 55 and 56 of CDR2, and 56 to 62 of the framework region FRill.
`The combining site is formed by the association of the remaining five
`hypervariable regions. The principal feature of the site is a shallow groove
`15 X 6 X 6 A deep, bordered by residues from the H and L chains. Fab
`Newm binds several haptens at this site with affinity constants ranging from
`103 to 105 M-1. A derivative of vitamin K 1 binds with the higher affinity and
`a crystallographic investigation has demonstrated that the menadione ring
`system binds in the shallow groove with the phytyl chain draped along the
`surface, making contact with a number of residues from the light and heavy
`chains (5). The number of contacts provided by the phytyl chain can
`probably account for the difference in binding between menadione and the
`vitamin K 1 derivative (103 vs 1.7 X 1Q5 M-1). As noted above, no conforma(cid:173)
`tional change is observed upon the hapten binding.
`
`KoL The human lgG A. cryoglobulin Kol and its Fab both crystallize and
`both structures have been solved, the intact molecule at 3.5 A and the Fab,
`for which the combining site specificity is unknown, at 1.9 A resolution
`(106). The crystals of the intact molecule display an unusual form of disor(cid:173)
`der, described above, that prevents visualization of the Fe.
`The Fab crystallizes well and has provided a detailed high resolution
`structure. The L chain conformation is quite similar to that of Fab Newm,
`except for the presence of the seven additional residues around CDR2.
`However, in the H chain, CDR3 is eight residues longer than Newm (and
`six longer than McPC603), and these additional residues fold into the
`combining site and fill it completely, thus obliterating the groove observed
`in Newm. This combining site is rich in aromatic side chains, being filled
`largely by Trp (47H, 52H, 90L and 108H), Tyr (35H, 35L and 97L), and
`His (59H).
`In both the crystals of the intact molecule and the Fab, the same contact
`is made between the hypervariable surface and the hinge segment, Cys221-
`Cys230, the light chain C terminus G1u212-Ser214, and the residues 133-
`
`PFIZER EX. 1058
`Page 13
`
`

`

`94
`
`DAVIES & METZGER
`
`138 and 196-199 of Cui of a neighboring molecule. This contact involves
`considerable surface area and it has been estimated that 1314A2 of the Kol
`hypervariable surface is excluded from solvent. The contact is tight .and
`closely packed and involves hydrophobic interactions as wei~ a~ salt h~ks
`and hydrogen bonds. Marquart et a! (105) suggest that thts mterac~ton
`could be a prototype antibody-antigen interaction and might be responstble
`for the cryo properties of this molecule.
`
`ANTIBODY COMBINING SITES Data on antibody combining sites have
`been comprehensively reviewed by Givol (76). Here, we only highlight a few
`structural topics.
`What common features, if any, will the three-dimensional structures of
`the combining sites share? Givol (76) notes that the sizes of these sites are
`comparable to those of some enzymes. Lysozyme, for example, has a groove
`that will accommodate a hexasaccharide, and this is believed to represent
`about the upper limit in size for this kind of antigen (88, 102). The binding
`of antidextrans can be divided into two classes: end-binders like W3129,
`which bind only the nonreducing end of the dextran; and middle-binders
`like QUPC52, which bind in the middle of the chain to runs of six glucose
`units. It has been suggested that the former type of antibody might have
`a pocketlike site, whereas the latter might be more likely to have a lengthy
`groove (33, 89).
`Since three-dimensional structures are known for but two Fabs with
`known binding specificities, only a limited picture can be obtained from
`direct observation. It is nevertheless suggestive that one of these structures,
`McPC603, has a pocket for binding PC, whereas the other, Newm, has a
`shallow groove where the menadione binding site is located. Again, if we
`were to argue from analogy with enzymes we might expect a pocket or
`groove in most antibodies to provide specificity. However, specificity can
`also be produced by complementarity between two interacting surfaces
`without necessarily invoking grooves or pockets, and significant contribu(cid:173)
`tions to the free energy of interaction can come from exclusion of hydro(cid:173)
`phobic groups from contact with water. This kind of specificity, like the
`interactions between subunits in a multisubunit protein, can be quite precise
`and can be destroyed by single amino acid changes. For instance, not only
`is it necessary to maintain complementarity of two interacting surfaces, but
`there also needs to be a suitable juxtaposition of oppositely charged groups
`forming salt bridges as well as appropriate disposition of hydrogen bond
`donors and receptors. Accordingly, although antibodies specific for small
`antigenic determinants will probably have a groove or pocket, other anti(cid:173)
`bodies specific for an array of amino acids such as epitopes on the surface
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`ANTIBODY FUNCTION
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`95
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`of a protein need not necessarily have these features. The Kol protein
`combining site, although similar in some respects to Newm, has its groove
`partially filled with aromatic amino acid side chains and it is significant that
`this site interacts strongly with the C-terminal portion of another Fab in
`what could be a prototype antibody/protein antigen complex.
`The availability of monoclonal antibodies to specific antigens through
`hybridoma technology should provide a significant increase in the diversity
`of antibodies studies by X-ray diffraction. In particular, they offer the
`opportunity to study interactions with ligands that are larger than simple
`haptens and that could completely fill the combining site. One example is
`an anti-influenza virus neuraminidase that has already been crystallized
`(37). More of these anti-protein antibodies need to be studied, both alone
`and complexed with antigen, to obtain structural comparisons with other
`studies (12, 57, 164). Other monoclonal antibodies to polysaccharides such
`as a 1 ~ 6 linked dextran should clarify the mechanism of binding for linear
`antigens (90) and the effects of amino acid changes on specificity.
`
`MODEL BUILDING STUDIES OF Fv Since in the last 10 years high reso(cid:173)
`lution structures have been determined for only three different Fab's, and
`only two (M603 and Newm) have known binding specificities, it appears
`that X-ray crystallography can only provide a small fraction of three(cid:173)
`dimensional structures for interesting antibody combining sites. There is
`also the difficulty that only some Fab's can be induced to crystallize. An
`alternative approach to direct X-ray analysis is to utilize the knowledge
`available from crystallography together with known amino acid sequences
`to construct models of the Fv's of interesting antibodies. The general prob(cid:173)
`lem of protein folding, i.e. how a polypeptide chain several hundred amino
`acid residues long folds into its final globular form, is being extensively
`investigated. However, it is most unlikely that it will soon be possible to
`predict correctly the final folded protein structure based on sequence. Nev(cid:173)
`ertheless, the forces that contribute to the stability of a protein continue to
`be studied and are being refined. There is also an increased appreciation of
`the dynamic aspects of protein structure.
`The problem of predicting antibody combining site structures is a special
`case with some features that make it an attractive candidate for investiga(cid:173)
`tion by molecular modeling. The similarity of variable domain structures
`is quite remarkable (4, 120-122), which indicates a strong conservation of
`the three-dimensional structure of the framework part of the variable do(cid:173)
`mains. Also, the most variable parts of the V domains occur largely at one
`end of the domain in the hypervariable loops. As a result, a comparison of
`the sequence with that of V doma~ns of known structure could, in optimal
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`96
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`DAVIES & METZGER
`
`cases, directly lead to a preliminary model that could then be refined by a
`suitable energy minimization technique. The problem becomes more com(cid:173)
`plicated when there are amino acid insertions and deletions that change the
`length of the hypervariable loops. The new loops could be copied wherever
`possible from similar size loops in proteins whose structures have been
`determined. The VL: VL dimers Rei and Au (60) provide an example of very
`similar three-dimensional structures associated with loops of the same
`length, although there are 18 amino acid differences in each domain. A
`different point of view would be inferred from the L chain dimer of Meg
`(58) where corresponding hypervariable loops do not preserve the local
`twofold symmetry of the V domains but have different conformations as a
`result of interaction with neighboring molecules in the crystal.
`The earliest modeling studies involved the 2,4-dinitrophenol-binding
`mouse myeloma protein MOPC315. The combining site of MOPC315 has
`been discussed (135) relative to the structure of Newm. Padlan et al (124)
`constructed a molecular model for MOPC315 that utilizes the framework
`structure of M603 and the hypervariable loops derived from other V regions
`that have CDR's of similar length whose structure had been determined by
`X-ray diffraction. An extensive nuclear magnetic resonance investigation of
`MOPC315 (54, 55, 186) and its interaction with ligands has led to a refined
`model that is similar to the original model, but has a different orientation
`for the DNP binding site (89). The crystal structure of the Fv of MOPC315
`is being investigated and should ultimately provide a basis for evaluating
`these models (6). Subsequently, Davies & Padlan (40) constructed a model
`for the homogeneous rabbit antibody (BS5) to type III pneumococcal poly(cid:173)
`saccharide. Potter et al (136) presented a model for the inulin-binding
`myeloma protein EPC109. More recently, Stanford & Wu (167) have con(cid:173)
`structed a backbone model for MOPC325, and Feldmann et al (62) have
`proposed models for J539 and included a proposal for the binding of hex(cid:173)
`asaccharide. The crystal structure for J539 has been determined at 4.5 A
`and the atomic resolution structure is under investigation so that it should
`soon be possible to test this model.
`None of the models described above has been subjected to any form of
`energy minimization. They may give an approximate general, low resolu(cid:173)
`tion picture of the combining sites particularly where they illustrate some
`striking insertion or deletion, as in EPC109. However, they are unlikely to
`be accurate to better than several angstroms for the backbone atoms, and
`could be quite incorrect in positioning the amino acid side chains. Until, for
`at least a few cases, they are compared with the results of X-ray diffraction
`these models should be regarded as being quite hypothetical and should be
`treated with caution. In the case of MOPC315, the structure investigation
`by Padlan et al (124) was consistent with the known chemical data from
`
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`ANTIBODY FUNCTION
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`97
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`affinity labeling and did lead to the discovery of an error in the sequence
`determination, but these correlations derive from coarse rather than fine
`detail in the model. Since single amino acid changes can produce large
`effects on structure, it is perhaps optimistic at this stage to expect to define
`an antibody site with reasonable precision. Certainly, some powerful form
`of energy minimization will be necessary to ensure that the models pro(cid:173)
`duced do at least satisfy the basic requirement of stereo-chemistry. How(cid:173)
`ever, satisfactory prediction also requires a larger library of known
`structures of antibodies to a greater variety of antigens.
`
`THE H: L ASSOCIATION For the combinatorial mechanism for generat(cid:173)
`ing antibody diversity to be reasonably effective, most light chains should
`have the ability to combine with most heavy chains. This requirement has
`been examined both in vitro and in vivo.
`The y-L interaction has been shown to obey second-order kinetics (7, 13,
`22, 69) and has a high affinity with Ka > 1010 M-1 (13). When the competi(cid:173)
`tive association of autologous and heterologous pairs of chains (i.