COLD SPRING HARBOR SYMPOSIA
`ON QUANTITATIVE BIOLOGY
`
`VOLUME XLI
`
`Origins of Lymphocyte Diversity
`
`COLD SPRINC HARBOR LAHOI{ATOHY
`
`1977
`
`PFIZER EX. 1116
`Page 1
`
`

`

`COLD SPRING HAHBOH SYMPOSIA ON (~UANTITATIVE BIOLOGY
`VOLUME XLI
`
`"J 1977 by The Cold Spring Harbor Laboratory
`International Standard Book Number 0-879G9-040-2 (clothbound)
`Library of Congress Catalog Card Number 34-8174
`
`Printed in the United States oj'Amerim
`All riuhts reserved
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`I 'I' >) fl NATIONAL LIBRARY fJr Mf.OICINE
`~ETHESDA, MARYLAND 20014
`
`Follll!!nl in J<J]J "-1'
`
`RECINALD C. IIARRIS
`
`Direr/or oflht' liiolo,~iml l.alumtlon JIJ2·1/o fl)](j
`
`J'n•vio11s Symj10sirt Vo/11 1111'1
`
`I (1933) Surface Phenomena
`II ( 1934) Aspects of Growth
`III (1935) Photochemical Reactions
`IV !1936) Excitation Phenomena
`V (1937) Internal Secretions
`VI (1938) Protein Chemistry
`VII !1939) Biological Oxidations
`VIII !1940) Permeability and the Nature of Cell Membranes
`IX !1941) Genes and Chromosomes: Structure and Organization
`X (1942) The Relation of Hormones to Development
`XI (1946) Heredity and Variation in Microorganisms
`XII !1947) Nucleic Acids and Nucleoproteins
`XIII (1948) Biological Applications of Tracer Elements
`XIV (1949) Amino Acids and Proteins
`XV !1950) Origin and Evolutionof Man
`XVI !1951) Genes and Mutations
`XVII (1952) The Neuron
`XVIII (1953) Viruses
`XIX (1954) The Mammalian Fetus: Physiological Aspects of De(cid:173)
`velopment
`XX !1955) Population Genetics: The Nature and Causes of Genetic
`Variability in Population
`
`XXI !1956) Genetic Mechanisms: Structure and Function
`XXII (1957) Population Studies: Animal Ecology and Demography
`XXIII (1958) Exchange of Genetic Material: Mechanism and Con-
`sequences
`XXIV (1959) Genetics and Twentieth Century Darwinism
`XXV (1960) Biological Clocks
`XXVI (1961) Cellular Hegulatory Mechanisms
`XXVII (1962) Basic Mechanisms in Animal Virus Biology
`XXVIII !1963) Synthesis and Structure of Macromolecules
`XXIX (196·1) Human Genetics
`XXX !1965) Sensory Receptors
`XXXI (1966) The Genetic Code
`XXXII 0967) Antibodies
`XXXIII (1968) Replication of DNA in Microorganisms
`XXXIV (1969) The Mechanism of Protein Synthesis
`XXXV 0970) Transcription of Genetic Material
`XXXVI 0971) Structure and Function of Proteins at the Three-
`dimensional Level
`XXXVII !1972) The Mechanism of Muscle Contraction
`XXXVIII 0973) Chromosome Structure and Function
`XXXIX 11974) Tumor Viruses
`XL 0975) The Synapse
`
`The Symposium Volumes are published by the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, and
`may be purchased directly from the Laboratory or through booksellers. Price of Volume XLI-2-part set $GO.OO !inc.
`postage). May be purchased only as a complete set. Price subject to change without notice.
`
`PFIZER EX. 1116
`Page 2
`
`

`

`Contents
`
`Part l
`
`Symposium Participants
`Foreword
`
`Introduction
`
`The Common Sense of Immunology N. K. Jerne
`
`LYMPHOCYTE FUNCTION
`
`1'-cell Marlwrs and Differentiation
`
`'l'hymopoietin and Bursopoietin: Induction Signals Regulating Early Lympho(cid:173)
`cyte Differentiation G. Goldstein, M. Scheid, E. A. Boyse, A. Brand and D. G.
`Gilmour
`/. L. Weissman,
`Normal and Neoplastic Maturation of 'I'-lineage Lymphocytes
`S. Baird, R. L. Gardner, V. E. Papaioannou and W. Raschke
`Regulation of Cellular and Humoral Immune Responses by '!'-cell Subclasses
`H. Cantor and E. A. Boyse
`Surface Markers and Functional Relationships of Cells Involved in Murine
`B-lymphocyte Differentiation L. A. Herzenberg, L. A. Herzenberg, S. J.
`l3lacli, M. R. Lolwn, K. Olwmura, W. van der Loo, B. A. Osborne, D. Hewgill,
`-1. W. Goding, G. Gutman and N. L. Warner
`An Unusual Kappa Immunoglobulin Antigen Present on the Membrane ofT and
`B Lymphocytes A. B. Gottlieb, M. Engelhard, H. G. Kunlwl and S. M. Fu
`Rat 'I'hy-1 Antigens from Thymus and Brain: Their Tissue Distribution, Purifica(cid:173)
`tion, and Chemical Composition A. F. Williams, A. N. Barclay, M. Letarte(cid:173)
`Muirhead and R .. ]. Morris
`Specialized DNA Polymerases in Lymphoid Cells D. Baltimore, A. E. Silverstone,
`P. C. Kung, 1'. A. Harrison and R. P. McCaffi·ey
`Studies on the Interactions between Viruses and Lymphocytes B. R. Bloom,
`A. Senih, G. Stoner, G. Ju, M. Nowalwwshi, S. Kano and L. Jimenez
`
`Helper and Suppressor 1' Cells and Their Products
`The Hermaphrocyte: A Suppressor-Helper 'I' Cell R. K. Gershon, D. D. Eardley,
`K. F. Naidorf and W. Ptah
`Suppressor 'I' Cells in Tolerance to Non-self and Self Antigens A. Basten, R. Lob(cid:173)
`lay, E. Chia, R. Gallard and H. Pritchard-Briscoe
`Tolerance: Two Pathways of Negative lmmunoregulation in Contact Sensitivity
`to DNFB H. N. Claman, S. D. Miller and J. W. Moorhead
`
`vii
`
`v
`XV
`
`1
`
`5
`
`9
`
`23
`
`33
`
`47
`
`51
`
`63
`
`73
`
`85
`
`93
`
`105
`
`PFIZER EX. 1116
`Page 3
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`

`

`viii
`
`CONTENTS
`
`Current Concepts of the Antibody Response: Heterogeneity of Lymphoid Cells,
`Interactions, and Factors M. Feldmann, P. Beverley, P. Erb, S. Howie, S.
`Kontiainen, A. Maoz, M. Mathies, I. McKenzie and J. Woody
`Suppressive and Enhancing T-cell Factors as /-region Gene Products: Properties
`and the Subregion Assignment 7'. Tada, M. Taniguchi and C. S. David
`
`B-cell Differentiation and Commitment
`
`.}. J. T. Owen,
`
`In Vitro Studies on the Generation of Lymphocyte Diversity
`R. K. Jordan, J. H. Robinson, U. Singh and H. N. A. Willcox
`Studies of Generation of B-cell Diversity in Mouse, Man, and Chicken M.D.
`Cooper, J. F. Kearney, P. M. Lydyard, C. E. Grossi and A. R. Lawton
`Ontogeny of Murine B Lymphocytes: Development of Ig Synthesis and of Reac(cid:173)
`tivities to Mitogens and to Anti-Ig Antibodies F. Melchers, .J. Andersson
`and R. A. Phillips
`Development and Modulation of B Lymphocytes: Studies on Newly Formed B
`Cells and Their Putative Precursors in the Hemopoietic Tissues of Mice
`M. C. Ra(f
`Induction of Immunoglobulin Synthesis in Abelson Murine Leukemia Virus(cid:173)
`transformed Mouse Lymphoma Cells in Culture B. J. Weimann
`The Interplay of Evolution and Environment in B-cell Diversification N. R.
`Klinman, N. H. Sigal, E. S. Metcalf, S. K. Pierce and P. J. Gearhart
`Synthesis of Multiple Immunoglobulin Classes by Single Lymphocytes B. Per(cid:173)
`nis, L. Forni and A. L. Luzzati
`Immunoglobulin Receptors on Murine B Lymphocytes E. S. Vitetta, J. Cambier,
`J. Forman, J. R. Kettman, D. Yuan and J. W. Uhr
`Functional and Structural Characterization of Immunoglobulin on Murine B
`Lymphocytes R. M. E. Parhhouse, E. R. Abney, A. Bourgois and H. N. A.
`Willcox
`Origin and Differentiation of Lymphocytes Involved in the Secretory IgA Re(cid:173)
`sponse J. J. Cebra, P .• ]. Gearhart, R. Kamal, S. M. Robertson and J. Tseng
`Mechanism of B-cell Activation and Self-Non-self Discrimination G. Moller
`Growth and Maturation of Single Clones of Normal Murine T and B Lymphocytes
`In Vitro J. Andersson, A. Coutinho, F. Melchers and T. Watanabe
`Hapten-specific B Lymphocytes: Enrichment, Cloning, Receptor Analysis, and
`Tolerance Induction G. J. V. Nossal, B. L. Pihe, J. W. Stoclwr, .]. E. Layton
`and J. W. Goding
`Regulation of Clonal B-lymphocyte Proliferation by Anti-immunoglobulin or
`Anti-Ia Antibodies P. W. Kincade and P. Ralph
`Cellular and Molecular Interactions in Control ofB-cell Immunity and Tolerance
`E. Diener, C. Shiozawa, B. Singh and K.-C. Lee
`
`Receptors
`
`Lymphocyte Surface Immunoglobulins: Evolutionary Origins and Involvement
`in Activation J. J. Marchalonis, .}. M. Decher, D. DeLuca, .}. M. Moseley,
`P. Smith and G. W. Warr
`Antigen-binding, Idiotypic Receptors from T Lymphocytes: An Analysis of Their
`Biochemistry, Genetics, and Use as Immunogens To Produce Specific Im(cid:173)
`mune Tolerance H. Binz and H. Wigzell
`On the Structure of the T-cell Receptor for Antigen U. Krawinhel, M. Cramer,
`C. Berch, G. Hiimmerling, S. J. Blach, K. Rajewslly and K. Eichmann
`The Immune Response to Staphylococcal Nuclease: A Probe of Cellular and
`Humoral Antigen-specific Receptors D. H. Sachs, J. A. Berzofshy, C. G.
`Fathman, D. S. Pisetshy, A. N. Schechter and R. H. Schwartz
`Functional Characterization of Rabbit Lymphocytes Carrying Fe Receptor P.-A.
`Cazenave, D. Juy and C. Bona
`Structural and Functional Heterogeneity of Fe Receptors H. M. Grey, C. L.
`Anderson, C. H. Heusser, B. K. Borthistle, K. B. Von Eschen and J. M. Chiller
`
`113
`
`119
`
`129
`
`139
`
`147
`
`159
`
`163
`
`165
`
`175
`
`185
`
`193
`
`201
`217
`
`227
`
`237
`
`245
`
`251
`
`261
`
`275
`
`285
`
`295
`
`307
`
`315
`
`PFIZER EX. 1116
`Page 4
`
`

`

`CONTENTS
`
`lX
`
`THE MAJOR HISTOCOMPATIBILITY COMPLEX
`
`Structure of the Molecular Products of the MHC
`
`Structure of HL-A A and B Antigens Isolated from Cultured Human Lympho(cid:173)
`cytes J. L. Strominger, D. L. Mann, P. Parham, R. Robb, T. Springer and
`C. Terhorst
`Structural Studies of (3 2-Microglobulin-associated and Other MHC Antigens
`P. A. Peterson, H. Anundi, B. Curman, L. Klareslwg, S. Kvist, L. Ostberg,
`/,. Rash, L. Sandberg and K Sege
`Comparative Chemical Analyses and Partial Amino Acid Sequences of the Heavy
`Chains of HL-A Antigens E. Appella, N. Tanigalli, 0. Henrillsen, D. Press(cid:173)
`man, D. F. Smith and T. Fairwell
`Structul'al Differences between Parent and Variant H-2K Glycoproteins from
`Mouse Strains Carrying H-2 Gene Mutations S. G. Nathenson, .]. L. Brown,
`B. M. Ewenstein, T. Nisizawa, D. W. Sears and J. H. Freed
`Structure of Murine Histocompatibility Antigens B. A. Cunningham, R. Hen(cid:173)
`ning, R. J. Milner, K Resile, J. A. Ziffer and G. M. Edelman
`Structural Studies of H-2 and TL Alloantigens J. W. Uhr, E. S. Vitetta, J. Klein,
`M. D. Poulih, D. G. Klapper and ,J. D. Capra
`Chemical Characterization of Products of the H-2 Complex J. Silver, J. M.
`Ceclw, M. McMillan and L. Hood
`Human Ia Antigens-Purification and Molecular Structure D. Snary, C. Bam(cid:173)
`stable, W. F. Boclrner, P. Goodfellow and M. ,], Crumpton
`Chemiral and Immunological Characterization of l-IL-A-linked B-lymphocyte
`Alloantigens 7'. A. Springer, J. F. Kaufman, L. A. Siddoway, M. Giphart,
`D. L. Mann, C. Terhorst and J. L. Strominger
`The Guinea Pig MHC: Functional Significance and Structural Characterization
`B. D. Schwartz, A.M. Kash and E. M. Shevach
`Partial Amino Acid Sequences of MHC Products J. Silver
`Analysis of Lymphocyte Surface Antigen Expression by the Use of Variant Cell
`Lines R. Hyman and I. Trowbridge
`
`323
`
`331
`
`341
`
`343
`
`351
`
`363
`
`369
`
`379
`
`387
`
`397
`405
`
`407
`
`PFIZER EX. 1116
`Page 5
`
`

`

`Model-building Studies of Antigen-binding Sites:
`'The 1-lapten-binding Site of MOPC-315
`
`E. A. PADLAN, D. R DAVIES, I. PECHT,* D. GIVOL* AND c. WRIGHT"!"
`Laboratmy of" Molecular Biology, National Institute of" Arthritis, Metabolism and Digestive Diseases, National Institutes of"
`Ilmlth, Bcthesd~r, J'vfm:yland 20014; ·''Department of" Chcmiml Immunology, The Weizmann Institute of" Science, Relwvot
`'
`Ismel; T Laborat01:y oj"J'v!olccular Biophysics, Department of" Zoology, Oxford University, Oxf'ord, England
`
`The molecular basis for the structural diversity
`of antibody combining sites has become apparent
`through the recent X-ray diffraction studies on
`several immunoglobulin <lg) fragments (see Davies
`et al. 1975a,b for a review). These structures reveal
`that the combining site is formed by bringing to(cid:173)
`gether the three hypervariable regions rwu and
`Kabat 1970) of v~. and of VII to form a continuous
`complementarity-providing surface. A quantitative
`comparison of the tertiary structures of a number
`of variable domains from both light and heavy
`chains has demonstrated that their nonhypervari(cid:173)
`able or framework regions are very similar, with the
`principal diflcrences occurring in the hypervariable
`loops (Padlan and Davies 1975).
`In view of this structural invariance, the im(cid:173)
`munoglobulin variable region can be regarded as
`consisting of a rigid framework to which are at(cid:173)
`tached the hypervariable loops. These loops are
`not large, generally consisting of at most 17 resi(cid:173)
`dues; in addition, the configurations of a number of
`difl'erent hypervariable loop regions are already
`known from X-ray difl'raction. Thus it might be
`possible to construct by model building new loop
`regions (and hence the Ig binding sites) from a
`knowledge of the sequences alone. Since it is clearly
`impractical to determine by X-ray difl'raction the
`structures of all the interesting antibodies, this
`model-building method oilers an attractive alter(cid:173)
`native approach when sequence data are available.
`We have examined the potential usefulness of this
`possibility by attempting to build the V regions of
`the rabbit anti-type-III polysaccharide antibody
`!Davies and Padlan 1976) and of protein MOPC-
`315, a mouse immunoglobulin with dinitrophenyl
`rDNP)-binding specificity
`!Eisen et al. 1968),
`based on the known three-dimensional structure
`of McPC-603 Fab' (Padlan et al. 1973, 1974; Segal
`et al. 1974a,b) together with the amino acid se(cid:173)
`quences of these immunoglobulin Fab's.
`In this paper, we describe the results of two
`independent attempts to construct the model of
`MOPC-315, one at the National Institutes of
`Health and the other at the Weizmann Institute.
`The two models were ultimately merged to give a
`final model that is presented and is discussed in
`terms of the known binding specificity, the kinetic
`mapping of the binding site (Pecht et al. 1972a,b;
`
`Haselkorn et al. 1974), and the results of affinity
`labeling with various reagents (Goetzl and Metzger
`1970a,b; Givol et al. 1971; Haimovich et al. 1972).
`We discuss some of the limitations and the potential
`of this method of model building, the assumptions
`that have to be made, and some of the additional
`information that will be needed to put this tentative
`approach on a firmer footing.
`
`MATERIALS AND METHODS
`
`Sequence Alignment
`
`The amino acid sequences of the V 11 domains of
`proteins McPC-603 (Rudikoff and Potter 1974)
`and MOPC-315 (Francis et al. 1974; L. Hood,
`M. Margolies, D. Givol and R. Zakut, unpubl.)
`were aligned for maximum sequence homology
`and structural analogy, as presented in Table la.
`The sequence of New V 11 (Poljak et al. 1974) is
`included for comparison. A similar alignment of
`the 603 (Segal et al. 1974a) and 315 V~. !Dugan et al.
`1973) sequences is given in Table lb. However,
`only the sequence of the first 49 residues is known
`for 603, and the residues given for the rest of the
`sequence are those which most frequently occur in
`mouse kappa chains (McKean et al. 1973). Included
`in Table lb are the sequences of the V~. domains of
`REI <Epp et al. 1974), Meg (Fett and Deutsch
`1974), and New (Chen and Poljak 1974) proteins.
`The sequence numbering schemes used in these
`tables are those obtained from the original refer(cid:173)
`ences. Whenever a residue position is referred to in
`the text, however, the quoted number corresponds
`to the 315 sequence. The hypervariable regions of
`the light chain are: Ll, residues 23-36; L2, residues
`52-58; and L3, residues 91-99; and those of the
`heavy chain are: H1, residues 31-36; H2, residues
`49-66; and H3, residues 99-104.
`
`Model Building
`
`Model 1 was constructed at the Weizmann Insti(cid:173)
`tute from Nicholson molecular models (Labquip,
`Reading, England), which have a scale of 1 em-~ 1
`A, and CPK space-filling models (1.25 em = 1 Al.
`Model 2 and the final model were constructed at
`the National Institutes of Health from Kendrew
`
`G27
`
`PFIZER EX. 1116
`Page 6
`
`

`

`628
`
`PADLAN ET AL.
`
`(2 em = 1 AJ molecular models mepetition Engi(cid:173)
`neers, Cambridge, England).
`The general principle used for model building was
`to c.onstruct first the framework part of the variable
`regiOn, b.ased on the structure of 603 Fab'. The
`hypervanable loop~ were then constructed, leaving
`the structure as l~ttle changed as possible except
`when forced by ammo acid insertions and deletions
`An attempt was made to maximize the structurai
`stability within eacl~ loop by forming hydrogen
`bonds ";hene~er possible and maintaining the phi
`and psi peptide angles within reasonable limits
`(Ramaknshnan and Ramachandran 1965 ). The
`interactions between loops were then maximized
`leaving no large holes in the domain interior whil~
`minimizing sterie hindrance between groups:
`The L1 regions in the kappa chains 60:3 and REI
`a:e simple lo_ops, . whereas the corresponding re(cid:173)
`gwns are hehcal m New (Poljak ct a!. 1974) and
`Meg (Schiffer et a!. 1973), which are both lambda
`chains. Since the light chain of protein a 15 is of the
`lambda type and since this region has the same
`n.umber of r.esidues as New and Meg with no gross
`differences m the nature of the amino acid side
`chains, it is most likely that this region in 315 will
`have a similar configuration to that found in New
`and Meg. Accordingly, the L1 loop in :315 was built
`to conform as closely as possible to the correspond(cid:173)
`ing region in the lambda chains with the aid of the
`ato~ic coordinates of the Meg backbone kindly
`provided by M. Schiffer and coworkers ( pers.
`comm.).
`L2 was built by assigning to it the same back(cid:173)
`bone conformation as in protein 603. In this region,
`603 and REI are not significantly different rPadlan
`and Davies 1975). Moreover, kappa and lambda
`light chains have the same number of residues in
`this part of the molecule may hoff 1972); an excep(cid:173)
`tion is New ('fable 1b), which has a seven-residue
`deletion in this part of the molecule. The L2 region
`in Meg appears to have the same structure as 603
`<Edmundson et a!. 1974). Thus it is reasonable to
`assume that 315 L2 will have the same configura(cid:173)
`tion as that of 603.
`L3 was built essentially as in 603, the main dif(cid:173)
`ference being the possibility of forming more hy(cid:173)
`drogen bonds in 315. The L3 loop in :315 was built
`as two, fully antiparallel, segments, the loop in 60:3
`being less regular. Although the sequence of 60:3
`is not known in this region, the six mouse kappa
`chains sequenced so far <McKean et a!. 1973) all
`have a proline at position 94. The possible occur(cid:173)
`rence of this residue in 603 could explain the fewer
`hydrogen bonds formed in 603 L3.
`Protein 315 has one more residue than 603 in H 1
`<Table 1a). Originally, the sequence of 315 H1 con(cid:173)
`tained a lysine in position 35 (Francis eta!. 1974).
`Structurally, this residue is analogous to Met 34
`of603 since both occur two positions from the struc(cid:173)
`turally invariant Trp 37. In 603, Met 34 is buried in
`
`the domain interior. Its replacement by a lysine
`side chain would cause a significant rearrangement
`of the II 1 backbone in order to expose the charged
`amino group to solvent. A reexamination of the
`sequence of this part of:l15 led to the assignment of
`a Trp instead of the Lys in position 35 rL. Hood.
`M. Margolies, D. Givol and H. Zakut, unpubl. '·
`A Trp side chain can easily he accommodated in the
`domain
`interior without significantly changing
`the configuration from that observed in 603.
`The alignment shown in Table 1a leads us to con(cid:173)
`clude that the additional residue in H1 of :315 can
`be best accommodated in the exposed loop at the
`beginning of II 1. The glycine at position 32 permi:_s
`the construction of a sharp turn in this part of 31'-'·
`H2 has three more residues in 603 than in 315,
`whereas :315 and New arc of the same length in this
`region. An initial model of :J 15 H2 was built by
`simply excising three residues from the end of the
`60:3 II2 loop. Minor adjustments were then made t_c>
`make the :nfi II2 loop resemble as closely as possi(cid:173)
`ble the corresponding region in New <Poljak et <1l.
`1974).
`Basically the same procedure was employed in
`building the :3Hi II:l region, which has the same
`length as New and which is two residues shorter
`than 60:3. Here, however, the C-terminal segment
`of :l15 I I:3 was made to approximate the correspond(cid:173)
`ing region in GO:l, rather than in New. The difler(cid:173)
`encc between G0:3 and New, aside from the two(cid:173)
`residue insertion in 60:3, is the configuration of the
`segment immediately preceding the phenylalanine
`at position 105, which almost always contains a
`large hydrophobic residue rDayhotr 1972). In 60:3.
`the side group of Trp 104a, which is structurallY
`analogous to Leu 10:3 of :315, projects into the
`hapten-binding cavity. On the other hand, the back(cid:173)
`bone of this segment in New appears to be diflerent
`rPoljak et al. 1974 ). In view of the greater structural
`similarity of residues 10:3-105 of 315 (Leu-Tyr(cid:173)
`Phe) to those of G0:3 ITrp-'l'yr-Phe) rather than to.
`those of New IGiy-Cys-IIc), the configuration of
`60:3 was followed in building this segment of 315.
`However, it should be kept in mind that an alterna(cid:173)
`tive configuration might be that observed in New.
`As soon as a tentative model of the binding site
`was completed, a likely site for DNP-binding wa::;.
`located using the criteria that the nitro groups ot
`the DNP moiety must be hydrogen-bonded to the
`protein and that the stacking van der Waals inter-.
`action between the DNP ring and the side group ot
`Trp 93 (L) must be maximal. The model was th~n
`adjusted to accommodate the DNP-hapten wlult:
`maintaining the general stability of the various loop
`structures. Further adjustments were then made to
`ensure the feasibility of labeling Tyr 33 (L) and
`Lys 52 !II) by specific affinity reagents rGivol et al.
`1971; Haimovich et a!. 1972). The adjustment~
`made to the tentative model to accommodate tht'
`ligand were minor and involved only slight displace-
`
`PFIZER EX. 1116
`Page 7
`
`

`

`Table I. Amino Acid Sequences of V 11 Domains of Proteins McPC-603
`and MOPC-:315
`
`(a) Ali;;nment of V11 Sequences
`
`McPC-GO:l
`MOI'C-:l15
`New
`
`0
`0
`4
`0
`2
`0
`4
`0
`2
`0
`1 Glu-Val -Lys-Leu-Val-Glu-Ser-Gly-Gly -Gly
`1 Asp- Val -Gin-Leu -Gln-Glu -Ser -Gly -Pro -Gly
`I Pea- Val -Gin-Leu -Pro-Glu -Ser -Gly -Pro -Glu
`
`4
`0
`4
`0
`0
`0
`0
`0
`4
`0
`ll Leu- Val -Gin-Pro -Gly-Gly -Ser -Leu-Arg -Leu
`II Leu-Val-Lys-Pro-Ser-Gln-Ser-Leu-Ser -Leu
`11 Leu- Val -Ser -Pro -Gly-Glx -Thr-Leu-Ser -Leu
`
`0
`4
`4 01 4 0
`0
`4
`0
`21 Ser -Cys -Ala-'I'hr -Ser -Gly -Phe-Thr-Phe -Scr
`21 Thr-Cys -Ser-Val-'I'hr-Gly-Tyr-Ser -Ile -Thr
`21 Thr -Cys -Thr-Gly -Ser -Thr- Val-Ser -Thr -Phe
`c
`c
`3
`4
`2
`4
`2
`4
`0
`-Phe-'I'yr -Met-Glu -Trp- Val-Arg -Gin
`31 Asp-
`31 Ser -Gly -Tyr-Phe-Trp-Asn -'l'rp-Ile -Arg -Gin
`31 Ala-
`-Val-Tyr-Ile -Val-Trp-Val-Arg-Gln
`c
`c
`4
`4
`2
`0
`0
`0
`0
`2
`40 Pro -Pro -Gly-Lys -Arg-Leu-Glu-Trp-Ile -Ala
`40 Phe -Pro -Gly-Asn-Lys-Leu -Glu -Trp -Leu -Gly
`40 Pro -Pro -Gly-Arg-Gly-Leu -Glu-Trp-Ile -Ala
`
`0
`2
`0
`0
`0
`0
`0
`2
`4
`50 Ala -Ser -Arg-Asn-Lys-Gly -Asn-Lys -Tyr -Thr
`50 Phe- lie -Lys-Tyr -Asp-Gly-
`-Ser
`50 'l'yr- Val -Phe-'l'yr -His -Gly-
`-Thr
`
`0 01
`4
`0
`0
`3
`0
`2
`filibThr-Glu -Tyr-Ser -Ala-Ser - Val-Lys -Gly -Arg
`57 Asx- !Tyr , Gly) Asx -Pro - Ser -Leu- Lys- Asn - Arg
`57 Ser -Asp -Thr-Asp-Thr-Pro -Lcu-Arg-Ser -Arg
`
`3
`0
`0
`0
`0
`2
`0
`4
`0
`4
`68 Phc-Ile - Val-Scr-Arg -Asp-Thr-Ser -Gin -Ser
`68 Val-Ser -Ile -Thr -Arg-Asp -Thr-Ser -Glu -Asn
`68 Val -Thr -Met-Leu- Val-Asn -Thr-Ser -Lys -Asn
`
`0
`4
`0
`0
`4
`0
`4
`4
`0
`78 Ile -Leu -'l'yr-Leu-Gln-Met -Asn-Ala -Leu -Arg
`78 GIn -!'he - Phe- Leu- Lys- Leu -Asp- Ser -Val - Thr
`78 Gin -Phe -Scr-Leu-Arg-Leu -Ser -Ser- Val -Thr
`c
`4
`4
`4
`3
`2
`0
`0
`c
`c
`88 Ala -Glu -Asp-Thr -Ala-Ile -Tyr -Tyr -Cys -Ala
`88 Thr-Glx -Asx-Thr-Ala-Thr -Tyr-Tyr-Cys -Ala
`88 Ala -Ala -Asp- Thr- Ala- Val - Tyr- Tyr- Cys -Ala
`c
`c
`c
`c
`c
`0
`0
`2
`3
`:3
`98 Arg-Asn -Tyr-Tyr -Gly-Ser
`-Thr-Trp-Tyr -Phe
`-Leu-Tyr -Phe
`98 Gly -Asp -Asn-Asp-His-
`98 Arg-Asx -Leu-lie -Ala-
`-Gly-Cys -lie
`14 c
`0
`4
`0
`2
`4
`0
`4
`lOG Asp- Val -Trp-Gly -Ala-Gly -Thr-Thr- Val -Thr
`lOG Asp -Tyr -Trp-Gly -Gln-Giy -Thr-Thr-Leu -Thr
`lOG Asx- Val -Trp-Gly -Gln-Gly -Ser -Leu- Val -Thr
`
`4
`0
`0
`IIG Val -Ser -Ser
`llG Val -Ser -Ser
`116 Val-Ser -Ser
`
`(a) Above each residue in the Mci'C-603 sequence is its structural location designated
`by: 0, completely exposed to solvent; I, mainly exposed; 2, partly exposed, partly buried
`in the domain interior; :l, mainly buried; 4, completely buried; or C, in contact with the
`homologous domain. The numbers at the left alongside the sequences correspond to the
`sequence number of the first residue in each row as obtained from the original publica(cid:173)
`tions.
`
`629
`
`PFIZER EX. 1116
`Page 8
`
`

`

`MePC-603
`REI
`MOPC-315
`Meg
`New
`
`Table I. ( eontinuedJ
`
`lbi Alignment of' v~. sequence'S
`
`4
`0
`4
`0
`3
`0
`()
`()
`1 Asp-Ile - Val-Met-Thr-Gln -Ser -Pro -Ser
`1 Asp-lle -Gln-Met-Thr-Gln -Ser -Pro -Ser
`1 Pea -Ala- Val- Val-Thr-Glu -Glu-
`-Ser
`1 Pea -Ser -Ala-Leu-Thr-Gln -Pro -Pro-
`1 Pea -Ser -Val- Leu -Thr- Gin -Pro- Pro-
`
`0
`-Ser
`-Ser
`-Ala
`-Ser
`-Ser
`
`()
`4
`0
`0
`()
`()
`0
`4
`()
`4
`11 Leu-Ser - Val-Ser -Ala-Gly -Glu -Arg- Val -Thr
`11 Leu-Ser -Ala-Ser- Val-Gly -Asp-Arg- Val -Thr
`10 Leu-Thr -Thr-Ser -Pro -Gly -Gly -Thr-IVal, Ilel
`10 Ala -Ser -Gly-Ser -Leu-Gly -Gin -Ser- Val -Thr
`10 Val -Ser -Gly-Ala -Pro -Gly -Gin -Arg- Val -Thr
`
`0
`()
`27 Asx -Ser
`28
`30
`30
`27e
`
`()
`:l
`0
`()
`()
`4
`0
`4
`()
`4
`21 Met-Scr -Cys-Lys -Ser -Ser -Glx -Ser -Leu -Leu
`21 Ile -Thr -Cys-Gln-Ala -Scr -Gln-
`20 Leu-Thr -Cys-Arg-Ser -Ser -Thr-Gly -Ala- Val
`20 Ile -Ser -Cys-Thr-Gly-Thr-Ser -Ser -Asp -Val
`20 Ilc -Scr -Cys-Thr-Gly -Ser -Ser -Ser -Asn -lie
`c
`c
`c
`c
`e
`4
`3
`-Gly-Asx-Glx -Lys -Asx-Phe-Leu -Ala
`-Asp-Ile -Ile -Lys-Tyr-Lcu -Asn
`-Thr -Thr -Ser -Asn-Tyr -Ala -Asn
`-Gly -Gly -Tyr -Asn-Tyr- Val -Ser
`-Gly -Ala -Gly -Asn-His- Val - Lys
`c
`c
`c
`c
`0
`0
`0
`3
`4
`35 Trp -Tyr -Glx-IG]x)-Lys-Pro -Gly -Glx- Pro -Pro
`35 Trp-Tyr -Gln-Gln-Thr-Pro -Gly-Lys-Ala -Pro
`:37 Trp -Ile -Glx-Glx -Lys -Pro -Asx-IIis -Leu -Phc
`37 Trp -Tyr -Gin-Gin -His -Ala -Gly -Lys -Ala -Pro
`34 Trp -Tyr -Gin-Gin -Leu-Pro -Gly -Thr -Ala -Pro
`c
`c
`()
`1
`()
`1
`1
`4
`4
`0
`45 Lys -Leu -Leu-Ile
`- Tyr - X - X - X - X - X
`45 Lys -Leu -Leu-lie
`-Tyr -Glu -Ala -Scr -Asn -Leu
`47 Thr-Gly -Leu-Ile
`-Gly -Gly -Thr-Scr -Asp -Arg
`47 Lys- Val - Ilc -Ilc
`-Tyr -Glu- Val -Asn-Lys -Arg
`44 Lys -Leu -Leu- Ilc
`-Phc-IIis -Asn-Asn-Ala -Arg
`
`() 41 () 14 ()
`0
`()
`55 X - X -Gly- Val -Pro -Ala -Arg-Phc-Scr
`55 Gin -Ala -Gly- Val-Pro -Scr -Arg-Phc-Sc1·
`57 Ala -Pro -Gly- Val-Pro- Val -Arg-I'hc-Scr
`57 Pro -Ser -Gly- Val-Pro -Asp-Arg-I'hc-Scr
`61
`-I'hc-Scr
`
`4
`-Gly
`-Gly
`-Gly
`-Gly
`-Val
`
`()
`4
`()
`4
`()
`2
`()
`()
`4
`()
`65 Scr -Giy -Ser -Arg-Thr -Asp- I'hc-Thr-Lcu -Thr
`65 Ser -Gly -Scr-Gly-Thr-Asp-Tyr-Thr-Phc -Thr
`67 Scr -Leu -Ile -Gly -Asp-Lys -Ala -Ala -Leu -Thr
`67 Ser -Lys -Ser -Gly -Asn-Thr -Ala -Scr -Leu -Thr
`64 Ser -Lys -Ser-Gly-Scr -Sc1· -Ala-Thr-Lcu -Ala
`
`() 1 2 4
`()
`2
`2
`()
`()
`4
`75 Ilc -Asx -Pro- Val -Glx -Ala -Asx--Asp- Val -Ala
`75 Ile -Scr -Scr-Lcu-Gln -Pro -Glu--Asp-Ilc -Ala
`77 Ile -Thr -Gly-Ala -Glx -Thr -Glx -Asp-Asp -Ala
`77 Val -Ser -Gly-Leu-Gln -Ala -Glu-Asp-Glu -Ala
`74 Ile -Thr -Gly-Leu-Gln -Ala -Glu-Asp-Giu -Ala
`c
`c
`c
`()
`()
`2
`4
`4
`4
`e
`85 Thr-Tyr -Phc-Cys- X - X - X - X - X - X
`85 Thr -Tyr -Tyr-Cys -Gin -Gin -Tyr -Gin -Scr -Leu
`87 Mct-Tyr -Phc-Cys-Ala-Leu-Trp-Phc-Arg -Asx
`87 Asp-Tyr -Tyr-Cys-Ser -Scr -Tyr-Glu-Gly -Scr
`84 Asp-Tyr -Tyr-Cys -Gin -Scr -Tyr -Asp-Arg -Scr
`()
`c
`c
`()
`e
`()
`4
`4
`3
`- X -X- X -Phc-Gly-Gly-Gly-Thr -Lys
`
`95
`
`630
`
`PFIZER EX. 1116
`Page 9
`
`

`

`MODELS OF ANTIGEN-BINDING SITES
`
`631
`
`Table I. (continued)
`
`I b) Alignment of V, sequences
`
`-Pro -Tyr-Thr-I'he-Gly-Gln-Gly-Thr -Lys
`D5
`H7
`-His -Phe- Val-Phe-Gly -Gly -Gly -Thr -Lys
`97 Asp -Asn -Phe- Val -Phe-Gly -Thr-Gly -Thr -Lys
`94
`-Leu -Arg- Val-Phe-Gly -Gly -Gly -Thr -Lys
`
`0
`0
`3
`0
`4
`10,1 Leu -Glu -lie -Lys -Arg
`104 Leu-Gin -lie -Thr-Gly
`lOG Val-Thr -Val-Leu-Gly
`107 Val-Thr -Val-Leu-Gly
`105 Leu-Thr -Val-Leu-Arg
`
`I b) Sec footnote to"· The Mci'C-603 sequence is known only to residue 49; for the rest
`of the Hequencc, the most frequently occurring residue in other mouse kappa chains I Me(cid:173)
`l< can ct al. IH7:li is given fi>r each position except for the hypervariable residues, which
`are simply designated by X. Ala :!4, Thr 85, and Gly 100 in 603 project into the V11 :V, inter(cid:173)
`face but are not in actual contact with V11 ; this structural classification is designated by
`the lower case c.
`
`ments of main-chain atoms and rotation of side
`groups about single bonds.
`Atomic coordinates of the :Jl5 hypervariable
`residues were measured from the model and ad(cid:173)
`justed using Diamond's model-building program
`miamond 1966). In building the DNP-hapten into
`the model, the nitro groups were assumed to be
`coplanar with the phenyl ring. Although the crystal
`structure of2,4-dinitroaniline has not been reported,
`theoretical arguments (Pauling 1948) would pre(cid:173)
`dict a planar molecule. This is consistent with the
`observations of Trotter <Trotter 1961; Trotter and
`Williston 1966) on the crystals of various nitro(cid:173)
`phenyl compounds; Trotter found small departures
`from coplanarity but attributed these to intermo(cid:173)
`lecular forces in the crystal. In DNP-lysine and its
`derivatives, a hydrogen bond was assumed between
`one oxygen of the 2-nitro group and the NH of the
`E-arnino group of the lysine, thus fixing the position
`of the CE.
`
`RESULTS
`
`Sequence Comparisons and Correlation with
`Structure
`
`Included in Table 1a,b is the structural classifi(cid:173)
`cation of each residue, as observed in 603, according
`to whether it is buried in the domain interior, ex(cid:173)
`posed to solvent, or involved in the contact with
`the homologous domain.
`
`ComJmrison of frameworh residues. As expected,
`the residues that are completely or mainly buried
`in the domain interiors are more conserved than
`those that are completely or mainly exposed to
`solvent. In V11 , 16 of the 29 residues that are com(cid:173)
`pletely or mainly buried are identical in 315 and
`60:3. Of the buried residues that are diflerent, a
`Val in :315 replaces a Thr <position 24), a Tyr re(cid:173)
`places a Phe (position 27), and 2 Leu replace an lie
`rposition 48) and a Met rposiiion s:n in GO:l. Of the
`
`residues that are completely or mainly
`exposed to
`solvent, 18 of 44 are identical 5 invol
`S
`ve er-Thr
`·
`. .
`'
`mterchanges (positiOns 21
`')5 28 3· 0
`d
`' ~ '
`, an 71) a
`'
`Lys replaces an Arg (position 44) and Gl
`'
`'
`..
`a
`ure-
`I
`p aces a Gin (positiOn 76). The residues I·n 1 d .
`vo ve In
`beta bends (Venkatachalam 1968) are
`"t
`'fh b d
`qui e con-
`d
`-1-
`e en around position 14-15 ·
`serve .
`r
`IS taCI I(cid:173)
`tated by the sequence Pro-Gly in 603· Pro 14 ·
`1
`IS a SO
`.
`,
`'
`·
`present m 315. The other hairpin bend arou d
`41 4') . d
`t .
`n posi-
`wn
`- ~ IS ue to the sequence Pro-Gly h" h ·
`IC
`IS
`.
`W
`present m both 315 and 603.
`The lack of co~plete sequence information for 603
`precludes a detarled correlation of the v~. sequences
`with structure. On the basis of the tentative se(cid:173)
`quence shown in Table 1b, 17 of the 26 complete!
`or mainly buried residues are identical in 315 an~
`603 V~.. Of the buried residues that are different in
`315 and 603, a Thr in 315 replaces a Val in 603 at
`position 12 and a Leu replaces a Met at position 20.
`Of the completely or mainly exposed residues, 16 of
`41 are identical, and 2 Thr for Ser interchanges oc(cid:173)
`cur at positions 11 and 21. The Gly at position 16
`which is involved in a beta bend, is present in both
`315 and 603, the sharp bend in 315 being further
`facilitated by Pro 15. Another bend around positions
`42-43 involves the sequence Pro-Gly in 603; Pro is
`also present at position 42 in 315. The additional
`residue at position 8 or 9 (Table 1b) produces a kink
`in this region in kappa chains (Epp et al. 1974; Segal
`et al. 1974a,b). However, the a-carbons of residues 8
`and 10 are only about 5 A apart, so that the excision
`of this additional residue requires only minor struc(cid:173)
`tural alterations. The substitutions at positions 45
`and 46 result in one more potential hydrogen bond
`in 315 compared to 603.
`The replacement of an interior residue that in(cid:173)
`volves a change in side-group size is generally
`accompanied by a compensatory substitution else(cid:173)
`where so that no large voids are created in the do(cid:173)
`main interiors. For example, in V11 , the lie <315)
`for Phe (603) substitution at position 29 is balanced
`
`PFIZER EX. 1116
`Page 10
`
`

`

`632
`
`PADLAN ET AL.
`
`by the Phe 1315) for Leu (603) change at position 79.
`The cluster involving Met 83, Leu 86, and Val 114
`in 603 is replaced by the grouping of Leu 83, Val86,
`and Leu 114 in 315. Furthermore, the rep