Pfizer v. Genentech
`IPR201(cid:26)-01488(cid:3)
`Genentech Exhibit 202(cid:25)
`
`

`

`
`
`Ourlisting of sequences will be kept up to date. investigators are invited to send additional
`sequence data when accepted for publication. Send two copies of the manuscript together with a
`letter of acceptance from a journalto:
`Dr. E.A. Kabat
`National institutes of Health
`Building 8, Room 126
`9000 Rockville Pike
`Bethesda, Maryland 20892
`tt would be extremely helpful if you can send us your sequence data on magnetic tapes orfloppy
`diskettes or a clean copy of the sequences.Thefile formats should be such that they can be read
`by a generic word processor.
`When published, three reprints should be provided.
`lf any published sequences have been overlookedorif any errors are found, please bring them to
`our attention.
`
` ||'
`
`eGRRESASE
`
`ova
`
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`vil
`
`INTRODUCTION
`
`Our earlier “Variable Regions of lmmunoglobulin Chains” (1), the second edition “Sequences
`of Immunoglobulin Chains”’ (2) and thethird edition “Sequencesof Proteins of Immunological Interest”
`(3) have been further expanded herein to include amino acid and nucleotide sequences of precursors,
`variable regions, constant regions, J-chain, 89-microglobulins, antigens of the major histocompatibility
`complex (HLA, H-2, la, DR) as well as of Thy-1, compiement, T-lymphocyte receptors for antigens, other
`T-cell antigens of the immunoglobulin superfamily, interleukins and various other proteins related to
`immunefunctions. The identification and sequencing of clones obtained using recombinant DNA
`techniques has yielded nucleotide sequencesof signal, variable, and constant regions of immunoglo-
`bulins (4,5), and these nucleotide sequences have beentranslated into amino acid sequences.Instances
`of the latter have been includedin the tables of amino acid sequences and are indicated by an apostrophe
`followed by CL after the nameof the clone. We have continued to use the PROPHET Computer System
`of the Division of Research Resources, NationalInstitutes of Health (6,7) to tabulate the sequences.
`
`in compiling the data we havetried to be as up-to-date as possible and have included only
`sequences which have been published or which have been accepted for publication. Residues which
`have not been definitely determined have been excluded. It should be remembered that sequences
`are often publishedin review articles without detailed documentary evidence. These have often been
`revised. We havelisted such revisions in the notes in many instances; others can readily be found by
`comparison with sequences in previous editions.
`
`Since the preparation of camera-ready copyfor printing the pagesis carried out in sequence
`from page 1 in batches, the amino acid sequences were set several months before the nucleotide
`sequences. We have continued to include new nucleotide sequencesup to the point at which camera-
`ready copy for them hadto be set, but translated amino acid sequences were not able to be included.
`Thus many nucleotide sequences appearwithout translation. When antibodyactivities were known,
`they have beenlisted at the end of the nucleotide sequences and are included in the index.
`
`When doubts arise as to the validity of any residue in a sequence, the original reference
`should be examinedto ascertain whetherdefinitive evidence for the sequence has been provided. We
`have sent the amino acid and nucleotide sequencesas stored in the computer to the original authors
`for verification. If so verified, this is denoted by ‘(checked by author)” at the end of each reference.
`Exceptfor the earliest sequences, the date on which the checked sequence wasreturned to us is given.
`Wheneverpossible, nucleotide sequences from GenBank (8) have been used. Programsfor converting
`a GenBank sequenceto the codon formatof our tables have been developed. The correctnessof the
`table sequence has beenverified by converting backinto the linear form and comparing with GenBank.
`Whenthis has been done the sequenceis listed as “(from GenBank)’. If the sequences were entered
`by us from theliterature and then checked with GenBank,this is indicated by ‘(checked with GenBank)’.
`We have entered many nucleotide sequences which were not available from GenBank.In general, we
`have notincluded stretches of sequence such as enhancers, switch regions and introns for which no
`codification of the nucleotide sequences is as yet agreed upon with respect to function, etc. Much
`information about such stretches may be foundin references 9, 10.
`
`It is also possible, by examining the numbers of sequences at the end of each table and the
`summary tables, to evaluate the probability that a given amino acid at a given position may not be
`correct. This is most readily done for the framework residuesof the V-region and for the C-region; in the
`complementarity-determining regions this is moredifficult because of the high variability.
`
`Amino Acid Sequences
`
`The first column in each table gives the residue number. Except for complemeni, T-cell surface
`antigens and miscellaneousproteins, the second column is a tabulation of invariant residues. Since
`exceptions to invariance are found, the frequency,if less than 1.0 and greater than or equal to 0.95,
`is indicated alongside the residue listed as invariant; when only a single sequenceis available, this
`is not given. Each sequenceis tabulated in each subsequent column. Dashes (-—-) indicate that no
`amino acid is present at that position and that the sequence continues. In ali instances residues
`
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`

`vili
`
`considered uncertain by the authors have not been includedin the table. In some instances the symbol
`# is used to indicate that several aminoacid residues were found in one position, and these residues
`are listed in the notes. The four columnsat the end of each table give:
`
`1.
`2.
`3.
`4.
`
`the number of residues sequenced at that position,
`the number of different amino acids found at that position,
`the numberof times the most common amino acid occurred and that amino acid in parentheses, and
`the variability.
`
`Variability is calculated (11) as:
`
`Variability =
`
`Number of different amino acids occurring
`at a given position
`Frequencyof the most common aminoacid
`at that position
`
`An invariant position would have a variability of one; if 20 amino acids occurred with equal
`frequency,the variability would be 20 divided by 0.05 equals 400. If, for example, four different amino
`acids Ser, Asp, Pro, and Thr occurred at a given position, and of 100 sequences available at that position,
`Ser occurred 80 times, the variability would be 4/0.8 = 5. When any of the amino acid residues
`sequenced were notidentified completely and arelisted as Glx (or Asx), two values, separated by a
`colon, are givenin thelast three columns.Thefirst value in eachof these columns is calculated assuming
`that only one of the two possibilities, e.g., Glu or Gin (or Asp or Asn) occurred, while the second considers
`that both were present and maximizes variability. In the variability plots, the norizontal bars indicate
`the two values.
`
`When two or more amino acids are most common and occur with equal frequency, they are
`tabulated as a note, and the symbol + is used in the nextto last column. If no sequence data have been
`reported for any position, there are no entriesin the last four columns. Variability is not calculated for
`insertionsorif only a single sequence is known. Whenthetranslated sequence of aclone corresponds
`to a previously listed sequenceof a plasmacytoma from which it was prepared, only one sequence
`is listed so that the variability computations are not affected, and a noteis included.
`
`Ifa given sequenceis associated with any antibody activity,this is indicated by an asterisk alongside
`the protein heading, andthe antibody specificities are given in a separate list with binding constants
`if available. The noteslist the a-allotypes for the rabbit heavy chain V-region and the b-allotypes for
`the constant domainof the rabbit kappalight chain. A key reference to the sequenceis given; generally
`the most recent reference sinceit is usually the most nearly complete, but often several references
`are included, especially whenrevisions of a sequence have been made. Notes are now of two types:
`general notes abouta table indicated by the symbol #, and specific notes indicated by the sequence
`number.
`
`Signal Sequences
`
`The signal (precursor) amino acid sequencesof immunoglobulin chainsarelisted in three tables:
`one for kappalight chains, onefor lambdalight chains, and one for heavy chains. They were obtained
`either by direct sequencing of signal proteins (12-14) or by translating nucleotide sequences from DNA
`clones. Signal segments range from 17-29 aminoacid residuesin length and are thus numbered from
`-29 to -1. Genomic DNA clones contain introns of varying length that interrupt the coding sequence
`of the precursor within the codonforposition -4, and in rare casesfor position -6. Thus, the L-gene
`encodesthe leader peptide to position -4 and the 5’ end of the V-gene codesfor positions -4 to -1.
`
`The signal-amino acid sequences of the T-cell receptors for antigens, B2-microglobulins,
`major histocompatibility complex proteins, and complement componentsare listed in separate tables.
`
`By conformational energy calculations, the core V,, hydrophobic Leu-Leu-Leu-Trp-Val-Leu-Leu-
`Leu (MOPC321, MOPC63) exists in an alpha helical conformation, terminated by chain reversal confor-
`mationsin the four C-terminalresidues Tro-Val-Pro-Gly; the four amino terminal residues are compatible
`with the alpha helix (15).
`
`Variable Region Sequences
`
`Thevariable regions (16) of immunoglobulins have been shownto contain hypervariable segments
`in theirlight (11 ,17-23) and heavy (22,24-27) chains,of which certain residues have beenaffinity labeled
`(28-41). Three hypervariable segments of light chain were delineated from a statistical examination
`Page 4
`Page 4
`
`
`

`

`of sequences of human V,, human V), and mouseV,, light chains aligned for maximum homology
`(11,22). These and the three corresponding segments of the heavy chains (22,26,27) were hypothesized
`(11,22) to be the complementarity-determining regions or segments (CDR) containing the residues
`which make contact with various antigenic determinants, and this has been verified by X-ray diffraction
`studies at high resolution (42-67). The rest of the V-region constitutes the framework (11,22,66-68).
`itis convenientto identify the framework segments (FR1, FR2, FR3, and FR4) and the complementarity-
`determining segments (CDR1, CDR2, and CDR3) with the three CDRsseparating the four FRs. The
`residue numbers for these segments are as follows:
`
`Light Chain
`
`Heavy Chain
`
`Segment
`
`FR1
`
`CDR1
`
`FR2
`
`CDR2
`
`1-23 (with an occasional
`residue at 0, anda
`deletion at 10 in V) chains)
`
`24-34 (with possible
`insertions numbered
`as 27A,B,C,D,E,F)
`
`35-49
`
`50-56
`
`1-30 (with an occasional
`residue at 0)
`
`31-35 (with possible
`insertions numbered
`as 35A,B)
`
`36-49
`
`50-65 (with possible
`insertions numbered
`as 52A,B,C)4
`
`66-94 (with possible
`insertions numbered
`as 82A,B,C)
`
`95-102 (with possible
`insertions numbered as
`100A,B,C,D,E,F,G,H,I,J,K)
`
`103-113
`
`FR3
`
`57-88
`
`CDR3
`
`FR4
`
`89-97 (with possible
`insertions numbered as
`95A,B,C,D,E,F)
`
`98-107 (with a possible
`insertion numbered
`as 106A)
`
`* In the rabbit, Mage et al. (69) consider position 65 in Vj to be in FR3, sinceit is allotype related.
`
`In the tables of V-regions, the FR and CDR are separated by horizontallines for convenience
`in reading. One mouse kappalight chain, MPC 11, has an extra segment of 12 amino acid residues
`betweenposition 1 and the signal sequence (70). Several chains have internal deletions.
`
`in the tables, the V-genesforthe light chains code to amino acid position 95, and the Jminigenes
`from position 97 to 107 for lambda and 108 for kappalight chains. Position 96 is usually the site of V-J
`joining by recombination and ray be coded partly by the V-geneandpartly by the J-minigene. Because
`the site of V-J recombination could occur at different positions within a codon, different amino acid
`residues mayresult at this position. We have changed the location of the inserted residues from 97A-
`F (2) to 95A-F, sinceit makesforbetter alignment by confining chainsofdifferent lengthsto the V-gene
`region. In V,, chains, J1 and J2 were used 5 to 10 times more frequently than J4 and J5 (71).
`
`The V-genesfor the heavy chains code up to amino acid position 94 and are followed by the D-
`and J-minigenes. Becauseof the extensivevariation in the lengths of D-minigenes,the exact boundary
`between D andJis not always located at the same amino acid position. In addition, the lengths of the
`J encoded amino acid sequences vary by a few amino acid residues. Moreover, the process of D-J
`joining appearsto involve insertionsof extra nucleotides between V and D and between D and J, termed
`the N region (72-76) andcorrelates with the appearanceofterminal deoxytransferasein B cells (75).
`Theoriginal numbering system for the heavy chains has therefore beenretained. Wysockiet al. (76)
`have provided some evidence suggesting a non-randomorigin for the Vi-Dy junction, perhaps a
`minigene, rather than random addition of the N nucleotides.
`
`It has become evidentthat a critical understanding of the architecture of antibody combiningsites
`and the genetics of the generationof diversity and of antibody complementarity will depend to a great
`extent on the evaluation of a large number of sequencesofthe variable regions and especially of the
`complementarity-determining segmentsoflight and heavy chainsof immunoglobulinsofdifferent spe-
`cies. Ability to locate residuesin the site making contact with antigenic determinants (77) and to predict
`(87,78-82) the structures of antibody combining sites will depend heavily upon such sequences.
`Figures 1 and 2 are stereoviewsof the a-carbon skeletonsof the four Fv regions for which high
`resolution X-ray structures have been determined, NEWM (44), KOL (62), MCPC603 (47, 48, 63), and
`J539 (64). The residues in the CDRs are shownassolid circles. In Fig. 1 the combiningsite is at the
`
`Page 5
`Page 5
`
`

`

` MCPC603
`
`FIG. 1. Stereodrawings of the a-carbon skeletonsof four Fv regions studied crystallographically. Top to bottom:
`NEWM(43,44,49,59), KOL (62), MCPC603(47, 48, 53, 55, 63), J539(64). Coordinates for NEWM, KOLfrom the
`Protein Data Bank (Bernstein et al. 1977, J. Mol. Biol. 112:532-544); for MCPC603 and J539 courtesy of David
`R. Davies. The stereodrawingsof Figures 1,2,3 and 4 were prepared by Dr. Eduardo Padian.
`V,_ is on theleft, V,4 antheright. Thefirst and last residues of each chain as well as several other residues are
`shown as open circles for reference; residues of the CDRs are shownassolid circles. The Fv’s are aligned by
`least-squares superposition using the program ALIGN (G.H. Cohen, NIH); the stereoplots were prepared using
`the program PLUTO (S. Motherwell, Cambridge, England). The view shownis with the combiningsite at the top.
`With a stereo viewerit is possible to see two adjacent modelsat the sametime, so that a comparison may be made
`in three dimensions.
`
`Page 6
`Page 6
`
`

`

`
`
`FIG. 2. Same as Fig. 1, but looking into the combining site; models perpendicular to those of Fig. 1.
`
`Page 7
`
`

`

`
`
`REI
`
`Loc
`
`
`RHE-2
`
`Page 8
`Page 8
`
`RHE-1
`
`FIG. 3. Stereodrawingsof the a-carbon skeletons of four Bence Jones V;_ dimers studied crystallographically.
`Top to bottom: MCG(42,46,50), REI (45, 51, 57), LOC(61), RHE(60). The bottom view is of RHEwithits twofold
`axis toward the top of the page.Theleft V,is orientedlike the V,_ of the Fv’s in Fig. 1. The view shownis like that
`of Fig. 1. Coordinates for MCG, REI, RHE from the Protein Data Bank; for LOC,courtesy of Dr. Marianne Schiffer.
`
`

`

`MCG
`
`LOG
`
`
`
`xii
`
`RHE-1
`
`RHE-2
`
`FIG. 4. Same as Fig. 3. but looking into the combining site. Models perpendicular to those of Fig. 3. The bottom
`view of RHE is looking into its twofold axis.
`
`Page 9
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`
`

`

`xiv
`
`top; the view of Fig. 2 is perpendicularto Fig. 1 and is looking into the combining site. The different
`orientations of the loops containing the complementarity-determining regions provide some insight
`into how specificity of various sites mightdiffer (67,82). If the amino acid side chains were included,
`the differences would become much more detailed.
`
`Figures 3 and 4 are stereoviewsof the four V,_ dimers, Bence Jones proteins, for which high reso-
`lution X-ray structures are available, MCG (42,46,50), REI (45, 51, 57), LOC (61) and RHE (60). The
`V_ chains eachcontribute fourf-strandsto the V_-V_ or Vy-Vinteraction whereasthe Vj chains each
`providefive (65). Thus, althoughin a Bence Jones V,_ dimer one V_ assumestheposition of Vy (50),
`nevertheless the absence of one f-strandin each V_ may makethesites of V,_ dimers less specific
`than those of the Fab fragments. This is supported by the finding that the V, dimer of MCG binds a
`wide variety of ligands whereas noligand which binds has yet been found for the MCG protein (83).
`
`A recenthigh resolution x-ray crystallographic study (84) of a crystalline complexof lysozyme
`with a monocional anti-lysozyme showsthat contact between lysozyme and antibody occur on a rather
`flat surface with the interactions largely due to protruberances and depressions formed by the amino
`acid side chains producing a tightly packed region of interaction. The lysozyme determinant involves
`two noncontiguousstretches, residues 18 to 27 and 116 to 119 ofits polypeptide chain. All six CDRs
`of the antibody and two residues outside the CDRs but adjacentto the CDRs, Tyr 49 in Vand Thr
`30 in V4, make contactwith the lysozyme. Ten of the 17 contacting residuesare in Vy. Four of the 10
`contacting Vy residues and three of the seven contacting residues in V,_ are in the corresponding
`CDR3s. Table 1 lists the residues on the anti-lysozyme and on lysozyme which are in contact. These
`findings,if and to the extent applicable to anti-carbohydratesites, with respectto interactionsof side
`chains on essentially flat surfaces could have substantial implications for our understanding of these
`antigen-antibody interactions. However, the J539 site would appear to be some type of groove
`complementaryto atetrasaccharide. Unfortunately thusfar the crystal form has not allowed the ligand
`to enterthe site (64). Figure 5 is a stereoviewof the lysozyme-antilysozyme carbon skeleton showing
`the region of interaction.
`
`Bence Jones dimer LOC (61) has a convex (male) bindingsite quite different from the usual Bence
`Jones dimers MCG and REL.
`If such a male type combining site were to be found for an Fab, the
`possibility would have to be considered that a reciprocal type of antigen-antibodyinteraction might occur
`in which the side chains of the CDRswouldfit into a groove or depression on the surface of an antigen.
`The possibility has been noted that interactions might occurwith the CDRsof the two faces of the
`projecting convexsite (61). RHE also has a quite distinct type of binding site based on a unique V|-
`V,_ interaction. The basis for such differencesis not understood but could contribute a new parameter
`to site complementarity and diversity,
`The sequence data may be used to make rough screens of anew sequencefor homology with
`the V-region. If the sequence to be compared is alignedwith the large V-region summary tables, one
`can ascertain whether any homology exists. If homologyinvolves the less frequently occurring residues,
`they can be foundin the individual tables and homology evaluated.
`The variable region a-group ailotypes and allotype a-negative rabbit Vj, chains have been
`correlated with certain amino acids in FR1 and FR3asfollows (69):
`
`Allotype
`
`Amino Acid Position
`
`
`
`FRI
`
`5
`glu
`
`8
`gly
`
`i0)06120C
`ARG val
`
`Vij at
`
`Vij a2
`
`LYS GLU gly
`
`PHE lys
`
`18 6S 6 67
`THR thr
`pro
`gy
`phe
`pro
`gly
`gly
`ser
`ASP THR
`
`FR3
`
`74
`thr
`
`70
`ser
`
`7
`lys
`
`75
`[-*
`
`76
`[]
`
`87
`85
`84
`THR GLU thr
`
`GLN thr
`SER SER THR ARG ASN GLU asn ala
`ser
`GLY ala
`ala
`ala
`
`gly
`
`gly
`
`phe
`thr
`phe
`
`ser
`ser
`ser
`
`flys
`flys
`ser
`
`thr
`ser
`ala
`
`[-
`[-]
`gin
`
`ala
`{[-}
`2
`[-]
`asn ala
`
`ala
`2
`ala
`
`thr
`MET
`thr
`
`giu
`Vi a3®
`Vi, at00 glu
`Vy &
`gu
`val
`
`gly
`gly
`gly
`
`ASP val
`gly
`val
`gl
`val
`
`flys
`gin
`gin
`
`ala
`ala
`gly
`glu
`thr
`
`ser
`ser
`gly
`ser
`
`4 Square brackets indicate gaps to maximize homology. Allotype related residues are in capitals.
`
`> In some a3-like genes, codonsfor amino acids 75 and 76 were found (85).
`
`Page 10
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`
`

`

`XV
`
`In Figs. 1 and 2, the location of the allotypic regions may clearly be seen to be on the outside of
`VH awayfrom the combiningsite. Residues 13 and 65 of Vy are numbered andwill facilitate location
`of the Va allotypes. The few cDNA sequences (69)available have provided no evidence as yet that
`germ line sequences encoding latent allotypes may exist in some rabbits. Antisera to rabbit Vya
`allotypes crossreact with human IgG (86), various other species of IgM and IgG, and with the Galapagos
`shark 7S immunoglobulin and correlate with the N-terminal amino acid sequence (87).
`
`There are substantial species differences between the human, rat and rabbit C,, allotypes.
`The amino acid sequencesofrabbit C,, allotypic determinants K-1, b4, b5 and b9 differ at 47 of 106
`positions,the differences occuringin clusters; the K-2 basisotypediffers at three additional positions
`(88) whereas the humanC,, allotypes differ by two positions (89) and the rat R1-1a and R1-1b differ at
`11 positions (90).
`
`TABLE1
`
`Antibody Residues Involved in Contact with Lysozyme
`
`[
`Antibody residues
`Lysozymeresidues in contact
`
`Light Chain
`CDRi1
`
`FR2
`CDR2
`CDR3
`
`30
`His
`32
`Tyr
`49
`Tyr
`50
`Tyr
`Phe 91
`Trp
`92
`Ser 93
`
`Leu 129
`Leu 25, Gln 121, lle 124
`Gly 22
`Asp 18, Asn 19, Leu 25
`Gln 121
`Gin 121, lle 124
`Gin 121
`
`CDR2
`
`
`
`Science 233:747-753; courtesy of Dr. Roberto Poljak and Science. Copyright 1986 by the AAAS.)
`
`Heavy Chain
`FRi Thr=30 Lys 116, Gly 117
`
`
`CDRI1
`Gly
`31
`Lys 116, Gly 117
`Tyr
`32
`Lys 116, Gly 117
`Trp
`52
`Gly 117, Thr 118, Asp 119
`Gly
`53
`Gly 117
`Asp 54
`Gly 117
`Arg
`99 (96
`Arg 21, Gly 22, Tyr 23
`)
`Asp 100 {
`Gly 22, Tyr 23, Ser 24, Asn 27
`97)
`Tyr 107 (98)
`Thr 118, Asp 119, Val 120, Gin 121
`Arg 102 (99)
`Asn 19, Gly 22
`
`
`CDR3
`
`Sequencepositions are numberedas in this book except for V}y CDR3, where the numbers are
`givenin parentheses; the others are sequential. (From (84), Amit, Mariuzza, Phillips, and Poljak (1986)
`
`FIG. 5. Stereo diagram of the Ca skeleton of the complex. Fab is shown (upperright) with the heavy andlight
`chains with thick and thin bonds, respectively. The lysozyme active site is the cleft containing the label HEL.
`Antibody-antigen interactions are most numerous between lysozyme and the heavy chain CDRloops.(From (84).
`Amit, Mariuzza, Phillips, and Poljak (1986) Science 233:747-753; courtesy of Dr. Roberto Poljak and Science.
`Copyright 1986 by the AAAS.)
`
`Page 11
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`
`

`

`
`
`it has proven extremely useful, except for mouse V,, chains, to order the Vi and Vy sequences
`into sets (68) such that all chains with identical FR1 arelisted together, the set with the most members
`beinglisted first. Chains differing in sequence from this set by a single residueare thenlisted in order
`of substitution, beginning at residue 22forlight chains and residue 30 for heavy chains and proceeding
`in decreasing position numberto residue 1. These are then followed by chains with two amino acid
`differences, againlisting in the same decreasing order, and followed by chains with three amino acid
`substitutions, etc. Amino acid residuesdiffering from the major FR1 sequence are givenin lower case
`letters, so that one can readily see the patternof substitution.In this ordering procedure, missing residues
`are treated as potentially different from the main sequence. If residues are missing at position 23 in
`the light chain and position 22 in the heavy chain, they are assumedto be Cys to preserve the essential
`V-domain structure. Finally, sequences which are incomplete in FR1 are given. Within a given FR1
`set, identical FR2 sets are also listed together.
`
`The human V,, rearranged and germ-line genesofall four subgroups have been sequenced
`(91-95). HumanV,IV has but a single germ-line gene (92, 93); thus somatic mutation must play a
`dominantrole in the utilization of this gene.V,,1l genes are characterized by a much longerintron between
`the signal and V-region (94) than the other subgroups.Unlike the mouse the human V,1V,ll, and V,-lll
`genesare not separated in the genome, buta large section of the V,, focus has been duplicated; both
`sections existing as two non-allelic clusters, containing eight and six genes of all three subgroups(91,95).
`All genes are in the 5’->3’ orientation. These findings have necessitated reclassification of some
`incomplete sequences (RPMI-6410’CL).
`
`Thetables of mouseV,,light chains have beenrearranged(96,97). in previous editions (1-3), mouse
`V,, light chains werelisted in one table with the length from residue 1 to Trp 35 specified. They have
`now been separated into eight tables. Thefirst six tables vary from 41 to 34 residues by the different
`lengths of CDA1(residues 27A-F); the sixth also lacks residue 28 and the seventhis also missing residue
`22 in FR1. If residues 1 through 35 have not been determined completely, the sequences are listed
`inthe eighth table, unless they show good homology to more complete sequences in one of the other
`tables. In each table, the group numberis given below the nameof the chain. When residues 1 through
`35 have not been determined, the earlier group designation based on residues 1-23 (96) is given in
`square brackets [ ]. However, for each table the same principles have been used in ordering the chains.
`in all instances, residues 1-35 for the largest group are givenin capitals. Variations from this sequence
`are in lower case, beginning at residue 34 and proceeding toward residue 1 as in the other tables.
`
`The mouse V4 sequences have beenrevised to take into accounttheir division into families by
`Diidrop (98) based on amino acid sequences and of Brodeur and Riblet (100) based on nucleotide
`sequencesof completely sequenced V-regions. We have however retained the earlier classification
`of Vy chains into subgroups but have subdivided the subgroupstolist the families as follows:
`Antibody specificities found
`FamilyBrodeur and Riblet
`(100, 101)
`Winter et al (102)
`
`Dildrop (98)
`Dildrop et al (99)
`
`Subgroup 1A
`B
`
`Ars, DNP, HEL, DIG, poly-GA
`2-PHEOX, HEL
`
`Subgroup A a(1+3)a(1>6)DEX, RNA,Ars, a(1>6)DEX, DIG,
`HEL, ldAc38, GAT
`
`B
`
`NP, GAT, DIG, PC, IdAc38, 2-PHEOX
`
`Cc
`Subgroup iH A
`B
`
`G
`D
`Subgroup V A
`B
`
`GAT, GA, H-2K-k
`PC, DNP, HEL, DNA
`B(1>6)GAL, a(1+6)DEX, NACMAN, f(2-6)FRU, GAT,
`STR-A(p-DGicNAc)
`B(21)FRU, STR-A, SRBC, GAT
`DIG, H-2kK-k, SRBC
`ARS CRI+, ARS CRE
`ARS, ARS CRI +
`
`,
`
`Miscellaneous
`
`ARS, DNA, HEL, H-2K-k, GAT, DIG, 2-PHEOX, CEA
`
`3
`2
`8
`
`5
`
`1
`
`7
`4
`
`6
`1
`
`1
`
`5
`
`V4436-60
`VyQs2
`V443609,
`MV31
`
`Vys558
`VMU-T,
`VGANS-8
`
`V,4J606
`V,48107
`V4J558,
`Vi4X24
`V4J606
`J558
`
`J558
`
`Viy7183
`
`For newly sequenced V4 regions, the nucleotide or amino acid sequence homologies have been
`compared with the previously classified sequences.
`
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`Page 12
`
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`

`xvii
`
`itis evident that antibody specificities for a given antigen fall into several of these subgroups and
`that many framework residues which are characteristic of the subgroupsare sufficiently different to
`indicate that different germ-line genes maygive rise to antibodies of a given specificity (103).
`
`The classification given is in better accord with the amino acids (98) than with the nucleotides
`(100), since the latter used probes whereas the former was based on complete sequences. Tworabbit
`germ-line a-negative V;, clones showed the greatest nucleotide sequence homology, 70.4-79.4%to
`Vi X24, Vy 7183, V4 J606, and V}4 S107 (100) and considerably less homologyto the other families.
`
`Each table of mouse V,, and Vj4 chainsis followedbya list of strains other than BALB/c.
`
`The membersof identical FR and identical CDR sets are givenin the notes. Membersof individual
`FR1 sets may be associatedwith different FR2 sets, etc. (68), suggesting independent assortmentof
`FR sets.
`
`A sequenceidentical to the FR2 sequenceofthe light chain of McPC603 has been foundin two
`human V,1, one human V, IV, 31 mouse V,, (14 NZB and 17 BALB/c), one each mouseV,,1 and V,.
`VL and 15 rabbit V, sequences, and thus has been preservedfor about 80 million years (104). It and
`the corresponding loap of the heavy chain are seen at the bottom centerof each of the four stereofigures
`(Fig. 1); L40 and H41 are numberedtofacilitate its location. Despite its preservation, there are 12 other
`FR2 sets in the mouse and8in the rabbit with sequence variation which mayinvolve 13 of the 15 posi-
`tions, only Trp 35 and Gin 39 being invariant. Theloopis in a relatively open position so that substitutions
`are readily permitted (104,63). The evidence of assortment of FR segments (68) suggested the
`hypothesis that the V-region was codedfor by sets of minigenes for the FR and CDR segments and
`that these minigenes were assembled somatically during embryogenesis. FR2 of Vi, also shows
`substantial preservation, one set having six mouse and eight rabbit chains of identical amino acid
`sequence. It is extraordinary that one human V4lll genomic clone, Vi426 (105), and a rabbit CDNA clone
`(106), were identical in nucleotide sequence of the codonsfor amino acids 36 through 47, differences
`being seen in codons for amino acids 48 and 49.
`
`Since only FR sets were used in demonstrating the assortment (68), it would be independentof
`and would be seen whetheror not any CDR residues assorted with any FR. The early cloning studies
`(4,5) showedthat the genes codingfor residues comprising FR1 almost through CDR3 of mouse V) and
`V,, light chains were assembledin twelve-day-old mouse embryo DNA, and each was followed by an
`intervening sequence;in one VI clone, two residues of CDR3 were included with FR4 (107). In an adult
`V, myeloma,the genes coding for the entire V-region were assembled (108). Thus, the minigene coding
`for FR4plus the last two residues of CDR3 (107), termed the J segment(108), had been joined to the
`rest of the V-region between the twelfth day of embryoniclife and the adult, and thus was added somat-
`ically by recombination.Milstein (17),in his original description of humanV,. groups, had pointed outthat
`subgroup associated residues extendedonly through residue 94 and that it seemedasif frequent cross-
`ing over occurred beyondresidue 94. Weigert efal. (109), in studying the V,, 21 group in NZB myelamas,
`assorted the last two or three residues of CDR3 together with FR4 and suggested that this would
`contribute to the generationof diversity. With rabbit light chains,it was possible to assort the individual
`FR and CDR segments considering FR4, plus the last two residues of CDR3, as a J-minigene (110).
`
`The subsequent demonstration and sequencingoffive J nucleotide segments from BALB/c, each
`separated by an intervening sequencein the mouse genome, supported the assortmentdata (68) and
`established J as a minigene. Four of the J segments encoded amino acid residues 96-108, but J3 ap-
`peared to be a pseudogene (111,112). In mouse Vy four J-(113-115), in human Vj six J-(4 16), in rat V,
`six J-(117,118), in humanV,, five J-(119), and in mouse V)four J-minigenes (120,121) were recognized;
`J,4 may not be functional. In rabbits of b4 and b5 allotypes,there arefive V,, J-minigenes (122, 123);
`only J2 beingfunctional. There appearto be two or more J, minigenes that can be expressed with the
`b9 allotype and three potentially functional J,-s are found associated with the K2 isotype (124, 125).
`
`Unlike the findings forlight chains, the mouse genomic V} and Jj, segments did not encode
`complete V-regions (113,114). Segments coding for five and for 14 amino acids, most of CDR3, were
`missing. Subsequently, genomic DNAs capable of encoding three, four, and six amino acids were
`located, using as a probe an incompletely rearranged D-J segmentincluding the flanking signal
`sequences (126-128). Some of these mouse D-minigenes coded for as many as six aminoacids of
`CDR3, matchesof 9 to 18 nucleotides having been found.
`
`Four human D-minigenes have been sequenced (129), but their presumed coding regions did
`not correspond to any CDR3. However, one ViJIIl human genomic clone (105) contained fourteen
`nucleotides in CDR2 which completely matched the D2 minigene coding segment (130). Another human
`Vill genomic clone (131) matched at 13 of these 14 nucleotides, the first of the fourteen nucleotides
`being c instead of g (131). Other instances were a match of 12 nucleotides in mouse (132) and a
`rabbit CDNA (133). The amino acids codedfor by these segments (130) were present as residues 53-56
`
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`

`
`
`xvili
`
`in two fractions of typeIII rabbit antipneumococcal antibody from one rab