Proc. Nat. Acad. Sci. USA
`Vol. 71, No. 3, pp. 845-848, March 1974
`
`Variable Region Sequences of Five Human Immunoglobulim Heavy Chains
`of the VHTTI Subgroup: Definitive Identification of Four Heavy
`Chain Hypervariable Regions
`(myeloma proteins/amino acid sequences/antibody combining site)
`
`J. DONALD CAPRA AND J. MICHAEL KEHOE
`
`Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, 10 East 102 Street, New York, N.Y. 10029
`
`kappa), and Tur (IgAl kappa) were isolated from serum or
`plasma by zone electrophoresis on polyvinyl copolymer
`("Pevicon") (8). After further purification by gel filtration
`chromatography, they were reduced with 0.1 M fl-mercapto-
`ethanol and alkylated with iodoacetamide. The heavy
`and light chains were separated by gel filtration in propionic
`acid (9, 10).
`Fragment Preparation: Heavy chains were treated with
`cyanogen bromide (11) and the resulting individual frag-
`ments purified by gel filtration chromatography in 5 M guani-
`dine * HCl. Three proteins (Tei, Zap, and Tur) yielded a
`large N-terminal fragment comprising residues 1-85. Pro-
`teins Was and Jon, which contain a methionine residue at
`position 34, gave fragments comprising residues 1-34 and
`35-85. Since all human IgG myelomas have a methionine at
`position 252, Tei and Was yielded a large fragment comprising
`residues 86-252. In protein Jonj however, an additional
`methionine was present at position 111. Consequently two
`distinct fragments comprising residues 86-111 and 112-252
`were obtained from this protein. IgAl proteins contain a
`methionine at residue 426 (12) so proteins Zap and Tur both
`yielded a very large fragment composed of residues 86-426.
`Sequencing Procedure. Positions 1-85: On both the intact
`heavy chain as well as on the 1-85 fragment, proteins Tei,
`Zap, and Tur were sequenced 60 steps on the automated se-
`quencer (13, 14). Tryptic peptides were prepared and sepa-
`rated on Dowex 50X4 with a pyridine-formate buffer system.
`In proteins Tei and Zap two invariant peptides were aligned
`by homology alone (70-74 and 75-78) while in protein Tur,
`
`12 3 4 56
`H
`H H
`
`7
`I-I
`
`8
`H
`
`9
`
`10
`I
`
`6.0 -
`OZ 5.0-
`W~ 4.0-
`
`Communicated by Henry G. Kunkel, November 5, 1973
`
`ABSTRACT
`The variable regions of five human immu-
`noglobulin heavy chains of the VHIII subgroup have been
`totally sequenced. Three of the heavy chains belonged to
`the IgG class and two to the IgA class. Examination of
`these sequences, and comparison with additional pub-
`lished heavy chain sequences, showed that a total offour
`hypervariable regions is characteristic of human heavy
`chain variable regions.
`The relatively conserved character of large segments of
`the heavy chain variable region was very evident in these
`studies. The conserved segments, which are those sections
`located outside the hypervariable regions, comprise ap-
`proximately 65% of the total heavy chain variable region.
`The following general structural pattern for antibody
`molecules emerges from this and related studies: an over-
`all combining region superstructure is provided by the
`more conserved segments while the refinements of the
`active site specificity are a function of hypervariable re-
`gions.
`The antibody combining site is now believed to reside ex-
`clusively in the variable regions of the heavy and light poly-
`peptide chains of the immunoglobulin molecule. Evidence
`is accumulating from several laboratories which indicates
`that hypervariable regions within the variable region are
`directly involved in the antibody combining site as well as
`being responsible, at least in part, for the idiotypic determi-
`nants of myeloma proteins and specific antibodies (1-5).
`The existence of three hypervariable regions in the vari-
`able region of human immunoglobulin heavy chains has been
`established by previous studies from this laboratory. Resi-
`dues 31-37 were described as the first hypervariable region
`of the heavy chain (6), and, after fragmentation of IgG
`heavy chains with cyanogen bromide, two additional hyper-
`variable regions were localized between residues 86-91 and
`101-110 (7).
`We have now completed the amino acid sequence from
`residues 41 to 84 of the three VHIII proteins originally re-
`ported (6, 7) as well as the complete V region sequence of
`two IgA proteins with VHIII variable regions. The data
`make apparent an additional area of sequence hypervari-
`ability between residues 51 and 68, thus supporting the ob-
`servations of Cebra et al. made on pooled guinea-pig immuno-
`globulins (5). When these data on VHIII proteins are in-
`cluded with that available for proteins of the VHI and VHII
`subgroups and analyzed by the method of Wu and Kabat (3),
`four distinct areas of sequence hypervariability are observed.
`MATERIALS AND METHODS
`Myeloma Proteins. Tei (IgG1 kappa, Gm az), Was (IgG1
`kappa, Gm az), Jon (IgG3 lambda, Gm g), Zap (IgAl
`
`845
`
`0 fm
`
`FIG. 1.
`
`0o20 30
`hydrolysate of the amino terminal (1-85) cyanogen bromide
`f a chain from protein Tuir. Peptides were isolated
`fragent
`from a Dowex 50X4 column and characterized and analyzed as
`described in the test.
`
`40
`
`50
`
`60
`
`70
`
`80
`
`Representative ion exchange chromatogram of tryptic
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1092 Page 1 of 4
`
`

`
`846
`
`Immunology:
`
`Capra and Kehoe
`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`GLU VAL GoN LEU VAL GLU
`
`20
`10
`SER GLY GLY GLY LEU VAL GLN PRO GLY GLY SER LEU ARG LEU SER CYS ALA ALA SER
`
`Tei
`
`Was
`
`Joh
`
`zap
`
`Tur
`
`Tei
`
`Was
`
`Jon
`
`Zap
`
`Tur
`
`Tei
`
`Was
`
`Jon
`
`LYS
`
`ALA
`
`ASP
`
`LEU
`
`LEU
`
`30
`GLY PHE THE PHE SEE
`
`UE
`
`SER
`
`_ ASP
`
`SER ALA VAL TYR (
`I
`ALA TRP MET LYS r
`
`ET
`
`40
`50
`1 TRP VAL ARG GLN ALA PRO GLY LYS GLY LEU GLU TRP VAL
`
`I I
`
`1 I
`
`THR SER ARG PHE r
`
`ARG VAL LEU SER SER i
`
`60
`GLY TRP ARG TYR GLU GLY SER SER LEU THE HIS TWi ALA
`
`70
`VAL SER VAL ow GLY ARG PHE TER
`
`ILE SER ARG ASK
`
`ALA
`
`VAL
`
`LYS
`
`Go
`
`GLU ALA _
`
`ASH SER
`
`PHE
`
`ASP TH_ AS8
`
`VAL
`
`GIo
`
`VAL VAL GLU LYS ALA PHE
`
`ASN _
`
`ASN
`
`Zap
`
`Tur
`
`Tei
`
`Was
`
`Jon
`
`Zap
`
`Tur
`
`Te i
`
`Was
`
`Jon
`
`Zap
`
`Tur
`
`GLU PHIE
`
`VAL GI
`
`AA ILE SER
`
`ASP
`
`ALA
`
`-_
`
`SER GLY
`
`LEU ASN ALA
`
`ASN LEU
`
`PHE
`
`ALA ___
`
`80
`too
`90
`ASP SER LYS AS1N THR LEU TYR LEU GUI MET LEU SER LEU GLU PRO GLK ASX U ALA VAL TYR TYR CYS ALA ARG
`
`-
`
`ASN ARG
`
`ALA
`
`ILE
`
`VAL
`
`U
`
`ASN THR GLY
`
`ALA
`
`GUI ALA
`
`LEU
`
`110
`120
`VAL THR PRO ALA ALA ALA SER LEU THR PHE SER ALA VAL TRP GLY GLI GLY THR LEU VAL THR
`
`PHE ARG GIN PRO PHE VAL GLI
`
`[
`
`_
`
`PHE ASP
`
`PHE
`
`VAL VAL SER UHR [
`
`THR ARG -GLY GLY TYR [
`
`] SER MET ASP
`
`]
`
`ASP
`
`PRO
`
`SER
`
`LEU SER VAL
`
`VAL [
`] ALA PHE ASP
`LYS
`SER
`FIG. 2. The amino-acid sequence of the variable regions of five human immunoglobulin heavy chains.
`
`the isolation of chymotryptic peptides established the se-
`quence unambiguously. In all cases, tryptic peptides were
`sequenced in the automated sequencer, often using 4-sulfo-
`phenylisothiocyanate (Pierce Chemical) on the lysine pep-
`tides (15). In proteins Was and Jon, which contained cyano-
`gen bromide fragments 1-34 and 35-85, the first 60 residues
`were established by automated sequencing of the intact
`heavy chain. Thus, in these two proteins, sequencing
`cyanogen bromide fragment 1-34 was superfluous since its
`composition agreed with the previously determined sequence.
`
`Fragment 35-85 of proteins Was and Jon was sequenced 35
`and 40 residues respectively; this, together with the C-
`terminal tryptic peptides mentioned above gave the com-
`plete sequence for this section. Residues 86-121: In proteins
`Tei, Was, Zap, and Tur the sequence was established by a
`continuous automated run of 45 steps from residue 86 into
`the CH1 domain. In both Zap and Tur, tryptic digestion and
`isolation of the resulting peptides confirmed a few question-
`able positions. In protein Jon, residues 86-111 were obtained
`disulfide linked to residues 1-34 after cyanogen bromide
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1092 Page 2 of 4
`
`

`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`Human Immunoglobulin Heavy Chains
`
`847
`
`GAP
`
`GAP
`
`GAP
`
`70
`
`60-
`
`50-
`
`:2 40-
`
`X 30
`
`I0-
`
`digestion. This sequence was obtained by difference since
`residues 1-34 were known from the initial study of the
`intact heavy chain. Jon fragment 112-253 was subjected to a
`long sequencer run which definitely established the sequence
`of residues 112-121 as well as providing sequence data into
`the CH1 domain.
`Ion Exchange Chromatography. An example of a Dowex
`50X4 chromatogram is shown in Fig. 1 for a tryptic digest
`of the Tur 1-85 fragment; 6.5-ml fractions were collected and
`0.1 ml of each fraction analyzed by the fluorescamine pro-
`cedure initially described by Udenfriend et al. (16). Ninhydrin
`analysis was also performed after alkaline digestion of 0.5-ml
`aliquots. In most analyses, only the fluorescamine procedure
`was employed since it was much more sensitive. As shown in
`Fig. 1, 10 fractions were pooled. Each was subjected to amino-
`acid analysis and several useful peptides were isolated and
`sequenced. T-1 (Asn Thr Leu Tyr Leu Gln Hsr) (79-85), T-3
`(Asn Asp Ser Lys) (75-78), T-7 (Gly Leu Gly Trp Val Ser
`Gly Arg) (46-53), and T-10 (Phe Thr Ile Ser Arg) (70-74).
`
`RESULTS AND DISCUSSION
`The amino-acid sequences of the variable regions of the
`five human myeloma proteins is displayed in Fig. 2. The
`variability-factor values determined by the method of Wu
`and Kabat (3) for these as well as all the other human V
`region sequences available is shown in Fig. 3. These calcula-
`tions were based on 25 sequences from residues 1 to 34, 11
`sequences from residues 35 to 85, and 14 sequences from
`residues 86 to 122. Previous to this study there were only six
`published complete V region sequences, all but one (Nie) of
`the VHI (Eu) or VHII (Daw, Cor, He, Ou) subgroup (for
`references see legend to Fig. 3). With five additional VHIII
`sequences the variability within and between subgroups can
`now be compared more meaningfully. In addition, with the
`availability of 11 complete sequences and several fragments,
`the Wu-Kabat plot becomes more statistically significant.
`A discussion of the sequences can be conveniently divided
`into those sections of the V region which are relatively con-
`stant (1-30, 38-50, 69-83, 92-100, and 111-121), and the
`hypervariable regions (31-37, 51-68, 84-91, and 101-110).
`About 65% of the variable region of the heavy chain shows
`limited variation. In fact, there are 17 positions (14%) which
`have been absolutely invariant in all human heavy chains
`regardless of their V region subgroup assignment. Certain
`positions are subgroup specific since at these positions all,
`or nearly all, of the members of one subgroup have a particular
`amino acid, while members of the other subgroup contain a
`different amino acid.
`Utilizing the four available VHII
`proteins, positions 3, 9,16, 17, 19, 21, 23, 28, 29, 39, 42, 46, 50,
`80, 81 and 82 appear to be subgroup specific. As noted
`previously, no subgroup specific residues are identifiable in
`the C terminal portion of the V region (7). There are thus
`33 positions (27%) in the V region which are either invariant
`or subgroup specific. A comparison with the published se-
`18), pooled immuno-
`quences of myeloma proteins (17,
`globulins (5, 19, 20), and specifically purified antibodies (5,
`21-23) from lower species, indicates that the particular
`amino acids found at these positions are characteristic of a
`wide variety of mammals and have been faithfully conserved
`during evolution. Such residues may have extremely im-
`portant attributes for variable region function such as, for
`
`10
`
`20 30 40
`
`60 70 80 90 100 110
`POSITION
`FIG. 3.
`Variability-factor values for the sequences shown in
`Fig. 1 as well as several other published sequences (36) deter-
`mined according to the method of Wu and Kabat (3).
`
`120
`
`example, the provision of a distinct backbone structure which
`is crucial to antibody function.
`As can be seen on inspection of Figs. 2 and 3, about a third
`of the heavy chain variable region can be considered "hyper-
`variable." These regions deserve special consideration be-
`cause of their specific implications for the formation of the
`antibody combining site, the nature of idiotypic determinants,
`and various theoretical conceptions of the origin of antibody
`diversity.
`In light chains, affinity labels have been localized near or
`within hypervariable regions (23-25), thus providing direct
`support for the general concept that hypervariable regions
`participate directly in the antibody-combining site. For the
`heavy chain, recent work has also been consistent with this
`idea. For example, Ray and Cebra localized affinity labels to
`the first (31-37) and the fourth (101-110) heavy chain
`hypervariable regions (26), Haimovich et al. (27) localized
`an affinity label to residue 54 of the mouse myeloma protein
`315 (which has anti-dinitrophenol activity), and Press and
`coworkers have localized affinity labels at or near the fourth
`hypervariable region in rabbit antibodies (28). Therefore,
`although the primary structure and affinity labeling studies
`of these proteins was being carried out independently, and
`even in different laboratories in many instances, there is a gen-
`eral implication from the experimental observations that the
`same regions of the molecule which show the highest degree
`of sequence variation are near or part of those particular re-
`gions of the heavy chain where affinity labels have been local-
`ized.
`A second piece of evidence linking the antibody combining
`site to the hypervariable regions has come from comparisons
`of sequences obtained from pooled immunoglobulin heavy
`chains with those of specifically purified antibody heavy
`chains. Sequence analyses of rabbit (29), guinea pig (5), and
`other mammalian heavy chain pools (19), indicate that a
`definitive sequence cannot be obtained within those regions
`which have been identified as hypervariable on the basis
`of studies with myeloma proteins. However, when specifically
`purified antibodies are studied, a single major sequence can be
`determined, as has been shown most definitively by Cebra and
`his coworkers (5).
`Additional support for the functional significance of hyper-
`variable regions has been provided by current notions con-
`cerning the tertiary structure of the immunoglobulin mole-
`cule. Crystallographic analysis of human immunoglobulins
`has now advanced to the point where it has been possible
`to assign the residues which may line a "pocket" within the
`
`PETITIONER'S EXHIBITS
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`Exhibit 1092 Page 3 of 4
`
`

`
`848
`
`Immunology:
`
`Capra and Kehoe
`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`immunoglobulin molecule which presumably represents the
`combining site itself (30, 31). In each instance, the major
`residues which line the pocket are associable with hyper-
`variable regions.
`In addition, the conformational models
`generated by the nearest neighbor calculations of Kabat and
`Wii (32) place hypervariable regions in close association
`with the putative combining site.
`There is also growing evidence that at least some of the
`hypervariable regions are involved in the idiotypic determi-
`nants of myeloma proteins and antibodies. Cross idiotypic
`specificity among the cold agglutinins (33) and the anti-
`gamma globulins (34) is believed to be related to the com-
`bining site. In at least two distinct anti-gamma globulin
`molecules, the hypervariable regions show striking sequence
`similarities (7, 35).
`The genetic origin of hypervariable regions remains un-
`clear. The variability within heavy chain hypervariable
`regions seems more marked than that of light chain hyper-
`variable regions. Of the I1 proteins which have now had their
`V regions completely sequenced, if one considers the 43
`hypervariable positions of the heavy chain, there are no two
`proteins which have more than 10 residues in common. It
`seems likely that hundreds, or even thousands, of proteins
`would have to be sequenced in order to find two which are
`identical if no preselection bias (such as selection by idiotypic
`antisera or for combining specificity) is involved. This im-
`plies either that there are a very large number of germ
`line genes or that somatic processes are necessary to explain
`the diversity in the heavy chain hypervariable regions.
`Regardless of their origin, the hypervariable regions clearly
`play a crucial role in the antigen binding function of immuno-
`globulin molecules.
`We thank Dr. Henry Kunkel for the subclass and genetic typ-
`ing of the myeloma proteins. Bonnie Gerber, Ellen Bogner and
`Donna Atherton rendered invaluable technical assistance. This
`work was aided by grants from the National Science Foundation
`(GB 17046) and the U.S. Public Health Service (AI 09810) and a
`Grant-in-Aid from the New York Heart Association. J.D.C. is the
`recipient of National Institutes of Health Career Development
`Award 6-K4-GM-35, and J.M.K. is an Established Investigator
`of the American Heart Association.
`Milstein, C. (1967) Nature 216, 330-332.
`1.
`Franek, F. (1969) Symposium on Developmental Aspects of
`2.
`Antibody Formation and Structure, Prague.
`3. Wu, T. T. & Kabat, E. A. (1970) J. Exp. Med. 132, 221-250.
`Capra, J. D., Kehoe, J. M., Winchester, R. & Kunkel, H. G.
`4.
`(1971) Ann. N.Y. Acad. Sci. 190, 371-381.
`Cebra, J. J., Ray, A., Benjamin, D. & Birshtein, B. (1971)
`Progr. Immunol. (First International Congress of Immunol-
`ogy), 269-284.
`Capra, J. D. (1971) Nature New Biol. 230, 61-63.
`Kehoe, J. M. & Capra, J. D. (1971) Proc. Nat. Acad. Sci.
`USA 68, 2019-2021.
`
`6.
`7.
`
`5.
`
`8.
`9.
`
`10.
`
`11.
`
`12.
`
`15.
`
`16.
`
`18.
`
`19.
`
`20.
`
`21.
`22.
`
`23.
`
`24.
`25.
`
`Kunkel, H. G. (1954) Methods Biochem. Anal. 1, 141-155.
`Fleischman, J. G., Porter, R. R. & Press, E. M. (1963)
`Biochem. J. 88, 220-228.
`Capra, J. D. & Kunkel, H. G. (1970) J. Clin. Invest. 49,
`610-621.
`Gross, E. & Witkop, B. (1962) J. Biol. Chem. 237, 1856-
`1863.
`Chuang, C. Y., Capra, J. D. & Kehoe, J. M. (1973) Nature
`244, 158-160.
`13. Edman, P. & Begg, F. (1967) Eur. J. Biochem. 1, 80-91.
`Capra, J. D. & Kunkel, H. G. (1970) Proc. Nat. Acad. Sci.
`14.
`USA 67,87-92.
`Inman, J. K., Hannon, J. E. & Appella, E. (1972) Biochem.
`Biophys. Res. Commun. 46, 2075-2081.
`Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leim-
`gruber, W. & Wiegele, M. (1972) Science, 178, 881-882.
`17. Kehoe, J. M. & Capra, J. D. (1972) Proc. Nat. Acad. Sci.
`USA 69,2052-2055.
`Bourgois, A., Fougereau, M. & de Preval, C. (1972) Eur. J.
`Biochem. 24, 446-455.
`Capra, J. D., Wasserman, R. W. & Kehoe, J. M. (1973) J.
`Exp. Med. 138, 410-427.
`Mole, L. E., Jackson, S. A., Porter, R. R. & Wilkinson, J. M.
`(1971) Biochem. J. 124, 301-318.
`Fleischman, J. B. (1973) Immunochemistry 10, 401-407.
`Strosberg, A. D., Jaton, J. C., Capra, J. D. & Haber, E.
`(1972) Fed. Proc. 31, 771.
`Goetzl, E. J. & Metzger, H. (1970) Biochemistry 9, 1267-
`1278.
`Franek, F. (1971) Eur. J. Biochem. 19, 176-183.
`Chesebro, B. & Metzger, H. (1972) Biochemistry 11, 766-
`771.
`Ray, A. & Cebra, J. J. (1972) Biochemistry 11, 3647-3657.
`26.
`27. Haimovich, J., Eisen, H. N., Hurwitz, E. & Givol, D. (1972)
`Biochemistry 11, 2389-2397.
`Press, E. M., Fleet, G. W. J. & Fisher, C. E. (1971) in
`Progress in Immunology, ed. Amos, B. (Academy Press,
`New York), p. 233.
`Cebra, J. J., Givol, D. & Porter, R. R. (1968) Biochem. J.
`107,69-70.
`Schiffer, M., Girling, R. L., Ely, K. R. & Edmundson, A. B.,
`personal communication.
`Poljak, R. J. (1973) Abstracts of Ninth International Con-
`gress ofBiochemistry-Stockholm, p. 31.
`32. Kabat, E. A. & Wu, T. T. (1972) Proc. Nat. Acad. Sci. USA
`69, 960-964.
`Williams, R. C., Kunkel, H. G. & Capra, J. D. (1968)
`Science 161, 379-381.
`34. Kunkel, H. G., Agnello, V., Joslin, F. G., Winchester, R. J.
`& Capra, J. D. (1973) J. Exp. Med. 137, 331-342.
`Capra, J. D. & Kehoe, J. M., unpublished observations.
`(a) Edelman, G. M., Cunningham, B. A., Gall, W. E., Gott-
`lieb, P. D., Rutishauser, U., & Waxdal, M. J. (1969) Proc.
`Nat. Acad. Sci. USA 63, 78-85; (b) Press, E. M. & Hogg, N.
`M. (1970) Biochem. J. 117, 641-660; (c) Cunningham, B. A.,
`Pflumm, M. N., Rutishauser, U. & Edelman, G. M. (1969)
`Proc. Nat. Acad. Sci. USA 64, 997-1003; (d) Wikler, M.,
`Kohler, H., Shinoda, T. & Putnam, F. W. (1969) Science 163,
`75-78; (e) Ponstingl, H., Schwarz, J., Reichel, W. & Hilsch-
`mann, N.(1970) Hoppe-Seyler's Z. Physiol. Chem. 351, 1591.
`
`28.
`
`29.
`
`30.
`
`31.
`
`33.
`
`35.
`36.
`
`PETITIONER'S EXHIBITS
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`Exhibit 1092 Page 4 of 4