`
`·. · U1 715272 · .
`JPRESS MAIL LABEL NO. 859937585
`DATE MAILED: June 14, 1991
`GENENTECH, INC.
`
`
`
`460 Point San Bruno Boulevard, South San Francisco, CA 94080
`(415) 266-1000
`
`Docket No. 709
`
`
`
`NEW APPLICATION TRANSMITTAL
`
`r·
`
`SIR:
`
`
`
`
`
`
`Transmitted herewith for filing is the patent application of lnvantor(sl:
`PAUL J. CARTER ET AL.
`
`
`
`
`
`Title: IMMUNOGLOBULIN VARIANTS
`
`
`
`CERTIFICATION UNDER 37 CFR §1.10
`
`I hereby certify that this New Application and the docunents referred to es enclosed herein ere being deposited with the United
`
`
`
`
`
`
`
`Mell Ing Lebel Mell Post Office To Addressee" States Postal Service on this date June 14, 1991, In en envelope bearing "Express
`
`
`
`
`
`
`
`Nurt>er 859937585 addressed to: Patent Application, Honorable Comnlssloner of etents nd T ad ks, ashlngton, D.C. 20231.
`
`Carolyn R, Adler
`
`(Name of person malling paper)
`
`Enclosed are:
`
`1.· The papers required for filing date under CFR § 1.53(b):
`
`
`
`
`
`.1filL Pages of specification (Including claims); _L Sheets of drawings Lformal / .lL informal)
`
`.2L Declaration/Oath/Power of Attorney
`
`
`_ Assignment of the invention to GENENTECH, INC.
`4. Fee Calculation
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`16 -20"
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`. \ndep. Claims
`
`8 -3 ..
`
`I Basic Fee
`I
`x S60.00
`·s200.ooI
`
`$630
`
`630.
`
`300.
`
`tNll!ber F !led INuroer Extra
`-
`I
`* 5
`depe ndent c:lalm.<s>, If any
`Hllltlple
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`11
`Recordlng Assignment [$8.001 Total Fees Enclosed
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`8 sfypljcate sheet is
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`1'0. Information Disclosure Statement
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`11. .2L . Return Receipt Postcard
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`. . . . . . $930.00
`
`14, 1991
`Dated _June
`
`�,�/(,�
`Name: CarotvnRAd1er
`
`Registration No. 32,324
`
`. .
`
`·Pfizer v. Genentech
`IPR20I 7-01489
`
`
`
`I
`
`
`
`
`
`Genentech Exhibit 2032
`
`
`
`we
`O”
`mm v., mm .
`
`‘
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`W 715272
`
`ADS
`
`HU4D5
`
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`
`Anneal hu‘VL or lrmVH oligomers to pAKl template
`
`
`
`l. Ligate
`
`2. Isolate assembled oligomers
`3. Anneal to pAKl template (Xhol ", Stu! +)
`4. Extend and 1i gate
`
`‘ Xhol \
`
`1. Transform E. ‘coli
`
`\
`
`.
`
`2. Isolate phagernid pool
`3. Enrich for hufi and huVH(XhoI,+ Stul‘)
`
`4 Sequence verify
`
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`
`
`
`
`
`
`
`/
`
`Percentofcontrolcell
`proliferation
`
`
`
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`
`
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`
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`
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`
`
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`W152i?
`
`_
`DOCKET 709 .
`EXPRESS MAlL NO. 359937585
`
`MAILED 14 JUNE 1991
`
`
`
` @/ IMMUNOGLOBULIN VARIANTS
`
`Field of the invention
`
`This invention relates to-methods for the preparation and use of
`_
`variant antibodies and finds application particularly in the fields of
`
`immunology and cancer diagnosis and therapy.
`
`Background gf the invention
`
`Naturally occUrring antibodies (immunoglobulins) comprise two
`heavy chains linked together by di3ulfide bonds and two light chains, one
`light chain being linked to each of the heavy chains by disulfide bonds. Each
`heavy chain has at one end a variable domain (VH) followed by a number of
`constant domains.
`.Each light chain has a variable domain (VL) at one end
`and a constant domain at its other end; the constant domain of the light
`
`chain is aligned with the first constant domain of the heavy chain, and the
`
`light chain variable demain is aligned with the variable domain 'of the heavy
`chain; Particular amino acid residues are believed to form an interface
`between the light and heavy chain variable domains, see erg. Chothia eta/L,
`J. Mol. Biol. 186:651~663 (1985); Novotny and Haber, Proc. Nat/j Acad. Sci.
`
`10
`
`15
`
`30
`
`
`
`
`
`0
`
`‘ O
`
`USA‘8224592—4596 (1985).
`I
`The constant domains are not
`
`involved directly in binding the
`
`antibody to‘an antigen, but are involved in various effector functions, such
`
`as participation of the antibody in antibody-dependent cellular cytotoxicity.
`The variable domains of each pair of light and heavy chains are involved
`directly in binding the antibody to the antigen. The domains of natural light
`and heavy chains have the same general structure, and each domain
`
`comprises four framework (FR) regions, whose sequences are somewhat
`
`conserved,
`connected
`by'
`three hyper-variable
`or
`complementarity
`determining regions (CDRs) (see Kabat, E. A. et al.,. Seqdences of Proteins
`of Immunological Interest, National Institutes ‘of Health, Bethesda, MD, '
`
`(1987)). The four framework regions largely adopt a fi-sheet conformation
`and theCDRs form loops connecting, and'in some cases forming part Of, the
`
`B-sheet structure. The‘CDRs in each chain are held in close proximity by the .
`framework regionsand, with the CDRs from the other chain, contribute to
`
`the formation of the antigen binding site.
`
`Widespread use has been made of monoclonal antibodies,
`
`_
`
`particularly those derived from rodents including mice, however they are
`
`frequently antigenic in human clinical use.) For example, a major limitation in
`the clinical use of rodent monoclonal antibodies is an anti-globulin response
`
`during therapy (Miller, R. A. eta/., Blood 62:988-995 (1983); Schroff, R. W.
`
`et a/., Cancer Res. 452879885 (1985)).
`
`The art has attempted to overcome this problem by constructing
`
`"chimeric" antibodies in which an animal antigen-binding variable domain is
`
`coupled to a human constant domain (Cabilly er a/., U.S. patent No.
`4,816,567; Morrison, 8. L. et 3]., Proc. Natl. Acad. Sci. USA 81:6851-6855
`(1984); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neube‘rger, M.
`S. er al., Nature 314:268-270 (1 985)). .The term "chimeric" antibody is used
`herein to describe a polypeptide comprising at least the antigen binding
`
`portion of an antibody molecule linked to at least part of another protein '
`(typically an immunoglobulin constant domain).
`‘
`The isotype of the human constant domain maybe selected to tailor
`
`10
`
`15
`
`'20
`
`3'0
`
`
`
`the chimeric antibody for participation in antibody-dependent cellular
`
`cytotoxicity (ADCC) and complement-dependent cytotoxicity (see 9.9.
`BrUggemann, M. era/.,J. Exp. Med. 166:135)-1361 (1987); Riechmann, L.
`et al., Nature 332:323-327 (1988); Love at a/., Methods in Enzyma/ogy
`
`’
`
`178515-5527 (1989); Bindon et a/., J. Exp. Med. 1682127442 (1988).
`
`In the typical embodiment, such chimeric antibodies contain about
`
`one third rodent (or other non—human species) sequence and thus are capable
`
`I of eliciting a significant antiglobulin response in humans. For example, in the
`
`case of
`
`the murine anti--CD3 antibody, OKT3, much of
`
`the resulting
`
`anti—globulin response is directed against the variable region rather than the
`
`constant region (Jaffers, G. J. et a/;, Transplantation 41:572-578 (1986)).
`
`in a further effort
`
`to resolve the antigen binding functions 'of
`
`antibodies and to minimize the use of heterologous sequences in human
`antibodies, Winter and colleagues (Jones,‘P. T. e! 51., Nature 321522-5253
`(1986); Riechmann, L. at al., Nature 332:323-327 (1988);'Verhoeyen, M. et
`
`a/., Science 239:1534-1536 (1988))have substituted rodent CDRs or CDR
`
`sequences for the corresponding segments of a human antibody. As used
`
`the term "humanized" antibody is an embodiment of chimeric
`herein,
`antibodies wherein substantially less than an intact human variable domain
`
`has been substituted by the corresponding sequence from a_ non-human
`species.
`lnvpractice, humanized antibodies are typically human antibodies in
`
`which some CDR residues and possibly some FR residues are substituted by
`residues from analogous sites in rodent antibodies.
`.
`‘
`
`The therapeutic promise of this approach is supported by the clinical
`
`efficacy of a humanized antibody specific for the CAMPATH-l antigen with
`
`two non-Hodgkin lymphoma patients, one of whom had previously‘developed
`
`an anti-globulin response to the parental rat antibody_(Ri_echmann, L. et al.,
`Nature 332:323-327 (1988); Hale, G. et al., Lancet izl 394-1399 (1988)).
`A murine antibody to the interleukin 2 receptor has also recently been
`humanized (Queen, c. e: al., Proc. Natl. Acad. sci. USA 86110029le033
`(1989)) as a potential immunosuppressive reagent.
`(Additional references
`
`related to humanization of antibodies include Co at al., Proc. Natl. Acad. Sci.
`Hu
`
`10
`
`15
`
`20
`
`30
`
`
`
`
`
`10
`
`15
`
`20
`
`(1321
`USA 88:2869~2873 (1991); German at al., Proc. Natl. Acad. Sci.
`88:4181-4185 (1991); Daugherty etal., NucleicAcids Research 1 9(9):2471 —
`247611991); Brown et al., Proc. Natl. Acad. Sci. USA 88:2663-2667
`(1991); Junghans et al., cancer Research 50214954502 (1990).
`
`in some cases, substituting CDRS'from rodent antibodies for the
`
`hUman CDRs inhuman frameworks is sufficient to‘transfer high antigen
`
`binding affinity (Jones, P. T. etal., Nature 321 :522-525 (1986); Verhoeyen,
`M. et al., Science 239:1534~1536 (1988)), whereas in-other cases it has
`been necessary to additionally replace one (Riechmann, L. et al., Nature ‘
`
`332:323-327 (1988)) or several (Queen, C. etal., Proc. Natl. Acad. Sci. USA
`86:10029-1003'3 (1989)) framework regidn (Ffilresidues. See also Co etl
`al. , supra.
`For a given antibody a small number of FR residues are anticipated
`to be important for antigen binding. Firstly for example, certain antibodies
`
`have been shown to contain a few FR residues which directly contact antigen
`in crystal structures of antibody-antigen complexes (e.g., reviewed in Davies,
`D R. et al., Ann. Rev Biochem. 59:439-479 (1990)). Secondly,a number
`
`of FR residues have been proposed by Chothia, Lesk and colleagues (Chothia,
`.81 Lesk, A. M, J. Mol BiOl 1:96901-917 (1987); Chothia, C. et al.,
`Nature 342:877—883 (1989); Tramontano, A. ' at al.,
`J.
`,Mol. Biol.
`215.:175-182 (1990)) as critically affecting the conformation of particular
`
`CDRs and thus their contribution to antigen binding. See also Margolies er
`
`'al., Proc. Natl. Aca-drSC/Z USA 72:2180‘2184 (1975).
`
`It
`is‘ also 'known that,_in a few instances, an antibody variable
`' domain (either VH or VL) may contain glycosylation sites, and that this
`glycosylation may improve
`or
`abolish antigen binding, Pluckthun,
`
`Biotechnology 92545-51 (199-1); Spiegelberg' et 8]., Biochemistry 9:4217-
`4223 (1970); Wallic et al., J. Exp. Med. 168:1099-1109 (1988); Sox et al,
`
`Proc. Natl. Acad. Sci. USA 66:975-98211970); Margni et al,, Ann. Rev.
`
`30
`
`Immunol. 6:535—554 (1988). Ordinarily, however, glycosylation has no
`
`- influence on the antigen—binding properties of an antibody, Pluckthun, supra,
`
`(1991).
`
`10
`
`
`
`
`
`I The three—dimensional structure-ofimmunoglobulin chains has been
`
`studied, and crystal structures for intact immunoglobulins, (for a variety of
`
`immunoglobulin fragments, and for antibody-antigen complexes have been
`
`published (see e.g., Saul et al., Journal of Biological Chemistry 25:585~97
`1(1978); ' Sheriff etal., Proc. Natl. Ac-ad. Sci. USA 84:8075-79 (1987); Segal'
`era/q Proc. Natl. Acad. Sci. USA 71:4298-4302 (1974); Eppv er al.,
`'Biochemistry 14(22):4943-4952 (1975); Marquart
`er al., J. Mol. Biol.
`141 :369—391 (1980); Furey er al., J. Mo/.'Bio/. 1 67:661-692 (1983): Snow
`and Amzel, Protein: Structure, Function, and Genetics 1:267—279, Alan'R.
`Liss, lnc. pUbs. (1986); Chothia and Lesk,J_. Mol. Biol. 196:901-917 (1987’);-
`Chothia etal., Nature 342:877—883 (1989); Chothia et’al, Science 233:755-
`58 (1986); HUber et al., Nature 264:415—420 (1976); Bruccoleriet al.,
`Nature 335:564-568 (1988) and Nature 336:266 (1988);. Sherman at a/.;
`Journal of Biological Chemistry 263:4064-4074 (1988); Amzel and Poliak,
`Ann, Rev. Biochem. 482961-67 (1979); Silverton’et al., Proc. Natl. Acad.
`
`Sci. USA 74:5140-5144 (1977); and Gregory et al., Molecular Immunology
`24:821-829 (1937).
`lt
`is known that the function of an antibody is
`dependent on its
`three dimensional
`structure, and that amino acid
`substitutions can change the three-dimensional structure of‘ an'antibody,
`Snow and Amzel, supra,
`It has previously been shown that the antigen
`binding (affinity of a humanized antibddy canlbe increased by mutagenesis
`
`based upon molecular modelling (Riechmann, L. et al., Nature 332:323-327
`
`(1988); Queen, C. et al., Proc. Natl. Acad. Sci. USA 86:10029-10033
`
`(1989)).
`
`Humanizing an antibody-with retention of high affinity'for antigen
`and other desired biological activities is at present difficult to achieVB u‘sing
`
`currently available procedures. Methods are needed for rationalizing the
`
`selection of sites for substitution in preparing such antibodies and thereby
`
`increasing the efficiency of antibody humanization.
`The prom—oncogene HERZ
`(human epidermal growth factor_
`receptor 2) encodes a protein tyrosine kinase (p‘l85HER2) that is related’to
`and somewhat homologous to the human epidermal growth factor-receptor
`
`5
`
`' 11
`
`10
`
`15
`
`20
`
`30
`
`
`
`
`
`\‘l
`
`
`
`‘10
`
`15
`
`20
`
`30
`
`O ,
`
`r
`
`‘
`
`i
`
`0
`
`(see Coussens, L. et a/., Science 230:1132-1139 (1985); Yamamoto, T. et‘
`al., Nature 319:230-234 (1986); King, C. R. at al., Science 229:974—976 g
`(1985)). HERZ is also known "in the field as c-erbB-Z, and sometimes by the
`name of the rat'homolog, neu. Amplification and/or overexpression of H532
`is associated with multiple human malignancies and appears to be integrally
`involved in progression of 25-30% of human breast and ovarian cancers
`(Slamon, D. J. et a/.,'Science 235:177-182 (1987), Slamon, D. J. et al.,
`Science 244:707-712 (1989)). Furthermore, the extent of amplification is
`
`inversely’correlated with the observed median patient survival time (Slamon,
`
`R
`’
`supra, Science 1989).
`The murine monoclonal antibody known as muMAb4D5 (Fendly, B.
`M. et a/., Cancer Res. 50:1550-1558- (1990)), directed against
`the
`extracellular domain (ace) of p185HER2, specifically inhibits the growth of
`tumor cell lines overexpressing p1 BSHERQ in monolayer culture or in soft agar
`
`(Hudziak, R. M. etal.', Molec. Cell. Biol. 9:1 1 65-1 172 (1989); Luca, R. or a/.,
`
`Science 249:1552—1555 (1990)). MuMAb4DS also has the potential of
`enhancing tumor cell sensitivity to tumor necrosis factor, an important
`effector molecule in macrophagemediated tumor cell cytotoxicity (Hudziak,
`supra, 1989; Shepard, H. M." I and Lewis, G. D. J. Clinical Immunology:
`8:333-395 (1 988)). Thus muMAb4D§ has potential for clinical intervention
`in and imaging of carcinomas in which p185HERZ is overexpressed. The
`muMAb4'D5 and its uses are describedbin copending U.S. patent applications
`
`07/143,912 and 07/147,461, and in corresponding PCT application WO
`
`89106692 published 27 July 1989. .This murine antibody was deposited
`with the ATCC and designated ATCC CRL 10463.
`l-loWever, this antibody
`may be immunogenic in humans.
`’
`I
`g
`It istherefore an object of this invention to provide methods for the
`preparation or antibodies which are less antigenic in humans than non-human
`antibodies but have desired antigen binding and other characteristics and
`activities.» _
`It is a further object of this invention to provide methods for the
`
`efficient humanization of antibodies, i.e. selecting non-human amino acid
`
`1.2
`
`
`
`O '
`
`‘
`
`‘
`
`0
`
`residues for importation intoa human antibody background sequence in such
`a‘fashion as to retain or
`improve the affinity of the'non-human donor
`antibOdy for a given antigen.
`‘
`lt
`is another object of
`this invention to provide humanized
`antibodies capable of binding p185HER’2,
`,
`.
`Other objects, features, and characteristiCs of the present invention
`
`'
`
`will became apparent upon consideration of the following description and the
`appended claims.
`
`_
`
`*5
`
`'
`
`10
`
`§gmmary 9f the Invention
`
`The objects of'thisiinvention are accomplished, by a method for
`making a humanized antibody comprising amino acid sequence of an import,
`non-human antibody and 'a human antibody, comprising the steps of:
`I
`
`15
`
`.
`
`'
`
`a.
`
`b.
`
`c.
`
`d.
`
`8.
`
`f.
`
`' 20
`—
`
`4
`
`.
`
`-
`
`'
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`25
`
`_
`
`V
`
`I
`
`'
`
`‘30
`
`,
`
`'
`
`.
`
`obtaining the amino acid sequences of at least a portion.
`of an import antibody variable domain and of a censensus
`human variabie domain;
`.
`,
`.
`i
`identifying Complementarity Determining Region (CDR)
`amino acid sequences in the import and the human
`variable domain sequences:
`substituting an import CDR amino aCid sequence for the
`corresponding human CDR amino'acid sequence;
`
`aligning the amino acid sequences of a Framework Region
`
`(FR) of the import antibody and the corresponding FR of
`the consensus antibodyr
`
`identifying import antibody FR residues in the‘aligned FR
`
`sequences that are non—homologous to thecorresponding
`
`consensus antibody-residues;
`determining if the non-homologous import amino acid
`
`residue is reasonably expected to have at least one of the
`following effects:
`1.
`non—covalently binds antigen directly,
`
`13
`
`
`
`
`
`2.
`
`3.
`
`V
`
`interacts with a CDR; or
`
`participates in the VL - VH interface; and
`
`g.
`
`for any non—homologous
`
`import antibody amino acid
`
`residue which is reasonably expected to have at least one -
`
`of
`
`these effects,
`
`substituting that
`
`residue for
`
`the
`
`corresponding amino acid residue in the consensus
`antibody FR'sequence.
`.
`i
`Optionally, the method of this invention comprises the additional
`.
`steps‘of determining if any non~homologous residues identified in step (elare
`exposed. on the surface of the domain or buried within it, and if the residue
`
`isexposed buthas none of theeffects identified in step (i), retaining the
`Consensus residue.
`
`Additionally, in certain embodiments the method of this invention .
`
`comprises the feature wherein" the corresponding consensus antibody “
`residues identified in step (9) above are selected from the group consisting
`of 4L, 35L, 36L, 38L, 43L, 44L, 46L,_‘58L, 62L, 63L, 64L, 65L,” 66L, 67L,
`68L, 69L, 70L, 71L, 73L, 85L; 87L, 98L, 2H, 4H, 24H, 36H,'37i-l, 39H,_ ‘
`
`43H, 45H, 49H, 58H, 60H, 68H. 69H, 70H, 73H, 74H, 75H, 76H,-78H,
`
`91 H.192H, 93H, and 103H (utilizing the numbering system set forth in Kabat,
`
`E. A. et al., Sequences of Proteins of Immunological Interest (National
`institutes-of Health, Bethesda, MD, 1987)).
`
`in certain embodiments, the method of this invention comprises the
`
`additional steps of searching either or both of the import, non-human and the
`
`-
`
`“umwynw'
`——-——-.~,
`M
`N
`MM» ,,
`consensus variable domain sequences for glycosylation sites, determining if
`the glycosylation is reasonablywexpected to be important for the desired
`.-..__..., a...
`,,,...,...w—".~-... ,,
`antigen binding and biological activity of thema, determining if the
`glycosylation‘site binds to antigen or changes a side chain of an amino acid
`residue that binds to antigen, or if the glycosylation enhances or weakens
`antigen binding, or is important for maintaining antibody affinity).
`If the
`
`import sequence bearstheglycosylation site, it is preferred to substitute that
`site for the corresponding residuesin the consensus human sequence if the
`glycosylation site is reasonably expected to be important.
`If only the
`
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`
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`
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`consensus sequence, and not the import, bears the glycosylation site, it is
`
`preferred to eliminate-that glycosylation site or substitute therefor the '
`
`corresponding amino acid residues from the import seguence.
`
`l l
`
`‘\
`
`Another embodiment of this invention comprises aligning import
`
`antibody and the consensus antibody FR sequences,
`
`identifying import
`
`A antibody FR residues which are non-homologous with the aligned consensus
`
`FR sequence,and for each such non-homologous import antibody FR residue.
`
`determining if‘ the corresponding consensus antibody residue represents a
`
`residue which is'highly conserved across all species at that site, and if it is
`so conserved, preparing a humanized-antibody which comprises the
`consensus antibody amino acid residue at that site.,
`I
`3 Certain alternate embodiments of the methods of this invention
`
`comprise obtaining the amino acid sequence of at least a portion of an
`
`import, non—human antibody variable domain having a CDR and aim,
`
`' obtaining the amino acid sequence of at least a portion of a consensus
`human antibody variable domain having a CDR and ‘3 FR, substituting the
`
`non-hUman CDR for the human CDR in the consensus human antibody
`variable domain, and then substituting
`an amino acid residue for the.
`consensus amino acid residue .at at least one of the following sites:
`a.
`(in the FR of the variable domain of the light chain) 4L,
`35L, 36L, 38L, 43L, 44L, 53L, 46L, 62L, 63L, 64L, 65L,
`
`b.
`
`.
`
`66L, 67L, 68L, 59L, 70L, 71L,_73L, 85L, 87L. 98L,_ or
`(in the FR of the variable domain of the heavy chain) 2H,
`4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H,
`68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H,'92H,V
`
`-
`
`93H, and 103H.
`"
`in preferred embodiments, the non-COR residue substituted at the consensus
`FR site is the residue found at the corresponding location of the non—human
`
`_
`antibody.
`Optionally, this just-recited embodiment comprises the additional
`'
`steps of following the method steps'appearing at the beginning of this
`
`summary and determining. whether a particular amino acid residue can
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`O ,
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`. O
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`reasonably be expected to have undesirable effects.
`This invention also relates to a humanized antibody'comprising the
`
`CDR sequence of an import, non-human antibody and the FR sequence of a
`human antibody, wherein an amino acid residue within the human FR
`sequence located at any one. of the sites 4L, 35L, 36L, 38L, 43L, 44L, 461.,
`581., 62L, 63L, 64L, 55L, 66L, 67L, 68L, 69L, 70L, 7711., 73L, 85L, 87L,
`98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 68H, 69H.
`70H, 73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H has been
`
`I
`
`the residue
`in preferred embodiments,
`substituted by another residue.
`, substituted at the human FR site is the residue found at the corresponding
`
`»
`
`location of the non-human antibody from which the non-human CDR was ‘
`
`obtained.
`
`In other embodiments, no human FR residue other than those set
`
`forth in this group hasbeen substituted;
`
`~ This invention also encompasses specific humanized antibody.
`variable domains, and isolated polypeptides having homology With the
`following sequences.
`
`lD N0. 1, which is the light chain variable demain of a
`1. SEC).
`humanized version of muMAb4D5:
`DlOMTOSPSSLSASVGDRVTlTCRASODVNTAVAWYOQKPGKAP
`
`KLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLOPEDFATYYCQOHY
`TTPPTFGOGTKVEIKRT
`
`2. SEO. 10 NO. 2, which is the heavy chain variable domain of a ‘
`
`_
`
`humanized version of muMAb4D5):
`
`. _EVOLVESGGGLVQPGGSLRLSCAASGFMKDTYlHWVROAPGKGLE
`
`WVARlYPTNGYTRYADSVKGRFTlSADTSKNTAYLOMNSLRAEDT '
`AVYYCSRWGGDGFYAMDVWGOGTLVTVSS
`\n
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`20‘
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`30
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`antibody variable domain amino acid sequence for use in the preparation of
`
`in another aspect, this invention provides a consensus human
`
`humanized antibodies, methods for obtaining, using, and storing a computer
`
`representation of such a consensus sequence, and computers comprising the
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`1‘6
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`
`
`
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`sequence data of such a sequence.
`
`ln one embodiment, the following
`
`consensus human antibody variable domainiamino acid sequences are
`
`provided:
`
`SEQ.
`
`lD NO. 3 (light chain):
`
`DIQMTQSPSSLSASVGDRVTITCRAS'ODVSSYLAWYQOKP'GKAPK
`
`LUYAASSLESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOQYN
`
`SLPYTFGOGTKVEIKRT, and
`
`SEO. le NO. 4 (heavy chain):
`
`EVOLVESGG_GLVOPGGSLRLSCAASGFTFSDYAMSWVRQAPGKG
`LEWVAVISENGGYTfiYADSVKGRFTlSIADTSKNT‘AYLOMNSLBAE
`'DTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
`A
`'
`
`_Brief Desgrigtign of the Drawings
`
`FIGURE :1A shows the comparison of the VL domain amino acid
`residues of muMAb4DE, huMAb4DS, and a consensus human sequence (Fig.
`
`'1A, SEOJD NO. 5, SEO. )0 NO. 1 and SEQ. ID NO. 3, respectively). FIGURE
`18 shows the comparison between the VH domain amino acid residues of the
`muMAb4d5, huMAinDS, and a consensus human sequence (Fig, 18, SEQ.
`
`ID NO. 6, SE0. “3 NO. 2-and SEQ. ID NO. 4, respectively). Both Figs 1A and
`
`18 use the generally accepted numbering scheme from Kabat, E. A., et 8].,
`Sequences of Proteins of Immunological Interest (National
`institutes of
`
`In both Fig. 1A and Fig. is, the, CDR
`Health, Bethesda, MD (1987)).
`residues determined according to a standard sequence‘definition (as in Kabat,
`
`E. A. et aI., Sequences of Proteins of Immunological Interest (National
`V Institutes or Health, Bethesda, MD, 1987))
`are~ indicated by the first
`
`underlining beneath the sequences, and the CDR residues determined
`
`according‘to a structural definition (as in Chothia, C. & Lesk, A. M., J. Mol.
`
`Biol. 196:901-917 (1987)) are indicated by the second, lower underlines.
`
`11
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`17
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`30
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`
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`pg
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`4/ The mismatches betweenWshown by the vertical lines.
`
`FIGURE 2 shows a scheme for humanization of muMAb4D5 VL and
`
`VH by gene conversion mutagenesis.
`FlGURE 3 shows the inhibition of SK- BR- 3 proliferation by MAb4DS
`variants. Relative cell proliferation was determined as described (Hudziak, R.
`
`M. et a/., Molec. Cell. Biol. 9:1165-1172 (1989)) and datalaverage of
`triplicate determinations) are presented as a percentage of results with
`untreated cultures for muMAb4D5 (l), huMAb4DS—8 (n) and huMAb4DS- 1
`(l)-
`FIGURE 4 shows a stereo view of a—carbon tracing for model of
`huMAb4DS8 VL and VH The CDR residues (Kabat, E. A. et aI, Sequences
`’ of Proteins of Immunology/cal Interest (National lnstitutes of Health, Bethesda,
`
`MD, 1987)) are shown in bold andside chains of VH residues A71, T73,
`
`A78, 593, Y102 and VL residues Y55 plus R66 (see Table 1) are shown.
`
`Detailed Description of the Invention
`
`Definitions
`
`In general,
`
`the following words or phrases have the indicated-
`
`definitions when used in the description, examples, and claims:
`
`The murine monoclonal antibody known as muMAb4D§ (Fendly,8
`M. er al., Cancer Res. 50:1550 1558 (1990)) is directed against
`the
`extracellular domain (ECD) of p185HER2. The muMAb4DS and its uses are
`
`applications 07/143,912 and
`co‘pending U.S. patent
`described in
`, 07/147,461, and in corresponding PCT application WO 89/06692 published
`' 27VJuly 1989. This murine antibody was deposited with the ATCCIZ and
`designated ATCC ,CRL 10463.
`In this description and claims, the terms
`muMAb4D5, chMAb4D5 and huMAb4D§ represent murine,'chimeri2ed and'
`humanized versions of the monoclonal antibody 405, respectively.'. __
`A humanized antibody for the purposes herein is an immunoglobulin
`amino acid sequence variant or fragment thereof which is capable of binding
`to a predetermined antigen and which comprises a FR region having
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`substantially the amino acid sequence of a human immunoglobulin and a CDR
`having
`substantially
`the
`amino. acid
`sequence
`of
`a
`non-human
`
`immunoglobulin.
`
`In general, the humanized antibody will comprise substantially all of
`
`at least one, and typically two, variable domains (Fab)
`
`in which all or
`
`"substantially all of the CDR regions correspond to those of a non-human
`immunoglobulin and all or substantially all of the FR regions are those of a,
`human immunoglobuiin consensus sedtience. The humanized antibody
`
`optimally also will comprise at least aportion of an immundglobulin constant
`
`the
`typically that of a human immunoglobulin. Ordinarily,
`region (Fc),
`antibody will contain both the light chain as well as at least the variable
`domain of a heavy chain. The antibody also may include the CH1, hinge,
`
`CH2, CH3, and CH4 regions of the heavy chain.
`The humanized antibody will be selected from any class of
`
`'
`
`immunoglobulins,
`
`including lgM,
`
`lgG, IgD,
`
`lgA and lgE, and any isotype,
`
`including lgG1,
`
`lgGZ, lgGa and lgG4. Usually the constant domain is a ‘
`
`complement fixing constant domain where it is desired that the humanized
`antibody exhibit cytotoxic activity, and the class is typically lgG,". Where
`such cytotoxic activity is not desirable, the constant domain may be of the
`lgG, class. The humanized antibody may comprise sequences. from more ,
`
`than one class or isotype, and selecting particular constant domains to
`
`optimize desired effector functions is within the ordinary skill in the art“
`
`The. FR and CDR regions of the humanized antibody need not
`
`correspond precisely to the parental sequences, e.g., the impert CDR or the
`
`consensus FR may be mutagenized by substitution, insertion or deletion of
`
`a residue so that the CDR or FR residue at that site does not correspond to.
`
`either the consensus or the import antibody. Such mutations, however, will
`
`not be extensive. Usually, at least 75% of the humanized antibody residues
`
`will correspond to those of the parental FR and CDR sequences, more often
`
`30
`
`' 90%, and most preferably greater than 95%.
`
`in general, humanized antibodies prepared by the method» of this
`
`invention are produced by a process of analysis of the parental sequences
`
`13
`
`'19 ,
`
`
`
`
`
`_ and various conceptual humanized products using three dimensional models
`of
`the
`parental
`and
`humanized
`sequences.
`Three
`dimensional
`
`immunoglobulin models are commonly available and, are familiar to those
`' skilled in the art. Computerprograms are available which illustrate and
`
`display probable three dimensional conformational structures of selected ‘
`candidate immunoglobulin sequences.
`inspection of these displays permits
`analysis of the likely role of the residues in the functioning of‘the candidate
`immunoglobulin sequence, i.e., the analysis of residues that influence the
`ability of the candidate immunoglobulin to bind its antigen.
`Residues that influence antigen binding are defined to be residues .
`
`that are substantially responsible for the antigen affinity or antigen specificity '
`of a candidate immunoglobulin, in a positive or a negative sense. The object
`
`here is toselect FR residues from the consensus and import sequence so that
`the desired immunoglobulin characteristic is achievedfi Such desired
`
`characteristics include increases in affinity and greater specificity for’the
`
`target antigen, although it is conceivable that in some Circumstances the
`opposite. effects might be desired.- In general, the 008 residues are directly .
`and most substantially involved in influencing antigen binding (although not '
`all CDR residues aie so involved and therefore need not be substituted-into ‘
`
`the consensus sequence). However, FR residues also have a significant
`effect and can exert their influence in at least three ways: They may .
`
`'
`
`noncovalently directly bind to antigen, they may interact with CDR residues
`and they may affect the interfacebetween the heavy and light chains.
`
`A residue that noncovalently directly binds to antigen is one that,
`
`is reasonably expected to noncovalently
`by three dimensional analysis,
`directly bind to antigen. Typically, it is necessary to impute the position ’of
`
`antigen from the spatial location of neighboring CDRs and the dimensionsand
`structure of the target antigen. 'In general, only these humanized antibody
`'residues that are capable of
`forming salt bridges, hydrogen bonds, or
`' hydrophobic interactions are. likely to be involved in non--COvalent antigen
`.._\4
`binding, however residues which are separated spatially by 3.2Angstroms )
`
`_fl_-- -- H
`\. M
`Such residues typically are the
`“/“—— -——~«MW
`or less may also non--covalently interact.
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`relatively larger amino acids, ngninw Antigen—
`binding FR residues also typically will have side chains that are oriented into
`an envelope surrounding the solvent oriented-face of a CDR which extends
`about 7 Angstroms into the solvent from the CDR domain and about 7
`Angstroms on either side of the CDR domain, again as visualized by three
`dimensional modeling.
`‘
`..
`A residue that in