`
`01 715272
`.
`\
`.... ~PRESS 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
`
`SIR:
`
`NEW APPLICATION TRANSMITTAL
`
`Transmitted herewith for filing is the patent application of lnventor(s): PAUL J. CARTER ET AL.
`
`Title:
`
`IMMUNOGLOBULIN VARIANTS
`
`CERTIFICATION UNDER 37 CFR § 1. 10
`
`I hereby certify that this New Application end the docunents referred to as enclosed herein are being deposited with the United
`States Postal Service on this date June 14, 1991, in an envelope bearing "Express Mail Post Office To Addressee" Mailing Lebel
`Nllli>er 859937585 addressed to: Patent Application, Honorable Conmissloner of atents nd Tad
`ks, ashington, D.C. 20231.
`Carolyn R. Adler
`(Name of person mail1ng paper)
`
`Enclosed are:
`
`The papers required for filing date under CFR § 1.53(b):
`.1Q§_ Pages of specification (including claims); ~ Sheets of drawings L formal I ..1L informal)
`.lL Declaration/Oath/Power of Attorney
`_ Assignment of the invention to GENENTECH, INC.
`
`Fee Calculation
`
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`
`Total Fees Enclosed
`
`Payment of Fees
`.lL Charge Account No. 07-0630 in the amount of$_. A duplicate of this transmittal Is attached.
`.lL Authorization to Charge Additional Fees
`The Commissioner is hereby authorized to charge any additional fees (or credit any overpayment) associated
`with this communication and which may be required under 37 CFR § 1.16 or § 1.17 to Account No. 07-0630.
`A duplicate sheet is attached.
`
`10.
`11 .
`
`Information Disclosure Statement
`. Return Receipt Postcard
`
`.lL
`
`Dated June 14. 1991
`
`L
`
`By: ~,e, l!dv
`
`Name: CarOIVJ1RAd1er
`Registration No. 32,324
`
`L
`
`:)..
`3.
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`4.
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`7.
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`8.
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`9.
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`Nuiber FI led
`16 - 20 =
`8 - 3 =
`Multiple dependent claim(s), if any
`
`j
`
`Total Claims
`
`. lndep. Claims
`
`-
`*If less than zero, enter 11011 •
`Recording Assignment [$8.00J
`
`Nllli>er Extra
`- *
`* 5
`
`Basic Fee
`S630
`
`630.
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`300.
`
`Rate
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`x $20.00
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`x S60.00
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`$200.00
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`$
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`. . • . • . $930.00
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`1 of 389
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`Celltrion, Inc., Exhibit 1094
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`
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`
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`
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`2 of 389
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`Celltrion, Inc., Exhibit 1094
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`FIGURE lB: Vu DOMAIN
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`•
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`01 715272
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`405
`
`HU405
`
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`40
`30
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`
`3 of 389
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`Celltrion, Inc., Exhibit 1094
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`\ .
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`F /GURE,, 2
`Anneal hu VL or huV8 oligomers to pAKl template
`3' ..J.-..--A.-..J.-.. ..J.,_.. ..J.-..-->.J 5'
`.....
`--····
`
`•
`
`~1 715272
`
`1. Ligate
`2. Isolate assembled oligomers
`3. Anneal to pAKl template (Xhof-, Stu!+)
`4. ·Extend and ligate
`
`,
`
`Xhol
`
`1. Transfonn E.coli
`2. Isolate phagemid pool
`3. Enrich for hu\L and huVH(Xho /~Stu!-)
`4. Sequence verify
`
`pAK2
`
`4 of 389
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`Celltrion, Inc., Exhibit 1094
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`
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`
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`
`4
`
`12
`8
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`16
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`5 of 389
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`Celltrion, Inc., Exhibit 1094
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`6 of 389
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`Celltrion, InC., Exhibit 1094
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`6 of 389
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`Celltrion, Inc., Exhibit 1094
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`ft:115272
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`DOCKET 709
`EXPRESS MAIL NO. 859937585
`MAILED 14 JUNE 1991
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`- s
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`IMMUNOGLOB.ULIN VARIANTS
`
`Field of the Invention
`
`This invention relates to methods for the preparation and use of
`variant antibodies and
`finds application particularly in the
`immunology and cancer diagnosis and therapy.
`
`fields of
`
`Background of the Invention
`
`Naturally occurring antibodies (immunoglobulins) comprise two
`heavy chains linked together by disulfide 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 <Vt) 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 domain 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 e.g. Chothia et al.,
`J. Mo/. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. A cad. Sci.
`
`1
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`7 of 389
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`Celltrion, Inc., Exhibit 1094
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`USA 82:4592-4596 (1985).
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`•
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`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.
`
`5
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`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
`
`10
`
`determining regions (CDRs) (see Kabat, E. A. et al., Sequences of Proteins
`
`of Immunological Interest, National Institutes of Health, Bethesda, MD,
`
`(1987)). The four framework regions largely adopt a P-sheet conformation
`
`and the CD Rs form loops connecting, and in some cases forming part of, the
`
`P-sheet structure. The CDRs in each chain are held in close proximity by the
`
`15
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`framework regions and, with the CDRs from the other chain, contribute to
`
`the formation of the antigen binding site.
`
`Widespread use has been made of monoclonal antibodies,
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`particularly those derived from rodents including mice, however they are
`
`frequently antigenic in human clinical use. For example, a major limitation in
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`20
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`the clinical use of rodent monoclonal antibodies is an anti-globulin response
`
`during therapy (Miller, R. A. eta/., B/ood62:988-995 (1983}; Schroff, R. W.
`
`et al., Cancer Res. 45:879-885 (1985}).
`
`The art has attempted to overcome this problem by constructing
`
`"chimeric" antibodies in which an animal antigen-binding variable domain is
`
`25
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`coupled to a human constant domain (Cabilly et al., U.S. patent No.
`
`4,816,567; Morrison, S. L. eta/., Proc. Natl. Acad. Sci. USA 81:6851-6855
`(1984); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neuberger, M.
`
`S. et al., Nature 314:268-270 (1985)). The term "chimeric" antibody is used
`
`herein to describe a polypeptide comprising at least the antigen binding
`
`30
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`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 may be selected to tailor
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`2
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`the chimeric antibody for participation
`
`•
`
`in antibody-dependent cellular
`
`cytotoxicity (ADCC) and complement-dependent cytotoxicity (see e.g.
`
`Bruggemann, M. etal.,J. Exp. Med. 166:1351-1361 (1987); Riechmann, L.
`
`et al., Nature 332:323-327 ( 1988); Love et al., Methods in Enzymology
`
`- 5
`
`-178:515-527 (1989); Bindon eta/., J. Exp. Med. 168:127-142 (1988).
`
`In the typical embodiment, such chimeric antibodies contain about
`
`one third rodent (or other non-human species) sequence and thus are capable
`
`of eliciting a significant anti-globulin response in humans. For example, in the
`
`case of the murine anti-CD3 antibody, OKT3, much of the resulting
`
`10
`
`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. eta/., Nature 321:522-525
`
`15
`
`( 1986); Riechmann, L. et al., Nature 332:323-327 (1988); V~rhoeyen, M. et
`
`al., Science 239: 1534-1536 (1988)) have substituted rodent CDRs or CDR
`
`sequences for the corresponding segments of a human antibody. As used
`
`herein, the term "humanized" antibody is an embodiment of chimeric
`
`antibodies wherein substantially less than an intact human variable domain
`
`20
`
`has been substituted by the corresponding sequence from a non-human
`
`species. In practice, 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 the-rapeutic promise of this approach is supported by the clinical
`
`25
`
`efficacy of a humanized antibody specific for the CAMPA TH-1 antigen with
`
`two non-Hodgkin lymphoma patients, one of whom had previously developed
`
`an anti-globulin response to the parental rat antibody (Riechmann, L. et al.,
`
`Nature 332:323-327 (1988); Hale, G. et al., Lancet i:1394-1399 (1988)).
`
`A murine antibody to the interleukin 2 receptor has also recently been
`
`30
`
`humanized (Queen, C. et al., Proc. Natl. Acad. Sci. USA 86:10029-10033
`
`(1989)} as a potential immunosuppressive reagent. Additional references
`
`related to humanization of antibodies include Co et al., Proc. Natl. A cad. Sci.
`
`3
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`9 of 389
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`Celltrion, Inc., Exhibit 1094
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`15
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`20
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`25
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`30
`
`•
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`••
`
`USA 88:2869-2873 (1991); Gorman et al., Proc. Natl. Acad. Sci. USA
`88:4181-4185 (1991 ); Daugherty eta/., Nucleic Acids Research 19(9):2471-
`2476 (1991); Brown et al., Proc. Natl. Acad. Sci. USA 88:2663-2667
`(1991); Junghans eta/., Cancer Research 50:1495-1502 (1990).
`In some cases, substituting CDRs ·from rodent antibodies for the
`human CDRs in human frameworks is sufficient to transfer high antigen
`binding affinity (Jones, P. T. et al., 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. et al., Proc. Natl. Acad. Sci. USA
`86: 10029-10033 (1989)) framework region (FR) residues. See also Co et
`al., supra.
`For a given antibody a small number of FR resi~ues 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. eta/., Ann. Rev. Biochem. 59:439-473 (1990)). Secondly, a number
`of FR residues have been proposed by Chothia, Leskand colleagues (Chothia,
`C. & Lesk, A. M., J. Mo/. Biol. 196:901-917 (1987); Chothia, C. et al.,
`Nature 342:877-883 (1989); Tramontano, A. ·et al., J. Mo/. Biol.
`215.:175-182 (1990)) as critically affecting the conformation of particular
`CDRs and thus their contribution to antigen binding. See also Margolies et
`al., Proc. Natl. Acad. Sci. 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, Pluckthu11,
`Biotechnology 9:545-51 (1991); Spiegelberg et al., Biochemistry 9:4217-
`4223 (1970); Wallie et al., J. Exp. Med. 168:1099-1109 (1988); Sox et al.,
`Proc. Natl. Acad. Sci. USA 66:975-982 (1970); Margni et al., Ann. Rev.
`lmmunol. 6:535-554 (1988). Ordinarily, however, glycosylation has no
`influence on the antigen-binding properties of an antibody, Pluckthun, supra,
`(1991).
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`4
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`10 of 389
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`The three-dimensional structure of immunoglobulin 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
`(1978); Sheriff et al., Proc. Natl. Acad. Sci. USA 84:8075-79 (1987); Segal
`et al., Proc. Natl. Acad. Sci. USA 71:4298-4302 (1974); Epp et al.,
`Biochemistry 14(22):4943-4952 (1975); Marquart et al., J. Mo/. Biol.
`141:369-391 (1980); Fureyeta/.,J. Mo/. Biol. 167:661-692 (1983); Snow
`and Amzel, Protein: Structure, Function, and Genetics 1 :267-279, Alan R.
`Liss, Inc. pubs. (1986); Chothia and Lesk, J. Mo/. Biol. 196:901-917 (1987);
`Chothia eta/., Nature 342:877-883 (1989); Chothia eta/., Science 233:755-
`58 (1986); Huber et al., Nature 264:415-420 (1976); Bruccoleri et al.,
`Nature 335:564-568 (1988) and Nature 336:266 (1988); Sherman et al.,
`Journal of Biological Chemistry 263:4064-4074 (1988); Amzel and Poljak,
`Ann. Rev. Biochem. 48:961-67 (1979); Silverton eta/., Proc .. Natl. Acad.
`Sci. USA 74:5140-5144 (1977); and Gregory eta/., Molecular Immunology
`24:821-829 (1987).
`It is known that the function of an antibody is
`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 antibody can be 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)).
`
`dependent on
`
`Humanizing an antibody with retention of high affinity for antigen
`and other desired biological activities is at present difficult to achieve using
`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 proto-oncogene HER2
`(human epidermal growth factor
`receptor 2) encodes a protein tyrosine kinase (p195HER2> that is related to
`and somewhat homologous to the human epidermal growth factor receptor
`
`s
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`•
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`{see Coussens, l. eta/., Science 230:1132-1139 (1985); Yamamoto, T. et
`
`. s
`
`al., Nature 319:230-234 {1986); King, C. R. et al., Science 229:974-976
`
`(1985)). HER2 is also known in the field as c-erbB-2, and sometimes by the
`
`name of the rat homolog, neu. Amplification and/or overexpression of HER2
`
`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 al., 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,
`
`10
`
`supra, Science 1989).
`
`15
`
`20
`
`The murine monoclonal antibody known as muMAb405 (Fendly, B.
`
`M. et al., Cancer Res. 50:1550-1558 (1990)), directed against the
`extracellular domain (ECO) of p 185HER2, specifically inhibits the growth of
`tumor cell lines overexpressing p185HER2 in monolayer culture or in soft agar
`(Hudziak, R. M. eta/., Malec. Cell. Biol. 9:1165-1172 (1989); Lupu, R. eta/.,
`
`Science 249: 1552-1555 (1990)). MuMAb405 also has the potential of
`
`enhancing tumor cell sensitivity to tumor necrosis factor, an important
`
`effector molecule in macrophage-mediated tumor cell cytotoxicity (Hudziak,
`
`supra, 1989; Shepard, H. M. and Lewis, G. D. J. Clinical Immunology
`
`8:333-395 (1988)). Thus muMAb4D5 has potential for clinical intervention
`in and imaging of carcinomas in which p1 g5HER2 is overexpressed. The
`muMAb4D5 and its uses are described in copending U.S. patent applications
`
`07 /143,912 and 07 /14 7,461, and in corresponding PCT application WO
`
`89/06692 published 27 July 1989. This murine antibody was deposited
`
`25
`
`with the ATCC and designated ATCC CRL 10463. However, this antibody
`
`may be immunogenic in humans.
`
`It is therefore an object of this invention to provide methods for the
`
`preparation of antibodies which are less antigenic in humans than non-human
`
`antibodies but have desired antigen binding and other characteristics and
`
`30
`
`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
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`6
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`•
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`residues for importation into a 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.
`It is another object of this invention to provide humanized
`antibodies capable of binding p1 s5HER2.
`
`-
`
`5
`
`Other objects, features, and characteristics of the present invention
`will become apparent upon consideration of the following description and the
`appended claims.
`
`10
`
`Summary of the Invention
`
`15
`
`20
`
`25
`
`30
`
`The objects of this invention 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:
`a.
`obtaining the amino acid sequences of at least a portion
`of an import antibody variable domain and of a consensus
`human variable domain;
`identifying Complementarity Determining Region (CDR)
`
`b.
`
`c.
`
`d.
`
`e.
`
`f.
`
`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 antiJ,lody;
`identifying import antibody FR residues in the aligned FR
`seque!'lces that are non-homologous to the corresponding
`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,
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`7
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`13 of 389
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`Celltrion, Inc., Exhibit 1094
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`•
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`g.
`
`2.
`interacts with a CDR; or
`3.
`participates in the VL - VH interface; and
`for any non-homologous import antibody amino acid
`residue which is reasonably expected to have at lea.st one
`of these effects, substituting
`that residue
`for the
`corresponding· amino acid residue in the consensus
`antibody FR sequence.
`Optionally, the method of this invention comprises the additional
`steps of determining if any non-homologous residues identified in step (e) are
`exposed on the surface of the domain or buried within it, and if the residue
`is exposed but has none of the effects identified in step (f), 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 (e) above are selected from the group consisting
`of4L,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,37H, 39H,
`43H, 45H,49H, 58H, 60H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H,
`91 H, 92H, 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
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`.consensus variable domain s_:qu~~~~!~~ ~lycosyla~on !it~-~· d.etermining if
`the glycosylation is rea~~i:t~~y ex~~cted to be important for the desired
`antigen binding and biological activity of the antibody (i-.e~-:-determining ifthe ·
`glyc~s~lati~n ~-ite binds to anti.gen 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 bears the glycosylation site, it is preferred to substitute that
`---
`--------------------
`" . . -
`site for the corresponding residues in the consensus human sequence if the
`glycosylation site is reasonably expected to be important.
`If only the
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`ft consensus sequence, and not the import, bears the glycosylation site, it is
`! 1
`preferred to eliminate that glycosylation site or substitute therefor the
`\ corresponding amino acid residues from the import sequence.
`Another embodiment of this invention comprises aligning import
`antibody and the consensus antibody FR sequences, identifying import
`. 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.
`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 a FR,
`obtaining the amino acid sequence of at least a portion of a consensus
`human antibody variable domain having a CDR and a FR, substituting the
`non-human CDR for the human CDR in the consensus human antibody·
`variable domain, and then substituting an amino acid residue f9r 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,58L,46L,62L,63L,64L,65L,
`66L, 67L,68L,69L, 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,
`
`b.
`
`68H~69H,70H,73H,74H,75H,76H,78H,91H,92H,
`93H, and 103H.
`In preferred embodiments, the non-CDR 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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`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, 46L,
`58L, 62L, 63L, 64L, 65L, 66L, 67l, 68L, 69L, 70l, 71 L, 73L, 85L, 87l,
`98L,2H,4H,24H,36H,37H,39H,43H,45H,49H,58H,60H,68H,69H,
`70H, 73H, 74H, 75H, 76H, 78H, 91 H, 92H, 93H, and 103H has been
`substituted by another residue.
`In preferred embodiments, the 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 has been substituted.
`This invention also encompasses specific humanized antibody
`variable domains, and isolated polypeptides having homology with the
`following sequences.
`
`1. SEO. ID NO. 1, which is the light chain variable domain of a
`humanized version of muMAb4D5:
`DIOMTOSPSSLSASVGDRVTITCRASODVNTAVAWYOOKPGKAP
`KLLIYSASFLESGVPSRFSGSRSGTDFTL TISSLOPEDFATYYCOQHY
`TTPPTFGOGTKVEIKRT
`
`2. SEO. ID NO. 2, which is the heavy chain variable domain of a
`humanized version of muMAb4D5):
`
`EVOLVESGGGL VOPGGSLRLSCAASGFNIKDTYIHWVROAPGKGLE
`WVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLOMNSLRAEDT
`AVYYCSRWGGDGFYAMDVWGQGTL VTVSS
`
`In another aspect, this invention provides a consensus human
`antibody variable domain amino acid sequence for use in the preparation of
`humanized antibodies, methods for obtaining, using, and storing a computer
`representation of s~ch a consensus sequence, and computers comprising the
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`sequence data of such a sequence.
`In one embodiment, the following
`consensus human antibody variable domain amino acid sequences are
`provided:
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`SEO. ID NO. 3 (light chain):
`DIOMTOSPSSLSASVGDRVTITCRASODVSSYLAWYOOKPGKAPK
`LUY AASSLESGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCQQYN
`SLPYTFGOGTKVEIKRT, and
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`SEO. ID NO. 4 (heavy chain):
`
`EVOLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVROAPGKG
`LEWVAVISENGGYTRY ADSVKG RFTI SADTSKNT A YLOMNSLRAE
`DTAVYYCSRWGGDGFYAMDVWGOGTL VTVSS
`
`Brief Description of the Drawings
`
`FIGURE 1 A shows the comparison of the VL domain amino acid
`residues of muMAb405, huMAb4D5, and a consensus human sequence (Fig.
`1 A, SEO.ID NO. 5, SEO. ID NO. 1 and SEO. ID NO. 3, respectively) .. FIGURE
`
`1 B shows the comparison between the VH domain amino acid residues of the
`muMAb4d5, huMAb4D5, and a consensus human sequence (Fig. 18, SEO.
`ID NO. 6, SEQ. ID NO. 2 and SEQ. ID NO. 4, respectively). Both Figs 1A and
`1 Buse the generally accepted numbering scheme from Kabat, E. A., et al.,
`Sequences of Proteins of Immunological Interest (National Institutes of
`Health, Bethesda, MD (1987)).
`In both Fig. 1 A and Fig. 18, the CDR
`residues determined according to a standard sequence definition (as in Kabat,
`E. A. et al., Sequences of Proteins of Immunological Interest (National
`Institutes of 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. Mo/.
`Biol. 196:901-917 ( 1987)) are indicated by the second, lower underlines.
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`{._,,r The mismatches betwee~shown by the vertical lines.
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`FIGURE 2 shows a scheme for humanization of muMAb4D5 VL and
`VH by gene conversion mutagenesis.
`FIGURE 3 shows the inhibition of SK-BR-3 proliferation by MAb4D5
`variants. Relative cell proliferation was determined as described (Hudziak, R.
`M. et al., Molec. Cell. Biol. 9: 1165-1172 ( 1989)) and data (average of
`triplicate determinations) are presented as a percentage of results with
`untreated cultures for muMAb4D5 (I), huMAb4D5-8 (n) and huMAb4D5-1 (I).
`FIGURE 4 shows a stereo view of a-carbon tracing for model of
`huMAb4D5-8 VL and VH . The CDR residues (Kabat, E. A. et al., Sequences
`of Proteins of Immunological Interest (National Institutes of Health, Bethesda,
`MD, 1987)) are shown in bold and side chains of VH residues A71, T73,
`A78, S93, 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 muMAb4D5 {Fendly, B.
`M. et al., Cancer Res. 50:1550-1558 (1990)) is directed against the
`extracellular domain {ECO) of p1 asHER2. The muMAb4D5 and its uses are
`described
`in copending U.S. patent applications 071143,912 and
`07 /14 7 ,461, and in corresponding PCT application WO 89/06692 published
`27 July 1989. This murine antibody was deposited with the ATCC and
`designated ATCC CRL 10463.
`In this description and claims, the terms
`muMAb4D5, chMAb4D5 and huMAb4D5 represent murine, chimerized and
`humanized versions of the monoclonal antibody 4D5, 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.
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`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 immunoglobulin consensus sequence. The humanized antibody
`optimally also will comprise at least a. portion of an immunoglobulin constant
`region (Fe), typically that of a human immunoglobulin. Ordinarily, the
`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, lgD, lgA and lgE, and any isotype,
`including igG 1, lgG2, lgG3 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 1 • Where
`such cytotoxic activity is not desirable, the constant domain may be of the
`lgG2 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 import CDR or the
`consensus FR may be mutagenized by substitutio'1, 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
`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
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`and various conceptual humanized products using three dimensional models
`
`of
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`the parental and humanized sequences.
`
`Three dimensional
`
`immunoglobulin models are commonly available and are familiar to those
`
`skilled in the art. Computer programs are available yvhich 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.
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`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 to select FR residues from the consensus and import sequence so that
`
`the desired immunoglobulin characteristic is achieved. Such desired
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`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 CDR residues are directly
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`and most svbstantially involved in influencing antigen binding (although not
`
`all CDR residues are so involved and therefore need not be substituted into
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`the cons