),
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`\
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`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
`
`NEW APPLICATION TRANSMITTAL
`
`SIR:
`
`Transmitted herewith for filing is the patent application of lnventor(s):
`
`Title:
`
`IMMUNOGLOBULIN VARIANTS
`
`PAUL J. CARTER ET AL.
`
`CERTIFICATION UNDER 37 CFR § 1. 10
`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 T ad ks, ashington, D.C. 20231.
`
`I hereby certify that this New Application end the docunents referred to as enclosed herein are being deposited with the United
`
`Carolyn R. Adler
`(Name of person mail1ng paper)
`
`The papers required for filing date under CFR § 1.53(b):
`
`Enclosed are:
`L
`3. _ Assignment of the invention to GENENTECH, INC.
`.1Q§_ Pages of specification (including claims); � Sheets of drawings L formal I ..1L informal)
`:).. .lL Declaration/Oath/Power of Attorney
`
`4. Fee Calculation
`
`CLAIMS AS FILED
`
`j
`
`Total Claims
`
`. lndep. Claims
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`8 -
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`Nllli>er Extra
`
`- *
`* 5
`
`Nuiber FI led
`16 - 20 =
`3 =
`-
`*If less than zero, enter 11011•
`Recording Assignment [$8.00J
`
`Multiple dependent claim(s), if any
`
`Basic Fee
`
`S630
`630.
`300.
`
`Rate
`
`x $20.00
`x S60.00
`$200.00
`
`$
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`7.
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`8.
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`Payment of Fees
`
`Total Fees Enclosed
`
` $930.00
`.
`•
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`•
`.
`.
`.lL Charge Account No. 07-0630 in the amount of$_. A duplicate of this transmittal Is attached.
`9. .lL Authorization to Charge Additional Fees
`11 . .lL
`10.
`
`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.
`
`By: �,e, l!dv
`
`Name: CarOIVJ1RAd1er
`
`Registration No. 32,324
`
`Information Disclosure Statement
`
`. Return Receipt Postcard
`
`Dated June 14. 1991
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`lll 715272
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`OIVMTQSHKFMSTSVGORVSITCKASQOVNTAVAWYQQKPGHSPKLLIYSASFRYT
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`
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`405
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`60
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`2 of 389
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`FIGURE lB: Vu DOMAIN
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`01 715272
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`3 of 389
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`\ .
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`•
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`�1 715272
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`F /GURE,, 2
`Anneal hu VL or huV8 oligomers to pAKl template
`.....
`--····
`3' ..J.-..--A.-..J.-.. ..J.,_.. ..J.-..-->.J 5'
`2. Isolate
`3. Anneal to pAKl template
`
`1. Ligate
`
`assembled oligomers
`(Xhof-, Stu!+)
`
`4. ·Extend and ligate
`
`,
`
`1. Transfonn E.coli
`2. Isolate phagemid pool
`3. Enrich for hu\L and huVH(Xho /�Stu!-)
`4. Sequence verify
`
`Xhol
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`pAK2
`
`4 of 389
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`80
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`
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`
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`
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`
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`
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`[MAb4D5 variant] µg/ml
`
`16
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`5 of 389
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`ft:115272
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`41
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`DOCKET 709
`
`EXPRESS MAI L NO. 8 5 9 9375 8 5
`MAI LED 1 4 JUNE 1991
`
`-
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`s
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`r
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`<!);;;
`
`IMMUNOGLO B.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 fields of
`
`immunology and cancer diagnosis and therapy.
`
`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
`
`30
`
`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. 186:6 51-663 (198 5); Novotny and Haber, Proc. Natl. A cad. Sci.
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`1
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`USA 82:4 592-4 596 (198 5).
`
`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
`
`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 com plementarity
`
`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 a re held in close proximity by the
`
`15
`
`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,
`
`particularly those derived from rodents including mice, however they are
`
`frequently antigenic in human clinical use. For example, a major limitation in
`
`20
`
`the clinical use of rodent monoclonal antibodies is an anti-globulin response
`
`during therapy ( Miller, R. A . eta/., B /ood62:988-9 9 5 (19 83}; Schroff, R. W.
`
`et al. , Cancer Res. 4 5:879-8 8 5 (198 5}).
`
`The art has attempted to overcome this problem by constructing
`
`25
`
`"chimeric" antibodies in which an animal antigen-binding variable domain is
`
`4,816,567; Morrison, S . L. et a/., Proc. Natl. Acad. Sci. USA 81:68 51-685 5
`
`coupled to a human constant domain (Cabilly et al. , U . S . patent No.
`
`(19 84); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neuberger, M.
`
`S. et al., Nature 314:268-270 (198 5)). The term "chimeric" antibody is used
`
`herein to describe a polypeptide comprising at least the antigen binding
`
`30
`
`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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`•
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`•
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`the chimeric antibody for participation
`
`in antibody-dependent cellular
`
`cytotoxicity
`
`(ADCC) and complement-dependent cytotoxicity
`
`(see e.g.
`
`Bruggemann, M. e tal . ,J . Exp. Med. 1 66:1 351- 1 3 6 1 ( 1 987); R iechmann, L.
`
`et al . , Nature 332:323-327 ( 1 9 88); Love e t al. , Methods in Enzymol ogy
`
`-1 78:515-527 ( 1 989); Bindon e t a/ . , J. Exp. Med. 1 68:1 2 7-1 42 ( 1 988).
`
`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, O KT3, m uch of the resulting
`
`anti-globulin response is directed against the variable region rather than the
`
`constant region (Jaffers, G. J. et a/;, Transp lan tation 41 :5 72-578 (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 ta/ . , Nature 321 :522-525
`
`( 198 6); Riechmann, L. et al., Nature 332:323-327 ( 1 988); V�rhoeyen, M. e t
`
`al., Sci ence 2 3 9 : 1 534-1 536 ( 1 988)) 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
`
`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
`
`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. e t al. ,
`
`Nature 332:323-327 ( 1 988); Hale, G. e t al. , L ancet i: 1 394-1 399 ( 1 988)).
`
`A murine antibody to the interleukin 2 receptor has also recently been
`
`humanized (Queen, C. et al . , Proc. Natl. Acad. Sci. USA 86:1 0029-1 0033
`
`(1989)} as a potential immunosuppressive reagent. Additional references
`
`related to humanization of antibodies include Co e t al. , Proc. Natl . A cad. Sci.
`
`3
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`,
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`-
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`s
`
`•
`
`••
`
`USA 8 8:2869-2873 (1991); Gorman et al. , Proc. Natl. Acad. Sci. USA
`88:4181-418 5 (19 91 ); Daugherty eta/. , Nucleic Acids Research 19 (9):2471-
`2476 (19 91); Brown et al. , Proc. Natl. Acad. Sci. USA 8 8:2663-2667
`
`(19 91); Junghans et a/., Cancer Research 50:1495-1502 (19 90).
`
`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
`
`10
`
`332:323-327 ( 198 8)) or several (Queen, C. et al. , Proc. Natl. A cad. Sci. USA
`
`al. , supra.
`
`86: 10029-10033 (19 89)) framework region (FR) residues. See also Co et
`
`For a given antibody a small number of FR resi�ues are anticipated
`
`to be important for antigen binding . Firstly for example, certain antibodies
`
`15
`
`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 a/. , Ann. Rev. Biochem. 59:439-473 (19 90)). Secondly, a number
`
`of FR residues have been proposed by Chothia, Lesk and colleagues (Chothia,
`
`20
`
`C. & Lesk, A. M., J. Mo/. Biol. 196:901-917 (1987); Chothia , C. et al.,
`Nature 342:877-883
`(198 9); Tramontano, A. ·et al. , J. Mo/. Biol.
`
`215.:17 5-182 (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
`
`25
`
`domain (either VH or VL) may contain glycosylation sites, and that this
`
`g lycosylation 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 (19 88); Sox et al. ,
`
`Proc. Natl. A cad. Sci. USA 66:975-982 (1970); Margni et al. , Ann. Rev.
`
`30
`
`lmmunol. 6:535-5 54 (1988). Ordinarily, however, glycosylation has no
`
`influence on the antigen-binding properties of an antibody, Pluckthun, supra,
`
`(19 91).
`
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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 25:58 5-97
`
`. 5
`
`(1978); Sheriff et al. , Proc. N atl. Acad. Sci. USA 84:8075-79 (19 87); Segal
`
`et al. , Proc. Natl. Acad. Sci. USA 71:4298-4302 (1974); Epp e t al. ,
`
`Biochemistry 14(22):4943-49 52 (1975); Marquart et al. , J. Mo/. Biol.
`
`141:369-391 (1980); Furey et a/. , J. Mo/. Biol. 167:661-692 (1983); Snow
`
`and Amzel, Protein : Structure , Function , and G enetics 1 :267-279, Alan R .
`
`10
`
`Liss, Inc. pubs. (19 86); Chothia and Lesk, J. Mo/. Biol. 196:901-917 (19 87);
`
`Chothia eta/. , Nature 342:877-883 (19 89); Chothia eta/. , Science 233:75 5-
`
`5 8 (1986); Huber et al. , Nature 264:415-420 (1976); Bruccoleri et al. ,
`
`Nature 335:564-568 (1988) and Nature 336:266 (19 88); Sherman et al. ,
`
`Journal of Biological Chemistry 263:4064-4074 (1988); Amzel and Poljak,
`
`15
`
`Ann. Rev. Biochem. 48:961-67 (1979); Silverton et a/. , Proc .. Natl. Acad.
`
`20
`
`25
`
`30
`
`Sci. USA 74:5140-5144 (1977); and Gregory et a/. , Molecular Immunology
`
`24:821-829 (1987).
`
`It is known that the function of an antibody is
`
`its three dimensional structure, and that amino acid
`dependent on
`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 m utagenesis
`
`based upon molecular modelling (Riechmann, L. et al. , Nature 332:323-327
`
`(19 8 8); Queen, C . et al. , Proc. Natl. Acad. Sci. USA 86:10029-10033
`
`(19 8 9)).
`
`H umanizing 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 pre paring 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 (p19 5HER2> that is related to
`and somewhat homologous to the human epidermal growth factor receptor
`
`s
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`{see Coussens, l. eta/., Science 230:1132-1139 (1985); Yamamoto, T. et
`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,
`
`
`
`supra, Science 1989).
`
`B.
`The murine monoclonal antibody known as muMAb405 (Fendly,
`
`
`
`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
`
`
`
`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
`
`activities.
`
`It is a further object of this invention to provide methods for the
`
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`efficient humanization of antibodies, i.e. selecting non-human amino acid
`
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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
`
`antibodies capable of binding p 1 s5HER2.
`
`invention to provide humanized
`
`-
`
`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
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`
`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 o f a t least a portion
`
`of an import antibody variable domain and of a consensus
`
`human variable domain;
`
`b.
`
`identifying Complementarity Determining Region (CDR)
`
`amino acid sequences in .the import and the human
`
`variable domain sequences;
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`c.
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`d .
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`substituting an import CDR amino acid sequence for the
`
`corresponding human CDR amino acid sequence;
`
`aligning the amino acid sequences o f a Framework Region
`
`(FR) of the import antibody and the corresponding FR of
`the consensus antiJ,lody;
`
`e.
`
`identifying import antibody FR residues in the aligned FR
`
`seque!'lces that are non-homologous to the corresponding
`
`consensus antibody residues;
`
`f.
`
`determining if the non-homologous import amino acid
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`residue is reasonably expected to have at least one of the
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`following effects:
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`1 .
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`non-covalently binds antigen directly,
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`2.
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`3 .
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`interacts with a CDR; or
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`participates i n the VL - VH interface; and
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`5
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`g .
`
`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 c onsensus
`
`antibody FR sequence.
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`Optionally, the method of this invention comprises the additional
`
`steps of determining if any non-homologous residues identified in step (e) are
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`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
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`consensus residue.
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`Additionally, in certain embodiments the method of this i nvention
`
`comprises the feature wherein the corresponding consensus antibody
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`residues identified in step (e) above are selected from the group consisting
`
`of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L, 67L,
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`68L, 69L, 70L, 7 1 L, 73L, 85L, 87L, 98L, 2H, 4H , 24H , 36H, 37H, 39H,
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`43H, 45H, 49H, 58H, 60H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H,
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`91 H, 92H, 93H, and 1 03H (utilizing the numbering system set forth in Kabat,
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`20
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`E. A. et al. , Sequences of Proteins of Immunological Interest (National
`
`Institutes of Health, Bethesda, MD, 1 987)).
`
`In certain embodiments, the method of this invention comprises the
`
`additional steps of searching either or both of the import, non-human and the
`
`f'
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`.
`the glycosylation is rea��i:t��y ex��cted to be important for the desired
`antigen binding and biological activity of the antibody (i
`ite binds to anti
`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
`
`consensus variable domain s_:qu����!�� �lycosyla�on !it�-�· d.etermining if
`.e�-:-determining ifthe ·
`.gen or changes a side chain of an amino acid
`glyc�s�lati�n �-
`" ..
`---
`--------------------
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`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.
`\
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`Another embodiment of this invention comprises aligning import
`
`antibody and the consensus antibody FR sequences, identifying
`
`i mport
`
`. 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 a lternate 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 F R of the variable domain of the light chain) 4L,
`
`b .
`
`35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L,
`
`66L, 67L, 68L, 69L, 70L, 7 1 L, 7 3L, 8 5L, 8 7 L, 98L, or
`
`(in the FR of the variable d omain of the heavy chain) 2H,
`4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H,
`68H�69H , 70H , 73H, 74H, 75H, 76H, 78H , 9 1 H, 92H,
`
`93H, and 1 03H.
`
`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.
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`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
`
`" 5
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`sequence located at any one of the sites 4L, 3 5 L, 36L, 38L, 43L, 44L, 46L,
`
`58L, 62L, 63L, 64L, 65L, 66L, 67l, 68L, 69L, 70l, 7 1 L, 73L, 8 5L, 8 7l,
`
`98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H , 58H, 60H, 68H, 69H,
`
`70H, 73H, 74H, 7 5 H , 76H, 78H, 91 H , 92H, 93H, and 1 03 H has been
`
`substituted by another residue.
`
`In preferred embodiments, the residue
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`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
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`varia ble domains, and
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`isolated polypeptides having homology with the
`
`following sequences.
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`20
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`25
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`1 . SEO. ID NO. 1 , which is the light chain variable domain of a
`
`humanized version of muMAb4D5:
`
`D I O MTOSPSS LSASVG DRVTITCRASODVNTAVAWYOOKPG KAP
`
`KLLIYSASFLESGVPSRFSGSRSGTDFTL TISSLOPEDFATYYCOQHY
`
`TTPPTFGOGTKVEIKRT
`
`2. S EO. ID NO. 2, which is the heavy chain variable domain of a
`
`humanized version of muMAb4D5):
`
`EVOLVESGGGL VOPGG SLRLSCAASGFNIKDTYIHWVROAPGKGLE
`
`WVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLOMNSLRAEDT
`
`AVYYCSRWGGDGFYAMDVWGQGTL VTVSS
`
`30
`
`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 s�ch a consensus sequence, and computers comprising the
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`sequence data of such a sequence.
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`In one embodiment, the following
`
`consensus human antibody variable domain amino acid sequences are
`
`provided:
`
`SEO. ID N O . 3 (light chain):
`
`DIOMTOSPSSLSASVGDRVTITCRASODVSSYLAWYOOKPGKAPK
`
`LUY AASSLESGVPSRFSGSGSGTDFTL TISSLOPEDFATYYCQQYN
`
`SLPYTFGOGTKVEIKRT, and
`
`SEO. I D N O . 4 (heavy chain):
`
`EVOLVESGGG LVQPGG SLRLSCAASGFTFSDYAMSWVROAPGKG
`
`LEWVAVISENGGYTRY ADSVKG R FTI 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 N O . 5 , SEO. I D 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. 1 8, SEO.
`
`ID NO. 6 , SEQ. ID N O . 2 and SEQ. ID N O . 4, respectively). Both Figs 1 A and
`
`1 B use the generally accepted numbering scheme from Kabat, E. A . , et al. ,
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`s ·
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`(1 987)).
`
`In both Fig . 1 A and Fig . 1 8, 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, 1 987)) are indicated by the first
`
`30
`
`underlining beneath the sequences, and the CDR residues determined
`
`according to a structural definition (as in Chothia, C . & Lesk, A . M . , J. Mo/.
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`Biol. 1 96:901 -91 7 ( 1 987)) are indicated by the second , lower underlines.
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`{._,,r The mismatches betwee�shown by the vertical lines.
`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: 1 1 65-1 1 72 ( 1 989)) 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
`
`of Proteins of Immunological Interest (National Institutes of Hea lth, Bethesda,
`
`huMAb4D5-8 VL and VH . The CDR residues (Kabat, E. A. et al. , Sequences
`MD, 1 987)) are shown in bold and side chains of VH residues A7 1 , T73,
`A78 , S93, Y1 02 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, a nd claims:
`
`The murine monoclonal antibody known as muMAb4D5 {Fendly, B.
`
`M. et al. , Cancer Res. 50: 1 550-1 558 ( 1 990)) is directed against the
`
`extracellular domain {ECO) of p1 asHER2. The muMAb4D5 and its uses are
`
`described
`
`in copending U.S. patent applications 0711 43,9 1 2 and
`
`07 /1 4 7 ,461 , and in corresponding PCT application WO 8 9/06692 published
`
`27 July 1 989. This m urine antibody was deposited with the ATCC and
`
`designated ATCC CRL 1 0463.
`
`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
`the amino acid sequence of a non-human
`
`having substantially
`
`immunoglobulin.
`
`In general, the humanized antibody will comprise substantially all of
`
`at least one, and typically two, variable d omains (Fab) in which a ll or
`
`substantially all of the CDR regions correspond to those of a non-human
`
`immunoglobulin a nd 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 immunog lobulin. Ordinarily, the
`
`antibody will contain both the light chain as well as at least the variable
`
`domain of a heavy chain. The antibody a lso may include the C H 1 , 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 lgG1• 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 d omains 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 9 5 % .
`
`In general, humanized antibodies prepared by the method o f 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 m odels
`
`of
`
`the parental and humanized sequences.
`
`Three d imensional
`
`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 d isplays 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 a ntigen 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 sequenc