,..
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`J.
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`\ ij1 715272
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`_);PRESS MAIL LABEL NO. 059937585
`DATE MAILED: June 14, 1991
`
`460 Point San Bruno Boulevard. South San Francisco. CA 94080
`
`GENENTECH. INC.
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`
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`(415)266-1000
`
`Docket No. M
`
`SIR:
`
`
`
`NEW APPLICATION TRANSMIITAL
`
`
`
`
`
`
`
`
`Transmitted herewith for filing is the patent application of lnventor(sl:
`PAUL J. CARTER ET Al.
`
`
`
`Title: IMMUNOGLOBULIN VARIANTS
`
`
`
`CERTIFICATION UNDER 37 CFR §1.10
`
`I hereby certify that this New Appl !cation and the docunents· referred to es ·enclosed herein are being deposited with the United
`
`To Addressee" Hall Ing Label States Postal Service on this date June 14, 1991, In en envelope bearing 11Expres.s Mall Post Office
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`
`
`
`
`Nl.<lt>er 859937585 addressed to: Patent Application, Honorable COIM!lssloner of atents nd T ad ks, ashfngton, O.C. 20231.
`
`Carolyn R, Adler
`(Name of person mailing paper)
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`Enclosed are:
`1. The papers required for filing date under CFR § 1.53(b):
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`Total Claims
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`7.
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`Dated .June 14, 1991
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`L_
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`av,�t(,�
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`Name: CarolvnRAd1er
`
`Registration No. 32,324
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`. .
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`· Pfizer v. Genentech
`IPR2017-01488
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`1
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`
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`Genentech Exhibit 2032
`
`

`

`we
`0”
`FIGURE 1A: VL DOMAIN
`,
`
`I
`
`W 715272
`
`4D5
`
`HU4D5 .
`
`HUVLKI'
`
`50
`4o
`30
`'
`20
`10
`DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGHSPKLLIYSASFRYT
`IIIIII
`IIIII
`III!
`I
`DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLES
`DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES
`—.—-—-—-————¢-.—-
`—--—--—
`. ~——.—.——-
`
`4D5
`
`HU4D5
`
`HUVLKI
`
`100
`’ 90
`80
`7o
`60
`GVPDRFTGNRSGTDFTFTiSSVQAEDLAVYYCQQHYTTPPTFGGGTKLEIKRA
`I
`:
`I
`I
`GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
`GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT
`__..-—.——o--—
`
`13
`
`
`
`r/
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`

`

`0‘
`
`at
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`FIGURE 13: v“ DOMAIN
`'
`
`[N 715279
`N
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`405
`
`HU4D5
`
`HUVgIII
`
`A
`so
`40
`30
`20 ,
`'
`10 A
`EVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIYPTN
`I
`I
`I
`I
`I
`I
`I
`I
`EVQLVESGGGLVQPGGSLRLSCAAsGFNIKDTYIHWVRQAPGKGLEWVARIYPTN
`III
`IIII
`I
`IIII
`III
`III!
`IIIII
`EVQLVESGGGLVQPGGSLRLSCARSGFTFSDYAMSWVRQAPGKGLEWVAVISENG
`-‘--—~--—-
`—-—--—
`~c—--—-———
`,
`4.....-—
`
`Vh-CDRl
`
`I
`
`Vh-CDRZ
`
`'
`
`4D5
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`~HU4D5
`
`60
`
`70
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`80
`
`ABC
`
`90
`
`IOOABC
`
`GYTRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYW
`IIIIIIII
`I
`III
`II
`IIIIIIII
`III
`II
`' GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVW
`III IIIIII
`IIIIII
`
`HUVHIII
`
`SDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVW
`~—.....—..—.—..-_-
`~———-—_‘.—_-——
`
`-
`
`405
`
`110
`.
`P GQGASV’I‘VSS
`I
`I
`I
`I
`
`HU4D5
`
`, GQGTLVTVSS
`
`HUVHIII
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`GQGTLVTVSS
`
`
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`

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`
`FIGURE 2
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`'
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`-
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`07715272
`
`Anneal hu‘VL or huVH oligomers to pAKl template
`
`
`
`1. Ligate
`
`2. Isolate assembled oligomers
`3. Anneal to pAKl template (Xhol “‘, StuI")
`4. ' Extend and 1i gate
`
`> X120] .\
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`1. Transform E. 'coli
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`\
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`I
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`2 Isolate phagemid pool
`3. Enrich for hu\i and huVH(XhoI,* Stul")
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`4. Sequence verify
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`XhoI
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`
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`

`

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`/
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`Percentofcontrolcell
`proliferation
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`[MAb4D5 variant] pg/ml
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` EXPRESS MAlL NO. 859937585
`
`MAILED 14 JUNE 1991
`
`#3415272
`
`DOCKET 709 .
`
`10
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`15
`
` fi/ IMMUNOGLOBULIN VARlANTS
`
`Field of the invention
`
`This invention relates toimethods for the preparation and use of
`_
`variant antibodies and finds application particularly in the fields of
`
`immunology and cancer diagnosis and therapy.
`
`‘20,
`
`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 (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/Q
`J. MOI. Biol. 186:651—663 (1985); Novotny and Haber, Proc. Natl; Acad. Sci.
`
`30
`
`
`
`

`

`(1318245924596 (1985).
`
`The constant domains are not
`
`involved directly in binding the
`
`antibody toan antigen, but are involved in various effector functions, such
`
`' as participation of the antibody in antibody-dependent cellular cytotoxicity.
`The variable domains of each pairof 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.,» Sequences of Proteins
`of Immunological Interest, National Institutes of Health, Bethesda, MD,’
`
`(1987)). The four framework regions largely adopt a B-sheet conformation
`
`and the ,CDRs form loops connecting, and'in some cases forming part Of, the
`
`B-sheet structure. TheCDRs 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
`
`10
`
`15
`
`520'
`
`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. et al., Blood 62:988-995 (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
`
`coupled to a human constant domain (Cabilly et al., U.S. patent No.
`4,816,567; Morrison, 5. L. eta‘L, Proc. Natl. Acad. Sci. USA 81:6851-6855
`(1984); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neube‘rger, M.
`S. etal. , Nature 314:268-270 (1985)). ,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
`
`

`

`the chimeric antibody for participation in antibody-dependent cellular
`
`cytotoxicity (ADCC) and complement-dependent cytotoxicity (see e.g.
`Brflggemann, M. etal., J. Exp. Med. 166:1351—1361 (1987); Riechmann, L.
`et al., Nature 332:323-327 (1988); Love et al.~, Methods in Enzyme/09y
`
`I
`
`1782515527 (1989); Bindon et al., 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
`
`V of eliciting a significant antiglobulin response in humans. For example, in the
`case of
`the murine anti-C03 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 séquencesin human
`antibodies, Winter and colleagues (JonesgP. T. et .51., Nature 321:522-525
`(1986); Riechmann, L. eta/., Nature 332:323-327 (1988))Verhoeyen, M. et
`
`a/., Science 239:1534-1 536 (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. 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 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 a/., Lancet i:1394-1399 (1988)).
`A murine antibody to the interleukin 2 receptor has also recently been
`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 eta/., Proc. Natl. Acad. Sci.
`x,‘\J
`
`10
`
`15
`
`20
`
`30
`
`
`
`

`

`10
`
`15
`
`20
`
`USA 88:2869-2873 (1991); Gorman et al., Proc. Natl. Acad. Sci. USA
`88:4181-41 85 (1991); Daugherty etal., NucleicAcids Research 1 9(9):247 1 —
`2476(1991): Brown et al., Proc. Natl. Acad. Sci. USA 88:2663-2667
`(1991); Junghans et al., Cancer Research 50:1495-1502 (1990).
`
`in some cases, substituting CDRsfrom rodent antibodies for the
`
`human CDRs in human frameworks is sufficient to‘transfer high antigen
`
`binding affinity (Jones, P. T. etal., Nature 321 :522-525 (1986); Verhoeyen,
`M. et 31., science 239:1534-1536 (1988)), whereas in-other cases it has
`been necessary to additionally replace one (Riechmann, L. et al., Nature 9
`
`332:323—327 (1988)) or several (Queen, C. et al., Proc. Natl. Acad. Sci. USA
`86:10029-1003‘3 (1989)) framework region (Emresidues. See also Co et-
`al. , sup/a.
`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,
`9. 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,
`C. & Lesk, A M, J Mol. Biol. 19.6901 -917 (1987); Chothia, C. et al,,
`Nature 342:877-883 (1989); Tramontano, A. 'et al.,
`J. Mol. Biol.
`215.:1754'82 (1990)) as critically affecting the conformation of particular
`
`CDRs and thus their contribution to antigen binding. See also Margolies et
`
`al,, Proc. Natl. Aca‘dfis‘ci. 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 9:545-51 (199-1 ); Spiegelberg' et al, Biochemistry 9:4217-
`4223(1970);Walliceta/.,J Exp Med. 168: 1099-1109(1988): Sox eta/U ‘
`
`Proc. Natl. Acad. Sci. USA 66:975-982l1970); 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'
`
`
`
`

`

`' The three—dimensional structure ofimmunoglobulin chains has been
`
`studied, and crystal structures for intact ir'nmunoglobulins, for avari'ety of
`
`immunoglobulin fragments, and for antibody-antigen complexes have been
`
`published (see 9.9.. Saul et al., Journal of Biological Chemistry” 25:585~97
`(1978); I Sheriff eta/., Proc, Natl. Acad. Sci. USA 84:8075-79 (1987); Segal'
`ei'al, Proc. Natl. Acad. Sci. USA 71:42984302 (1974); Epp. er al.,
`‘Biochemistry 14(22):4943-4952 (1975); Marquart
`et al., J. Mol. Biol.
`141:369-391 (1980); i—‘urey er a/., J. Mol.'Bio/. 167:661-692 (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 er al., Nature 264:415420 (197-6); Bruccoleriet al.,
`Nature 335:564-568 (1988) and Nature 336:266 (meantsherman' er a/.,-
`Journal of Biological Chemistry 263:4064-4074 (1988); Amzel and Pol-jaky
`Ann. Rev. Biochem. 48:961-67 (1979); Silverton 'et al... Proc. Natl. Acad.
`
`Sci. USA 74:5140-5144 (1977); and Gregory et al., Molecular Immunology
`24:821-829 (1987).
`it
`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 A'mzel, 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 achieve 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 proto—oncogene HERZ
`(human epidermal growth factor
`receptor 2) encodes a protein tyrosine kinase (p18‘5HER2) that is relatedto
`andsomewhat homologous to the human epidermal growth factorreceptor
`
`5
`
`11
`
`10
`
`15
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`20
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`30
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`

`\“.
`
`
`
`O y
`
`t
`
`‘
`
`-
`
`0
`
`(see Coussens, L. et a/., Science 230:1132-1139 (1985); Yamamoto, T. et‘
`3]., Nature 319:230-234 (1986): King, C. R. et a/., Science 229:974—976 ’
`(1985)). HERZ 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 HERZ-
`is associated with multiple human malignanciesand appears to be integrally
`involved in progression of 25-30% of human breast and ovarian cancers
`(Slamon, D. J. at a/.,-Science 235:177482 (1987), Slamon, D. J. at 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
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`30
`
`The murine monoclonal antibody known as muMAb4DS (Fendly, B.
`M. et a/., Cancer Res. 50:1550-1558‘ (1990)), directed against
`the
`extracellular domain (ECD) of p185HER2, specifically inhibits the growth of
`tumor cell lines overexpressing p1 85'“.R2 in monolayer culture or'in soft agar
`
`(Hudziak, R; M. etal.‘, Molec. Cell. Biol. 9:1 .1 65-1 1 72 (1989); Lupu‘, a. e: a/.,
`
`Science 249:1552~1555 (1990)). MuMAb4D5 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 '.
`85333-395 (1988)). Thus muMAb4DS has potential for clinical intervention
`in and imaging of carcinomas in which p185HER2 is overexpressed. The
`muMAb4'DS and its uses are described'in copending U.S. patent applications
`
`07/143,912 and 07/147,461. 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.
`'
`g
`i
`_
`It istherefore an object of this invention to provide methods for the
`preparation Of antibodies which are less antigenic in humans than nonhuman
`
`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 hum’anization of antibodies, Le. selecting non‘human amino acid
`
`112
`
`

`

`residues for importation intoe human antibody background sequence in such
`' *a‘fashion as to retain or
`improve the affinity of the'non-human donor
`
`antibbdy for a given antigen.
`
`it
`is another object of
`this invention to provide humanized
`antibodies capable of binding p185HER2.
`.
`
`Other objects, features, and characteristics of the present invention
`will become apparent upon consideration of the following description and the
`appended claims.
`
`mm r
`
`i he Invention
`
`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:
`
`l
`
`a.
`
`b.
`
`c.
`
`d.
`
`3.
`
`f.
`
`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)
`amino acid sequences in the import and the human
`variable domain sequences;
`I
`I
`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 antibodyi
`
`identifying import antibody FR residues in the'eligned FR
`
`sequences that are non—homologous to thecorresponding
`
`consensus antibodyresidues;
`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
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`

`

`2.
`
`3.
`
`,
`
`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.
`.
`t
`
`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
`
`isiexposed but‘has none of thereffects identified in step if), retaining the
`Consensus residue.
`
`Additionally, in certain embodiments the method of this invention I
`
`comprises the feature wherein the corresponding consensus antibody '
`residues identified in step is) 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,'37l-l, 39H,» '
`
`43H, 45H, 49H, 58H, 60H, 68H, 69H, 70H, 73H, 74H, 75H, 76H,’78H,
`
`91H,‘92H, 93H, and 103H (utilizing the numbering system set forth in Kabat,
`
`E. A. er ‘31., 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
`
`.
`
`..._ Mm
`m
`mum—Ivan.» u—w-
`--_
`”N
`My...“a. .,
`consensus variable domain sequences for glycosylation sites, determining if
`M. _.,..~._._.,
`
`the glycosylation is reasonablywexpected to be important for the desired
`“.mmmsx v.4
`antigen bindingand biological activity of theantibody (i.e,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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`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.
`
`i i
`
`,i
`
`Another embodiment of this invention comprises aligning import
`
`antibody and the consensus antibody FR sequences,
`
`identifying» import
`
`. antibody FR residues which are nonohomologous 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 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 3 FR, substituting the
`
`non-hUman CDR for the human CDR in the consensus human antibody
`
`an amino acid residue for the.
`variable domain, and then substituting
`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, 981., or
`
`b.
`
`‘
`
`(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,l
`
`-
`
`93H, and 103H.
`
`'
`
`"
`
`ln preferred embodiments, the nomCDR 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
`'
`I
`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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`. .
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`.
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`~ .
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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, 381., 431., 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, 91H, 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 hasbeen substituted;
`
`’
`
`v This invention also encompasses specific humanized antibody-
`variable domains, and isolated polypeptides having. homology ’with the
`following sequences.
`
`' 1. SEO.1D N0. 1, which is the light chain variabie domain of a
`humanized version of muMAb4DS:
`
`DIOMTOSPSSLSASVGDRV‘TiTCRASODVNTAVAWYOQKPGKAP
`
`KLLIYSASFLESGVPSRFSGSRSGTDFTLTISSLOPEDFATYYCOOHY
`TTPPTFGOGTKVEIKRT
`
`2. SEQ. ID NO. 2, which is the heavy chain variable domain of a '
`
`_
`
`humanized-version of muMAb405):
`_
`. EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYiHWVROAPGKGLE
`
`WVARiYPTNGYTRYADSVKGRFTISADTSKNTAYLOMNSLRAEDT ‘
`
`AVYYCSRWGGDGFYAMDVWGOGTLVTVSS
`.n
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`
`antibody variable domain amino acid sequence for use in the preparation of
`
`ln 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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`sequence data of such a sequence.
`
`In one embodiment, the following
`
`consensus human antibody variable domainamino acid sequences are
`
`provided:
`
`SEQ. ID NO. 3 (light chain):
`
`DIOMTOSPSSLSASVGDRVTITCRAS‘ODVSSYLAWYOOKPGKAPK
`
`LLIYAASSLESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOOYN
`
`SLPYTFGOGTKVEIKRT, and
`
`seem NO. 4 (heavy chain):
`
`EVOLVESGGGLVOPGGSLRLSCAASGFTFSDYAMSWVBOAPGKG
`LEWVAVISENGGYTFIYADSVKGRFTI‘SVADTSKNT‘AYLOMNSLRAE
`iDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS
`‘
`’
`
`Brief Description of the Drawings
`
`FIGURE :1A shows the comparison of the VL domain amino acid
`residues of muMAb4DS, huMAb4DS, and a consensus human sequence (Fig.
`
`~‘I A, SEOJD NO. 5, SEQ. ID NO. 1 and SEQ. ID NO. 3, respectively). 'FIGURE
`18 shows the comparison between the VH domain amino acid residues of the
`muMAb4d5, huMAb4‘DS, and a consensus human sequence (Fig. 18, SEQ.
`
`ID NO. 6, SEQ. ID NO. 2-and SEQ. ID NO. 4, respectively). Both Figs 1A and
`
`18 use the generally accepted numbering scheme from Kabat, E. A., et a/.,
`Sequences of Proteins of Immunological Interest (National Institutes of
`Health, Bethesda, MD (1987)).
`In both Fig. 1A and Fig. 18, the CDR
`residues determined according to a standard sequence-definition (as in Kabat,
`
`E. A., et aI., Sequences of Proteins of Immunological Interest (National .
`i Institutes Of Health, Bethesda, MD, 1987)) ate 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. MOI.
`
`Biol. 196901917 (1987)) are indicated by the second, lower underlines.
`
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`’4
`
`a) The mismatches betweenWshown by the vertical lines.
`
`FlGURE 2 shows a scheme for humanization of muMAb4DS 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 a/., Molec. Cell Biol. 9:1165—1172 (1989)) and data (average of
`
`’
`
`triplicate determinations) are presented as a percentage of results with
`untreated cultures for muMAb4DS (l), huMAb4DS—8 (n) and huMAb405- 1 (I).
`FlGURE 4 shows a stereo view of a—carbon tracing for model of
`huMAb4DS-8 VL and VH. The CDR residues (Kabat, E. A. er a/., Sequences
`' of Proteins of ImmunologIca/ Interest (Nationai institutes of Health, Bethesda,
`
`MD, 1987)) are shown in bold andside chains of VH residues A71, T73,
`
`A78, 893, 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 monoclonai antibody known as muMAb4D§ iFendly, B
`M. et a/., Cancer Res 50:1550 1558 (1990)) is directed against
`the
`extracellular domain (ECD) of p1 SSHERZ. The muMAb4DS and its uses are
`
`described in
`co‘pending U.S_. patent
`applications 07/143,912 and
`, O7/147,461, and in corresponding PCT application WO 89/06692 published
`
`‘ 27.July 1989. This murine antibody was deposited with the ATCC; and
`
`In this description and ciaims, the terms
`designated ATCC ,CRL 10463.
`muMAb4D5, chMAb4DS and huMAb4D5 represent murine,'chimerized 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 PR 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
`
`I
`
`' 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 seq’Uence. ~The humanized antibody
`
`optimally also will comprise at least aportion of an immunoglobulin constant
`region (Fc),
`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, IgA and lgE, and any isotype,
`
`including lgGl, lgGZ,
`
`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,'. Where
`such cytotoxic activity is not desirable, the Constant domain may be of the
`lgG, class. The humanized antibody may comprise sequences. from more a
`
`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 COR regions of‘ the humanized antibody need not
`
`correspond precisely to the parental sequences, 9.9., the import 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 C-DR sequences, more often
`
`'
`
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`
`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
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`
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`

`'
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`O
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`
`_ 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. Computer programs 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 ofthe 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 substantiallyresponsible 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 achieved. ’ Such desired
`characteristics include increases in affinity and greater specificity-forlthe
`target antigen, although it is conceivable that in some Circumstances the
`opposite. effects might be desired.- in general, the CDR residues are directly _
`and most substantially involved in influencing antigen binding (although not '
`all CDR residues aie so involved and therefore need not be substit‘utedinto ‘
`
`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 interface-between the heavy and light chains.
`
`A residue that noncovalently directly binds to antigen is one that,
`by three dimensional analysis,
`is reasonably expected to noncovalently
`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 those humanized antibody
`' residues that are capable of
`forming salt bridges, hydrogen bonds, or
`‘ hydrophobic interactions are likely to be involved in nonCovalent antigen _\.
`binding, however residues which are separated spatially by 32Angstroms)
`
`flexed—W _-
`\ ,
`or less may also non--covalently interact. Such, residues typically are the
`
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`
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`
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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.
`I}
`A residue that int