`

`A TENT NUMBER
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`(ONE SUHCLASS PER BLOCK)
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`
`PFIZER EX. 1502
`Page 2
`
`

`

`

`

`

`

`.... Rlle~X OF CLA:MS
`
`. Allowed
`(Through numeral) Canceled
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`PFIZER EX. 1502
`Page 5
`
`

`

`

`

`

`

`

`

`•
`
`405
`
`H0405
`
`50
`40
`30
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`I
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`405
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`
`100
`90
`80
`70
`60
`GVPDRFTGNRSGTOFTFTISSVQAEOLAVYYCQQHYTTPPTFGGGTKLEIKRA
`l
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`I
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`I
`I
`I I
`I
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`GVPSRFSGSGSGTOFTLTISSLQPEOFATYYCQQYNSLPYTFGQGTKVEIKRT
`
`PFIZER EX. 1502
`Page 9
`
`

`

`• PJ:GIIRB ~( J:I()DJ:B
`
`•
`
`PCT!US 92/05126
`N/14&~&
`
`405
`
`HU405
`
`HUVa:I:I:I
`
`50 A
`40
`30
`20
`10
`EVQLQQSGPELVKPGASLKLSCTASGFN:IKDTY:IHWVKQRPEQGLEW:IGR:IYPTN
`I
`I
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`I l l 1111
`I 1111
`I l l 1111
`I 1111
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENG
`
`405
`
`HU405
`
`HUV8 :III
`
`100ABC
`90
`80 ABC ~
`70
`60
`GYTRYOPKFQOKAT:ITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFYAMDYW
`11111111
`I l l II
`I
`I
`I
`11111111
`I
`I
`I l l II
`I
`GYTRYADSVKGRFT:ISADTSKNTAYLQMNSLRAEOTAVYYCSRWGGDGFYAMDVW
`II
`I
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`I
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`I
`II 111111
`It
`I
`I
`I
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`SOTYYADSVKGRFTISROOSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVW
`
`405
`
`HU405
`
`110
`GQGASVTVSS
`I
`I
`I
`I
`GQGTLVTVSS
`
`HUVa:I:I:I
`
`GQGTLVTVSS
`
`PFIZER EX. 1502
`Page 10
`
`

`

`

`

`

`

`

`

`

`

`-?T!US 92/-0512 6
`. 0~/14~20&,
`
`I'XGURB 6A
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`30
`20
`10
`QVQLQQSGPELVKPGASVKISCKTSGYTFTE
`·*** ·** **·**·*···** ********
`MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCATSGYTFTE
`20
`30
`40
`50
`10
`
`80
`70
`60
`50
`40
`YTMHWMKQSHGKSLEWIGGFNPKNGGSSHNQRFMDKATLAVDKSTSTAYM
`******·*· **·***··*·******·********· *··**********
`YTMHWMRQAPGKGLEWVAGINPKNGGTSHNQRFMDRFTISVDKSTSTAYM
`60
`70
`80
`90
`.
`100
`
`130
`120
`110
`100
`90
`ELRSLTSEOSGIYYCARWRGLNYGFDVRYFOVWGAGTTVTVSSASTKGPS
`•• ** ·**···********************** •• ************
`QMNSLRAEDTAVYYCARWRGLNYGFDVRYFOVWGQGTLVTVSSASTKGPS
`110
`120
`130
`140
`150
`
`180
`170
`160
`150
`140
`VFPLAPSSKSTSGGTAALGCLVKOYFPEPVTVSWNSGALTSGVHTFPAVL
`****** *·*** ·************************************
`VFPLAPCSRSTSESTAALGCLVKOYFPEPVTVSWNSGALTSGVHTFPAVL
`160
`170
`180
`190
`200
`
`230
`220
`210
`200
`190
`QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVOKKVEPKSCOKTH
`************** **··***** ***·********** ** * *
`QSSGLYSLSSVVTVTSSNFGTQTYTCNVOHKPSNTKVDKTVERKCC---v
`210
`220
`230
`240
`
`240
`280
`270
`260
`250
`TCPPCPAPELLGGPSVFLFPPKPKOTLMISRTPEVTCVVVDVSHEOPEVK
`*******
`··*************************************·
`ECPPCPAPP-VAGPSVFLFPPKPKDTLMISRTPEVTCVVVOVSHEOPEVQ
`250
`260
`270
`280
`290
`
`H52H4.-160·
`
`pH52-8.0
`
`330
`320
`310
`300
`290
`FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
`*******·*************·***·********·***************
`FNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQOWLNGKEYKCKVS
`300
`310
`320
`330
`340
`
`PFIZER EX. 1502
`Page 15
`
`

`

`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`380
`370
`360
`350
`340
`NKALP~IEKTISKAKGQPREPQVYTLPPSREEMT~QVSLTCLVKGFY~­
`**·***********·******•****************************
`NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTANQV~LTCLVKGFYP
`350
`360
`370
`390
`38~
`
`430
`420
`410
`400
`390
`SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
`**********************·***************************
`SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRliQO-. .;NVFS
`400
`410
`420
`430
`440
`
`450
`440
`CSVMHEALHNHYTQKSLSLSPGK
`***********************
`CSVMHEALHNHYTQKSLSLSPGK
`450
`460
`
`PFIZER EX. 1502
`Page 16
`
`

`

`.. .
`
`I':IGURB IB
`
`H52L6-158
`
`30
`20
`10
`DVQMTQTTSSLSASLGDRVTINCRASQDINN
`*·****· ******·****** *********
`pH52-9.0 MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQDINN
`20
`30
`40
`50
`10
`
`80
`70
`60
`50
`40
`H52L6-158 YLNWYQQKPNGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLDQE
`*********
`• ***************************·****·*· *
`YLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPE
`60
`70
`80
`90
`100
`
`pH52-9.0
`
`130
`120
`110
`100
`90
`H52L6-158 DIATYFCQQGNTLPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`*·***·*******************************************
`DFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`110
`120
`130
`140
`150
`
`pH52-9.0
`
`180
`170
`160
`150
`140
`H52L6-158 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`**************************************************
`VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`160
`170
`180
`190
`200
`
`pH52-9.0
`
`H52L6-158
`
`pH52-9.0
`
`·210
`200
`190
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`*********************************
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`210
`220
`230
`
`PFIZER EX. 1502
`Page 17
`
`

`

`

`

`'!US 92/0512 6
`
`to the formation of the antigen binding site.
`Widespread use has been made of monoclonal antibodies, particularly those derived
`
`from rodents including mice, however they are frequently antigenic in human clinical use. For
`
`example, a major limitation in the clinical use of rodent monoclonal antibodies is an
`
`5
`
`anti-globulin response during therapy (Miller, R. A. eta/., B/ood62:988-995 (1983); Schroff,
`
`R. W. eta/., 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 eta/., U.S. patent No. 4,816,5-67; Morrison, S. l. eta/., Proc. Nat/. Acad. Sci. USA
`
`10
`
`81:6851-6855 (1984); Boulianne, G. L. eta/., Nature 312:643-646 (1984); Neuberger, M.S.
`
`eta/., Nature 314:268-270 {1985)). The term "chimeric" antibody is used herein to describe
`
`a polypeptide comprising at least the antigen binding portion of an antibody molecule linked
`to at least part of another protein (typically an immunoglobulin constant domain).
`
`The isotype of the human constant domain may be selected to tailor the chimeric
`antibody
`for participation
`in ·antibody-dependent cellular cytotoxicity
`(ADCC) and
`
`15
`
`complement-dependent cytotoxicity (see e.g. Bruggemann, M. et a/., J. Exp. Med.
`
`166:1351-1361 (1987); Riechmann, L. eta/., Nature 332:323-327 (1988); love et al.,
`
`Methods in Enzymology 178:515-527 (1989); Bindon eta/., J. Exp. Med. 168:127-142
`(1988).
`
`20
`
`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 anti-globulin response is directed against the variable region rather than the
`constant region (Jaffers, G. J. eta/., Transplantation 41:572-578 (1986)).
`
`25
`
`In a further effort to resolve the antigen binding functions of antibodies and to minimize
`
`the use ofheterologous sequences in human antibodies, Winter and colleagues (Jones, P. T.
`
`et a/., Nature 321:522-525 (1986); Riechmann, L. et a/., Nature 332:323-327 (1988);
`
`Verhoeven, M. eta/., 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
`
`30
`
`"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.
`
`PFIZER EX. 1502
`Page 19
`
`

`

`•
`
`3
`
`~"IUS 9 2 I 0 5 1 2 6
`
`The therapeutic 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. et al., Nature 332:323-327 (1988}; Hale, G. et al., Lancet
`
`5
`
`i: 1394-1399 ( 1988)). A murine antibody to the interleukin 2 receptor has also recently been
`
`humanized (Queen, C. eta/., Proc. Nat/. Acad. Sci. USA 86:10029-10033 (1989)) as a
`
`potential immunosuppressive reagent. Additional references related to humanization of
`
`antibodies include Co eta/., Proc. Nat/. Acad. Sci. USA 88:2869-2873 (1991 }; Gorman eta/.,
`
`Proc. Nat/. Acad. Sci. USA 88:4181-4185 (1991 ); Daugherty eta/., Nucleic Acids Research
`
`10
`
`19(9}:2471-2476 (1991 ); Brown eta/., Proc. Nat/. Acad. Sci. USA 88:2663-2667 (1991 );
`
`Junghans et a/., 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
`
`15
`
`321:522-525 (1986); Verhoeyen, M. eta/., Science239:1534-1536 (1988)), whereas in other
`cases "it has been necessary to · additionally replace one (Riechmann, L. et al., Nature
`(Queen, C. et a/., Proc. Nat/. Acad. Sci. USA
`332:323-327
`(1988)) or several
`
`86:10029-10033 (1989)) framework region (FR} residu~s. See also Co eta/., supra.
`
`For a given antibody a small number of FR residues are anticipated to be important for
`
`antigen binding. Firstly for example, certain antibodies have been shown to contain a few FR
`
`20
`
`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, Lesk and colleagues (Chothia, C. &
`
`Lesk, A. M., J. Mol. Bioi. 196:901-917 (1987); Chothia, C. et al., Nature 342:877-883
`
`(1989); Tramontano, A. eta/., J. Mol. Bioi. 215:175-H~2 (1990)) as critically affecting the
`
`25
`
`conformation of particular CDRs and thus their contribution to antigen binding. See also
`
`Margolies eta/., Proc. Nat/. 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, Pluckthun, Biotechnology 9:545-51 (1991 ); Spiegelberg eta/., Biochemistry 9:4217-
`
`30
`
`4223 ( 1970); Wallie eta/., J. Exp. Med. 168:1 099-11 09 ( 1988); Sox eta/., Proc. Nat/. A cad.
`
`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 ).
`
`The three-dimensional structure of immunoglobulin chains has been studied, and crystal
`
`PFIZER EX. 1502
`Page 20
`
`

`

`•
`
`'IUS 92/ 0 5 1 2 6
`
`structures for intact immunoglobulins, for a variety of immunoglobulin fragments, and for
`
`antibody-antigen complexes have been published (see e.g., Saul eta/., Journal of Biological
`
`Chemistry 25:585-97 (1978); Sheriff eta/., Proc. Nat/. Acad. Sci. USA 84:8075-79 (1987);
`
`Segal eta/., Proc. Nat/. Acad. Sci. USA 71:4298-4302 (1974); Epp eta/., Biochemistry
`
`5
`
`14(22):4943-4952 (1975); Marquart eta/., J. Mol. Bioi. 141:369-391 {1980); Furey eta/.,
`
`J. Mol. Bioi. 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. Mol. Bioi. 196:901-
`
`917 (1987); Chothia eta/., Nature 342:877-883 (1989); Chothia et al., Science 233:755-58
`
`( 1986); Huber et a/., Nature 264:415-420 ( 1976); Bruccoleri et a/., Nature 335:564-568
`
`10
`
`(1988) and Nature 336:266 (1988); Sherman eta/., JournalofBiologica/Chemistry 263:4064-
`
`4074 (1988); Amzel and Poljak, Ann. Rev. Biochem. 48:961-67 (1979); Silverton et al., Proc.
`
`Nat/. 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 dependent on its three
`
`dimensional structure, and that amino acid substitutions can change the three-dimensional
`
`15
`
`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. eta/., Proc.
`
`Nat/. Acad. Sci. USA 86: 1 0029-1 0033 ( 1989)).
`
`Humanizing an antibody with retention of high affinity for antigen and other desired
`
`20
`
`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 (p185HER2) that is related to and somewhat homologous to the human
`
`25
`
`epidermal growth factor receptor (see Coussens, L. et al., Science 230:1132-1139 (1985);
`
`Yamamoto, T. eta/., Nature 319:230-234 {1986); King, C. R. eta/., 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
`
`30
`
`and ovarian cancers (Siamon, D. J. eta/., Science 235:177-182 (1987), Slamon, D. J. eta/.,
`
`Science 244:707-71 2 ( 1 989)). Furthermore, the extent of amplification is inversely correlated
`
`with the observed median patient survival time (Siamon, supra, Science 1989).
`
`The murine monoclonal antibody known as muMAb405 (Fendly, B. M. et a/., Cancer
`Res. 50:1550-1558 (1990)), directed against the extracellular domain CECD) of p185HER2,
`
`PFIZER EX. 1502
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`•
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`ecT/US 9 2 I 0 5 1 26
`
`5
`specifically inhibits the growth of tumor cell lines overexpressing p185HER2 in monolayer
`culture or in soft agar {Hudziak, R. M. eta/., Malec. Cell. Bioi. 9:1165-11 72 ( 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
`
`5
`
`macrophage-mediated tumor cell cytotoxicity {Hudziak, supra, 1989; Shepard, H. M. and
`
`lewis, G. D. J. Clinical Immunology 8:333-395 (1988)). Thus muMAb405 has potential for
`clinical intervention in and imaging of carcinomas in which p185HER2 is overexpressed. The
`muMAb4D5 and its uses are described in PCT application WO 89/06692 published 27 July
`
`1989. This murine antibody was deposited with the ATCC and designated ATCC CAL 10463.
`
`10
`
`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 efficient humanization
`
`15
`
`of antibodies, i.e. selecting non-human amino acid residues for importation into a human
`
`. antibody background sequence in such a fashion as to retain or improve the affinity of the non(cid:173)
`
`human donor antibody for a given antigen .
`
`. It is another object of this invention to provide humanized antibodies capable of binding
`p 185HER2.
`Other objects, features. and characteristics of the present invention will become
`
`20
`
`apparent upon consideration of the following description and the appended claims.
`
`Summary of the Invention
`
`25
`
`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 variable domain;
`
`30
`
`b.
`
`identifying Complementarity Determining Region (CDR) amino acid sequences
`
`in the import and the human variable domain sequences;
`
`c.
`
`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
`
`d.
`
`PFIZER EX. 1502
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`•
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`,T/US 9 2 I 0 5 1 2 6
`
`antibody and the corresponding FR of the consensus antibody;
`
`5
`
`10
`
`e.
`
`identifying import antibody FR residues in the aligned FR sequences that are
`
`non-homologous to the corresponding consensus antibody residues;
`
`f.
`
`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,
`
`2.
`
`interacts with a CDR; or
`participates in the vl- VH interface; and
`for any non-homologous import antibody amino acid residue which is reasonably
`
`3.
`
`g.
`
`expected to have at least 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
`
`15
`
`(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 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,37H,39H,43H,
`
`20
`
`45H,49H,58H,60H,67H,68H,69H,70H,73H,74H,75H,76H,78H,91H,92H,93H,and
`"'
`1 03H (utilizing the numbering system set forth in Kabat, E. A. eta/., 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 consensus variable domain
`
`25
`
`sequences for glycosylation sites, determining if the glycosylation is reasonably expected to
`
`be important for the desired antigen binding and biological activity of the antibody (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 bears the glycosylation
`
`30
`
`site, it is preferred to substitute that site for the corresponding residues in the consensus
`human if the glycosylation site is reasonably expected to be important. If only the 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
`
`sequence.
`
`PFIZER EX. 1502
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`

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`•
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`~~CT/US 9 2 I 0 5 1 2 6-
`
`7
`Another embodiment of this invention comprises aligning import antibody and the
`
`consensus antibody FR sequences, identifying import antibody FR residues which are non(cid:173)
`
`homologous with the aligned consensus FR sequence, and for each such non-homologous
`
`import antibody FR residue, determining if the corresponding consensus antibody residue
`
`5
`
`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
`
`10
`
`having a CDR and a FR, obtaining the amino acid sequence of at least a portion of a consensus
`
`15
`
`20
`
`antibody variable domain having a CDR and a FR, substituting the non-human CDR for the
`
`human CDR in the consensus antibody variable domain, and then substituting an amino acid
`
`residue for the consensus amino acid residue at at least one of the following sites:.
`(in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L,
`a.
`44L, 58l, 46L, 62L, 63L, 64L, 65L, 66l, 67L, 68L, 69L, 70l, 71 L, 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,67H,68H,69H,70H,73H,74H,75H,76H,
`78H, 91 H, 92H, 93H, and 103H.
`In preferred embodiments, the non-CDR residue substituted at the consensus FR site is the
`
`b.
`
`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 reasonably be expected to have undesirable effects.
`
`25
`
`This invention also relates to a humanized antibody comprising the CDR sequence of
`
`30
`
`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,71l,73l,85L,87L,98L,
`2H,4H,24H,36H,37H,39H,43H,45H,49H,58H,60H,67H,68H,69H,70H,73H,74H,
`75H, 76H, 78H, 91 H, 92H, 93H, and 1 03H 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.
`
`PFIZER EX. 1502
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`

`

`~T/US 9 2 I 0 5 1 2 6
`
`This invention also encompasses specific humanized antibody variable domains, and
`
`•
`
`isolated polypeptides having homology with the following sequences.
`
`1. SEQ. ID NO. 1, which is the light chain variable domain of a humanized version of
`
`muMAb4D5:
`
`5
`
`Dl QMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLESGVP
`
`SRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
`
`2. SEQ. ID NO.2, which is the heavy chain variable domain of a humanized version of
`
`muMAb4D5):
`
`10
`
`EVQLVESGGGL VQPGGSLRLSCAASGFNIKDTYIHWVROAPGKGLEWVARIYPTNGYTR
`
`Y ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDVWGQGTLV
`
`TVSS
`
`In another aspect, this invention provides a consensus antibody variable domain amino
`
`IS
`
`acid sequence for use in the preparation of humanized antibodies, methods for obtaining,
`
`using, and storing a computer representation of such a consensus sequence, and computers
`
`comprising the sequence data of such a sequence.
`
`In one embodiment, the following
`
`consensus antibody variable domain amino acid sequences are provided:
`
`20
`
`SEQ. I'D NO. 3 (light chain):
`
`DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIY AASSLESGVP
`
`SRFSGSGSGTDFTL TISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT, and
`
`SEQ. ID NO. 4 (heavy chain):
`
`25
`
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSDY AMSWVROAPGKGLEWVAVISENGGYT
`
`RYADSVKGRFTISADTSKNTA YLQMNSLRAEDTAVYYCSRWGGDGFY AMDVWGQGTL
`
`VTVSS
`
`30
`
`Brief Description of the Drawings
`
`FIGURE 1A shows the comparison of the VL domain amino acid residues of
`
`muMAb4D5, huMAb4D5, and a consensus sequence (Fig. 1 A, SEQ.ID NO. 5, SEQ. ID NO. 1
`
`and SEQ. ID NO. 3, respectively). FIGURE 1 B shows the comparison between the VH domain
`
`PFIZER EX. 1502
`Page 25
`
`

`

`•
`
`,TIUS 9 2/ 0 5 1 2 6
`
`.
`.
`.
`.
`~"'MAb~
`ammo ac1d res1dues of the FRl:JMAb4dS, huMAb4D5, and a consensus sequence (F1g. 1 B, SEQ.
`
`ID NO. 6, SEQ. 10 NO. 2 and SEQ. 10 NO. 4, respectively). Both Figs 1A and 1 B use the
`
`generally accepted numbering scheme from Kabat, E. A., eta/., Sequences of Proteins of
`
`Immunological Interest (National Institutes of Health, Bethesda, MD (1987)). In both Fig. 1 A
`
`5
`
`and Fig. 1 B, the CDR residues determined according to a standard sequence definition (as in
`
`Kabat, E. A. et a/., 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. Mol. Bioi. 196:901-917 (1987)) are indicated by the second, lower underlines. The
`
`10
`
`mismatches between genes are 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 a/., Malec. Cell. Bioi.
`
`15
`
`20
`
`f
`
`25
`
`9:1165-1172 ( 1989)) and data (average of triplicate determinations) are presented as a
`{o)
`(~
`percentage of r@sults with untreated cultures for muMAb4D5 p.r. huMAb4D5-8 (1'0 and
`A
`(0)
`,{
`
`huMAb4D5-1 j.l-t. "
`
`FIGURE 4 shows a stereo view of a-carbon tracing for a model of huMAb4D5-8 VL and
`
`VH. The CDR residues (Kabat, E. A. eta/., 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, 593, Y102 and VL residues Y55 plus R66 (see Table 3) are shown.
`
`FIGURE 5 shows an amino acid sequence comparison of VL (top panel) and VH (lower
`
`panel) domains of the murine anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby eta/., J.
`I
`Exp. Med. 175, 217-225 (1992) with a humanized variant of this antibody (huxCD3v~). Also
`
`shown anfc_Q~s~n~us_~~_q~ences (most commonly occurring residue or pair of residues) of the
`
`most abundant human subgroups, namely VL K 1 and VH Ill upon which the humanized
`
`sequences are based (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th
`
`edition, National Institutes of Health, Bethesda, MD, USA (1991 )). The light chain sequences-(cid:173)
`muxCD3, huxCD3~and huKI--correspond to SEO.ID.NOs 16, 17, and 18, respectively. The
`.
`I
`~
`heavy chain sequences--muxCD3, huxCD3v)hmd huKI--correspond to SEO.ID.NOs 19,$, and
`21, respectively. Residues which differ between muxCD3 and huxCD3~are identified by an
`asterisk ( *), whereas those which differ between humanized and consensus sequences are
`identified by a sharp sign (#). A bullet <•> denotes that a residue at this position has been
`found to contact antigen in one or more crystallographic structures of antibody/antigen
`
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`

`

`iT/US 9 2 I 0 5 1 2 6
`
`IO
`
`complexes (Kabat eta/., 1991; Mian, I. S. eta/., J. Mol. Bioi. 217,133-151 (1991)). The
`
`•
`
`location of CDR residues according to a sequence definition (Kabat et a/., 1991 ) and a
`
`structural definition (Chothia and Lesk, supra 198 7) are shown by a line and carats (") beneath
`
`the sequences, respectively.
`
`5
`
`FIGURE 6A compares murine and humanized amino acid sequences for the heavy chain
`
`of an anti-CD18 antibody. H52H4-160 (SEQ. 10. NO. 22) is the murine sequence, and pH52-
`
`8.0 (SEQ. ID. NO. 23) is the humanized heavy chain sequence. pH52-8.0 residue 1435 is the
`
`final amino acid in the variable heavy chain domain VH, and residue 144A is the first amino
`acid in the constant heavy chain domain CH 1.
`FIGURE 68 compares murine and humanized amino acid sequences for the light chain
`
`of an anti-CD18 antibody. H52L6-158 (SEQ. ID. NO. 24) is the murine sequence, and pH52-
`
`9.0 (SEQ. ID. NO. 25) is the humanized light chain sequence. pH52-9.0 residue 128T is the
`
`final amino acid in the light chain variable domain VL, and residue 129V is the first amino acid
`
`in the light chain constant domain CL.
`
`.Detailed Description of the Invention
`
`10
`
`15
`
`Definitions
`
`In general, the following words or phrases have the indicated definitions when used in
`
`20
`
`the description, examples, and ·claims:
`
`The murine monoclonal antibody known as muMAb405 (Fendly, B. M. et a/., Cancer
`Res. 50:1550-1558 (1990)) is directed against the extracellular domain (ECO) of p185HER2.
`The muMAb4D5 and its uses are described in PCT application WO 89/06692 published 27 July
`
`1989. This murine antibody was deposited with the ATCC and designated ATCC CRL 10463.
`
`25
`
`In this description and claims, the terms muMAb4D5, chMAb405 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 substantially the amino acid sequence of a human
`
`30
`
`immunoglobulin and a CDR having substantially the amino acid sequence of a non-human
`
`immunoglobulin.
`
`Generally, a humanized antibody has one or more amino acid residues introduced into
`
`it from a source which is non-human. These non-human amino acid residues are referred to
`
`herein as "import" residues, which are typically taken from an "import" antibody domain,
`
`II
`
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`

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`W/US 9 2 I 0 5 12 6
`
`l \
`
`particularly a variable domain. An import residue, sequence, or antibody has a desired affinity
`
`•
`
`and/or specificity, or other desirable antibody biological activity as discussed herein.
`
`In general, the humanized antibody will comprise substantially all of at least one, and
`typically two, variable domains (Fab, Fab', F(ab') 2 , Fa be, Fv) in which all or substantially all
`of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially
`
`5
`
`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
`
`10
`
`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 lgG1, lgG2, lgG3 and lgG4. Usually the
`
`constant domain is a complement fixing consta