•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`immunoglobulin, wherein the sequence of
`
`the acceptor
`
`immunoglobulin heavy chain variable
`
`region framework
`
`consensus sequence of human immunoglobulin heavy chain
`
`variable
`
`region
`
`frameworks.
`
`40. First and second
`
`encoding heavy and light chain variable
`
`a humanized
`
`immunoglobulin having complementarity
`
`regions
`
`(CDRs) from a donor
`
`immunoglobulin and
`
`and light chain
`
`variable region frameworks from human ace ptor immunoglobulin
`
`heavy and light chains, which humanized mmunoglobulin
`
`specifically
`
`within about
`
`an affinity constant
`
`immunoglobulin, wherein said
`
`humanized immunoglob
`
`comprises one or more amino
`
`acids from the donor
`
`heavy chain
`
`framework
`
`outside the Kabat and Chothia
`
`wherein
`
`the donor amino
`
`acids substitute for
`
`in the acceptor
`
`immunoglobulin heavy chain f amework, and each of these said
`
`donor amino acids :
`
`( I ) is adjacent to
`
`the donor immunoglobulin sequence,
`
`or
`
`(II} contains an
`
`within a distance of 6 ANGSTROM of a
`
`CDR in said humanized mmunoglobulin.
`
`- 6 -
`
`Board Assigned Page #973
`
`PFIZER EX. 1095
`Page 1120
`
`

`

`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`41. A humanized immunoglobulin having complementa ity
`
`determining regions (CDRs)
`
`from
`
`a donor
`
`immunoglobuli
`
`heavy and light chain variableregion
`
`frameworks from
`
`acceptor immunoglobulin heavy and light chain framewo
`
`which
`
`humanized immunoglobul in specifically binds to an
`
`with an
`
`affinity constant of at least 107 M- 1 and no
`
`four - fold
`
`that of the donor immunoglobulin,
`
`about
`
`sequence
`
`of the humanized
`
`i mmunoglobul i n
`
`n variable region
`
`framework is at least 65% identical
`
`sequence of the donor
`
`immunoglobulin heavy chain variable
`
`framework and
`
`comprises at least 70
`
`acceptor human
`
`acid sequence .
`
`identical
`
`to an
`
`variable region amino
`
`42. A
`
`h
`
`noglobulin according to claim 41
`
`whi ch is an antibody
`
`two light chain/heavy chain
`
`dimers.
`
`43.
`
`immunoglobulin having complementarity
`
`from
`
`a donor
`
`immunoglobulin and
`
`heavy and l i ght
`
`ariable region
`
`frameworks
`
`from acceptor
`
`immunoglobulin
`
`and
`
`light chain frameworks, which
`
`humanized i mmunoglo ulin spec ifically binds to an antigen with
`
`an affinity
`
`of at
`
`least about 10 8 W 1 and no
`
`- 7 -
`
`Board Assigned Page #97 4
`
`PFIZER EX. 1095
`Page 1121
`
`

`

`

`

`

`

`

`

`•
`
`•
`
`DOCKET NO.: CARP-0057
`
`PATENT
`
`REMARKS
`
`Newly added claims 32-40 have been copied from claims
`
`in Queen et al., U.S. Patent No. 5,693,761. Claims 41-48 have
`been copied from claims in Queen et al., u.s. Patent No.
`
`5,693,762. Copies of both patents are enclosed. Applicants are
`
`i n compliance with 35 USC §135(b } since both Queen patents were
`
`issued on December 2, 1997.
`
`Respectfully submitted,
`
`, ~AM/~~rr~~
`
`Vad.1s J.\. '~~tin
`Registration No. 19,386
`
`WOODCOCK WASHBURN KURTZ
`MACKIEWICZ & NORRIS LLP
`One Liberty Place - 46th Floor
`Philadelphia, PA 19103
`(215} 568-3100
`
`- 11 -
`
`Board Assigned Page #978
`
`PFIZER EX. 1095
`Page 1125
`
`

`

`

`

`

`

`

`

`

`

`

`

`.
`
`DAcJc5d~IMJIPml~llllll~llll
`United States Patent {191
`Adair et al.
`
`1111 Patent Number:
`[45 1 Date of Patent:
`
`5,859,205
`Jan. 12, 1999
`
`[54] HUMANISED ANTIBODIES
`
`(75]
`
`Inventors: .John Robert Adair, High Wy..:omb~:;
`Diljeet Singh Athwal, Lomkm; Jobn
`Spencer Emtage, Mnrlow, all of United
`Kingdom
`
`[73) Assignee: Cclltcch Limited, Berkshire, United
`Kingdom
`
`(21] Appl. No.: 303,569
`
`(22) Filed:
`
`Sep. 7, 1994
`
`Related U.S. Application Dnta
`
`[63) Continuation of Scr. N(}. 743,329, Sep. 17, 1991, abandoned.
`
`[30]
`
`Foreign A11plication Priority Data
`
`(G.BJ United Kingdom ................... 8928874
`
`Dec. 21, 1989
`Int. CJ.6
`(51)
`.• : ..... ....... .................................... A61K 39/395
`[52) U.S. Cl ....................................... 530/387.3; 530/387.1
`[58] Field of Search ........................... 530131\7.1, 387.3,
`53U!38H.22, H67, 1\64
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,348 .• 376
`
`9/1982 Goldenberg .
`
`FOREI GN PATENT DOCUMENTS
`
`0239400A2
`AI 0323306
`0 328 404 i\1
`0 365 2(9 A2
`U -103 !56 Al
`WO 89/07452
`WO 90/07861
`WO 92104381
`wo 92/11018
`WO 92115683
`WO 92!Hi553
`
`3/1987
`711989
`8/1989
`411990
`12/1990
`8/1989
`711990
`3/1992
`7/1992
`9/1992
`10/1992
`
`Enropean Pat. Off ..
`Europ~an Pat. OU . .
`European Pat. Off ..
`European Pat. Off ..
`European .l'at. Off ..
`WJPO .
`Wli:'O .
`WlPO.
`W JPO.
`W lPO.
`W!PO .
`
`OTHER PUBLICATIONS
`Cbotl:tia, Cyrus et al (Dec. 1989) Nnmre., "Conformations of
`Im munoglobulin Hypervariable Regions", vol. 342, pp.
`877-883.
`Queen, C. et al (Dec. 1989) Proceedings of the National
`Academy of Sciences, "A Humanized Antibody That Binds
`!O lnterlcukin 2 Receptor" vol. 86, pp. 10029- 10033.
`Riecbmann et al (Mar. 1988) Nature, "Reshaping Human
`Antibodies for Tilerapy," vol. 332, pp. 323- 327.
`Roberts et al, "Generation of Antibody with Enhanced
`Affinity and Specificity for its Antigen by Protein Engineer(cid:173)
`ing" Nature, 328(20):731- 734, Aug., 1987.
`Verhoeyen i:l al, "Reshaping Human Antibodies: Grafting an
`AntilysO?.yme Activity", Science, 239:1534-36 Mar. 2">,
`1988.
`Jones et a!., " Replacing the <Xlmplementarity-Dctermining
`Regions in a Human Antibody with those from a Mouse",
`Nature, 321:522-525, 1986.
`Ward et al., "Binding activities or a Repertoire of Single
`Immunoglobulin Variable Domains Secreted from Esclieri(cid:173)
`cl!ia Coli", Nature, 341:544-546, 1989.
`Primary Examiner-Donald E. Adams
`Auorney, Agem, or Finn- Woodcock W~shburn Kurtz
`Mackiewicz & Norris
`[57)
`
`ABSTRACT
`
`CDR-grafted antihody heavy and light chains compri.>e
`acceptor framework and donor antigen b inding regions, the
`heavy chains compri'ling donor residues al at. least one of
`positions (6, 23) and/or (24, 48) and/or (49, 71) andtor (73,
`75) and/or (76) and/or (78) and (88) and/or (91). The
`CDR-grafted light chains comprise donor residues at at least
`one of positions (1) and/or (3) and (46) and/or (47) or at at
`least one of positions (46, 48, 58) and (71). Tbe CDR·graftecl
`antibodies are preferably humanised antibodies, having non
`human, e.g, rodent, donor and human acceptor frameworks,
`and may be used for in vivo therapy and d iagnosis. A
`generally applicable protocol is disclosed [or obtaining
`CDR·grafted antibodies.
`
`8 Clai mR, 18 Drawing Sheets
`
`Carter Exhibit 2024
`Carter v. Adair
`Interference No. 105,744
`
`Board Assigned Page #984
`
`PFIZER EX. 1095
`Page 1131
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 1 of 18
`
`5,859,205
`
`1 GAATTCCCAA AG ACAAA~tg g~ttttc~~g tgc~gntttt cngcttcctg
`51 ctnntcngtg cctcngtcnt nntntccngn ggncnnnttg ttctcncccu
`101 gtctccngcn ntcntgtctg cntctccngg ggngnnggtc nccnt gncct
`151 gcngt gccng ctcn~gtgtn ngt t ncntgn ~ctgg tnccn gcngnngtcn
`201 ggcncct ccc cc ~n~~gntg gntttntgnc ~cntccnnnc tggc t tctgg
`251
`~gtccctgct cncttc~ggg gcngtgggtc tgggncctct tnctctctcn
`301 c~ntcngcgg cntgg~ggct gnngntgctg cc~cttnttn ctgccngcng
`351
`·tggngt ngtn ~cc~ttc nc gttcggctcg gggncnnngt t ggnnntnnn
`401
`ccgggctgnt nctg c nccn~ c t gtntcc~t ctt ccc~c cn t c cngtg~gc
`451
`~gtt~ncn tc tggnggtgcc tcngtcgtgt gcttcttgnn c~ncttctnc
`501 cccn~ngncn tc~ntgtcn~ gtggnngntt g~tggcngtg ~~cgncnn nn
`551
`t ggcgtcctg nncngt tggn ctgntcnggn c~gcnnngnc ~gcncc tncn
`601 gcntgngcng cnccct cncg ttgnccnngg ncgngtntgn ~ c gncn t nnc
`65 1 ngctntncct gtg~ggccnc t cncnngncn tcnncttcnc ccnttgt cnn
`701 gngcttcnnc nggnntg~gt gtTAGAGACA AAGGTCCTGA GACGCCACCA
`751 CCAGCTCCCA GCTCCATCCT ATCTTCCCTT CTAAGGTCTT GGAGGCTTCC
`801 CCACAAGCGC t TACCACTGT TGCGGTGCTC TAAACCTCCT CCCACCTCCT
`851 TCTCCTCCTC CTCCCTTTCC TTGGCTTTTA TCATGCTAAT ATTTGCAGAA
`901 AATATTCAAT AAAGTGAGTC TTTGCCTTGA AAAAAAAAAA AAA
`<SEQ ID N0 :4)
`
`FIG. Ja
`
`1 MDFQVQI FSF LL ISASV I IS RGOQIVLTQSP AIMSASPGEK VTMTCSASSS
`51 VSYMNWYQQK SGTSPKRWIY DTSKLASGVP AHFRGSGSGT SYSLTISGME
`101 AEDAATYYCQ QWSSNPFTFG SGTKLEINRA DTAPTVS IFP PSSEQLTSGG
`151 ASVVCFLNNF YPKDINVKWK
`IDGSERQNGV LNSWTDQDSK DSTYSMSS TL
`201
`TL TKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC* <SEQ ID N0: 5)
`
`FIG. 1 b
`
`Board Assigned Page #985
`
`PFIZER EX. 1095
`Page 1132
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 2 of 18
`
`5,859,205
`
`1 GAATTCCCCT CTCCACAGAC ACTGAAAACT CTGACTCAAC ATGGAAAGGC
`51 ACTGGATCTT TCTACTCCTG TTGTCAGTAA CTGCAGGTGT CCACTCCCAG
`101 GTCCAGCTGC AGCAGTCTGG GGCTGAACTG GCAAGACCTG GGGCCTCAGT
`151 GAAGATGTCC TGCAAGGCTT CTGGCTACAC CTTTACTAGG TACACGATGC
`201 ACTGGGTAAA ACAGAGGCCT GGACAGGGTC TGGAATGGAT TGGATACATT
`251 ATTCCTAGCC GTGGTTATAC TAATTACAAT CAGAAGTTCA AGGACAAGGC
`301 CACATTGACT ACAGACAAAT CCTCCAGCAC AGCCTACATG CAACTGAGCA
`351 GCCTGACATC TGAGGACTCT GCAGTCTATT ACTGTGCAAG ATAT TATGAT
`401 GATCATTACT GCCTTGACTA CTGGGGCCAA GGCACCACTC TCACAGTCTC
`451 CTCAGCCAAA ACAACAGCCC CATCGGTCTA TCCACTGGCC CCTGTGTGTG
`501 GAGATACAAC TGGCTCCTCG GTGACTCTAG GATGCCTGGT CAAGGGTTAT
`551 TTCCCTGAGC CAGTGACCTT GACCTGGAAC TCTGGATCCC TGTCCAGTGG
`601 TGTGCACACC TTCCCAGCTG TCCTGCAGTC TGACCTCTAC ACCCTCAGCA
`651 GCTCAGTGAC TGTAACCTCG AGCACCTGGC CCAGCCAGTC CATCACCTGC
`701 AATGTGGCCC ACCCGGCAAG CAGCACCAAG GTGGACAAGA AAATTGAGCC
`801 ACCTCTTGGG TGGACCATCC GTCTTCATCT TCCCTCCAAA GATCAAGGAT
`851 GTACTCATGA TCTCCCTGAG CCCCATAGTC ACATGTGTGG TGGTGGATGT
`901 GAGCGAGGAT GACCCAGATG TCCAGATCAG CTGGTTTGTG AACAACGTGG
`951 AAGTACACAC AGC TCAGACA CAAACCCATA GAGAGGATTA CAACAGTACT
`1001 CTCCGGGTGG TCAGTGCCCT CCCCATCCAG CACCAGGACT GGATGAGTGG
`1051 CAAGGAGTTC AAATGCAAGG TCAACAACAA AGACCTCCCA GCGCCCATCG
`1101 AGAGAACCAT CTCAAAACCC AAAGGGTCAG TAAGAGCTCC ACAGGTATAT
`1151 GTCTTGCCTC CACCAGAAGA AGAGATGACT AAGAAACAGG TCACTCTGAC
`1201 CTGCATGGTC ACAGACTTCA TGCCTGAAGA CATTTACGTG GAGTGGACCA
`1251 ACAACGGGAA AACAGAGCTA AACTACAAGA ACACTGAACC AGTCCTGGAC
`1301 TCTGATGGTT CTTACTTCAT GTACAGCAAG CTGAGAGTGG AAAAGAAGAA
`1351 CTGGGTGGAA AGAAATAGCT ACTCCTGTTC AGTGGTCCAC GAGGGTCTGC
`1401 ACAATCACCA CACGACTAAG AGCTTCTCCC GGACTCCGGG TAAATGAGCT
`1451 CAGCACCCAC AAAACTCTCA GGTCCAAAGA GAGACCCACA CTCATCTCCA
`1501
`TGCTTCCCTT GTATAAATAA AGCACCCAGC AATGCCTGGG ACCATGTAAA
`1551 AAAAAAAAAA AAAGGAATTC
`<SEQ ID N0 :6)
`
`FIG. 2a
`
`Board Assigned Page #986
`
`PFIZER EX. 1095
`Page 1133
`
`

`

`U.S. Patent
`
`J an. 12, 1999
`
`Sheet 3 of 18
`
`5,859,205
`
`DKT 3 HEAVY CHAIN PROTEIN SEQUENCE DEDUCE D FROM DNA SEQUENCE
`
`1 MERH~ I FLLL LSVTAGVHSQ VQLQQSGAEL ARPGASVKMS CKASGYTFTR
`51
`Y TMH~VKQRP GQGLE~ I GYI NPSRGYTNYN QKFKDKATLT TDKSSSTAYM
`101 QLSSLTSEDS AVYYCARYYD DHYCLDY ~GQ GTTLTVSSAK TTAPSVYPLA
`151
`PVCGDTTGSS VTLGCLVKGY FPEPVT L T~N SGSLSSGVHT FPAVLQSDLY
`201 TLSSSVTVTS ST~PSQSITC NVAHPASSTK VDKKIEPRGP TI KPCPPCKC
`251
`PAPNLLGGPS VF IFPPKIK O VLMI SLSP IV TCVVVDVSED DP DVQ I S ~FV
`301 NNVEVHTAQT QTHREDYNST LRVVSALP IQ HQD~MSGKEF KCKVNNKDLP
`351 AP IERTI SKP KGSVRAPQVY VLPPPEEEMT KKQVTLTCMV TDFMPED IYV
`EWTNNGKTEL NYKNTEPVLD SDGSYFMYSK LRVEKKNWVE RNSYSCSVVH
`401
`451 EGLHNHHTTK SFSR TPG K ~ CSEQ ID N0: 7)
`
`FIG. 2b
`
`Board Assigned Page #987
`
`PFIZER EX. 1095
`Page 1134
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 4 of 18
`
`5,859,205
`
`RES TYPE
`Okt3vl
`REI
`
`RES TYPE
`Okt3vl
`REI
`ID N0 :8)
`
`RES TYPE
`Okt3vl
`REivl
`
`42
`
`23
`N N
`N
`N
`NN
`SBspSPESssBSbSsSssPSPSPsPSsse*s*p*P iAI SsSe
`QIVLTQSPAI MSASPGEKVTMTCSASS .SVSYMNWYQQKSGT
`DI QMTQSPSSLSASVGDRVT ITCQASQDIIKYLNWYQQl PGK
`I
`I
`CDRl
`CDRl
`
`<LOOP)
`<KABAT)
`
`*******
`***********
`
`56
`
`85
`
`N NN
`*ls iPp leesesssSBEsePsPSBSSEsPspsPsseesSPePb
`SPKRWIYDTSKLASGVPAtlFRGSGSGTSYSL TISyMEAEDAAT
`APKLLIYEASNLQAGVPSRFSGSGSGTD1T[ TISSLQPEDlAT <SEQ
`
`I
`
`? I
`
`? ?
`******* CDR2 <LOOP/KABAT)
`
`1 08
`1 02
`Pi PIPies**iPIIsPPSPSPSS
`YYCQQWSSN PF TFG~GTK LEI NR <SEQ ID N0:29)
`YYCQQYQSLPYTFGQGTKLQIIR <SEQ ID N0:9)
`?
`?
`CDR3 <LOOP)
`CRD3<KABAT)
`
`******
`*********
`
`FIG. 3
`
`Board Assigned Page #988
`
`PFIZER EX. 1095
`Page 1135
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 5 of 18
`
`5,859,205
`
`RES TYPE
`Okt3h
`KOL
`
`43
`32 35 N39
`23 26
`NN N
`SESPsASBssSAsSSsSpSpSPsPSEbSBssBeP iAPlpiesss
`QVQLQQ~GAELARPGASVKMSCKASGYTF T RY TMNHWVKQRPGQ
`qvQLVESGGGYVQPGBSLRLSCSSSGFlFSSYAMYWVRQAPGK
`"'"'
`"'
`
`CDRl <LOOP)
`******
`***** CDRl <KABAT)
`
`89
`N N N 82nbc
`65
`60
`52n
`RES TYPE IIe ipppAssssssssApsApSSsbSpseSsSseSpApSpsSBssSAePb
`Okt3vh
`GLEWIGYINPSRGY TNTNQKFKRKATLTTDKSSSTAYMQLSSLTSEDSAV
`KOL
`GLEWV~ I IWDDGSDQHYADSVKGRFT I SRD~SKN TL[LQMDSLEPEDT~V
`? ? ? ?
`??
`?
`CDR2
`<LOOP)
`************
`******************* CDR2
`<KABAT)
`
`RES TYPE
`Okt3vh
`KOL
`
`113
`1 07
`92 N
`PiP IE i ssss i i i sssb i b i *E I PI P*spSBSS
`<SEQ ID N0 :30)
`YYCARYYDDHY . .. . .. . CLDYWGQGTTLTVSS
`<SEQ ID NO:l O>
`Y[CARDGGHGFCSSASCFGPDYWGQGTEVTVSS
`***************** CRD4 <KABAT/LOOP)
`
`FIG. 4
`
`Board Assigned Page #989
`
`PFIZER EX. 1095
`Page 1136
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 6 of 18
`
`5,859,205
`
`OKT 3 HEAVY CHA IN CDR GRAF TS
`
`1. gh341 nnd derivntives
`
`Dkt3vh
`gH341
`gH341A
`
`gH34IE
`gH34lliE
`gH341*
`gH341D
`gH34l liE
`gH341 C
`
`gH341*
`gH341liE
`gH341B
`gH341*
`gH341*
`gH341*
`KOL
`
`35 39 43
`26
`1
`QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQ
`QVQLVESGGGVVQOGRSLRLSCSSSGYT FTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`
`QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCKASGY TFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCKASGY TFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`QVQLV~SGGGVVQPGRSLRLSCKASGY TFTRYTMHWVRQAPGK
`
`QVQLVQSGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`QVQLVESGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`QVQLVESGGGVVQPGRSLRLSCSSSGYTFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`QVQLVESGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`QVQLVQSGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`QVQLVESGGGVVQPGRSLRLSCSSSGFIFSSYAMYWVRQAPGK
`
`JA 178
`JA185
`
`JAI98
`JA207
`JA209
`JA197
`JA199
`JA184
`
`JA203
`JA205
`JA 183
`JA2 04
`JA2 06
`JA208
`
`FIG. 5a
`
`Board Assigned Page #990
`
`PFIZER EX. 1095
`Page 1137
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 7 of 18
`
`5,859,205
`
`GLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLR
`GLEW IGYINPSRGYTNYNQKVKDRFT ISTDKSKNTAFLQMDSLR
`GLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNT8FLQMDSLR
`GLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKNT[FLQMDSLR
`GLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTLFLQMDSLR
`GLEWVAYINPSRGYTNYNQKFKDRFTI SRDNSKNTLFLQMDSLR
`
`GLEWIGYINPSRGYTNYNOKVKDRFT ISTDKSKSTAFLQMDSLR
`GLEWIGYINPSRGYTNYNOKVKDRFTISTDKSKSTAFLQMDSLR
`GLEWIGYINPSRGYTNYNOKVKDRFT ISTDKSKSTAFLQMDSLR
`GLEW IGY INPSRGYTNYNOKVKDRFTISTDKSKSTAFLQMDSLR
`GLEW IGY INPSRGYTNYNOKVKDRFTISTDKSKSTAFLQMDSLR
`GLEWIGYINPSRGYTNYNOKVKDRFTISTDKSKNTAFLQMDSLR
`GLEWVA IIWDDGSDQHYADSVKGRFTISRDNSKNTLFLQMDSLR
`
`JA178
`JA 185
`
`JA198
`JA207
`JA209
`JA197
`JA199
`JA184
`
`JA207
`JA205
`JA183
`JA204
`JA206
`JA208
`
`FIG. 5b
`
`Board Assigned Page #991
`
`PFIZER EX. 1095
`Page 1138
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 8 of 18
`
`5,859,205
`
`113
`102
`95
`84
`SEDSAVYYCARYYDDHY .... .. . CLDYWGQGTTLTVSS
`Okt3vh
`PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH341
`gH341A PEDT8VY1CARYYDDHY .... . .. CLDYWGQGTTLTVSS
`
`PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH341E
`PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH341•
`PEDTGVYFCARYYDDHY . ... ... CLDYWGQGTTLTVSS
`gH341D
`gH341 • PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH341 * PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH34 1C PE DTGVYFCARYYDDHY ... .... CLDYWGQGTTLTVSS
`
`SEQ ID NO :
`
`JA178
`JA185
`
`JA 198
`JA207
`JA197
`JA209
`JA199
`JA184
`
`30
`12
`
`13
`14
`15
`16
`17
`18
`
`gH341* PEDTAVYYCARYYDDHY . . . .... CLDYWGQGTTLTVSS
`gH34 1* PE DT8VYXCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH34 18 PEDT8VYXCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH34 1* PE DTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH34 1• PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`gH34 1• PEDTGVYFCARYYDDHY .... ... CLDYWGQGTTLTVSS
`PE DTGVYFCARDGGHGFCSSASCFGPDYWGQGTPVTVSS
`KOL
`
`JA203
`19
`JA2 05 20
`JA183
`21
`JA204
`22
`JA206
`23
`JA208
`24
`1 0
`
`FIG. 5c
`
`Board Assigned Page #992
`
`PFIZER EX. 1095
`Page 1139
`
`

`

`U.S. Patent
`
`J an. 12, 1999
`
`Sheet 9 of 18
`
`5,859,205
`
`OKT3 LIGHT CHAIN CDR GRAFTING
`
`l. gL22l ond der ivat ives
`
`Okt3v l
`gL22l
`gL22lA
`gL22lB
`gL22lC
`REI
`
`Okt3v l
`gL22l
`gL22lA
`gL22lB
`gL22lC
`REI
`
`42
`34
`24
`1
`QIVLTQSPAOMSASPGEKVTMTCSASS .SVSYMNWYQQKSGT
`DIQMTQSPSSLSASVGDRVT ITCSASS.SVSYMNWYQQTPGK
`QIVMTQSPSSLSASVGDRVT ITCSASS.SVSYMNWYQQTPGK
`QIVMTQSPSSLSASVGDRVT ITCSASS.SVSYMNWYQQTPGK
`DIQMTQSPSSLSASVGDRVT ITCSASS.SVSYMNWYQQTPGK
`DIQMTQSPSSLSASVGDRVT ITCQASQDIIKYLNWYQQTPGK
`
`85
`56
`50
`43
`SPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAAT
`APKLLI YDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIAT
`APKRWIYDTSKLASGVPSRFSGSGSGTDYTFT ISSLQPEDIAT
`APKRWIYDTSKLASGVPSRFSGSGSGTDYTFT ISSLQPEDIAT
`APKRWIYDTSKLASGVPSRFSGSGSGTDYTFT ISSLQPEDIAT
`APK0CI YEASNLQAGVPSRFSGSGSGTDYTFT ISSLQPEDIAT <SEQ ID N0 :8)
`
`Okt3vl
`gL22l
`gL22l A
`gL22lB
`gL22lC
`REI
`
`108
`96
`91
`86
`YYCQQWSSNPFTFGSGTKLEINR
`YYCQQWSSNPETFGQGTKLQITR
`YYCQQWSSNPETFGQGTKLQ ITR
`YYCQQWSSNPETFGQGTKLQ ITR
`YYCQQWSSNPETFGQGTKLQ ITR
`YYCQQYQSLPYTFGQGTKLQITR
`
`CDR'S ARE UNDERLINED
`
`<SEQ ID N0:29)
`<SEQ ID N0:25)
`<SEQ ID N0:26 >
`(SEQ I D NO: 27>
`<SEQ ID N0 :28>
`<SEQ ID N0:9)
`
`FRAMEWORK RESIDUES INCLUDED IN THE GENE ARE DOUBLE UNDERLINED
`
`FIG. 6
`
`Board Assigned Page #993
`
`PFIZER EX. 1095
`Page 1140
`
`

`

`

`

`

`

`

`

`

`

`

`

`

`

`

`

`OKT3 COMPETITION
`MURIE REF STD vs. CDR GRAFTED OKT3
`
`0.35
`
`0.30
`
`0 .25
`
`w
`w
`e:::
`~ 0.20
`..........
`
`0
`z
`:::>
`0
`CD
`
`0.15 ~
`
`0.10
`
`0.05
`
`\
`\
`\
`\
`
`+ .
`
`'+
`
`----t:r--- MEDIA CNTRL
`REF IN MEDIA
`-.. ·+· ··· CDR 221 x 178
`#1
`.tr-- CDR 221 X 178.
`#2
`
`*
`
`0.00
`0.001
`
`I
`
`I
`
`0.01
`
`0 . 1
`
`1
`
`10
`
`(COMPETING ANTIBODY) nM
`
`* PROTE IN CONCENTRATIONS
`APPROXIMATE (ELISA)
`
`FIG. 12
`
`Board Assigned Page #1001
`
`0 .
`rJJ •
`~
`.....
`~·
`~ .....
`
`~
`"""
`t-.l
`....
`~
`
`00 =(cid:173)~
`~ .... ... -.l
`'""' ... ~
`
`Q
`
`...
`Ul
`00
`Ul
`~ ...
`N
`0
`Ul
`
`PFIZER EX. 1095
`Page 1148
`
`

`

`OKT3 COMPETITION
`MURIE REF STD vs. CDR GRAFTED OKT3
`
`v' / ~,< \7.
`
`o
`
`P,
`
`vr"-
`
`,~-,',Q,'Q
`
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`
`\ \b\
`
`\0~
`
`0.35
`
`0.30
`
`0 . 25
`
`0 .20
`
`0.15
`
`0.10
`
`0.05
`
`1.&.1
`1.&.1
`0::
`L.r-
`
`'-.
`0 z
`:::>
`0
`Cl
`
`00
`•
`
`~ .
`~ = ....
`~ = ....
`
`-= := ....
`'N -:g
`
`'C
`
`___s;J--- REF ST D
`
`---o--- CDR 221 C x 185
`
`{PROTEIN CONCENTRATION APPROXIMATE)
`
`00
`t:r'
`~
`
`~ -oe
`
`Q
`
`~ ....
`oe
`
`'
`0.00
`t
`0.001
`
`I
`
`I I iiiij
`I
`0.01
`
`i
`
`I
`
`i I '"I
`0.1
`
`I
`
`I
`
`I
`
`I IIIII
`
`I
`
`1
`
`I
`
`I
`
`I
`
`I
`
`I I
`
`I IIIII
`10
`
`(COMPETING ANTIBODY) nM
`
`• PROTE IN CONCENTRATIONS
`APPROXIMATE (ELISA]
`
`FIG. 13
`
`Board Assigned Page #1 002
`
`Ul
`-.
`00
`Ul
`...
`\C
`N
`Q
`Ul
`
`PFIZER EX. 1095
`Page 1149
`
`

`

`5,859,205
`
`1
`HUMANISED ANTIDODIES
`
`This is a continuation of application Ser. No. 07n43,329,
`filed Sep. 17, 1991, now abandoned.
`
`FIELD OF THE INVENTION
`
`The present invention relates to humanised antibody
`molecules, to processes for their production using recombi(cid:173)
`nant DNA technology, and to their therapeutic uses.
`The term "humanised antibody molecule" is used to
`describe a molecule having an antigen binding site derived
`from an immunoglobulin from a non-human species, and
`remaining immunoglobulin-derived parts of the molecule
`being derived from a human immunoglobulin. The antigen
`binding site typically comprises complementarity determin(cid:173)
`ing regions (CDRS) which determine the binding specificity
`of the antibody molecule and which are carried on appro(cid:173)
`priate framework regions in the variable domains. There are
`3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and
`light chain variable domains.
`In the description, reference is made to a number of
`publications by number. The publications are listed in
`numerical order at the end of the description.
`
`BACKGROUND OF THE INVENTION
`
`Natural immunoglobulins have been known for many
`years, as have the various fragments thereof, such as the Fab,
`(Fab')2 and Fe fragments, which can be derived by enzy(cid:173)
`matic cleavage. Natural immunoglobulins comprise a gen(cid:173)
`erally Y-shaped molecule having an antigen-binding site
`towards the end of each upper arm. The remainder of the
`structure, and particularly the stem of the Y, mediates the
`effector functions associated with immunoglobulins.
`Natural immunoglobulins have been used in assay, diag(cid:173)
`nosis and, to a more limited extent, therapy. However, such
`uses, especially in therapy, were hindered until recently by
`the polyclonal nature of natural immunoglobulins. A signifi(cid:173)
`cant step towards the realisation of the potential of immu(cid:173)
`noglobulins as therapeutic agents was the discovery of
`procedures for the production of monoclonal antibodies
`(MAbs) of defined specificity (1).
`However, most MAbs are produced by hybridomas which
`are fusions of rodent spleen cells with rodent myeloma cells.
`They are therefore essentially rodent proteins. There are
`very few reports of the production of human MAbs.
`Since most available MAbs are of rodent origin, they are
`naturally antigenic in humans and thus can give rise to an
`undesirable immune response termed the HAMA (Human
`Anti-Mouse Antibody) response. Therefore, the use of
`rodent MAbs as therapeutic agents in humans is inherently
`limited by the fact that the human subject will mount an
`immunological response to the MAb and will either remove
`it entirely or at least reduce its elfectiveness. In practice,
`MAbs of rodent origin may not be used in patients for more
`than one or a few treatments as a HAMA response soon
`develops rendering the MAb ineffective as well as giving
`rise to undesirable reactions. For instance, OKD a mouse
`IgG2a!k MAb which recognises an antigen in the T-cell
`receptor-CD3 complex has been approved for use in many
`countries throughout the world as an iromunosuppres.sant in
`the treatment of acute allograft rejection [ Chatenoud et al (2)
`and Jeffers et at (3)]. However, in view of the rodent nature
`of this and other such MAbs, a significant HAMA response
`which may include a major anti-idiotype component, may
`build up on use. Clearly, it would be highly desirable to
`
`2
`diminish or abolish this undesirable HAMA response and
`thus enlarge the areas of use of these very useful antibodies.
`Proposals have therefore been made to render non-human
`MAbs Jess antigenic in humans. Such techniques can be
`5 generically termed "humanisation" techniques. These tech(cid:173)
`niques typically involve the use of recombinant DNA tech(cid:173)
`nology to manipulate DNA sequences encoding the polypep(cid:173)
`tide chains of the antibody molecule.
`Early methods for humanising MAbs involved production
`10 of chimeric antibodies in which an antigen binding site
`comprising the complete variable domains of one antibody
`is linked to constant domains derived from another antibody.
`Methods for carrying out such chimerisation procedures are
`described in EP0120694 (Celltech Limited), EP0125023
`(Genentech Inc. and City of Hope), EP-A-0 171496 (Res.
`15 Dev. Corp. Japan), EP-A-0 173 494 (Stanford University),
`and WO 86/01533 (Celltech Limited). This latter Celltech
`application (WO 86/01533) discloses a process for preparing
`an antibody molecule having the variable domains from a
`mouse MAb and the constant domains from a human
`20 immunoglobulin. Such humanised chimeric antibodies,
`however, still contain a significant proportion of non-human
`amino acid sequence, i.e. the complete non-human variable
`domains, and thus may still elicit some HAMA response,
`particularly if administered over a prolonged period [Begent
`25 et al (ref. 4)).
`In an alternative approach, described in EP-A-0239400
`(Winter), the complementarity determining regions (CDRs)
`of a mouse MAb have been grafted onto the framework
`regions of the variable domains of a human immunoglobulin
`30 by site directed mutagenesis using long oligonucleotides.
`The present invention rela tes to humanlsed antibody mol(cid:173)
`ecules prepared according to this alternative approach, i.e.
`CDR-grafted humanised antibody molecules. Such CDR(cid:173)
`grafted humanised antibodies are much less likely to give
`35 rise to a HAMA response than humanised chimeric antibod(cid:173)
`ies in view of the much lower proportion of non-human
`amino acid sequence which they contain. The earliest work
`on humanising MAbs by CDR-grafting was carried out on
`MAbs recognising synthetic antigens, such as the NP or NIP
`40 antigens. However, examples in which a mouse MAb rec(cid:173)
`ognising lysozyme and a rat MAb recognising an antigen on
`human T-cells were humanised by CDR-grafting have been
`described by Verhoeyen et a! (5) and Riechmaon et al (6)
`respectively. The preparation of CDR-grafted antibody to
`45 the antigen on human T cells is also described in WO
`89/07452 (Medical Research Council).
`In Riechmann et al/Medical Research Council it was
`found that transfer of the CDR regions alone [as defined by
`Kabat refs. (7) and (8)] was not sufficient to provide sa tis-
`50 factory antigen binding activity in the CDR-grafted product.
`Riechmann et at found that it was necessary to convert a
`serine residue at position 27 of the human sequence to the
`corresponding rat phenylalanine residue to obtain a CDR(cid:173)
`grafted product having improved antigen binding activity.
`55 This residue at position 27 of the heavy chain is within the
`structural loop adjacent to CDIU . A further construct which
`additionally contained a human serine to rat tyrosine change
`at position 30 of the heavy chain did not have a significantly
`altered binding activity over the humanised antibody with
`60 the serine to phenylalanine change at position 27 alone.
`These results indicate that changes to residues of the human
`sequence outside the CDR regions, in particular in the
`structural loop adjacent to CDRl, may be necessary to
`obtain effective an tigen binding activity for CDR-graCted
`65 antibodies which recognise more complete antigens. Even
`so the binding affinity of the best CDR-grafted antibodies
`obtained was still significantly less than the original MAb.
`
`Board Assigned Page #1003
`
`PFIZER EX. 1095
`Page 1150
`
`

`

`5,859,205
`
`3
`Very recently Queen et al (9) have described the prepa(cid:173)
`ration of a humanised antibody that binds to the interleukin
`2 receptor, by combining the CDRs of a murine MAb
`(anti-Tac) with human immunoglobulin framework and con(cid:173)
`stant regions. The hu man framework regioos were chosen to 5
`maximise homology with the aoti-Tac MAb sequence. In
`addition computer modelling was used to identify frame(cid:173)
`work ami no acid re,.;idues which were likely to interact with
`the C DRs or antigen, and mouse amino acids were used at
`these positions in the humanised antibody.
`In WO 90!07861 Queen et al propose four criteria for
`designing humanised immunoglobulins. The fust criterion is
`to use as the human acceptor the framework from a particu-
`lar human immunoglobulin that is unusually homologous to
`th.: non-human donor immunoglobulin to be humaoised, or 15
`to use a consensus framework from many human antibodies.
`'l11e second criterion is to use the donor amino acid rather
`than the acceptor if the human acceptor residue is unusual
`and the donor residue is typical for human sequences at a
`specific residue of the framework. The third criterion is to 20
`usc the donor framework amino acid residue rather than the
`acceptor at positions immediately adjacent to the CDRs. The
`fourth criterion is to use the donor amino acid residue at
`framework positioos at which the amino acid is predicted to
`bave a side cbain atom within about 3 A of the CDRs in a 25
`three-dimensional immunoglobulin model and to be capable
`o( interacting with the antigen or with the CDRs of the
`humanised immunoglo bulin. It is proposed that criteria two,
`thn:t: or four may be applied in addition or alteroatively to
`criterion one, and may be applied singly or in any combi- 30
`nation.
`WO 90!07861 describes in detail the preparation of a
`single CDR-grafted humanised antibody, a humanised anti(cid:173)
`body having specificity for the p55 Tac protein of the IL-2
`receptor. The combination of all four criteria, as above, were
`employed in designing this humanised antibody, the variable
`n:gion frameworks of the human antibody Eu (7) being used
`as acceptor. In the resultant humanised antibody the donor
`CDRs were as defined by Kabat et al (7 and 8) and in
`addition the mouse donor residues were used in place of tbe 40
`human acceptor residues, at positions 27, 30, 48, 66, 67, 89,
`91, 94, 103, 104, 105 and 107 in the heavy chain S EQ ID
`N0:31 and at positions 48, 60 and 63 in the light chain, of
`the variable region frameworks. The bumanised anti-Tac
`antibody obtained is reported to have an affinity for p55 of 45
`3xl0~M-J, about one-third of that of the murine MAb.
`We have further investigated the preparation of CDR(cid:173)
`grafted humaniscd an tibody molecules and have identified a
`hierarchy of positions within the framework of the variable
`regions (i.e. outside both the Kabat CDRs and structural
`loops of the variable regions) at which the amino acid
`identities of the residues arc important for obtaining CDR(cid:173)
`grafted products with satisfactory binding affinity. This bas
`enabled us to establish a protocol for obtaining satisfactory
`CDR-grafted products which may be applied very widely
`irrespective of the level of homology between the donor
`immunoglobulin and acceptor framework. The set of resi(cid:173)
`dues which we have identified as being of critical impor(cid:173)
`tance does not: coincide with the residues identified by
`Queen et a! (9).
`
`35
`
`4
`residues at at least one of positions 6, 23 and/or 24, 48 and/or
`49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.
`In preferred embodiments, the heavy chain framework
`comprises donor residues at positions 23, 24, 49, 71,73 and
`78 or at positions 23,24 and 49.1be residues at positions 71,
`73 and 78 of the heavy chain framework are preferably
`either all acceptor or all donor residues.
`In particularly preferred embodiments the heavy chain
`fram.:work additionally comprises donor residues at one,
`t O some or all of positions 6, 37, 48 and 94. Alw it is
`particularly preferred that residues at positions of the heavy
`chain framework which arc commonly conserved across
`species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106
`and 107, if not conserved between donor and acceptor,
`additionally comprise donor residues. Most preferably the
`heavy chain framework additionally comprises donor resi-
`dues at positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103,
`104, 106 and 107.
`In addition the heavy chain framework optionally com(cid:173)
`prises donor residues at one, some or all of positions:
`1 and 3,
`72 and 76,
`69 (if 48 is different between donor and acceptor),
`38 and 46 (if 48 is the donor residue),
`80 and 20 (if 69 is the donor residue),
`67,
`82 and 18 (if 67 is the donor residue),
`91,
`88, and
`any one or more of 9, 11, 41, 87, 108, 110 and 112.
`In the first and other aspects of the present invention
`reference is made to CDR-grafted antibody products com(cid:173)
`prising acceptor framework and donor antigen binding
`regions. It will be: appreciated that the invention is widely
`applicable: to tbe C DR-grafting of antibodies in general.
`Thus, the donor and acceptor antibodies may be derived
`from animals of the same species and even same antibody
`class or sub-e t as.~. More usually, however, the donor and
`acceptor antibodies arc derived from animals of different
`species. Typically the donor antibody is a non-human
`antibody, such as a rodent Mi\b, and the acceptor antibody
`is a human antibody.
`In the first and other aspects of the present invention, the
`donor antigen binding region typically comprises at least
`one CDR from the donor antibody. Usually the donor
`antigen binding region comprises at least two and preferably
`all thrt:t: CDRs of each of the heavy chain and/or light chain
`50 varia bit: regions. l11e CDRs may comprise the Kaba t CDRs,
`the structural loop CDRs or a composite of the Kabat and
`structural loop CORs and any combination of any of these.
`Preferably, the antigen binding regions of the CDR-grafted
`hearty chain variable domain comprise CDRs corresponding
`ss to the Kabat COils at COR2 (residues 50-65) and CDR3
`(residues 95-100) and a composite of the Kabat and struc(cid:173)
`tural loop CDRs at CDRJ (residues 26-35).
`T he residue designations given above and elsewhere in
`the present application are numbered according to the Kaba t
`60 numbering [refs. (7) and (8)]. Thus the residue designations
`do not always correspond directly with the linear numbering
`of the amino acid residues. The aCtual linear amino acid
`sequence may contain fewer or additional amino acids than
`in the strict Kabat numbering correspon