United States Patent [19]
`Adair et al.
`
`[54] HUMANISED ANTIBODIES
`
`[75] Inventors: John Robert Adair, High Wycombe;
`Diljeet Singh AthWal, London; John
`Spencer Emtage, Marlow, all of United
`Kingdom
`
`[73] Assignee: Celltech Limited, Berkshire, United
`Kingdom
`
`[21] Appl. No.: 303,569
`[22]
`Filed:
`Sep. 7, 1994
`
`Related US. Application Data
`
`[63] Continuation of Ser. No. 743,329, Sep. 17, 1991, abandoned.
`[30]
`Foreign Application Priority Data
`
`Dec. 21, 1989 [GB]
`
`United Kingdom ................. .. 8928874
`
`[51] Int. Cl.6 ................................................. .. A61K 39/395
`[52] US. Cl. .................................... .. 530/387.3; 530/387.1
`[58] Field of Search ............................ .. 530/387.1, 387.3,
`530/38822, 867, 864
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,348,376
`
`9/1982 Goldenberg.
`
`FOREIGN PATENT DOCUMENTS
`
`0239400 A2 3/1987 European Pat. Off. .
`A1 0323806 7/1989 European Pat. Off. .
`0 328 404 A1 8/1989 European Pat. Off. .
`0 365 209 A2 4/1990 European Pat. Off. .
`0 403 156 A1 12/1990 European Pat. Off. .
`WO 89/07452 8/1989 WIPO .
`WO 90/07861
`7/1990 WIPO .
`WO 92/04381
`3/1992 WIPO .
`WO 92/11018 7/1992 WIPO .
`WO 92/15683
`9/1992 WIPO .
`WO 92/16553 10/1992 WIPO .
`
`US005859205A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,859,205
`Jan. 12, 1999
`
`OTHER PUBLICATIONS
`Chothia, Cyrus et al (Dec. 1989) Nature, “Conformations of
`Immunoglobulin 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
`to Interleukin 2 Receptor” vol. 86, pp. 10029—10033.
`Riechmann et al (Mar. 1988) Nature, “Reshaping Human
`Antibodies for Therapy,” vol. 332, pp. 323—327.
`Roberts et al, “Generation of Antibody with Enhanced
`Af?nity and Speci?city for its Antigen by Protein Engineer
`ing” Nature, 328(20):731—734, Aug., 1987.
`Verhoeyen et al, “Reshaping Human Antibodies: Grafting an
`AntilysoZyme Activity”, Science, 239:1534—36 Mar. 25,
`1988.
`Jones et al., “Replacing the complementarity—Determining
`Regions in a Human Antibody with those from a Mouse”,
`Nature, 321:522—525, 1986.
`Ward et al., “Binding activities of a Repertoire of Single
`Immunoglobulin Variable Domains Secreted from Escheri
`chia Cali”, Nature, 341:544—546, 1989.
`Primary Examiner—Donald E. Adams
`Attorney, Agent, or Firm—Woodcock Washburn KurtZ
`MackiewicZ & Norris
`[57]
`ABSTRACT
`CDR-grafted antibody heavy and light chains comprise
`acceptor framework and donor antigen binding regions, the
`heavy chains comprising donor 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). 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). The CDR-grafted
`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 diagnosis. A
`generally applicable protocol is disclosed for obtaining
`CDR-grafted antibodies.
`
`8 Claims, 18 Drawing Sheets
`
`BIOEPIS EX. 1189
`Page 1
`
`

`

`US. Patent
`
`Jan. 12, 1999
`
`Sheet1,0f18
`
`5,859,205
`
`GAATTCCCAA AGACAAAotg gottttcoog tgcagotttt cagcftcctg
`ttctcaccco
`ctaatcagtg cctcagtcat ootatccaga ggocaoottg
`ggaguoggtc
`catctccogg
`gtctccagca
`atcatgtctg
`actggtacca
`ctcoogtgtu
`ogttocatgu
`gcogtgccug
`acatccuuac
`ccuaoagotg
`gatttutgac
`ggcocctccc
`cocttcoggg
`QCOQtQQQtC
`ogtccctgct
`cuatcogcgg
`catgguggct
`guogotgctg
`uccattcuc
`-tggogtagt0
`gttcggctcg
`ccgggctgot
`ctgtotccot
`tcagtcgtgt
`ugttoocotc
`gtggoogott
`cccaaogoca
`ctgotcoggo
`tggcgtcctg
`ttguccoogg
`gcotgogcog
`tcacuogucu
`agctatacct
`gtTAGAGACA
`gagcttcaac
`CCAGCTCCCA
`ATCTTCCCTT
`
`1
`
`51
`
`101
`
`151
`
`801
`
`851
`
`301
`
`351
`
`401
`
`451
`
`501
`
`551
`
`601
`
`651
`
`701
`
`751
`
`801
`
`851
`
`901
`
`octgcoccoa
`tggoggtgcc
`tcaotgtcuo
`oacogttggo
`cuccctcocg
`gtgaggccac
`oggoatgagt
`GCTCCATCCT
`
`occutgocct
`gcogaagtco
`tggcttctgg
`toctctctca
`
`ctgccagcog
`tggaautaaa
`tccogtgogc
`caocttctoc
`
`aocgacoaoo
`agcacctaca
`ucgacataac
`ccattgtcoa
`GACGCCACCA
`
`tgggocctct
`ccocttutto
`
`gggacoaugt
`cttcccncco
`
`gcttcttgoo
`Qatggcogtg
`cogcoaagac
`acgugtotge
`tcuucttcac
`
`AAGGTCCTGA
`
`CTAAGGTCTT
`
`GGAGGCTTCC
`
`CCACAAGCGC
`
`tTACCACTGT
`
`TGCGGTGCTC
`
`TAAACCTCCT
`
`CCCACCTCCT
`
`TCTCCTCCTC
`
`CTCCCTTTCC
`
`TTGGCTTTTA
`
`TCATGCTAAT
`
`ATTTGCAGAA
`
`AATATTCAAT
`
`AAAGTGAGTC
`
`TTTGCCTTGA
`
`AAAAAAAAAA
`
`AAA
`
`(SEO
`
`ID ND:4)
`
`FIG. Ia
`
`MDFQVQIFSF
`
`LLISASVIIS RGDQIVLTQSP AIMSASPGEK
`
`VTMTCSASSS
`
`51
`
`VSYMNWYQQK
`
`SGTSPKRWIY DTSKLASGVP AHFRGSGSGT
`
`SYSLTISGME
`
`101
`
`151
`
`801
`
`AEDAATYYCQ
`
`QWSSNPFTFG SGTKLEINRA DTAPTVSIFP
`
`PSSEQLTSGG
`
`ASVVCFLNNF
`
`YPKDINVKWK IDGSERQNGV LNSWTDQDSK
`
`DSTYSMSSTL
`
`TLTKDEYERH
`
`NSYTCEATHK TSTSPIVKSF NRNECx
`
`(SEQ ID ND 5)
`
`FIG. 1b
`
`BIOEPIS EX. 1189
`
`PageZ
`
`BIOEPIS EX. 1189
`Page 2
`
`

`

`U.S. Patent
`
`Jan. 12, 1999
`
`Sheet 2 0f 18
`
`5,859,205
`
`GAATTCCCCT
`ACTGGATCTT
`GTCCAGCTGC
`GAAGATGTCC
`ACTGGGTAAA
`ATTCCTAGCC
`CACATTGACT
`GCCTGACATC
`GATCATTACT
`[ITCAGCCAAA
`GAGATACAAC
`TTCCCTGAGC
`TGTGCACACC
`GCTCAGTGAC
`AATGTGGCCC
`ACCTCTTGGG
`GTACTCATGA
`GAGCGAGGAT
`AAGTACACAC
`CTCCGGGTGG
`CAAGGAGTTC
`AGAGAACCAT
`GTCTTGCCTC
`CTGCATGGTC
`ACAACGGGAA
`TCTGATGGTT
`CTGGGTGGAA
`ACAATCACCA
`CAGCACCCAC
`TGCTTCCCTT
`AAAAAAAAAA
`
`[ITCCACAGAC
`TCTACTCCTG
`AGCAGTCTGG
`TGCAAGGCTT
`ACAGAGGCCT
`GTGGTTATAC
`ACAGACAAAT
`TGAGGACTCT
`SCCTTGACTA
`ACAACAGCCC
`TGGCTCCTCG
`CAGTGACCTT
`TTCCCAGCTG
`TGTAACCTCG
`ACCCGGCAAG
`TGGACCATCC
`TCTCCCTGAG
`GACCCAGATG
`AGCTCAGACA
`TCAGTGCCCT
`AAATGCAAGG
`[ITCAAAACCC
`CACCAGAAGA
`ACAGACTTCA
`AACAGAGCTA
`CTTACTTCAT
`AGAAATAGCT
`CACGACTAAG
`AAAACTCTCA
`GTATAAATAA
`AAAGGAATTC
`
`ACTGAAAACT
`TTGTCAGTAA
`[SGCTGAACTG
`[ITGGCTACAC
`GGACAGGGTC
`TAATTACAAT
`CCTCCAGCAC
`GCAGTCTATT
`CTGGGGCCAA
`CATCGGTCTA
`GTGACTCTAG
`GACCTGGAAC
`TCCTGCAGTC
`AGCACCTGGC
`CAGCACCAAG
`GTCTTCATCT
`CCCCATAGTC
`TCCAGATCAG
`CAAACCCATA
`CCCCATCCAG
`TCAACAACAA
`AAAGGGTCAG
`AGAGATGACT
`TGCCTGAAGA
`AACTACAAGA
`GTACAGCAAG
`ACTCCTGTTC
`AGCTTCTCCC
`GGTCCAAAGA
`AGCACCCAGC
`
`CTGACTCAAC
`CTGCAGGTGT
`GCAAGACCTG
`[ITTTACTAGG
`TGGAATGGAT
`CAGAAGTTCA
`AGCCTACATG
`ACTGTGCAAG
`GGCACCACTC
`TCCACTGGCC
`GATGCCTGGT
`TCTGGATCCC
`TGACCTCTAC
`CCAGCCAGTC
`GTGGACAAGA
`TCCCTCCAAA
`ACATGTGTGG
`CTGGTTTGTG
`GAGAGGATTA
`CACCAGGACT
`AGACCTCCCA
`TAAGAGCTCC
`AAGAAACAGG
`CATTTACGTG
`ACACTGAACC
`CTGAGAGTGG
`AGTGGTCCAC
`GGACTCCGGG
`GAGACCCACA
`AATGCCTGGG
`
`ATGGAAAGGC
`CCACTCCCAG
`GGGCCTCAGT
`TACACGATGC
`TGGATACATT
`AGGACAAGGC
`CAACTGAGCA
`ATATTATGAT
`TCACAGTCTC
`CCTGTGTGTG
`CAAGGGTTAT
`TGTCCAGTGG
`ACCCTCAGCA
`CATCACCTGC
`AAATTGAGCC
`GATCAAGGAT
`TGGTGGATGT
`AACAACGTGG
`CAACAGTACT
`[SGATGAGTGG
`GCGCCCATCG
`ACAGGTATAT
`TCACTCTGAC
`GAGTGGACCA
`AGTCCTGGAC
`AAAAGAAGAA
`GAGGGTCTGC
`TAAATGAGCT
`CTCATCTCCA
`ACCATGTAAA
`
`101
`151
`201
`251
`301
`351
`401
`451
`501
`551
`601
`651
`701
`801
`851
`901
`951
`1 001
`1 051
`1 1 01
`1 151
`1201
`1251
`1301
`1351
`1401
`1451
`1501
`1551
`
`FIG.
`
`2a
`
`BIOEPIS EX. 1189
`Page 3
`
`

`

`US. Patent
`
`Jan. 12, 1999
`
`Sheet3 0f18
`
`5,859,205
`
`DKT 3 HEAVY CHAIN PRDTEIN SEQUENCE DEDUCED ERDM DNA SEQUENCE
`
`1
`
`51
`
`101
`
`151
`
`801
`
`851
`
`301
`
`351
`401
`
`451
`
`MERHWIFLLL
`
`LSVTAGVHSQ
`
`VQLOQSGAEL
`
`ARPGASVKMS
`
`CKASGYTFTR
`
`YTMHWVKQRP
`
`GQGLEWIGYI
`
`NPSRGYTNYN
`
`QKFKDKATLT
`
`TDKSSSTAYM
`
`QLSSLTSEDS
`
`AVYYCARYYD
`
`DHYCLDYWGQ
`
`GTTLTVSSAK
`
`TTAPSVYPLA
`
`PVCGDTTGSS
`
`VTLGCLVKGY
`
`FPEPVTLTWN
`
`SGSLSSGVHT
`
`FPAVLQSDLY
`
`TLSSSVTVTS
`
`STWPSQSITC
`
`NVAHPASSTK
`
`VDKKIEPRGP
`
`TIKPCPPCKC
`
`PAPNLLGGPS
`
`VFIFPPKIKD
`
`VLMISLSPIV
`
`TCVVVDVSED
`
`DPDVQISWFV
`
`NNVEVHTAGT
`
`QTHREDYNST
`
`LRVVSALPIQ
`
`HQDWMSGKEF
`
`KCKVNNKDLP
`
`APIERTISKP
`
`KGSVRAPOVY
`
`VLPPPEEEMT
`
`KKGVTLTCMV
`
`TDFMPEDIYV
`
`EWTNNGKTEL
`
`NYKNTEPVLD
`
`SDGSYFMYSK
`
`LRVEKKNWVE
`
`RNSYSCSVVH
`
`EGLHNHHTTK
`
`SESRTPGKX (SEQ ID ND=7)
`
`FIG. 2b
`
`BIOEPIS EX. 1189
`
`Page4
`
`BIOEPIS EX. 1189
`Page 4
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 4 0f 18
`
`5,859,205
`
`1
`
`NN
`
`N
`
`83
`
`N
`
`48
`
`N
`
`N
`
`SBspSPESSSBSbSSSSSPSPSPSPSSse%s%p*PiAISSSe
`QIVLTQSPAIMSASPGEKVTMTCSASS.SVSYMNWYQOKSGT
`DIQMTQSPSSLSASVGDRVTITCQASQDIIKYLNWYQQIPGK
`7
`
`7 C
`
`DRl
`
`(LDDP)
`
`xxxx**x
`
`CDRl
`
`(KABAT)
`
`xxxxxxxxx%*
`
`N
`
`NN
`
`56
`
`85
`
`*IsiPpIeesesssSBEsePsPSBSSESPspsPsseeSSPePb
`SPKRWIYDTSKLASGVPAHFBGSGSGTSYSLTISQMEAEDAAT
`APKLLIYEASNLQAGVPSRFSGSGSGTDYTETISSLQPEDLAT (SEQ
`
`?
`
`??
`
`?
`
`?
`
`XXXXXX¥
`
`CURB (LDUP/KABAT)
`
`RES TYPE
`Dkt3vl
`REI
`
`RES TYPE
`Dkt3vl
`PEI
`ID ND:8)
`
`RES TYPE
`
`PiPIPies%%iPIISPPSPSPSS
`
`108
`
`108
`
`Dkt3vl
`REIvl
`
`YYCQQWSSNPFTFG§GTKLEINR (SEQ ID ND=89)
`YYCQQYQSLPYTFGQGTKLQIIR (SEQ ID ND 9)
`?
`?
`
`xx**%x
`
`CDRB (LUUP)
`
`xxxx%%%Xx
`
`CRD3(KABAT)
`
`FIG. 3
`
`BIOEPIS EX. 1189
`
`PageS
`
`BIOEPIS EX. 1189
`Page 5
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 5 0f 18
`
`5,859,205
`
`NN N
`
`83 86
`
`38 35 N39
`
`43
`
`RES TYPE
`DktBh
`KDL
`
`SESPSASBSSSASSSSSDSDSPSPSEbSBSSBQPiAPIpieSSS
`QVQL00§GAELARPGASVKMSCKASGYTFTRYTMNHWVKQRPGQ
`QVQLVESGGGMVOPGBSLRLSC§§SGFLFSSYAMYWVRQAPGK
`o
`77
`
`x%x*%x
`
`CDPI
`
`(LDDP)
`
`*XX%%
`
`CDRl
`
`(KABAT)
`
`580
`
`60
`
`65
`
`N N
`
`N
`
`880bc
`
`89
`
`RES TYPE IIQIDppASSSSSSSSApSADSSSbSpSQSSSSGSDApSpSSBSSSAGPb
`DRthh
`GLENIGYINPSRGYTNTNQKFKBKATLTTDKSSSTAYMQLSSLTSEDSAV
`KDL
`GLENVAIIWDDGSDQHYADSVKGRFTISRDNSKNTLELQMDSLEPEDTQV
`??
`?
`?
`T
`?
`?
`
`x%%x*%xxxxx*
`
`*xxxxx%xxxxxxxxx*xx
`
`CURB
`
`CURB
`
`(LUDP)
`
`(KABAT)
`
`98 N
`
`107
`
`113
`
`RES TYPE PiPIEissssiiisssbibi*EIPIP%SpSBSS
`Dkt3vh
`YYCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`(SEQ ID ND 30)
`
`KDL
`
`(SEQ ID ND=10)
`YECARDGGHGECSSASCFGPDYWGQGTEVTVSS
`xx%xx*%x¥xxxxx%%% CRD4 (KABAT/LDDP)
`
`FIG. 4
`
`BIOEPIS EX. 1189
`
`Page6
`
`BIOEPIS EX. 1189
`Page 6
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 6 0f 18
`
`5,859,205
`
`DKT 3 HEAVY CHAIN CDR GRAFTS
`
`1. gh341 and derivatives
`
`1
`
`86
`
`35
`
`39
`
`43
`
`Dkt3vh
`
`QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQ
`
`QVQLVESGGGVVQDGRSLRLSCSSSGYTFTRYTMHWVRQAPGK
`gH341
`gH341A QVQLVQSGGGVVQPGRSLRLSCEQSGYTFTRYTMHWVRQAPGK
`
`JA178
`JA185
`
`QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`gH341E
`gH341X QVQLVQSGGGVVQPGRSLRLSCEESGYTFTRYTMHWVRQAPGK
`gH341% QVQLVQSGGGVVQPGRSLRLSCEfiSGYTFTRYTMHWVROAPGK
`gH341D QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGK
`gH341X QVQLVQSGGGVVQPGRSLRLSCRKSGYTFTRYTMHWVRQAPGK
`9H341C QVQLVQSGGGVVQPGRSLRLSCKESGYTFTRYTMHWVROAPGK
`
`QVQLVQSGGGVVQPGRSLRLSCSQSGYTFTRYTMHWVRQAPGK
`gH341*
`OVQLVESGGGVVQPGRSLRLSCSQSGYTFTRYTMHWVRQAPGK
`gH341*
`gH341B QVQLVESGGGVVQPGRSLRLSCSSSGYTFTRYTMHWVRQAPGK
`gH341% DVQLVQSGGGVVQPGRSLRLSCSQSGYTFTRYTMHWVROAPGK
`gH341% QVQLVESGGGVVQPGRSLRLSCSASGYTFTRYTMHWVRQAPGK
`gH341*
`QVQLVQSGGGVVQPGRSLRLSCSESGYTFTRYTMHWVRQAPGK
`KDL
`QVQLVESGGGVVQPGRSLRLSCSSSGFIFSSYAMYWVRQAPGK
`
`JA198
`JA8D7
`JA209
`JA197
`JA199
`JA184
`
`JA803
`JAEUS
`JA183
`JA204
`JABU6
`JA208
`
`FIG. 5a
`
`BIOEPIS EX. 1189
`
`Page7
`
`BIOEPIS EX. 1189
`Page 7
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 7 0f 18
`
`5,859,205
`
`44
`
`50
`
`65
`
`83
`
`Dkt3vh
`
`GLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMOLSSLT
`
`GLEWVAYINPSRGYTNYNQKFKDRFTISRDNSKNTLFLQMDSLR
`gH341
`gH341A GLEWLQYINPSRGYTNYNQKVKQRFTISLDESK§TQFLQMDSLR
`
`JA178
`JA185
`
`GLEWLQYINPSRGYTNYNQKVKQRFTISLDESKgTQFLQMDSLR
`gH341E
`GLEWLQYINPSRGYTNYNQKVKDRFTISngSKNTQFLQMDSLR
`gH341x
`GLEWLQYINPSRGYTNYNQKVKQRFTISRDNSKNTQFLQMDSLR
`9H341x
`gH341D GLEWLQYINPSRGYTNYNQKVKDRFTISngSKNTLFLQMDSLR
`gH341x
`GLEWLQYINPSRGYTNYNQKVKDRFTISRDNSKNTLFLQMDSLR
`9H341C GLEWVAYINPSRGYTNYNQKFKDRFTISRDNSKNTLFLQMDSLR
`
`gH341% GLEWLQYINPSRGYTNYNDKVKDRFTISLDgSKgTQFLQMDSLR
`9H341x GLEWLQYINPSRGYTNYNDKVKDRFTISlD58K§TQFLQMDSLR
`9H34IB GLEWLQYINPSRGYTNYNDKVKDRFTISLDgSKgTQFLQMDSLR
`gH341% GLEWLQYINPSRGYTNYNDKVKDRFTISLD§SK§TQFLQMDSLR
`gH341*
`GLEWIGYINPSRGYTNYNDKVKQRFTISIDKSK§TQFLQMDSLR
`gH341x
`GLEWIEYINPSRGYTNYNUKVKDRFTISZDESKNTEFLQMDSLR
`KDL
`GLEWVAIIWDDGSDQHYADSVKGRFTISRDNSKNTLFLQMDSLR
`
`JA198
`JA807
`JA809
`JA197
`JA199
`JA184
`
`JA807
`JABUS
`JA183
`JA204
`JAEO6
`JA808
`
`FIG. 5b
`
`BIOEPIS EX. 1189
`
`Page 8
`
`BIOEPIS EX. 1189
`Page 8
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 8 0f 18
`
`5,859,205
`
`84
`
`95
`
`102
`
`113
`
`SEQ ID ND:
`
`Dkt3vh
`
`gH341
`gH341A
`
`gH341E
`gH341%
`gH341D
`gH341x
`gH341*
`9H341C
`
`gH341*
`gH341*
`gH341B
`gH341X
`gH341*
`gH34l*
`KDL
`
`SEDSAVYYCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`PEDTQVYlCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`JA178
`
`JA185
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`JA198
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`PEDTéVYlCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTQVYLCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTQVYlCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`JA807
`
`JA197
`
`JA209
`JA199
`
`JA184
`
`JA203
`JAEOS
`
`JA183
`JA204
`JA806
`
`PEDTGVYFCARYYDDHY ....... CLDYWGQGTTLTVSS
`
`JA808
`
`PEDTGVYFCARDGGHGFCSSASCFGPDYWGQGTPVTVSS
`
`30
`18
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`80
`21
`
`28
`83
`
`24
`
`10
`
`FIG. 5c
`
`BIOEPIS EX. 1189
`
`Page9
`
`BIOEPIS EX. 1189
`Page 9
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 9 0f 18
`
`5,859,205
`
`UKT3 LIGHT CHAIN CDR GRAFTING
`
`1.
`
`QLEBI and derivatives
`
`1
`
`24
`
`34
`
`48
`
`Ukthl
`
`QIVLTQSPADMSASPGEKVTMTCSASS.SVSYMNWYQQKSGT
`
`DIQMTQSPSSLSASVGDRVTITCSASS.SVSYMNWYQQTPGK
`
`gLBBI
`QLBEIA
`QIXMTQSPSSLSASVGDRVTITCSASS.SVSYMNWYQQTPGK
`gLBBIB QIXMTQSPSSLSASVGDRVTITCSASS.SVSYMNWYQOTPGK
`DIQMTQSPSSLSASVGDRVTITCSASS.SVSYMNWYQQTPGK
`9L221c
`REI
`
`DIQMTQSPSSLSASVGDRVTITCQASQDIIKYLNWYQOTPGK
`
`Dkt3vl
`
`gLBEI
`gLBBlA
`gLBElB
`gLBElC
`REI
`
`Dkt3vl
`
`QLEBI
`gLBBlA
`gLBElB
`gLBElC
`REI
`
`43
`
`50
`
`56
`
`85
`
`SPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAAT
`APKLLIYDTSKLASGVPSRFSGSGSGTDYTETISSLQPEDIAT
`
`APKBEIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIAT
`APKBEIYDTSKLASGVPSRESGSGSGTDYTETISSLQPEDIAT
`APKEEIYDTSKLASGVPSRESGSGSGTDYTFTISSLQPEDIAT
`APKLLIYEASNLQAGVPSRFSGSGSGTDYIETISSLQPEDIAT
`
`(SEQ ID NUI8)
`
`108
`96
`91
`86
`YYCQQWSSNPFTEGSGTKLEINR
`YYCQQWSSNPETEGQGTKLOITR
`YYCQQWSSNPETEGQGTKLQITR
`
`YYCQQWSSNPETEGQGTKLQITR
`
`YYCQQWSSNPETFGQGTKLQITR
`YYCOQYQSLPYTFGQGTKLQITR
`
`(SE0
`(SE0
`(SEO
`
`(SEQ
`(SEO
`
`(SEQ
`
`ID ND=B9)
`ID ND 85)
`
`ID ND 26)
`
`ID NU=E7)
`ID ND=E8)
`
`ID ND 9)
`
`CDR’S ARE UNDERLINED
`
`FRAMEWURK RESIDUES INCLUDED IN THE GENE ARE DDUBLE UNDERLINED
`
`FIG.
`
`6‘
`
`BIOEPIS EX. 1189
`
`PagelO
`
`BIOEPIS EX. 1189
`Page 10
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 10 of 18
`
`5,859,205
`
`m? x 88 IT
`2: x 2% I?
`
`32 X URN iul -
`
`
`
`
`
`mm? X OPNN ILQII lOm
`
`Ari/5% | 6555 C85 -
`
`><mm< @2525 \OON
`
`
`
`l2:
`
`|o9
`
`AlISNI-liNl BONBOSHHO?Ii
`
`u .Uhm
`
`82 2: E
`
`
`
`ABEECV 58:5
`
`________ _ ________ _ ________ _ ________ _ _ O
`
`P F.
`
`BIOEPIS EX. 1189
`Page 11
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 11 of 18
`
`5,859,205
`
`mw_ x 0am |<|
`
`
`w? x QPNN II!
`
`
`he x “INN IOI
`
`QEXSNNLHT :2:
`
`
`
`__ ____________________c
`
`
`
`ABEBCV 6852
`
`0mm ooN om’ o0? on o
`
`% 6h“
`
`
`
`><mm< @2285
`
`
`
`Ari/‘ml: | BEES C83
`
`ow?
`
`I 0N?
`
`AiISNEIlNI HONEIOSEIHOFTH
`
`BIOEPIS EX. 1189
`Page 12
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 12 of 18
`
`5,859,205
`
`law? x QFNNIIIQ
`
`NlmwF x QPNNIUIM
`
`
`
`Flmww x 0FNN|0|< I
`
`Rm? x UFNNLQIm I
`.mwP x OFNNIOIQ h
`
`
`
`
`><wm< @2503
`
`
`
`Ari/‘E1 | 6296 C85
`
`I ooN
`
`AilSNHlNI EIONBOSHHO?I-I
`
`
`
`@5235 6852
`
`0mm 00w om: o0? cm 0
`
`m. 62
`
`_________________________GOP
`
`BIOEPIS EX. 1189
`Page 13
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 13 of 18
`
`5,859,205
`
`OKTS — GRAFTED HEAVY CHAINS
`BINDING ASSAY
`(Mean Channel — HPALL’s)
`AB
`CF H
`
`D G
`
`2 4|. 1
`
`0 O O O O 5 O 5
`
`
`
`C_mzm;z_ mozmommmozi
`
`111111
`
`X144 A
`C X 185 B
`C X 199 C
`C X 204 D
`C X 205 E
`C X 207 F
`+221C X 208 G
`—A—221C X 209 H
`
`O
`
`.1
`
`llllllllllllln]Illlln‘llllll'll
`10
`100
`1000
`1
`ANTIBODY (ng/’ru be)
`
`I I I I I I I
`
`I I I I I I I
`
`I I I I I I I
`
`I I I I I I J
`
`24
`E4
`84
`24
`24
`E4
`24
`
`I I I I I I I
`
`48
`48
`48
`48
`48
`48
`48
`
`I I I I I I I
`
`49
`49
`49
`49
`49
`49
`49
`
`I I I I I I I
`
`71
`71
`71
`7
`7
`
`I I I I I I I
`
`73
`73
`73
`73
`73
`
`I I I I I I I
`
`I I I I I I I 23
`23
`23
`23
`4
`
`FIG. 10a
`
`BIOEPIS EX. 1189
`Page 14
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 14 of 18
`
`5,859,205
`
`3
`
`T W - K I
`
`2 1 1
`
`
`
`O ______ ____________ O
`
`O 5 O 5
`nu 0 O O O
`
`_ m .|%
`
`e H
`
`$6M ,
`GEO -
`HAI .1
`AAH I
`mNh -1
`.lWn '0
`WYP H2
`E$_ .%
`FDo .IO
`
`H :8 |
`
`L |
`
`C M |%
`M \.l H2
`m |%
`
`12222222
`
`42222222
`
`CCCCCCC
`
`VAXXVAXXVA X
`
`
`
`48900000 41122222
`
`5945789
`
`ABCDEFGH
`
`ANTIBODY (ng/’ru be)
`
`—<>—<204> e,__
`—-—-<199> 6,23
`2 > 6,23
`1
`> 6,23
`2
`> 6,23
`4 x 144
`
`I I I I I I I
`
`24
`24
`24
`E4
`84
`24
`24
`
`I I I I I I I
`
`48
`48
`48
`48
`48
`48
`48
`
`I I I I I I l
`
`49
`49
`49
`49
`49
`49
`49
`
`I I I I I I I
`
`71
`71
`71
`7
`7
`
`I I I I I I I
`
`73
`73
`73
`3
`3
`
`I J I I I J I
`
`I I I I I I I
`
`FIG. 10b
`
`BIOEPIS EX. 1189
`Page 15
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 15 of 18
`
`5,859,205
`
`OKT3 — GRAFTED HEAVY CHAINS
`BINDING ASSAY
`(Mean Channel — HPALL’s)
`
`>- 200
`5
`E
`5 150
`‘I
`g 100
`5
`g 50
`a:
`3
`
`LL
`
`0 I
`.1
`
`——~—221c x 183 A
`—><—221C X 184 B
`+210 X185 0
`+221C X 198 D
`+221C X 205 E
`+221C X 205 F
`—A—221c x 206 o
`
`lllllll'l II1I|||| Illlllll Illllq
`1
`10
`100
`1000
`ANTIBODY (ng/fube)
`
`--*—(183) _,_,___, ,48,49,71,73,76,78,88,91,
`—l—(205) _,__,84,48,49,71,73,76,78,88,91,
`
`—*—-(206) _,__,24,48,4 , 1,73,76,78,__,_
`+(203) 6,__,24,48,49,71,73,76,78,88,9T:
`——<>—(185) 6,E3,24,48,49,71,73,7E>,78,88,91,
`——I—(198) 6,23,84,48,49,71,73,76,78, ___l.__l
`
`FIG. 11a
`
`BIOEPIS EX. 1189
`Page 16
`
`

`

`US. Patent
`
`Jan. 12,1999
`
`Sheet 16 0f 18
`
`5,859,205
`
`OKTS - GRAFTED HEAVY CHAINS
`BINDING ASSAY
`(Mean Channel — HPALL’s)
`
`
`
`A
`B
`g
`E
`G
`
`A
`e
`G
`E
`D
`
`175
`
`>51
`E
`E 150
`
`a 125
`E
`a
`F1
`
`O :
`
`0
`
`o
`
`50
`100
`150
`200
`ANTIBODY (ng/fube)
`
`250
`
`_I__
`
`——+——(183)
`_,_, __, 48 49 71, 73 76 78 88 91
`—-*—-(805) _,__,84, 48 49 71 73 76 78 88 91
`——*—-(184) 6 83 84 __, _,__ __,__,__,
`,__,
`-—fi——(206)
`E4, 48 49 71 73 76 78,__,__,
`-—D——(803) 6,__ 84, 48 49 71 73 76 78 88 91,
`-—¢-(185) 6,23,84,48,49,71,73,76,78,88,91,
`——I——(198) 6,83,84,48,49,71,73,76,78,_ ;__J
`
`FIG. 11b
`
`BIOEPIS EX. 1189
`
`Pagel7
`
`BIOEPIS EX. 1189
`Page 17
`
`

`

`U.S. Patent
`
`Jan. 12,1999
`
`Sheet 17 of 18
`
`5,859,205
`
`E: x SN ~50
`
`i
`
`
`
`
`
`mkxo 00.7030 100
`
`
`
`ZOCCMEEOQ m._.v_O
`
`N N .0?‘
`
`_ - _=____ - _ _____h- _
`
`
`
`2C 068:? 025E000
`
`P00
`_00.0
`
`|III1||III|IIIIIIIIIIIIIIIIIII
`0 LO
`LO
`0
`0
`LO
`“f
`O
`N)
`N
`‘
`0
`D O
`0
`0
`O
`
`00.0
`
`33am / ownos
`
`BIOEPIS EX. 1189
`Page 18
`
`

`

`US. Patent
`
`Jan. 12, 1999
`
`Sheet 18 0f 18
`
`5,859,205
`
`
`
`AMH<2201m¢<zoz<mHzMozoozaHOmmv
`mw_xUFNNmoo1.01:0—0
`
`0km“Em|D|0N0
`
`
`ofio
`
`00.0
`
`00.0
`
`
`
`ZO_.E.H._n_200murxo
`
`
`
`22000E<momoo.m>0._.mHE0E0:
`
`mmd
`
`0M0
`
`0N0
`
`
`
`
`
`TEEN;m._.<2_x0mn_n_<2:A>00m_._.z<OZEhEEOOV
`
`
`
`mzo_._.<w_._.zmozooZEHOWE*0——F05.0900.0
`
`0N.QNK
`
`3an / GNnOEl
`
`BIOEPIS EX. 1189
`
`Page 19
`
`BIOEPIS EX. 1189
`Page 19
`
`

`

`1
`HUMANISED ANTIBODIES
`
`5,859,205
`
`This is a continuation of application Ser. No. 07/743,329,
`?led Sep. 17, 1991, noW abandoned.
`
`FIELD OF THE INVENTION
`
`The present invention relates to humanised antibody
`molecules, to processes for their production using recombi
`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
`ing regions (CDRS) Which determine the binding speci?city
`of the antibody molecule and Which are carried on appro
`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 Fc fragments, Which can be derived by enZy
`matic cleavage. Natural immunoglobulins comprise a gen
`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
`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. Asigni?
`cant step toWards the realisation of the potential of immu
`noglobulins as therapeutic agents Was the discovery of
`procedures for the production of monoclonal antibodies
`(MAbs) of de?ned speci?city
`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 effectiveness. 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, OKT3 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 immunosuppressant in
`the treatment of acute allograft rejection [Chatenoud et al (2)
`and Jeffers et al
`HoWever, in vieW of the rodent nature
`of this and other such MAbs, a signi?cant HAMA response
`Which may include a major anti-idiotype component, may
`build up on use. Clearly, it Would be highly desirable to
`
`10
`
`15
`
`25
`
`35
`
`45
`
`55
`
`65
`
`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 less antigenic in humans. Such techniques can be
`generically termed “humanisation” techniques. These tech
`niques typically involve the use of recombinant DNA tech
`nology to manipulate DNA sequences encoding the polypep
`tide chains of the antibody molecule.
`Early methods for humanising MAbs involved production
`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-O 171496 (Res.
`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
`immunoglobulin. Such humanised chimeric antibodies,
`hoWever, still contain a signi?cant proportion of non-human
`amino acid sequence, ie the complete non-human variable
`domains, and thus may still elicit some HAMA response,
`particularly if administered over a prolonged period [Begent
`et al (ref.
`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
`by site directed mutagenesis using long oligonucleotides.
`The present invention relates to humanised antibody mol
`ecules prepared according to this alternative approach, i.e.
`CDR-grafted humanised antibody molecules. Such CDR
`grafted humanised antibodies are much less likely to give
`rise to a HAMA response than humanised chimeric antibod
`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
`antigens. HoWever, eXamples in Which a mouse MAb rec
`ognising lysoZyme and a rat MAb recognising an antigen on
`human T-cells Were humanised by CDR-grafting have been
`described by Verhoeyen et al (5) and Riechmann et al (6)
`respectively. The preparation of CDR-grafted antibody to
`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 de?ned by
`Kabat refs. (7) and
`Was not suf?cient to provide satis
`factory antigen binding activity in the CDR-grafted product.
`Riechmann et al 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
`grafted product having improved antigen binding activity.
`This residue at position 27 of the heavy chain is Within the
`structural loop adjacent to CDR1. A further construct Which
`additionally contained a human serine to rat tyrosine change
`at position 30 of the heavy chain did not have a signi?cantly
`altered binding activity over the humanised antibody With
`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 CDR1, may be necessary to
`obtain effective antigen binding activity for CDR-grafted
`antibodies Which recognise more complete antigens. Even
`so the binding affinity of the best CDR-grafted antibodies
`obtained Was still signi?cantly less than the original MAb.
`
`BIOEPIS EX. 1189
`Page 20
`
`

`

`5,859,205
`
`3
`
`Very recently Queen et al (9) have described the prepa-
`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-
`stant regions. The human framework regions were chosen to
`maximise homology with the anti-Tac MAb sequence. In
`addition computer modelling was used to identify frame-
`work amino acid residues which were likely to interact with
`the CDRs 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 first criterion is
`to use as the human acceptor the framework from a particu-
`lar human immunoglobulin that is unusually homologous to
`the non-human donor immunoglobulin to be humanised, or
`to use a consensus framework from many human antibodies.
`The 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
`use 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
`
`10
`
`15
`
`20
`
`framework positions at which the amino acid is predicted to
`have a side chain atom within about 3 A of the CDRs in a
`
`25
`
`three-dimensional immunoglobulin model and to be capable
`of interacting with the antigen or with the CDRs of the
`humanised immunoglobulin. It is proposed that criteria two,
`three or four may be applied in addition or alternatively to
`criterion one, and may be applied singly or in any combi-
`nation.
`
`WO 90/07861 describes in detail the preparation of a
`single CDR-grafted humanised antibody, a humanised anti-
`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
`region 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 the
`human acceptor residues, at positions 27, 30, 48, 66, 67, 89,
`91, 94, 103, 104, 105 and 107 in the heavy chain SEQ ID
`NO:31 and at positions 48, 60 and 63 in the light chain, of
`the variable region frameworks. The humanised anti-Tac
`antibody obtained is reported to have an affinity for p55 of
`3x109M'1, about one-third of that of the murine MAb.
`We have further investigated the preparation of CDR-
`grafted humanised antibody 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 are important for obtaining CDR-
`grafted products with satisfactory binding affinity. This has
`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-
`dues which we have identified as being of critical impor-
`tance does not: coincide with the residues identified by
`Queen et al (9).
`
`SUMMARY OF THE INVENTION
`
`in a first aspect the invention provides a
`Accordingly,
`CDR-grafted antibody heavy chain having a variable region
`domain comprising acceptor framework and donor antigen
`binding regions wherein the framework comprises donor
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`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. The 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
`framework additionally comprises donor residues at one,
`some or all of positions 6, 37, 48 and 94. Also it
`is
`particularly preferred that residues at positions of the heavy
`chain framework which are 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-
`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-
`prising acceptor framework and donor antigen binding
`regions. It will be appreciated that the invention is widely
`applicable to the CDR-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-class. More usually, however, the donor and
`acceptor antibodies are derived from animals of different
`species. Typically the donor antibody is a non-human
`antibody, such as a rodent MAb, 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 three CDRs of each of the heavy chain and/or light chain
`variable regions. The CDRs may comprise the Kabat CDRs,
`the structural loop CDRs or a composite of the Kabat and
`structural loop CDRs and any combination of any of these.
`Preferably, the antigen binding regions of the CDR-grafted
`hearty chain variable domain comprise CDRs corresponding
`to the Kabat CDRs at CDR2 (residues 50—65) and CDR3
`(residues 95—100) and a composite of the Kabat and struc-
`tural loop CDRs at CDR1 (residues 26—35).
`The residue designations given above and elsewhere in
`the present application are numbered according to the Kabat
`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 corresponding to a shortening
`of, or insertion into, a structural component, whether frame-
`work or CDR, of the basic variable domain structure. For
`example, the heavy chain variable region of the anti-Tac
`
`BIOEPIS EX. 1189
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`BIOEPIS EX. 1189
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`5,859,205
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`5
`
`antibody described by Queen et al (9) contains a single
`amino acid insert (residue 52a) after residue 52 of CDR2 and
`a three amino acid