FIG. 38
`
`0.8
`
`~ g 0.6
`;:;;
`..,,
`.....
`c.>
`~
`
`0.2
`
`N ... CIII
`
`O 120
`
`140
`
`160
`
`HFR 1
`111 111
`
`I
`
`I I I
`
`CDR HI
`I I tutu•
`
`HFR 2
`111
`
`180
`RESIDUE POSITION
`CDR H2
`tttttttttttt
`
`11 11
`
`200
`
`220
`
`HFR 3
`
`111
`
`Ill I
`
`CDR HJ
`ttttttttttttttttt
`
`(SW ID NO: 13)
`glb2 QVQWQSGTELARPGASVRLSCKASGYTmFGIT··WYKQRT<J,lGLt"'WIGEIFPGNS··KTYYAERFKGXATLTADXSS'l'l'AYMQLSSLTSEDSAVYFCAREIR·············OO
`(SiX) ID NO: 14)
`lfdl QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVN··WVRQPPGKGLMOOWGOO···lfl'DYNSALKSRLSISKDNSK9JVFLKMNSLHTDDTARYYCARERDYR1·········IIDK;
`(SEX) ID 00: 15)
`2hfl ·VQLQQSGAELMKIQ.SVK!SCKASGY'ITSDYWIE··WVKQRPGiGLOOGEILroSG-·S'l'NYHERFKGXATF'l'AIYl'SSSTAYMQLNSLTSEDOOVYYCUIGNYDF··········IG«;
`(SW ID 00: 16)
`Jhfm DVQLQESGPSLVKPSQ'l'LSL'ltS1fl'GDSITSDYWS··WIRXFPGN"RLEYMGYVSYSG--·Sl'Y'fNPSLXSR!SI'l'Rl)'.l'SKNQYYLDLNSVffll1l'A'l'YYCANWOO··········-·DOO
`(SW ID NO: 17 l
`2fbj EVXLLESGOOLVQPGGSLKLSCAASGFDFSKYWMS··WVRO'PGKGLEWIGEIHPDSG--TMTPSLI<DKFIISRDNAXHSLYIJllSKVRSEDl'ALYYCARLHYYGYH·-·-··-·AYWG
`(S'F.Xl ID NO: 18)
`2fb4 EI/QLVQSGGGVVQR:RSLRLSCSSSGFIFSSYAMY··WVRQAPGKGIJ.WAIIWDDGS··J)JHYAl)SVJCGRFSRNOOJOO'LFL(J(!)SLRPEinGVYFCARimlGFCSSASCFGPIJYli«i
`2mcp EVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYII.E--WVROPPGKRLEWIAASRNJCGN'lYfflYSASVKGRFIVSRI7l'SQSIL YI.{1!NALRAEDl.'AIYYCARNYYGS'MF ······D'OO ( SW ID NO: 19 I
`7 f ab ·VQLEQSGroLVRPSQTLSLTCTVSGTSFDDYYST··WVRQPPGRGLEWIGYVFYHG-··TSm'IJl'PLRSRVMVN'l'SKNQFSLRLSSVTAAMAVYYCARNLIAGCI •••• -···D'OO
`( SB.) ID NO: 20)
`4f ab EVKLDE'l.'Ql;LVQPGRPMJCLSCVASGFTFSDYWMN··WVRQSPEKGLEWVAQ!RNKPYNYETYYSDSVKGRFTISRDOOKSSVYI,()4NNLRVEDl(;lYYC'roSYYGl!· --··· • •• ·IJYli«i
`(SEQ ID NO: 21)
`lfl9 QVQLKESGAELI/MSSSVKMSCKASGY'!'f'i'SYGVN··WVKQRPGOO!,EWIGYINFGKG··YLSYNEKFKGK'I'l'L'IVDRSSSTAYMOLRSLTSEDSAVYrCAASF'fGGSDLAVYYF--DSWG
`(Sa} ID NO: 22)
`( sm ID NO: 2 3 I
`Hao !.VQLQQSGVELVRAGSSVXMSC:OSGYTITSNGIN · ·WVKQRPG;X;LEWIGYNNPGNG-· m.YNEKFXGKT!'L'IVDXSSSTAYMQLRSLTSEDSA VYrCAASEYYC-GSYKF • • ···DYWG
`ldfo EVOLVESC:.(',GLVOPGRS!JLSCMSGF'i'FNDYAMH··WVRM?GKGL:i.WISGI3WDSS··SiG'!'ADSVXGlU'TISRDNAKNSLY'L<MlSLmDMALYYCV'i<GRDY'!DSC'iGYFTVi\F!)iWG
`!SEO ID NO: 241
`
`m
`"U
`0
`
`! ... g
`), ...
`
`PFIZER EX. 1002
`Page 501
`
`

`

`FIG. 4A
`Light Chain Sequences
`
`N .... co
`
`1 N901L
`
`2 KOL
`
`3 N901L/KOL
`
`4 KV2F$HUMAN
`[most identical
`5 N901L/KV2F
`[CDR grafted]
`6 KV4B$HUMAN
`(most identical
`7 N901L/KV4B
`(Resurfaced)
`
`1 N901L
`
`2 KOL
`
`3 N901L/KOL
`
`4 KV2F$HUMAN
`[most identical
`5 N901L/KV2F
`[CDR grafted)
`6 KV4B$HUMAN
`[most identical
`7 N901L/KV4B
`[Resurfaced]
`
`10
`20
`30
`70
`50
`40
`60
`------+
`---------+---------+--- ------+---------+ ---------+----- ----+--
`:DVLMTQTPLSLPVSLGDQASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`
`:QSVLTQPPSASG-TPGQRVTISC SGTSSNIGS----STVN WYQQLPGMAPKLLIY RDAMRPS GVPDRFSG
`I •
`I
`I
`I
`:QVLMTQTPSSLPVTLGOQASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`
`seq)
`
`:DVVMTQSPLSLPVTLGQPASISC RSSQSLVYSDGNTY-LN WFOQRPGQSPRRLIY KVSNRDS GVPDRFSG
`*
`•
`I
`I
`11
`:DVLMTQSPLSLPVTLGQPASISC RSSQIIIHSDGNTY-LE WFOQRPGQSPRLLIY KVSNRFS GVPDRFSG
`
`surf)
`
`:DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYOQKPGQPPKLLIY WASTRES GVPDRFSG
`*
`I
`I
`:DVLMTQTPDSLPVSLGDRASISC RSSQIIIHSDGNTY-LE WFLQKPGQSPKLLIY KVSNRFS GVPDRFSG
`(
`Ll
`]
`[ L2
`
`100
`90
`80
`110
`---------+---------+------ ---+------- --+-----
`:SGSGTDFTLMISRVEAEDLGVYYC FQGSH--VPHT FGGGTKLEI-
`
`(SEQ ID NO: 25)
`
`:SKSGASASLAIGGLQSEDETDYYC AAWDVSLNAYV FGTGTKVTVL
`*
`I
`I
`I
`:SGSGTSFTLAISRVEAEDEGVYYC FQGSH--VPHT FGGGTKLEI-
`
`( 44)
`
`(SEQ ID NO: 26)
`
`(104)
`
`(SEQ ID NO: 27)
`
`seq]
`
`:SGSGTDFTLKISRVEAEDVGVYYC MQGTH--wswr FGQGTKVEIK.
`I
`I
`•
`:SGSGTDFTLKISRVEAEDVGVYYC FQGSH--VPHT FGGGTKVEI-
`
`( 87)
`
`(SEQ ID NO,: 28)
`
`(101)
`
`(SEQ ID NO: 29)
`
`surf)
`
`:SGSGTDFTLTISSLQAEDVAVYYC OQYDT---IPT FGGGTKVEIK
`
`( 71)
`
`(SEQ ID NO: 30)
`
`:SGSGTOFTLMISRVEAEDLGVYYC FQGSH--VPHT FGGGTKLEI-
`I
`[
`L3
`
`(109)
`
`(SEQ ID NO: 31)
`
`m
`"V
`0
`
`! ....
`:!!
`
`0
`0)
`
`PFIZER EX. 1002
`Page 502
`
`

`

`FIG. 48
`Heavy Chain Sequences
`
`1 N901H
`
`2 KOL
`
`3 N901H/KOL
`
`120
`150
`160
`170
`180
`140
`130
`--+---------+---------+------- --+---- -----+-------- -+---------+ -------
`:DVQLVESGGGLVQPGGSRKLSCAASGFTFS SFGMH-- WVRQAPEKGLEWVA YISSGSF--TIY HADTVKG
`
`:EVQLVQSGGGVVQPGRSLRLSCSSSGFIFS SYAMY-- WVRQAPGKGLEWVA IIWDDGS--DQH YADSVKG
`I
`I
`I II
`I
`I
`II
`I
`:EVQLVESGGGVVQPGRSLRLSCAASGFIFS SFGMH-- WVRQAPGKGLEWVA YISSDGF--TIY HADSVKG
`
`4 G36005
`[most identical
`5 N901H/G36005
`[CDR grafted)
`6 PL0123
`(most identical
`7 N901H/PL0123
`[Resurfaced)
`
`N
`N
`C,
`
`seq)
`
`:QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH-- WVRQAPGKGLEWVA VISYDGS--NKY YADSVKG
`j
`I
`I
`I 11
`I
`I
`:QVQLVESGGGVVQPGRSLRLSCAASGFTFS SFGHH-- WVRQAPGKGLEWVA YISSGSF--TIY YADSVKG
`
`:EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYWMS-- WVRQAPGKGLEWVA NIKQDGS--EKY YVDSVKG
`surf I I
`11
`I
`I
`:EVQLVESGGGLVQPGGSLRLSCAASGFTFS SFGMH-- WVRQAPGKGLEWVA YISSGSF--TIY HADSVKG
`I
`[
`I
`H2
`[ Hl
`
`1 N901H
`
`2 KOL
`
`3 N901H/KOL
`
`4 G36005
`[most identical
`5 N901H/G36005
`[CDR grafted]
`6 PL0123
`[most identical
`7 N901H/PL0123
`[Resurfaced)
`
`190
`210
`240
`220
`200
`230
`--+--------· +---------+--------- +--- -----+------ ---+------
`:RFTISRDNPKNTLFLQMTSLRSEDTAMYYCAR MRKGYAM--------DY WGQGTTVTVS
`
`:RFTISRNDSKNTLFLQMDSLRPEDTGVYFCAR DGGHGFCSSASCFGPDY WGQGTPVTVS
`I*
`•
`:RFTISRDDPKNTLFLQMTSLRSEDTAMYYCAR MRKGYAM--------DY WGQGTTVTVS
`
`seq)
`
`:RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DRKDWGWALF-----DY WGQGTLVTVS
`I
`I
`I
`I
`I
`.
`:RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR MRKGYAM--------DY WGQGTLVTVS
`
`surf]
`
`:RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR ----------------- ---------(cid:173)
`
`:RFTISRDNAKNTLFLQMTSLRAEDTAMYYCAR MRKGYAM--------DY WGQGTTVTVS
`I
`·
`[
`H3
`
`(SEO ID NO: 32)
`
`( 77)
`
`(SEQ ID NO: 33)
`
`(106)
`
`(SEQ ID NO: 34)
`
`( 89)
`
`(SEQ ID NO: 35)
`
`(103)
`
`(SEQ ID NO: 36)
`
`( 74)
`
`(SEQ ID NO: 37)
`
`(110)
`
`(SEQ ID NO: 38)
`
`m
`'V
`C,
`
`! ....
`
`C, en
`~
`
`PFIZER EX. 1002
`Page 503
`
`

`

`EP O 592 106 A1
`
`y
`
`_ EP 0 592108 A1
`
`FIG. 5
`
`X
`
`221
`
`PFIZER EX. 1002
`Page 504
`
`

`

`~
`
`RMSD
`
`1.4
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0
`
`••
`
`I
`
`./ ..
`
`.. .
`:
`:"
`.
`.
`\
`~
`.
`.
`~
`.
`\
`'
`
`FIG. 6
`
`m
`'V
`0
`
`is ....
`
`0
`0)
`
`~
`
`. .
`.
`..
`·"·
`.
`.
`..
`... ·
`'
`..
`.....
`;
`...
`•
`
`.•.•. ..,,. -
`
`...... -
`
`,.,
`
`....•. ,
`·,.
`'
`.
`;
`'·
`' .
`..
`...
`. .
`.·
`'
`",!
`,.
`'
`'•, . \
`. .
`,·.
`·····•··
`',
`•..
`..•..
`·,
`.'
`'•,
`........
`!
`•...
`··•·••
`..
`-
`..,,
`··:•.
`!
`.
`. -~.
`.
`. .. .
`.•.. . ...... ""·
`)' .
`! ....
`•.
`-...
`•.•
`.,,..
`/
`...
`··"''·..
`. .. ·.-.·
`... '
`:··
`....,, •.•. /"-.<'
`·•.
`·-;~-...
`••••
`.,.,.,.....,;..._/
`.,, .. /···
`... ,-, ·'
`. . .
`·...---:~·-:, .......• , .... ~:.;·.-·•·•''-··.
`...... -......
`,· ....,.. -~···
`_.,
`•.
`.. ..
`. .. .. .. .. ..
`....... .. .. .
`.....
`. . ....
`:: ... ·
`...... -·······
`.....
`·,
`,.,·
`···•·••
`........ :::.
`········--..·:.:·.,,.,·::
`,. ~ ........ .
`.....
`.
`. .. ········, ..... ······ ·····
`.................. ···...
`..
`..... .
`·-·
`. . ....
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`16
`
`18
`
`Residue number in strand
`
`PFIZER EX. 1002
`Page 505
`
`

`

`EP O 592106 A1
`
`Knowl~i:e base
`1. Brookhaven databank
`2. Antibody sequence database
`3. Antibody structure database
`4. Loop database
`I
`
`I Analysis I
`
`I
`Framework
`construction
`.
`Modelling
`protocol for
`each CDR
`
`.
`
`I
`
`I
`
`ab-inilio
`construction
`
`canonical
`construction
`
`I
`Database &
`ab-initio
`construction
`I
`
`I
`
`I
`
`•
`Ana!!'.!is and validation
`1. Energy screening
`2. FIitering
`3. Torsional clustering
`4. Comparison with canonical structures
`
`I
`Loop
`
`insertion .
`
`Energy
`minimisation
`I
`FINAL
`MODEL
`
`223
`
`I
`
`Database
`construction
`
`I
`
`FIG. 7
`
`PFIZER EX. 1002
`Page 506
`
`

`

`EP O 592106 A1
`
`\ ~ d .~
`~
`L3 ~ ~
`/
`
`L2
`
`Hl
`
`~ .~ -
`
`3D6
`
`36-71
`
`Dl.3
`
`Gloop-2
`
`FIG. 8
`
`224
`
`PFIZER EX. 1002
`Page 507
`
`

`

`EP O 592108A1
`
`EP 0 592 108 A1
`
`225
`
`.
`
`(.!)
`LL
`
`PFIZER EX. 1002
`Page 508
`
`

`

`CD
`(1)
`
`EP O 592106 A1
`
`1A8o1295oPE
`
`(!)
`
`lJ...
`
`226
`
`PFIZER EX. 1002
`Page 509
`
`

`

`u
`m .
`
`EP 0 592 106 A1
`
`(!)
`LL
`
`EP O 592106 A1
`
`227
`
`PFIZER EX. 1002
`Page 510
`
`

`

`0
`(j)
`
`EP 0 592 108 A1
`
`0 -LL
`
`EP O 592106 A1
`
`228
`
`PFIZER EX. 1002
`Page 511
`
`

`

`60
`
`50
`
`40
`Number or
`loops
`
`30
`
`20
`
`10
`
`EP O 592106 A1
`
`:, :.: :
`.....
`:.::-:
`; :• ...
`...
`...
`
`....
`
`··. :•
`
`. ;.
`.
`.. · .. '
`
`. ...
`
`.....
`...
`
`0 1 2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19
`
`Loop length
`
`FIG. IQ
`
`.229
`
`PFIZER EX. 1002
`Page 512
`
`

`

`EP O 592106A1
`
`European Patent
`Office
`
`EUROPEAN SEARCH REPORT
`
`EP
`
`93 30 7051
`
`DOCUMENTS CONSIDERED TO BE RELEVANT
`
`IMMUNOLOGY
`D,A MOLECULAR
`vol. 28, no. 4/5, 1991, GB
`pages 489 - 498
`PADLAN A E 'POSSIBLE PROCEDURE FOR
`REDUCING THE IMMUNOGENICITY OF ANTIBODY
`VARIABLE DOMAINS WHILE PRESERVING THEIR
`LIGAND-BINDING PROPERTIES'
`• Materials and Methods, p.490, 491,
`Tables 1-3 •
`
`D,A WO-A-9 109 967 (CELLTECH LIMITED)
`11 July 1991
`* p. 5, second paragraph, p. 6 second
`11Rat1ona1 11 pp. 19-23 *
`paragraph,
`EP-A-0 519 596 (MERCK & CO. INC.)
`23 Oece~ber 1992
`* Claims *
`
`P,A
`
`Relennt
`to claim
`
`a.ASSIFICATION 01' THE
`APl'UCAUON (lat. 0.9 )
`C12N15/13
`Cl2Nl5/62
`C07K15/00
`C12P21/08
`
`C07K
`
`.. _.,_
`
`I
`
`_.,_..... .,1111 _
`
`ICalllaR
`l1----MU-NI_C_H _____ ....__ __ 1_2_J_A_N_U_AR_Y_19_9_4 __ .....___ __ G_er_m_i_n_ar_i_o_c_. __ ---1
`I
`T : lllmry ar prlndple lllldalJIDa .. , 111-111111
`CATEGORY OJ' OTD> DOCUMENTS
`a
`E: arllar paunt ,1-, fiut pallllsW on, ar
`ll'llr 11111 flllll& ate
`X : pullcularly Nltftllt If tum llmle
`g
`Y : parlladuly nl-t if comlllne,1 wltll 111111d11r
`D : ,IDCllllllDt dtd ID Ille application
`~
`~ of lbesamecat911
`L 1 ~ c:ltei fwattiar-
`ll!
`A: latmolatlc:al ~
`- ""
`iiil
`0
`,I : · - - llf Ille -
`patat family, CDITISpGll,lflla
`0 : 111111-11Tltltll ~DSIIN
`"'
`ti.__ ____ ___________ _ ______ _ ______ _ __,
`P : IDt--i.t• - - -
`olacluuat
`0
`
`l
`
`230
`
`PFIZER EX. 1002
`Page 513
`
`

`

`®
`
`©
`
`Europalsches Patentamt
`
`European Patent Office
`
`Office europ6en des brevets
`
`1111111111111111 ll~I Ill~ IH 111111111111111 ~Ill lllll lllll 111111111111
`0 620 276 A1
`
`@) Publication number:
`
`EUROPEAN PATENT APPLICATION
`
`@ Application number: 94104042.0
`
`@ Date of filing: 21.12.90
`
`@ Int. c1.s, C12N 15/13, C07K 15/28,
`A61 K 39/395, G01 N 33/577
`
`This application was filed on 16 - 03 - 1994 as a
`divisional application to the application
`mentioned under INID code 60.
`
`E) Applicant: CELL TECH LIMITED
`216 Bath Road
`Slough Berkshire SL 1 4EN (GB)
`
`The applicant has subsequently filed a sequence
`listing and declared, that it includes no new
`matter.
`
`@ Priority: 21.12.89 GB 8928874
`
`@ Date of publication of application:
`19.10.94 Bulletin 94/42
`® Publication number of the earlier application in
`accordance with Art.76 EPC: 0 460 167
`® Designated Contracting States:
`AT BE CH DE DK ES FR GB GR ITU LU NL SE
`
`@ Inventor: Adair, John Robert
`23, George Road,
`Stokenchurch
`High Wycombe, Buckinghamshire, HP14 3RN
`(GB)
`Inventor: Athwal, Dlljeet Singh
`33 Casella Road
`New Cross Gate, London SE17 (GB)
`Inventor: Emtage, John Spencer
`49 Temple Mill Island
`Temple Marlow, Buckinghamshire, SL71SQ
`.(GB)
`
`@) Representative: Mercer, Christopher Paul et al
`Carpmaels & Ransford
`43, Bloomsbury Square
`London WC1A 2RA (GB)
`
`@ Humanised antibodies.
`
`© 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/or (91). The CDR-grated 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 residues
`and human acceptor frameworks, and may be used for in vivo therapy and diagnosis. A generally applicable
`protocol is disclosed for obtaining GOA-grafted antibodies.
`
`... cc
`co " N
`0
`N co
`0
`A. w
`
`Rank Xerox (UK) Business Services
`13. 10/3.09/3.3.41
`
`PFIZER EX. 1002
`Page 514
`
`

`

`EP O 620 276 A1
`
`1 GAATrCccAA ACAcAAAotg anttttsons tacosotttl coacttssta
`51 ctontsaeta cctsoatsnt oototesnsn qqocaaattg ttctcaccca
`101 gtctccagca atcatgtctg catctccagg ggagaaggtc accatgacct
`151 gcagtgccag ctcaagtgta agttacatga actggtacca gcagaagtca
`201 ggcacctccc ccaaaagatg gatttatgac acatccaaac tggcttctgg
`.251 aqtccctgct cacttcaggg gcagtgggtc tgggacctct tactctctca
`301 caatcagcgg catggaggct gaagatgctg ccacttatta ctgccagcag
`tggagtagta acccattcac gttcggctcg gggacaaagt tggaaataaa
`351
`401 ccgggctgat actgcaccaa ctgtatccat cttcccacca tccagtgagc
`451 agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac
`501 cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa
`551
`tggcgtcctg aacagttgga ctgatcagga cagcaaagac agcacctaca
`601 gcatgagcag caccctcacg ttgaccaagg acgagtatga acgacataac
`6!,l agctatacct gtgaggccac tcacaagaca tcaacttcac ccattgtcaa
`701 gagcttcaac aggaatgagt gtTACACACA AACCTCCTGA GACCCCACCA
`7 51 CCAGCTCCCA GCTCCATCCT. ATCTTCCCTT CTAAGGTCTT GGACGC'ITCC
`801 CCACAAGC.GC tTAC.CACTGT TGCGGTGCTC tAAACCTCCT CCCACCTCCT
`851 TCTCCTCCTC CTCCCTTTCC TTGGCTTTTA TCATGCTAAT ATTTGCAGAA
`
`901 AATATTCAAT AAAGTGAGTC TTTCCCTTGA A••••••••• AAA
`Fig. 1 (a)
`
`1 MpfQYQIFSf I J JSASYJIS RGOIVLTQSP AIMSAS~EK VTMTCSASSS
`51 VSYMNWYQQK SGTSPKRWIY DTSKLASGVP Al!FRGSGSGT SYSLTISGM:£
`101 AEDAATYYCQ QWSSNPFTFG SGTKL!:Il:IRA OTAPTVSIFP PSSEQLTSGG
`151 ASVVCFLNNF YPl<OIIIVltWlC IDGSERQNGV LNSh"Tl)QDSK DSTYSMSSTL
`201 TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC•
`Fig. ,Cb)
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`Field of the Invention
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`The present invention relates to humanised antibody molecules, to processes for their production using
`recombinant DNA technology, and to their therapeutic uses.
`The term "humanised antibody molecule" in 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 determining regions (CDRs) which determine the binding specificity of the antibody
`molecule and which are carried on appropriate framework regions in the variable domains. There are 3
`10 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
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`20
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`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 enzymatic cleavage. Natural im(cid:173)
`munoglobulins comprise a generally 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, diagnosis 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 significant step towards the realisation of the potential of immunoglobulins as therapeu(cid:173)
`tic agents was the discovery of procedures for the production of mon.oclonal antibodies (MAbs) of defined
`25 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
`30 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
`35 giving rise to undesirable reactions. For instance, OKT3 a mouse lgG2a/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 (3)(cid:173)
`]. 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
`40 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 techniques typically involve the
`use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the
`45 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-0 171496 (Res. Dev.
`50 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
`humanized 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,
`55 particularly if administered over a prolonged period [Bagent 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 by site directed mutagenesis using long oligonucleotides. The present invention
`
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`relates to humanized antibody molecules prepared according to this alternative approach, i.e. CDR-grafted
`humanised antibody molecules. Such CDR-grafted humanized antibodies are much less likely to give rise to
`a HAMA response than humanised chimeric antibodies in view of the much lower proportion of non-human
`amino acid sequence which they contain.
`The earliest work on humanizing MAbs by CDR-grafting was carried out on MAbs _recognizing synthetic
`antigens, such as the NP or NIP antigens. However, examples in which a mouse MAb recognizing 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
`defined by Kabat refs. (7) and (8)] was not sufficient to provide satisfactory 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 significantly 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
`20 antibodies which recognise more complex antigens. Even so the binding affinity of the best CDR-grafted
`antibodies obtained was still significantly less than the original MAb.
`Very recently Queen et al (9) have described the preparation 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 constant regions. The human framework regions were chosen to maximise homology with
`the anti-Tac MAb sequence. In addition computer modelling was used to identify framework amino acid
`residues which wore 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 particular human immunoglobulin that is
`30 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 framework positions at which the amino acid is predicted to have a
`side chain atom within about 3 A of the CDRs in a 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 combination.
`WO 90/07861 describes in detail the preparation of a single CDR-grafted humanised antibody, a
`humanised antibody 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 humanized antibody, the variable region frame(cid:173)
`works of the human antibody Eu (7) being used as acceptor. In ttie 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
`45 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 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 3 x 109 M- 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
`55 of residues which we have identified as being of critical importance does not coincide with the residues
`identified by Queen et al (9).
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`Summary of the Invention
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`35
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`Accordingly, in a first aspect the invention provides a CDR-grafted antibody heavy chain having a
`variable region domain comprising acceptor framework and donor antigen binding regions wherein the
`framework comprises 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.
`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 residues at positions 2,
`15 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.
`In addition the heavy chain framework optionally comprises donor residues at one, some or all of
`positions:
`1 and 3,
`72 and 76,
`20 69 Of 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),
`25 91,
`as. 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 comprising 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
`4-0 CDR-grafted heavy 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 structural loop CDRs at
`CDR1 (residues 26-35).
`The residue designations given above and elsewhere in the present application are numbered accord(cid:173)
`ing to the Kabat numbering [refs. (7) and (8)]. Thus the residue designations do not always correspond
`45 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 framework or CDR: of the basic variable domain structure.
`For example, the heavy chain variable region of the anti-Tac 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 insert (residues
`50 82a, 82b and 82c) after framework residue 82, in the Kabat numbering. The correct Kabat numbering of
`residues may be determined for a given antibody by alignment at regions of homology of the sequence of
`the antibody with a "standard" Kabat numbered sequence.
`The invention also provides in a second aspect a CDR-grafted antibody light chain having a variable
`region domain comprising acceptor framework and donor antigen . binding regions wherein the framework
`comprises donor residues at at least one of positions 1 and/or 3 and 46 and/or 47. Preferably the CDR
`grafted light chain of the second aspect comprises donor residues at positions 46 and/or 47.
`The invention also provides in a third aspect a CDR-grafted antibody light chain having a variable region
`domain comprising acceptor framework and donor antigen binding regions wherein the framework com-
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`prises donor residues at at least one of positions 46, 48, 58 and 71.
`In a preferred embodiment of the third aspect, the framework comprises donor residues at all of
`positions 46, 46, 58 and 71.
`In particularly preferred embodiments of the second and third aspects, the framework additionally
`comprises donor residues at positions 36, 44, 47, 85 and 87. Similarly positions of the light chain framework
`which are commonly conserved across species, i.e. positions 2, 4, 6, 35, 49, 62, 64-69, 98, 99, 101 and
`102, if not conserved between donor and acceptor, additionally comprise donor residues. Most preferably
`the light chain framework additionally comprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49,
`62, 64-69, 85, 87, 98, 99, 101 and 102.
`In addition the framework of the second or third aspects optionally comprises donor residues at one,
`some or all of positions:
`1 and 3,
`63,
`60 (if 60 and 54 are able to form at potential saltbridge),
`15 70 (if 70 and 24 are able to form a potential saltbridge),
`73 and 21 (if 47 is different between donor and acceptor),
`37 and 45 (if 47 is different between donor and acceptor),
`and
`any one or more of 10, 12, 40, 80, 103 and 105.
`Preferably, the antigen binding regions of the CDR-grafted light chain variable domain comprise CDRs
`corresponding to the Kabat CDRs at CDR1 (residue 24-34), CDR2 (residues 50-56) and CDR3 (residues 89-
`97).
`The invention further provides in a fourth aspect a CDR-grafted antibody molecule comprising at least
`one CDR-grafted heavy chain and at least one CDR-grafted light chain according to the first and second or
`first and third aspects of the invention.
`The humanised antibody molecules and chains of the present invention may comprise: a complete
`antibody molecule, having full length heavy and light chains; a fragment thereof, such as a Fab, (Fab')2 or
`FV fragment; a light chain or heavy chain monomer or dimer; or a single chain antibody, e.g. a single chain
`FV in which heavy and light chain variable regions are joined by a peptide linker; or any other CDR-grafted
`Jo molecule with the same specificity as the original donor antibody. Similarly the GOA-grafted heavy and light
`chain variable region may be combined with other antibody domains as appropriate.
`Also _the heavy or light chains or humanised antibody molecules of the present invention may have
`· attached to them an effector or reporter molecule. For instance, it may have a macrocycle, for chelating a
`heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure. Alternatively, the
`35 procedures of recombinant DNA technology may be used to produce an immunoglobulin molecule in which
`the Fe fragment or CH3 domain of a complete immunoglobulin molecule has been replaced by, or has
`attached thereto by peptide linkage, a functional non-immunoglobulin protein, such as an enzyme or toxin
`molecule.
`Any appropriate acceptor variable region framework sequences may be used having regard to
`40 class/type of the donor antibody from which the antigen binding regions are derived. Preferably, the type of
`acceptor framework used is of the same/similar class/type as the donor antibody. Conveniently, the
`framework may be chosen to maximise/optimise homology with the donor antibody sequence particularly at
`positions close or adjacent to the CDRs. However, a high level of homology between donor and acceptor
`sequences is not important for application of the present invention. The present invention identifies a
`45 hierarchy of framework residue positions at which donor residues may be important or desirable for
`obtaining a CDR-g