`
`US 6,407,213 Bl
`
`-continued
`
`82
`
`va l Val Thr Va l Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr
`215
`220
`225
`
`Cys Asn Va l Asp His Lys Pro Ser Asn Thr Ly s Val As p Lys Th r
`230
`235
`240
`
`val Glu Arg Lys cys cys val Glu cys Pro Pro cys Pro Ala Pro
`245
`250
`255
`
`Pro Val Al a Gly Pro ser val Phe Leu Phe Pro Pro Lys Pro Lys
`260
`265
`270
`
`Asp T hr Leu Met Ile Ser Ar g T hr Pro Glu Val Thr Cys Val Val
`275
`280
`285
`
`val Asp Val ser His Gl u Asp Pro Gl u Va l Gln Phe Asn Trp Tyr
`290
`295
`300
`
`Va l Asp Gly Met Glu Va l His Asn Ala Lys Thr Lys Pro Arg Glu
`305
`3 10
`315
`
`Glu Gln Phe Asn ser Thr Phe Arg Val Va l ser Val Leu Th r Val
`320
`325
`3 30
`
`Val His Gln Asp Tr p Leu ASn Gly Lys Gl u Tyr Lys cys Lys Val
`335
`340
`345
`
`Ser Asn Ly s Gly Leu Pro Al a Pro Ile Glu Ly s Thr lle Ser Ly s
`350
`355
`360
`
`Thr Lys Gly Gl n Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
`365
`370
`375
`
`Ser Ar g Glu Glu Met Thr Ly s Asn Gln Va l Se r Leu Thr Cys Leu
`380
`385
`390
`
`Va l Lys Gly Phe Tyr Pro Ser Asp I le Al a val Glu Trp Glu Ser
`395
`400
`405
`
`ASn Gly Gln Pro Glu Asn ASn Tyr Lys Thr Thr Pro Pro Met Leu
`410
`4 15
`420
`
`Asp Ser Asp Gly Se r Phe Phe Leu Ty r Ser Ly s Leu Thr Val Asp
`425
`430
`435
`
`Lys Ser Arg Trp Gln Gl n Gl y Asn Val Phe Ser Cys Ser Val Met
`440
`445
`450
`
`His Glu Ala Leu His Asn His Ty r Thr Gln Ly s Ser Leu Ser Leu
`455
`460
`465
`
`Ser Pro Gly Lys
`469
`
`(2) INFORMATION FOR S EQ ID N0:24:
`
`(i) SEQUENCE CHARACTERISTICS:
`(A) LENGTH: 214 amino acids
`(B) TYPE: Amino Aci d
`(D) TOPOLOGY: Linear
`
`(xi) SEQUENCE DESCR IPTION : SEQ IO N0: 24 :
`
`ASp Va l Gln Met Thr Gl n Thr Th r Ser Ser Leu ser Ala ser Leu
`1
`5
`1 0
`15
`
`Gly Asp Ar g Va l Th r Ile Asn Cys Ar g Al a Se r Gln Asp Ile Asn
`25
`~
`~
`
`Asn Ty r Leu Asn Trp Tyr Gln Gln Ly s Pro Asn Gly Thr Val Ly s
`35
`40
`45
`
`Leu Leu I l e Tyr Tyr Thr Ser Th r Leu His Ser Gl y Va l Pro Ser
`w
`55
`~
`
`Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr I le
`65
`70
`75
`
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`83
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`US 6,407,213 Bl
`
`-continued
`
`84
`
`Ser Asn Le u Asp Gln Glu Asp Ile Ala Thr Ty r Phe Cys Gl n Gln
`H
`85
`~
`
`Gly Asn Thr Leu Pr o Pro Tbr Ph e Gly Gly Gly Thr Lys Val Glu
`95
`1 00
`1 05
`
`Ile Lys Arg Thr Val Ala Ala Pro ser Va l Phe I le Phe Pro Pro
`110
`115
`120
`
`Ser Asp Glu Gln Le u Lys Ser Gly Thr Ala Ser Va l Val Cys Le u
`125
`130
`135
`
`Leu Asn Asn Phe Tyr Pro Ar g Glu Ala Lys Val Gln Trp Lys Val
`140
`1 45
`150
`
`Asp Asn Ala Leu Gln ser Gl y Asn ser Gln Glu ser val Thr Glu
`155
`160
`165
`
`Gln Asp Ser Lys Asp Ser Thr Ty r Ser Leu Ser Ser Thr Leu Thr
`170
`175
`180
`
`Leu ser Lys Ala Asp Tyr Glu Lys His Lys va l Tyr Ala cys Glu
`185
`190
`195
`
`Val Th r His Gln Gly Leu Ser ser Pro Val Thr Lys Ser Ph e Asn
`200
`205
`210
`
`Ar g Gly Glu Cy s
`214
`
`(2) INFORMAT ION FOR SEQ ID N0:25:
`
`(i) SEQUENCE CHARACTERISTICS :
`(A) LENGTH : 233 amino acids
`(B) TYPE : Amino Aci d
`( D) TOPOLOGY : Linea r
`
`(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
`
`Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Tbr Al a Thr
`1
`5
`10
`15
`
`Gl y Val His ser Asp Ile Gln Met Thr Gln ser Pro ser ser Le u
`25
`~
`~
`
`Ser Ala Ser Va l Gly Asp Ar g Val Tbr I le Thr Cys Ar g Al a Ser
`35
`40
`45
`
`Gln Asp I le Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
`55
`~
`~
`
`Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu His Ser
`65
`70
`75
`
`Gly Va l Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr
`80
`85
`90
`
`Thr Leu Thr I l e Ser ser Leu Gln Pro Gl u Asp Phe Ala Thr Tyr
`9 5
`1 00
`1 OS
`
`Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro Thr Phe Gly Gln Gly
`110
`115
`1 20
`
`Thr Lys Val Glu Ile Lys Arg Th r Val Ala Ala Pro Ser Va l Phe
`125
`1 30
`135
`
`Ile Phe Pro Pro Ser Asp Gl u Gln Leu Lys Ser Gly Thr Al a Ser
`140
`145
`150
`
`Va l Val Cys Leu Leu Asn Asn Phe Ty r Pro Arg Glu Al a Lys Va l
`155
`160
`165
`
`Gin Trp Lys Val Asp Asn Ala Leu Gl n Ser Gly Asn Ser Gl n Gl u
`175
`180
`170
`
`Ser Val Thr Glu Gln Asp Ser Ly s Asp Ser Thr Ty r Ser Leu Ser
`185
`190
`195
`
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`85
`
`US 6,407,213 Bl
`
`-continued
`
`86
`
`Ser Th r Le u Thr Leu Ser Lys Al a Asp Tyr Glu Lys Hi s Lys Va l
`200
`205
`2 10
`
`Tyr Ala Cy s Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Th r
`220
`225
`2 15
`
`Lys ser Phe Asn Arg Gly Gl u c ys
`230
`233
`
`(2) INFORMAT ION FOR S EQ ID N0 :26:
`
`(i) SEQUENCE CHARACTERISTICS :
`(A) LENGTH: 122 amino aci ds
`(B) TYPE : Amino Acid
`( D) TOPOLOGY : Linea r
`
`(xi) SEQUENCE DESCRIPTI ON: SEQ ID N0 :26:
`
`Glu Val Gin Leu Val Glu Ser Gl y Gly Gl y Le u Va l Gl n Pro Gly
`5
`1 0
`1 5
`
`Gl y Ser Leu Ar9 Leu Ser Cys Ala Al a Ser Gl y Tyr Ser P he Thr
`20
`25
`30
`
`Gl y Ty r Thr Met Asn Tr p Val Ar9 Gin Al a Pro Gly Lys Gl y Le u
`35
`40
`45
`
`Glu Trp Va l Al a Leu I le ASn Pro Tyr Lys Gl y Va l Thr Th r Tyr
`55
`~
`~
`
`Ala Asp Ser Val Lys Gly Ar9 Ph e Thr I le Ser Val Asp Lys Ser
`65
`70
`75
`
`Lys Asn Thr Al a Tyr Leu Gl n Met Asn Ser Le u Arg Ala Gl u Asp
`8 5
`H
`~
`
`Thr Ala Val Tyr Tyr Cys Ala Ar9 Ser Gly Tyr Tyr Gl y Asp Ser
`1 00
`1 OS
`95
`
`Asp Trp Tyr Phe Asp Va l Trp Gl y Gin Gl y Thr Leu Val Th r Va l
`120
`110
`115
`
`ser ser
`122
`
`50
`
`We claim:
`1. A humanized antibody variable domain compnsmg
`non-human Complementarity Determining Region (CDR) 45
`amino acid residues which bind an antigen incorporated into
`a human antibody variable domain, and further comprising
`a Framework Region (FR) amino acid substitution at a site
`selected from the group consisting of: 4L, 38L, 43L, 44L,
`58L, 62L, 65L, 66L, 67L, 68L, 69L, 73L, 85L, 98L, 2H, 4H,
`36H, 39H, 43H, 45H, 69H, 70H, 74H, and 92H, utilizing the
`numbering system set forth in Kabat.
`2. The humanized variable domain of claim 1 wherein the
`substituted residue is the residue found at the corresponding
`location of the non-human antibody from which the non(cid:173)
`human CDR amino acid residues are obtained.
`3. The humanized variable domain of claim 1 wherein no
`human Framework Region (FR) residue other than those set
`forth in the group has been substituted.
`4. The humanized variable domain of claim 1 wherein the
`human antibody variable domain is a consensus human 60
`variable domain.
`5. The humanized variable domain of claim 1 wherein the
`residue at site 4L has been substituted.
`6. The humanized variable domain of claim 1 wherein the
`residue at site 38L has been substituted.
`7. The humanized variable domain of claim 1 wherein the
`residue at site 43L has been substituted.
`
`8. The humanized variable domain of claim 1 wherein the
`residue at site 44L has been substituted.
`9. 'lbe humanized variable domain of claim 1 wherein the
`residue at site 58L has been substituted.
`10. The humanir..ed variable domain of claim 1 wherein
`the residue at site 62L bas been substituted.
`11. The humanized variable domain of claim 1 wherein
`the residue at site 65L has been substituted.
`12. The humanized variable domain of claim 1 wherein
`the residue at site 66L has been substituted.
`13. The humanized variable domain of claim 1 wherein
`the residue at site 67L has been substituted.
`14. The humanized variable domain of claim 1 wherein
`55 the residue at site 68L bas been substituted.
`15. The humanized variable domain of claim 1 wherein
`the residue at site 69L has been substituted.
`16. The humanized variable domain of claim 1 wherein
`the residue at site 73L has been substituted.
`17. The humanized ''ariable domain of claim 1 wherein
`the residue at site 85L has been substituted.
`18. The humanized variable domain of claim 1 wherein
`the residue at site 98L has been substituted.
`19. The humanized variable domain of claim 1 wherein
`65 the residue at site 2H has been substituted.
`20. The humanized variable domain of claim 1 wherein
`the residue at site 4H has been substituted.
`
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`87
`21. The humanized variable domain of claim 1 wherein
`the residue at site 36H has been substituted.
`22. The humanized variable domain of claim 1 wherein
`the residue at site 39H bas been substituted.
`23. The humanized variable domain of claim 1 wherein
`the residue at site 43H has been substituted.
`24. The humanized variable domain of claim 1 wherein
`the residue at site 45H has been substituted.
`25. The humanized variable domain of claim 1 wherein
`the residue at site 69H has been substituted.
`26. The humanized variable domain of claim 1 wherein
`the residue at site 70H has been substituted.
`27. The humanized variable domain of claim 1 wherein
`the residue at site 74H has been substituted.
`28. The humanized variable domain of claim 1 wherein
`the residue at site 92H has been substituted.
`29. An antibody comprising the humanized variable
`domain of claim 1.
`30. An antibody which binds p185HERZ and comprises a
`humanized antibody variable domain, wherein the human(cid:173)
`ized an tibody variable domain comprises non-human
`Complementarity Determining Region (CDR) amino acid
`residues which bind p185HEm incorporated into a human
`antibody variable domain, and further comprises a Frame(cid:173)
`work Region (FR) amino acid substitution at a site selected
`from the group consisting of: 4L, 38L, 43L, 44L, 46L, 58L, 25
`62L, 65L, 66L, 67L, 68L, 69L, 73L, 85L, 98L, 2H, 4H, 36H,
`39H, 43H, 45H, 69H, 70H, 74H, 75H, 76H, 78H and 92H,
`utilizing the numbering system set forth in Kabat.
`31. The antibody of claim 30 wherein the substituted
`residue is the residue found at the corresponding location of
`the non-human antibody from which the non-human CDR
`amino acid residues are obtained.
`32. The antibody of claim 30 wherein no human Frame(cid:173)
`work Region (FR) residue other than those set forth in the
`group has been substituted.
`33. The antibody of claim 30 wherein the human antibody
`variable domain is a consensus human variable domain.
`34. The antibody of claim 30 wherein the residue at site
`4L has been substituted.
`35. The antibody of claim 30 wherein the residue at site
`38L has been substituted.
`36. The antibody of claim 30 wherein the residue at site
`43L bas been substituted.
`37. The antibody of claim 30 wherein the residue at site
`44L has been substituted.
`38. The antibody of claim 30 wherein the residue at site
`46L has been substituted.
`39. The antibody of claim 30 wherein the residue at site
`58L bas been substituted.
`40. The antibody of claim 30 wherein the residue at site
`62L bas been substituted.
`41. The antibody of claim 30 wherein the residue at site
`65L has been substituted.
`42. The antibody of claim 30 wherein the residue at site
`66L bas been substituted.
`43. The antibody of claim 30 wherein the residue at site
`67L has been substituted.
`44. The antibody of claim 30 wherein the residue at site
`68L has been substituted.
`45. The antibody of claim 30 wherein the residue at site
`69L bas been substituted.
`46. The antibody of claim 30 wherein the residue at site
`73L has been substituted.
`47. The antibody of claim 30 wherein the residue at site
`85L has been substituted.
`48. The antibody of claim 30 wherein the residue at site
`98L has been substituted.
`
`88
`49. The antibody of claim 30 wherein the residue at site
`2H has been substituted.
`50. The antibody of claim 30 wherein the residue at site
`4H has been substituted.
`51. The antibody of claim 30 wherein the residue at site
`36H bas been substituted.
`52. The antibody of claim 30 wherein the residue at site
`39H has been substituted.
`53. The antibody of claim 30 wherein the residue at site
`tO 43H bas been substituted.
`54. The antibody of claim 30 wherein the residue at site
`45H has been substituted.
`55. The antibody of claim 30 wherein the residue at site
`69H has been substituted.
`56. The antibody of claim 30 wherein the residue at site
`70H bas been substituted.
`57. The antibody of claim 30 wherein the residue at site
`74H has been substituted.
`58. The antibody of claim 30 wherein the residue at site
`20 75H has been substituted.
`59. The antibody of claim 30 wherein the residue at site
`76H bas been substituted.
`60. The antibody of claim 30 wherein the residue at site
`78H has been substituted.
`61. The antibody of claim 30 wherein the residue at site
`92H has been substituted.
`62. A humanized antibody variable domain comprising
`non-human Complementarity Determining Region (CDR)
`amino acid residues which bind an antigen incorporated into
`30 a consensus human variable domain, and further comprising
`an amino acid substitution at a site selected from the group
`consisting of: 4L, 38L, 43L, 44L, 46L, 58L, 62L, 65L, 66L,
`67L, 68L, 69L, 73L, 85L, 98L, 2H, 4H, 36H, 39H, 43H,
`45H, 69H, 70H, 74H, 75H, 76H, 78H and 92H, utilizing the
`35 numbering system set forth in Kabat.
`63. A humanized antibody which lacks immunogenicity
`compared to a non-human parent antibody upon repeated
`administration to a human patient in order to treat a chronic
`disease in that patient, wherein the humanized antibody
`40 comprises non-human Complementarity Determining
`Region (CDR) amino acid residues which bind an antigen
`incorporated into a human antibody variable domain, and
`further comprises an amino acid substitution at a site
`selected from the group consisting of: 4L, 38L, 43L, 44L,
`45 46L, 58L, 62L, 65L, 66L, 67L, 68L, 69L, 73L, 85L, 98L,
`2H, 4H, 36H, 39H, 43H, 45H, 69H, 70H, 74H, 75H, 76H,
`78H and 92H, utilizing the numbering system set forth in
`Kabat.
`64. A humanized variant of a non-human parent antibody
`so which binds an antigen and comprises a human variable
`domain comprising the most frequently occurring amino
`acid residues at each location in all human immunoglobulins
`of a human heavy chain immunoglobulin subgroup wherein
`amino acid residues forming Complementarity Determining
`55 Regions (CDRs) thereof comprise non-human antibody
`amino acid residues, and further comprises a Framework
`Region (FR) substitution where the substituted FR residue:
`(a) noncovalently binds antigen directly; (b) interacts with a
`CDR; (c) introduces a glycosylation site which affects the
`60 antigen binding or affinity of the antibody; or (d) participates
`in the Vc V H interface by affecting the proximity or orien(cid:173)
`tation of the V L and V H regions with respect to one another.
`65. The humanized variant of claim 63 which binds the
`antigen up to 3-fold more in the binding affinity than the
`65 parent antibody binds antigen.
`66. A humanized antibody heavy chain variable domain
`comprising non-human Complementarity Determining
`
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`
`10
`
`89
`Region (CDR) amino acid residues which bind antigen
`incorporated into a human antibody variable domain, and
`further comprising a Framework Region (FR) amino acid
`substitution at a site selected from the group consisting of:
`24H, 73H, 76H, 78H, and 93H, utilizing the numbering
`system set forth in Kabat.
`67. The humanized variable domain of claim 66 wherein
`the substituted residue is the residue found at the corre(cid:173)
`sponding location of the non-human antibody from which
`the non-human CDR amino acid residues are obtained.
`68. The humanized variable domain of claim 66 wherein
`no human Framework Region (FR) residue other than those
`set forth in the group bas been substituted.
`69. The humanized variable domain of claim 66 wherein
`the human antibody variable domain is a consensus human
`variable domain.
`70. The humanized variable domain of claim 66 wherein
`the residue at site 24H has been substituted.
`71. The humanized variable domain of claim 66 wherein
`the residue at site 73H has been substituted.
`72. The humanized variable domain of claim 66 wherein
`the residue at site 76H has been substituted.
`73. The humanized variable domain of claim 66 wherein
`the residue at site 78H has been substituted.
`74. The humanized variable domain of claim 66 wherein 25
`the residue at site 93H has been substituted.
`75. The humanized variable domain of claim 66 which
`further comprises an amino acid substitution at site 71H.
`76. The humanized variable domain of claim 66 which
`further comprises amino acid substitutions at sites 71H and 30
`73H.
`77. The humanized variable domain of claim 66 which
`further comprises amino acid substitutions at sites 71H, 73H
`and 78H.
`
`20
`
`90
`78. An antibody compnsmg the humanized variable
`domain of claim 66.
`79. A humanized variant of a non-human parent antibody
`which binds an antigen, wherein the humanized variant
`comprises Complementarity Determining Region (CDR)
`amino acid residues of the non-human parent antibody
`incorporated into a human antibody variable domain, and
`further comprises Framework Region (FR) substitutions at
`heavy chain positions 71H, 73H, 78H and 93H, utilizing the
`numbering system set forth in KabaL
`80. A humanized antibody variable domain comprising
`non-human Complementarity Determining Region (CDR)
`amino acid residues which bind an antigen incorporated into
`a human antibody variable domain, and further comprising
`a Framework Region (FR) amino acid substitution where the
`JS substituted FR residue:
`(a) noncovalently binds antigen directly;
`(b) interacts with a CDR; or
`(c) participates in the Yc V H interface by affecting the
`proximity or orientation of the V L and V n regions with
`respect to one another, and wherein the substituted FR
`residue is at a site selected from the group consisting of:
`4L, 38L, 43L, 44L, 58L, 62L, 65L, 66L, 67L, 68L, 69L,
`73L, 85L, 98L, 2H, 4H, 24H, 36H, 39H, 43H, 4SH,
`69H, 70H, 73H, 74H, 76H, 78H, 92H and 93H, utiliz(cid:173)
`ing the numbering system set forth in Kabat.
`81. The humanized variable domain of claim 80 wherein
`the substituted residue is the residue found at the corre(cid:173)
`sponding location of the non-human antibody from which
`the non-human CDR amino acid residues are obtained.
`82. The humanized variable domain of claim 80 wherein
`no human Framework Region (FR) residue other than those
`set forth in the group has been substituted.
`
`* * * * *
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`BEST AVAILABLE COPY PCT/GB90/0l017
`-
`l
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`
`HtJKANISED ANTIBODIES
`
`F~eld of the Invention
`The present 1nvention relates ~o h~ised antibody
`molecules, to processes for their production ua1ng
`recombLnant DNA technoloqy, and to theLr therapeutic usee.
`
`The term •hnmaoiaed antibody molecule" ia aaed to describe
`a molecule havinq an antigen binding site derived from an
`~oqlobulin from a non-human species, and remoininq
`~oglobul~-derived parts of the molecule being der1ved
`from a human i=munoglobulin.
`The antigen binding s1te
`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 J
`CDRs (CDR!, 'CDR2 and CDR3) in each of the heavy and liqht
`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 jmmunoglohulins 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 immunoglobulins comprise a
`generally Y-shaped molecule havinq ·an antiqe.n-bi.ndi.nq 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 immunoqlobulins.
`
`Natural immunoqlobulins have been us.ed in assay, diaqnosis
`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
`
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`of t D"'nmoqlohu.U.na aa therapeutic agenta waa the ~covery
`of proce<lu.re.a for the producti.on of monoclonal. antibodies
`(~a) of defined apecifici.ty (1).
`
`HoWever, moat MAbs are produced by hybridomaa whi.ch are
`fasiona of rodent spleen cella with rodent myeloma
`cella.
`They are therefore essentia11y rodent proteins.
`There are very f~ reports of the production of human MAba.
`
`Since most available HAbs are of rodent oriq:i.n, they are
`naturally antigenic i.n humans and tbua can gi.ve rise to an
`undesirable i mmnne response termed the BAMA (Duman
`Anti-Mouse Antibody) response.
`Therefore, the use of
`rodent MAbs as therapeutic agents in humans is inherently
`l~ted by the fact that the human subject will mount an
`Lmmunological response to the MAb and will either remove
`it entirely or at least reduce its effectiveness.
`Xn
`practice, M.Abs of rodent origin may not be used in
`patients for more than one or a few treatments as a BAMA
`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 co~ntries· throughout the worrd ·
`as an immunosuppressant in the treat=ent of acute
`allograft rejection (Chatenoud et al (2) and Jeffe~s et al
`However, i.n view of the rodent nature of thi.s and
`( 3) ] •
`other such KAbs, a significant BAMA response which may
`include a majo.r anti-idiotype component, may build up on
`uae.
`c~~arly, it would be highly desirable to djmjn;sh
`or abolish this undesirable BAHA response and thus enlarge
`the areas of use of these very useful antibodies.
`
`Proposals have therefore been made to render non-human
`MAbs lesa antigenic in hWDAns.
`Such techniques can be
`geiJerieally termed •hnman.i sation• techniques.
`These
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`technJ.quea typical.ly .involve the use of recombinant DNA
`technology to manipulate DNA sequences encoding the
`polypeptide chaine of the antibody molecule.
`
`Early methods for hnmani sing HAba involved production of
`chimeric antibodies in which an antigen bindinq site
`comprising the complete variable domains of one antibody
`is linked to constant domains derived from another
`antLbody. Methods for carry~g out such chimerisat~on
`procedures are described in BP0120694 (Celltech L~ted),
`EP012502l (Genentech me. and City of Hope), BP-A-0 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) ~scloses a
`process for preparinq an antibody molecule havinq the
`variable domains from a mouse MAb and the constant domains
`from a human immunoglobulin.
`Such humaniaed chimeric
`antibodies, however, still ·c~ntain a siqnificant
`proportion of non-human amino acid sequence, i.e. the
`complete non-human variable domains, and thus may still
`elicit some RAMA response, particularly if administered
`over a prolonged period (Bagent at 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 domain• o£ a human L=munoglobulin
`by site directed mutagenesis usLng long oligonucleotides.
`The present invention relates to humanised antibody
`molecules prepared according to this alternative approach,
`i.e. CDR-grafted humariised aptibody molecules.
`Such
`CDR-grafted humanised antibodies are much less likely to
`give rise to a HAMA response than humanised chimeri~
`antibodies in view of the much loWer proportion of
`non-human amino acid sequence which they contain •
`
`. '
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`The earliest work on bnment si.nq NAbs by CDR-qra£ti.ng was
`carried out on HAbs reeoqni.si.nq synthetJ.c antigens, such
`as the NP or Nl:P antigens .
`However, ex•mples J.n whi.cb a
`mouse HAb recoqnisi.nq lysozyme and a rat ~ recoqnisi.ng
`an antigen on huma.a T-cells were httman~sed by CDR-qra£tJ.ng
`have been described by Verhoeyen et al ( 5) and Riechmenn
`et al (6) respectively.
`The preparation of CDR-grafted
`antibody to the antigen on bwaa.a 'f' cell.s is also described
`in WO 89/07452 (Me~al Research CouncL!).
`
`rn RJ.ecbmana et ~/Medic&! Research Council it was found
`that transfer of the CDR regions alone (as defi.ned by
`Kabat refs. (7) and (8)} was not sufficient to provide
`satisfactory antigen bi.nding activi.ty in the CDR-qrafted
`product.
`Riecbm•nn 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 havinq improved antiqen
`binding activity.
`This residue at position 27 of the
`heavy chain is within the structur&l loop adjacent to
`CDR!.
`A further construct which additionally contained a
`human serine to rat tyrosine chanqe at position 30 of the
`heavy chain did not have a significantly altered binding
`activity over the hnmanised antibody witb the ser.ine to
`phenylalanine change at position 27 alone.
`These results
`indicate that changes t .o residues of the human sequence
`outside the CDR regions, in particular in the structural
`l.oop adjacent to CDRl, may be necessary to obtain
`effective antigen bindinq activity for CDR-grafted
`Even
`antibodies which recognise more complex antigens .
`ao . tbe b;nd1ng affinity of the .best CDR-grafted antibodies
`obtained was still. aiqnifi.cantly leas than the original
`MAb.
`
`Very recently Queen et al (9) have described the
`preparation of a hnmaniaed antibody that binds to the
`
`\
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`J.nter~aulc.in 2 receptor, by combLn.i.ng tha CDRa of a marine
`MAb ( &.llti-Tac) with hWDAD
`i mmgaoglobul.in framework and
`constant regions.
`The -human framework regions were
`chosen to maximise h~logy with the anti-Tac HAb
`sequence.
`In addition computer model~ing was used to
`identify framework amino acid residues which ware lLkely
`to interact with the CDRa or antigen, and mouse amino
`acids ware used at these positions in the hmaaaiaed
`Alltihody.
`
`In WO 90/07861 Quean at al propose four criteria for
`desiqning humaniaed immunoqlobulins.
`The first criterion
`is to use as the human acceptor the framework from a
`particu~ar .human Lmmunoqlobulin that is unusu~ly
`·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 uae the donor
`amino acid rather than the acceptor if the human acceptor
`r .esidue is unusual. and the donor residue is typical. for
`human sequences at a specific residue of the framework .
`The third criterion ia to use the donor framework amLno
`acid residue rather than the acceptor at positions
`immediately adjacent to the CDRs.
`The fourth criterion
`is to use the donor amino acid residua at framework
`positions at whl.ch the a.m.lno acid is .predicted to. have a ·
`si.de chai.n atom withi.n about 3 A of the CORa in a'
`three-d.imenaionu immunoglobulin model and to be . capab~e
`of interacting with the antigen or with the CDRs of the
`bnmanj sed .immunoqlobulin.
`It is proposed that criteria
`two, three or four may be applied in addition or
`alternati.vely to criterion one·, and may be appli.ed singly
`,or in any combination.
`
`WO 90/078·61 describes i.n detai.~ the preparati.on of a
`sinqle CDR-grafted humaniaad antibody, a humanised
`antibody havinq specificity for the p55 Tac proteLn of the
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`The c:ambina.ti.on of al.1 four criteri..a, as
`IL-2 receptor.
`above, were employed J.n desiqn.i.Dq th.ia humani sed a..uti.bociy,
`the variable reqion frameworks of the human antibody Eu
`(7) be.inq used 48 ac:c:e~tptor.
`l:n the resultant hnmeniae<l
`antibody the donor CD~ a were as defined by Kabat et al ( 7
`and 8) and .in add.ition the mouse donor residues were used
`in place of the human acceptor re~idues, at position• 27,
`30, 4a, 66, 67, 89, 91, 94, 103, 104, lOS and 101 in the
`heavy cha..i.n and at positions 48, 60 and 63 in the liqht
`cha.i.n, of the variable reqion frameworbl.
`The hu.m.&ni.sed
`anti-'l'ac antibody obtained i.a reported to have an affJ..ni.ty
`for pSS of 3 x 109 Mll, about one-third of that of the
`murine HAb.
`
`We have further i.nvesti.qated the preparation of CDR(cid:173)
`grafted humani.sed antibody molecules and have identified a
`Mera.rchy of positions wi.thi.n the framework of the
`variable reqions (i.e. outside both th~ Kabat CORa and
`st~ctural loops of the variable reqi.ons) at whlch the
`ami.no acid identities of the ·residues are important for
`obtaining CDR-grafted products with satisfactory bindinq
`affi.n.ity.
`This ha.s enabled us to establish a protocol
`for obtaininq satisfactory CDR-qrafted products which may
`be applied very widely .irrespective of the l.evel of
`homoloqy between the donor immunoqlobu1.in and acceptor
`framework.
`The set of residues which we have identified
`as beinq of critical importance does not coincide with the
`residues identified by Queen et al. (9).
`
`Snmmacy of the Invention
`Accordingl.y, in a first aspect the invention provides a
`CDR-grafted antibody heavy chain havinq a variabl.e reqion
`domain comprising acceptor framework and donor antiqen
`bindinq 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.
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`ID pre£erred embcKU menta 1 the heavy C~ £ram.ework
`campr1aea donor res1dnes at poa1tiona 23, 24, 49, 71, 73
`and 78 or at pos.itionli 23, 24 and 49.
`The residues at
`positions 71, 73 and 78 of tbe heavy chain framework are
`preferably either all acceptor or all donor residues.
`
`In particularly pre£erred embodiments the heavy ch~
`framework additionally compr1aea d~nor residue• at one,
`soma or all of positions 6, 37, 48 and gc·. Also it .is
`particularly preferred that residnea at positions of the
`heavy chain framework wh.ic~ 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 reaidues. Moat
`preferably the heavy chain framework additionally
`comprises donor residues at positions 2, 4, 6, 25, 36, 37,
`39, 47, 48, 93, 94, 103, 104, 106 and 107.
`
`In addition the heavy chain fr&JD.ework optionally comprises
`donor residues at one, some or all of posit ions:
`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 firat and other aspects of the present invention
`reference is made to CDR-grafted antLbody products
`comprising acceptor framework and donor antiqe~ binding
`regions.
`It will be appreciated that the invention is
`widely applicable to the CDR-qraftinq of antibodies in
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`geueral..
`Thus, the donor and acceptor an~ea may be
`derived £rom antmala of the same species and eveu same
`anti.bod.y cl.aas or . aub-claaa. More uaua.ll.y, however, the
`donor and acceptor an~~·· are derived from anim•ls o£
`different species. Typically the donor antLbody ia a
`non-huma..n antibody, such aa a rodent MAb, and the acceptor
`anti.body J.a a hum&D antibody.
`
`In the first ADd other aspects of the present invention,
`the donor antigen binding region typical.l.y comprises at
`least one CDR from the donor antibody. Osual.ly · the donor
`antigen binding reqion comprises at l.eaat two and
`preferabl.y all three CDRs of each of the heavy chain
`and/or light chaLn variable regions.
`The CORa may
`comprise the Kabat CDR•, the structural. ·loop CORa or a
`composite of the Kabat and structural. loop CDRs and any
`combination of an~ of these.
`Preferabl.y, the antigen
`binding regions of the CDR-grafted heavy chain variable
`domain comprise CORa corresponding to the Kabat CORa at
`COR2 (residues 50-65) ~d CORJ (residues 95-100) and a
`composite of the Kabat and structural. l.oop CORa at COR!
`(residues 26-35).
`
`The residue designations given above. and el.setoi'here .in ~he
`present appl.i.cation are numbered according to the Kabat
`nu.mberi.nq [r