`USOO7375193B2
`
`0
`
`(12) United States Patent
`(10) Patent N0.:
`US 7,375,193 B2
`
`Baca et a1.
`(45) Date of Patent:
`May 20, 2008
`
`(54) ANTI-VEGF ANTIBODIES
`
`(75)
`
`Inventors: Manuel Baca, Black Rock (AU);
`James A. Wells, Burlingamc, CA (US);
`Leonard G. Presta, San Francisco, CA
`(US); Henry B. Lowman, El Granada,
`CA (US); Yvonne NIan-Yee Chen, San
`Mateo, CA (US)
`
`W0
`\VO
`
`W0
`W0
`WO
`W0
`W0
`
`W0 92/ 18619
`“'0 92/2265}
`
`WO 94/04679
`W0 94/10202
`96/30046
`W0 98/45331
`WO 98/45332
`
`10/1992
`12/1992
`
`3/1994
`5/1994
`10/1996
`10/1998
`10/1998
`
`(73) Assignee: ginentech, Inc., South San Francisco,
`(US)
`,
`_
`.
`,
`,
`,
`( 7,: ) NOUCC:
`SlleCCT to any (115913191913 the term (‘me5
`patent is extended or adjusted under 35
`U~S~C~ 15403) by 0 days.
`
`(21) Appl. No.2 11/537,015
`
`(22)
`
`Filed:
`
`5613- 291 2006
`
`(65)
`
`Prior Publication Data
`US 2007/0248610 A1
`Oct. 25, 2007
`
`Related US. Application Data
`(60) Continuation Of application NO. 10/974,591, filed on
`Oct. 26, 2004, now Pat. No. 7,297,334, which is a
`continuation of application NO. 09/723,752, filed on
`Nov. 27, 2000, now Pat. No. 7,060,269, which is a
`division of application No. 08/908,469, filed on Aug.
`6, 1997, now Pat. NO. 6,884,879,
`‘
`,
`,
`,
`,
`PIOVISIOnal application NO‘ 60/126’446’ 1'1le on Apr.
`7, 1997~
`
`(60)
`
`(51)
`
`Int- Cl-
`(2006-01)
`C0 7K 16/00
`(52) US. Cl.
`...............................
`53 0/3811; 530/3873;
`530/38885; 424/1301; 435/3201; 536/2353
`(58) Field of Classification Search ............. 530/3871,
`530/3873. 388.85; 424/1301; 435/3201;
`536/2353
`.
`.
`.
`See applleatlon file for complete search history.
`References Cited
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`/2000 Jardieu et a1.
`4/2000 Carter et a1.
`4(2005 53” er al-
`6/2006 Ba” at 31'
`1/2007 Baca et a1.
`3/2002 Baca et a1.
`
`,
`.............. 530/3871
`
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`
`EP
`EP
`RP
`E},
`(.113
`GB
`WO
`W0
`
`07.39400
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`451216 BI
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`~
`(commuc‘i)
`Primary ExamineriDavid J. Blanchard
`Assistant Examiner Parithosh K. Tungaturthi
`
`(57)
`
`ABSTRACT
`
`Humanized and variant anti-VEGF antibodies and various
`
`uses therefor are disclosed. The anti-VEGF antibodies have
`
`
`
`strong binding a inities for VEGF; inhibit VEGF-indueed
`proliferation of endothelial cells in vitro; and inhibit tumor
`.
`.
`,
`grOWth 1“ V‘VO'
`
`22 Claims, 16 Drawing Sheets
`
`Pfizer v. Genentech
`|PR2017—01488
`Genentech Exhibit 2042
`
`Pfizer v. Genentech
`IPR2017-01488
`Genentech Exhibit 2042
`
`

`

`US 7,375,193 B2
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`Page 2
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`
`* cited by examiner
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 1 of 16
`
`US 7,375,193 B2
`
`A452
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`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 2 of 16
`
`US 7,375,193 B2
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 3 of 16
`
`US 7,375,193 B2
`
`“
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`(5)
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 4 of 16
`
`US 7,375,193 B2
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`*
`
`EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA
`
`31112.30
`
`EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWIRQA
`
`A413. E
`
`hulf)
`
`80
`7O
`60
`50 a
`PGKGLKWMGWINTYTGEPTYAADFKRRFTFSLETSASTAYL
`* *
`* *** ** *
`
`PGKGLEWVGWINTYTGEPTYAADFKRRFTISRDNSKETLYL
`
`$1112. 3 G
`
`
`PGKGLEWVGWINTYTGEPTYAADFKRRFT:QLDTSASTVYL
`
`Akififl
`
`hLEZfl
`
`110
`lOOabcdef
`90
`abs
`Q1SNLKNDDTATYFCARYPHYYGSSHWYFDVWGAGTTVTVSS
`*7???
`*7'rir
`it
`7%
`:k
`75:
`1k
`
`(8an N09)
`
`QMNSLRAEDTAVYYCARYFfinySSHWYFDVWGQGTLVTVSS
`
`(mama N0114)
`
`E1112. 3 U
`
`QMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSS
`
`(SEQJD N<Z§:16)
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 5 of 16
`
`US 7,375,193 B2
`
`WW\‘
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 6 of 16
`
`US 7,375,193 B2
`
`Stu
`
`
`
`S‘di
`
`pimA
`
`QBRSQZ cm
`
`TRANSFORM E. coii
`
`+M1 3541K)? HELPER PHAGE
`
`FABn—pfil FUSEON
`x”
`
`\
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 7 of 16
`
`US 7,375,193 B2
`
`GAATTCAACT TCTCCATACT TTGGATAAGG;MARACAGAC ATGAAAAATC TCATTGCTGA
`
`60
`
`GTTGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC TTCGCAATAT GGCGCAAAAT
`
`120
`
`GACCAACAGC GGTTGATTGA TCAGGTAGAG GGGGCGCTGT ACGAGGTAAA GCCCGATGCC
`
`180
`
`AGCATTCCTG ACGACGATAC GGAGCTGCTG CGCGATTACG TAAAGAAGTT ATTGAAGCAT
`
`246
`
`CC TCGTCAGT AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT
`
`300
`
`TATAGTCGCT TTGTTTTTAT TTTTTAATGT ATTTGTAACT AGAATTCGAG CTCGGTACCC
`
`360
`
`GGGGATCCTC TAGAGGTTGA GGTGATTTT ATG AAA AAG AAT ATC GCA TTT CTT
`
`413
`
`Net Lys Lya Ash Ila Ala Phe Leu
`«23
`~20
`
`CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT ACA AAC GCG TAC GCT GAT
`
`461
`
`Lea Ala 8e: Met The Val Pha Se: Ila Ala Th: ASh Ala Tl: Ala Aap
`«15
`m10
`m5
`1
`BEGIN stII SIGNAL SEQUENCE
`ATC GAG TTG ACC CAG TCC CCG AGC TCC CTG TCC GCC TCT GTG GGC GAT
`
`509
`
`Ila Gln Leu Th: Gln Se: Pra Se: Se: Leu Se: Ala Se: Val Gly A39
`5
`10
`15
`
`AGG GTC ACC ATC ACC TGC AGC GCA AGT CAG GAT ATT AGC AAC TAT TTA
`
`557
`
`Arg Val Th: Tle Th: Cys Se: Ala Se: Glh Asp Tle Se: Aan Ty: Leu
`20
`25
`30
`BEGIN LIGHT CHAIN
`AAC TGG TAT CAA CAG AAA CCA GGA AAA GCT GCG AAA CTA CTG ATT TAC
`
`605
`
`Asa Trp Ty: Gln Gln Lys Prw Gly Lya Ala Pra Lys Lea Len Ila Ty:
`35
`40
`45
`
`TTC ACC TCC TCT CTC CAC TCT GGA GTC CCT TCT CGC TTC TCT GGA TCC
`
`653
`
`?he Th: Se: Se: Leu His Se: Gly Val Pro Se: Arq Pha Se: Gly Se:
`50
`55
`60
`65
`
`GGT TCT GGG ACG GAT TAC ACT CTG ACC ATC AGC AGT CTG CAG CCA GgA
`
`701
`
`Gly Se: Gly Th: Asp Ty: Th: Leu Th: Ile Se: Se: Lea Gln Pra Glu
`70
`75
`80
`
`GAC TTC GCA ACT TAT TAC TGT CAA CAG TAT AGC ACC GTG CCG TGG ACG
`
`749
`
`Asp Phe Ala Th: Ty: Ty: Cya Gln Gln Ty: Se: Th: Val Pro Trp Th:
`85
`g0
`95
`
`TTT GGA CAG GGT ACC AAG GTG GAG ATC AAA CGA ACT GTG GCT GCA CCA
`
`797
`
`The Gly Gln Gly Th: Lys Val Glu Ila Lys Arg Th: Val Ala Ala Prg
`100
`105
`110
`
`TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG GAG TTG AAA TCT GGA ACT
`
`845
`
`Se: Val The Tle Pha Pre PIG Se: Asp Glu Gln Leu Lys Se: Gly Th:
`115
`120
`125
`
`CT TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA
`
`893
`
`Ala Se: Val Val Cys Leu Leu Asa Asa Phe Ty: Pro Arr Glu Ala Lys
`130
`135
`140
`145
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 8 of 16
`
`US 7,375,193 B2
`
`GTA GAG TGG AAG GTG GAT AAG GGC GTG CAA TCG GGT
`
`Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
`150
`155
`
`AGT GTG AGA GAG GAG GAG AGG AAG GA
`
`G AGC ACG TAC
`
`Ser Val Thr Glu Gln Asp Ser Lys As
`165
`17
`
`p Ger Thr Tyr
`O
`
`AAG TGG
`Asn Ser
`
`AGG CTC
`Ser Leu
`175
`
`GAG GAG
`Gln Glu
`169
`
`AGC AG“
`Sat Ser
`
`941
`
`889
`
`AGC GTG AGG GTG A§G AAA GGA GAG TAG GAG AAA GAG
`
`AAA GTG
`
`TAG GGG
`
`1037
`
`Thr Lea Thr Lea Ser Lys Ala Asp Tyr Glu Lys His
`189
`185
`
`Lys Val
`
`Tyr Ala
`
`TGC GAA GTC ACC CAT GAG GGC CT: AGC TCG CCC GTC
`
`ACA AAG
`
`AGC TTC
`
`1085
`
`Cys Glu Val Th: His Gln Gly Leu Sex Ser Pro Val Th: Lys Ser Phe
`195
`200
`205
`
`AAC AGG GGA GAG T4T T AAGCTGATCC TGTAGGCGGG AGG”ATGGTG
`
`1131
`
`Asn Arg Gly Glu Cys 0 CA (SEQJIENEA130)
`210
`
`GGCCTAGTAG GGAAGTAGTG GTAAAAAGGG TATCTAGAGG TTGAGGTGAT TTT ATG
`Met
`—23
`
`BEGIN StlI SIGNAL SEQUENCE
`AAA AAG AAT ATC GCA TTT CTT CTT GCA TGT ATG TTC
`GTT TTT
`TCT ATT
`Val Phe
`Sex Ila
`U10
`
`Lys Lys Asn Ila Ala Phe Leu Lea Ala Ser Met Phe
`129
`_15
`
`1187
`
`1235
`
`GGT ACA AAG GCG TAG GGT GAG GTT GAG CTG GTG GAG
`
`TCT ‘GC
`
`GGT GGG
`
`1283
`
`Ala Thr Asn Ala Tyr Ala Glu Val Gln Lea Val Glu
`m5
`1
`5
`
`Sex Gly
`
`Gly Gly
`10
`
`CTG GTG GAG GCA GGG GGG TGA GT‘ GGT TTG TCG TGT
`
`Leu Val Gln Tro Gly Gly Ser Lea Arg Lea Ser Gys
`15
`2@
`BEGIN HEAVY CHAIN
`TAT ACC TTC ACC AAC TAT GGT ATG AAC TGG ATC CGT
`
`Tyr Thr Phe Th: Asn Tyr Gly Met Asn Trp Ile Arg
`30
`35
`
`GCA GGT
`Ala Ala
`
`GAG GGC
`Gln Ala
`40
`
`TCT GG:
`
`1331
`
`Sex Gly
`25
`
`GCG GGT
`Pro le
`
`1379
`
`AA; GGC CTG GAA TGG GTT GGA TGG ATT AAC ACG TAT
`
`ACG lG“
`
`Lys Gly Leu Glu Trp Val Gly Trp Ile Asa Tbr Tyr
`45
`50
`
`Th: Gly
`55
`
`GAA CGG
`Glu Pro
`
`1427
`
`ACC TAT ICT GCG GAT TTC AAA GET CGT TTT ACT ATA
`
`Thr Tyr Ala A‘a Asp Phe Lys Arg Arg Phe Thr Ile
`60
`65
`70
`
`TCT GGA
`Sex Ala
`
`GAG AGG
`
`Asp Thr
`
`1475
`
`TGG AGG AAG ACA GTT TAG GTG GAG ATG AAC AGG CTG
`
`GGG GGT
`
`GAG GAG
`
`1523
`
`Ser Ser Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
`75
`80
`85
`
`Arg Ala
`
`Glu Asp
`90
`
`ACT GCC GTG TAT TAG TGT GGA AAG TAG CGG GAG TAT
`
`TAT GGG
`
`Thr Ala Val Tyr Tyr Cys Ala Lys Tyr PrQ His Tyr
`95
`196
`
`Tyr Gly
`
`1571
`
`AGC AG‘
`Set Ser
`105
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 9 of 16
`
`US 7,375,193 B2
`
`CAC TGG TAT TTC GAG GTC TGG GGT CAA GGA ACG CTG GTG AGC GTG TCC
`
`1619
`
`His Trp Tyr Fae Asp Val Trp Gly Gl_n Gly Thr Leu Val Thr Val Ser
`110
`115
`128
`
`TCG GCC TCC AGC AAG GG
`
`CCA TCG GTC TTC CCC CTG GCA CC
`
`TCC TCC
`
`1667
`
`Ser Ala Ser Tar Lys Gly Pro Ser Val Phe Pro Leu Ala PrQ Ser Ser
`125
`130
`135
`
`AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC AAG GAG
`
`5a.: \3 H {.71
`
`Lys Ser Thr Ser Gly le Thr Ala Ala Len Gly Cys Lea Val Lys Asp
`140
`145
`150
`
`TAG TTC CCC GAA GCG GTG ACG GTG TCG TGG AAC TCA GGC GCG CTG ACG
`
`1
`
`-1 31 5.1.3
`
`Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lea Thr
`155
`166
`165
`170
`
`AGC GGC GTG CAC AGC TTC CCG GCT GTC CTA GAG TCC TCA GGA ETC TAG
`
`1811
`
`Ser Gly Val His Thr Phe Pro Ala Val Len Gln Ser Ger Gly Lea Tyr
`175
`180
`185
`
`TCG CTC AGC AGC GTG GTG ACC GTG GCC TCC AGC AGC TTG GGG ACC CAG
`
`1859
`
`Ser Leu Ser Ser V l Val Thr Val Pro Ser Ser Ser Lsu Gly Thr Gln
`190
`125
`209
`
`ACG TAG ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTC GAG
`I»
`Thr Tyr lle Cys Asa Val Asn His Lvs Pro Sex Asa Thr Lys Val Asp
`205
`210
`215
`
`5-: K0 0 \3
`
`AAG AAA GTT GAG CCC AAA TCT TGT GAG AAA ACT CAC CTG TAG AGT GGC
`
`1955
`
`Lys Lys Val Glu Pro Lys Sex Cys Asp Lys Thr His Lsu
`220
`225
`239
`
`* Ser Gly
`
`GGT GGG TCT GGT TGC GGT GAT TTT GAT TAT GAA AAG ATG GGA AAC GCT
`
`h) C (2) £1.)
`
`Gly Gly Sex Gly Ger Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala
`235
`249
`245
`250
`END LIGHT CHAIN
`BE
`AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT GAA AAC GCG CTA GAG TCT
`
`2051
`
`Asa Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asa Ala Leu G1
`255
`260
`265
`
`GIN g3p DOMAIN
`GAG GCT AAA GGC AAA CTT GAT TCT GTC GCT ACT GAT TAG GGT GCT GCT
`
`2099
`
`Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tvr Gly Ala Ala
`270
`275
`289
`
`ATC GAT GGT TTC ATT GGT GAC GTT TCC GGC CTT GCT AAT GGT AAT GGT
`
`2147
`
`Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Lea Ala Asn Gly Asa Gly
`285
`290
`295
`
`GCT ACT SGT GAT TTT GCT GGC TCT AAT TCC CAA ATG GCT CAA GTC GGT
`
`2195
`
`Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly
`300
`305
`31G
`
`GAG GGT GAT AAT '
`UJHma fiW
`
`Asp Gly Asp Asa
`315
`
`CCT TTA ATG AAT AAT TTC CGT CAA TAT TTA CCT
`
`2243
`
`PrQ Leu Met Asn Asn Phe Arg Gln Tyr Lea Pre
`326
`325
`330
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 10 of 16
`
`US 7,375,193 B2
`
`TCC
`SeT
`
`CTC
`Leu
`
`CCT
`Pro
`
`_ Val
`
`GAA
`Glu
`
`TGT
`
`CGC
`
`Cys
`
`Arg
`
`CCT
`Pro
`340
`
`TTT
`Phe
`
`GTC
`Val
`
`TTT AGC
`Phe Ser
`
`AAA
`
`Lys
`
`CCA
`Pro
`
`TAT
`
`Ty:
`
`TCT
`Ser
`
`ATT
`Ile
`
`Phe
`
`GAT
`
`TGT
`
`GAG
`
`AAA
`
`Asp
`
`Cys
`$55
`
`Asp
`
`Lys
`
`ATA
`Ile
`
`AAC TTA
`Asn Leu
`358
`
`Ala Gly
`345
`
`TTC
`Phe
`
`CGT
`
`Arg
`
`GCT SGT
`
`2291
`
`GGT
`
`Gly
`
`GTC
`Val
`
`TTT
`HmTV
`365
`
`TTT
`Phe
`
`CTT
`Leu
`
`TTA
`Leu
`
`TAT
`
`Ty:
`376
`
`v
`
`' GCC
`T Ala
`
`ACC
`Thr
`
`TTT
`Phe
`
`ATG TAT
`
`STA TTT
`
`MH:®TVMfi%e
`375
`
`M £4.)
`
`(.3) KO
`
`M i4.) 00 ~J
`
`ATA
`GCT AAC
`Ala Asa Ile
`
`TTT
`"he:
`.2..'\.‘
`
`TCT
`Sex
`
`ACG
`Thr
`380
`AGTTCTTTTG
`
`ATGGAGCCGG
`
`CTG
`Leu
`385
`GCTAGCGCCG CCCTATACCT TGTCTGCCTC
`END GB FROTEIN
`GCCACCTCGA
`CCTGAATGGA
`
`CGT
`
`AAT AAG
`
`Arg
`
`Asn Lys
`
`GAG
`Glu
`
`AGCCGGCGGC
`
`TCT
`
`TAATCATGCC
`
`Ser OC* (SEQJ£)N{THXD
`390
`CCCGCGTTGC
`
`GTCGCGGTGC
`
`ACCTCGCTAA
`
`CGGATTCACC
`
`ACTCCAAGAA
`
`TTGGAGCCAA
`
`TCAATTCTTG
`
`CGGAGAACTG
`
`TGAATGCGCA
`
`AACCAACCCT
`
`TGGCAGAACA
`
`TATCCATCGC
`
`GTCCGCCATC
`
`TCCAGCAGCC
`
`GCACGCGGCG
`
`CATCTCGGGC
`
`AGCGTTGGGT
`
`CCTGGCCACG
`
`GGTGCGCATG
`
`ATCGTGCTCC
`
`TGTCGTTGAG
`
`GACCCGGCTA
`
`GCGAACGTGA
`
`GGCTGGCGGG
`
`GTTGCCTTAC
`
`TGGTTAGCAG
`
`AATGAATCAC
`
`CGATACGCGA
`
`AGCGACTGCT
`
`GCTGCAAAAC
`
`GTCTGCGACC
`
`TGAGCAACAA
`
`CATGAATGGT
`
`CTTCGGTTTC
`
`CGTGTTTCGT
`
`AAAGTCTGGA
`
`AACGCGGAAG
`
`TCAGCGCCCT
`
`GCACCATTAT
`
`GTTCCGGATC
`
`2§13
`
`TGCATCGCAG
`
`GATGCTGCTG
`
`GCTACC TGT
`
`GGAACACCTA
`
`CATCTGTATT
`
`AACGAAGCGC
`
`TGGCATTGAC
`
`CCTGAGTGAT
`
`TTTTCTCTGG
`
`TCCCGCCGCA
`
`TCCATACCGC
`
`CAGTTGTTTA
`
`CCCTCACAAC
`
`GTTCCRGTAA
`
`CCGGGCATGT
`
`TCATCATCAG
`
`TAACCCGTAT
`
`GTGAGCATC
`
`CTCTCTCGTT
`
`JCATCGGTAT
`
`CATTACCCCC
`
`ATGAACAGAA
`
`ATTCCCCCTT
`
`ACACGGAGGC
`
`EM)
`
`‘4') \i 4.1.)
`
`3033
`
`3093
`
`3153
`
`ATCAAGTGAC
`
`CAAACAGGAA
`
`AAAACCGCCC
`
`TTAACATGGC
`
`CCGCTTTATC
`
`AGAAGCCAGA
`
`CATTAACGCT
`
`TCTGGAGAAA
`
`CTCAACGAGC
`
`TGGACGCGGA
`
`TGAACAGGCA
`
`GACATCTGTG
`
`AATCGCTTCA
`
`CGACCACGCT
`
`GATGAGCTTT
`
`ACCGCAGGAT
`
`CCGGAAATTG
`
`TAAACGTTAA
`
`TATTTTGTTA
`
`AAATTCGCGT
`
`TAAATTTTTG
`
`TTAAATCAGC
`
`TCATTTTTTA
`
`ACCAATAGGC
`
`CGAAATCGGC
`
`AAAATCCCTT
`
`ATAAATCAAA
`
`AGAATAGACC
`
`GAGATAGGGT
`
`TGAGTGTTGT
`
`TCCAGTTTGG
`
`AACAAGAGTC
`
`CACTATTAAA
`
`GAACGTGGAC
`
`TCCAACGTCA
`
`AAGGGCGAAA
`
`AACCGTCTAT
`
`CAGGGCTATG
`
`GCCCACTACG
`
`TGAACCATCA
`
`CCCTAATCAA
`
`GTTTTTTGGG
`
`GTCGAGGTGC
`
`CGTAAAGCAC
`
`TAAATCGGAA
`
`CCCTAAAGGG
`
`AGCCCCCGAT
`
`TTAGAGCTTG
`
`F
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 11 0f 16
`
`US 7,375,193 B2
`
`AGGGGGAAAG
`
`CC‘CUGAACG
`
`TGGCEAGAAA
`
`f“ {‘1
`Tia; 53AGGGAAG
`
`AAAGCGAAAG
`
`GAGCGGGCGC
`
`TAGGGCGCTG
`
`GCAAGTGTAG
`
`CGGTCACGCT
`
`GCGCGTAACC
`
`~CCGCGCTTAA
`
`TGCGCCGCTA
`
`CAGGGCGCGT
`
`CCGGATCCTG
`
`CGTCGCGCGT
`
`- AGGGTGAAAA
`
`LCTCTGACAC
`
`ATGCAGCTCC
`
`CGGAGACGGT
`
`CAGAGGTTGT
`
`CTGTAAGCGG
`
`A“GLCC'GG‘‘G
`
`O32'GACAA(I)
`
`('1C
`
`CGTCAGGGCG
`
`CGTCAGCGGG
`
`TGTTGGCGGG
`
`TGTCGGGGCG
`
`CAGCCATGAC
`
`CCAGTCACGT
`
`AGCGATAGCG
`
`GAGTGTATAC
`
`TGGCTTAACT
`
`ATGCGGCATC
`
`AGAGCAGATT
`
`GTACTGAGAG
`
`””ALCA”AT
`
`GCGGTGTGAA
`
`TAG CGCACA
`
`GATGCGTAAG
`
`GAGAAAATAC
`
`CGCATCAGGC
`
`GCTCTTCCGC
`
`TTCCTCGCTC
`
`ACTGACTCGC
`
`TGCGCTCGGT
`
`CGTTCGGCTG
`
`
`
`CG GAGCGGCi ('2
`
`TATCAGCTCA
`
`CTCAAAGGCG
`
`GTAATACGGT
`
`TATCCACAGA
`
`ATCAGGGGAT
`
`AACGCAGGAA
`
`AGgACATGTG
`
`AGGAAAAGGC
`
`CAGCAAAAGG
`
`CCAGGAACCG
`
`TAAAAAGGCC
`
`GCGTTGC TGG
`
`CGTTTTTCCA
`
`TAGGCTCCGC
`
`CCCCCTGACG
`
`AGCATCACAA
`
`AAATCGACGC
`
`TCAAGTCAGA
`
`GGTGGCGAAA
`
`CCCGACAGGA
`
`CTATAAAGAT
`
`ACCAGGCGTT
`
`TCGCCCTGGA
`
`4353
`
`
`
`‘GLTCCC TCG””CLT“”CG
`
`TETTCCGACG
`
`CTGCCGCTTA
`
`CCGGATACCT
`
`GTCCGCCTTT
`
`4413
`
`CAGTTCGGTG
`
`CTCCCTTCGG
`
`GAAGCGTGGC
`
`GCTTTCTCAT
`
`AGCTCACGCT
`
`GTAGGTATCT
`
`4473
`
`TAGGTCGTTC
`
`GCTCCAAGCT
`
`GGGCTGTGTG
`
`CACGAACCCC
`
`CCGTTCAGCC
`
`GCCTTATCCG
`
`GTAACTATCG
`
`TCTTGAGTCC '
`
`iva a ammE
`
`GAGA"CGACTT
`
`ATGGCCACTG
`
`4593
`
`GCAGCAGCCA
`
`CTGGTAACAG
`
`GATTAGCAGA
`
`
`
`(I)(“1 C3C:C?E1?P3C3- TAGGCGGTGC
`
`TACAGAGTTC
`
`TTGAAGTGGT
`
`GGC CTAACTA
`
`CGGCTACACT
`
`AGAAGGACAG
`
`TATTTGGTAT
`
`“TGCGCTCTG
`
`CTGAAGCCAG
`
`TTACCTTCGG
`
`AAAAAGAGTT
`
`GTAGCTCTT
`
`GATCCGGCAA
`
`ACAAACGACC
`
`GCTGGTAGCG
`
`GTGGTTTTTT
`
`TGTTTGCAAG
`
`CAGCAGATTA
`
`CGGGCAGAAA
`
`AAAAGGATCT
`
`CAAGAAGATC
`
`CTTTGATCTT
`
`TTCTACGGGG
`
`TGTGACGCTC
`
`AGTGGAACGA
`
`AAACTCACGT
`
`TAAGGGATTT
`
`TGGTCATGAG
`
`ATTATGAAAA
`
`AGGATCTTCA
`
`CCTAGATCCT
`
`TTTAAATTAA
`
`AAATGAAGTT
`
`TTAAATCAAT
`
`CTAAAGTATA
`
`TATGAGTAAA
`
`CTTGGTCTGA
`
`CAGTTACCAA
`
`TGCTTAATCA
`
`GTGAGGCACC
`
`TATCTGAGCG
`
`ATCTGTCTATT
`
`TGGTTCATC
`
`CATAGTTGCC
`
`G TGGTGTAGAT
`
`AACTACGATA
`
`CGGGAGGGCT
`
`mACCATCTGG
`
`CCCCAGTGCT
`
`GCAATGATAC
`
`CGCGAGACCC
`
`ACGCTCACCG
`
`GCTCCAGATT
`
`TATCAGCAAT
`
`AAACCAGCCA
`
`GCCGGAAGGG
`
`CCGAGCGCAG
`
`AAGTGGTCCT
`
`GL'AACTTTAT
`
`CCGCCTCCAT
`
`CGAGTCTATT
`
`§§GE
`
`8
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 12 0f 16
`
`US 7,375,193 B2
`
`AATTGTTGCC
`
`GGGAAGCTAG
`
`AGTAAGTAGT
`
`TCGCCAGTTA
`
`ATAGTTTGCG
`
`CAACGTTGTT
`
`GCCATTGCTG
`
`CAGGCATCGT
`
`GGTGTCACGC
`
`TCGTCGTTTG
`
`GTATGGCTTC
`
`ATTCAGCTCC
`
`GGTTCCCAAC
`
`GATCAAGGCG
`
`AGTTACATGA
`
`TCCCCCATGT
`
`TGTGCAAAAA
`
`AGCGETTAGC
`
`CTCCGATCGT
`
`TGTCAGAAGT
`
`i
`
`AAG
`
`H1 x3E2
`
`GCCG
`
`CAGTGTTATC
`
`ACTCATGGTT
`
`TGCATAATTC
`
`TCTTACTGTC
`
`ATGCCATCCG
`
`TAAGATGCTT
`
`TTCTGTGACT
`
`CAACCAAGTC
`
`ATTCTGAGAA
`
`TAGTGTATGC
`
`GGCGACCGAG
`
`CCGGCGTCAA
`
`CACGGGATAA
`
`TACCGCGCCA
`
`CATAGCAGAA
`
`CTTTAAAAGT
`
`GCTCATCATT
`
`GGAAAACGTT
`
`CTTCGGGGCG
`
`AAAACTCTCA
`
`AGGATCTTAC
`
`CGCTGTTGAG
`
`ATCCAGTTCG
`
`ATG':AACGCA
`
`CTCGTGCACC
`
`CAACTGATCT
`
`TCAGCATCTT
`
`TTACTTTCAC
`
`CAGCGTTTCT
`
`GGGTGAGCAA
`
`AAACAGGAAG
`
`GCAAAATGCC
`
`GCAAAAAAGG
`
`GAATAAGGGC
`
`GACACGGAAA
`
`TGTTGAATAC
`
`TCATACTCTT
`
`CCTTTTTCAA
`
`TATTATTGAA
`
`GCATTTATCA
`
`GGGTTATTGT
`
`CTCATGAGCG
`
`GATACATATT
`
`TGAATGTATT
`
`TAGAAAAATA
`
`AACAAATAGG
`
`GGTTCCGCGC
`
`ACATTTCCCC
`
`GAAAAGTGCC
`
`ACCTGACGTC
`
`TAAGAAACCA
`
`TTATTATCAT
`
`GACATTAACC
`
`TATAAAAATA
`
`GGCGTATCAC
`
`GAGGCCCTTT
`
`CGTCTTCAA
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 13 of 16
`
`US 7,375,193 B2
`
`10
`DIQMTQSPSS
`
`
`DIQHHQSPSS
`DIQE QSPSS
`919$ QSPSS
`
`' QSFSS
`
`50
`GKAPKVLIYF
`
`GKAFKELIYF
`GKAPKVLEYF
`GKAPKVLIYF
`GKAPKVLIYF
`
`20
`LSASVGDRVT
`LSASVGDRVT
`LSASVGDRVT
`LSASVGDRVT
`LSASVGDRVT
`
`60
`TSSLHSGVPS
`TSSLHSGVFS
`TSSLHSGVPS
`TSSLHSGVPS
`TSSLHSGVPS
`CIDER—3.2
`
`100
`9C3
`EDFATYYCQQ YSTVPWTFGQ
`EDFATYYCQQ YSTVPWTFGQ
`EDFATYYCQQ YSTVFWTFGQ
`EDFATYYCQQ YSTVPWTFGQ
`EDFATYYCQQ YSTVPWTFGQ
`CDR-LS
`
`40
`30
`NYLNWYQQKP
`ITCSASQDIS
`NYLNWYQQKP
`TTCSASQDTS
`NYLNWYQQKP
`ITCSASQDIS
`NYLNWYQQKP
`ETCSASQDES
`
`NYLNWYQQKP
`
`DR~L£
`
`8O
`FTLTISSLQP
`
`RFSGSGSGTD
`RFSGSGSGTD
`RFSGSGSGTD
`RFSGSGSGTD
`RFSGSGSGTD
`
`110
`GTKVEIKRTV
`GTKVEIKRTV
`GTKVEIKRTV
`GTKVEIKRTV
`GTKVEIKRTV
`
`YTLTISSLQ?
`FTLTISSLQP
`FTLTISSLQP
`
`(SEQJD MM)
`(SEQJD Nona)
`(3mm) 310303)
`(SEQJD NOz’iOS)
`(SEQJD 11mm,
`
`H
`
`
`
`g
`
`10
`EVQLVESGGG
`EVQLVESGGG
`EVQLVESGGG
`EVQLVESGGG
`EVQLVESGGG
`
`20
`LVQPGGSLRL
`LVQPGGSLRL
`LVQPGGSLRL
`LVQPGGSLRL
`LVQPGGSLRL
`
`40
`NYGMNWVRQA
`
`NYGMNWERQA
`
`30
`SCAASGYTFT
`SCAASGYTFT
`SCAASGYTFT
`NYGMNWERQA
`SCAASGYTFT
`NYGMNWVRQA
`SCAASGYTFT
`NY65NWVRQA
`CDR-H i,
`7%
`AADFKRRFTF
`
`Ram—12
`RfiBifi
`iHZBGifi
`Y9101
`Y0192
`
`Ruby—12
`M 331 .6
`H3036
`370101
`373192
`
`mm- 3 2
`M8115»
`13523036
`370101
`3/0192
`
`Nab}? 2
`MBI.6
`15523036
`Yfilfil
`“31’0192
`
`Ram-v12
`MB‘Lfi
`szasfi
`you);
`ymgz
`
`Hahn:
`MBLG
`Hmum
`3:010:
`Y0192
`
`50
`PGKGLEWVGW
`PGKGLEWVGW
`PGKGLEWVGW
`PGKGLEWVGW
`PGKGLEWVGW
`
`90
`LQMNSLRAED
`LQMNSLRAED
`LQMNSLRAED
`LQMNSLRAED
`LQMNSLRAED
`
`8f)
`
`SLDTSKSTAY
`fidmfifi":
`sgangg m
`SLDTSKSTAY
`
`60
`INTYTGEPTY
`INTYTGEPTY
`AADFKRRFTE
`AADFKRRFTF
`INTYTGEPTY
`AADFKRRFTF
`INTYTGEPTY
`AADFKRRFTF
`INTYTGEPTY
`My
`CUR—H2
`(7BR ~7
`100
`TAVYYCAKYP
`TAVYYCAKYP
`TAVYYCAKYP
`TAVYYCAKYP
`TAVYYCAKYP
`
`113
`HYYGSSHWYF
`HYYGSSHWYF
`HYYGSSHWYF
`HYYGSSHWYF
`HYYGSSHWYF
`CDR&{§
`
`DVWGQGTL
`DVWGQGTL
`DVWGQGTL
`DVWGQGTL
`DVWGQGTL
`
`(8mm NOz7)
`(SEQJD 310102)
`(SEQJD NG:I()4)
`(SEQ.1D 510306)
`(8mm NOz’iGE)
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 14 of 16
`
`US 7,375,193 B2
`
`F(ab)—l'2
`N024N1
`Y02383
`Y03LNI
`YOSII
`
`Fmbyiz
`YOZNNI
`NOZNNB
`WEB—i
`YN3I7
`
`wa}12
`Y0243—1
`IN23N3
`NGSNNI
`IN3I7
`
`Ham-42,
`Y02¢N1
`NOZNNS
`YN3NN1
`YN3I7
`
`4O
`30
`20
`10
`NYINNYQQNN
`DIQNTQSNSS LSASVGDRV‘T
`NYLNWYQQKP
`:
`NINE QSPSS LSASVGDRVT
`a NNLNNNQQNN
`LSNSNGNNVI ”Hg .
`" NYLNWYQQKP
`LSASNGNNVI
`a
`LSASVGDRVT ITCSASQNIS NYLNWYQQKP
`CDNL}
`?0
`
`50
`
`60
`
`8o
`
`
`
`GKAPKVLIYF INSLNNGVNN NNNGNGNGIN NILIISSLQN
`GKAPKVLIYF INSNNNGVNN RFSGSGSGTD FTLTISSLQP
`GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP
`GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQF
`GKAPKVLIYF ISSLNSGNNS RFSGSGSGTD IILIISSLQN
`CDRJfl
`
`90
`
`100
`
`110
`
`EDFATYYCQQ NSIVNNINGQ GINNNINNIN
`EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV
`EDFATYYCQQ ISIVNNINGQ GINNNINNIV
`NNNAIINCQQ YSTVPWTFGQ GINNNINNIN
`EDFATYYCQQ YSTVPWTFGQ GINVNINNIN
`CDRdfi
`
`(SNQIDINJN)
`(SEQJD N02199)
`(SEQIDNN