`
`93B
`
`2
`
`US0073751
`
`United States Patent
`(12)
`(10) Patent No.:
`US 7,375,193 B2
`Bacaet al.
`(45) Date of Patent:
`May20, 2008
`
`
`(54) ANTI-VEGF ANTIBODIES
`
`(75)
`
`Inventors: Manuel Baca, Black Rock (AU);
`James A. Wells, Burlingame, CA (US);
`Leonard G. Presta, San Francisco, CA
`(US); Henry B. Lowman, E! Granada,
`CA (US); Yvonne Man-Yee Chen, San
`Mateo, CA (US)
`
`Wo
`WO
`wo
`WO
`wo
`WO
`wo
`
`WO 92/18619
`WO 92/22653
`WO 94/04679
`WO 94/10202
`96/30046
`WO 98/45331
`WO 98/45332
`
`10/1992
`12/1992
`3/1994
`5/1994
`10/1996
`10/1998
`10/1998
`
`(73) Assignee: Genentech, Ine., South San Francisco,
`(us)
`.
`.
`.
`.
`.
`.
`(*) Notice:
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 11/537,015
`
`(65)
`
`Prior Publication Data
`US 2007/0248610 Al
`Oct. 25. 2007
`‘
`Related U.S. 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.
`.
`_.
`.
`(60) Provisional application No. 60/126,446,filed on Apr.
`7, 1997.
`
`(51)
`
`Int. Cl.
`(2006.01)
`CO7K 16/00
`(52) U.S. Ch. oe eeceenereeees
`§30/387.1; 530/387 .3;
`530/388.85; 424/130.1; 435/320.1; 536/23.53
`(58) Field of Classification Seareh ............. 530/387.1,
`530/387.3, 388.85; 424/130.1; 435/320.1;
`536/23.53
`or
`:
`See application file for complete search history.
`References Cited
`U.S. PATENT DOCUMENTS
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`Primary Examiner—David J. Blanchard
`Assistant Examiner Parithosh K. Tungaturthi
`
`(56)
`
`aePOL A
`5,580,723 A
`6,037,454 A
`6,054,207 A
`6,884,879 BI
`7,060,269 Bl
`7,169,901 B2*
`2002/0032315 Al
`
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`4/2005 Bacaet al.
`/
`6/2006 Baca et al.
`1/2007 Bacaetal. ....... 530/387.1
`3/2002 Bacaet al.
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`EP
`GB
`GB
`WO
`WO
`
`0239400
`0451216
`451216 BI
`1325 932 Bl
`2188638
`2268744
`91/0996
`WO 91/09967
`
`9/1987
`1/1996
`1/1996
`4/2005
`10/1987
`12/1994
`1/1991
`T/A99L
`
`(57)
`
`ABSTRACT
`
`Humanized and variant anti-VEGF antibodies and various
`
`uses therefor are disclosed. The anti-VEGF antibodies have
`
`
`
`strong binding affinities for VEGI; inhibit VEGI'-induced
`proliferation of endothelial cells in vitro; and inhibit tumor
`:
`.
`growth in vivo.
`
`22 Claims, 16 Drawing Shects
`
`Pfizer v. Genentech
`IPR2017-01488
`Genentech Exhibit 2042
`
`Pfizer v. Genentech
`IPR2017-01488
`Genentech Exhibit 2042
`
`

`

`US 7,375,193 B2
`
`Page 2
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`

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`
`* cited by examiner
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 1 of 16
`
`US 7,375,193 B2
`
`A464
`
`40
`30
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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
`
`180600
`
`740000 —
`
`1OGG9
`
`@
`
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`
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`
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`@ NOADDBITION
`
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`
`.
`
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`
`20000
`
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`
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`
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`
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`
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`
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`
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`VEGF
`VEGF
`VEGF
`(5)
`(0.5)
`(5)
`(0.5)
`(5)
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 4 of 16
`
`US 7,375,193 B2
`
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`PGRGLEWVGWINTYTGRPTYAADFPRRREITISLDTSASTVYL
`
`A461
`
`bu2.0
`
`11¢
`1O0abcedef
`ao
`abe
`QISNLANDDTATYFCARY PHYYGSSHWYFDVWGAGTTVTVSS
`kK RK
`kk
`OO
`*
`*
`*
`
`(SEQID NO:9)
`
`OMNSLRARDTAVYYCARY PHY YGSSHWYFDVWGOGTLVTVSS
`
`(SEOID NO14)
`
`huZ.iG
`
`OMNSLRARDTAVYYCARY PHYYGSSHWYPDVWGOGTLVTVSS
`
`(SEQID NO:16)
`
`
`
`

`

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

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 6 of 16
`
`US 7,375,193 B2
`
`stil
`
`sti “pBR322 or
`
`Amp’
`
`TRANSFORM E.colf
`
`+M1ISKO7 HELPER PHAGE
`
`FAB-pIIl FUSION
`
`ooh,
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 7 of 16
`
`US 7,375,193 B2
`
`GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC TCATTGCTGA
`
`69
`
`GITGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC TICGCAATAT GSOCGCAAAAT
`
`120
`
`GACCAACAGC GGTTGATTIGA TCAGGTAGAG GGGGCGCTGT ACGAGGTAAA GCCCGATGCC
`
`186
`
`AGCATTCCTG ACGACGATAC GGAGCTGCT& CGCGATTACG TAAAGAAGTT ATTGAAGCAT
`
`240
`
`CCTCGTCAST AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT
`
`306
`
`TATAGTCGCT TIGTTTITAT TITTTAATGT ATTTGTAACT AGAATTCGAS CYCGGTACC?
`
`360
`
`GGGGATCCTC TAGAGGTTGA GGTGATTTT ATG AAA AAG AAT ATC GCA TIT CTT
`Met Lys Lys Asn Ile Ala Phe Leu
`~23
`~20
`
`CTT GCA TCT ATG TIC GIT TIT TCT ATT GCT ACA AAC GCG TAC GCT GAT
`Leu Ala Ser Met Phe Vai Phe Ser Tle Ala Thr Asn Ala Tyr Ala Asp
`-15
`~10
`“5
`1
`BEGIN stii SIGNAL SEQUENCE
`ATC CAG TTG ACC CAG TCC CCG AGC TCC CTG TCC GCC TCT GTS Gaoc GAT
`Tle Gin Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Giy Asp
`5
`19
`15
`
`AGG GTC ACC ATC ACC TGC AGC GCA AGT CAS GAT ATT AGC AAC TAT TTA
`Arg Val Thr Ile Thr Cys Ser Ala Ser Gin Asp Ile Ser Asn Tyr Leu
`20
`25
`30
`BEGIN LIGHT CHAIN
`AAC TGG TAT CAA CAG AAA CCA GGA AAA GCT CCG AAA CTA CTG ATT TAC
`Asn Trp Tyr Gin Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
`35
`40
`45
`
`CAC TCT GGA GTC CCT PCT Cac TIC TCT GGA Tce
`TTC ACC TCC TCT CTC
`Phe Thr Ser Ser Leu Bis Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
`59
`33
`60
`65
`
`GGT TCT GGG ACG GAT TAC ACT CTG ACC ATC AGC AGT CTG CAG CCA GAA
`Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu
`70
`75
`80
`
`GAC TTC GCA ACT TAT TAC TGT CAA CAG TAT AGC ACC GTG CCG TGG ACG
`Asp Phe Ala Thr Tyr Tyr Cys Gin Glin Tyr Ser Thr Val Pro Trp Thr
`85
`90
`95
`
`TTT GGA CAG GGT ACC AAG GTG GAG ATC AAA CGA ACT GTG GCT GCA CCA
`Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
`109
`105
`110
`
`TC? GTC TTC ATC TTC CCG CCA TCT GAT GAGS CAG TTG AAA TCT GGA ACT
`Ser Val Phe Tle Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
`115
`120
`125
`
`SCT TCT oT? GiG TGC CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA
`Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
`139
`135
`149
`145
`
`Fl
`
`413
`
`461
`
`509
`
`557
`
`605
`
`653
`
`702
`
`749
`
`797
`
`845
`
`893
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 8 of 16
`
`US 7,375,193 B2
`
`GTA CAG TGG AAG GTG GAT AAC GcC CTC CAA TCG GGT BAC TCC CAG GAG
`&
`Val Gln Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu
`150
`155
`160
`
`AGT GTC ACA GAG CAG GAC AGC AAG GA
`Ser Val Thr Glu Gin Asp Ser Lys As
`165
`17
`
`C AGC ACC TAC AGC CTC AGC AGC
`p Ser Thr Tyr Ser Len Ser Ser
`0
`
`ACC CPG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC
`Thr Lea Thr Len Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
`1890
`185
`190
`
`TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC ACA AAG AGC TIC
`Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
`195
`200
`205
`
`AAC AGG GGA GAG TGT T AAGCTGATCC TCTACGCCGG ACGCATCGTG
`Asn Arg Gly Glu Cys 0 ¢* GSEQID NO:130)
`210
`
`GCCCTAGTAC GCAACTAGTC GTAAAAAGGG TATCTAGAGG TIGAGGTGAT TIT ATG
`Met
`-23
`
`BEGIN stIIT SIGNAL SEQUENCE
`GCA TCT ATG TIC GIT TTT TCT ATT
`AAA AAG AAT ATC GCA TTT CTT CTT
`Lys Lys Asn Ile Ala Phe Leu Len Ala Ser Met Phe Val Phe Ser Ile
`~20
`“15
`~10
`
`GCT ACA AAC GCG TAC GCT GAG GTT CAG CTG GTG GAG TCT GGC GGT GGC
`Ale Thr Asn Ala Tyr Ala Glu Vel Gin Leu Val Glu Ser Gly Gly Gly
`~5
`1
`5
`16
`
`CTG GTG CAG CCA GGG GGC TCA CTC CGT TTG Tec TGT GCA GCT TCT Gee
`Leu Val Gln Pro Gly Gly Ser Len Arg Leu Ser Cys Ala Ala Ser Gly
`15
`20
`25
`BEGIN HEAVY CHAIN
`TAT ACC TTC ACC AAC TAT GGT ATG AAC TGG ATC CGT CAG GCC CCG GGT
`Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Tle Arg Gln Ala Pro Gly
`30
`35
`40
`
`AAG GGC CTIG GAA TGG GTT GGA TGG ATT AAC ACC TAT ACC GGT GAA CCG
`Lys Gly Leu Glu Trp Val Gly Trp Tle Asn Thr Tyr Thr Gly Glu Pro
`45
`5G
`55
`
`02
`ACC TAT GOT GCG GAT TTC AAA Cot Cat TTT ACT ATA TCT GCA GAC ACC
`Bei
`wl
`Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Ile Ser Ala Asp Thr
`ab
`65
`70
`
`Td108inkyo3
`
`AGC ABC ACA GIT TAC CTG CAG ATG AAC AGC CTG CGC GCT GAG GAC
`Ser Asn Thr Val Tyr Leu Gln Met Asn Ser Len Arg Ala Glu Asp
`&O
`B5
`9
`
`ACT GCC GicC TAT TAC TGT GCA AAG TAC CCG CAC TAT TAT GGG AGC AGC
`Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser
`95
`100
`195
`
`
`
`941
`
`989
`
`1037
`
`1085
`
`1131
`
`1187
`
`2235
`
`1283
`
`1331
`
`1379
`
`1427
`
`1475
`
`1523
`
`1571
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 9 of 16
`
`US 7,375,193 B2
`
`CAC TGG TAT TTC GAC GTC TGG GGT CAA GGA ACC CTG GTC ACC STC Te
`His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser
`116
`115
`1290
`
`TCC TCC
`CCA TCG GTC TRC CCC CTG GCA CCC
`TOG GCC TCC ACC AAG GGC
`Ser Ala Ser Tar Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
`125
`130
`135
`
`AAG AGC ACC TCT GGG GGC ACA GCG GCC CPG GGC TGC CTG GTC AAG GAC
`Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
`140
`145
`159
`
`TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC
`Tyr Phe Pro Glu Pro Val Thre Val Ser Trp Asn Ser Gly Ala Leu Thr
`155
`169
`165
`179
`
`AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC
`Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
`175
`189
`185
`
`ToC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG
`Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gla
`180
`195
`200
`
`ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG STC GAC
`Thr Tyx Tle Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
`205
`210
`215
`
`AAG AAA GTP GAG CCC AAA TCT TGT GAC AAA ACT CAC CTC TAG AGT GGC
`Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu
`* Ser Gly
`220
`225
`239
`
`GGT GGC TCT GGT TCC GGT GAT TIT GAT TAT GAA AAG ATG GCA AAC GCT
`Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala
`235
`240
`245
`259
`END LIGHT CHAIN
`BE
`AAT AAG GGG GCT ATG ACC GAA AAT GCC GAT GAA AAC GCG CTA CAG TCT
`Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Glin Ser
`255
`260
`265
`
`GIN g3p DOMAIN
`GAC GCT AAA GGC AAA CTT GAT TCT GTC GCT ACT GAT TAC GGT GCT GCT
`Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala
`270
`275
`280
`
`ATC GAT GGT TTC ATT GGT GAC GTT TcC GGC CTT GCT AAT GGT AAT GGT
`Tle Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly
`285
`290
`295
`
`GCT ACT GGT GAT TIT GCT GGC TCT AAT TcC CAA ATG GCT CAA GTC GGT
`Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly
`300
`305
`319
`
`GAC GGT GAT AAT ¢
`Asp Gly Asp Asn
`315
`
`me QOryiD
`
`CCT TPA ATG AAT AAT TPC CGT CAA TAT TTA CCT
`Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro
`320
`325
`330
`
`
`
`161%
`
`1667
`
`ay ~~ he iF
`
`1
`
`ws an had
`
`1811
`
`185%
`
`1907
`
`2955
`
`2003
`
`2051
`
`2099
`
`2147
`
`2195
`
`2243
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 10 of 16
`
`US 7,375,193 B2
`
`TCC
`Ser
`
`CTC
`Leu
`
`cor
`PEO
`
`CAA
`Gin
`
`Teg Grr
`° Val
`
`GAA
`Glu
`
`Cys Al
`
`ect
`Pro
`340
`
`TTT
`Phe
`
`GTC
`Val
`
`TTT
`Phe
`
`AGC
`Ser
`
`Gct
`Ala G
`345
`
`AAA
`
`LYS
`
`CCA
`Pro
`
`TAT
`
`TYE
`
`GAA
`Glu
`350
`
`TCT
`Ser
`
`APT
`Tle
`
`GAP
`Asp
`
`Phe
`
`' GAC
`Asp
`
`ABA
`
`LYS
`
`ATA
`Tle
`
`AAC
`Asn
`
`GGT
`Gly
`
`GTC
`Val
`
`TET
`
`365
`
`GCG
`Ala
`
`TT?
`Phe
`
`CTT
`Leu
`
`TTA
`Leu
`
`TAT
`
`Tyr
`376
`
`' gcc
`i Ala
`
`ACC
`Thr
`
`TTT
`Phe
`
`ATG
`Met
`375
`
`TTC
`Phe
`
`TTA
`Leu
`360
`
`TAT
`
`Tyr
`
`OTA
`Val
`
`GCT
`Ala
`
`AAC
`ASH
`
`ATA
`Ile
`
`TIT
`haPoe
`
`TcT
`Ser
`
`ACG
`The
`380
`AGTTCTITPITG
`
`ATGGAGCCGS
`
`CTG
`Leu
`385
`GCTAGCGCCG CCCTATACCT TGTCTGcete¢
`END G3 PROTEIN
`BOCACCTCGA
`CCTGAATGGA
`
`Cor
`Arg
`
`AAT
`ASG
`
`AAG
`Livs
`
`GAG
`Glu
`
`AGCEGGCGGC
`
`FAATCATGCC
`TCT
`Ser OC* (SEQID NO:100)
`390
`CCCECGTTGC
`
`GTCGECGGTGC
`
`ACCTCGCTAA
`
`COGATICACC
`
`ACTCCAAGAA
`
`TTGGAGCCAA
`
`TCAATTCTTG
`
`CGGAGAACTG
`
`TGAATGCGCA
`
`AACCAACCCT
`
`TGGCAGAACA
`
`TATCCATCGC
`
`GTCCGCCATC
`
`TCCAGCAGCE
`
`GCACGCGGCG
`
`CATCTCGGGC
`
`AGCGTTGGGT
`
`COTGGCCACG
`
`GGOTGCGCATGS
`
`ATCGTGCTCC
`
`TOTCGTTGAG
`
`GACCCGGCTA
`
`AATGAATCAC
`
`CGATACGCGA
`
`GCGRACGTGA
`
`GGCTGGCGES
`
`GTTGCCTTAC
`
`TGGTTAGCAS
`
`AGCGACTGCT
`
`GCTGCAAAAC
`
`GTCTGCGACE
`
`TGAGCAACAA
`
`CATGAATGGT
`
`CTTCGGTTTC
`
`CGOTGTTICGT
`
`ARAGTCTIGGA
`
`AACGCGGAASG
`
`TCAGCGCCCT
`
`GCACCATTAT
`
`GTTCCGGATC
`
`TGCATCGCAG
`
`GATGCTGCTG
`
`GCTACCCTGT
`
`GGAACACCTA
`
`CATCTGTATT
`
`BRACGAAGCGC
`
`TGGCATTGAC
`
`CCOTGAGTGAT
`
`TITTCTCTGG
`
`TCCCGCCGCA
`
`TCCATACCGC
`
`CAGTTGTITA
`
`CCCTCACAAC
`
`GTTCCAGTAA
`
`CCGGGCATGT
`
`TCATCATCAG
`
`TAACCCGTAT
`
`GIGAGCATC
`
`CTCTCTCGTT
`
`TCATCGGTAT
`
`CATPACCCCC
`
`ATGAACAGAA
`
`ATTCCOCCTT
`
`ACACGGAGGC
`
`ATCAAGTGAC
`
`CAAACAGGAA
`
`AABRACCGCOC
`
`TTAACATGGC
`
`COSCTTITATC
`
`AGAAGCCAGA
`
`CATTAACGCT
`
`TCTGGAGAAA
`
`CTCAACGAGC
`
`TOGACGCOGA
`
`TGAACAGGCA
`
`GACATCTGTG
`
`KATCGCTTCA
`
`CGACCACGCT
`
`GATGAGCTTT
`
`ACCGCAGGAT
`
`CCGGABATTS
`
`TAAACGTTAA
`
`TATTTTGTTA
`
`ABATTCGCGT
`
`TABATTTITTS
`
`TTAARATCAGC
`
`TCATTTITTA
`
`ACCAATAGGEC
`
`CGABATCGGE
`
`ABAATCCCTT
`
`ATARATCAAA
`
`AGAATAGACC
`
`GAGATAGGCT
`
`TCAGTCTTGT
`
`TOCAGTTITGSG
`
`AACAAGAGTC
`
`CACTATTAAA
`
`GAACGTGGAC
`
`TCCAACGTCA
`
`AAGGGCGAAA
`
`BACCGTCTAT
`
`CAGGGCTATG
`
`GCCCACTACG
`
`TGAACCATCA
`
`COCTAATCAA
`
`GITTITTGSG
`
`GTCGAGGTGC
`
`CGOTABAGCAC
`
`TAAATCOGAA
`
`COCTAAAGGG
`
`AGCCOCCGAT
`
`TTAGAGCTES
`
`
`F,
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 11 of 16
`
`US 7,375,193 B2
`
`ACGGGGAAAG
`
`COGGCGAACG
`
`TGGCGAGARA
`
`GGAAGGGAAG
`
`AAAGCGAAAG
`
`GASCGEGGCGEC
`
`TAGGGCGCTG
`
`GCAAGTGTAG
`
`CGGPCACGCT
`
`GCGCGTAACC
`
`» CCGCGCTTAA
`
`TGCGCCGCTA
`
`CAGGGCGCGT
`
`COGGATCCTG
`
`OQ CTCGCGCGT
`
`> ACGGTGAAAA
`
`COTCTGACAC
`
`ATGCAGCTCC
`
`CGCAGACGGT
`
`CACAGCTTGT
`
`CTGTAAGCGG
`
`ATGCCGGGAG
`
`COTCAGGGECG
`
`CGPCAGCEGG
`
`TGTTGGCGGG
`
`TETCEGGSCG
`
`CAGCCATGAC
`
`' AGCGATAGCG
`
`GAGTGTATAC
`
`TGGCTTAACT
`
`ATGCGGCATC
`
`AGAGCAGATT
`
`GTACTGAGAG
`
`TGCACCATAT
`
`GCGGTOTGAA
`
`TACCGCACA
`
`GATGCGTAAG
`
`GAGAAAATAC
`
`COCATCAGGC
`
`GCTCTTCCGC
`
`PTCOTCECTC
`
`ACTGACTCGC
`
`PGCGCTCGGT
`
`COTPCGGCTG
`
`CGGCGAGCGG
`
`TATCAGCTCA
`
`CTCARAGECG
`
`GTAATACGGT
`
`TATCCACAGA
`
`ATCAGGGGAT
`
`AACGCAGGAA
`
`AGAACATGTG
`
`AGCAAAAGGC
`
`CAGCAAAAGG
`
`CCAGGAACCG
`
`TAARAAGGCE
`
`GCOTTGCTGG
`
`CGTTTTICCA
`
`TAGGCTCCGC
`
`COCCCTGACG
`
`AGCATCACAA
`
`ABATCGACGC
`
`TCAAGTCAGA
`
`GGTGGCGAAA
`
`COCGACAGGA
`
`CTATAAAGAT
`
`ACCAGGCGTT
`
`TCCCCCTGGA
`
`AGC PCCCTCG
`
`TECGCTCPCCY
`
`PETTCCGACC
`
`CTGCCGCTIA
`
`CCGGATACCT
`
`GtceesccTtTrT
`
`GTAGGTATCT
`
`CAGTTCOGTG
`
`CTCOCTTICSS
`
`GAAGCCETGGC
`
`GCTPTCTCAT
`
`AGCTCACGCT
`
`TAGGTCGTTC
`
`GCTCCAAGCT
`
`GGGCTGTGTG
`
`COGTTCAGCC
`
`CGACCGCTGC
`
`GCCPTATCCG
`
`GTAACTATCG
`
`TCTPGAGTCC J
`
`GACACGACTT
`
`ATCGCCACTG
`
`GCAGCAGCCA
`
`CTGGTAACAG
`
`GATTAGCAGA
`
`3 TAGGCGGTGC
`
`TACAGAGTTC
`
`TTGAAGTGGT
`
`GGCCTAACTA
`
`CGGCTACACT
`
`AGAAGGACAG
`
`TATTTGGTAT
`
`OTECGCTCTG
`
`CTGAASCCAG
`
`TTACCTTICGG
`
`ASARAGAGTT
`
`GTAGCICTT
`
`GATCCGGCAA
`
`ACABACCACC
`
`GCTGGTAGCG
`
`CTIGGTITITT
`
`PGTPTGCAAG
`
`CAGCAGATTA
`
`CGCGCAGAAA
`
`AAAAGGCATCT
`
`CAAGAAGATC
`
`CTTTGATCTT
`
`TICTACGGGG
`
`TCTGACGCTC
`
`AGTGGAACGA
`
`ABACTCACGT
`
`TAAGGGAPTT
`
`TOGTCATGAG
`
`ATTATCARBRA
`
`AGGATCTTICA
`
`CCTAGATCCT
`
`TPTAAATTAA
`
`AAATGAAGTT
`
`TTAAATCAAT
`
`CTAAAGTATA
`
`TATGAGTAAA
`
`CTIGGTCTGA
`
`CAGTTACCAA
`
`TGOCTTAATCA
`
`GTGAGGCACC
`
`TATCTCAGCG
`
`ATCTGICTAT
`
`TTCGTICATC
`
`CATAGTTGCC
`
`TGACTCCCCG
`
`TCGTGTAGAT
`
`AACTACGATA
`
`CGGGAGGGCT T
`
`3 COCCAGTGCT
`
`GCAATGATAC
`
`COCGAGACCC
`
`ACGCTCACCG
`
`GCTCCAGATT
`
`TATCAGCAAT
`
`ABACCAGCCA
`
`GCCGGAAGGG
`
`CCGACCGCAG
`
`AAGTGGTCCT
`
`GCAACTTTAT
`
`COGCCTCCAT
`
`CCAGTCTATT
`
`FIG.
`
`
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 12 of 16
`
`US 7,375,193 B2
`
`AATTGTTGCE
`
`GGGAAGCTAG
`
`AGTARGTAST
`
`TCOGCCAGTTA
`
`ATAGTTTGCG
`
`CAACGTTIGT®
`
`SCCATTGCTG
`
`CAGGCATCGT
`
`GGTGTCACGC
`
`TCGTCGTTTG
`
`GTATGGCTIC
`
`ATTCAGCTCC
`
`GGTTCCCAAC
`
`GATCAAGGCG
`
`AGTTACATGA
`
`* PETGCAAAAA
`
`AGCOGTTAGC
`
`TCOTICGGTC
`
`CTCCGATCGT
`
`TOTCAGAAGT
`
`, CAGTGTTATC
`
`ACTCATGGT?
`
`ATGGCAGCAC
`
`TGCATAATTC
`
`PTCTTACTGTC
`
`ATGCCATCCG
`
`TAAGATGCTT
`
`PTCTGTGACT
`
`GGTGAGTACT
`
`CAACCAAGTC
`
`ATICTGAGAA
`
`TAGTGTATGC
`
`GGCGACCGAG
`
`COGGCGTCAA
`
`CACGGGATAA
`
`TACCGCGCCA
`
`CATAGCAGAA
`
`CTT TAAAAGT
`
`GCTCATCATT
`
`GGAAAACGTT
`
`CTTCGGEGCG
`
`BRAAACTCTCA
`
`AGGATCTTAC
`
`CGCTGTTGAG
`
`ATCCAGTTCG
`
`ATGTAACCCA
`
`CTCGTGCACC
`
`CAACTGATCT
`
`TCAGCATCTT
`
`PTACTTTCAC
`
`CASCOTTTCT
`
`GGOGTGAGCAA
`
`ABACAGGAAG
`
`GCAARATGCE
`
`GCAAAAAAGG
`
`GAATAAGGGC
`
`GACACGGASA
`
`TOTTGAATAC
`
`TCATACTCTT
`
`COTPPTTTCAA
`
`TATTATTGAA
`
`GCATTTATCA
`
`GGGTPATTSGT
`
`GATACATATT
`
`TOAATGTATT
`
`TAGAAAAATA
`
`AACAAATASS
`
`GGTTCCGCEC
`
`ACAPTTTCCCE
`
`GAABRAGTGCC
`
`ACCTGACGTC
`
`TAAGAARCCA
`
`PTATTATCAT
`
`GACAPTAACC
`
`TATAAAAATA
`
`GGCGTATCAC
`
`GAGGCCCTTT
`
`CGTCTTCAA
`
`
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 13 of 16
`
`US 7,375,193 B2
`
`10
`DIUMTOSPSS
`
`DIOMTOSPSS
`DIQUTOSPSS
`DIQLIFOSPSS
`UTOSPSS
`
`
`50
`GRAPRVLIYF
`GRAPREILTYF
`GKAPKVLIYF
`GKAPKVLIYF
`GRAPRVLIYE
`
`20
`LSASVGDRVT
`LSASVGDRVE
`LSASVGDRVT
`LSASVGDRVT
`LSASVGDRVT
`
`60
`TSSLHSGVPS
`TSSLAHSGVPS
`TSSLHSGVPS
`TSSLHSGVES
`TSSLHSGVES
`CDR-L2
`
`100
`90
`EDFATYYCOO YSTVPWTFGO
`EDFATYYCOO YSTVPWTFGO
`EDPATYYCOG YSTVPWTFGO
`EDFATYYCOOG YSTVPWTFGO
`EDFATYYCOQ YSTVPWTFGO
`CDR-L3
`
`306
`ITCSASODIS
`TTCSASODIS
`ITCSASODIS
`TTCSASODIS
`
`40
`NYLNWYOORP
`NYLNWYOOKP
`NYLNWYOOKP
`NYLNWYOQOKP
`
`NYDNWYOQQKP
`
`REFSGSGSSTD
`RFSGSGSGTD
`RFSGSGSGTD
`REF SGSGSGTD
`REF SGSGSGTD
`
`110
`GTRVETRRTV
`GTRVETRKRTV
`GTRVETRRTV
`GTRVETRRTV
`GTRVETRERTV
`
`FrLTISSLOP
`
`cirrtsetop
`
`LETLSSLOP
`FTLTISSLOP
`FILTISSLOP
`
`(SEQUID NO:8)
`(SEQLID NO:101)
`(SEO.ID NC
`103)
`{SEQ.ID NO:105)
`(SEQ.AD NO:107)
`
`|
`
` 9
`
`Feab)-12
`MBI.6
`H2305.6
`YoIOt
`¥O192
`
`Fiab)-12
`MBL.6
`H2305.6
`YOO!
`yo192
`
`Plab)-12
`MBL&
`H2305.6
`YO101
`yo192
`
`F(ab}-12
`MB1.6
`H2305.6
`Yoo)
`YO192
`
`Plab)-12
`MBL.6
`H2305.6
`YOIOI
`YO192
`
`Frab)-12
`MBL6
`H2305.6
`YO1O!
`YO192
`
`30
`SCAASGYTFT
`SCAASGYTFTL
`SCAASGYTFT
`SCAASGYTFT
`
`40
`NYGMNWVROA
`
`NYGMNWOROA
`NYGMNWVROA
`
`NyGunnken
`SCAASGYTET eeNWVRDA
`
`id
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`
`20
`LVOPGGSLREIL
`LVOPGGSLRE
`LVOPGGSLRIL
`LVOPGGSLRL
`LVOPGGSLRIL
`
`SQ
`PGRGLEWVGW
`PGRKGLEWVGW
`PGRGLEWVGW
`PGRGLEWVGW
`PGRGLEWVGW
`
`60
`INTYTGEPTY
`INTYTGEPTY
`INTYTGEPTY
`INTYTGEPTY
`INTYTGEPTY
`
`CDR-H1
`80
`70
`AADFRKRRFTF
`SLDTSkSTAY
`AADFKRRFT §
`ITSSN
`SADTSEN
`RADFKRRETF
`AADFRRRFTF
`SLDTSESTAY
`AADFRKRRFTE
`F_SLDTSKSTAY
`CDR-7
`
`90
`LOMNSLRAED
`LOMNSLRARD
`LOMNSLRAED
`LOMNSLRAED
`LOMNSLRARD
`
`TAVYYCARYP
`TAVYYCARYP
`TAVYYCARYP
`TAVYYCARKYP
`TAVYYCARYP
`
`L1i6
`HYYGSSAWYF
`HYYGSSHWYF
`HYYSSSHAWYF
`HYYGSSHAWYF
`HYYGSSAWYF
`CDR-H3
`
`DVWGOGTL
`DVWGOOGTL
`DVWGOGTE
`DVWGOGTL
`DVWGOGTE
`
`(SEO.ID NO:7)
`(SEQ.ID NO:102)
`(SEQ.ID NO: 104)
`(SEQ.ID NO:106)
`(SEQ.ID NOQ:083
`
`
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 14 of 16
`
`US 7,375,193 B2
`
`10
`DIQMTQSPSS
`DIGETOSPSS
`
`
`
`20
`LSASVGDRVT
`LSASVGDRV?P
`LSASVGDRVT TTCR
`LSASVGDRV?
`LSASVGDRVT
`
`39
`LTCSASQDIS
`
`
`
`4Q
`NYLNWYOOQKP
`NYLNWYOOKP
`LS NYLNWYOOKP
`; NYLNWYOORP
`ITCSSASODIS NYLNWYOQORP
`CDR-L1
`80
`70
`60
`50
`TSSLHSGVPS
`GRAPRVLIYE
`RPSGSGSGTD FITLTISSLOP
`GKAPRVLIYE
`TSSLHSGVPS
`RFSGSGSGTD FTLTISSLOP
`GRAPEVLIYP TS
`SLHSGVPS
`RFSGSGSGTD PILTISSLOP
`GKAPEVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLOP
`GRAPKVLIYE TSSLHSGVPS RFSGSGSGTD FTLTISSLOP
`CDR-L2
`
`110
`1006
`990
`EDFATYYCOO YSTVPWIFGO GTKVEIKRTV
`EDFATYYCOQQ YSTVPWTFGO GTKVEITKRTV
`EDFATYYCOOQ YSTVPWEFGO GTKVEIKRTV
`EDFATYYCOO YSTVPWTFGO GTEVEIKRTY
`EDFATYYCGOQ YSTVPWIFGO GPKVEIKRTV
`CDR-L3
`
` (SEQ.ID NO:8)
` (SEQ.ID NO:109)
`(SEQ.ID NO:1ED
` (SEGIDNO:113)
`(SEQ.ID NO:115)
`
`Fi &
`
`70
`
`10
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`EVOLVESGGG
`
`26
`LVOQPGGSLREL
`LVOPGGSLREL
`LVOPGGSERE
`LVOPGGSLRE:
`LVOPGGSLRE
`
`40
`30
`SCAASGYTFT NYGMNWVROQA
`SCAASGYDIFT HYGMNWVROA
`SCAASGYTET NYGHINWVROA
`SeaSCAASGY
`YGMNWVROA
`YGMNWVROA
`CDRa
`
`F(ab)-12
`¥O243-1
`YO238-3
`YO313-1
`YO317
`
`F(ab)-12
`0243-1
`YO238-3
`YO313-1
`yO317
`
`F(ab)-12
`¥O243-1
`YO238-3
`YO313-1
`YOR
`
`Flab)-12
`0243-1
`Y¥O238-3
`YO313-1
`¥O317
`
`F(ab)- 12
`¥O243-1
`YO238-3
`YO313-1
`YOR17
`