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`US007060269B 1
`
`c12) United States Patent
`Baca et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 7,060,269 Bl
`Jun.13,2006
`
`(54) ANTI-VEGF ANTIBODrns
`
`(75)
`
`Inventors: Manuel Baca, Foster City, CA (US);
`James A. Wells, Burlingame, CA (US);
`Leonard G. Presta, San Francisco, CA
`(US); Henry B. Lowman, El Granada,
`CA (US); Yvonne Man-yee Chen, San
`Mateo, CA (US)
`
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ohhis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 697 days.
`
`(21) Appl. No.: 09/723,752
`
`(22) Filed:
`
`Nov. 27, 2000
`
`Related U.S. Application Data
`
`(62) Division of application No. 08/908,469, filed on Aug.
`6, 1997, now Pat. No. 6,884,879.
`
`(51)
`
`Int. Cl.
`A61K 39/395
`(2006.01)
`(52) U.S. Cl. ............................... 424/133.1; 424/156.1;
`530/387.3; 530/388.85
`(58) Field of Classification Search ............. 424/130.1,
`424/133.1, 135.1, 141.1, 155.1, 156.1; 530/387.1,
`530/387.3, 388.1, 388.24, 388.8, 388.85
`See application file for complete search history.
`
`(56)
`
`References Cited
`
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`(Continued)
`
`Primary Examiner-Larry R. Helms
`(74) Attorney, Agent, or Firm-Steven X. Cui; Genentech,
`Inc.
`
`(57)
`
`ABSTRACT
`
`Humanized and variant anti-VEGF antibodies and various
`uses therefor are disclosed. The anti-VEGF antibodies have
`strong binding affinities for VEGF; inhibit VEGF-induced
`proliferation of endothelial cells in vitro; and inhibit tumor
`growth in vivo.
`
`2 Claims, 16 Drawing Sheets
`
`Regeneron Exhibit 1023.001
`
`

`

`US 7,060,269 Bl
`Page 2
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`
`Regeneron Exhibit 1023.002
`
`

`

`US 7,060,269 Bl
`Page 3
`
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`
`* cited by examiner
`
`Regeneron Exhibit 1023.003
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 1 of 16
`
`US 7,060,269 Bl
`
`Variable Heavy
`
`A4.6.l
`
`EIQLVQSGPELKQPGETVRISCKASGYTFTNYGMNWVKQAPGKGLKWMG
`* ** *
`*
`*** * *
`* *
`*
`F(ab)-12 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVG
`* ** * *
`*
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS
`l
`10
`20
`30
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`A4.6.l
`
`WINTYTGEPTYAADFKRRFTFSLETSASTAYLQISNLKNDDTATYFCAK
`*** ***
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`* **** *** *** *
`* * * * *
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`50
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`60
`70
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`90
`
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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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`
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`
`Regeneron Exhibit 1023.004
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 2 of 16
`
`US 7,060,269 Bl
`
`N
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`
`Regeneron Exhibit 1023.005
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 3 of 16
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`Regeneron Exhibit 1023.006
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 4 of 16
`
`US 7,060,269 Bl
`
`Tumor Weight (gm)
`
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`Fig. 4
`
`Regeneron Exhibit 1023.007
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 5 of 16
`
`US 7,060,269 Bl
`
`VLdomain
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`10
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`
`Regeneron Exhibit 1023.008
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 6 of 16
`
`US 7,060,269 Bl
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`Regeneron Exhibit 1023.009
`
`

`

`U.S. Patent
`
`Jun.13,2006
`
`Sheet 7 of 16
`
`US 7,060,269 Bl
`
`stn
`
`Stll
`
`phoA
`
`gm (249-406)
`
`Transf onn E. coli
`
`+ MI3K07 helper phage
`
`Fab-plll fusion
`
`Fig. 7
`
`Regeneron Exhibit 1023.010
`
`

`

`1 GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC TCATTGCTGA GTTGTTATTT AAGCTTTGGA GATTATCGTC ACTGCAATGC
`CTTAAGTTGA AGAGGTATGA AACCTATTCC TTTATGTCTG TACTTTTTAG AGTAACGACT CAACAATAAA TTCGAAACCT CTAATAGCAG TGACGTTACG
`101 TTCGCAATAT GGCGCAAAAT GACCAACAGC GGTTGATTGA TCAGGTAGAG GGGGCGCTGT ACGAGGTAAA GCCCGATGCC AGCATTCCTG ACGACGATAC
`AAGCGTTATA CCGCGTTTTA CTGGTTGTCG CCAACTAACT AGTCCATCTC CCCCGCGACA TGCTCCATTT CGGGCTACGG TCGTAAGGAC TGCTGCTATG
`
`-23
`
`201 GGAGCTGCTG CGCGATTACG TAAAGAAGTT ATTGAAGCAT CCTCGTCAGT AAAAAGTTAA TCTTTTCAAC AGCTGTCATA AAGTTGTCAC GGCCGAGACT
`CCTCGACGAC GCGCTAATGC ATTTCTTCAA TAACTTCGTA GGAGCAGTCA TTTTTCAATT AGAAAAGTTG TCGACAGTAT TTCAACAGTG CCGGCTCTGA
`301 TATAGTCGCT TTGTTTTTAT TTTTTAATGT ATTTGTAACT AGAATTCGAG CTCGGTACCC GGGGATCCTC TAGAGGTTGA GGTGATTTTA TGAAAAAGAA
`ATATCAGCGA AACAAAAATA AAAAATTACA TAAACATTGA TCTTAAGCTC GAGCCATGGG CCCCTAGGAG ATCTCCAACT CCACTAAAAT ACTTTTTCTT
`M etLysLysAsn
`Begin stII signal sequence
`401 TATCGCATTT CTTCTTGCAT CTATGTTCGT TTTTTCTATT GCTACAAACG CGTACGCTGA TATCCAGTTG ACCCAGTCCC CGAGCTCCCT GTCCGCCTCT
`ATAGCGTAAA GAAGAACGTA GATACAAGCA AAAAAGATAA CGATGTTTGC GCATGCGACT ATAGGTCAAC TGGGTCAGGG GCTCGAGGGA CAGGCGGAGA
`-19
`IleAlaPhe LeuLeuAlas erMetPheva lPheserile AlaThrAsnA laTyrAlaAs pileGlnLeu ThrGlnSerP roSerSerLe userAlaser
`Begin light chain
`"
`501 GTGGGCGATA GGGTCACCAT CACCTGCAGC GCAAGTCAGG ATATTAGCAA CTATTTAAAC TGGTATCAAC AGAAACCAGG AAAAGCTCCG AAACTACTGA
`CACCCGCTAT CCCAGTGGTA GTGGACGTCG CGTTCAGTCC TATAATCGTT GATAAATTTG ACCATAGTTG TCTTTGGTCC TTTTCGAGGC TTTGATGACT
`15 ValGlyAspA rgValThril eThrCysser AlaSerGlnA spileSerAs nTyrLeuAsn TrpTyrGlnG lnLysProGl yLysAlaPro LysLeuLeuile
`
`601
`
`49
`
`701
`
`82
`
`801
`
`115
`
`901
`
`149
`
`TTTACTTCAC CTCCTCTCTC CACTCTGGAG TCCCTTCTCG CTTCTCTGGA TCCGGTTCTG GGACGGATTA CACTCTGACC ATCAGCAGTC TGCAGCCAGA
`AAATGAAGTG GAGGAGAGAG GTGAGACCTC AGGGAAGAGC GAAGAGACCT AGGCCAAGAC CCTGCCTAAT GTGAGACTGG TAGTCGTCAG ACGTCGGTCT
`TyrPheTh rserserLeu HisserGlyV alProSerAr gPheSerGly SerGlySerG lyThrAspTy rThrLeuThr IleserSerL euGlnProGlu
`AGACTTCGCA ACTTATTACT GTCAACAGTA TAGCACCGTG CCGTGGACGT TTGGACAGGG TACCAAGGTG GAGATCAAAC GAACTGTGGC TGCACCATCT
`TCTGAAGCGT TGAATAATGA CAGTTGTCAT ATCGTGGCAC GGCACCTGCA AACCTGTCCC ATGGTTCCAC CTCTAGTTTG CTTGACACCG ACGTGGTAGA
`AspPheAla ThrTyrTyrc ysGlnGlnTy rSerThrVal ProTrpThrP heGlyGlnGl yThrLysval GluileLysA rgThrValAl aAlaProSer
`GTCTTCATCT TCCCGCCATC TGATGAGCAG TTGAAATCTG GAACTGCTTC TGTTGTGTGC CTGCTGAATA ACTTCTATCC CAGAGAGGCC AAAGTACAGT
`CAGAAGTAGA AGGGCGGTAG ACTACTCGTC AACTTTAGAC CTTGACGAAG ACAACACACG GACGACTTAT TGAAGATAGG GTCTCTCCGG TTTCATGTCA
`ValPheileP heProProse rAspGluGln LeuLysSerG lyThrAlaSe rValValCys LeuLeuAsnA snPheTyrPr oArgGluAla LysValGlnTrp
`GGAAGGTGGA TAACGCCCTC CAATCGGGTA ACTCCCAGGA GAGTGTCACA GAGCAGGACA GCAAGGACAG CACCTACAGC CTCAGCAGCA CCCTGACGCT
`CCTTCCACCT ATTGCGGGAG GTTAGCCCAT TGAGGGTCCT CTCACAGTGT CTCGTCCTGT CGTTCCTGTC GTGGATGTCG GAGTCGTCGT GGGACTGCGA
`LysValAs pAsnAlaLeu GlnSerGlyA snSerGlnGl uSerValThr GluGlnAspS erLysAspSe rThrTyrser LeuserserT hrLeuThrLeu
`
`1001
`
`182
`
`GAGCAAAGCA GACTACGAGA AACACAAAGT CTACGCCTGC GAAGTCACCC ATCAGGGCCT GAGCTCGCCC GTCACAAAGA GCTTCAACAG GGGAGAGTGT
`CTCGTTTCGT CTGATGCTCT TTGTGTTTCA GATGCGGACG CTTCAGTGGG TAGTCCCGGA CTCGAGCGGG CAGTGTTTCT CGAAGTTGTC CCCTCTCACA
`serLysAla AspTyrGluL ysHisLysva lTyrAlaCys GluValThrH isGlnGlyLe userserPro ValThrLyss erPheAsnAr gGlyGluCys
`
`Fig. SA
`
`:::a
`CD
`(C
`CD
`::l
`CD .,
`0
`::l
`m
`><
`::l" -· C"
`;::::;
`
`~
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`0
`~
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`
`e •
`
`r:J).
`•
`~
`~
`~
`
`~ = ~
`
`2'
`? ....
`
`tH
`N
`0
`0
`O'I
`
`~
`
`('D
`
`QO
`
`rJ1 =(cid:173)
`('D ....
`0 ....
`....
`
`O'I
`
`d r.,;_
`
`'N
`0--,
`
`---l = 0--, =
`\C = """'
`
`

`

`1101
`
`215
`
`1201
`
`'l'MGC'l'GA'l'C CTCTACGCCG GACGCATCGT GGCCC.'J.W~A CGGMCTM:? GlG!rAil't.AMGG GlrATCTAGAG GTTGAGGTGA TTTTATGMA AAGAATA'l'CG
`ATTCGACTAG GAGATGCGGC CTGCGTAGCA CCGGGATCAT GCGTTGATCA GCATTTTTCC CATAGATCTC CAACTCCACT AAAATACTTT TTCTTATAGC
`OC*
`-23 MetLys LysAsnileAla
`Begin stII signal sequence
`CATTTCTTCT TGCATCTATG TTCGTTTTTT CTATTGCTAC AAACGCGTAC GCTGAGGTTC AGCTGGTGGA GTCTGGCGGT GGCCTGGTGC AGCCAGGGGG
`GTMAGAAGA ACGTAGATAC AAGCMAMA GATAACGATG TTTGCGCATG CGACTCCAAG TCGACCACCT CAGACCGCCA CCGGACCACG TCGGTCCCCC
`PheLeuLe uAlaSerMet PheValPheS erileAlaTh rAsnAlaTyr AlaGluValG lnLeuValGl userGlyGly GlyLeuValG lnProGlyGly
`Begin heavy chain
`1301 CTCACTCCGT TTGTCCTGTG CAGCTTCTGG CTATACCTTC ACCAACTATG
`GTATGAACTG GATCCGTCAG GCCCCGGGTA AGGGCCTGGA
`GAGTGAGGCA ·AACAGGACAC GTCGAAGACC GATATGGAAG TGGTTGATAC
`CATACTTGAC CTAGGCAGTC CGGGGCCCAT TCCCGGACCT
`17 SerLeuArg LeuSercysA laAlaSerGl yTyrThrPhe ThrAsnTyrG
`lyMetAsnTr pileArgGln AlaProGlyL ysGlyLeuGl
`
`-17
`
`ATGGGTTGGA
`TACCCAACCT
`UTrpValGly
`1401 TGGATTAACA CCTATACCGG TGAACCGACC TATGCTGCGG ATTTCAAACG TCGTTTTACT ATATCTGCAG ACACCTCCAG CAACACAGTT TACCTGCAGA
`ACCTAATTGT GGATATGGCC ACTTGGCTGG ATACGACGCC TAAAGTTTGC AGCAAAATGA TATAGACGTC TGTGGAGGTC GTTGTGTCAA ATGGACGTCT
`SO TrpileAsnT hrTyrThrGl yGluProThr TyrAlaAlaA spPheLysAr gArgPheThr IleSerAlaA spThrSerSe rAsnThrVal TyrLeuGlnMet
`
`1501 TGMCAGCCT GCGCGCTGAG GACACTGCCG TCTATTACTG TGCAAAGTAC CCGCACTATT ATGGGAGCAG CCACTGGTAT TTCGACGTCT GGGGTCAAGG
`ACTTGTCGGA CGCGCGACTC CTGTGACGGC AGATAATGAC ACGTTTCATG GGCGTGATAA TACCCTCGTC GGTGACCATA AAGCTGCAGA CCCCAGTTCC
`·84
`AsnserLe uArgAlaGlu AspThrAlaV alTyrTyrcy sAlaLysTyr ProHisTyrT yrGlySerSe rHisTrpTyr PheAspValT rpGlyGlnGly
`1601 AACCCTGGTC ACCGTCTCCT CGGCCTCCAC CAAGGGCCCA TCGGTCTTCC CCCTGGCACC CTCCTCCAAG AGCACCTCTG GGGGCACAGC GGCCCTGGGC
`TTGGGACCAG TGGCAGAGGA GCCGGAGGTG GTTCCCGGGT AGCCAGAAGG GGGACCGTGG GAGGAGGTTC TCGTGGAGAC·CCCCGTGTCG CCGGGACCCG
`117 ThrLeuVal ThrValSerS erAlaSerTh rLysGlyPro SerValPheP roLeuAlaPr oSerserLys SerThrSerG lyGlyThrAl aAlaLeuGly
`1701 TGCCTGGTCA AGGACTACTT CCCCGAACCG GTGACGGTGT CGTGGAACTC AGGCGCCCTG ACCAGCGGCG TGCACACCTT CCCGGCTGTC CTACAGTCCT
`ACGGACCAGT TCCTGATGAA GGGGCTTGGC CACTGCCACA GCACCTTGAG TCCGCGGGAC TGGTCGCCGC ACGTGTGGAA GGGCCGACAG GATGTCAGGA
`150 CysLeuValL ysAspTyrPh eProGluPro ValThrValS erTrpAsnse rGlyAlaLeu ThrSerGlyV alHisThrPh eProAlaVal LeuGlnSerSer
`1801 CAGGACTCTA CTCCCTCAGC AGCGTGGTGA CCGTGCCCTC CAGCAGCTTG GGCACCCAGA CCTACATCTG CAACGTGAAT CACAAGCCCA GCAACACCAA
`GTCCTGAGAT GAGGGAGTCG TCGCACCACT GGCACGGGAG GTCGTCGAAC CCGTGGGTCT GGATGTAGAC GTTGCACTTA GTGTTCGGGT CGTTGTGGTT
`184 GlyLeuTy rSerLeuSer SerValValT hrValProse rSerSerLeu GlyThrGlnT hrTyrileCy sAsnValAsn HisLysPros erAsnThrLys
`
`1901
`
`217
`
`GGTCGACAAG AAAGTTGAGC CCAAATCTTG TGACAAAACT CACCTCTAGA GTGGCGGTGG CTCTGGTTCC GGTGATTTTG ATTATGAAAA GATGGCAAAC
`CCAGCTGTTC TTTCAACTCG GGTTTAGAAC ACTGTTTTGA GTGGAGATCT CACCGCCACC GAGACCAAGG CCACTAAAAC TAATACTTTT CTACCGTTTG
`ValAspLys LysValGluP roLysSerCy sAspLysThr HisLeuAM*S erGlyGlyGl yserGlySer GlyAspPheA spTyrGluLy sMetAlaAsn
`end light chain Begin g3p domain
`CGAAAATGCC GATGAAAACG CGCTACAGTC TGACGCTAAA GGCAAACTTG ATTCTGTCGC
`GCTTTTACGG CTACTTTTGC GCGATGTCAG ACTGCGATTT CCGTTTGAAC TAAGACAGCG
`rGluAsnAla AspGluAsnA laLeuGlnSe rAspAlaLys GlyLysLeuA spSerValAl
`
`TACTGATTAC GGTGCTGCTA
`ATGACTAATG CCACGACGAT
`aThrAspTyr GlyAlaAlaile
`
`2001 GCTAATAAGG GGGCTATGAC
`CGATTATTCC CCCGATACTG
`250 AlaAsnLysG lyAlaMetTh
`
`2101 TCGATGGTTT CATTGGTGAC GTTTCCGGCC TTGCTAATGG TAATGGTGCT ACTGGTGATT TTGCTGGCTC TAATTCCCAA ATGGCTCAAG TCGGTGACGG
`AGCTACCJ>.AA GTAACCACTG CAAAGGCCGG AACGATTACC ATTACCACGA TGACCACTAA AACGACCGAG ATTAAGGGTT TACCGAGTTC AGCCACTGCC
`AspGlyPh eileGlyAsp ValSerGlyL euAlaAsnGl yAsnGlyAla ThrGlyAspP heAlaGlySe rAsnSerGln MetAlaGlnV alGlyAspGly
`
`284
`
`2201 TGATAATTCA CCTTTAATGA ATAATTTCCG TCAATATTTA CCTTCCCTCC CTCAATCGGT TGAATGTCGC CCTTTTGTCT TTAGCGCTGG TAAACCATAT
`ACTATTAAGT GGAAATTACT TATTAAAGGC AGTTATAAAT GGAAGGGAGG GAGTTAGCCA ACTTACAGCG GGAAAACAGA AATCGCGACC ATTTGGTATA
`317 AspAsnSer ProLeuMetA snAsnPheAr gGlnTyrLeu ProserLeuP roGlnServa lGluCysArg ProPheValP heSerAlaGl yLysProTyr
`
`Fig. SB
`
`:::a
`CD
`(C
`CD
`::l
`CD .,
`0
`::l
`m
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`::l"
`C"
`;::::;
`
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`
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`
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`
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`
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`
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`N
`0
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`
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`
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`
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`
`rJ1 =(cid:173)
`('D ....
`0 ....
`....
`
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`
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`
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`0--,
`
`---l = 0--, =
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`
`

`

`2301 GAATTTTCTA TTGATTGTGA CAAAATAAAC TTATTCCGT.G GTCTCTTTGC GTTTCTT/1!,TA TATGTTGCCA CCTTTATGTA TGTATTTTCT ACGTTTGCTA
`CTTAAAAGAT AACTAACACT GTTTTATTTG AATAAGGCAC CACAGAAACG CAA:AGAA:A:P:T A'I'll:t:AACGGT GGAAATACAT ACATAAAAGA TGCAAACGAT
`350 GluPheSerI leAspCysAs pLysileAsn LeuPheArgG lyValPheAl aPheLeuLeu TyrValAlaT hrPheMetTy rValPheSer ThrPheAlaAsn
`2401 ACATACTGCG TAATAAGGAG TCTTAATCAT GCCAGTTCTT TTGGCTAGCG CCGCCCTATA CCTTGTCTGC CTCCCCGCGT TGCGTCGCGG TGCATGGAGC
`TGTATGACGC ATTATTCCTC AGAATTACrA CGGTCAAGAA AACCGATCGC GGCGGGATAT GGAACAGACG GAGGGGCGCA ACGCAGCGCC ACGTACCTCG
`384
`IleLeuAr gAsnLysGlu seroc• (~eQ. \i::> NO'· ,co)
`end 93 protein
`2501 CGGGCCACCT CGACCTGAAT GGAAGCCGGC GGCACCTCGC TAACGGATTC ACCACTCCAA GAATTGGAGC CAATCAATTC TTGCGGAGAA CTGTGAATGC
`GCCCGGTGGA GCTGGACTTA CCTTCGGCCG CCGTGGAGCG ATTGCCTAAG TGGTGAGGTT CTTAACCTCG GTTAGTTAAG AACGCCTCTT GACACTTACG
`2601 GCAAACCAAC CCTTGGCAGA ACATATCCAT CGCGTCCGCC ATCTCCAGCA GCCGCACGCG GCGCATCTCG GGCAGCGTTG GGTCCTGGCC ACGGGTGCGC
`CGTTTGGTTG GGAACCGTCT TGTATAGGTA GCGCAGGCGG TAGAGGTCGT CGGCGTGCGC CGCGTAGAGC CCGTCGCAAC CCAGGACCGG TGCCCACGCG
`2701 ATGATCG'lt'GC TCCTGTCGTT GAGGACCCGG CTAGGCTGGC GGGGTTGCCT TACTGGTTAG CAGAATGAAT CACCGATACG CGAGCGAACG TGAAGCGACT
`TACTAGCACG AGGACAGCAA CTCCTGGGCC GATCCGACCG CCCCAACGGA ATGACCAATC GTCTTACTTA GTGGCTATGC GCTCGCTTGC ACTTCGCTGA
`2801 GCTGCTGCAA AACGTCTGCG ACCTGAGCAA CAACATGAAT GGTCTTCGGT TTCCGTGTTT CGTAAAGTCT GGAAACGCGG AAGTCAGCGC CCTGCACCAT
`CGACGACGTT TTGCAGACGC TGGACTCGTT GTTGTACTTA CCAGI\AGCCA AAGGCACAAA GCATTTCAGA CCTTTGCGCC TTCAGTCGCG GGACGTGGTA
`2901 TATGTTCCGG ATCTGCATCG CAGGATGCTG CTGGCTACCC TGTGGAACAC CTACATCTGT ATTAACGAAG CGCTGGCATT GACCCTGAGT GATTTTTCTC
`ATACAAGGCC TAGACGTAGC GTCCTACGAC GACCGATGGG ACACCTTGTG GATGTAGACA TAATTGCTTC GCGACCGTAA CTGGGACTCA CTAAAAAGAG
`3001 TGGTCCCGCC GCATCCATAC CGCCAGTTGT TTACCCTCAC AACGTTCCAG TAACCGGGCA TGTTCATCAT CAGTAACCCG TATCGTGAGC ATCCTCTCTC
`ACCAGGGCGG CGTAGGTATG GCGGTCAACA AATGGGAGTG TTGCAAGGTC ATTGGCCCGT ACAAGTAGTA GTCATTGGGC ATAGCACTCG TAGGAGAGAG
`3101 GTTTCATCGG TATCATTACC CCCATGAACA GAAATTCCCC CTTACACGGA GGCATCAAGT GACCAAACAG GAAAAAACCG CCCTTAACAT GGCCCGCTTT
`CAAAGTAGCC ATAGTAATGG GGGTACTTGT CTTTAAGGGG GAATGTGCCT CCGTAGTTCA CTGGTTTGTC CTTTTTTGGC GGGAATTGTA CCGGGCGAAA
`3201 ATCAGAAGCC AGACATTAAC GCTTCTGGAG AAACTCAACG AGCTGGACGC GGATGAACAG GCAGACATCT GTGAATCGCT TCACGACCAC GCTGATGAGC
`TAGTCTTCGG TCTGTAATTG CGAAGACCTC TTTGAGTTGC TCGACCTGCG CCTACTTGTC CGTCTGTAGA CACTTAGCGA AGTGCTGGTG CGACTACTCG
`3301 TTTACCGICAG GATCCGGAAA TTGTAAACGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC
`AAATGGCGTC CTAGGCCTTT AACATTTGCA ATTATAAAAC AATTTTAAGC GCAATTTAAA AACAATTTAG TCGAGTAAAA AATTGGTTAT CCGGCTTTAG
`3401 GGCAAAA'JI'CC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG
`CCGTTTTAGG GAATATTTAG TTTTCTTATC TGGCTCTATC CCAACTCACA ACAAGGTCAA ACCTTGTTCT CAGGTGATAA TTTCTTGCAC CTGAGGTTGC
`3501 TCAAAGGGCG AAAAACCGTC TATCAGGGCT ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG
`AGTTTCCCGC TTTTTGGCAG ATAGTCCCGA TACCGGGTGA TGCACTTGGT AGTGGGATTA GTTCAAAAAA CCCCAGCTCC ACGGCATTTC GTGATTTAGC
`3601 GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG
`CTTGGGATTT CCCTCGGGGG CTAAATCTCG AACTGCCCCT TTCGGCCGCT TGCACCGCTC TTTCCTTCCC TTCTTTCGCT TTCCTCGCCC GCGATCCCGC
`3~01 CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCCGGATC CTGCCTCGCG CGTTTCGGTG
`GACCGTTICAC ATCGCCAGTG CGACGCGCAT TGGTGGTGTG GGCGGCGCGA ATTACGCGGC GATGTCCCGC GCAGGCCTAG GACGGAGCGC GCAAAGCCAC
`3801 ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT TGTCTGTAAG CGGATGCCGG GAGCAGACAA GCCCGTCAGG GCGCGTCAGC
`TACTGCCACT TTTGGAGACT GTGTACGTCG AGGGCCTCTG CCAGTGTCGA ACAGACATTC GCCTACGGCC CTCGTCTGTT CGGGCAGTCC CGCGCAGTCG
`Fig. BC
`
`:::a
`CD
`(C
`CD
`::l
`CD .,
`0
`::l
`m
`
`>< ::l" -· C"
`
`;:::::;:
`
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`0
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`0
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`
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`
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`•
`~
`~
`~
`
`~ = ~
`
`2'
`? ....
`
`tH
`N
`0
`0
`O'I
`
`~
`
`('D
`
`0
`
`rJ1 =(cid:173)
`('D .... ....
`0 ....
`....
`
`O'I
`
`d r.,;_
`
`---l = 0--, = 'N
`\C = """'
`
`0--,
`
`

`

`3901 GGGTGTTGGC GGGTGTCGGG'GCGCAGCCAT GACCCAG'tCA CGTAGCGATA 'GCG;GAGTGTA TACTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA
`CCCACAACCG CCCACAGCCC CGCGTCGGTA CTGGGTCAGT GCATCGCTAT CGCCTCACAT ATGACCGAAT TGATACGCCG TAGTCTCGTC TAACATGACT
`4001 GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAAA TACCGCATCA GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC
`CTCACGTGGT ATACGCCACA CTTTATGGCG TGTCTACGCA TTCCTCTTTT ATGGCGTAGT CCGCGAGAAG GCGAAGGAGC GAGTGACTGA GCGACGCGAG
`4101 GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA
`CCAGCAAGCC GACGCCGCTC GCCATAGTCG AGTGAGTTTC CGCCATTATG CCAATAGGTG TCTTAGTCCC CTATTGCGTC CTTTCTTGTA CACTCGTTTT
`4201 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC
`CCGGTCG'l!'TT TCCGGTCCTT GGCATTTTTC CGGCGCAACG ACCGCAAAAA GGTATCCGAG GCGGGGGGAC TGCTCGTAGT GTTTTTAGCT GCGAGTTCAG
`4301 AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA
`TCTCCACCGC TTTGGGCTGT CCTGATATTT CTATGGTCCG CAAAGGGGGA CCTTCGAGGG AGCACGCGAG AGGACAAGGC TGGGACGGCG AATGGCCTAT
`4401 CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT
`GGACAGGCGG AAAGAGGGAA GCCCTTCGCA CCGCGAAAGA GTATCGAGTG CGACATCCAT AGAGTCAAGC CACATCCAGC AAGCGAGGTT CGACCCGACA
`4501 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG
`CACGTGC'll'TG GGGGGCAAGT CGGGCTGGCG ACGCGGAATA GGCCATTGAT AGCAGAACTC AGGTTGGGCC ATTCTGTGCT GAATAGCGGT GACCGTCGTC
`4601 CCACTGG'l!'AA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA CAGTATTTGG
`GGTGACCATT GTCCTAATCG TCTCGCTCCA TACATCCGCC ACGATGTCTC AAGAACTTCA CCACCGGATT GATGCCGATG TGATCTTCCT GTCATAAACC
`4701 TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC
`ATAGACGCGA GACGACTTCG GTCAATGGAA GCCTTTTTCT CAACCATCGA GAACTAGGCC GTTTGTTTGG TGGCGACCAT CGCCACCAAA AAAACAAACG
`4801 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA
`TTCGTCG'l!'CT AATGCGCGTC TTTTTTTCCT AGAGTTCTTC TAGGAAACTA GAAAAGATGC CCCAGACTGC GAGTCACCTT GCTTTTGAGT GCAATTCCCT
`4901 TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC
`AAAACCAIGTA CTCTAATAGT TTTTCCTAGA AGTGGATCTA GGAAAATTTA ATTTTTACTT CAAAATTTAG TTAGATTTCA TATATACTCA TTTGAACCAG
`5001 TGACAGT'll'AC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG
`ACTGTCAATG GTTACGAATT AGTCACTCCG TGGATAGAGT CGCTAGACAG ATAAAGCAAG TAGGTATCAA CGGACTG