`
`,I.
`
`···~--..
`
`W088/09344
`
`PCT/US88/01737
`
`11/31
`
`so
`60
`40
`30
`20
`10
`GATCCCGAGGTTATGCTGGTTGAATCTGGTCCACTACTGATGGAACCTGGTGCGTCCCTG
`D P E V M L V E S G G V L M E P C C S L
`seal
`Ecoo
`
`110
`100
`90
`80
`70
`120
`AAGCTGAGCTG'l'GCTGCTAGCGGCTTCACGTTCTCTCC'l'TACGCCATGTCTTGGGTCCGT
`K L S C A A S G F T F S R Y A M S W V R
`NheI
`Pf'lMI
`Esp I
`
`180
`170
`160
`150
`140
`130
`CAGACTCCCGAGAAGCGTCTAGAGTGGGTCCCGACGATATCTTCl'GGTGGT'l'CGAACACT
`Q T P E K R L E W V A T
`I S S G G S N T
`BspMII
`XbaI
`NruI
`EcoRV
`AsulI
`
`240
`230
`220
`210
`200
`190
`TACTATCCAGACAGTG'l'GAAGCGTCCAT'l'CACGATCTCTCGAGACAACGCTAAGAACACG
`Y Y P D S V K G R F T
`I S R D N A K N T
`XhoI
`
`300
`290
`280
`270
`260
`250
`TTGTACCTGCAAATGTCTTCTCTACGTAGTGAAGATACTGCTATGTACTACTGTGCACGT
`L Y L Q M S S L R S E D T A M Y Y C A R
`snaBI
`.
`ApaLI
`BspMI+
`
`·J60
`350
`· 340
`330
`320
`310
`CCTCCACTGATCTCACTAGTTGCTGAT'l'ATGCCATGGATTATTGGGGTCATGGTGCTAGC
`P P L
`I S L V A D Y A M D Y W G H G A S
`SpeI
`NcoI
`NheI
`
`·420
`370
`410
`400
`390
`380
`GTTACTGTGAGCTCTGGTGGCGGTGGGTCGGGCGGTGGTGGCTCGGGTGGCGGCGGATCG
`V T V S S G G G G S G G G G S G G G G S
`sacI
`
`480
`470
`460
`450
`440
`430
`· GATATCGTTATGACTCAGTCTCATAAGTTCATGTCCACTTCTGTTGGTGACCGTGTTTCT
`DIV MT Q S H.K FM S
`'l' S VG DR VS
`EcoRV
`BstEII
`
`540
`530
`.
`520
`510
`.
`500
`490
`ATCACTTGTAAGGCCAGCCAGCATGTGGGTGCTGCTATCGCATGGTATCAGCAGAAGCCC
`I T C K A S Q D V G A A
`I A W Y Q Q K P
`PflMI
`Sma
`
`590
`580
`600
`570
`560
`550
`CGGCAGTCTCC'l'AAGCTGCTGATCTACTGGCCGTCGACTCGTCATACTGGTGTCCCGGAT
`G Q S P K L L
`I Y W A S T R H T G V P D
`I
`SalI
`
`660
`650
`640
`630
`620
`610
`CGTTTCACTGGGTCCGGATCAGCTAC'l'GAT'l'TCAC'l'CTGACTA'1"1"1'CGAACGT'l'CAGTCT
`R F T G S G S G T
`I> F T L T
`I S N V Q S
`AsuII
`BspMII
`
`690
`680
`670
`720
`710
`•
`700
`GATGACCTGGCTGATTACTTCTGCCAGCAATATTCCGGGTACCC'l'CTGACTTTCGGTGCC.
`DD LADY F C Q Q·Y S GYP LT F GA
`SspI
`KpnI
`Nae
`Fl GI. ctE
`
`750
`740
`730
`GGCACTAAACTCGAGCTGAAGTAACTGCAG
`.L K *
`G T K L E
`I
`XboI
`
`PstI
`
`BIOEPIS EX. 1002
`Page 3001
`
`
`
`W088/09344
`
`20
`10
`Met Lys Ala Ile Phe Val Leu Lys Gly Ser Leu Asp Arg Asp Leu Asp Ser Arg Leu Asp
`ATG AAA GCA ATT TTC GTA CTG AAA GGT TCA CTG GAC AGA GAT CTG GAC TCT CGT CTG GAT
`Bg1II
`
`40
`30
`Leu Asp Val Arg Thr Asp His Lys Asp Leu Ser Asp His Leu Val Leu Val Asp Leu Ala
`CTG GAC GTT CGT ACC GAC CAC AAA GAC CTG TCT GAT CAC C'l'G GTT CTG G'l'C GAC C'l'G GCT
`Bell
`SalI
`so
`60
`Arg Asn Asp Leu Ala Arg Ile Val Thr Pro Gly Ser Arg Tyr Val Ala Asp Leu Glu Phe
`CGT AAC GAC CTG GCT CGT ATC G'l"l' ACT CCC GGG TC'l' CGT TAC G'1"l' GCG GA'l' C'1'G GAA TTC
`SmaI
`EcaRI
`
`Asp
`GAT
`
`I= I e,.. 10 A
`
`Eco RI
`SspI/
`
`pD312
`4801'bp
`
`Afll[
`
`F"l6i. JO 5
`
`BIOEPIS EX. 1002
`Page 3002
`
`
`
`W088/09344
`
`PCT/US88/01737
`
`-
`
`5
`
`- 4
`
`
`
`FlC:n. ll
`
`':)
`
`94- -
`...
`... -
`67-
`~ 29- -
`~ 43-
`~ 20.1- -
`t4.4- -
`
`~b /3 I
`a !r
`- --
`
`0 1
`
`2
`
`3
`
`..
`
`D V O L O E S G P G L V K P S O S L S L T C S V T G Y S I T
`S G Y F W N W l R O F P G N K L E W L G F I K Y D G S N Y G
`N P S L K N R V S I T R D T S E N Q F F L K L D S V T T A T
`YYCAGDNDHLYFDYWGQGTTLTVS
`
`G G G G S G G G G S G G G G S
`
`·O AV VT OE SALT TSP G GT VILT CR SST GA V.T
`~ S N Y A N W I O E K P D H L F T G L I G G T S N R A P G V
`PVRFSGSL I GDKAALTITGAOTEDDAMYFC
`ALWFRNHFVFGGGTXVTVLG
`
`FIG. 9C
`
`BIOEPIS EX. 1002
`Page 3003
`
`
`
`W088/09344
`
`PCT/US88/01737
`
`-26-tOsFv
`--- 26-tO Fab
`0Di9011n
`A Acetyl Strcpttanthidin
`• Ouabain
`
`I
`
`II
`/I
`
`I
`4001
`I
`so
`~
`~
`95 60
`~
`~ i
`~ 40f-
`
`,, II
`, , , ,.
`----74
`I,,
`, ,
`,,
`,
`,,
`,,
`, ,
`,,
`
`I
`I
`I
`I
`I
`I
`I
`I
`
`I
`
`I
`I
`
`1/
`11
`II
`II
`II
`II
`II
`,,
`I
`,,,
`,,
`l)I
`,,,
`,,
`, /
`,
`
`,.,,,,
`ti"
`
`20
`
`0
`
`tO" ..
`
`I
`i0 .. 0
`
`10·•
`
`10·•
`
`10·'
`
`-to·•
`INHIBITOR CONCENTRATION tM.J
`
`10·1 •
`
`...
`
`FIC:n. tJ..
`
`BIOEPIS EX. 1002
`Page 3004
`
`
`
`WOSS/09344
`
`PCT/US88/01737
`
`•
`•
`
`5
`
`O
`
`-8
`
`-fO -9
`-7 · -6
`-5 -4
`LOG 1./NBOIJN[) [)/GOXIN CONCENTRATION /Ml
`f"I Gi. l:,A
`
`8
`
`r
`
`~ 4
`~
`~
`· cS 2
`i
`~
`ij OL---'-..-::::~..J.-~-~~--=--~--1
`-e
`-6
`-9
`-1
`-10
`-11
`LOG UNBOUND l)/GOXIN CONCENTRAnON !Ml
`F•Gi. 13B
`
`BIOEPIS EX. 1002
`Page 3005
`
`
`
`W088/09344
`
`PCT/US88/01737
`
`•
`
`60
`50
`40
`30
`20
`10
`GAATTCATGGCTGACAACAAA'l"l'CAACAAGGAACAGCAGAACGCGTTCTACGAGATC'l"l'G
`E F M A D N X F H K E Q Q H A F Y E
`I L
`EcoRI
`MluI
`BqlII
`XlDnI
`
`90
`80
`120
`110
`100
`. 70
`CACCTGCCGAACCTGAACGAAGAGCAGCGTAACGGC'l"l'CATCCAAAGC'l"l'GAAGGATGAG
`H L P H L N E E Q R N G F
`I Q S L K D E
`BspMI+
`.
`HindIII
`
`180
`170
`160
`150
`140
`130
`CCCTCTCAGTCTGCCAATCTGCTAGCGGATGCCAAGAAACTGAACGATGCGCAGGCACCG
`P S Q S A N L L A D A K K L N D A Q A P
`NheI
`FspI
`
`240
`.
`230
`220
`210
`190
`200
`AAATCGGATCAGGGGCAATTCATGGCTGACAACAAA'l'TCAACAAGGAACAGCAGAACGCG
`X S D Q G Q F M A D N K F N K E Q· Q N A
`MluI
`XmnI
`
`300
`290
`280
`270
`260
`250
`TTCTACGAGATCTTGCACC'l'GCCGAACCTGAACGAAGAGCAGCGTAACGGC'l"l'CATCCAA
`FYE IL H LP.NL NEE QR NG F I Q
`BglII
`BspMI+
`H
`
`360
`3SO
`340
`330
`320
`310
`AGCTTGAAGGATGAGCCCTCTCAGTCTGCGAATCTGCTAGCGGATGCCAAGAAACTGAAC
`S L K D E P S Q S A N L
`L A D A K K
`L
`·N
`indIII
`NheI
`
`380
`370
`GATGCGCAGGCACCGAAATCGGATCC
`D A Q A P K S D P
`FspI
`Ba111BI
`
`r
`
`F \ ~. I~
`
`BIOEPIS EX. 1002
`Page 3006
`
`
`
`W088/09344
`
`PCT/US88/01737
`
`(BABS)-
`
`~'i/31
`
`70
`30
`20
`10
`60
`50
`40
`GGATCCGGTAACTCTGACTCTGAATGCCCGCTGAGCCACGACGCGTACTGCCTGCACGACGGTGTTTGCATGTAC
`G S G H S D S E C P L S B D G Y C L B D G V C M Y
`BaJllHI
`BsmI+
`. EspI
`
`~
`
`145
`135
`125
`115
`105
`95
`85
`ATCGAAGCTCTGGACAAATACGCATGCAACTGCGTTGTAGGCTACATCGGTGAGCGCTGCCAGTATCGCGATCTG
`I E A L D K Y A C H C V V G Y I G E R C Q Y R D L
`SphI
`NruI
`
`170
`160
`AAATCGTGGGAGCTGCGTTAACTGCAG
`K W W E L R *
`HpaX PstI
`
`F'lua. 15A
`
`I)
`
`BIOEPIS EX. 1002
`Page 3007
`
`
`
`W088/09344
`
`PCT{US88/01737
`
`(BABS)-
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTGGCGACCCGTCCAAGGACTCCAAAGCTCAGGTTrCTGCTGCCGAAGCTr.GT
`G S G G D P S K D S K A Q V S A A E A G
`BamHI
`
`120
`110
`100
`70
`90 ""'
`80
`ATCAC'l'GGCACCTGGTATAACCAACTGGGGTCGACT'l."l'CA'l"l'GTGACCGCTGGTGCGGAC
`I T G T W Y N Q L ~ S T F
`I V T A G A D
`Sall
`
`180
`170
`160
`150
`140
`130
`GGAGC'l'C'l'GACTGGCACC'l'ACGAATCTGCGGTTGGTAACGCAGAATCCCGCTACGTACTG
`G A L T G T Y E S A V G N A E S R Y V L
`SacI
`.
`SnaBI
`
`240
`230
`220
`210
`200
`190
`ACTGGCCGTTATGACTCTGCACCTGCCACCCATGCC'l'CTGGTACCGCTCTGGGCTGGACT
`T G R Y D S A P A T D G S G T A L G W T
`BspMI+
`KpnI
`
`300
`290
`280
`250
`270
`260
`GTGGC'l'TGGAAAAACAAC'l'ATCGTAATGCGCACAGCGCCAC'l'ACGTGGTC'l'GGCCAATAC
`V A W K N N Y R N A H S A T T W S G Q Y
`Bal I
`FspI
`DraIII
`PflMI
`BstXI.
`
`360
`350
`340
`330
`320
`310
`GTTGGCGGTGCTGAGGCTCGTATCAACACTCAGTGGC'l'GTTAACATCCGGCAC'l'ACCGAA
`V G G A E A R
`I N T Q W L L T S G T T E
`DraIII
`HpaI
`
`420
`410
`400
`390
`38~
`370
`. GCGAATGCATGGAAATCGACACTAGTAGGTCATGACACCTTTACCAAAGT'l'AAGCCTTC'l'
`A N A W K S T L V G H D T F T K V K P S
`SpeI
`BsmI+
`NsiI
`
`480
`470
`460
`450
`440
`430
`GCTGC'l'AGCAT'l'GATGC'l'GCCAAGAAAGCAGGCGTAAACAACGGTAACCCTCTAGACGCT
`A AS I DA AK KA G V N.N GNP L DA
`NheI
`BstEII XbaI
`
`490
`500
`GTTCAGCAATAACTGCAG
`V Q Q *
`
`PstI
`
`FlC:n. \SB.
`
`BIOEPIS EX. 1002
`Page 3008
`
`
`
`WOSS/09344
`
`(BABS)-
`
`PCT/US88/01737
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTGTACGTAGCTCCTCTCGCACTCCGTCCGATAAGCCGGTTGCTCATGTAGTT
`G S G V R S S S R T P S D K P V A H V V
`BamHI
`snaBI
`
`120
`110
`100
`70
`90
`80
`GCTAACCCTCAGGCAGAAGGTCAGCTTCAGTGGCTGAACCGTCGCGCTAACGCCCTGCTG
`A N P Q A E G Q L Q W L N R R A N A L L
`MstII
`BglI
`
`180
`170
`160
`150
`140
`130
`GCAAACGGCGTTGAGCTCCGTGATAACCAGCTCGTGGTACCTTCTGAAGGTCTGTACCTG
`A N G V E L R D N Q L V ·V P S E G L Y L
`Sac I
`PflMI
`KpnI
`
`240
`210
`220
`210
`200
`190
`ATCTATTCTCAAGTACTGTTCAAGGGTCAGGGCTGCCCGTCGACTCATGTTCTGCTGACT
`I Y S Q V L F K G Q G C P S T H V L L T
`ScaI
`SalI
`
`300
`290
`280
`270
`260
`250
`CACACCATCAGCCGTATTGCTGTATCTTACCAGACCAAAGTTAACCTGCTGAGCGCTATC
`H T
`I S R
`I A V S Y Q T K V N L L S A
`I
`HpaIBspMI+ Eco47III
`EspI
`
`360
`350
`340
`330
`320
`310
`AAGTCTCCGTGCCAGCGTGAAACTCCCGAGGGTGCAGAAGCGAAACCATGGTATGAACCG
`KS PC QR ET PEG A.EA KP WYE P
`NcoI
`
`420
`410
`400
`390
`380
`370
`ATCTACCTGGGTGGCGTATTTCAACTGGAGAAAGGTGACCGTCTGTCCGCAGAAATCAAC
`I Y L G G V F Q L E K G D R L S A E
`I N
`BstEII
`
`460
`450
`440
`480
`470
`430
`CGTCCTGACTATCTAGATTTCGCTGAATCTGGCCAGGTGTACTTCGGTATTATCGCACTG
`R P D Y L D F A E S G Q V Y F G I
`I A L
`XDaI
`BalI
`
`•
`
`e
`
`490
`TAACTGCAG
`* PstI
`
`Fl6'. rsc.
`
`BIOEPIS EX. 1002
`Page 3009
`
`
`
`W088/09344
`
`(BABS) -
`
`PCT/US88/01737
`
`so
`60
`40
`30
`20
`10
`GGATCCGGTGCTGATCAGCTGACTGACGAGCAGATCGCTGAATT'l'AAAGAGGCT'l'TCTCT
`G S G A D Q L T D E Q
`I A E F K E A F S
`BamHI
`BclIPYUZI
`DraI
`
`120
`110
`100
`70
`90
`80
`CTGTTTGACAAAGACGGTGACGGTACCATCACTACCAAAGAGCTCGGCACCGTTATGCGC
`L F D K D G D G T
`I T T K E L G T V M R
`SacI
`l<pnI
`FspI
`
`180
`170
`160
`150
`140
`130
`AGCCTTGGCCAGAACCCGACTGAAGCTGAATTGCAGGACATGATCAACGAAGTCGACGCT
`S L G Q N P T E A E L Q D M I N E V D A
`Bal I
`Bel!
`Sal I
`
`240
`230
`220
`210
`200
`190
`GACGGTAACGGCACCATCGATTTTCCGGAATTTCTGAACCTGATGGCGCGCAAGATGAAA
`D G N G T
`I D F P E F L N L M A R X M K
`ClaI
`BspMII
`BssHII
`
`300
`250
`290
`280
`270
`260
`GACACTGACTCTGAAGAGGAACTGAAAGAGGCCTTCCGTGTT'l'TCGACAAAGACGGTAAC
`D T D S E E E L K E A F R V F O K D G N
`stuI
`
`350
`360
`340
`330
`320
`310
`GGTTTCATCTCGGCCGCTGAACTGCGTCACGTTATGACTAACCTGGGTGAAAAGCTTACT
`G F
`I S A A E L R H V M T N L G E K L T
`EagI
`HindIII
`
`420
`410
`400
`390
`380
`370
`GACGAAGAAGTTGACGAAATGATTCGCGAAGCTGACGTCGATGGTGACGGCCAGGTTAAC
`D E E V O E M I R E A D V D G D G Q V N
`XmnI
`NruI
`AatII
`~paI
`
`4SO
`440
`430
`TACGAAGAGTTCGTTCAGGTTATGATGGCTAAGTAACTGCAG
`Y E E F V Q V M M A K *
`
`PstI
`
`FICn. ISD
`
`•
`
`BIOEPIS EX. 1002
`Page 3010
`
`
`
`W088/09344
`
`(BABS)-
`
`...........
`PCTfUS88/01737
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTGGAGGCTCTCTGGGCTCTCTGACTATTGCCGAACCGGCAATGATTGCTGAA
`I A E P A M
`I A E
`G S G G G S L G S L T
`BglI
`BamHI
`Bsm
`
`6
`
`120
`110
`90
`80
`70
`100
`TGCAAGACTCGTACCGAAGTCTTCGAGATCTCTCGTCGTCTGATCGATCGCACTAATGCC
`CKTRTE VF E IS RRL I DRTNA
`I+
`BglII
`ClaI
`Bs
`PvUI
`
`160
`150
`140
`130
`180
`170
`AACTTCCTGGTATGGCCGCCGTGCGTCGAGGTACAACGCTGCTCCGGGTGTTGCAACAAT
`N F L V W P P C V E V Q R C S G C C N N
`tXI
`
`240
`230
`220
`210
`200
`190
`CGTAACGTTCAATGTCGACCGACTCAAGTCCAGCTGCGTCCGGTCCAAGTCCGCAAAATC
`R N V Q C R P T Q V Q L R P V Q V R K
`I
`Sall
`PvuII
`
`300
`290
`280
`270
`260
`250
`GAGATTGTACGTAAGAAACCGATCTTTAAGAAGGCCACTGTTACTCTGGAAGACCATCTG
`E
`I V R K K P
`I F K K A T V T L E D H L
`SnaBI
`
`350
`340
`330
`320
`310
`GCATGCAAATGTGAGACTGTAGCGGCCGCACGTCCAGTTACTTAACTGCAG
`A C K C E T V A A A R P V T *
`SphI
`EagI
`NotI
`
`PstI
`
`Fl"1. ISE-
`
`•
`
`~ .
`
`BIOEPIS EX. 1002
`Page 3011
`
`
`
`W088/09344
`
`· PCT/US88/0~737
`
`(BABS)-
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTATATTCCCCAAACAATACCCAATTATAAACT'l'TACCACAGCGGGTGCCACT
`G S G I F P K Q Y P
`I
`I N F T T A G A T
`BamHI
`
`120
`110
`100
`70
`90
`80
`GTGCAAAGCTACACAAACTTTATCAGAGCTGTTCGCGGTCGTTTAACAACTGGAGCTGAT
`V Q SY TN F IR A V.R GR LT T GAD
`
`180
`170
`160
`150
`140
`130
`GTGAGACATGAAATACCAGTGTTGCCAAACAGAGTTGGTTTGCCTATAAACCAACGGTTT
`P V L P N R V G L P
`V R H E
`I
`I N Q R F
`
`220
`240
`230
`210
`200
`190
`ATTTTAGTTGAACTCTCAAATCATGCAGAGCTTTCTGTTACATTAGCGCTGGATGTCACC
`I L V E L S N
`ff A E L S V T L A L D V T
`Eco47III
`
`300
`290
`280
`270
`260
`250
`AATGCATATGTGGTCGGCTACCGTGCTGGAAATAGCGCATATTTCTTTCATCCTGACAAT
`N A Y V V G Y R A G N S A Y F F H P D N
`NdeI
`NsiI
`
`360
`350
`340
`330
`320
`310
`CAGGAAGATGCAGAAGCAATCACTCATCTTTTCACTGATGTTCAAAATCGATATACATTC
`Q E D A E A
`I T H L F T O V Q N R Y T F
`ClaI
`
`420
`410
`400
`380
`370
`390
`GCCTTTGGTGGTAATTATGATAGACTTGAACAACTTGCTGGTAATCTGAGAGAAAATATC
`A F G G N Y D R L E Q L A G N L R E N
`I
`
`480
`470
`.460
`450
`·440
`430
`GAGTTGGGAAATGGTCCACTAGAGGAGGCTATCTCAGCGCTTTATTATTACAGTACTGGT
`E L G N G P L E E A
`I S A L Y Y Y S T G
`Seal
`Eco47III
`
`540
`530
`520
`510
`500
`490
`GGCACTCAGCTTCCAACTCTGGCTCGTTCCTTTATAATTTGCATCCAAATGATTTCAGAA
`G T Q L P T L A R S F
`I
`I C
`I Q M I S E
`
`6.00
`560
`590
`580
`570
`550
`GCAGCAAGATTCCAATATATTGAGGGAGAAATGCGCACGAGAATTAGGTACAACCGGAGA
`A A R F Q Y I E G E M R T R
`I R Y N R R
`FspI
`Bgl
`
`•
`
`~
`
`BIOEPIS EX. 1002
`Page 3012
`
`
`
`WOSS/09344
`
`(BABS)-
`
`PCT/US88/01737
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTGCTCCGACTTCTAGCTCTACTAAGAAAACTCAGCTTCAGC'l'GGAACACCTG
`G S G A P T S S S T K K T Q L Q L E H L
`BamHI
`PvUII
`
`120
`110
`70
`100
`90
`80
`CTGCTGGACCTTCAGATGATCCTGAACGGTATCAACAACTACAAGAACCCGAAACTGACT
`L L D L Q M I L N G
`I N N Y K N P K L T
`
`180
`170
`160
`150
`140
`130
`CGTATGCTGACTTTCAAATTCTACATGCCGAAGAAAGCTACCGAACTGAAACACCTTCAG
`R M L T F K F Y M P K K A T E L K H L Q
`
`240
`230
`220
`210
`.
`200
`190
`·TGCCTGGAAGAAGAACTGAAGCCGCTGGAGGAAGTACTGAACCTGGCTCAGTCT.AA>~.AC
`C L E E E L K P L E E V L N L A Q S K N
`scaI
`
`ioo
`290
`2so
`270
`260
`2so
`TTCCACCTGCGTCCGCGTGACCTGATCAGCAACATCAACGTAATCGTTCTAGAACTTAAA.
`F H L R P R D L
`I S N
`I N V
`I V L E L K
`BclI
`XbaI
`
`360
`350
`340
`330
`320
`310
`GGCTCTGAAACTACCTTCATGTGCGAATACGCTGACGAAACTGCTACCATCGTAGAATTT
`GS ET T FMC E·Y ADE TAT IVE F
`
`420
`390
`380
`370
`410
`400
`CTGAACCGTTGGATCACCTTCTGCCAGTCTATCATCTCTACTCTGACTTAACTGCAG
`I S T L T *
`·c Q S
`L N R W I T F
`I
`
`PstI
`
`PICrl. 1Sua
`
`BIOEPIS EX. 1002
`Page 3013
`
`
`
`W088/09344
`
`(BABS)"".
`
`PCT/US88/01737
`
`60
`50
`40
`30
`20
`10
`GGATCCGGTGCTGACAACAAATTCAACAAGGAACAGCAGAACGCGTTCTACGAGATCTTG
`G S G A O N K F N K E Q Q N A F Y E
`I L
`BamHI
`MluI
`BglII
`XmnI
`
`120
`110
`100
`90
`80
`70
`CACCTGCCGAACCTGAACGAAGAGCAGCGTAACGGCTTCATCCAAAGCTTGAAGGATGAG
`H L P N L N E E Q R N G F
`I Q S L K O E.
`BspMI+
`HinclIII
`
`180
`170
`150
`160
`140
`130
`CCCTCTCAGTCTGCGAATCTGCTAGCGGATGCCAAGAAACTGAACGATGCGCAGGCACCG
`P S Q S A N L L A D A K K L N D A Q A ~
`NheI
`FspI
`
`240
`230
`220
`210
`200
`190
`AAATCGGATCAGGGGCAATTCATGGCTGACAACAAATTCAACAAGGAACAGCAGAACGCG
`K S D Q ~ Q F M A D N K F N K E Q Q N ~
`MluI
`XmnI
`
`300
`270
`260
`250
`290
`280
`TTCTACGAGATCTTGCACCTGCCGAACCTGAACGAAGAGCAGCGTAACGGCTTCATCCAA
`F Y E
`I L H L P N L N E E Q R N G F
`I Q
`BglII
`BspMI+
`H
`
`360
`350
`340
`330
`320
`310
`AGCTTGAAGGATGAGCCCTCTCAGTCTGCGAATCTGCTAGCGGATGCCAAGAAACTGAAC
`S L K D E P S Q S A N L L A D A K K L N
`indIII
`NheI
`Fl&i. t5H
`
`380
`370
`GATGCGCAGGCACCGAAATAACTGCAG
`D A Q A P K *
`FspI
`
`PstI
`
`:. ..
`
`BIOEPIS EX. 1002
`Page 3014
`
`
`
`1
`
`r
`
`INTERNATIONAL SEARCH REPORT
`International Application No. PCI'/US88/01737
`I. CLASSIFICATION OF SUBJECT MATTER (If several classification symbols apply, lndicale all) a
`According to International Patent Classillcalion (IPC) or to bolh National Classification and IPC
`rPC{4):co7K 13/oo, C12P 21/00, C12N 15/00, C07H 15/12
`u.s. er.. .• . . 530L287, 435/68, 435/172.3, 536/27
`
`II. FIELDS SEARCHED
`
`Classification System
`
`530/387,388
`
`Classification Symbols
`935/6,9,10,11,15,22,23,24,59,60
`
`Minimum Documentation Searched 7
`
`U.S.
`
`435/68,170,172.3, 240.1
`
`536/27
`
`Documentation Searched other than Minimum Documentation
`to the !;xtent that such Documents are Included in the Fields Searched a
`
`Chemical Abstract Data Base (CAS) 1967-1988; BIOSIS DATA BASE 1969-
`J988 ffrn~: ~tigen, binding,
`site,synthetic,biosynthesis,
`ee a ac en.
`Ill. DOCUMENTS CONSIDERED TO BE RELEVANT9
`Category•
`Citation of Document, 11 with Indication, where appropr1ate, of lhe relevant passages 12
`y
`
`Relevant to Claim No. "·
`1-48
`
`u .s., A, 4,642,334 (MOORE ET AL)
`10 February 1987. See. abstract
`and columns 2, 3, 25 and 26.
`
`y
`
`y
`
`BIOCHEMISTRY, Volume 17 issued
`1978, September (Washington, o.c.,
`U.S.A.), (M.S. ROSEMBLATT ET AL.)
`uisolation of an active variable-
`domain fragment from a homogeneous
`rabbit antibody heavy chain,
`physiochemical and immunological
`properties", See pages 3877-3882,
`See particularly page 3877.
`
`BIOCHEMISTRY, Volume 19; issued
`1980, August (Washington, D.C.
`U.S.A.), (P.H. EHRLICH 'ET AL),
`"Isolation of an active heavy-chain
`variable domain from a homogeneous
`rabbit antibody by cathepsin
`B digestion of the aminoethylated
`heavy cbainu See pages 4091-4096.
`See particularly page 4091.
`
`1-48
`
`1-48
`
`• Special categories of cited documents: 10
`"A" document deflnlng.the general state of the art which Is not
`considered to be of particular relevance
`·E" earller document but published on or efter the international
`filing date
`"L" document which may throw doubts on priority claim(s) or
`which la cited to establish the publication date ol another
`citation or other special reason (as speclfled)
`"0" document referring to an oral disclosure, use, exhibition or
`other means
`"P" document published prior to lhe International filing date but
`later than the priority date claimed
`
`"TM later document published after the International flllng date
`or priority date and not In conflict with the appllcatlon but
`cited to understand the principle or theory underlying the
`invention
`"X" document of partlcular relevance: the claimed invention
`cannot be considered novel or cannot be considered to
`involve an Inventive step
`"Y" document of partlcular relevance: the claimed Invention
`cannot be considered to Involve an Inventive slap whan the
`document Is combined with one or more other such docu•
`ments; such combination being obvious to a pctraon skllled
`in the art.
`"d. .. document member of the seme patent family
`
`IV. CERTIFICATION
`Date of the Actual Completion of the International Search
`17 August 1988
`lntarnatlonal Searching Authority
`
`ISA/US
`
`Date of Malling of this International Search Report
`
`O 8 SEP 1988
`I
`l S ig~ Authorized 0~
`·Ri ~ e -AR,\
`
`·
`
`·
`
`BIOEPIS EX. 1002
`Page 3015
`
`
`
`r
`
`PCT/OSl:38/01737
`
`E
`
`r
`
`Attachment to PCT/ISA/210
`II. Field Searched·
`
`Keywords continued
`
`immunoglobulin, specificity, variable, region, domain,
`chimeric, heavy, light, Fv, antibody, antibodies,
`cancer, tumor, treatment.
`
`t
`
`BIOEPIS EX. 1002
`Page 3016
`
`
`
`111. DOCUMENTS CONSIDERED TO BE RELEVANT
`
`International Application No
`PCT/USR7Y01737
`(CONTINUED FROM THE SECOND SHEET)
`
`'
`
`I Relevant to Claim No
`1-48
`
`Category•
`
`Citation of Document, with indication, where appropriate, ol the relevant passages
`
`y
`
`y
`
`y
`
`y
`
`y
`
`'
`
`PROCEEDINGS NATIONAL ACADEMY OF
`SCIENCES, U.S.A., Volume 81,
`issued 1984, November (Washington,
`D.C., U.S.A.), (MORRISON ET AL.),
`"Chimeric human antibody molecules:
`mouse antigen binding domains with
`human constant region domains",
`See pages 6851-6855.~
`See particularly pages
`6851 and 6855.
`
`PROCEEDINGS NATIONAL ACADEMY OF
`SCIENCES U.S.A., Volume 84,
`issued 1987, January (Washington,
`D.C., U.S.A.), CL.K. SUN ET AL
`"Chimeric antibody with human
`constant regions and mouse
`variable regions directed against
`carcinoma-associated antigen
`17-A", See pages 214-218. See
`particularly page 214.
`
`PROCEEDINGS NATIONAL ACADEMY
`SCIENCES U.S.A., Volume 82,
`issued 1985, May (Washington,
`D.C., U.S.A.), (S. OHNO ET AL),
`"Antigen-binding specificities of
`antibodies are primarily determined
`by seven residues of VH", See pages
`2945-2949. See·particularly page
`2945.
`
`CHEMICAL ABSTRACTS, Volume 107,
`No. 5, issued 1987, August 3
`(Columbus, Ohio, UoS.A.), A.P.
`RICHARDSON ET AL., "The radiolabeling
`of antibodies for use in tumor
`location and treatment", See page 330
`column 1, the abstract no. 35697n,
`Med. Sci. Res. 1987, 15(7),
`343-7 (Eng.)
`
`CHEMICAL ABSTRACTS, Volume 105,
`No. 5, issued 1986, August 4
`(Columbus, Ohio, U.S.A.),
`R. ARNON "Immuno targeted
`chemotherapy in cancer research",
`See page 1, column 2, abstract no.
`34903y. Proc. FEBS Congr. 16th
`1984 (Pub. 1985). B,
`565-70 (Eng.)
`
`Form PCT/ISA/210 (mnraaheetJ (Rav.11-87)
`
`1-48
`
`1-48
`
`1-48
`
`1-48
`
`BIOEPIS EX. 1002
`Page 3017
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`
`
`r
`
`111. DOCUMENTS CONSIDERED TO BE RELEVA~T
`
`Category • I
`
`lnlemational Application No.
`
`PCTIUS88/01737·
`(CONTINUED FROM THE SECOND SHEEn
`
`I Relevant to Claim No
`
`y
`
`y
`
`y
`
`y
`
`! y
`
`E
`
`Cilalion of Document. with indicalion. ·where- appropriate. of the- relevant passages
`
`CHEMICAL ABSTRACTS, Volume 106,
`No. 13, issued 1987, March 30
`(Columbus, Ohio O.S.A.l,
`H.J. THIESEN ET AL "Selective
`Killing of human bladder cancer
`cells by combined treatment with
`A and B chain riciri antibody
`conjugates", See page 37, column 1,
`abstract no. ·9S759g. Cancer
`Res., 1987, 47(2), 419-423 Cl!lng.)
`
`CHEMICAL ABSTRACTS, Volume 105,
`No. 11, issued 1986, September 15
`(Columbus, Ohio, U.S.A.),
`M.S. NEUBERGER "Novel antibodies
`by DNA transfection", see page
`475, column 2, abstract no.
`95609d,. Adv. Imniunopharmacol. 3.,
`Proc. Int. Conf. 3rd 1985 (Pub. 1986),
`317-327 (Eng.)
`
`WO, A, WO 86/0090 DAW
`..
`3 January 1986- (03.01.86)
`See pages 1-16, See particularly
`pages·2~9.
`
`PROCEEDINGS NATIONAL ACADEMY OP
`SCIENCES, U.S.A~ Volume 81,
`issued 1984, June (Washington, D..C.
`U~S.A.), CS •. CABILLY ET AL.)
`"Generation of antibody activity
`from immunoglobulin polypeptide
`chains produced in Escherichia
`coli" See pages 3273-3277.
`See particularly·pages 3273 and 3275.
`
`WO, A, WO 86/01533 DAW
`13 March 1986 (13.03.86)
`See pages 1-3.2. See particularly
`pages 3-8.
`
`U .. S. A, 4,704,692 (R.C. LADNER
`)
`3 November 1987. See abstract and
`columns 2 and 14.
`
`-1-48
`
`1-48
`
`1-48
`
`1-48
`
`ll
`l-10,
`12-48
`
`(-48
`
`-
`
`i
`I
`
`I
`
`•
`
`f
`
`FormPCTIISA/210(8lllraS'-J(Rav.1t-87)
`
`BIOEPIS EX. 1002
`Page 3018
`
`
`
`PCT
`
`WORLD INTELLECTIJAL.PROPERTY ORGANIZATION
`International Bureau
`,
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER 1HE PATENT COOPERATION TREATY (PCl).
`WO 91/07500
`
`(SI) International Patent Oasslficatioo 5 :
`C12P 21/08, C12N 15/13, S/10
`A61K 39/395, GOIN 33/574
`
`Al
`
`(II} International Publication Number:
`
`(43) International Publlcadon Date: ·
`
`30 May 1991 (30.05.91)
`
`(21) International Application Number:
`
`PCT/GB90/01755
`
`(22) Iotematlooal F1Iiog Date:
`
`14 November 1990 (14. l 1.90)
`
`(74) Agent: BUTLER, David. John; Patent Division, Unilever
`pie. Unilever House, Blackfriars, London EC4P 4BQ
`(GB) .
`
`(30) Priority data:
`8926045.9
`9019552.0
`
`17 November 1989(17.11.89) · GB
`7 September 1990 (07.09.90) GB
`
`(81) Designated States: Au; BG, CA, FI, HU, JP, KR, NO,
`RO, SU; US.
`
`(71) Appllcant (for AU CA only): UNILEVER PLC [GB/GB]; Published
`Wilh internalional search report.
`Unilever House, Blackfriars, London EC4P 4BQ (GB).
`Wilh amended claim.r. . ·
`
`(71) Applicant (for all designated States ·except AU CA US): UN(cid:173)
`ILEVER NV [NL/NL]; Burgemeester s' Jacobplein ·1,
`NL-Rotterdam (NL).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): VERHOEYEN, Martine,
`Elisa [BE/GB]; l Tintagel Close, Manor Farm Estate,
`Rushden NNIO ONP (GB).
`
`(54) 11tle: SPECIFIC BINDING AGENTS
`
`(57) Abstract
`
`A reshaped human antibody or reshaped human antibody fragment having specificity for human placental alkaline phos(cid:173)
`phatase (PLAP) is produced by transferring the complementarity determining regions (CDRs) from a murine anti-PLAP hybrido(cid:173)
`. ma cell line Hl7E2 into a human antibody variable region framework. The reshaped molecule can be used in the treatment or di-
`agnosis of cancer.
`·
`
`'
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`•
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`BIOEPIS EX. 1002
`Page 3019
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`
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pap of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BP
`BG
`BJ
`BR
`CA
`CF
`cc
`CH
`Cl
`CM
`DE
`DK
`ES
`
`Allltria
`A111tralia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Bonhl
`Brazil
`Canada
`Cenll'al African Repubru:
`Congo
`Switrcrland
`C6le d'Ivoire
`ea..__
`Germany
`Denmad.
`Spain
`
`Fl
`FR
`CA
`CB
`CN
`CR
`HU
`rr
`JP
`KP
`
`Finland
`France
`Gabon
`United Kin(ldom
`Guinea
`Greece
`Hungary
`Italy
`Japan
`Ocmoc:ratic Peoplo.:'s Rupublic
`or Kore:a
`KR
`Republic or Korea
`Ucc:ht&:ns!Cin
`LI
`LK
`Sri Lanka
`LU
`IAJxcmbourg
`MC
`Monaco
`MC Madepsrar
`
`ML
`Mall
`Mongolia
`MN
`MR
`Mauritania
`MW Malawi
`NL
`Nctllcrlands
`NO
`Norway
`Poland
`PL
`RO
`Romania
`Sudan
`SD
`S8
`Swc:den
`Senegal
`SN
`Soviet Union
`SU
`TD
`Cbad
`Togo
`TG
`us
`United Slates of America
`
`•
`
`BIOEPIS EX. 1002
`Page 3020
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`WO 91/07SOO.
`
`. PCT /GB90/017SS
`
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`- 1 -
`
`SPECIFIC BIHDING AGENTS
`
`This invention relates to specific.binding agents,
`and in particular to polypeptides containing·amino acid
`sequences that bind specifically to other proteinaceous or
`non-proteinaceous materials. The invention most
`particularly concerns the production of suchspecific
`binding agents by genetic engineering.
`
`Antibody structure
`
`Natural antibody molecules consist of two identical
`heavy-chain and two identical light-~hain polypeptides,
`25 which are covalently linked bydisuiphide bonds. Figure
`13 of the accompanying drawings diagramatically:represents
`the typical structure of an antibody of the IgG class.
`.
`Each of the chains is folded into severai discrete
`.
`domains. The N-terminal domains of all.the chains are
`variable in sequence and therefore called the variable
`. . .
`.
`. .
`. .
`regions (V-regions) • The v-regions · of one heavy (VH) and .
`one light chain (VL) associate to form the antigen-binding
`site. The module formed by the combined VH and VL domains
`is referred to as the Fv (variable fragment) of the'
`
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`BIOEPIS EX. 1002
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`PCT/GB90/01755
`
`- 2 -
`
`antibody. The c-terminal ends of both heavy and light
`chains are more conserved in sequence and chains are more
`conserved in sequence and therefore referred to as the
`constant regions. Heavy chain constant regions are
`composed of several domains, eg. the heavy chain of the
`gamma-isotype (IgG) consists of three domains (CHl, CH2,
`CH3) and a hinge region which connects the CHl and CH2
`domains. The hinges of the two heavy chains are
`covalently linked together by disulphide bridges. Light
`chains have one constant domain which packs against the
`~'"Hl domain. The constant regions of the antibody molecule
`are involved in effector functions such as complement
`lysis and clearing by Antibody Dependant Cell Cytotoxicity
`(ADCC). Classical digestion of an antibody with the
`protease papain yields three fragments. One fragment
`contains the CH2 and CHJ domains and, as it crystallises
`easily, was called the Fe fragment. The other two
`fragments were designated the Fab (antigen-binding)
`fragments, they are.identical and contain the entire light
`chain combined with the VH and CHl domain. When using
`pepsin, the proteolytic cleavage is such that the two Fabs
`remain connected via the hinge and form the (Fab) 2
`fragment. Each of the domains is represented by a
`separate exon at the genetic level.
`
`The variable regions themselves each contain J
`clusters of hypervariable residues, in a framework·of more
`conserved sequences. These hypervariable regions interact
`with the antigen, and are called the Complementarity
`Determining Regions (CDRs). The more conserved sequences
`are called the Framework Regions (FRs). See Kabat et al
`(1987). X-ray studies of antibodies have shown that the
`CDRs form loops which protrude from the top of the
`molecule, whilst the FRs provide a structural beta-sheet
`framework.
`
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`W091/07500
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`PCT/GB90/017SS
`
`-
`
`3
`
`-
`
`Modified antibodies
`
`In one embodiment, the invention relates to so-called
`"reshaped" or "altered" human antipodies,. ie.
`immunoglobulins having essentially human constant and
`framework regions but in which the complementarity
`determining regions (CDRs) correspond to those found in a
`non-human immunoglobulin, and also to corresponding
`reshaped antibody fragments.
`
`The general principles by which such reshaped·human
`antibodies and fragments may be produced are now
`well-known, and referen~e can be made to Jories et al
`(1986), Riechmann et al (1988}, Verhoeyen et al· (1988),
`and EP-A-239400 (Winter). A comprehensive list of
`relevant l·iterature references is provided later· in this
`specification.
`
`Reshaped human antibodies and fragm¢n~s · .have ·
`particular utility in the in-vivo diagnosis and treatment
`of human ailments because the essentially human.proteins
`are less likely to induce undesirable adversereactions
`when they are administered to a human patient, and
`the desired specificity conferred by the CDRs can be
`raised in a host animal, such as a: 'mouse, frbm which
`antibodies of selected specificity can be obtained more
`readily. The variable region genes can be cloned from the
`non-human antibody, and the CDRs grafted into a human
`variable-region framework by genetic engineering
`techniques to provide the reshaped human antibody or
`fragment. To achieve this desirable result, it is
`necessary to identify and sequence at least. the CDRs.in
`the selected non-human antibody, and preferably the whole
`non-human variable region sequence, to allow
`
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`PCT/GB90/01755
`
`-
`
`4 -
`
`identification of potentially important CDR-framework
`interactions.
`
`summary of the invention
`
`The invention provides, as one embodiment, a
`synthetic specific binding polypeptide having specificity
`for human placental alkaline phosphatase (PLAP). By
`synthetic, we particularly mean that the polypeptide is
`produced by recombinant DNA technology, and to that extent
`at least is different from a naturally-occurring or
`naturally-induced specific binding agent having identical
`specificity. Alternatively, the synthetic polypeptide has
`been produced by artificially assembling a sequence of
`amino acids to produce a novel or nature-identical
`molecule. The synthetic polypeptide can be equivalent to
`an intact conventional antibody, or equivalent to a
`multiple or single-chain fragment of such an antibody, or
`can be simply a material that includes one or more
`sequences that confer the desired specific binding
`capability.
`
`The invention provides as an important embodiment, a
`reshaped human antibody, or a reshaped human antibody
`fragment, having anti-human placental alkaline phosphatase
`(PLAP) specificity.
`
`More particularly, the invention provides a reshaped
`human antibody or reshaped human antibody fragment, having
`anti-human placental alkaline phosphatase specificity,
`containing one or more of the CDRs depicted in Figures 1
`and 2 of the accompanying drawings. Preferably, the
`reshaped antibody or fragment of the invention contains
`all 3 of the CDRs depicted in Figure 1 of the accompanying
`drawings, in a human heavy chain variable region
`
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`W091/07SOO
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`(
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`framework. Alternatively, or in addition, the reshaped
`antibody or fragment of the invention contains all 3 of
`the CDRs depicted in Figure 2 of the accompanying
`drawings, in a human light chain variable region
`framework.
`
`.
`
`.
`Another embodiment of the invention is a reshaped
`antibody or reshaped antibody fragment . containing a
`_ _
`protein sequence as depicted in Figure 10 and/or Figure 11
`of the accompanying drawings.
`
`.
`
`Other important embodiments of the invention are an
`expression vector incorporating a DNA sequence as depicted
`in Figure 10 and/ or Figure ii of the_. accompanying
`drawings, and an expression vector_incorpora:ting·a DNA
`sequence encoding one or more of the.protein sequences
`designated as being a CDR in Figure· 1 and/ or. -F'igure. 2 of·
`the accompanying drawings.
`
`"'
`An important aspect of the invention is a stable host
`cell line containing a foreign gene that causes the host
`cell line to produce a specific binding agent according to(cid:173)
`the invention. This can be a stable hostcell<line
`containing a foreign gene that encodes at least one of the
`amino acid sequences designated · as being a CDR · in Figure· 1
`an~/or Figure 2 of the accompanying drawings, .together
`with a protein framework that enables the encoded amino
`acid sequence when expressed to function as a·coR having
`specificity for PLAP.
`
`The invention particularly provides an. immortalised
`mammalian cell line, or a yeast, or other e\lkaryotic cell,· .
`or a prokaryotic·cell such as a bacterium, producing a
`reshaped antibody or fragmentaccording to the invention.
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`Another important aspect of the invention is a
`synthetic specific binding agent, reshaped human antibody
`or reshaped human antibody fragment, having specificity
`equivalent to that of the gamma-1, kappa anti-PLAP
`monoclonal antibody secreted by murine hybridoma cell line
`H17E2.
`
`The invention also provides two novel plasmids,
`pSVgptHu2VHPLAP-HuigG1 and pSVneoHuVkPLAP-HuCk, and these
`plasmids can be used in the production of a synthetic
`specific binding agent,