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`a2) United States Patent
`US 7,374,758 B2
`(10) Patent No.:
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`*May20, 2008
`(45) Date of Patent:
`Papadopoulosetal.
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`US007374758B2
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`(54) MODIFIED CHIMERIC POLYPEPTIDES
`WITH IMPROVED PHARMACOKINETIC
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`PROPERTIES AND METHODS OF USING
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`THEREOF
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`(75)
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`Inventors: Nicholas J. Papadopoulos,
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`Lagrangeville, NY (US); Samuel Davis.
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`New York, NY (US); George D.
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`Yancopoulos, Yorktown Heights, NY
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`(US)
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`(*) Notice:
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`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
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`Tarrytown, NY (US)
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`Subject to any disclaimer,the term of this
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 489 days.
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`This patent is subject to a terminal dis-
`claimer.
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`(21) Appl. No.: 11/016,503
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`(22)
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`(65)
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`Filed:
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`Dec. 17, 2004
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`Prior Publication Data
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`US 2005/0245447 Al
`Nov. 3, 2005
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`Related U.S. Application Data
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`(62) Division of application Na. 10/009,852, filed as appli-
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`cation No. PCT/US00/14142 on May 23, 2000, now
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`Pat. No. 7,070,959.
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`(60) Provisional application No. 60/138,133. filed on Jun.
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`8, 1999.
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`(51)
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`Int. Cl.
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`(2006.01)
`AGIK 38/18
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`(2006.01)
`CO7K 14/71
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`(2006.01)
`C12N 15/62
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`(32) US. Che ccc ene 424/134.1; 424/192.1;
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`514/2; 514/23; 530/350; 536/23.4
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`(58) Field of Classification Search 0.0.0.0... None
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`See application file for complete search history.
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`References Cited
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`ligand binding and mayinitiate a signal transduction cascade”, The
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`EMBO Journal 15(18):4919-4927.
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`* cited by examiner
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`Primary Examiner—Christine J Saoud
`Assistant Examiner
`Jon M Lockard
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`(74) Attorney, Agent, or Firm—Valeta Gregg, FE
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`wnq.
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`(57)
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`ABSTRACT
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`Modified chimeric polypeptides with improved pharmaco-
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`kinetics are disclosed. Specifically, modified chimeric F1t1
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`receptor polypeptides that have been modified in such a way
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`as to improve their pharmacokinetic profile are disclosed.
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`Also disclosed are methods of making and using the modi-
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`fied polypeptides including but not limited to using the
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`modified polypeptides to decrease or inhibit plasma leakage
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`and/or vascular permeability in a mammal.
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`3 Claims, 55 Drawing Sheets
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`CELLTRION - EXHIBIT 1010
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`CELLTRION - EXHIBIT 1010
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`U.S. Patent
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`May20, 2008
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`Sheet 1 of 55
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`US 7,374,758 B2
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`woeacetylated Fit-1(1-3)-Fe
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`U.S. Patent
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`May20, 2008
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`Sheet 2 of 55
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`US 7,374,758 B2
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`U.S. Patent
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`May20, 2008
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`Sheet 3 of 55
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`US 7,374,758 B2
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`U.S. Patent
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`May20, 2008
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`Sheet4 of 55
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`U.S. Patent
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`May20, 2008
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`Sheet 5 of 55
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`US 7,374,758 B2
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`Fig.6A.
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`123 45 67 8
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`Sheet6 of 55
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`US 7,374,758 B2
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`U.S. Patent
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`May20, 2008
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`Sheet7 of 55
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`US 7,374,758 B2
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`Fig.8.
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`U.S. Patent
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`May20, 2008
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`Sheet 8 of 55
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`US 7,374,758 B2
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`U.S. Patent
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`May20, 2008
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`Sheet 9 of 55
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`US 7,374,758 B2
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`Fig.10A.
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`
`
`
`
`190
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TSG TOT TIS CCT GAA ATG GIG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GOO
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACC AGA AAC GGA CIT TAC CAC TCA TIC CTT TOS CTT TCC GAC TOG TAT TGA TIT AGA COG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Trp Ser Leu Bro Glu Met Val Ser Lys Glu Ser Glu Arg Deu Ser Ile Thr Lys Ser Ala>
`
`
`
`
`
`
`
`*
`
`260
`*
`
`
`
`
`
`270
`*
`
`
`*
`
`
`
`250
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`‘TST GGA AGA AAT GGC AKA CAA TIC TGC AGT ACT TTA ACC TIG AAC ACA GCT CAA GCA AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA COT TCT TIA COG TTT GIT AAG ACG TCA TGA AAT TGG AAC TTG TGT CGA GTT CGT TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Gly Arg Asn Gly Ilys Gln Phe Cys Ser Tor Leu Thar Leu Asn Thr Ala Gln Ala Asn>
`
`*
`
`
`
`280
`*
`
`*
`
`
`250
`*
`
`300
`*
`
`
`
`
`*
`
`
`
`310
`t
`
`
`*
`
`320
`*
`
`
`
`
`*
`
`
`330
`*
`
`
`*
`
`340
`t
`
`
`
`
`*
`
`
`
`350
`*
`
`
`360
`*
`
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`GTG TIGA COG AAG ATG TOG ACG TTT ATA GAT OGA CAT GGA TGA AGT Tre Tre TIC CIT Tor
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr>
`
`*
`
`
`370
`i
`
`
`*
`
`
`390
`ry
`
`*
`
`
`
`420
`
`
`
`
`
`&20
`4
`
`
`400
`390
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA TOT GOA ATC TAT ATA TTT ATT AGT GAT ACK GOT AGA COT TIC GTA GAG ATG TAC AST
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT AGA CGT TAG ATA TAT. AAA TAA TCA CTA TGT CCA TCT GGA AAG CAT CIC TAC ATG TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ser Ala [le Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`*
`
`
`
`
`
`*
`
`
`440
`*
`
`
`
`
`450
`*
`
`*
`
`
`
`a
`
`
`460
`*
`
`
`
`
`*
`
`
`470
`*
`
`
`
`
`460
`*
`
`*
`
`
`
`430
`a
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA ATC COC GAA ATT ATA CAC ATG ACT GAA GGA ASG GAS CIC GC ATT CCC TSC OGG GIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT TAG GGG CTT TAA TAT GIG TAC TGA CIT CCT TCC CIC GAG CAG TAA GGG ACG GOC CAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Tle Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val>
`
`*
`
`
`
`*
`
`
`
`520
`500
`490
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TIT CCA CTT GAC ACT TTG ATC CCT GAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGC AGT GGA TIG TAG TGA CAA TIGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAS GGA CTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Sex Pro Asn Tie Thr Val Thr Leu Lys Lys Phe Pro Lev Asp Thr Leu Ile Pro Asp>
`
`
`
`529
`*
`
`*
`
`
`+t
`
`
`
`
`
`es
`
`
`
`530
`
`
`
`
`
`540
`*
`
`t
`
`
`
`€
`€
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 10 of 55
`
`
`
`US 7,374,758 B2
`
`Fig.10B.
`
`*
`
`
`*
`
`
`
`
`&
`
`*
`
`
`
`
`
`
`
`570
`*
`
`
`*
`
`
`
`
`580
`*
`
`*
`
`
`
`
`
`
`
`AAA CGC ATA ATC TGG GAC
`
`
`
`
`
`TTT GCG TAT TAG ACC CTG
`
`
`
`
`
`
`Gly Lys Arg Ile Ile Trp Asp
`
`AGT AGA
`
`
`
`
`TA TCT
`
`
`Ser Arg
`
`
`
`
`
`
`
`AAG GSC TH ATC ATA TCA
`
`
`
`
`
`
`TIC CCG AAG TAG TAT AGT
`
`
`
`
`
`
`Lys Gly Phe Ile Ile Ser
`
`
`
`*
`
`
`
`600
`590
`*
`t
`
`
`
`AAT GCA ACG TAC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`TTA CGT TGC ATS TIT
`
`
`
`
`
`
`Asn Ale Thr Tyr Lys>
`
`610
`*
`
`
`
`*
`
`620
`«
`
`
`
`630
`*
`
`
`
`t
`
`640
`&
`
`
`
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA GCA
`ACA GTC AAT GGG CAT TIG
`ATA GGG CTT CTS ACC ToT
`TAT AAG ACA AAC TAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TAT CCC GAA GAC TGG ACA
`CTT CGT
`ATA THC TET TIG ATA
`TGT CAG TTA CCC GTA AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu
`Glu Ala
`Ile Gly Leu Leu Thr Cys
`Thr Val Asn Gly His Leu
`Tyr Lys Thr Asn Tyr>
`
`
`
`650
`*
`
`
`r
`
`
`
`
`660
`t
`
`
`*
`
`
`
`*
`
`680
`«
`
`
`
`
`
`690
`*
`
`700
`
`
`
`670
`*
`*®
`‘
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA ATC
`ATA GAT GTC CAA ATA AGC
`ACA CAT CGA CAA ACC AAT
`TGT GTA GCT GTT TGG TTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGT TAG
`TAT CTA CAG GTT TAT TG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Ile
`Thr His Arg Gln Thr Asn
`Ile Asp Val Gln Ile Ser
`
`x
`
`720
`t
`
`
`
`710
`*
`
`
`
`t
`
`
`
`
`
`
`ACA CCA COC CCA GC
`
`
`
`
`
`TGT GGT GOS GST CAG
`
`
`
`
`
`
`Thr Pro Arg Pro Val>
`
`
`
`
`
`740
`*
`
`*
`
`760
`*
`
`
`
`770
`Cg
`
`
`
`780
`*
`
`
`
`*
`
`730
`*
`
`7
`
`750
`*
`
`
`*
`
`
`TTA CTT AGA GGC CAT ACT
`CTT Gre
`
`
`
`
`
`
`
`
`
`
`
`
`
`CIC AAT TGT ACT GCT ACC
`GAA CAG
`AAT GAA TCT CCG GTA TGA
`GAG TTA ACA TGA CGA TGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Val
`Leu Leu Arg Gly His Thr
`Leu Asn Cys Thr Ala Thr
`
`*
`
`ACT CCC TIG AAC ACG
`
`
`
`
`NGA GGG AAC TIG TGC
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Pro Leu Asn Thr>
`
`
`
`*
`
`800
`*
`
`
`
`810
`*
`
`
`
`840
`830
`820
`790
`*
`*
`*
`*
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`GAT GAA AAA AAT AAG AGA
`Tac CCT
`GCT TCC GTA AGG CGA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AGA GIT CAA ATG ACC TGG AGT
`CTA CTT TIT TTA TIC TCT
`TCT CAA GIT TAC TGG ACC TCA ATG GGA
`CGA AGG CAT TCC GCT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tyx Pro
`Ala Ser Val Arg Arg>
`Asp Glu Lys Asn Lys Arg
`Arg Val Gin Met Thr Trp Ser
`
`
`
`
`
`
`
`850
`*
`
`
`
`«
`
`
`
`500
`B70
`880
`860
`
`*
`*
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`AAC ATA TTC TAC AGT GTT
`CTT ACT ATT GAC AAA
`caT Gece
`AAT TCC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CGA ATT GAC CAA AGC
`GTA CGG
`TTA AGG
`TTG TAT AAG ATG TCA CAA
`GAA TGA TAA CTG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`GCT TAA CTIG GTT TCG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Ser
`His Ala
`Leu Thr Ile Asp Lys>
`Arg Ile Asp Gin Ser
`Asn Ile Phe Tyr Ser Val
`
`
`
`
`
`850
`*
`
`*
`
`
`
`*
`
`910
`*
`
`
`
`*
`
`
`
`
`
`ATG CAG AAC AAA GAC
`TAC GIC TITG TIT CTS
`
`
`
`
`
`
`
`
`Met Glin Asn Lys Asp
`
`920
`*
`
`930
`*
`
`
`
`*
`
`5940
`*
`
`
`
`950
`t
`
`
`
`960
`*
`
`
`
`*
`*
`
`
`
`
`GGA CCA TCA TIC AAA
`CTT TAT
`AAA GGA
`‘ACT IGT CGT GTA AGG AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA ATA
`TTT CCT
`CCT GGT AGT AAG TIT
`TGA ACA GCA CAT TCC TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Tyr
`Thr Cys Arg Val Arg Ser
`Lys Gly
`Gly Pro Ser Phe Lys>
`
`*
`
`970
`*
`
`
`
`*
`
`980
`*
`
`
`
`*
`
`990
`
`*
`
`1000
`1010
`*
`*
`*
`*
`
`
`
`
`GAG CCC AAA TCT TCT
`GtG CAT
`ATA TAT
`GAT AKA GCA GGC CCG GGC
`TCT GTT AAC ACC TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TAT ATA
`CAC GTA
`CTA TTT CGT CCG G6C CCG
`CTC GGG TTT AGA ACA
`AGA CAA TIG TGG AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ser Val Asn Thr Ser
`Val His
`Glu Pro Lys Ser Cys>
`Tle Tyr
`Asp Lys Ala Gly Pro Gly
`
`
`
`
`
`1020
`*
`
`
`
`*
`
`*
`
`21030
`*
`
`
`
`1040
`
`*
`*
`
`
`GAC AAA ACT CAC ACA TGC CCA
`
`
`
`
`
`
`CTS TTT TGA GTG IGT ACG GGT
`
`
`
`
`
`
`
`
`
`
`
`
`Asp Lys Thr His Thr Cys Pro
`
`*
`
`1050
`*
`
`
`
`1060
`*
`
`
`
`1070
`*
`
`
`
`1080
`*
`
`
`
`t
`*
`*
`
`
`
`ce TSC
`GGG GGA COG TCA GTC
`CCA GCA CCT GAA CX CTIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGC ACG
`cee CCT GGC AGT CAG
`GGT CGT GGA CTT GAG GAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Ala Pro Glu Leu Leu
`Pro Cys
`Gly Gly Pro Ser Val>
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`
`Sheet 11 of 55
`
`
`
`US 7,374,758 B2
`
`Fig.10C.
`
`
`
`*
`
`1990
`*
`
`
`
`*
`
`1100
`
`e
`
`*
`
`
`
`1110
`*
`
`
`*
`
`1126
`
`*
`
`t
`
`1130
`J
`
`
`
`1140
`t
`
`
`
`*
`
`AAS
`TTC CTC TTC CCC CCA ARA CoC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAC ACC CTC ATG ATC TOC CGG ACC CCT GAG GTC ACA
`Tr
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTG TSG GAG TAC TAG AGG GCC TGG GGA CTC CAG TCT
`AAG GAG AAG GGG GST TIT GGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Lys
`Phe Leu Phe Pro Pro Lys Pro
`Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr>
`
`©
`
`i150
`*
`
`
`
`*
`
`1160
`*
`
`
`
`
`
`
`
`
`
`TCC GIG GTS GIG GAC STG AGT
`
`
`
`
`
`ACG CAC CAC Cac CTG Cac TOG
`
`
`
`
`
`
`Cys Val Val Val Asp Val Ser
`
`CAD
`
`
`
`His
`
`
`
`41180
`*
`
`
`
`*
`
`1200
`1170
`
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA GAC COT GAS GIC AAG TTC AAC TGS TAC GTG GAC
`
`
`
`
`
`
`
`
`
`
`
`
`CTT CTG GGa CTC CaG TTC AAG TTG ACC ATG Cac CTS
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Asp Pro Glu Val Lys Fhe Asn Trp Tyr Val Asp>
`
`1190
`*
`
`
`
`4
`
`t
`
`1220
`
`*
`
`
`1230
`
`
`
`1240
`
`*
`
`tf
`
`1250
`*
`
`
`
`1260
`t
`
`
`
`1216
`
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA AAS COG CGG GAG GAG CAG TAC AAC AGC ASG TAC
`AB
`os GTS GAG GIG CAT AAT GCC
`TST TIC Got GCC CK Cle GC ATS TTS TCS TSC ATG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CCG CAC CIC CAC GTA TTA CGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Vai Glu Val His Asn Ala
`Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr>
`
`Lys
`
`*
`
`1280
`*
`
`
`
`*
`
`1279
`
`*
`
`
`
`
`
`
`
`
`
`CGT GTG GTC acc GTC cic acc
`GCA CAC CAG TOG CAG GAG TGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Val
`Arg Val Val Ser Val Leu Thr
`
`1290
`
`*
`
`*
`
`*
`
`1300
`
`*
`
`*
`
`1310
`
`*
`
`
`
`
`
`
`
`
`
`*
`
`*
`
`1320
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTS Cac CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG
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`1350
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`
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`1350
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`Jyr Thr Leu Pro Pro Ser Arg
`Asp Glu
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`1440
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`1460
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`Val Lys Gly Phe Tyr Pro Ser Asp Ile
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`1490
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`AAC AAC TAC AAG ACC ACG CCT
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`Pro Val
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`1560
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`1570
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`
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`1600
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`ace
`AAG CTC ACC GTG GAC AAG AGC
`
`
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`CTG CCG AGS AAG AAG GAS ATS
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`Arg Trp Gln Gin Gly>
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`
`*
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`
`
`
`
`t
`
`€
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 12 of 55
`
`
`
`US 7,374,758 B2
`
`Fig.10D.
`
`
`
`
`
`1630
`*
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`1640
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`1660
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`
`
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`
`
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`
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`
`
`
`
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`Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 13 of 55
`
`
`
`US 7,374,758 B2
`
`
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`
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`
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 14 of 55
`
`
`
`US 7,374,758 B2
`
`
`
`
`
`
`
`
`
`
`
`
`
`
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`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 15 of 55
`
`
`
`US 7,374,758 B2
`
`Fig.13A.
`
`
`
`*
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`
`70
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`110
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`His Leu Gln Cys Arg
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`
`150
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`
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`
`160
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`180t
`
`
`
`170
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`GCC CAT ABA
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`Crt ccT
`
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`Glu Ala
`Ala His Lys>
`Gly
`
`
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`200
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`TGG TCT TYG CCT GAA ATG GIG AGT
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`GAA AGC GAA AGG CTG
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`Lys
`Glu Ser Glu Arg Leu
`Trp Ser Leu Pro Glu Met Val Ser
`
`210
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`
`
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`
`220
`
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`
`
`230
`
`
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`
`240*
`
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`ATA ACT
`
`
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`TIT AGA CGG
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`Ile Thr
`Lys Ser Ala>
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`ASC
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`ToS
`
`Ser
`
`
`270
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`
`
`
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`
`
`
`290
`
`
`
`300*
`
`*
`
`260
`*
`
`280
`250
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`
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`TGC
`AGT ACT TTA ACC TTS
`TGT GGA AGA AAT GGC AAA CAA TTC
`
`
`
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`ACA CCT TCT TIA CCG TIT GIT AAG
`TCR TGA AAT TGG AAC
`
`
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`
`
`
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`Ser Thr Leu Thr Leu
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`Cys Gly Arg Asn Gly Lys Gln Phe
`
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`CAA GCA ARC
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`TGT CGA
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`
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`Gln Ala Asn>
`Thr Ala
`
`Bae.
`
`
`
`
`
`*
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`
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`
`320
`310.
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`AAG GAA ACA
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`
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`Ser
`Leu Ala Veal Pro Thr
`Lys Lys
`Lys Glu Thr>
`His Thr Gly Phe Tyr Ser Cys Lys
`
`330
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`
`
`
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`
`340
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`
`350
`
`
`
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`360*
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`
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`380
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`
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`Giu Ser Ala Ile Tyr Ile Phe [le
`
`390
`
`*
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`
`400
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`TTC
`GTA GAG
`GAT ACA GGT AGA CCT
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`
`
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`CAT CTc
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`CTA TOT CCA TCT GGA
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`Vel Giu
`
`
`
`
`
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`
`Asp Thr Gly Arg Pro
`
`410
`*
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`
`420
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`
`
`
`
`TAC ATG TCA
`
`
`
`
`
`
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`
`Met Tyr Ser>
`
`460
`Ci
`
`
`
`*
`
`480
`*
`
`
`
`TGC OGG CTT
`
`
`ACG GCC CAA
`
`
`
`
`
`
`Cys Arg Val>
`
`470
`450
`430
`
`
`*
`*
`*
`*
`
`
`
`
`
`
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`
`ATT ccc
`STC
`ACT GAA GGA AGG GAG CTC
`GAA ATC CCC GAA ATT ATA CAC ATG
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`Tle Pro
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`Glu Tle Pro Glu Ile Ile His Met
`Thr Glu Gly Arg Glu Leu
`
`+ T
`
`*
`
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`
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`
`440
`*
`
`
`
`t
`
`©
`
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`
`510
`*
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`*
`
`520
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`
`
`
`*
`
`530
`
`
`
`540
`*
`
`*
`
`490
`500
`
`
`*
`*
`
`
`
`
`GAC
`TTA AAA AAG TTT CCA CIT
`ACT TTS ATC CCT GAT
`ACS TCA CCT AAC ATC ACT GTT Act
`
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`AAT TIT TIC AAA GST GAA CTG
`TGA AAC TAG GGA CTA
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`
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`
`
`Thr Ser Pro Asn Ile Thr Val Thr
`Thr Leu Tle Pro Asp>
`Leu Lys Lys Phe Pro Leu Asp
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 16 of 55
`
`
`
`US 7,374,758 B2
`
`Fig.13B.
`
`
`
`*
`
`550
`*
`
`
`
`580
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`
`
`
`
`CCT TT GCG TAT TAG ACC CTG TCA
`TT TIC COG AAG TAG TAT AGT TTA CGT TCC ATG MrT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ser
`Gly Lys Arg Tle Ile Trp Asp Ser
`arg LyS Gly Phe Ile Ile
`Asn Ala Thr Tyr Lys>
`
`
`
`
`
`$70
`*
`
`
`
`
`*
`
`
`
`$30
`*
`
`
`
`
`600
`cf]
`
`t
`
`610
`*
`
`
`
`
`a
`
`
`
`
`630
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TTS
`GAA ATA GGG CIT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT
`CIT TaT CCC GAA GaCc
`TSG ACA CTT
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAT
`CGT TGT CAG TTA CCC GTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu
`Glu Ile Gly Leu Lew
`Thr Cys Glu Ala Thr Val Asn Gly His
`
`*
`
`
`
`640
`*
`
`
`*
`
`
`650
`*
`
`
`
`
`660
`t
`
`*
`
`
`
`
`
`TAT AAG ACA AAC TAT
`
`
`
`
`
`ATA TTC TOT THG ATA
`
`
`
`
`
`
`Tyr Lys Thr Asn Tyr>
`
`
`690
`700
`*
`*
`J
`
`
`
`
`CTC ACA CAT CGA CAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACC AAT ACA
`ATC ATA GAT GTC CAA ATA
`GAS TGT GTA GCT GIT
`
`
`
`
`
`
`
`
`
`
`
`(GG TTA ToT
`TAG TAT CTA CAG GTT TAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Asn Thr
`Leu Thr His Arg Gin
`Tle Tle Asp Val Gln Tle
`730
`*
`
`670
`°
`
`
`
`*
`
`td
`
`*
`
`*
`
`
`
`
`
`*
`
`
`
`
`
`
`
`710
`*
`
`
`
`720
`7
`*
`
`
`
`
`
`
`
`ACA CCA C&C CCA GTC
`
`
`
`
`
`
`TGT GGT GCG GGT CAG
`
`
`
`
`
`
`Thr Pro Arg Pro Val>
`
`
`
`aa.
`
`
`
`
`
`Ser
`
`
`
`750
`+
`
`*
`
`
`
`760
`*
`
`4
`
`
`
`*
`
`
`
`
`AAA TTA CTT AGA GGC
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAT ACT CTT
`GTC CIC AAT TGT ACT GCT
`GTA TGA GAA
`
`
`
`
`
`
`
`
`
`
`
`
`TIT AAT GAA TCT COG
`CAG GAG TTA ACA TGA CGA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Thr Leu
`Lys Leu Leu Arg Gly
`Val Leu Asn Cys Thr Ala
`
`*
`
`
`
`
`
`7790
`780
`*
`t
`
`
`
`
`
`
`
`
`ACT CCC TTG AAC ACG
`
`
`
`
`
`TGA GGG AAC TIG TGC
`
`
`
`
`
`
`Thr Pro Leu Asn Thr>
`
`*
`
`750
`*
`
`
`
`
`
`
`
`AGA GTT CAA ATG ACC
`TCT CAA GIT TAC TGG
`
`
`
`
`
`
`
`
`Arg Val Gln Met Thr
`
`*
`
`
`
`
`
`
`
`TGG AGT TAC
`acC TCA ATG
`
`
`
`
`
`
`Trp Ser Tyr
`
`*
`
`
`
`.
`
`
`
`
`810
`820
`840
`*
`«
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`RAT TCC CAT GCC AAC
`CCT GAT GAA ATT GAC CAA
`
`
`
`
`
`
`
`
`TTA AGG GTA CGTIG
`GGA CTA CTT TAA CTG GTT
`
`
`
`
`
`
`
`
`
`
`
`Asn Ser His Ala Asn>
`Pro Asp Glu Ile Asp Gin
`
`
`
`830
`*
`
`*
`
`
`
`B50
`*
`*
`
`
`
`
`
`CTT AcT ATT
`ATA TIC TAC AGT GTT
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAC AAA ATG CAG AAC AAA
`TAT AAG ATG TCA CAA
`GAA TGA TAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CIG TIT TAC GTC TTG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Thr Ile
`Tle Phe Tyr Ser val
`ASD Lys Met Gln Asn Lvs
`
`
`
`*
`
`
`
`B70
`*
`
`*
`
`
`
`8B0
`*
`
`-
`
`3910
`
`*
`
`*
`
`
`
`*
`*
`
`
`
`
`GGA CCA ICA
`TST CST GTA AGG AGT
`
`
`
`
`
`
`
`
`
`
`
`
`TTC AAA TCT GrT AAC ACC
`ACA GCA CAT TCC TCA
`CCT GGT AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAG TTT AGA CAA TIG TGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Arg Val Arg Ser
`Gly Pro Ser
`Phe Lys Ser Val Asn Thr
`
`
`$30
`=
`
`
`940
`a
`
`
`
`900
`*
`
`
`890
`*
`*
`
`
`
`
`
`
`
`AAA GGA CTT TAT ACT
`
`
`
`
`
`
`TTT CCT GAA ATA TGA
`
`
`
`
`
`
`Lys Gly Leu Tyr Thr>
`
`
`560
`aT
`
`
`
`950
`*
`
`*
`
`
`
`
`
`
`
`GIG CAT ATA TAT GAT
`
`
`
`
`
`CAC GTA TAT ATA CTA
`
`
`
`
`
`
`Val His Tle Tyr Asp>
`
`1010
`*
`
`
`
`1020
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`TGC CCA CCG TSC CCA
`
`
`
`
`
`
`ACG GGT GS ACG GOT
`
`
`
`
`
`
`Cys Pro Pro Cys Pro»
`
`*
`
`
`
`990
`°
`
`1000
`*
`
`$70
`*
`
`
`
`*
`
`*
`
`*
`
`*
`
`
`
`
`
`AAA GCA GGC CCG GGC
`GAG CCC AAR
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TCT TGT GAC ABA ACT CAC
`TIT CoT CCG Got ccG
`cre GGG TTT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AGA ACA CTG TTT TGA GTG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Lys Ala Gly Pro Gly
`Glu Pro Lys
`Ser Cys Asp Lys Thr His
`1030
`
`
`
`1050
`1066
`*
`*
`*
`*
`*
`
`
`
`
`
`GCA CCT GAA CTC cCTG
`GGG GGA CCG
`
`
`
`
`
`
`
`
`
`
`
`
`
`TCA GTC TIC Cre TH coe
`CGT GGA CTT GAG GAC
`cee cer Goce
`
`
`
`
`
`
`
`
`
`
`
`
`AGT CAG AAG GAG AAG GGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`‘Ala Pro Glu Leu Leu
`Ser Val Phe Leu Phe Pro
`Gly Gly Pro
`
`
`
`1070
`a
`
`
`
`
`i080
`*
`*
`
`
`cca
`
`
`
`
`
`
`
`ABA CCC AAG GAC ACC
`
`
`
`
`
`
`
`TTT? GGG TIC CTG 166
`
`
`
`
`
`
`
`Pro
`Lys Pro Lys Asp Thr>
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 17 of 55
`
`
`
`US 7,374,758 B2
`
`
`Fig.13C.
`
`*
`
`
`
`*
`
`
`1090
`1100
`of
`*
`
`
`
`GAG
`CTC ATG AIX TCC CGG ACC CCT
`GTC ACA TSC GTS GIG GTG
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAG TAC TAG AGG GCC TGG GGA
`CAG TGT ACG CAC CAC CAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`cre
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu
`Val Thr Cys Val Val Val
`Leu Met Ile Ser Arg Thr Pro
`
`1110
`*
`
`
`
`*
`
`*
`
`1120
`*
`
`
`
`1130
`e
`
`
`
`*
`
`1140
`*
`
`
`
`*
`
`
`
`
`GAC GTG AGC CAC GAA GAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTS CAC TG GIG CIT CTS
`
`
`
`
`
`
`
`Asp Val Sex His Glu Asp>
`
`*
`
`1150
`*
`
`
`
`1160
`*
`«
`*
`
`
`
`GTG GAC GGC GTG GAS GTS
`TAC
`CCT GAG GTC AAG TTC AAC TGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATG
`GGA CTC CAG TIC AAG TTG Acc
`Cac CTG ccG CAC CTC CAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Glu Val Lys Phe Asn Trp
`Val Asp Gly Val Glu Val
`
`
`
`1170
`*
`
`
`
`*
`
`
`1180
`*
`
`
`
`
`
`1200
`.
`
`1190
`
`*
`*
`*
`
`
`
`
`CAT AAT GCC AAG ACA ARG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GTA TTA CGG TIC TOT TIC
`
`
`
`
`
`
`
`His Asn Ala Lys Thr Lys>
`
`1210
`*
`
`
`
`
`
`
`1230
`1220
`*
`*
`*
`t
`*
`
`
`
`
`
`
`ACG TAC CGT GIG GTC AGC
`CCG CGG GAG GAG CAG TAC AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TOG
`TSC ATG GCA CAC CAG TCG
`GGoe GCC CIR CIC GI ATG TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ser
`Val
`Thr Tyx Arg Val Val Ser
`Pro Arg Glu Glu Gin Tyr Asn
`
`
`
`1240
`*
`
`*
`
`_ 1250*
`
`
`
`1260
`*
`
`
`
`*
`
`ere Acc Git CNG cac
`
`
`
`
`
`
`
`
`
`
`GAG TGS CAS GAC GIG
`
`
`
`
`
`
`Leu Thr Val Leu His>
`
`1300
`1280
`*
`*
`*
`
`
`
`
`CAG GAC TGG CIG AAT GGC AAG
`TAC AAG TGC AAG GTC TCC
`
`
`
`
`
`
`
`
`
`
`
`GTC CTG Acc GAC TTA CCG TIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATG TIC ACG TTC CAG AGG
`
`
`
`
`
`
`Asn
`
`
`
`
`
`
`
`
`Glu Tyr Lys Cys Lys Val Sex
`Glin Asp Trp Leu Asn Gly Lys
`
`1270
`*
`
`
`
`*
`
`*
`
`
`
`1290
`*
`
`
`
`*
`
`
`
`*
`
`
`
`
`1320
`*
`
`
`
`*
`
`
`1310
`*
`
`AAA GCC CTC CCA GO
`
`
`
`
`
`
`TTT CGG GAG GGT CGG
`
`
`
`
`
`
`
`
`
`
`
`
`Lys Ala Leu Pro Ala>
`
`
`
`
`
`
`
`
`
`1330
`*
`
`
`
`*
`
`*
`
`1340
`*
`
`
`
`
`ccc ATX GAG AAA ACC ATC TCC
`GCC AAA GGG CAG CCC CGA
`
`
`
`
`
`
`
`
`
`
`
`
`
`OGG TTT CCC GTC G&G CCT
`GGG TAG CT TIT TGS TAG AGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Ile Glu Lys Thr Ile Ser
`Lys Ala Lys Gly Gin Pro Arg
`
`1350
`*
`
`*
`
`1360
`*
`
`*
`
`*
`
`1390
`*
`
`
`
`*
`
`1420
`1410
`1400
`
`
`*
`*
`*
`*
`*
`
`
`
`
`
`ACC AAG AAC CAG GiC AGC
`CTs CCC CCA TCC CGG GAT GAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGG TI TIG GTC CAG TCG
`GAC GGG GGT AGG GCC CTA CIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Thr Lys Asn Gin Val Ser
`Leu Pro Pro Ser Arg Asp Glu
`
`
`
`*
`
`1450
`®
`
`
`
`*
`
`1460
`*
`
`
`
`1480
`1470
`*
`*
`*
`
`
`
`
`GTG GAG TGG GAG AGC AAT
`GGt TT TAT CCC AGC GAC ATC
`
`
`
`
`
`
`
`
`
`
`
`cac Ci act Cl TOG TTA
`COG AAG ATA GGG TOG CTG TAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ala Val Glu Trp Glu Ser Asn
`Gly Phe Tyr Pro Ser Asp Ile
`
`
`
`*
`
`
`
`*
`
`«
`
`1510
`*
`
`
`
`1520
`*
`
`