(54)
`
`(75)
`
`Inventors: Nicholas J. Papadopoulos,
`Lagrangeville, NY (US); Samuel Davis,
`NewYork, NY (US): George D.
`Yancopoulos, Yorktown Heights, NY
`(US)
`
`(73)
`
`Assignee: Regeneron Pharmaceuticals, Inc..
`Tarrytown, NY (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the termofthis
`patent is extended or adjusted under 35
`U.S.C, 154(b) by 489 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.: 11/016,097
`
`(22)
`
`Filed:
`
`Dec. 17, 2004
`
`(65)
`
`(62)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Prior Publication Data
`
`US 2005/0163798 Al
`
`Jul. 28, 2005
`
`Related U.S. Application Data
`
`Divisionof application No. 10/009,852, filed as appli-
`cation No. PCT/US00/14142 on May23, 2000, now
`Pat. No. 7,070,959.
`
`Provisional application No. 60/138,133, filed on Jun.
`8, 1999,
`
`Int. Cl.
`(2006.01)
`A6IK 38/18
`(2006.01)
`CO7K 14/71
`(2006.01)
`CI2N 15/62
`US. Ch ooocccccccceeeeeseeeeeeeeees 424/134.1; 424/192.1;
`§14/2: 514/12; 530/350; 536/23.4
`Field of Classification Search ..........00.00.. None
`See applicationfile for complete search history.
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,712,380 A
`1/1998 Kendall et al.
`6.011,003 A
`1/2000 Charnock-Jones et al.
`6,100,071 A *
`8/2000 Davis-Smyth et al.
`..... 435/69.7
`2005/0043236 AL*®
`2/2005 Daly et al. .......
`.. 514/12
`
`2006/0058234 AL*
`3/2006 Daly et al. wc. 514/12
`FOREIGN PATENT DOCUMENTS
`
`WO
`
`W097/44453
`
`11/1997
`
`(12)
`
`United States Patent
`Papadopouloset al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,374,757 B2
`*May 20, 2008
`
`US007374757B2
`
`MODIFIED CHIMERIC POLYPEPTIDES
`WITH IMPROVED PHARMACOKINETIC
`PROPERTIES
`
`WO
`WO
`
`WO98'13071
`W099/03996
`
`4/1998
`1/1999
`
`OTHER PUBLICATIONS
`
`Terman, B. I., et al, “Identification of a new endothelial cell growth
`factor receptor tyrosine kinase”, Oncogene (1991) 6:1677-1683.
`Terman, B.L., et al, “Identification of the KDR tyrosine kinase as a
`receptor for vascular endothelial cell growth factor”, Biochem
`Biophys Res Comm (1992) 187(3):1579-1586,
`Tsutsumi. Y., et al. “PEGylation of interleukin-6 effectively
`increases
`its
`thrombopoietic
`potency”,
`Thrombosis
`and
`Haemostasis (1997) 77(1): 168-173.
`Dunca, R. and Spreafico, F., “Polymer Conjugates”, Drug Delivery
`Systems (1994) 27(4):290-306.
`Hileman, R.F., et al., “Glycosaminoglycan-protein interactions:
`definitions of consensus sites in glycosaminoglycan binding pro-
`teins”, BioEssays (1998) 20: 156-167.
`deVries, Carlie, et al., “The fms-like tyrosine kinase, a receptor for
`vascular endothelial growth factor”, Science (1992) 225:989-991.
`Sharifi, J., et al., “Lmproving monoclonal antibody pharmacokinet-
`ics via chemical modification”, Quart J Nucl Med (1998) 42:242-
`249.
`Jensen-Pippo, K.E., et al., “Enteral bioavailability of human granu-
`locyte colony stimulating factor conjugated with poly(ethylene
`glycol)”, (1996) Pharm Res 13(1):102-107,
`Tanaka, K., et al., “Characterization of the extracellular domain in
`vascular endothelial growth factor
`receptor-l
`(FIt-l Tyrosine
`kinase)”, (1997) Jpn J Cancer Res 88:867-876.
`Yang, JC., et al. “The use of polyethylene glycol-modified
`interleukin-2 (PEG-IL-2) in the treatmentof patients with metastatic
`renal cell carcinoma and melanoma”, (1995) Cancer 76(4): 687-
`694.
`Davis-Smyth, T., et al., 1996, “The second immunoglobulin-like
`domain of the VEGFtyrosine kinase receptor Flt-1 determines
`ligand binding and mayinitiate a signal transduction cascade”, The
`EMBO Journal 15(18):4919-4927,
`
`* cited by examiner
`
`Primary Examiner—Christine J Saoud
`Assistant Examiner—Jon M Lockard
`(74) Attorney, Agent, or Firm—Gregg Valeta, Esq.
`
`(57)
`
`ABSTRACT
`
`Modified chimeric polypeptides with improved pharmaco-
`kinetics are disclosed. Specifically, modified chimeric FItl
`receptor polypeptides that have been modified in such a way
`as to improve their pharmacokinetic profile are disclosed.
`Also disclosed are methods of making and using the modi-
`fied polypeptides including but not
`limited to using the
`modified polypeptides to decrease or inhibit plasma leakage
`and/or vascular permeability in a mammal.
`
`7 Claims, 55 Drawing Sheets
`
`CELLTRION- EXHIBIT 1022
`
`CELLTRION - EXHIBIT 1022
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 1 of 55
`
`US 7,374,757 B2
`
`Fig.1.
`
`1
`
`2
`
`3 45 6
`
`7
`
`8
`
`9
`
`10 9.3
`
`
`id
`
`5.2
`
`0.001
`
`0.01
`
`0.1
`
`1
`
`10
`
`100
`
`ug/ml
`—t— ITIE-2-Fe
`ofacetylated Fit-1(1-3)-Fe
`----O--- unmodified Fit-1(1-3)-Fe
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 2 of 55
`
`US 7,374,757 B2
`
`P=| SALT-WASH ed1
`
`13141516
`
`17181920
`
`VEGF
`Fit-1(1-3)-Fe
`rTIE-2-Fe
`Heparin
`
`+
` -
`-
`-
`
`+
`+
`-
`-
`
`+
`-
`+
`-
`
`+
`+acetylated
`-
`-
`
`+
`+ pegylated
`-
`
`+
`+
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 3 of 55
`
`US 7,374,757 B2
`
`0.01
`
`
`
`o oO
`Wi
`oo oe
`2 7— !— Tr
`rE
`ayi=
`wir
`BoD
`= 2
`G &
`oy
`
`EDEe 83 0
`
`Asueq jeondo
`
`
`
`
`
`—@—pegylatedFit-1(1-3)-Fc
`
`0.1
`
`pg/ml
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 4 of 55
`
`US 7,374,757 B2
`
`
`
`oO oO
`wi Of o
`aa
`own —~
`ee OO
`-rTT
`ae YT
`bi t=
`colt
`®OUVDH
`EEL OL
`33 88a
`EES Sw
`ecesgo-
`Sdaak
`
`
`
`0.5
`
`oO
`
`Te)
`nN
`
`A
`
`£—
`wo
`
`a@
`
`®= +
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 5 of 55
`
`US 7,374,757 B2
`
`Fig.6A.
`
`123 45 678 9
`
`10
`
`
`Fig.6B.
`
`11.12 13 14 15 1617 1819 20
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 6 of 55
`
`US 7,374,757 B2
`
`Fig.7
`
`—{}— rTIE-2-Fc
`sesteneeOne unmodified Fit-1(1-3)-Fe
`----C---- acetylated Fit-1(1-3)-Fe (10x)
`------/\----- acetylated Fit-1(1-3)-Fe (20X)
`---9--- acetylated Fit-1(1-3)-Fe (30X)
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 7 of 55
`
`US 7,374,757 B2
`
`Fig.8.
`
`
`O5ugml
`
`1.0yug/ml
`
`
`5.0 ug/ml
`
`580
`
`zB
`4goll
`
`a
`
`,
`
`180
`
`80
`
`380 > 2801},
`
`
`
`ool’ *10*20°30 40*5060 70 800
`
`|\«-- Degree of step-acetylation of Fit-1(1-3)-Fe protein —-->1
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`+
`
`VEGF
`0.2 ug/ml
`
`4+
`
`Unmodified
`Fit-1(1-3)
`-Fe
`
`Acetylated
`Fit-i(i-3)
`-Fe
`
`-
`
`7
`
`+
`
`~
`
`-
`
`*
`
`-
`
`*
`
`-
`
`t+
`
`-
`
`t+
`
`-
`
`F
`
`-
`
`Ft
`
`+
`
`Ft
`
`-
`
`+
`
`-
`
`+
`
` -
`
`+
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 8 of 55
`
`US 7,374,757 B2
`
`94-(€-
`
`(xor)
`
`
`
`(x09)94-(€-1)b-"paye|Ajeoe©
`
`94-(€-b)b-dpeye|Ajeoem
`
`
`
`94-(€-L)L-W4Ppeylpowun¥
`
`
` (x001)
`94-(€-L)L-apayeiAjeoeO( (x0z).04-(€-L)L-4peryejAjeoe@XOL)
`
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 9 of 55
`
`US 7,374,757 B2
`
`Fig.10A.
`
`t
`
`10
`*
`
`*
`
`20
`*
`
`*
`
`30
`*
`
`*«
`
`40
`*
`
`*
`
`50
`*
`
`*
`
`60
`*«
`
`ATG GTC AGC TAC TGG GAC ACC
`TAC CAG TCG ATG ACC CIG TGG
`Met Val Ser Tyr Trp Asp Thr
`
`GTc CIG
`CAG GAC
`Gly Val Leu
`
`TGC GCG CIG
`ACG CGC GAC
`Cys Ala Leu
`
`crc
`GAG
`Leu
`
`AGC TGT CTG CTT circ
`TCG ACA GAC GAA GAG
`Ser Cys Leu Leu Leu>
`
`*
`
`70
`*
`
`80
`*
`
`90
`
`*
`
`100
`*
`
`110
`*
`
`*
`
`120
`*
`
`ACA GGA TCT AGT TCA GGT TCA
`CCA AGT
`TGT CCT AGA TCA AGT
`Thr Gly Ser Ser Ser
`Gly Ser Lys
`
`TTA AAA
`AAT TTT
`Leu Lys
`
`GAT
`CTA
`Asp
`
`CCT GAA CTG
`GGA CTT GAC
`Pre Glu Leu
`
`TTA AAA
`AAT TIT
`Leu Lys
`
`GGCc
`CoG
`Gly
`
`ACC CAG
`TGG GTC
`Thr Gin>
`
`*
`
`130
`*
`
`*
`
`140
`*
`
`150
`
`*
`
`160
`*
`
`*
`
`170
`*
`
`180
`
`CAC ATC ATG CAA GCA
`
`_-GTG TAG TAC GTT CGT
`His Ile Met Gln Ala
`
`GoC CAG
`CccG GTC
`Gly Gin
`
`#98
`
`CTG CAT
`GAC GTA
`Leu His
`
`CAA TGC AGG
`GTT ACG TCC
`Gin Cys Arg
`
`Gly
`
`GAA GCA
`CTT CGT
`Glu Ala
`
`ccc
`CGG
`Ala
`
`CAT AAA
`GTA TIT
`His Lys>
`
`*
`
`190
`*
`
`*
`
`200
`*
`
`210
`
`.
`
`220
`*
`
`230
`_
`
`240
`
`TCT TIG CCT GAA ATG GIG
`TAC CAC
`AGA AAC GGA CTT
`Ser Leu Pro Glu
`Met Val Ser
`
`AAG GAA
`TTC CIT
`Lys Glu
`
`GAA AGG CTG
`CTT TCC GAC
`Glu Arg Leu
`
`ATA ACT
`TAT TGA
`Ile Thr
`
`TcT ccc
`AGA CGG
`Lys Ser Ala>
`
`Ser
`
`*
`
`270
`
`*
`
`280
`*
`
`290
`*
`
`300
`
`*
`
`250
`*
`
`260
`*
`
`GGA AGA AAT GGC
`CCT TCT TTA CCG
`Gly Arg Asn Gly
`
`AAA CAA
`TIT GIT
`Lys Gln
`
`TGC AGT
`ACG TCA
`Phe Cys Ser
`
`TTA ACC TIG
`AAT TGG AAC
`Leu Thr Leu
`
`*
`
`310
`*
`
`320
`*
`
`330
`
`*
`
`340
`*
`
`ACT GGC TIC
`TGA CCG AAG
`Thr Gly Phe
`
`TAC
`ATG
`
`asc Tec
`TCG ACG
`Ser Cys
`
`TAT CTA
`ATA GAT
`Lys Tyr Leu
`
`GTA CCT ACT
`CAT GGA TGA
`Val Pro Thr
`
`Baa.
`
`pao.
`
`ACA GCT
`TGT CGA
`Thr Ala
`
`350
`t
`
`AAG AAG
`TTC TIC
`Lys Lys
`
`370
`*
`
`380
`*
`
`390
`
`*
`
`400
`*
`
`410
`*
`
`GCA AAC
`CGT TIG
`Ala Asn>
`
`360
`
`GAA ACA
`CIT Tot
`Glu Thr>
`
`420
`
`*
`
`TCT
`AGA
`
`GCA ATC
`CGT TAG
`Ala Ile
`
`TAT
`ATA
`
`ATA TIT
`TAT AAA
`Ile Phe
`
`ATT AGT GAT
`TCA CTA
`Tle Ser Asp
`
`GGT AGA CCT
`CCA TCT GGA
`Gly Arg Pro
`
`GTA GAG
`CAT CTc
`Val Glu
`
`ATG
`Tac
`Met
`
`TAC AGT
`ATG TCA
`Tyx Ser>
`
`Phe
`
`430
`*
`
`440
`*
`
`450
`*
`
`*
`
`460
`*
`
`470
`*
`
`480
`
`ccc GAA
`GGG CTT
`Pro Glu
`
`ATT
`TAA
`Tle
`
`ATA CAC
`TAT GTG
`Tle His
`
`ATG
`TAC
`Met
`
`ACT GAA
`TGA CTT
`Thr Glu
`
`TAG
`Tle
`
`GGA AGG GAG CTC
`ccT TCC CIC GAG
`Gly Arg Glu Leu
`
`ATT CCC
`TAA GGG
`Ile Pro
`
`$88
`
`CGG GIT
`Gcc CAA
`Arg Val>
`
`val
`
`490
`
`500
`*
`
`510
`
`*
`
`520
`*
`
`530
`*
`
`540
`
`CCT AAC
`GGA TIG
`Pro Asn
`
`ATC
`TAG
`Tle
`
`ACT GTT
`TGA CAA
`Thr Val
`
`TTA AAA
`AAT TIT
`Thr Leu Lys
`
`Ser
`
`AAG TTT CCA CTT
`TTC AAA GGT GAA
`Lys Phe Pro Leu Asp Thr Leu
`
`ACT TIG
`TGA AAC
`
`GAC
`CTG
`
`ATC
`TAG
`Tle
`
`CCT GAT
`GGA CTA
`Pro Asp>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 10 of 55
`
`US 7,374,757 B2
`
`Fig.10B.
`
`*
`
`*
`
`*
`
`5
`
`*
`
`*
`
`570
`od
`
`*
`
`580
`*
`
`*
`
`590
`*
`
`*
`
`600
`4
`
`GGA AAA CGC ATA ATC
`CcT TIT GCG TAT TAG
`Gly Lys Arg Ile Ile
`
`TGG GAC AGT AGA AAG
`ACC CTG TCA TCT TIC
`Trp Asp Ser Arg Lys
`
`GGc
`CCG
`Gly
`
`TIC ATC ATA
`AAG TAG TAT
`Phe Ile fle
`
`TCA
`AGT
`ser
`
`AAT GCA ACG TAC AAA
`TTA CGT TGC ATG TTT
`Asn Ala Thr Tyr Lys>
`
`*
`
`610
`*
`
`*
`
`620
`*
`
`*
`
`630
`*
`
`*
`
`640
`«
`
`650
`*
`
`*
`
`660
`*
`
`GAA ATA GGG CTT CIG
`CTT TAT CCC GAA GAC
`Glu Ile Gly Leu Leu
`
`ACC TGT GAA GCA ACA
`TGG ACACTT CGT TGT
`
`Thr Cys Glu Ala Thr
`
`GcTc
`CAG
`Val
`
`AAT GGG CAT
`TTA CCC GTA
`Asn Gly His
`
`*
`
`670
`*
`
`*
`
`680
`*
`
`*
`
`690
`*
`
`*
`
`700
`*
`
`CTC ACA CAT .CGA CAA
`GAG TGT GTA GCT GIT
`
`Leu Thr His Arg Gin
`
`ACC AAT ACA ATC ATA
`TGG TTA TGT TAG TAT
`Thr Asn Thr Ile Ile
`
`GAT GTC CAA ATA
`CTA CAG GIT TAT
`
`Asp Val Gln Ile
`
`TAT AAG
`ATA TTC
`Tyr Lys
`
`ACA AAC TAT
`TCT TTG ATA
`
`Thr Asn Tyr>
`
`710
`*
`
`.
`
`720
`*
`
`ACA CCA
`TGT GGT
`Thr Pro
`
`ccc
`GCG
`Arg
`
`CCA GIC
`GGT CAG
`Pro Val>
`
`*
`
`730
`*
`
`*
`
`740
`*
`
`*
`
`750
`*
`
`*
`
`760
`*
`
`770
`
`*
`
`780
`*
`
`AAA TTA CTT AGA GGC
`TTT AAT GAA TCT CCG
`Lys Leu Leu Arg Gly
`
`CAT ACT CTT GTC CTC
`GTA TGA GAA CAG GAG
`His Thr Leu Val Leu
`
`AAT
`TTA
`Asn
`
`IGT ACT GCT
`ACA TGA CGA
`Cys Thr Ala
`
`acc
`TGG
`Thr
`
`AcT ccc
`TGA GG
`
`Thr Pro BRa
`
`AAC ACG
`TTG Toc
`Asn Thr>
`
`830
`
`840
`
`*
`
`790
`*
`
`*
`
`800
`*
`
`*
`
`810
`*
`
`*
`
`820
`*
`
`AGA GTT CAA ATG ACC
`TCT CAA GTT TAC TGG
`Arg Val Gln Met Thr
`
`TGG .AGT TAC CCT GAT
`ACC TCA ATG GGA CTA
`Trp Ser Tyr Pro Asp
`
`GAA
`ctt
`Glu
`
`AAA AAT AAG
`TTT TTA TIC
`Lys Asn Lys
`
`GcT Tcc
`CGA AGG
`Ala Ser
`
`CAT
`Val
`
`AGG CGA
`tcc GcT
`Arg Arg>
`
`*
`
`850
`*
`
`*
`
`860
`*
`
`*
`
`870
`*
`
`*
`
`880
`*
`
`850
`*
`
`900
`
`CGA ATT GAC CAA AGC
`GCT TAA CTIG GIT TCG
`Arg Ile Asp Gin Ser
`
`AAT TCC CAT GCC AAC
`TTA AGG GTA CGG TIG
`Asn Ser His Ala Asn
`
`ATA
`TAT
`Tle
`
`TTC TAC AGT
`AAG ATG TCA
`Phe Tyr Ser
`
`CTT ACT ATT
`GAA TGA TAA
`Leu Thr Ile
`
`GAC AAA
`CTG TIT
`Asp Lys>
`
`*
`
`910
`*
`
`*
`
`920
`.
`
`*
`
`930
`*
`
`*
`
`940
`*
`
`950
`*
`
`960
`
`ATG CAG AAC AAA GAC
`TAC GTC TIG TIT CIG
`Met Gln Asn Lys Asp
`
`AAA GGA CTT TAT ACT
`TTT CCT GAA ATA TGA
`Lys Gly Leu Tyr Thr
`
`CGT GTA AGG
`GCA CAT TCC
`Arg Val Arg
`
`GGA CCA TCA
`CCT GGT AGT
`Gly Pro Ser
`
`TTC AAA
`AAG TIT
`Phe Lys>
`
`TCA
`Ser
`
`*
`
`970
`*
`
`*
`
`980
`*
`
`*
`
`990
`*
`
`*
`
`1000
`*
`
`1010
`*
`
`TCT GTT AAC ACC TCA
`AGA CAA TIG TGG AGT
`Ser Val Asn Thr Ser
`
`GTG CAT ATA TAT GAT
`CAC GTA TAT ATA CTA
`Val His Ile Tyr Asp
`
`GCA GGC CCG
`CccT ccG GO
`Lys Ala Gly Pro
`
`coe
`COG
`Gly
`
`GAG ccc
`CTC GG
`Glu Pro
`
`Lys
`
`1020
`*
`
`*
`
`TCT TCT
`AGA ACA
`Ser Cys>
`
`*
`
`1030
`*
`
`*
`
`1040
`*
`
`1050
`*
`
`*
`
`*
`
`1060
`*
`
`1070
`*
`
`1080*
`
`*
`
`GAC AAA ACT CAC ACA
`CTG TIT TIGA GIG TGT
`Asp Lys Thr His Thr
`
`TGC CCA CCG TGC CCA
`ACG GGT GGC ACG GGT
`Cys Pro Pro Cys Pro
`
`GCA CCT GAA CTC
`CGT GGA CTT GAG
`Ala Pro Glu Leu
`
`CTG
`GAC
`Leu
`
`GGG GGA
`ecc ccT
`Gly Gly
`
`CCG
`GGCc
`Pro
`
`TCA GTC
`AGT CAG
`Ser Val>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 11 of 55
`
`US 7,374,757 B2
`
`Fig.10C.
`
`*
`
`1090
`*
`
`*
`
`1100
`*
`
`1110
`*
`
`*
`
`1120
`*
`
`*
`
`1130
`
`1140
`*
`
`*
`
`Tre cre TTC CCC CCA AAA CCC
`
`AAG GAG AAG GGG GGT TIT GGG
`Phe Leu Phe Pro Pro Lys Pro
`
`Lys
`
`GAC ACC CTC ATG ATC TCC
`CTG TGG GAG TAC TAG AGG
`Asp Thr Leu Met Ile Ser
`
`CGG
`6ce
`Arg
`
`acc ccT
`TGG GGA
`Thr Pro
`
`GAG GTC ACA
`CTC CAG TCT
`Glu Val Thr>
`
`*
`
`1150
`*
`
`*
`
`1160
`*
`
`1170
`.
`
`*
`
`1180
`*
`
`*
`
`TGC GTG GTG GTG GAC GTG AGC
`
`ACG CAC CAC CAC CTG CAC TCG
`Cys Val Val Val Asp ValSer
`
`GTG
`His
`
`GAA GAC CCT GAG GTC AAG
`CTT CTG GGA CTC CAG TIC
`
`Glu Asp Pro Glu Val Lys
`
`1190
`*
`
`1200
`*
`
`*
`
`AAC TGG
`TTG ACC
`Asn Trp
`
`TAC GTG GAC
`ATG CAC CIG
`Tyr Val Asp>
`
`BA.
`
`Phe
`
`*
`
`1210
`*
`
`*
`
`1220
`*
`
`1230
`*
`
`*
`
`1240
`*
`
`*
`
`1250
`*
`
`*
`
`1260
`*
`
`GGC GTG GAG GTG CAT AAT GCC
`CCG CAC CTC CAC GTA TTA CGG
`
`ACA AAG CCG CGG GAG GAG
`TGT TTC Goc Gec cre cr
`
`Gly Val Glu Val His Asn Ala
`
`Lys
`
`Thr Lys Pro Arg Glu Glu
`
`TAC AAC
`ATG TTG
`Tyr Asn
`
`AGC ACG TAC
`TOG TGC ATG
`Ser Thr Tyr>
`
`*
`
`1270
`*
`
`*
`
`1280
`*
`
`1290
`*
`
`*
`
`1300
`*
`
`*
`
`*
`
`1310
`®
`
`1320
`.
`
`*
`
`CGT GTG GTC AGC Git Clic ACC
`GCA CAC CAG TCG CAG GAG TGG
`Arg Val Val Ser Val Leu Thr
`
`Val
`
`CTG CAC CAG GAC TGG CTG
`GAC GTG GTC CTG ACC GAC
`Leu His Gin Asp Trp Leu
`
`AAT GGC AAG
`TTA CCG TIC
`Asn Gly Lys
`
`GAG TAC AAG
`CTC ATG TIC
`Glu Tyr Lys>
`
`1350
`*
`
`1370
`*
`
`*
`
`1330
`*.
`
`*
`
`1340
`*
`
`*
`
`*
`
`1360
`*
`
`1380
`*
`
`*
`
`TGC AAG GTC TCC AAC AAA GCC
`ACG TIC CAG AGG TG TIT CG
`Cys Lys Val Ser Asn Lys Ala
`
`CCA GCC CCC ATC GAG AAA
`GGT CGG GGG TAG CIC TIT
`Pro Ala Pro Ile Glu Lys
`
`*
`
`1390
`*
`
`1400
`
`*
`
`1410
`*
`
`*
`
`1420
`*
`
`*
`
`GGG CAG CCC CGA GAA CCA CAG
`ccc GIC GGGT CTT GGT GTc
`
`TAC ACC CIG CCC CCA TCC
`ATG TGG GAC GGG GGT AGG
`
`Gly Gln Pro Arg Glu Pro Gin
`
`Val
`
`Tyr Thr Leu Pro Pro Ser
`
`ATC TCC
`TAG AGG
`Tle Ser
`
`AAA GCC AAA
`TTT CGG TTT
`Lys Ala Lys>
`
`1430
`*
`
`1440
`*
`
`*
`
`GAT GAG
`CTA CTC
`Asp Glu
`
`CTG ACC AAG
`GAC TGG TIC
`Leu Thr Lys>
`
`*
`
`*
`
`1450*
`AAC CAG GTC AGC CTG
`TTG GTC CAG TCG GAC
`Asn Gln Val Ser Leu
`
`1460*
`AcC TGC
`TGG ACG
`Thr Cys
`
`1470
`*
`
`*
`
`1480
`*
`
`*
`
`1490
`*
`
`1500
`*
`
`GTIc AAA GoC TIC TAT CCC
`CAG TTT CCG AAG ATA GGG
`Val Lys Gly Phe Tyr Pro
`
`Leu
`
`GAC ATC
`CTG TAG
`
`Asp Ile
`
`GCC GTG GAG
`CGS cac cic
`Ala Val Glu>
`
`t
`
`1510
`*
`
`*
`
`1520
`*
`
`1530
`*
`
`*
`
`1540
`*
`
`*
`
`1550
`*
`
`1560
`*
`
`TGG GAG AGC AAT GGG
`ACC CTC TOG TTA CCC
`Txp Glu Ser Asn Gly
`
`CAG CCG
`GTc GGc
`Gln Pro
`
`AAC AAC TAC AAG ACC ACG
`TTG TIG ATG TIC TGG TGC
`Asn Asn Tyr Lys Thr Thr
`
`Glu
`
`ccc GTG
`GGG CAC
`Pro Val
`
`CTIG GAC TCC
`GAC CTG AGG
`Leu Asp Ser>
`
`*
`
`1570
`*
`
`1580
`
`*
`
`1590
`*
`
`*
`
`*
`
`1600
`*-
`
`1610
`
`1620
`
`*
`
`GAC Goce TCC TIC TIC
`CTG CCG AGG AAG AAG
`Asp Gly Ser Phe Phe
`
`cTc TAC
`GAG ATG
`Leu Tyr
`
`AAG CTC ACC GTG GAC AAG
`TTC GAG TGG CAC CTIG TIC
`Lys Leu Thr Val Asp Lys
`
`AGG TGG
`Tec Acc
`Ser Arg Trp
`
`CAG CAG GGG
`GT GTC ccc
`Gln Gin Gly:
`
`Ser
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 12 of 55
`
`US 7,374,757 B2
`
`Fig.10D.
`
`*
`
`1630
`*
`
`*
`
`1640
`*
`
`1650
`*
`
`*
`
`*
`
`1660
`*
`
`*
`
`1670
`*
`
`1680
`*
`
`*
`
`AAC GTC TIC TCA TGC TCC GIG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC
`TTG CAG AAG AGT ACG AGG CAC TAC GTA CTC CGA GAC GIG TIG GIG ATG TGC GU TIC TCG
`
`Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser>
`
`*
`
`1690
`*
`
`*
`
`1700
`*
`
`cic TC CTG TCT CCG GGT AAA TGA
`GAG AGG GAC AGA GGC CCA TIT ACT
`
`Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 13 of 55
`
`US 7,374,757 B2
`
`“EEBY
`
`eueiquUiaWwsues
`
`
`urewiopSuIeWWOp
`
`
`
`
`
`
`@SeUlyJEIN||a0eJ}U]q1Buipuiqpuebysejnjyjeoersyxyg
`
`Selthtv669c6
`
`
`
`
`zig\6SZ0Sz2489(0SS~=ZSh=OSKECOELONploeoul
`og
`
`
`
`NZN9NGNbNENe
`
`"ee16enbiun
`
`SNUILUI}-9
`
`s$s
`
`178SSSy7SSb-dSNE
`
`
`
`*78_Sy7®,§b-HASNe
`
`b-UdSNZ
`
`L-4SN9
`
`b-dSNS
`
`L-U4SNV
`
`
`
`
`
`
`

`

`U.S. Patent
`
`May20, 2008
`
`US 7,374,757 B2
`
`Sheet 14 of 55
`
`Fig.12A.
`
`oooogogg000000
`Seeoooescosgoss
`UFHAK Ora TH
`AyoytydoupAH
`
`SOOO O000OOO
`SS66666665556
`UFTANKOranyw
`Ayo1j1ydoupAH
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 15 of 55
`
`US 7,374,757 B2
`
`Fig.13A.
`
`tk
`
`*
`
`*
`
`*
`
`*-
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`ATG GTC AGC TAC TGS GAC ACC GGG GTC CTG CTIG TGC GCG CTIG CIC AGC TCT CIG CIT CIC
`TAC CAG TOG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAG
`Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu>
`
`*
`
`70
`*
`
`*
`
`80
`*
`
`*
`
`90
`*
`
`*
`
`100
`*
`
`.
`
`110
`*
`
`120
`*
`
`*
`
`ACA GGA TCT AGT TCA GGT TCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG
`(GT CCT AGA TCA AGT CCA AGT TTT AAT TIT CTA GGA CTT GAC TCA AAT TIT CCG 166 GTC
`Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln>
`
`*
`
`130
`*
`
`*
`
`140
`*
`
`*
`
`150
`*
`
`*
`
`160
`*
`
`*
`
`170
`*
`
`*
`
`180
`*
`
`Cac ATC ATG CAA GCA GGC CAG
`GIG TAG TAC GTT CGT CCG GTC
`His Ile Met Gln Ala Gly Gin
`
`#45 5 3 8
`
`CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA
`GTT ACG TCC CCC CTT CGT CG GTA TIT
`Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys>
`
`*
`
`190
`*
`
`*
`
`200
`*
`
`*
`
`210
`*
`
`*
`
`220
`*
`
`*
`
`230
`*
`
`a8
`TGG TCT TTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CIG AGC ATA ACT
`ACC AGA AAC GGA CTT TAC CAC TCA TIC CTT TCG CTT TCC GAC TCG TAT TGA
`frp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys
`
`*
`
`*
`
`*
`
`240
`
`300
`
`*
`
`250
`*
`
`*
`
`260
`*
`
`270
`*
`
`280
`*
`
`290
`*
`
`$54
`
`a8
`GGA AGA AAT GGC AAA CAA TIC TGC AGT ACT TTA ACC TTG AAC ACA GCT
`CCT TCT TTA CCG TIT GTT AAG ACG TCA TGA AAT TGG AAC TIG TCT CGA
`Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gln
`
`*
`
`310
`*
`
`*
`
`320
`*
`
`*
`
`330
`*
`
`*
`
`340
`*
`
`*
`
`350
`*
`
`360
`*
`
`4B gg°
`
`ACA
`CAC ACT GGC TIC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAG AAG
`TGT
`GTG TGA CCG AAG ATG TCG ACG TTT ATA GAT CGA CAT GGA TGA AGT TTC TIC
`His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr>
`
`*
`
`370
`*
`
`*
`
`380
`*
`
`«
`
`390
`*
`
`*
`
`400
`*
`
`*
`
`410
`*
`
`420
`*
`
`*
`
`GAA TCT GCA ATC TAT ATA TTT ATT AGT GAT ACA GGT AGA CCT TIC GTA GAG ATG TAC
`CTT AGA CGT TAG ATA TAT AAA TAA TCA CTA TGT CCA TCT GGA AAG CAT CTC TAC ATG
`Glu Ser Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`AGT
`TCA
`
`*
`
`430
`*
`
`*
`
`440
`*
`
`*
`
`450
`*
`
`*
`
`460
`*
`
`*
`
`470
`*
`
`*
`
`480
`*
`
`GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CIC GIC ATT CCC TGC CGG GIT
`CTT TAG GGG CTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA
`Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg vVal>
`
`*
`
`490
`*
`
`*
`
`500
`*
`
`*
`
`510
`*
`
`*
`
`520
`*
`
`*
`
`530
`*
`
`*«
`
`540
`*
`
`TTT CCA CTT GAC ACT TIG ATC CCT GAT
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA
`AB
`AAA GGT GAA CTG TGA AAC TAG GGA
`TGC AGT GGA TTG TAG TGA CAA TGA AAT TIT
`Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 16 of 55
`
`US 7,374,757 B2
`
`Fig.13B
`
`*
`
`550
`*
`
`*
`
`560
`.
`
`*
`
`570
`.
`
`*
`
`580
`*
`
`«
`
`590
`*
`
`600
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TIC ATC ATA TCA AAT GCA ACG TAC AAA
`CCT TIT GCG TAT TAG ACC CTIG TCA TCT TIC CCG AAG TAG TAT AGT TTA CGT TCC ATG TTT
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys>
`
`*
`
`610
`.
`
`*
`
`620
`*
`
`GAA ATA GGG CTT CTG ACC TGT GAA
`CTT TAT CCC GAA GAC TGG ACA CTT
`Glu Ile Gly Leu Leu Thr Cys Glu
`
`630
`*
`
`*
`
`640
`*
`
`650
`
`.
`
`660
`
`ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT
`TGT CAG TTA CCC GTA-AAC ATA TIC TGT TIG ATA
`Thr
`Leu
`Thr
`Val Asn Gly His
`Tyr Lys
`Asn Tyr>
`
`Fag*
`
`*
`
`670
`*
`
`=
`
`680
`*
`
`*
`
`690
`*
`
`*
`
`700
`*
`
`*
`
`710
`*
`
`*
`
`720
`*
`
`Circ ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC
`GAG TGT GTA GCT GIT TGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT GCG GGT CAG
`Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val>
`
`*
`
`730
`*
`
`*
`
`740
`*
`
`*
`
`750
`*
`
`*
`
`760
`*
`
`*
`
`770
`*
`
`*
`
`780
`*
`
`AAA TTA CTT AGA GGC CAT ACT CTT GIC CIC AAT IGT ACT GCT ACC ACT CCC TTG AAC ACG
`TTT AAT GAA TCT CCG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TIG TGC
`Lys Leu Leu Arg Gly His Tnr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`*
`
`790
`*
`
`*
`
`800
`*
`
`*
`
`810
`*
`
`*
`
`820
`*
`
`*
`
`830
`*
`
`840
`
`*
`
`GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC
`CAA GTT TAC TGG ACC TCA ATG GGA CTA CTT TAA CTG GTT TCG TTA AGS GTA CGS TIG
`Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Ile Asp Gln Ser Asn Ser His Ala Asn>
`
`850
`860
`870
`880
`890
`*
`*
`*
`*
`*
`*.
`*
`*
`*
`*
`TIC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA
`TAT AAG ATG TCA CAA GAA TGA TAA CIG TIT TAC GTC TIG TIT CIG TIT CCT
`
`*
`
`910
`*
`
`.
`
`920
`*
`
`930
`*
`
`*
`
`940
`*
`
`*
`
`950
`.
`
`900
`*
`*
`TAT ACT
`ATA TGA
`
`*
`
`960
`*
`
`Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Bea
`88 g54° d
`
`TGT CGT GTA AGG AGT GGA CCA
`ACA GCA CAT TCC TCA CCT GGT
`Cys Arg Val Arg Ser Gly Pro Ser
`
`AAA TCT GTT AAC ACC TCA GIG CAT ATA TAT GAT
`AGA CAA TTG TGG AGT CAC GTA TAT ATA CTA
`Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp>
`
`*
`
`370
`*
`
`co
`
`380
`*
`
`+
`
`990
`*
`
`*
`
`1000
`*
`
`*
`
`1010
`*
`
`1020
`*
`
`*
`
`AAA GCA GGC CCG GGC GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA
`TTT CGT CCG GGC CCG CTC GGG TTT AGA ACA CTIG TIT TGA GTG TGT ACG GGT GCC ACG GCT
`Lys Ala Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro>
`
`*
`
`1030
`*
`
`*
`
`1040
`*
`
`1050
`*
`
`*
`
`*
`
`1060
`*
`
`*
`
`1070
`*
`
`1080
`*
`
`*
`
`GCA CCT GAA CIC CTG GGG GGA CCG TCA GTc TIt Cie TIC CCC CCA AAA CCC AAG GAC ACC
`CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TIT GGG TIC CTG TGG
`Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 17 of 55
`
`US 7,374,757 B2
`
`Fig.13C.
`
`*
`
`1090
`*
`
`*
`
`1100
`*
`
`1110
`*
`
`*
`
`*
`
`1120
`*
`
`*
`
`1130
`*
`
`1140
`*
`
`*
`
`CTC ATG ATC TCC CGG ACC CCT GAG GIC ACA TGC GTG GIG GTG GAC GTG AGC CAC GAA GAC
`GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GIG CTT CTG
`Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp>
`
`*
`
`1150
`*
`
`*
`
`1160
`*
`
`1170
`*
`
`*
`
`*
`
`1160
`*
`
`*
`
`1190
`*
`
`1200
`*
`
`*
`
`CCT GAG GTC AAG TIC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG
`GGA CTC CAG TIC AAG TTG ACC ATG CAC CTG CCG CAC CTC CAC GTA TIA CGG TIC TCT TIC
`Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys>
`
`*
`
`1210
`*
`
`*
`
`1220
`*
`
`1230
`*
`
`*
`
`*
`
`1240
`*
`
`*
`
`1250
`*
`
`1260
`*
`
`*
`
`CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GIG GIC AGC GTC CTC ACC GIC CTIG Cac
`GGc Gcc CIc CIC GIC ATG TIG TOG TCC ATG GCA CAC CAG TCG CAG GAG TGG CAG GAC GIG
`Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His>
`
`*
`
`1270
`*
`
`=
`
`1280
`*
`
`1290
`*«
`
`*
`
`*
`
`1300
`*
`
`*
`
`1310
`.
`
`1320
`*
`
`*
`
`CAG GAC TGG CTGAAT GGC AAG GAG TAC AAG TGC AAG GIC TCC AAC AAA GCC CIC CCA GCC
`GTC CTG ACC GAC TTA CCG TIC CTC ATG TIC ACG TIC CAG AGG TIG TIT CGG GAG GGT CGG
`Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala>
`
`*
`
`*«
`
`*
`
`*
`
`*
`
`1330
`*
`
`*
`
`1340
`*
`
`1350
`*
`
`1360
`*
`
`1370
`*
`
`1380
`*
`
`ccc ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC
`GGG TAG CIC TIT TGG TAG AGG TIT CGG TIT CCC GIC GGG GCT CTT GGT GTC CAC ATG TGS
`Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr>
`
`*
`
`1390
`*
`
`*
`
`1400
`*
`
`1410
`*
`
`*
`
`*
`
`1420
`*
`
`*
`
`1430
`*
`
`1440
`*
`
`®
`
`CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG. AAC CAG GTC AGC CTG ACC TGC CTIG GIC AAA
`GAC GGG GGT AGG GCC CTA CIC GAC IGG TIC TIG GTC CAG TCG GAC TGG ACG GAC CAG TIT
`Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys>
`
`*
`
`1450
`*
`
`*
`
`1460
`*
`
`1470
`*
`
`*
`
`*
`
`1480
`*
`
`*
`
`1490
`*
`
`1500
`*
`
`*
`
`GGC TIC TAT CCC AGC GAC ATC GCC GIG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC
`CCG AAG ATA GGG TCG CTIG TAG CG CAC CIC ACC CTC TOG TTA CCC GTC Got CTC TIG TIG
`Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn>
`
`*
`
`1510
`*
`
`*
`
`1520
`*
`
`1530
`*
`
`*
`
`*
`
`1540
`*
`
`*
`
`1550
`*
`
`1560
`*
`
`*
`
`TAC AAG ACC ACG CCT CCC GTG CTG GAC TC GAC GGC TCC TK TIC CTC TAC AGC AAG CIC
`ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TIC GAG
`Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu>
`
`*
`
`1570
`*
`
`*
`
`1580
`*
`
`1590
`*
`
`*
`
`*
`
`1600
`*
`
`*
`
`1610
`*
`
`1620
`*
`
`*
`
`ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TIC TCA TGC TCC GIG ATG CAT GAG
`TGG CAC CIG TIC TCG TCC ACC GTC GIC CCC TIG CAG AAG AGT ACG AGG CAC TAC GTA CTC
`Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 18 of 55
`
`US 7,374,757 B2
`
`Fig.13D.
`
`1630
`*
`
`*
`
`1640
`*
`
`*
`
`1650
`*
`
`*
`
`1660
`*
`
`*
`
`1670
`®
`
`*
`
`GCT CIG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA
`CGA GAC GIG TTG GIG ATG TGC GIC TIC TCG GAG AGG GAC AGA GGC CCA TIT ACT
`Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 19 of 55
`
`US 7,374,757 B2
`
`Fig.14A.
`
`*
`
`*
`
`*
`
`*
`
`30
`
`40
`
`ATG GTC AGC TAC TGG GAC ACC
`TAC CAG TCG ATG ACC CTG TGG
`Met Val Ser Tyr Trp Asp Thr
`
`GGG
`ecc
`Gly
`
`GTC CTG
`GAC
`Leu
`
`Val
`
`GAC
`Leu
`
`*
`
`70
`*
`
`*
`
`80
`*
`
`90
`
`*
`
`ACA GGA TCT AGT TCC GGA GGT AGA
`
`TGT CCT AGA TCA AGG CCT CCA TCT
`Thr Gly Ser Ser Ser Gly Gly Arg
`
`TTc
`GGA AAG
`Pro Phe
`
`GTA
`
`Val
`
`*
`
`130
`*
`
`*
`
`140
`*
`
`ATA CAC ATG ACT GAA GGA AGG
`TAT GTG TAC TGA CTT CCT TCC
`Ile His Met Thr Glu Gly Arg
`
`GAG
`cTtc
`Glu
`
`150
`
`GTC
`CAG
`Val
`
`ATT
`TAA
`Ile
`
`*
`
`190
`*
`
`*
`
`200
`*
`
`ACT GTT ACT TTA AAA AAG TIT
`TGA CAA TGA AAT TIT TIC AAA
`Thr Val Thr Leu Lys Lys Phe
`
`Pro
`
`.
`
`250
`*
`
`*
`
`260
`*
`
`ATC
`TAG
`Ile
`
`#88
`
`TTA
`Asn
`
`GCG CTG
`cGc GAC
`Ala Leu
`
`cTc
`GAG
`Leu
`
`100
`
`ATG TAC
`TAC ATG
`
`Met Tyr Ser
`
`TGC CGG
`ACG GCC
`Cys Arg
`
`220
`
`ATC CCT
`TAG GGA
`Ile Pro
`
`*
`
`50*
`
`60
`*
`AGC TGT CTG CTT CTC
`TCG ACA GAC GAA GAG
`Ser Cys Leu Leu Leu>
`
`110
`*
`
`.
`
`GAA ATC
`CTT TAG
`Glu Ile
`
`Pro Glu
`
`120
`*
`
`ATT
`TAA
`Ile>
`
`180
`
`ATC
`TAG
`Ile>
`
`240
`
`160
`
`*
`
`170*
`
`a&*
`
`ACG TCA
`TGC AGT
`Thr Ser
`
`Pro
`
`230*
`
`GGA AAA
`ccT TIT
`Gly Lys
`
`Arg
`
`ATA
`TAT
`Tle
`
`ATC
`TAG
`Tle>
`
`280
`
`*
`
`290*
`
`GAA ATA
`CTT TAT
`Glu fle
`
`300
`
`a
`
`g88
`
`GAC AGT AGA AAG GCC TTC
`CTG TCA TCT TIC CCG AAG
`Asp Ser Arg Lys Gly Phe
`
`*
`
`310
`*
`
`*
`
`320
`*
`
`#88
`
`IGT GAA GCA
`ACA CTT CGT
`Cys Glu Ala
`
`ACA GTC AAT
`TGT CAG TTA
`Thr Val Asn
`
`GGG
`cece
`
`Gly
`
`*
`
`370
`*
`
`*
`
`380
`*
`
`Acc
`TGG
`Thr
`
`AAT ACA ATC
`TTA TGT TAG
`Asn Thr Ile
`
`ATA GAT GTC
`TAT CTA CAG
`Ile Asp Val
`
`ATA
`TAT
`Tle
`
`GIT
`Gin
`
`*.
`
`430
`*
`
`*
`
`440
`*
`
`ACT CTT GTC
`TGA GAA CAG
`Thr Leu Val
`
`CTC AAT TCT
`GAG TTA ACA
`Leu Asn Cys
`
`#88
`
`TCT
`
`#88
`
`*
`
`490
`*
`
`*
`
`500
`*
`
`TGG
`acc
`
`AGT TAC CCT
`TCA ATG GGA
`Trp Ser Tyr Pro Asp Glu Tie
`
`GAT GAA ATT
`CTA CTT TAA
`
`510
`
`AGC
`
`Ser
`
`CTG
`
`Asp Gln
`
`AAT
`TTA
`Asn
`
`CAT GCC
`GTA CGG
`His Ala
`
`Ser
`
`ATA TIC
`TAT AAG
`Ile Phe
`
`TAC
`ATG
`Tyr Ser
`
`Asn
`
`Val>
`
`ACG TAC
`TGC ATG
`Thr Tyr
`
`Lys
`
`Gly
`
`Leu>
`
`340
`
`350
`
`*
`
`360
`
`TAT
`ATA
`
`AAG
`
`Lys
`
`ACA AAC
`TGT TIG
`Thr Asn
`
`TAT
`ATA
`
`CTC ACA
`GAG TCT
`Leu Thr
`
`CAT
`GTA
`His
`
`400
`
`410
`*
`
`ccc CCA
`GCG GGT
`Arg Pro
`
`GTc
`
`Val
`
`AAA TTA
`TIT AAT
`Lys Leu
`
`BdRab°
`
`Leu
`
`460
`
`470
`*
`
`ACG
`
`TTG AAC
`AAC TIG
`Leu Asn
`
`AGA GTT
`TCT CAA
`Arg Val
`
`ATG
`TAC
`Glin Met
`
`520
`
`530
`*
`
`Gin>
`
`420
`
`Gly>
`
`480
`
`Thr>
`
`540
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 20 of 55
`
`US 7,374,757 B2
`
`Fig.14B.
`
`*
`
`550
`*
`
`*
`
`560
`*
`
`570
`*
`
`*
`
`580
`*
`
`*
`
`590
`.
`
`*
`
`600
`*
`
`ACT ATT GAC AAA ATG CAG
`TGA TAA CTG TET TAC GTC
`Thr Ile Asp Lys Met Gln
`
`g28
`
`GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT
`CTIG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA
`Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser>
`
`*
`
`610
`*
`
`*
`
`620
`*
`
`CCA TCA TIC AAA TCT GTT
`GGT AGT AAG TTT AGA CAA
`Pro Ser Phe Lys Ser Val
`
`dB
`Asn
`
`Gly
`
`*
`
`670
`*
`
`*
`
`680
`*
`
`CCC AAA TCT TGT GAC AAA
`GGG TTT AGA ACA CIG TIT
`Pro Lys Ser Cys Asp Lys
`
`act
`#8
`
`Glu
`
`*
`
`730
`*
`
`*
`
`740
`*
`
`GGA CCG TCA GTC
`CCT GGC AGT CAG
`Gly Pro Ser Val
`
`TTc cic
`AAG GAG
`Phe Leu
`
`Phe
`
`Gly
`
`*
`
`640
`*
`
`*
`
`650
`*
`
`660
`*
`
`*
`
`CAT ATA TAT GAT AAA GCA
`GTA TAT ATA CTA TTT CGT
`His Ile Tyr Asp Lys Ala
`
`Gec CCG GGC
`CCG GGC CCG
`Gly Pro Gly>
`
`*
`
`700
`*
`
`*«
`
`710
`*
`
`720
`*
`
`*
`
`CCA CCG TGC CCA GCA CCT
`GGT GGC ACG GGT CGT GGA
`Pro Pro Cys Pro Ala Pro
`
`GAA CTC CTG
`CTT GAG GAC
`Glu Leu Leu>
`
`760
`*
`
`*
`
`770
`*
`
`*
`
`780
`®
`
`ecc
`
`Pro
`
`AAG GAC ACC CTC ATG ATC TCC CG
`TTC CTG TGG GAG TAC TAG AGG GCC
`Lys Asp Thr Leu Met Ile Ser Arg>
`
`*
`
`ccT
`GGA
`Pro
`
`790
`*
`
`*
`
`800
`*
`
`810
`
`820*
`
`*
`
`830
`.
`
`cac
`Val
`
`Val
`
`*
`
`840*
`GTC AAG TIC
`CAG TIC AAG
`
`CAC GAA GAC CCT GAG
`GIG CTT CTG GGA cCIc
`His Glu Asp Pro Glu
`
`Asp
`
`Val
`
`*
`
`880
`*
`
`*
`
`890
`
`AAT
`TTA
`Asn
`
`AAG ACA AAG
`TIC TGT TIC
`Lys Thr Lys
`
`CCG CGG
`gcc ccc
`Pro Arg
`
`Val Lys Phe>
`
`*
`
`900*
`GAG GAG CAG
`crc cre Gitc
`Glu Glu Gin>
`
`940*
`
`*
`
`950
`
`.
`
`960
`.
`
`GTC CTG Cac
`CAG GAC GTG
`Val Leu His
`
`CAG GAC
`GTc CTG
`
`Gin Asp
`
`TGG CTG AAT
`ACC GAC TTA
`Trp Leu Asn>
`
`1000
`*
`
`*
`
`1010
`*
`
`1020
`*
`
`*
`
`4g CGG
`Asn Lys Ala
`
`CTC CCA GCC CCC ATC GAG AAA ACC
`GAG GGT CGG GGG TAG CTC TIT TGG
`Leu Pro Ala Pro Ile Glu Lys Thr>
`
`870
`
`GTA
`His
`
`930
`
`ctc
`
`Val
`
`990
`
`GAG GTC ACA
`CTc CAG TGT
`Glu Val Thr
`
`TGC GTG
`ACG CAC
`Cys Val
`
`850
`*
`
`.
`
`860
`*
`
`TAC GTG GAC GCC GTG
`ATG CAC CTG CCG CAC
`Tyr Val Asp Gly Val
`
`Glu
`
`910
`*
`
`*
`
`920
`*
`
`AGC ACG TAC CGT GIG
`TCG TGC ATG GCA CAC
`Ser Thr Tyr Arg Val
`
`Val
`
`970
`*
`
`*
`
`GAG TAC AAG
`cic ATG TIC
`Glu Tyr Lys
`
`980*
`TGC AAG
`ACG TTC
`Cys Lys
`
`Val
`
`BAR
`
`TAC
`ATG
`
`Gly
`
`*
`
`*
`
`1030
`*
`
`*
`
`1040
`*
`
`1050
`
`1060
`*
`
`*
`
`1070
`*
`
`1080
`*
`
`.
`
`ATC
`TAG
`Tle
`
`AGG
`ser
`
`AAA GCC AAA
`TIT CGG TIT
`Lys Ala Lys
`
`GGG CAG
`ccc GTC
`Gly Gln
`
`ccc
`
`Pro
`
`CGA
`GcT
`Arg
`
`GAA
`cTtT
`Glu
`
`GOT
`Pro
`
`GIG TAC ACC
`CAC ATG TGG
`Val Tyr Thr
`
`CTG CCC CCA TCC CGG
`GAC GGG GGT AGG GC
`Leu Pro Pro Ser Arg>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 21 of 55
`
`US 7,374,757 B2
`
`Fig.14C.
`
`*
`
`1090
`*
`
`*
`
`1100
`*
`
`GAT GAG CTG ACC AAG AAC CAG GTC
`CTA CTC GAC TGG TIC TIG GTC CAG
`Asp Glu Leu Thr Lys Asn Gln Val
`
`*
`
`1110
`*
`
`AGC CTIG
`TCG GAC
`Ser Leu
`
`1120
`*
`
`*
`
`1130
`*
`
`*
`
`1140
`*
`
`*
`
`ACC TGC CTG GTC AAA GGC TIC
`TGG ACG GAC CAG TIT CCG AAG
`Thr Cys Leu Val Lys Gly Phe
`
`TAT CCC AGC
`ATA GGG TCG
`Tyr Pro Ser>
`
`*
`
`1150
`*
`
`*
`
`1160
`*
`
`1170
`*
`
`*
`
`.
`
`1180
`*
`
`*
`
`1190
`*
`
`GAC ATC GCC GTG GAG TGG GAG AGC
`CTG TAG CGG CAC CTC ACC CTC TG
`
`AAT GGG
`TTA ‘CCC
`
`Asp Ile Ala Val Glu Trp Glu Ser
`
`Asn Gly
`
`CAG CCG GAG AAC AAC TAC AAG
`GTc GGc ci TIG TTIG ATG TIC
`Gln Pro Glu Asn Asn Tyr Lys
`
`1200
`*
`
`*
`
`ACC ACG CCT
`TGG TGC GGA
`Thr Thr Pro>
`
`*
`
`1210
`*
`
`*
`
`1220
`*
`
`ccc GTG CTG GAC TCC GAC Goce TCC
`GGG CAC GAC CTG AGG CTG CCG AGG
`Pro Val Leu Asp Ser Asp Gly Ser
`
`*
`
`1270
`*
`
`*
`
`1280
`*-
`
`AGG TGG CAG CAG GG AAC GTC TIC
`Tcc ACC Gic GTC CCC TIG CAG AAG
`
`Arg Trp Gln Gln Gly Asn Val Phe
`
`*
`
`1330
`*
`
`*
`
`1340
`*
`
`TAC ACG CAG AAG AGC CTC TC CTG
`ATG TGC GTC TIC TCG GAG AGG GAC
`Tyr Thr Gln Lys Ser Leu Ser Leu
`
`1230
`*
`
`*
`
`TIC TIC
`AAG AAG
`Phe Phe
`
`*
`
`1290
`*
`
`TCA TGC
`AGT ACG
`Ser Cys
`
`1350
`*
`
`r
`
`TCT CCG
`AGA GGC
`Ser Pro
`
`1240
`*
`
`*
`
`1250
`*
`
`*
`
`1260
`*
`
`*
`
`crc TAC AGC AAG CTC ACC GTG
`GAG ATG TCG TIC GAG TGG CAC
`Leu Tyr Ser Lys Leu Thr Val
`
`GAC AAG AGC
`CiIG TIC TcG
`Asp Lys Ser>
`
`1300
`*
`
`*
`
`1310
`*
`
`*
`
`1320
`*
`
`*
`
`TCC GIG ATG CAT GAG GCT CTG
`AGG CAC TAC GTA CTC CGA GAC
`Ser Val Met His Glu Ala Leu
`
`CAC AAC CAC
`GTG TIG GTG
`His Asn His>
`
`*
`
`GGT AAA TGA
`CCA TTT ACT
`Gly Lys ***>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 22 of 55
`
`US 7,374,757 B2
`
`Fig.15A.
`
`*
`
`10
`*
`
`*
`
`20
`*
`
`*
`
`30
`*
`
`*
`
`40
`*
`
`*
`
`50
`*
`
`*
`
`60
`*
`
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTIG TGC GCG CIG CTC AGC TCT CTG CTT CTC
`TAC CAG TCG ATG ACC CIG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAG
`Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu>
`
`*
`
`70
`*
`
`*
`
`80
`*
`
`*
`
`90
`*
`
`*
`
`100
`*
`
`*
`
`110
`*
`
`*
`
`120
`*
`
`ACA GGA TCT AGT TCC GGA GGT AGA CCT TIC GTA GAG ATG TAC AGT GAA ATC CCC GAA ATT
`TGT CCT AGA TCA AGG CCT CCA TCT GGA AAG CAT CTC TAC ATG TCA CTT TAG GGG CTT TAA
`Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile>
`
`*
`
`130
`.
`
`*
`
`140
`*
`
`*
`
`150
`*
`
`*
`
`160
`*
`
`*
`
`170
`*
`
`*
`
`180
`*.
`
`ATA CAC ATG ACT GAA GGA AGG GAG CTC GIC ATT CCC TGC CGG GTT ACG TCA CCT AAC ATC
`TAT GIG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA TGC AGT GGA TIG TAG
`Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile>
`
`*
`
`190
`*
`
`*
`
`200
`*
`
`.
`
`210
`.
`
`*
`
`220
`*.
`
`*
`
`230
`*
`
`*
`
`240
`*
`
`CTT GAC ACT TTG ATC CCT GAT GGA AAA CCC ATA ATC
`ACT GTT ACT TTA AAA AAG TIT
`a8
`GAA CTG TGA AAC TAG GGA CTA CCT TTT GCG TAT TAG
`TGA CAA TGA AAT TTT TTC AAA
`Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile>
`
`*
`
`250
`*
`
`*
`
`260
`*
`
`*
`
`270
`*
`
`*
`
`280
`*
`
`*
`
`290
`*
`
`*
`
`300
`*
`
`TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGG CTT CTG
`ACC CTG TCA TCT TIC CCG AAG TAG TAT AGT TIA CGT TGC ATG TIT CTT TAT CCC GAA GAC
`Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu>
`
`x
`
`310
`*
`
`*
`
`320
`*
`
`*
`
`330
`*
`
`*
`
`340
`*
`
`*
`
`350
`*
`
`*.
`
`360
`*
`
`ACC TGT GAA GCA ACA GIC AAT GGG CAT TTG TAT AAG ACA AAC TAT CIC ACA CAT CGA CAA
`TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TIG ATA GAG TGT GTA GCT GIT
`Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln>
`
`420
`410
`400
`390
`380
`370
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TIA CTT AGA GCC
`IGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT GCG GGT CAG TTT AAT GAA TCT COG
`Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly>
`
`*
`
`430
`*
`
`*
`
`440
`*
`
`*
`
`450
`*
`
`*
`
`460
`*
`
`*
`
`470
`*
`
`*
`
`480
`*
`
`CAT ACT CTT GTC CTC AAT TGT
`GTA TGA GAA CAG GAG TTA ACA
`His Thr Leu Val Leu Asn Cys
`
`#88
`
`GCT ACC ACT CCC TTG AAC ACG AGA GTT CAA ATG ACC
`CGA TGG TGA GGG AAC TIG TGC TCT CAA GT? TAC TGG
`Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr>
`
`*
`
`490
`*.
`
`*
`
`500
`*
`
`*
`
`510
`*
`
`*
`
`520
`«
`
`*
`
`530
`*
`
`540
`
`AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA CGA ATT GAC CAA AGC
`TCA ATG GGA CTA CTT TTT TTA TIC TCT CGA AGG CAT TCC GCT GCT TAA CTIG GTT TCG
`Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser>
`
`

`

`U.S. Patent
`
`May20, 2008
`
`Sheet 23 of 55
`
`US 7,374,757 B2
`
`Fig.15B.
`
`*
`
`550
`*
`
`*
`
`560
`*
`
`*
`
`570
`*
`
`*
`
`580
`*
`
`*
`
`590
`*
`
`600
`*
`
`*
`
`ACT ATT GAC AAA ATG CAG AAC AAA GAC
`AAT TCC CAT GCC AAC ATA TIC TAC AGT GIT
`gy
`TGA TAA CTG TTT TAC GTC TIG TIT CTG
`TTA AGG GTA CGG TTG TAT AAG ATG TCA
`Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp>
`
`*
`
`610
`*
`
`*
`
`620
`*
`
`*
`
`630
`*
`
`*
`
`640
`*
`
`*
`
`650
`*
`
`660
`*
`
`*
`
`AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TIC AAA TCT GTT AAC ACC TCA
`TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TIT AGA CAA TTG TGG AGT
`Lys Gly Leu Tyr Thr Cys Arg