US007070959B1
`
`(12)
`
`United States Patent
`Papadopouloset al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,070,959 B1
`*Jul. 4, 2006
`
`(54)
`
`(75)
`
`MODIFIED CHIMERIC POLYPEPTIDES
`WITH IMPROVED PHARMACOKINETIC
`PROPERTIES
`
`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) by0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.:
`
`10/009,852
`
`(22)
`
`PCTFiled:
`
`May23, 2000
`
`(86)
`
`PCTNo.:
`
`PCT/US00/14142
`
`§ 371 (c)Q),
`(2), (4) Date: Dee. 6, 2001
`
`(87)
`
`PCTPub. No.: WO00/75319
`
`PCT Pub. Date: Dee. 14, 2000
`
`Related U.S. Application Data
`
`Provisional application No. 60/138,133, filed on Jun.
`8, 1999,
`
`Int. Cl.
`(2006.01)
`C12N15/62
`(2006.01)
`C12N15/63
`(2006.01)
`CO7H 21/04
`(2006.01)
`CO7K 16/00
`(2006.01)
`CO7K 14/00
`UNS. Che cece 435/69.7; 435/70.1; 435/71.1;
`435/320. 1; 435/325; 435/252.3: 530/387.3;
`530/350; 536/23.4
`Field of Classification Search ............... 435/69.7,
`435/320.1, 70.1, 71.1: 530/387.3
`See applicationfile for complete search history.
`
`References Cited
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`5,712,380 A
`6.011,003 A
`6,100,071 A *
`
`1/1998 Kendall et al.
`1/2000 Charnock-Jones etal.
`8/2000 Davis-Smyth et al.
`..... 435/69.7
`
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`W0O97'44453
`11/1997
`WO098/ 13071
`4/1998
`WO099/03996
`1/1999
`
`OTHER PUBLICATIONS
`
`Herleyet al. (1999), Biochem. Biophys. Res. Comm. 262:
`731-738.*
`Terman,B. L., et al, “Identification of a new endothelial cell
`growth factor receptor tyrosine kinase”, Oncogene (1991)
`6:1677-1683.
`Terman, B.I., 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.
`Dunea, R. and Spreafico, F., “Polymer Conjugates”, Drug
`Delivery Systems (1994) 27(4):290-306.
`Hileman. R.E., et al., “Glycosaminoglycan-protein interac-
`tions: definitions of consensus sites in glycosaminoglycan
`binding proteins”, 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.
`antibody
`“Improving monoclonal
`Sharifi,
`J.,
`et
`al.,
`pharmacokinetics via chemical modification”, Quart J Nucl
`Med (1998) 42:242-249.
`Jensen-Pippo, K.E., et al., “Enteral bioavailability of human
`granulocyte colonystimulating 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-1
`(Flt-1 Tyrosine kinase)’, (1997) Jpn J Cancer Res 88:767-
`876.
`Yang, J.C., et al., “The use of polyethylene glycol-modified
`interleukin-2 (PEG-IL-2) in the treatment of patients with
`metastatic renal cell carinoma and melanoma”, (1995) Can-
`cer 76(4): 687-694.
`
`(Continued)
`
`Primary Examiner—Lorraine Spector
`Assistant Examiner—Jon M Lockard
`(74) Attorney, Agent, or Firm—Valeta Gregg, Esq.
`
`(57)
`
`ABSTRACT
`
`Modified chimeric polypeptides with improved pharmaco-
`kinetics are disclosed. Specifically, modified chimeric FIt1
`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.
`
`15 Claims, 55 Drawing Sheets
`
`CELLTRION- EXHIBIT 1023
`
`CELLTRION - EXHIBIT 1023
`
`

`

`US 7,070,959 B1
`Page 2
`
`OTHER PUBLICATIONS
`
`Davis-Smyth,T., et al., 1996, “The second immunoglobulin-
`like domain of the VEGFtyrosine kinase receptor Flt-1
`determines
`ligand binding and may initiate a
`signal
`transduction-cascade”, The EMBO Journal 15(18):4919-
`4927.
`Holash,J., et al. (2002) PNAS, vol. 99, No. 17, pp. 11393-
`11398.
`Heidaran, M.A., (1990) The Journal of Biological Chemis-
`try, vol. 265, No. 31, Issue of Nov. 5, pp. 18741-18744.
`
`Cunningham,S.A. et al., (1997) Biochemical and Biophysi-
`cal Research Communications, vol. 231, pp. 596-599.
`Fuh, G.et al. (1998) The Journal of Biological Chemistry,
`vol. 273, No. 18, Issue of May 1, pp. 11197-11204.
`Wiesmann, C. et al. (1997) Cell, vol. 91, pp. 695-704.
`Barleon, B. et al. (1997) The Journal of Biological Chem-
`istry, vol. 272, No. 16, pp. 10382-10388.
`Davis-Smyth, T. et al. (1998) The Journal of Biological
`Chemistry, vol. 273, No. 6, pp. 3216-3222.
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 1 of 55
`
`US 7,070,959 B1
`
`Fig.1.
`
`123 45 6
`
`7
`
`8
`
`9
`
`10 0.01
`
`.
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`1
`
`Lg/ml
`—O— ITIE-2-Fe
`smote acetylated Fit-1(1-3)-Fe
`----O---- unmodified Fit-1{1-3)-Fe
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 2 of 55
`
`US 7,070,959 B1
`
`|=
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`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 3 of 55
`
`US 7,070,959 B1
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`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 4 of 55
`
`US 7,070,959 B1
`
`=—
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`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 5 of 55
`
`US 7,070,959 B1
`
`Fig.6A.
`
`123 45 6
`nt of oe
`
`
`
`7 8 9
`
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`
`1112 13 14 15 1617 1819 20
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`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 6 of 55
`
`US 7,070,959 B1
`
`Fig.7.
`
`—{}— ITIE-2-Fc
`cnnsente© unmodified Fit-1(1-3)-Fe
`----O---- acetylated Fit-1(1-3)-Fe (10X)
`nananf----- acetylated Fit-1(1-3)-Fe (20X)
`---4#--- acetylated Fit-1(1-3)-Fe (30X)
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 7 of 55
`
`US 7,070,959 B1
`
`4801},
`
`0.5 pg/ml
`
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`
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`
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`50
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`100
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`+
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`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 8 of 55
`
`US 7,070,959 B1
`
`
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`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 9 of 55
`
`US 7,070,959 B1
`
`Fig.10A.
`
`*
`
`10
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`40
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`50
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`ATG GTC AGC TAC TGG GAC ACC GGG GIC CTG CIG TGC GGG CTIG CIC AGC TET CIG CIT cic
`TAC CAG TCG 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>
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`His Ile Met Gln Ala Gly Gln Thr Leu His Leu Gln Cys Arg Gly Glu Ale Ala His Lys>
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`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 10 of 55
`
`US 7,070,959 B1
`
`*
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`Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 11 of 55
`
`US 7,070,959 B1
`
`Fig.10C.
`
`*
`
`1090
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`*
`
`*
`
`1120
`*
`
`*
`
`*
`
`1130
`*
`
`1140
`*
`
`*
`
`TTC CTC TI CCC CCA AAA CCC
`AAG GAG AAG GGG GGT TTT GGG
`
`Phe Leu Phe Pro Pro Lys Pro
`
`AAG
`TIC
`
`Lys
`
`GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA
`CTG TGG GAG TAC TAG AGG GCC IGG GGA CTC CAG TGT
`Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr>
`
`*
`
`1150
`*
`
`*
`
`1160
`*
`
`.
`
`1170
`*
`
`*
`
`1180
`*
`
`*
`
`1190*
`
`1200
`*
`
`*
`
`TGC GIG GIG GIG GAC GIG AGC
`ACG CAC CAC CAC CIG CAC TCG
`Cys Val Val Val Asp Val Ser
`
`Cac
`
`His
`
`GAA GAC CCT GAG GTC AAG TIC AAC TGG TAC GIG GAC
`CTT CIG GGA CTC CAG TIC AAG TIG ACC ATG CAC CTG
`Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp>
`
`*
`
`1210
`*
`
`*
`
`1220
`*
`
`1230
`*
`
`*
`
`1240
`*
`
`®
`
`1250
`*
`
`*
`
`1260
`*
`
`*
`
`GGC GIG GAG GTG CAT AAT GCC
`CCG CAC CIC CAC GTA TIA CGG
`Gly Val Glu Val His Asn Ala
`
`Lys
`
`ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC
`1ST TIC GGC GCC CIC CTC GTC ATG TIG TCG TC ATG
`Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr>
`
`*
`
`1270
`*
`
`*
`
`1280
`*
`
`1290
`*
`
`*
`
`1300
`*
`
`*
`
`1310
`t
`
`*
`
`1320
`*
`
`*
`
`CGT GTG GTC AGC GTC CTIc ACC
`GCA CAC CAG TCG CAG GAG TGG
`Arg Val Vai Ser Val Leu Thr
`
`GTtc
`CAG
`Val
`
`CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG
`GAC GTG GTC CTG ACC GAC TTA CCG TIC CIC ATG TIC
`Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys>
`
`1330
`*
`
`1360
`*
`
`1370
`*
`
`*
`
`*
`
`1340
`*
`
`1350
`*
`
`*
`
`*
`
`*
`
`1380
`*
`
`*
`
`TGC AAG GTC TCC AAC AAA GCC
`ACG TIC CAG AGG TIG TIT CGG
`Cys Lys Val Ser Asn Lys Ala
`
`CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA
`GGT CGG GGG TAG CTC TIT TG TAG AGG TIT CGG TIT
`Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys>
`
`*
`
`13906.
`
`*
`
`1400*
`
`1410
`*
`
`*
`
`1420
`*
`
`*
`
`1430
`*
`
`*
`
`1440
`*
`
`*
`
`GGG CAG CCC CGA GAA CCA CAG
`CCC GTC G6 CCT CTT Got GTc
`Gly Gin Pro Arg Giu Pro Gin
`
`TAC AcC CTG CCC CCA TCC CG GAT GAG CTG ACC AAG
`ATG TGG GAC GGG GGT AGG GCC CTA CIC GAC TGG TIC
`Tyr Thr Levu Pro Pro Ser Arg Asp Glu Leu Thr Lys>
`
`Val
`
`*
`
`1450
`*
`
`*
`
`1460
`*
`
`1470
`*
`
`*
`
`1480
`*
`
`*
`
`1490
`®
`
`*
`
`1500
`*
`
`*
`
`AAC CAG GTC AGC CTIG ACC TOC
`TTG GIC CAG TCG GAC TGG ACG
`Asn Gln Val Ser Leu Thr Cys
`
`CTG
`GAC
`Leu
`
`GTc AAA GGC TIC TAT CCC AGC GAC ATC GCC GIG GAG
`CAG TTT CCG AAG ATA GGG TCG CTIG TAG CGG CAC CTC
`Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu>
`
`*
`
`1510
`*
`
`*
`
`1520
`*
`
`1530
`*
`
`*
`
`*
`
`1540
`*
`
`1550
`*
`
`*
`
`1560
`*
`
`*
`
`TSG GAG AGC AAT GGG CAG CCG
`AcC CTC TOG TTA CCC GTC GGC
`Trp Glu Ser Asn Gly Gln Pro
`
`AAC AAC TAC AAG ACC ACG CCT CCC GIG CIG GAC TCC
`TIG TIG ATG TIC TGG TGC GGA GGG CAC GAC CTG AG
`Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser>
`
`*
`
`1570
`*
`
`*
`
`1580
`*
`
`1590
`*
`
`*
`
`*
`
`1600
`*
`
`1610
`*
`
`*
`
`1620
`*
`
`*
`
`GAC GGC TCC TFC TIC CT TAC
`CTG CCG AGG AAG AAG GAG ATG
`
`Asp Gly Ser Phe Phe Leu Tyr
`
`AAG CTC ACC GIG GAC AAG AGC AGG TGG CAG CAG GGG
`TIC GAG TGG CAC CTG TIC TCG TCC ACC GTC GI CCC
`Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gln Gly>
`
`Ser
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 12 of 55
`
`US 7,070,959 B1
`
`Fig.10D.
`
`*
`
`1630
`*
`
`*
`
`1640
`*
`
`1650
`a
`
`*
`
`*
`
`1660
`*
`
`*
`
`1670
`2
`
`1680
`*
`
`*
`
`AAC GTC TIC TCA TGC ICC GIG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC
`T1G CAG AAG AGT ACG AGG CAC TAC GTA CIC CGA GAC GIG TIG GIG ATG TCC GTC TIC TG
`Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser>
`
`*
`
`1690
`*
`
`*
`
`1700
`*
`
`cre TCC CIG TCT CCG GGT AAA TGA
`GAG AGG GAC AGA GGC CCA TIT ACT
`Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 13 of 55
`
`US 7,070,959 B1
`
`BSE
`
`ulewop
`
`
`
`SSBUIyJEIN|JOOeIU}
`
`“LEBY
`
`
`
`eUuBIQWweWwsUesulewopqA.Hulpuigpuebiysejnyjaoesyxy
`
`
`
`
`
`‘SELv66926
`ZG\6SZ0SzZ289
`
`OBZ
`
`ossSbSPE0NZNQ9NSNbNENe
`
`"e’eteonbiun
`
`SNUILWI8}-0
`
`ss
`
`dS}}-174wewny
`
`
`
`LONploeoulwy
`
`bL-UssSNZ
`
`b-udsSN9
`
`L-HaASNS
`
`b-HAdSNP
`
`b-UdsNE
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 14 of 55
`
`US 7,070,959 B1
`
`|||eylene
`
` 80
`
`
`D| |
` Fig.12B.
`
`
`
`
`
`
`40 oocooocoeo
`
`60
`
`SS8SSssssss
`STON er Or Ao wy lo
`AyorydoupA}
`
`Utmeli)
`
`Ssseseesesss
`LOTON KF Or No FLO
`Ayouiydospsy
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 15 of 55
`
`US 7,070,959 B1
`
`Fig.13A.
`
`*
`
`30
`*
`
`*
`
`40
`.
`
`*
`
`50
`*
`
`*
`
`60
`*
`
`ATG GTC AGC TAC TGG GAC ACC GGG
`TAC CAG TCG ATG ACC CTIG TGS CCC
`Met Val Ser Tyr Trp Asp Thr Gly
`
`GTC CTG CIG TGC GCG CTG CTC
`CAG GAC GAC ACG C&C GAC GAG
`Val Leu Leu Cys Ala Leu Leu
`
`AGC TGT CTG CTT cic
`TCG ACA GAC GAA GAG
`Ser Cys Leu Leu Leu>
`
`*
`
`70
`3
`
`=
`
`80
`*
`
`*
`
`30
`*
`
`*
`
`100
`*
`
`*
`
`110
`+
`
`*
`
`120
`*
`
`ACA GGA TCT AGT TCA GGT TCA AAA
`TGT CCT AGA TCA AGT CCA AGT TTT
`Thr Gly Ser Ser Ser Gly Ser Lys
`
`TTA AAA GAT CCT GaaA CTG AST
`AAT TTT CTA GGA CTT GAC TCA
`Leu Lys Asp Pro Glu Leu Ser
`
`TTA AAA GGC ACC CAG
`AAT TTP CCG TGG GTC
`Leu Lys Gly Thr Gin>
`
`*
`
`130
`*
`
`*
`
`140
`*
`
`*
`
`150
`*
`
`*
`
`160
`*
`
`*
`
`170
`*
`
`*
`
`180
`*
`
`CAC ATC ATG CAA GCA GGC CAG ACA
`GTG TAG TAC GIT CGT CCG GIC TCT
`His Ile Met Glin Ala Gly Gln Thr
`
`CTG CAT CTC CAA TGC AGG GG
`GAC GTA GAG GTT ACG Tce ccc
`Leu His Leu Gln Cys Arg Gly
`
`GAA GCA GCC CAT AAA
`CTT CGT CGG GTA TIT
`Glu Ala Ala His Lys>
`
`*
`
`190
`*
`
`*
`
`200
`.
`
`*
`
`210
`*
`
`*
`
`220
`*
`
`230
`*.
`
`*
`
`*
`
`240
`*
`
`TGG TCT TIG CCT GAA ATG GTG AGT
`ACC AGA AAC GGA CTT TAC CAC TCA
`Trp Ser Leu Pro Glu Met Val Ser
`
`AAG GAA AGC GAA AGG CTG AGC
`TTC CTT ICG CTT TCC GAC TCG
`Lys Glu Ser Glu Arg Leu Ser
`
`ATA ACT AAA TCT GCC
`TAT TGA TIT AGA CGG
`fle Thr Lys Ser Ala>
`
`*
`
`250
`*
`
`*
`
`260
`*
`
`290
`*
`
`*
`
`300
`*
`
`270
`2
`
`*
`
`*
`
`280
`*
`
`*
`
`TGT GGA AGA AAT GGC AAA CAA TIC
`ACA CCT TCT TIA CCG TIT GIT AAG
`Cys Gly Arg Asn Gly Lys Gln Phe
`
`TGC AG? ACT TTA ACC TTG AAC
`ACG TCA TGA AAT TGG AAC TIG
`Cys Ser Thr Leu Thr Leu Asn
`
`ACA GCT CAA GCA AAC
`TGT CGA GIT CGT TIG
`Thr Ala Gln Ala Asm>
`
`*
`
`310
`*
`
`*
`
`320
`*
`
`*
`
`330
`*
`
`*
`
`340
`*
`
`350
`*
`
`*
`
`*
`
`360
`*
`
`CAC ACT GGC TIC TAC AGC TOC AAA
`GTG TGA CCG AAG ATG TCG ACG TIT
`His Thr Gly Phe Tyr Ser Cys Lys
`
`TAT CTA GCT GTA CCT ACT TCA
`ATA GAT CGA CAT GGA TGA AGT
`Tyr Leu Ala Val Pro Thr Ser
`
`AAG AAG AAG GAA ACA
`TTc TIC Tic CIT ToT
`Lys Lys Lys Glu Thr>
`
`*
`
`370
`*
`
`*
`
`380
`*
`
`*
`
`390
`*
`
`*
`
`400
`*
`
`*
`
`410
`*
`
`*
`
`420
`*
`
`GAA TCT GCA ATC TAT ATA TIT ATT
`CTT AGA CGT TAG ATA TAT AAA TAA
`
`Glu Ser Ala Ile Tyr Ile Phe Ile
`
`AGT GAT ACA GGT AGA CCT TIC
`TCA CTA TGT CCA TCT GGA AAG
`Ser Asp Thr Gly Arg Pro Phe
`
`GTA GAG ATG TAC AGT
`CAT CTC TAC ATG TCA
`Val Glu Met Tyr Ser>
`
`*
`
`430
`*
`
`t
`
`440
`*
`
`*
`
`450
`*
`
`*.
`
`460
`.
`
`470
`*
`
`=
`
`«x
`
`480
`*
`
`GAA ATC CCC GAA ATT ATA CAC ATG
`CTT TAG GGG CTT TAA TAT GTG TAC
`Glu Tle Pro Giu fle fle His Met
`
`ACT GAA GGA AGG GAG CIC GTC
`TGA CTT CCT TCC CTC GAG CAG
`Thr Glu Gly Arg Glu Leu Val
`
`ATT CCC TGC CG CGTT
`TAA GGG ACG GCC CAA
`Tle Pro Cys Arg Val>
`
`*
`
`490
`*
`
`*
`
`$00
`*
`
`*
`
`510
`*
`
`*
`
`520
`*.
`
`os
`
`$30
`*
`
`*
`
`540
`*
`
`ACT TTG ATC CCT GAT
`TTA AAA AAG TTT CCA CTT GAC
`ACG TCA CCT AAC ATC ACT GTT ACT
`TGA AAC TAG GGA CTA
`AAT TTT TIC AAA GGT GAA CTG
`TGC AGT GGA TIG TAG TGA CAA TGA
`Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 16 of 55
`
`US 7,070,959 B1
`
`Fig.13B.
`
`*
`
`550
`*
`
`*
`
`560
`*
`
`*
`
`570
`*
`
`*
`
`580
`*
`
`*
`
`590
`*
`
`*
`
`600
`*
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA
`CCT TTT GOG TAT TAG ACC CTG TCA TCT TIC CCG AAG TAG TAT AGT TTA CGT TCC ATG TIT
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys>
`
`*
`
`610
`*
`
`*
`
`620
`*
`
`*x
`
`630
`*
`
`*
`
`640
`*
`
`»*
`
`650
`*
`
`*
`
`660
`*
`
`GAA ATA GGG CTT CTG ACC TGT GAA GCA ACA GIC AAT GGG CAT TTIG TAT AAG ACA AAC TAT
`CTT TAT CCC GAA GAC IGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TIC TGT TIG ATA
`Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr>
`
`*
`
`670
`*
`
`*
`
`680
`*
`
`690
`*
`
`*
`
`«x
`
`700
`*
`
`*
`
`710
`*
`
`720
`*
`
`*
`
`CTc ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GIC
`GAG TGT GTA GCT GIT IGG TTA TGT TAG TAT CTA CAG GTT TAT TCG IGT 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 GTC CTC AAT TGT ACT GCT ACC ACT CCC TG AAC ACG
`TTT AAT GAA TCT COG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTIG TCC
`Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`
`
`840
`830
`820
`810
`800
`790
`
`.4 * . * * «x * * * * * *
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AGA GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC
`TCT CAA GIT TAC TGG ACC TCA ATG GGA CTA CTT TAA CTG GTT TCG TIA AGG GTA CGG TIG
`Arg Val Glin Met Thr Trp Ser Tyr Pro Asp Glu Ile Asp Gln Ser Asn Ser His Ala Asn>
`
`*
`
`850
`*
`
`*
`
`860
`*
`
`*
`
`870
`*
`
`*
`
`880
`=
`
`*
`
`890
`*
`
`*
`
`900
`*
`
`ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT
`. TAT AAG ATG TCA CAA GAA TGA TAA CTG TIT TAC GTC TIG TIT CTG TIT CCT GAA ATA TGA
`Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr>
`
`*
`
`910
`*
`
`*
`
`920
`*
`
`*
`
`930
`*
`
`*
`
`$40
`*
`
`*
`
`950
`*
`
`*
`
`$60
`*
`
`TGT CGT GTA AGG AGT GGA CCA TCA TIC AAA TCT GTT AAC ACC TCA GIG CAT ATA TAT GAT
`ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TIG TGG AGT CAC GTA TAT ATA CTA
`Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp>
`
`*
`
`970
`*
`
`t
`
`980
`%
`
`x
`
`990
`*
`
`*
`
`1000
`*
`
`*
`
`1010
`*
`
`1020
`*
`
`*
`
`AAA GCA GGC CCG GGC GAG CCC AAA ICT TGT GAC AAA ACT CAC ACA TGC CCA COG TGC CCA
`TIT CGT CCG GGC COG CTC GGG TIT AGA ACA CTIG TIT TGA GTG TGT ACG GGT GCC ACG GGT
`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 CTC CIG GGG GGA CCG TCA GTC TIC CTC TIc CCC CCA AAA CCC AAG GAC ACC
`CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GOT TIT GG TIC CIG TG
`Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 17 of 55
`
`US 7,070,959 B1
`
`Fig.13C.
`
`*
`
`1090
`*
`
`*
`
`1100
`*
`
`1110
`*
`
`*
`
`*
`
`1120
`*
`
`*
`
`1130
`*
`
`1140
`*
`
`*
`
`CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GIG GIG GIG GAC GIG AGC CAC GAA GAC
`GAG TAC TAG AGG GCC TGG GGACIC CAG TGT ACG CAC CAC CAC CTG CAC TCG GIG CTT CIG
`Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp>
`
`*
`
`1150
`*
`
`*
`
`1160
`*
`
`1170
`*
`
`*
`
`*
`
`1180
`*
`
`*
`
`1190
`*
`
`1200
`*
`
`*
`
`CCT GAG GTC AAG TIC AAC TGG TAC GIG GAC
`GGA CTC CAG TTC AAG TTG ACC ATG CAC CTG
`Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
`
`GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG
`CCG CAC CIC CAC GTA TTA CGG TIC TET TIC
`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
`Goce Gce Cic Cit GIc ATG TIG TG TCC ATG
`Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
`
`CGT GIG GIC AGC GTC CTC ACC GTC CTG CAC
`GCA CAC CAG TCG CAG GAG TGG CAG GAC GIG
`
`Arg Val Val Ser Val Leu Thr Val Leu His>
`
`1270
`*
`
`*
`
`1280
`*
`
`*
`
`1290
`*
`
`*
`
`1300
`*
`
`*
`
`*
`
`1310
`*
`
`1320
`®
`
`*
`
`CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG
`GTC CTG ACC GAC TTA CCG TIC CTC ATG TIC
`
`TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC
`ACG TTC CAG AGG TIG TIT CGG GAG GGT CG
`
`Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
`
`Cys Lys Val Ser Asn Lys Ala Leu Pro Ala>
`
`x
`
`1380
`®
`
`*
`
`1330
`*
`
`*
`
`1340
`*
`
`1350
`*
`
`*
`
`*
`
`1360
`.
`
`*
`
`1370
`*
`
`ccc ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC
`GGG TAG CTC TIT TG TAG AGG TIT GGG TTT CCC GI GSS CCT CTT GGT GTC CAC ATG TGG
`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 GIC AGC CTG ACC TGC CTG GIC AAA
`GAC GGG GGT AGG GCC CTA CTC GAC TGG TIC TIG GTC CAG TCG GAC TGG ACG GAC CAG TIT
`Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 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
`Acc cr TOG TTA CCC GTC GCC CTC TTG TIG
`CCG AAG ATA GGG TCG CTG TAG CGG CAC CTC
`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 TCC
`ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG
`Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
`
`GAC GGC TCC TIC TIC CTC TAC AGC AAG CIC
`CTG CCG AGG AAG AAG GAG ATG TOG TIC GAG
`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 CTG TIC TCG TCC ACC GIC GIC CCC TIG CAG AAG AGT ACG AGG CAC TAC GTACTC
`Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 18 of 55
`
`US 7,070,959 B1
`
`Fig.13D.
`
`«x
`
`1630
`*
`
`*
`
`1640
`*
`
`1650
`*
`
`*
`
`*
`
`1660
`*
`
`*
`
`1670
`*
`
`GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA
`CGA GAC GIG TIG GIG ATG TGC GTC 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
`
`Jul. 4, 2006
`
`Sheet 19 of 55
`
`US 7,070,959 B1
`
`Fig.14A.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`ATG GTC AGC TAC TGG GAC ACC GGG GIC CTG CTG TOC GGG CTG CT} ACC TET 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
`*
`
`«x
`
`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>
`
`*
`
`*
`
`200
`*
`
`210
`*
`
`*
`
`*
`
`220
`*
`
`*
`
`230
`*
`
`240
`*
`
`*
`
`190
`*
`
`ACT GTT ACT TTA AAA AAG TIT CCA CTT GAC ACT TIG ATC CCT GAT GGA AAA CGC ATA ATC
`TGA CAA TGA AAT TTY TIC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT TIT GCG TAT TAG
`Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile>
`
`250
`260
`270
`280
`230
`300
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`x
`*
`™GG GAC AGT AGA AAG GGC TIC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGG CTT CIG
`ACC CTG TCA TCT TIC COG AAG TAG TAT AGT TTA 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 Tle Gly Leu Leu>
`
`*
`
`310
`*
`
`*
`
`320
`*
`
`330
`*
`
`*.
`
`*
`
`340
`*
`
`*
`
`350
`*
`
`360
`*.
`
`*
`
`ACC TGT GAA GCA ACA GTC AAT GGG CAT TIG TAT AAG ACA AAC TAT CTC ACA CAT CGA CAA
`GG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TIC 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>
`
`*
`
`370
`*
`
`*
`
`380
`*
`
`390
`*
`
`*
`
`*
`
`400
`*
`
`*
`
`410
`*
`
`420
`*
`
`*
`
`ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTA CTT AGA GGC
`GG TTA IGT TAG TAT CTA CAG GTT TAT TCG TGT GGT GOG GGT CAG TTT AAT GAA TCT CCG
`Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly>
`
`*
`
`430
`*
`
`*
`
`440
`*
`
`450
`*
`
`*
`
`*
`
`460
`r
`
`*
`
`470
`*
`
`480
`*
`
`*
`
`CAT ACT CTT GIC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAA ATG ACC
`GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC ICT CAA GIT TAC TGG
`His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gin Met Thr>
`
`*.
`
`490
`*
`
`*
`
`500
`*
`
`*
`
`510
`*
`
`*
`
`520
`*
`
`*
`
`530
`*
`
`*
`
`540
`*
`
`TGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TIC TAC AGT GTT
`ACC TCA ATG GGA CTA CTT TAA CTG GIT TCG TTA AGG GTA CGG TIG TAT AAG ATG TCA CAA
`Trp Ser Tyr Pro Asp Glu Ile Asp Gin Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 20 of 55
`
`US 7,070,959 B1
`
`Fig.14B.
`
`*
`
`550
`*
`
`*
`
`560
`*
`
`*
`
`$70
`*
`
`*
`
`580
`*
`
`*
`
`S30
`*
`
`*
`
`600
`*
`
`CTT ACT ATT GAC AAA ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT
`GAA TGA TAA CTG TIT TAC GTC TIG TIT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA
`Leu Thr Ile Asp Lys Met Gin Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser>
`
`.
`
`610
`Sd
`
`*
`
`620
`*
`
`630
`*
`
`a
`
`*«
`
`640
`*
`
`*
`
`650
`*
`
`660
`*
`
`*
`
`GGA CCA TCA TIC AAA TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT AAA GCA GGC COG GCC
`CCT GGT AGT AAG TIT AGA CAA TIG TG AGT CAC GTA TAT ATA CTA TIT CGT CCG GGC CCG
`Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Gly Pro Gly>
`
`*
`
`670
`*
`
`*
`
`680
`*
`
`*
`
`690
`*
`
`*
`
`700
`*
`
`*
`
`710
`*
`
`720
`*
`
`*
`
`GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTIG
`CTC GGG TIT AGA ACA CTG TIT TGA GTG IGT ACG GGT GGC ACG GGT CGT GGA CTT GAG GAC
`Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu>
`
`*
`
`730
`*
`
`*
`
`740
`*
`
`*
`
`750
`*
`
`«x
`
`760
`*
`
`.
`
`770
`*
`
`°.
`
`780
`*
`
`GGG GGA CCG TCA GIC TIc CiIc Tic CCC CCA AAA CCC AAG GAC ACC CIC ATG ATC TCC CG
`ccc CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT GGG TIC CTG TGG GAG TAC TAG AGG GC
`
`Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg>
`
`*
`
`*
`
`*
`
`*
`
`*
`
`790
`*
`
`*
`
`800
`*
`
`810
`*
`
`820
`*
`
`830
`*
`
`840
`=
`
`ACC CCT GAG GTC ACA TGC GIG GIG GIG GAC GIG AGC CAC GAA GAC CCT GAG GTC AAG TIC
`TGG GGA CTC CAG IGT ACG CAC CAC CAC CTG CAC TCG GTG CTT CTG GGA CTC CAG TIC AAG
`Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe>
`
`x
`
`850
`*
`
`*
`
`860
`*«
`
`*
`
`870
`*
`
`*
`
`880
`*
`
`*
`
`890
`*
`
`*
`
`900
`*
`
`AAC TGG TAC GIG GAC GGC GTG GAG GIG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG
`TTG ACC ATG CAC CTG CCG CAC CIC CAC GTA TTA CGG TIC TGT TIC GoC GMC CI Cle Gr
`Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln>
`
`*
`
`910
`*
`
`*
`
`$20
`*
`
`*
`
`930
`*
`
`.
`
`$40
`*
`
`«
`
`$50
`*
`
`*
`
`960
`*
`
`TAC AAC AGC ACG TAC CGT GIG GTC AGC GIC CTC ACC GTC CIG CAC CAG GAC TGG CTG AAT
`ATG TIG TCG TGC ATG GCA CAC CAG TOG CAG GAG TGG CAG GAC GTG GTC CTG ACC GAC TIA
`Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn>
`
`*
`
`970
`*
`
`*
`
`980
`*
`
`x
`
`990
`*
`
`*
`
`1000
`*
`
`*
`
`1010
`*
`
`1020
`*
`
`*
`
`GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC
`ccc TIC CTC ATG TIC ACG TIC CAG AGG TIG TTT CGG GAG GGT CGG GGG TAG CIC TIT 166
`Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr>
`
`*
`
`1030
`*
`
`*«
`
`1040
`*
`
`1050
`*
`
`*
`
`«
`
`1060
`*
`
`*
`
`1070
`*.
`
`1080
`*
`
`*
`
`ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG
`TAG AGG TIT CG TIT CCC GIC GGG GT CTT GGT GTC CAC ATG TGG GAC GG GOT AG CCC
`Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 21 of 55
`
`US 7,070,959 B1
`
`Fig.14C.
`
`*
`
`1090
`*
`
`*
`
`1100
`*
`
`1110
`*
`
`*
`
`*
`
`1120
`*
`
`*
`
`1130
`*
`
`1140
`*
`
`*
`
`GAT GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTIG GIC AAA GGC TIC TAT CCC AGC
`CTA CTC GAC 1GG TIC TIG GTC CAG TCG GAC TGG ACG GAC CAG TIT CCG AAG ATA GGG TCG
`Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser>
`
`*«.
`
`1150
`*
`
`*
`
`1160
`*
`
`1170
`*
`
`*
`
`*
`
`1180
`*
`
`*
`
`1190
`*
`
`1200
`*
`
`*
`
`GAC ATC GCC GIG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT
`CTG TAG CGG CAC CTC ACC CIC TCG TTA CCC GIC GC CIC TIG TIG ATG TIC TG TGC GGA
`Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro>
`
`*
`
`1210
`*
`
`*
`
`12206
`*
`
`1230
`x
`
`*
`
`*
`
`1240
`*
`
`*
`
`1250
`*
`
`1260
`*
`
`*
`
`ccc GIG CIG GAC TCC GAC GGC TCC TIC TIC CIC TAC AGC AAG CTC ACC GIG GAC AAG AGC
`GGG CAC GAC CTG AGG CTIG COG AGG AAG AAG GAG ATG TCG TIC GAG TGG CAC CTIG TIC TCG
`Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser>
`
`3
`
`1270
`*
`
`*
`
`1280
`rr
`
`1290
`*
`
`*
`
`*
`
`1300
`*
`
`*
`
`1310
`*
`
`1320
`.
`
`*
`
`AGG TGG CAG CAG GGG AAC GIC TIC TCA TGC TCC GIG ATG CAT GAG GCT CTG CAC AAC CAC
`TCC ACC GTC GTC CCC TIG CAG AAG AGT ACG AGG CAC TAC GTA CTC CGA GAC GIG TIG GIG
`Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His>
`
`*
`
`1330
`*
`
`*
`
`1340
`*
`
`1350
`t
`
`*
`
`*
`
`TAC ACG CAG AAG AGC CIC TCC CTG TCT CCG GOT AAA TGA
`ATG TGC GTC TIC TCG GAG AGG GAC AGA GGC CCA TTT ACT
`Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`U.S. Patent
`
`Jul. 4, 2006
`
`Sheet 22 of 55
`
`US 7,070,959 B1
`
`Fig.15A.
`
`+
`
`10
`*
`
`*
`
`20
`*
`
`*
`
`30
`*
`
`*
`
`40
`*
`
`*
`
`50
`*
`
`*
`
`60
`*
`
`ATG GTC AGC TAC TGG GAC ACC GGG GIC CTG CTG TGC GCG CTG CTC ACC IGT CIG CTT Cic
`TAC CAG TCG 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 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 Iie Pro Glu [le>
`
`*
`
`130
`4
`
`*
`
`140
`*
`
`*
`
`150
`*
`
`*«
`
`160
`*
`
`*
`
`170
`*
`
`=
`
`180
`*
`
`ATA CAC ATG ACT GAA GGA AGG