USOO7374758B2
`
`(12) United States Patent
`Papadopoulos et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,374,758 B2
`*May 20, 2008
`
`(54)
`
`(75)
`
`(73)
`
`MODIFIED CHMERC POLYPEPTDES
`WITH IMPROVED PHARMACOKINETIC
`PROPERTIES AND METHODS OF USING
`THEREOF
`
`Inventors: Nicholas J. Papadopoulos,
`Lagrangeville, NY (US); Samuel Davis,
`New York, NY (US); George D.
`Yancopoulos, Yorktown Heights, NY
`(US)
`Assignee: Regeneron Pharmaceuticals, Inc.,
`Tarrytown, NY (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`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)
`(22)
`(65)
`
`(62)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Appl. No.: 11/016,503
`
`Filed:
`
`Dec. 17, 2004
`
`Prior Publication Data
`US 2005/0245447 A1
`Nov. 3, 2005
`
`Related U.S. Application Data
`Division of application No. 10/009,852, filed as appli
`cation No. PCT/US00/14142 on May 23, 2000, now
`Pat. No. 7,070,959.
`Provisional application No. 60/138,133, filed on Jun.
`8, 1999.
`
`Int. C.
`(2006.01)
`A6 IK 38/18
`(2006.01)
`C07K I4/7
`(2006.01)
`CI2N 5/62
`U.S. Cl. ............................... 424/134.1; 424/192.1;
`514/2; 514/23: 530/350,536/23.4
`Field of Classification Search ..................... None
`See application file for complete search history.
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`1/1998 Kendall et al.
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`2004/0265309 A1* 12/2004 Kandel et al. ........... 424,145.1
`2005/0175610 A1* 8/2005 Wiegand et al. ......... 424,145.1
`2005/0260203 A1* 11/2005 Wiegand et al. ......... 424,145.1
`2005/0281822 A1* 12/2005 Cedarbaum et al. ..... 424,145.1
`2005/0281831 A1* 12/2005 Davis-Smyth et al. ... 424/178.1
`2006/0210566 A1* 9, 2006 Holash et al. ........... 424,145.1
`2007/0037748 A1
`2/2007 Stahl et al. ................... 514/12
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`FOREIGN PATENT DOCUMENTS
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`OTHER PUBLICATIONS
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`Wells, J.A. (1990). Additivity of mutational effects in proteins.
`Biochemistry, 29(37):8509-8517.*
`Ngo et al. (1994). Computational complexity, protein structure
`prediction, and the Levinthal paradox. In Merz and Le Grand (Eds.)
`The Protein Folding Problem and Tertiary Structure Prediction.
`Birkhauser:Boston, pp. 491-495.*
`Herley et al. (1999). Characterization of the VEGF binding site on
`the Flt-1 receptor. Biochemical and Biophysical Research Commu
`nications. 262:731-738.
`Terman, B.I., 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
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`thrombopoietic potency'. Thrombosis and
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`Dunca, R. and Spreafico. F., “Polymer Conjugates'. Drug Delivery
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`Hileman, R.E., et al., “Glycosaminoglycan-protein interactions:
`definitions of consensus sites in glycosaminoglycan binding pro
`teins”, BioEssays (1998) 20:156-167.
`de Vries, Carlie, et al., “The fins-like tyrosine kinase, a receptor for
`vascular endothelial growth factor'. Science (1992) 225:989-991.
`Sharifi, J., et al., “Improving 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-1 (Flt-1 Tyrosine
`kinase)', (1997) Jpn J Cancer Res 88:867-876.
`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 carcinoma and melanoma', (1995) Cancer 76(4): 687
`694.
`Davis-Smyth, T., et al., 1996, “The second immunoglobulin-like
`domain of the VEGF tyrosine kinase receptor Flt-1 determines
`ligand binding and may initiate 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 Valeta Gregg, Esq.
`
`(57)
`
`ABSTRACT
`
`Modified chimeric polypeptides with improved pharmaco
`kinetics are disclosed. Specifically, modified chimeric Flt 1
`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.
`
`3 Claims, 55 Drawing Sheets
`
`Celltrion Exhibit - 1010
`Celltrion, Inc. v. Regeneron Pharmaceuticals, Inc.
`
`Pg. 001
`
`

`

`U.S. Patent
`
`May 20, 2008
`Sheet 1 of 55
`Fig.1.
`
`
`
`2 3 4 5 6 7 8 9 10
`
`US 7,374,758 B2
`
`0.001
`
`0.01
`
`O.
`
`1O
`
`100
`
`1
`ug/ml
`-OH- rTE-2-FC
`...a...... acetylated Flt-1 (1-3)-Fo
`----O---- unmodified Flt-1 (1-3)-Fo
`
`Pg. 002
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 2 of 55
`
`US 7,374,758 B2
`
`
`
`HSVM, IT\/S
`
`
`
`Mylan Exhibit 1010
`Mylan v. Regeneron, IPR2022-01225
`Page 3
`
`Pg. 003
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 3 of 55
`
`US 7,374,758 B2
`
`
`
`
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`so
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`
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`Pg. 004
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 4 of 55
`
`US 7,374,758 B2
`
`S his
`s
`O
`GS)
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`O O
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`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 5 of 55
`
`US 7,374,758 B2
`
`Fig.6A.
`
`1 2 3 4 5 6 7 8 9 10
`
`Fig.6B.
`
`1 12 13 4 5 16 17 1819 20
`
`
`
`Pg. 006
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 6 of 55
`
`US 7,374,758 B2
`
`
`
`Fig.7.
`
`rTIE-2-FC
`...KX. unmodified Flt-1 (1-3)-FC
`----O---. acetylated Flt-1 (1-3)-Fo (10X)
`---A----- acetylated Flt-1 (1-3)-FC (20X)
`-------. acetylated Flt-1 (1-3)-Fc (30X)
`
`Pg. 007
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 7 Of 55
`
`US 7,374,758 B2
`
`Fig.8.
`
`0.5 g/ml
`
`1.0g/ml
`
`
`
`480
`
`380
`
`18O
`
`80
`
`--Degree of step-acetylation of Flt-1 (1-3)-Fo protein
`-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`
`-
`
`-
`
`-
`
`-
`
`se
`
`r
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`y
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`
`t
`
`s
`
`so
`
`-
`
`-
`
`-
`
`-
`
`-
`
`VEGF
`0.2 ug/ml
`Unmodified
`Flt-1 (1-3)
`-FC
`Acetylated
`Flt-1 (1-3)
`
`Pg. 008
`
`

`

`U.S. Patent
`U.S. Patent
`
`May 20, 2008
`May20, 2008
`
`Sheet 8 of 55
`Sheet 8 of 55
`
`US 7,374,758 B2
`US 7,374,758 B2
`
`(
`
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`Pg. 009
`
`Pg. 009
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 9 of 55
`
`US 7,374,758 B2
`
`Fig.10A.
`
`O
`2)
`SO
`e
`t
`t
`t
`y
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CNS TGC GOG cTG CIC AGC TGT. CTG CTT CTC
`TAC CAG TCG AEG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAG
`Met Val Ser Tyr Trp Asp Thr Gly vall Leu Leu. Cys Ala leu Leu Ser Cys Leu. Leu Lleus
`
`6C
`
`30
`
`O
`
`70
`
`30
`90
`t
`t
`t
`w
`ACA GGA TCT AGF OA GST TOCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG
`TGT CCT AGA RA AGT CCA AGT TTT AAT TTT CIA GGA CTT GAC TCA AA TTT CCG TGG GTC
`Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pre G.u. Leu Ser Leu Lys Gly thr Glind
`
`OO
`
`10
`
`20
`
`3D
`
`140
`
`SO
`170
`BO
`r
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`t
`CAC ATC ATS CAA. G.C.A. GGC CAG ACA CTG CA CTC CAA TGC AGG GGG GAA GCA GCC CAF AAA
`GRG TAG TAC GT CGI CCG GTC GF GAC GIA GAG (GTT ACG ROC CCC CT CGT CGG GTA TTT
`His Ilie Met Gin Aa Gly Glin Thr Leu Riis Leu Gin Cys Arg Gly Glu Ala Ala is Lys)
`
`50
`
`90
`
`200
`
`2O
`
`220
`
`230
`
`r
`
`t
`
`r
`
`2O
`
`r
`
`TGG TOT TTG CCT GAA ARG GTG. AGF AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT, GCC
`ACC AGAAA GGA CTT TAC CAC TCA TTC CTT TOG CTT. TCC GAC TCG TATTGA TTT AGA CGG
`Frp Ser Lei Pro Glui et Wai Ser Lys giu Ser Giu Arg Leu Ser Lie Thr Lys Ser Ala)
`
`250
`260
`27C
`3OO
`t
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`r
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`r
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`TGF GA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC TTGAAC ACA GCT CAA GCA AAC
`ACA CCT TCT, TTA. COG T G T AAG ACG TCATA AA TGG AAC TG TGT CGA GT CGT TG
`Cys Giy Arg Asn Gly Iys Gin Phe cys Ser Thr Leu. Thr Leu Asn Thr Ale Gin Ala Asn
`
`290
`
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`
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`
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`
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`
`340
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`
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`
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`Etis Thr Giy Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Gli thr)
`
`39
`
`37
`r
`t
`GAA TCT, GCA ATC AT AA TT ATT AGT, GAT ACA GG AGA CCT TO GA GAG AG AC AGT
`CTTAGA CGT TAG ATAATAAATAATCA CTA TGT cca TCT GGA AAG CAT CTC TAC AIG TCA
`Glu Ser Ala Ile Tyr Ile the Ilie Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser?
`
`390
`
`400
`
`49
`
`23
`
`G30
`440
`A60
`470
`480
`ve
`-
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`y
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`it.
`r
`r
`GAA ATC CCC GAA AIT ATA CAC ATG ACT GAA SCA AGG GAG CTC GTC ATT CCC TGC CGGGTT
`CT TAG GGG CT TAA TAT GTG TA TGA CTT, CT, TCC CTC GAG CAG TAA. GGG ACG GCC CAA
`Giu Tile Pro Glu Ilie Ile His Met Thr Gu Giy Arg Giu. Leu Val Ile Pro Cys Arg Wall
`
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`
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`
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`
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`
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`
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`
`540
`
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`
`ACG TCA CCT AAC ATC ACT GT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT (GAT
`TGC AGT (GGA TING TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GA CTA
`Tir Ser Pro Asn. Ile Thr Wall Thr Leu Lys Lys Phe Pro Leu Asp Thir Leu Ilie Pro Asp
`
`Pg. 010
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 10 Of 55
`
`US 7,374,758 B2
`
`Fig.10B.
`
`560
`
`590
`550
`570
`600
`w
`t
`t
`t
`g
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`s
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA
`CCTTTT GCG TAT TAG ACC CTG TCA TCT TTC ccG AAG TAG TAT AGT TTA CGT TGC ATG TTT
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys)
`
`SBO
`
`60
`
`620
`
`630
`
`s
`
`640
`
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`
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`
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`
`GAA ATA GGG CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TIG TAT. AAG ACA AAC TAT
`CT TAT CCC GAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TTG ATA
`Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly. His Leu Tyr Lys Thr Asn Tyr>
`
`670
`
`5BO
`
`690
`
`700
`
`70
`
`720
`
`CIC ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC
`GAG TGT GIA GCT, GT TGG TTA TG TAG AT CIA. CAG GTT TAT TCG GT GGT GOGGGT CAG
`Leu Thr His Arg Glin Thr Asn Thr Ile Ile Asp Val Glin Ile Ser Thr Pro Arg Pro Wals
`
`730
`
`740
`
`750
`t
`t
`t
`AAA TTA. CTTAGA GGC CAT ACT. CTT, GTC CTC AAT TGT ACT, GCT ACC ACT CCCTTGAAC ACG
`TT AAT GAA CT COG GA GA GAA CAG GAG
`A ACA GA CGA TGG. TGA GGG AAC TG GC
`Lys Lieu Lieu. Arg Gly. His Thr Leu Val Leu Asn. Cys Thr Ala Thr Thr Pro Lieu. ASn Thr)
`
`760
`
`770
`
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`
`800
`
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`840
`t
`s
`s
`y
`y
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`t
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`t
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`TCT CAA GT TAC TGG ACC TCA ATG GGA CTA CT TTT TTA TTC TCT, CGA AGG CAT TCC GCT
`Arg Val Glin Met Thr Trp Ser Tyr Pro Asp Gu Lys Asn Lys Arg Ala Ser Val Arg Arg
`
`80
`
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`
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`880
`890
`900
`t
`s
`t
`s
`t
`s
`r
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`GC
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`Arg Ile Asp Glin Ser Asn. Ser His Ala Asn. Ile Phe Tyr Ser Val Leu Thr Ile Asp Lyss
`
`910
`
`920
`950
`r
`t
`s
`r
`ATG CAG AAC AAA GAC AAA GGA CT. At ACT GT CG GTA AGG AGT GGA COA. CAC AAA
`TAC GTC TTG. T.T. CTG TTT CC GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT
`Met Glin Asn Lys Asp Lys Giy Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys)
`
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`
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`
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`
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`
`990
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`AGA CAA TTG TGG AGT CAC GTA TAT AA CIA TT CGT CCG GGC CCG CTC GGG
`T AGA ACA
`Ser Val Asn. Thir Ser Val His Ile Tyr Asp Lys Ala Gly Pro Gly Glu Pro Llys Ser Cys>
`
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`
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`
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`t
`t
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`CTG TTT TGA GTG TGT ACG GGT GGC ACG GGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG
`Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gu Leu Leu Gly Gly Pro Ser Val)
`
`OSO
`
`O60
`
`OO
`
`1080
`
`Mylan Exhibit 1010
`Mylan v. Regeneron, IPR2022-01225
`Page 11
`
`Pg. 011
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 11 Of 55
`
`US 7,374,758 B2
`
`Fig.10C.
`
`iOSO
`
`LOC
`
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`'
`TTX, CTC TTC CCC COA. AAA CCC AAG GAC ACC Cric AG AC. TCC CGG ACC CC se GEC AA
`AAG GAG AAG GGG GGTTTT GGG TC CTG TGG GAG TAC AG. AGG GCC TGG GGA CTC CAG TGT
`Pine Leu Phe Pro Pro Llys Pro Lys Asp Tir Leu Met ille Ser Arg Thr Pro Glu Wall thre
`
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`ACG CAC CAC CAC CTG cAcroG Grs or crg GSA circ cag Trc AAG TTG ACC ATG CAC CTG
`Cys Wall Val Val Asp Wal Ser His Giu. As Pro Glu Wall Lys Ehe Asia Trp Tyr Wall Asp)
`
`21.
`
`22
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`
`230
`
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`
`1250
`
`250
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`r
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`t
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`GGC GTG GAG GIG CAT AAT Gcc AAG ACA AAG ccs cog GAG GAG CAG TAC AAC AGC ACG TAC
`CCG CAC CTC CAC GTA IA CGG TC G.
`C. GGC GCC C C CC GIC AIG G CG TeC ATG
`Gy Wai Giu Wall Eis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr
`
`2BO
`
`29
`
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`32.
`r
`r
`t
`CGT GTG GTC AGC GTC CTC ACC GNC CTG CAC CAG GAC GG CNG AA GGC AAG GAG AC AAG
`GCA CAC CAG TOG CAG GAG TSG CA
`AC.
`G. GC CG ACC GAC T
`COG TC CTC AIG TC
`Arg Val Val Ser Wall Let: Thr Val Lea His Glin Asp Trp Leu Asn. Gly Lys Glu Tyr EySP
`
`3CO
`
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`
`30
`
`330
`350
`350
`t
`t
`t
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`ge
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`TGC AAG GTC TCC AAC AAA GCC C CCA GCC CCC ATC GAG AAA ACC ACTCCAAA GCC AAA
`ACG TC CAG AGK TG TTT Ce GAG GT CGG GGG AG CTC TT NGG TAG AGG
`OGG TTT
`Cys Llys Wall Ser Asan Lys Alia Leu Pro Ala Pro Le Gu Lys Thr le Ser Lys Ala Lys?
`
`3O
`
`380
`
`1390
`
`O
`
`110
`1820
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`s
`r
`r
`s
`GGG CAG CCC CGA GAA CA CAG GIG AC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG
`CCC GTC GGG GCT. CTT GGT GTC CAC ATG TGG GAC GGG (GGT AGG GCC CA CTC GAC TGG TEC
`Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu Pro Pro Ser Arg Asp Gu Lieu. Thr Lyss
`
`30
`
`&O
`
`450
`
`O
`
`450
`1480
`t
`'
`t
`'
`t
`t
`AAC CAG GTC AC CTG ACC TGC CTG GTC AAA GGC TTC TAF CCC AGC GAC ATC GCC GTG GAG
`G GTC CAG TCG GAC TGG ACG GC CAG T COG AAG ATA GGG TCG G TAG CGG CAC CTC
`ASn Gin Val Ser Let Thr Cys Leu Val Lys Giy Phe Tyr Pre Ser Asp Ile Ala Wall Gill)
`
`i490
`
`SOD
`
`SO
`
`1S2O
`
`530
`
`1SO
`t
`TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CO.T CCC GTG CG GAC TCC
`ACC CTC TCG TTA. CCC GTC GGC CTC TTG TTG ATG TTC TGG TGC GGA. GGG CAC GAC CTG AGG
`Trp Gu Ser Asri Gly Gin Pro Giu. Asr Ash Tyr flys. Thir hr Pro Pro Wall Leu Asp Set
`
`SSO
`
`560
`
`SO
`
`SB
`
`SOO
`El
`SO
`-r
`s
`r
`ar
`t
`GAC GGC. TCC TTC TC CTC AC AGC AAG CC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG
`CTG CCG AGG AAG AAG GAG ATG TCG TC GAG (GG CAC CTG TC TCG TCC ACC GTC GTC CCC
`Asp Gly Ser Phe Phe Leu Tyr Ser Lys Lieu. Thr Val Asp Lys Ser Arg Trip Gln Gin Giy>
`
`59
`
`Pg. 012
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 12 of 55
`
`US 7,374,758 B2
`
`Fig.1OD.
`
`630
`
`l650
`
`1640
`1660
`r
`t
`w
`y
`w
`y
`AAC GTC TTC TCA TGC TCC GTG 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 GTG TIG GTG ATG TGC GTC TTC TOG
`Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Glin Lys Ser>
`
`670
`
`680
`
`TOO
`
`690
`w
`w
`st
`CTC. TCC CTG TCT CCG GGT AAA TGA
`GAG. AGG GAC AGA GGC CCA TT ACT
`Leu Ser Leu Ser Pro Gly Lys * * * >
`
`Pg. 013
`
`

`

`U.S. Patent
`U.S. Patent
`
`May 20, 2008
`May20, 2008
`
`Sheet 13 of 55
`Sheet 13 of 55
`
`US 7,374,758 B2
`US 7,374,758 B2
`
`
`
`
`SPU)JEIN|}ede1}uIBuipuiqpuebysepnyjsoesyx3]
`
`
`
`ulewopSUIBWOp
`
`SUBIQWOWSUBIL
`
`"EEIY
`
`08?
`
`NZNQ9NGSNeNENe
`
`‘e’e-eanbiun
`
`SNUILL8)}-9
`
`s§
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`StelIGkt=».peGzBCZNGSZOSZ789OSSZSPLONploeoujwy
`
`
`
`
`
`GHGI
`
`178SOSSSSL-W4SNE
`
`*7SS,78S&“YASNZ
`
`“dS]basNP
`
`b-HSNG
`
`Pg. 014
`
`Pg. 014
`
`
`
`
`
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 14 of 55
`
`US 7,374,758 B2
`
`
`
`|
`
`|
`
`|
`
`|
`
`|
`
`| O
`
`O
`s
`
`
`
`
`
`S
`
`|
`III
`|1||
`III
`|
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`S.
`
`S
`
`9
`
`C. C C C O O O O COO
`C C C C C C C C C C C
`ui rif cici - c. cici fui
`AloudolpAH
`
`O. O. O. O. O. O. O. O. O. C. O
`C C C C C C C C C C C
`Lort () () - O c car up
`AOLudolpAH
`
`Pg. 015
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 15 of 55
`
`US 7,374,758 B2
`
`Fig.13A.
`
`t
`"r
`t
`t
`t
`w
`g
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CEG CTG TGC GCG CG CTC AGC TGT CTG CTT CTC
`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 Lieu eu Leu)
`
`80
`
`70
`O
`20
`y
`t
`w
`ar
`t
`t
`t
`t
`ACA GGA TCT AGT TCA GGT TCA AAA TTA AAA GAT CCT GAA CTG AGT TA AAA GGC ACC CAG
`GT CCT AGA CA AGT CCA G T AA.
`T. CTA GGA CT GAC CA AA
`CCG GG GTC
`Thr Gly Ser Ser Ser Gly Ser Lys Lieu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln)
`
`90
`
`lOO
`
`30
`
`40
`50
`70
`80
`t
`h
`y
`r
`v
`t
`CAC ATC ANG CAA GCA GGC CAG ACA CTG CAT CTC CAA TGC Aeg GGG GAA GCA GCC CAT AAA
`GTG TAG TAC GT CGT CCG GC TGT, GAC GTA (SAG GIT ACG TCC CCC CT CGT CGG GATT
`His le Met Gin Ala Gly Gin Thr Lieu. His Leu Glin Cys Arg Gly. Glu Ala Alia Eiis Lys?
`
`SO
`
`9.
`t
`
`t
`
`2OO
`t
`
`2O
`
`220
`t
`
`t
`
`23
`
`240
`
`TGG TCT TIG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GCC
`ACC AGA AAC GGA CTT TAC CAC TCA TTC C. TCG CT, TCC GAC TCG TAT TGA TTT AGA CGG
`Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala)
`
`250
`250
`280
`300
`r
`t
`r
`s
`'t
`TGT GGA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC TXG AAC ACA GCT CAA GCA ARC
`ACA CCT TOT TTA CCG TT GT AAG ACG TCA AGA AAT TGG AACTTG TGT CGA GTT CGF TG
`Cys Gly Arg Asri Gly Lys Glin Phe Cys Ser Thr Lieu. Thir Lieu. Asn Thr Ala Glin Ala Asn’
`
`290
`
`270
`
`30.
`320
`340
`r
`g
`t
`r
`t
`r
`CAC ACT GGC TXC TAC AGC GC AAA FA CA, GCT GFA CC ACT TOA. AAG AAG AAG GAA ACA
`GTG Tea CCG AAG ATG TCG ACG TTT ATA GAT CGA CAT GGA TGA AG TC TTC TTC CT TGT
`His Thr Ghy Phe Tyr Ser Cys Lys Tyr Leu Alia Val Pro Thr Ser Lys Lys Lys Glu Thro
`
`350
`
`360
`
`330
`
`380
`
`390
`
`370
`OO
`t
`r
`'t
`s
`GAA TCT, GCA ATC AT ATA TTT ATT AGT, GAT ACA GGT AGA CCT TTC GTA GAG ATG TAC AGT
`CTTAGA CGT TAG ATA TAT AAA TAA TCA CTA TGT ccATCT GGA AAG CAT Crc TAC ATG TCA
`Glu Ser Ala Ile Tyr Ile Phe Ile Ser Asp Thr Giy Arg Pro Phe Val Giu Met Tyr Ser>
`
`410
`
`420
`
`440
`
`430
`450
`460
`470
`y
`ge
`r
`r
`t
`r
`t
`t
`GAA ATC CCC GAA ATT. ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TGC CGGGTT
`CT TAG GGG CIT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA
`Glu le Pro Gu Yie tie His Met Thr Gli gly Arg Giu. Leu Val tie Pro Cys Arg Wax
`
`4BO
`
`490
`SOD
`50
`r
`t
`y
`t
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TTT COA CT GAC ACT TTG ATC CCT GAT
`TGC AGT GGA TTG TAG TGA CAA TGA AAT TT TEC AAA GGT GAA CTG TGA AAC TAG GA CTA
`Thr Ser Pro Asn Ile Thr Wall Thr Leu Lys Lys Phe Pro Leu Asp Thr Lieu. Ile Pro Aspx
`
`S2O
`
`530
`
`510
`
`Pg. 016
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 16 of 55
`
`US 7,374,758 B2
`
`Fig.13B.
`
`S50
`
`560
`y
`
`t
`
`570
`w
`
`580
`t
`
`590
`y
`
`500
`
`y
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TO ATC ATA TCA AAT GCA ACG TAC AAA
`CCT TTT GCG TAT TAG ACC CTG TCA FCI
`EC CCG AS TAG AT AGT TRA CGT TGC ATG TTT
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr LyS)
`
`62O
`
`SO
`530
`640
`650
`r
`*
`t
`t
`t
`w
`GAA AA GGG CTT. CTG ACC TGST GAA GCA ACA GTC AAT GGG CAT TTG TAT. AAG ACA AACTAT
`CTT TAT CCC GAA GAC TGG ACA CT CGT GT CAG TTA. CCC GTA AACATA TTC TGT TTG ATA
`Glu Ilie Gly Leu Leu. Thir Cys G. Ala Thr Val Asn Gy. His Leu Tyr Lys Thr ASn Tyrs
`
`560
`
`68)
`
`57D
`690
`700
`710
`720
`r
`w
`e
`t
`t
`t
`t
`t
`CTC ACA CAT CGA CAA ACC AAT ACA AFC ATA (SAT GTC CAA ATA AGC ACA CCA CGC CCA GTC
`GAG TGT, GTA GCT GTT GG TA TG TAG TAT CTA CAG GTT TAT TCG TGT GGT GCG GGT CAG
`Leu Thr is Arg Glin Ehr Asn. Thr Ile Ile Asp Val Glin Ile Ser Thr Pro Arg Pro Wal)
`
`730
`
`740
`77
`80.
`e
`w
`r
`f
`t
`t
`t
`AAA TTA CTTAGA GGC CAT ACT CT, GTC CTC AAT TOST ACT GCT ACC ACT CCC TTGAAC ACG
`TTT AAT GAA TCT CCG GTA TGA GAA CAG GAG TA ACA TGA CGA TGG NGA GSG AACTTG TGC
`Lys Leu. Leu Arg Gly. His Thr Leu Vai Lieu. Asn. Cys Thr Ala Thr Thr Pro Leil ASn Third
`
`750
`
`760
`
`790
`r
`
`BDO
`t
`
`3)
`t
`
`82D
`
`8:30
`
`840
`Wr
`
`r
`
`AGA GTT CAA ATG ACC TGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC
`TCT CAA GTT TAC TGG ACC TCA AEG GGA CIA CT TAA CTG GT TCG TTA AGG GTA CGGTTG
`Arg Val Glin Met Thr Trp Ser Tyr Pro Asp Gu Ile Asp Glin Ser Asn. Ser His Alia ASn>
`
`BSO
`B50
`B9C
`t
`“A
`y
`r
`r
`e
`g
`vr
`As A NC TAC AGT, GT CIT ACT AIT GAC AAA ANG CAG AAC AAA GAC AAA GGA CT TAT ACT
`FAT AAG ATG TCA CAA GAA TGA TAA CTG TTT TAC GTC TTG TTT CTG TTT CCT GAA ATA TGA
`Ile the Tyr Ser Val Leu Thr Ile Asp Lys Met Glin Asn Lys Asp Lys Gly Leu Tyr Thr?
`
`B70
`
`BBO
`
`900
`
`90
`
`920
`r
`
`930
`
`s
`
`r
`
`94.
`
`t
`
`950
`
`950
`
`y
`
`TGT CGT GA AGG AGT GGA CCA TCA TTC AAA TCT, GTT AAC ACC TCA GTG CAT ATA TAT GAT
`ACA GCA CAT TCC TCA CCT GGT AGT AAS TTT AGA CAA TTG TGG AGT CAC GTATAT, ATA CTA
`CY's Arg Val Arg Ser Gly Rro Ser Phe Lys Ser Val Asn Thr Ser Val His le Yr Asp>
`
`970
`
`990
`
`98O
`OOO
`OO
`ve
`t
`r
`t
`AAA GCA GGC CCG GGC GAG CCC AAA TCT GT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA
`TTT CGT COG GGC CCG CTC GGG TTT AGA ACA CTG TLT TGA GTG GT ACG GGT GGC ACG GGT
`Lys Ala Giy Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro)
`
`020
`
`O3O
`1040
`50
`O60
`108
`r
`r
`t
`t
`r
`r
`t
`GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TNC CTC TYC CCC CCA AAA CCC AAG GAC ACC
`CGT (GGA CT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG (GGT TTT GGG TTC CTG TGG
`Ala Bro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr)
`
`O70
`
`Pg. 017
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 17 of 55
`
`US 7,374,758 B2
`
`Fig.13C.
`
`O90
`00
`ve
`wk
`r
`f
`w
`CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GIG GAC GTG AGC CAC GAA GAC
`GAG TAC TAG AGG GCC TGG GGA CTC CAG TOST ACG CAC CAC CAC CTG CAC TCG GTG CTT CTG
`Leu Met Ile Ser Arg Thr Pro Glu
`Val Thr Cys Val Val Val Asp Val Ser His Glu Asp>
`
`20
`
`ill.30
`
`ill40
`
`ill.0
`
`50
`
`200
`190
`60
`t
`t
`t
`y
`y
`w
`w
`t
`GNG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG
`CCT GAG GTC AAG TTC AAC TGG TAC
`CAC CVG CCG CAC CTC CAC GTA TTA CGG TTC TOST TTC
`GGA CTC CAG TTC AAG TTG ACC ATG
`Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys2
`Pro Glu Val Lys Phe Asn Trip Tyr
`
`170
`
`18O
`
`20
`
`220
`y
`r
`w
`t
`CTC ACC GTC CTG CAC
`CCG CGG GAG GAG CAG TAC AAC AGC
`ACG TAC CGT Gle GIC AGC
`GAG TAGG CAe GAC GTG
`NGC AIG GCA CAC CAG CG
`GGC GCC CTC CTC GTC ANG TTG TCG
`Leu Thr Val Leu. His>
`Thr Tyr Arg Val Val Ser
`Val
`Pro Arg Glu Glu Glin Tyr Asn Ser
`
`12:30
`y
`
`l240
`yr
`
`250
`
`1260
`
`270
`
`290
`
`30
`300
`28O
`t
`t
`r
`d
`t
`W
`AAA GCC CTC CCA GCC
`AAC
`TAC AAG GC AAG GC TCC
`CAG GAC TGG CTG AAT GGC AAG
`T CGG GAG (GGT CGG
`ATG TTC ACG TTC CAG AGG
`ITG
`GTC CTG ACC GAC TTA CCG TTC
`Tyr Lys Cys Llys Val Ser
`Lys Ala Leu Pro Ala)
`Gln Asp Trp Leu ASn Gly Lys Glu
`ASn
`
`320
`
`y
`
`1330
`
`1340
`
`CCC ATC GAG AAA ACC ANC CC
`GGG TAG CTC TTT GG TAG AGG
`Pro Ile Glu Lys Thr le Ser
`
`390
`
`400
`Wr
`CTG CCC CCA TCC CGG GA. GAG
`GAC GGG GGT AGG GCC. CTA CTC
`Leu Pro Pro Ser Arg Asp Giu Le
`
`360
`
`350
`t
`t
`r
`CCA CAG GTG TAC ACC
`GCC AAA GGG CAG COC CGA GAA
`GGT GTC CAC ATG TGG
`CGG TTT CCC GTC GGG GCT. CTT
`Pro Glin Val Tyr Thr)
`Ala Lys Gly Gin Pro Arg Glu
`
`370
`
`380
`
`g
`
`140
`y
`
`420
`y
`t
`ACC AAG AAC CAG GTC AGC
`TGG TTC TTG GTC CAG TCG
`Thr Lys Asn Glin Val Ser
`
`1440
`
`430
`y
`t
`ACC TGC CTG GTC AAA
`TGG ACG GAC CAG T
`Thr Cys Leu Val Lys2
`
`1450
`
`1460
`
`1470
`480
`s
`s
`s
`t
`CAG CCG GAG AAC AAC
`GG GAG GG GAG AGC AA
`GGC TTC AT CCC AGC GAC AC
`GTC GGC CTC TTG TTG
`CAC CTC ACC CTC TOG
`A
`CCG AAG AA GGG TCG CTG AG
`Glin Pro Glu ASn Asn)
`Val Glu Trip Glu Ser Asn Gly
`Gly Phe Tyr Pro Ser Asp Ile Ala
`
`490
`
`SOO
`y
`
`1510
`
`y
`
`TAC AAG ACC ACG CCT CCC GTG
`ATG TC SG GC GGA. GGG CAC
`Tyr Lys Thr Thr Pro Pro Val Leu
`
`530
`
`560
`1550
`540
`t
`t
`y
`r
`t
`r
`t
`CTC TAC AGC AAG CTC
`GAC TCC GAC GGC. TCC TC
`TTC
`GAG ATG CG TTC GAG
`CTNG AGG CTG CCG AGG AAG
`AAG
`Asp Ser Asp Gly Ser Phe
`Leu Tyr Ser Lys Lieu
`Phe
`
`570
`
`t
`
`1580
`gr
`
`t
`r
`w
`r
`CAT GAG
`TCC GTG ATG
`TGC
`CAG GGG AAC GTC TC CA
`ACC GTG GAC AAG AGC AGG TGG
`CAG
`GTA CTC
`AGG CAC TAC
`ACG
`GTC CCC g CAG AAG AG
`GTC
`TGG CAC CTG TTC TCG TCC ACC
`His Glud
`Thr Val Asp Llys Ser Arg Trip
`Gin Glin Gly Asn Val Phe Ser Cys Ser Val Met
`
`1590
`
`1600
`
`1610
`
`1620
`
`y
`
`Pg. 018
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 18 of 55
`
`US 7,374,758 B2
`
`Fig.13D.
`
`l640
`
`1650
`
`630
`1660
`w
`T
`r
`GCT. CTG CAC AAC CAC AC ACG CAG AAG AGC CTC. TCC CTG TCT CCG GGT AAA TGA
`CGA GAC GTG TTG GTG ATG TGC GTC TTC TCG GAG AGG GAC AGA GGC CCA TTT ACT
`Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys "''>
`
`570
`
`Pg. 019
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 19 of 55
`
`US 7,374,758 B2
`
`Fig.14A.
`
`y
`'
`y
`t
`g
`t
`st
`w
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTC
`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 Lleu. Cys Ala Leu Leu Ser Cys reli Lei Leu)
`
`BC)
`
`SO
`
`70
`lOO
`120
`t
`te
`g
`y
`r
`t
`ACA GGA TCT AGT TCC GGA GGST AGA CCT TTC GTA GAG ATG TAC AG GAA ATC CCC GAA ATT
`TGT CCT AGA TCA AGG CCT CCA TCT GGA AAG CAT CTC TAC ATG INCA CTT TAG GGG CTT TAA
`Thr Gly Ser Ser Ser Gly Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Iled
`
`O
`
`30
`
`140
`SO
`r
`r
`t
`t
`t
`ATA CAC ATVG ACT GAA GGA AGG GAG CTC GTC ATCCC GC CGG GT ACG TCA CCT AAC ATC
`TAT GTG TAC TGA CTT cr Tcc cTc GAG CAG TAA GGG ACG Gcc cAA TGc AGT GGATTG TAG
`Ile His Met Thr Glu Gly Arg Glu Leu Val Ilie Pro Cys Arg Val Thr Ser Pro ASn Ile>
`
`160
`
`70
`
`80
`
`190
`y
`
`200
`t
`
`2O
`t
`
`9.
`
`22O
`t
`
`y
`
`230
`
`240
`t
`
`ACT GTT ACT TTA AAA AAG TTT COA CTTGAC ACT TIG ATC ccT GAT GGA AAA CGC ATA ATC
`TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT, TTT GCG TAT TAG
`Thr Vai Thr Leu Lys Llys Phe Pro Leu Asp Ihr Lieu. Ile Pro Asp Gly Lys Arg Ile Iles
`
`260
`250
`270
`280
`g
`w
`t
`"A
`s
`s
`t
`t
`TGG GAC AGT AGA AAG GGC TTC ATC ATA CA AAT GCA ACG TAC AAA GAA ATA GGG CTT CTVg
`ACC CTG TCA TCT, TTC CCG AAG TAG TAT AG TA CGT TGC ATG TT, CT AT CCC GAA GAC
`Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu le Gly Leu Leu)
`
`290
`
`300
`
`30
`
`r
`
`320
`r
`
`330
`
`340
`r
`
`350
`1A
`
`360
`
`y
`
`ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CAT CGA CAA
`TGG ACA CTT COT TGT CAG TA CCC GTA AAC ATA Trc TGT TG ATA GAG TGT GTA GOT GIT
`Thr Cys Glu Ala Thr Val Asin Gly. His Leu. Tyr Lys Thr Asn Tyr Lieu. Thr His Arg Gins
`
`370
`
`38O
`
`390
`
`420
`400
`t
`r
`t
`e
`ACC AAT ACA AIC AIA GA GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTA. CTTAGA GGC
`TGG TTA TGT TAG TA CTA CAG GTT TAT TCG TGT GGT GCG GGT CAG TTT AAT GAA TCT CCG
`Thr Asn Thr Ile Ile Asp Val Glin Ile Ser Thr Pro Arg Pro Val Lys Leu Leu Arg Gly>
`
`40
`
`440
`
`430
`450
`460
`480
`w
`t
`e
`w
`t
`t
`t
`t
`CAT ACT CTT GTC CTC AAT GT ACT GCT ACC ACT CCC TTGAAC ACG. AGA GTT CAA AG ACC
`GA TGA GAA CAG GAG TTA ACA TGA CGA TGG GA. GGG AAC TTG TGC CT CAA GT TAC TGG
`His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Glin Met Thrd
`
`470
`
`490
`SOO
`50
`S2O
`530
`t
`r
`t
`t
`t
`t
`r
`st
`t
`TGG AGT TAC CCT GAT GAA ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT
`ACC TCA ATG GGA CTA CTT TAA CTG GTR TCG TTA AGG GTA CGG TTG TAT AAG ATG TCA CAA
`Trip Ser Tyr Pro Asp Glu Ile Asp Glin Ser Asn Ser His Ala Asn ille Phe Tyr Ser Val
`
`540
`
`Pg. 020
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 20 of 55
`
`US 7,374,758 B2
`
`Fig.14B.
`
`y
`
`t
`
`y
`
`'r
`
`t
`
`570
`
`SBO
`
`S90
`t
`
`r
`
`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 TTT TAC GTC TTG TTT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA
`Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr cys Arg Val Arg Ser>
`610
`
`640
`r
`
`r
`
`650
`
`660
`
`y
`
`620
`
`630
`
`w
`
`y
`
`GGA CCA TCA TTC AAA TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT. AAA GCA GGC CCG GGC
`CCT GGT AGT AAG TTT AGA CAA TTG TGG AGF CAC GTA TATATA CTA TTT 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
`
`580
`
`690
`
`'t
`
`y
`
`t
`
`700
`y
`
`70
`
`720
`
`ACT CAC ACA TGc CCA CCG TGc CCA
`GAG
`CCC AAA TCT TGT GAC AAA
`GCA CCT GAA CTC CTG
`CC
`GGG TTT AGA ACA CG TFT
`CGT GGA CTT GAG GAC
`TGA GTG TGT ACG GG, GGC ACG GG
`Pro Lys Ser Cys Asp Lys
`Thr His Thr Cys Pro Pro Cys Pro
`Glu
`Ala Pro Glu Lieu. Leu)
`
`730
`
`740
`
`750
`
`760
`
`770
`
`780
`
`GGA CCG TCA GTC TC CTC
`CCT GGC AGT CAG AAG GAG
`Gly
`Gly Pro Ser Val Phe Leu
`
`Ple
`
`t
`
`t
`
`w
`CCC CCA AAA CCC
`CTC ATG ATC. TCC CGG
`AAG GAC ACC
`GAG TAC TAG AGG GCC
`Trc CTG TGG
`GGG GGT TTT GGG
`Lys Asp Thr
`Leu Met le Ser Arg)
`Pro Pro Llys Pro
`
`790
`y
`
`w
`
`y
`
`BOO
`w
`
`CCT GAG GTC AcA TGc GTG
`GGA CTC CAG NGT ACG CAC
`Pro Glu Val Thr Cys Val
`Val
`
`80
`y
`
`820
`ye
`
`830
`g
`r
`CCT GAG GTC AAG TTC
`GAC GTG. AGC
`CAC GAA GAC
`GGA CTC CAG TTC AAG
`CIG CAC TCG
`GTG. CTT
`Pro Glu Val Lys Phe)
`Asp Val Ser
`His Gill
`
`840
`
`BSO
`
`B60.
`y
`
`TGG TAC GTG GAC GGC GTG
`ACC AG CAC CTG CCG CAC
`Trp Tyr Val Asp Gly Val
`Glu
`
`870
`
`BBO
`r
`CA. AAT GCC AAG ACA
`GTA ITA CGG TTC TXGT
`His Asn Ala Lys Thr
`
`890
`
`900
`
`'
`CCG CGG GAG GAG CAG
`GGC GCC CTC CTC GTC
`Pro Arg Glu Glu Gln2
`
`950
`
`960
`
`90
`
`92O
`
`t
`s
`AC
`AAC
`AGC ACG TAC CGT GYG
`ATG
`TIG
`TCG GC ATG GCA CAC
`Ser Thr Tyr Arg val
`Wall
`ASn
`
`CG
`Sea
`
`s
`
`970
`
`98O
`w
`w
`GAG TAC AAG GC AAG
`CTC ATG TTC ACG TTC
`Glu Tyr Lys Cys Lys
`Wall
`
`Gly
`
`Ar
`s
`TGG cTG AAT
`GTC CNG
`CAG GAC
`CC ACC
`ACC GAC TA
`CAG GAC
`GTG
`GTC CTG
`GAG GG
`Glin Asp
`Leu Thr
`Trp Leu Asn)
`His
`Wall Leu
`
`t
`
`OOO
`st
`AAA GCC
`CTC CCA
`TTT CGG
`GAG GGT
`Lys Ala
`Leu Pro
`
`OO
`
`020
`
`CCC ATC GAG AAA ACC
`GGG TAG CTC TTT TGG
`Pro Ile Glu Lys Thr)
`
`t
`
`g
`
`OTO
`w
`t
`t
`r
`y
`t
`AAA GCC AAA GGG CAG
`CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC
`TTT CGG TTT CCC GTC
`GGG GCT. CTT GGT GTC CAC ATG TGG GAC GGG
`Lys Ala Lys Gly Glin
`Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro
`Ser
`
`OSO
`
`1060
`
`1080
`
`CCA NCC CGG
`GGT AGG GCC
`Pro Ser Arg>
`
`ATC
`AG
`Ile
`
`Pg. 021
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 21 Of 55
`
`US 7,374,758 B2
`
`Fig.14C.
`
`1090
`OO
`O
`l2O
`t
`W
`y
`w
`y
`t
`t
`GAT GAG CTG ACC AAG AAC CAG GEC AGC CTG ACC GC CTG GTC AAA GGC TTC TAT CCC AGC
`CTA CTC GAC TGG TTC TTG GTC CAG TCG GAC TGG ACG GAC CAG TTT CCG AAG ATA GGG TCG
`Asp Glu Leu Thr Lys Asn Glin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser)
`
`40
`
`30
`
`SO
`
`60
`
`l70
`
`18O
`
`1.
`
`t
`
`y
`
`190
`s
`
`t
`
`200
`s
`
`w
`
`GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT
`CTG TAG CGG CAC CTC ACC CTCTCG TA CCC Grc GGC CTCTTG TTG ATG TTC TGG TGC GGA
`Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu. Asn Asn Tyr Lys Thr Thr Pros
`
`220
`
`230
`
`20
`1260
`s
`y
`y
`t
`s
`CCC GTG CTG GAC TCC GAC GGC. TCCTTC TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC
`GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TC GAG NGG CAC CTG TC TCG
`Pro Wall Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Llys Serd
`
`1240
`
`250
`
`270
`y
`
`t
`
`280
`y
`
`290
`y
`
`s
`
`300
`s
`
`y
`
`310
`
`1320
`
`r
`
`AGGTGG CAG CAG GGG AAC GTC TTC TCATGc TCC GTG ATG cAT GAG GCT. CTG CAC AAC CAC
`TCC ACC GTC GTC CCC ITG GAG AAG AGT ACG AGG CAC TAC GTA CTC CGA GAC GTG TTG GTG
`Arg Trip Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu. His Asn His>
`
`1330
`t
`
`340
`
`350
`s
`
`s
`
`TAC ACG CAG AAG AGC CTCTCC CTG TCT CCG GGT AAA TGA
`ATG TGC GTC TTC TCG GAG AGG GAC AGA. GGC CCA ITT ACT
`Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys * * * >
`
`Pg. 022
`
`

`

`U.S. Patent
`
`May 20, 2008
`
`Sheet 22 Of 55
`
`US 7,374,758 B2
`
`Fig.15A.
`
`s
`
`s
`
`20
`BO
`40
`SO
`6
`w
`t
`ATG GTC AGC Tac TGG GAc Acc GGG Grc CTG CTG TGc GCG cTG CTC AGC IGT CTG CT. 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 LeuP
`
`{
`
`t
`
`70
`8O
`90
`OO
`O
`2O
`t
`t
`*
`ACA GGA TCT AGT TOc GGA GGT AGA ccT TTC GTA GAG ATG TAC AGT GAA ATC CCC GAA Art
`TGT CCT AGA TCA AGG CCT CCA TCT GGA AAG CAF CC TAC ATG TCA CTT TAG GGG CTT TAA
`Thr Gly Ser Ser Ser Gly Gly Arg Pro Fhe Val Glu Met Tyr Ser Giu. Ile Pro Glu Iles
`
`t
`
`40
`
`SO
`
`BO
`t
`r
`t
`A. CCT AAC ATC
`ATA CAC AG AC GAA GA AGG GAG CTC GTC ATT CCC TGC CGG GT. ACG.
`TAT GTG TACGA C CCT TCC CNC GAG CAG AA GGG. ACG GCC CAA GC AG GGA TG TAG
`Ile His Met Thr Glu Gly. Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn. Ilex
`
`SO
`
`170