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`United States Patent
`(12)
`US 7,374,757 B2
`(10) Patent No.:
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`Papadopoulosetal.
`(45) Date of Patent:
`*May20, 2008
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`US007374757B2
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`(54) MODIFIED CHIMERIC POLYPEPTIDES
`WITH IMPROVED PHARMACOKINETIC
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`
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`PROPERTIES
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`(75)
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`Inventors: Nicholas J. Papadopoulos,
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`Lagrangeville, NY (US); Samucl Davis.
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`New York, NY (US); George D.
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`Yancopoulos, Yorktown Heights, NY
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`(US)
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`(*) Notice:
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`(73) Assignee: Regeneron Pharmaceuticals, Inc.,
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`Tarrytown, NY (US)
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`Subject to any disclaimer, the term ofthis
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`patent is extended or adjusted under 35
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`U.S.C. 154(b) by 489 days.
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`This patent is subject to a terminal dis-
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`claimer.
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`(21) Appl. No.: 11/016,097
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`(22)
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`Filed:
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`Dec. 17, 2004
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`(65)
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`Wo
`Wo
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`W098) 13071
`W099/03996
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`4/1998
`1/1999
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`OTHER PUBLICATIONS
`Terman, B. I., et al, “Identification of a newendothelial cell growth
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`factor receptor tyrosine kinase”, Oncogene (1991) 6:1677-1683.
`Torman, B.L., et al, “Identification of the KDR tyrosine kinase as a
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`receptor for vascular endothelial cell growth factor’. Biochem
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`Biophys Res Comm (1992) 187(3):1579-1586.
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`et al, “PEGylation of interleukin-6 effectively
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`increases
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`Haemostasis (1997) 77(1):168-173.
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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:
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`teins”, BioEssays (1998) 20:156-167.
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`deVries, Carlie, et al., “Thefms-like tyrosine kinase, a receptor for
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`vascular endothelial growth factor”, Science (1992) 225:989-991,
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`Sharifi, J., et al., “Lmproving monoclonal antibody pharmacokinet-
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`ics via chemical modification”, Quart J Nucl Med (1998) 42:242-
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`Jensen-Pippo, K.F., el al., “Enteral bioavailability of human granu-
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`locyte colony stimulating factor conjugated with poly(ethylene
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`glycol)’, (1996) Pharm Res 13(1):102-107.
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`‘Tanaka, K., et al., “Characterization of the extracellular domain in
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`vascular endothelial growth factor
`receptor-1 (Flt-1 Tyrosine
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`kinasey’, (1997) Jpn J Cancer Res 88:867-876.
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`glycol-modified
`J.C., et al., “The use of
`polyethylene
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`interleukin? (PEG-IL-2)inthetreatmentofpatientswithmetastatic
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`renal cell carcinoma and melanoma’, (1995) Cancer 76(4): 687-
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`Davis-Smyth, T., et al., 1996, “The second immunoglobulin-like
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`domain of the VEGF tyrosine kinase receptor Fit-1 determines
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`ligand binding and mayinitiate a signal transduction cascade”, The
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`EMBO Journal 15(18):4919-4927.
`
`
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`* cited by examiner
`
`
`
`
`Primary Examiner—Christine J Saoud
`
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`Assistant Examiner—Jon M Lockard
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`(74) Attorney, Agent, or Firm—Gregg Valeta, Esq.
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`ABSTRACT
`(7)
`Modified chimeric polypeptides with improved pharmaco-
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`kinetics are disclosed. Specifically, modified chimeric Fit!
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`receptor polypeptides that have been modified in such a way
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`as to improve their pharmacokinetic profile are disclosed.
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`‘Also disclosed are methods of making and using the modi-
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`fied polypeptides including but not limited to using the
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`modified polypeptides to decrease or inhibit plasma leakage
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`and/or vascular permeability in a mammal.
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`Prior Publication Data
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`US 2005/0163798 Al
`Jul. 28, 2005
`
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`
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`Related U.S. Application Data
`-
`as
`sons
`:
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`(62) Division ofapptiraonNo. 10/009,852, see as appli-
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`
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`cation No. PCT! - $00/14142 on May 23, 2000, now
`
`
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`Pat. No. 7,070,959.
`
`
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`
`(60) Provisional application No. 60/138,133. filed on Jun.
`
`
`8, 1999,
`:
`
`
`Int. Cl.
`
`
`(2006.01)
`A6IK 38/18
`
`
`
`(2006.01)
`CO7K 14/71
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`
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`CI2QN 15/62
`(2006.01)
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`
`(52) US. Ch ieeeccccccceecestteeetecsee
`424/134.1; 424/192.1;
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`514/2; 514/12; 530/350; 536/23.4
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`(58) Field of Classification Search .........0.0.000... None
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`See application file for complete search history.
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`References Cited
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`U.S. PATENT DOCUMENTS
`/
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`006 Cenaeta etal
`ett08 *
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`..... 435/69.7
`8/2000 Davis-Smyth et al.
`6.100.071 A *
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`2/2005 Daly etal. ........
`-§14/12
`200510043236 AL*
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`3/2006 Daly et al. vescecceseeen 514/12
`2006/0058234 AL*
`FOREIGN PATENT DOCUMENTS
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`(51)
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`(56)
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`wo
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`W097/44453
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`11/1997
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`7 Claims, 55 Drawing Sheets
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`CELLTRION- EXHIBIT 1022
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`CELLTRION - EXHIBIT 1022
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`U.S. Patent
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`May20, 2008
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`US 7,374,757 B2
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`Sheet 1 of 55
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`Fig.1.
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`4
`5
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`100
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`1
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`tug/mi
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`—(}— rTIE-2-Fe
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`ron fen acetylated Fit-1(1-3)-Fe
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`----O---~ unmodified Fit-1(1-3)-Fe
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`U.S. Patent
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`May20, 2008
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`Sheet 2 of 55
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`US 7,374,757 B2
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`SALT-WASH
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`100
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`1234
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`+
`VEGF
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`Fit-1(1-3)-Fe-
`rTIE-2-Fe
`-
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`Heparin
`-
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`567 8
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`9 101112
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`13141516
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`17181920
`21 22 23 24
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`+
`+
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`+ pegylated
`+
`-
`-
`-
`-
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`U.S. Patent
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`May20, 2008
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`Sheet 3 of 55
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`US 7,374,757 B2
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`U.S. Patent
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`May20, 2008
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`Sheet4 of 55
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`U.S. Patent
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`May20, 2008
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`Sheet 5 of 55
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`US 7,374,757 B2
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`9.3
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`123 45 6789 10
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`U.S. Patent
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`May20, 2008
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`Sheet6 of 55
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`US 7,374,757 B2
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`Fig.7
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`—_{}— ITIE-2-Fe
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`wone-e/\-~---- acetylated Fit-1(1-3)-Fe (20X)
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`---49--- acetylated Fit-1(1-3)-Fe (30X)
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`U.S. Patent
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`May20, 2008
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`Sheet7 of 55
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`US 7,374,757 B2
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`580
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`U.S. Patent
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`May20, 2008
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`Sheet 8 of 55
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`US 7,374,757 B2
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`U.S. Patent
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`May20, 2008
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`Sheet 9 of 55
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`US 7,374,757 B2
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`Fig.10A.
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`*
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`10
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`&
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`20
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`*
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`ATG GTC AGC TAC TGG GAC ACC GGG GIC CTIG CIG TCC GCG CIG Cit AGC TCT CTG CiT Cre
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`TAC CAG TCG ATG ACC CTIG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAG
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`Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu>
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`30
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`ACA GGA TCT AGT TCA GGT TCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG
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`TGT CCT AGA TCA AGT CCA AGT TIT AAT TTT CTA GGA CTT GAC TCA AAT TIT CCG TCG GIC
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`CAC ATC ATG CAA GCA GGC CAG ACA CTG CAT CIC CAA TGC AGG GGG GAA GCA GCC CAT AAA
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`His Ile Met Gln Ala Gly Gln Thr Leu His Leu Gin Cys Arg Gly Glu Ala Ala His Lys>
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`%GG TCT TIG CCT GAA ATG GIG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT AAA TCT GCC
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`ACC AGA AAC GGA CTT TAC CAC TCA TIC CIT TCG CTT TCC GAC TCG TAT TGA TIT AGA CGG
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`Trp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala>
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`*
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`*
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`«
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`260
`*
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`270
`*
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`
`250
`280
`
`*
`*
`*
`*
`
`
`
`
`TGT GGA AGA AAT GGC AAA CAA TIC TGC AGT ACT TTA ACC TIG AAC ACA GCT CAA GCA AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA CCT TCT TTA CCG TTT GTT AAG ACG TCA TGA AAT TGG AAC TIG TGT CGA GTT CGT TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gin Ala Asn>
`
`*
`
`290
`*
`
`
`
`*
`
`300
`*
`
`
`
`«x
`
`
`
`
`*
`
`320
`*
`
`
`
`330
`*
`
`
`
`x
`
`
`*
`
`
`
`340
`*
`
`
`
`*
`
`350
`*
`
`
`
`310
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAC ACT GGC TIC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAG AAG AAG GAA ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GTG TGA CCG AAG ATG TCG ACG TIT ATA GAT CGA CAT GGA TGA AGT TIC TIC TIC CIT IGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala val Pro Thr Ser Lys Lys Lys Glu Thr>
`
`360
`
`
`
`370
`*
`
`*
`
`380
`*
`
`390
`
`*
`
`*
`
`400
`*
`
`
`
`
`
`*
`*
`*
`
`
`
`
`
`
`
`GAA TCT GCA ATC TAT ATA TTT ATI AGT GAT ACA GGT AGA CCT TTC GTA GAG ATG TAC AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT AGA CGT TAG ATA TAT AAA TAA TCA CTA TGT CCA TCT GGA AAG CAT CIC TAC AIG TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ser Ala Tle Tyr Ile Phe Tle Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`410
`*
`
`
`
`420
`
`
`
`*
`
`430
`*
`
`
`
`*
`
`440
`*
`
`
`
`450
`*
`
`
`
`*
`
`460
`*
`
`
`
`*
`
`470
`*
`
`
`
`*
`*
`
`
`
`
`GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TH CGG GTT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT TAG GGG CTT TAA TAT GTG TAC TGA CFT CCT TCC CIC 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 Val>
`
`480
`*
`
`
`
`490
`*
`
`
`
`t
`
`500
`*
`
`
`
`*
`
`520
`510
`
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT GAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGC AGT GGA TIG TAG TGA CAA TGA AAT TIT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Tle Pro Asp>
`
`
`
`530
`*
`
`
`
`*
`
`540
`*
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`
`Sheet 10 of 55
`
`
`
`
`US 7,374,757 B2
`
`
`May20, 2008
`
`
`Fig.10B.
`
`550
`*
`
`*
`
`
`
`*
`
`
`560
`*
`
`
`*
`
`
`570
`
`*
`
`
`*
`
`
`580
`oe
`
`
`
`
`*
`
`
`
`
`590
`k&
`
`*
`
`600
`%
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TIC ATC ATA TCA AAT GCA ACG TAC AAA
`
`
`
`ccT TTT GCG TAT TAG ACC CTIG TCA TCT TIC CCG AAG TAG TAT AGT TTA CGT TOC ATG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile ser Asn Ala Thr Tyr Lys>
`
`*
`
`
`
`610
`*
`
`*
`
`
`
`620
`*
`
`*
`
`
`630
`
`*
`
`
`*
`
`
`
`640
`*
`
`
`
`650
`*
`
`*
`
`
`
`*
`
`
`
`
`
`660
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAR ATA GGG CTT CTIG ACC TGT GAA GCA ACA GTC AAT GGG CAT TIG TAT AAG ACA AAC TAT
`CTT TAT CCC GAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TIC TOT TTG ATA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ile Gly Leu Leu- Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr>
`
`
`
`
`
`
`
`720
`680
`670
`690
`710
`700
`
`
`
`
`
`*
`*
`*
`*
`*
`*
`6
`*
`*
`¥*
`*
`*
`
`
`
`
`
`
`CTC 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 TCT GOT GGG GGT CAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val>
`
`
`
`
`
`*
`
`730
`*
`
`
`
`*
`
`750
`740
`760
`
`*
`*
`«*
`
`
`
`
`
`
`
`
`
`
`BAA TTA CTT AGA GGC CAT ACT CTT GTC CIC AAT TGT 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 TTG TGC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`*
`
`
`
`*
`
`
`
`x
`
`
`
`770
`*
`
`*
`
`
`
`780
`*
`
`
`
`*
`
`
`
`*
`
`
`
`*
`
`
`
`
`
`790
`*
`
`*
`
`800
`
`*
`
`810
`*
`
`820
`*
`
`
`830
`*
`
`
`840
`*
`
`*
`
`
`*
`
`
`
`AGA GIT CAA ATG ACC TGG.AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA
`
`
`
`
`
`
`
`
`
`
`
`
`TCT CAA GTT TAC TGG ACC TCA ATG GGA CTA CTT TIT TIA TIC TCT CGA AGG CAT TCC GCT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg>
`
`«
`
`850
`*
`
`
`
`*
`
`860
`*
`
`*
`
`
`
`*
`
`
`880
`*
`
`*
`
`
`
`
`50
`*
`
`*
`
`$00
`870
`
`«*
`*
`
`
`
`
`
`CGA ATT GAC CAA AGC AAT TCC CAT GCC AAC ATA TIC TAC AGT GTT CIT ACT ATT GAC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GCT TAA CTG GIT TCG TTA AGG GTA CGG TTG TAT AAG ATS TCA CAA GAA TGA TAA CTIG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Arg Ile Asp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys>
`
`
`
`
`
`*
`
`910
`*
`
`
`
`*
`
`920
`
`*
`
`*
`
`930
`*
`
`
`
`
`*
`
`
`
`ATG CAG AAC AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TIC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TAC GIC TIG TIT CTIG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys>
`
`940
`*
`
`*«
`
`
`
`950
`*
`
`*
`
`
`
`960
`*
`
`*
`
`970
`*«
`
`
`
`*
`
`980
`*
`
`
`
`*
`
`990
`*
`
`
`
`*
`
`1000
`*
`
`
`
`
`1020
`*
`*
`
`
`
`
`
`
`
`
`
`TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT AAA GCA GGC CCG GGC GAG CCC AAA TCT TCT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AGA CAA TIG IGG AGT CAC GTA TAT ATA CTA TIT CGT CCG GGC CCG CTC GGG TTT AGA ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Gly Pro Gly Glu Pro Lys Ser Cys>
`
`1010
`*
`
`
`
`*
`
`*
`
`1030
`*
`
`*
`
`1040
`*
`
`
`
`1050
`*
`
`
`
`*
`
`
`1060
`*
`*
`*
`*
`
`
`
`
`
`GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTIG GGG GGA CCG TCA GTC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTG TIT IGA GTG TGT ACG GGT GGC ACG GGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ASp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val>
`
`1070
`*
`
`
`
`1080
`*
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`Fig.10C.
`
`
`
`Sheet 11 of 55
`
`
`
`
`
`US 7,374,757 B2
`
`*«
`
`«
`
`1100
`*
`
`
`
`1110
`*
`
`
`
`*
`
`
`®
`
`1120
`*
`
`
`
`*
`
`1130
`*
`
`
`
`1140
`*
`
`
`
`*
`
`
`1090
`
`*
`
`TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAG GAG AAG GGG GGT TIT GGG TIC CTG TGG GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr>
`
`*
`
`1150
`*
`
`
`
`*
`
`1160
`
`
`
`1170
`*
`
`
`
`*
`
`
`
`+
`
`
`1180
`*
`
`
`
`
`*
`
`
`
`1190
`*
`
`
`
`1200
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGC GIG GTG GTG GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TIC AAC TGG TAC GTG GAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACG CAC CAC CAC CTIG CAC TCG GIG CTT CIG GGA CTC CAG TIC AAG TTG ACC ATG CAC CIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Val Val Val Asp Val Ser His Glu Asp Pro-Glu Val Lys Phe Asn Trp Tyr Val Asp>
`
`*
`
`
`
`*
`
`1220
`*
`
`
`
`
`
`1210
`*
`
`
`
`
`
`
`
`GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAS GAG CAG TAC AAC AGC ACG TAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`CCG CAC CTC CAC GTA TTA CGG TIC TGT TIC GOC GCC CIM CK GTC ATG TTG TC TN ATG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Val Glu val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr>
`
`1230
`*
`
`
`
`*
`
`
`
`*
`
`
`
`1240
`*
`
`
`*
`
`1250
`*
`
`1260
`*
`
`*
`
`*
`
`
`
`1270
`«
`
`
`*
`
`1280
`
`*
`*
`
`
`
`
`
`
`
`CGT GTG GTC AGC GTC CTC ACC GTC CTIG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GCA CAC CAG TCG CAG GAG TGG CAG GAC GIG GIC CTG ACC GAC TTA CCG TIC CTC AIG TIc
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Arg Val Val Ser Val Leu Thr Val Leu His Glin Asp Trp Leu Asn Gly Lys Glu Tyr Lys>
`
`1290
`*
`
`
`
`1300
`*
`
`
`
`*
`
`
`
`1310
`*
`
`x
`
`
`
`1320
`*
`
`*
`
`
`
`
`
`*
`
`
`
`*
`
`1340
`*
`
`
`
`1350
`*
`
`
`
`*
`
`1360
`*
`
`
`
`*
`
`1330
`1370
`*
`*
`*
`
`
`
`TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC ABA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACG TTC CAG AGG TIG TIT CGG GAG GGT CGG GGG TAG CIC TIT TGG TAG AGG TIT CGG TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Jle Glu Lys Thr Ile Ser Lys Ala Lys>
`
`1380
`*
`
`t
`
`*
`
`1390
`*
`
`
`
`*
`
`
`1400
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGG CAG CCC CGA GAA CCA CAG GIG TAC ACC CTIG CCC CCA TCC CG GAT GAG CTG ACC AAG
`
`
`
`
`
`
`ccc GTC GGG GCT CTT GGT GTC CAC ATG TGG GAC GGG GGT AGG GCC CTA CTC GAC 166 TIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys>
`
`1410
`*
`
`
`
`*
`
`*
`
`1420
`*
`
`
`
`
`*
`
`
`
`1430
`*
`
`1440
`*
`
`*
`
`
`
`
`
`1450
`*
`
`
`*
`
`
`x
`
`1460
`*
`
`
`
`
`
`1470
`x
`
`
`*
`
`
`
`*
`
`1480
`*
`
`
`
`
`*
`
`
`
`1490
`*
`
`
`
`1500
`«
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA G6C TIC TAT CCC AGC GAC ATC GCC GIG GAG
`
`
`
`
`
`
`TTG GTC CAG TCG GAC TGG ACG GAC CAG TTT CCG AAG ATA GGG TCG CTG TAG CGS CAC CIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala val Glu>
`
`t
`
`
`
`
`1510
`*
`
`
`*
`
`1520
`*«
`
`
`
`1530
`*
`
`
`
`«
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`IGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CIG GAC TCC
`ACC CIC TOG TTA CCC GTC GGC CIC TYG TTG ATG TIC TGS TGC GGA GGG CAC GAC CTG AGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser>
`
`*
`
`
`
`1540
`*
`
`
`
`
`*«
`
`
`
`1550
`*
`
`1560
`*
`
`*
`
`
`
`*
`
`
`1570
`1580
`1590
`
`
`
`*
`*
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`GAC GGC TCC TIC TIC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTG CCG AGG AAG AAG GAG ATG TCG TIC GAG TGG CAC CTG TIC TCG TCC ACC GT GTC ccc
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly:
`
`1600
`*
`
`*
`
`1610
`*
`
`
`
`1620
`*
`
`
`
`*
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 12 of 55
`
`
`
`US 7,374,757 B2
`
`Fig.10D.
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAC GTC TTC 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 TOC GT TH NG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser>
`
`*
`
`1660
`*
`
`
`
`r
`
`1670
`*
`
`
`
`1680
`*
`
`
`
`*
`
`1630
`*
`
`
`
`x
`
`1640
`*
`
`
`
`1650
`*
`
`
`
`*
`
`1700
`*
`
`
`
`*
`
`1690
`*
`
`
`
`*
`
`cre Tc CiG TCT CCG GGT AAA TGA
`
`
`
`
`
`
`
`
`GAG AGG GAC AGA GGC CCA TTT ACT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Ser Leu Ser Pro Gly Lys ***>
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 13 of 55
`
`
`
`US 7,374,757 B2
`
`BEEL
`
`urewop
`
`
`
`SuIeLUOp
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`ASeUlyJE[N||e0eJ]U]
`
`SUBIGUBWSUeIE
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`NZN9NGNevNENe
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`
`
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`
`
`
`
`
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`
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`
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`
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`
`
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`
`
`Sheet 14 of 55
`
`
`
`US 7,374,757 B2
`
`
`
`
`
`Fig.12A. e2eoegoceeceooo
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`qgooeeeoe see
`
`
`
`
`TOA rK Ora TiO
`
`AyortydospAyH
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`
`SP SEooeooeoes
`
`
`
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`
`AyoiydoupAH
`
`

`

`
`U.S. Patent
`
`
`
`
`May20, 2008
`
`
`Fig.13A.
`
`&
`
`
`
`*
`
`
`
`x
`
`
`*«
`
`
`
`*
`
`
`
`Sheet 15 of 55
`
`
`
`
`US 7,374,757 B2
`
`
`
`*
`
`*
`
`
`
`*
`
`
`
`
`*
`
`
`
`*
`
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CIG CTC AGC TCT Clic CiT cre
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`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
`*
`
`*
`
`
`
`*
`
`
`100
`90
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA GGA TCT AGT TCA GGT TCA AAA TTA AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGT CCT AGA TCA AGT CCA AGT TTT AAT TTT CTA GGA CTT GAC TCA AAT TIT CCG IGG GTC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln>
`
`
`
`
`
`110
`*
`
`*
`
`
`
`120
`*
`
`
`
`*
`
`
`
`*‘
`
`140
`*
`
`
`
`
`
`
`150
`*
`
`*
`
`
`
`160
`*
`
`*
`
`
`
`*
`
`
`170
`*
`
`
`
`180
`6
`
`x
`
`*
`
`230
`*
`
`240
`*
`
`
`220
`190
`*
`*
`+
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`1GG TCT TTG CCT GAA ATG GIG AGT AAG GAA AGC GAA AGG CTIG AGC ATA ACT AAA TCT GCC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACC AGA AAC GGA CTT TAC CAC TCA TIC CTT TCG CTT TCC GAC TCG TAT TGA TIT AGA CGG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tsp Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Tle Thr Lys Ser Ala>
`
`130
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAC ATC ATG CAA GCA GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GIG TAG TAC GTT CGT CCG GTC TGT GAC GTA GAG GTT ACG TCC CCC CIT CGT CGG GTA TIT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Ile Met Gln Ala Gly Gln Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys>
`
`
`
`
`ad
`
`
`200
`*
`
`
`
`210
`*
`
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`*
`
`
`260
`*
`
`
`250
`«
`*
`«
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGT GGA AGA AAT GGC AAA CAA TIC TGC AGT ACT TTA ACC TTG AAC ACA GCT CAA GCA AAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACA CCT TCT TTA CCG TTT GTT AAG ACG TCA TGA AAT TGG AAC TIG TGT CGA GTT CGT TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr Ala Gln Ala Asn>
`
`*
`
`270
`*
`
`280
`*
`
`
`
`
`*
`
`
`*
`
`290
`*
`
`300
`*
`
`
`
`*
`
`
`
`
`
`310
`*
`
`*
`
`320
`*
`
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAC ACT GGC TIC TAC AGC TGC AAA TAT CTA GCT GTA CCT ACT TCA AAG AAG AAG GAA ACA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GTG TGA CCG AAG ATG TCG ACG TTT ATA GAT CGA CAT GGA TGA AGT TIC TTC TTC CTT TGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr>
`
`
`
`330
`*
`
`*
`
`
`
`340
`*x
`
`*
`
`*
`
`
`
`350
`*
`
`
`
`360
`*
`
`
`
`*
`
`
`
`
`380
`*
`
`*
`
`390
`*
`
`*
`
`
`
`
`
`410
`*
`
`*
`
`
`
`420
`*
`
`
`400
`370
`*
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA TCT GCA ATC TAT ATA TTT ATT AGT GAT ACA GGT AGA CCT TIC GTA GAG ATG TAC AGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT AGA CGT TAG ATA TAT AAA TAA TCA CTA TGT CCA TCT GGA AAG CAT CTC TAC ATG TCA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ser Ala Ile Tyr Ile Phe. Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser>
`
`
`
`*
`
`*€
`
`
`
`440
`t
`
`*«
`
`450
`x
`
`*
`
`
`430
`460
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC 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 Val>
`
`
`
`
`
`470
`*
`
`*
`
`*
`
`480
`*
`
`
`490
`520
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TYG ATC CCT GAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`TGC AGT GGA TIG TAG TGA CAA TGA AAT TIT TIC AAA GGT GAA CTG TGA AAC TAG GGA CTA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp>
`
`*
`
`
`*
`
`
`500
`*
`
`
`
`
`510
`*«
`
`
`*
`
`
`*
`
`
`
`t
`
`
`
`530
`*
`
`
`
`540
`*
`
`*
`
`
`
`

`

`
`U.S. Patent
`
`
`
`
`
`
`May20, 2008
`
`
`Fig.13B
`
`Sheet 16 of 55
`
`
`
`
`US 7,374,757 B2
`
`*
`
`
`
`550
`*
`
`
`
`
`*
`
`
`
`
`
`560
`*
`
`
`
`570
`*
`
`
`*
`
`
`*
`
`
`
`
`580
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGA AAA CGC ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CCT TIT GOG TAT TAG ACC CIG TCA TCT TIC CGG AAG TAG TAT AGT TIA CGT TOC ATG TTT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys>
`
`
`
`
`590
`*
`
`
`*
`
`600
`
`*
`
`
`ca
`
`
`
`610
`*
`
`*
`
`
`620
`*
`
`*
`
`
`
`630
`t
`
`*
`
`640
`*
`
`
`
`
`*
`
`
`
`650
`*
`
`660
`
`
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAA ATA GGG CTT CIG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CTT TAT CCC GAA GAC IGG ACA CTT CGT TGT CAG TTA CCC GTA-AAC ATA TIC TGT TTG ATA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr>
`
`*
`
`
`
`670
`*
`
`
`680
`
`*
`*
`*
`
`
`
`
`
`
`
`
`
`CTC ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAG TGT GTA GCT GTT 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>
`
`690
`*
`
`*
`
`700
`*
`
`
`
`
`*
`
`710
`*
`
`
`
`
`720
`
`
`
`
`
`*
`
`
`
`730
`*
`
`*
`
`740
`*
`
`
`
`
`
`
`750
`*
`
`*
`
`*
`
`760
`«
`
`
`
`
`
`
`770
`*
`
`*
`
`
`
`780
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAA TTA CTT AGA GGC CAT ACT CTY GTC CTC AAT TGT ACT GCT ACC ACT CCC TIG 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 Thr Leu Val Leu Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr>
`
`
`
`*
`
`
`
`790
`*
`
`*
`
`
`
`*
`
`810
`*
`
`*
`
`
`
`820
`800
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`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 ATG GGA CTA CTT TAA CTG GTT TCG TIA AGG GTA CGG TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Ile Asp Gln Ser Asn Ser His Ala ‘Asn>
`
`*
`
`
`
`830
`*
`
`
`
`840
`*
`
`
`*
`
`
`*
`
`850
`*
`
`*
`
`
`
`860
`*
`
`*
`
`870
`*
`
`*
`
`
`880
`*
`
`*
`
`
`900
`890
`*
`*
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATA TIC 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 CTIG TIT TAC GTC TIG TIT CiG TIT CCT GAA ATA TGA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyx Thr>
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`960
`“950
`940
`930
`920
`910
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`*
`“e
`
`
`
`
`
`
`
`
`
`TGT CGT GTA AGG AGT GGA CCA TCA TTC 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
`*
`
`
`
`*
`
`
`
`380
`*
`
`*
`
`
`
`990
`*
`
`*
`
`1000
`*
`«
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`AAA GCA GGC CCG GGC GAG CCC AAA TCT TGT GAC AAA ACT CAC ACATGC CCA CCG TGC CCA
`
`
`
`
`
`
`
`
`TTT CGT CCG GGC CCG CIC GGG TTT AGA ACA CTG TTT TGA GIG TGT ACG GGT GGC ACG GGT
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Lys Ala Gly Pro Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro>
`
`
`
`
`1010
`*
`
`
`
`
`*
`
`
`
`1020
`«
`
`
`
`
`*
`
`1030
`*
`
`
`
`*
`
`1040
`*
`
`
`
`1050
`*
`
`
`
`*
`
`«
`
`1060
`*
`
`
`
`*
`
`
`
`1070
`*
`
`
`
`1080
`*x
`
`*
`
`
`
`
`
`
`
`
`GCA CCT GAA CIC CTG GGG GGA CCG TCA GTC TTC C& TTC CCC CCA AAA CCC AAG GAC ACC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT 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
`
`
`Fig.13C.
`
`
`
`Sheet 17 of 55
`
`
`
`
`US 7,374,757 B2
`
`1090
`x
`
`
`
`*
`
`*
`
`1100
`tk
`
`
`
`
`
`
`1110
`*
`
`
`*
`
`
`
`1120
`*
`
`*
`
`
`*
`
`
`1130
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`cTc ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GIG GTG GIG GAC GTG AGC CAC GAA GAC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TG GTG CTT CIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp>
`
`
`
`1140
`*
`
`+
`
`
`*
`
`
`1150
`*
`
`*
`
`1160
`*
`
`
`
`1170
`*
`
`
`
`*
`
`*
`
`1180
`*
`
`
`
`
`
`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 CIC CAC GTA TIA CGG TIC TOT TIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys>
`
`1210
`*
`
`
`
`*
`
`1220
`*
`
`
`
`*
`
`
`
`
`
`
`
`
`
`CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GIC CIC ACC GTC CiG cac
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GGC GCC CTC CTC GTC ATG TIG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GAC GTG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His>
`
`1230
`*
`
`
`
`*
`
`
`
`
`1240
`*
`
`*
`
`
`
`
`1250
`*
`
`*
`
`1260
`‘
`
`*
`
`
`
`
`1270
`*
`
`*
`
`
`
`1280
`zy
`
`*
`
`
`1290
`*
`
`
`*
`
`
`
`1300
`*
`
`*
`
`
`
`
`
`1310
`*
`
`*
`
`
`
`1320
`*
`
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`CAG GAC TGG CTG. AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC
`
`
`
`
`
`
`
`
`
`
`
`GTC CTG ACC GAC TTA CCGG TIC CIC 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
`*
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`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 TTT OGG TIT CCC GIC GG GCT CIT 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>
`
`1360
`*
`
`1370
`*
`
`1380
`*
`
`*
`
`
`*
`
`1390
`*
`
`
`
`*
`
`1400
`*
`
`
`
`1410
`*
`
`
`
`*
`
`*
`
`1420
`*
`
`
`
`*
`
`1430
`*
`
`
`
`
`1440
`*
`
`
`
`
`*
`
`
`
`
`
`
`
`
`
`CTG cCC CCA TCC CGG GAT GAG CTG ACC AAG. AAC CAG GIC AGC CTG ACC TGC CTG GTC AAA
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GAC GGG GGT AGG GCC CTA CIC GAC 1GG 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 GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC
`
`
`
`
`
`
`
`
`
`
`
`
`CCG AAG ATA GGG TCG CTG TAG CGG CAC CIC ACC CTC TOG TIA CCC GTC Gee CTC TIG TIG
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn>
`
`1510
`*
`
`
`
`*
`
`1520
`*
`
`
`
`*
`
`1530
`*
`
`
`
`
`*
`
`1540
`*
`
`
`
`*
`
`
`
`1550
`*
`
`*
`
`
`
`
`1560
`|
`
`*
`
`
`
`
`
`
`
`
`
`
`
`TAC AAG ACC ACG CCT CCC GIG CIG GAC TCC GAC GGC TCC TIC TH CTC TAC AGC AAG CIC
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`ATG TTC TGG TGC GGA GGG CAC GAC CTIG 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
`*
`
`
`
`*
`
`
`*
`
`
`
`
`
`
`
`
`
`
`ACC GTG GAC AAG AGC AGG TGG C