(12) United States Patent (10) Patent No.: US 9,078,860 B2 Szkudlinski Et Al

US00907886OB2

(12) United States Patent (10) Patent No.: US 9,078,860 B2 Szkudlinski et al. (45) Date of Patent: Jul. 14, 2015

(54) VEGF ANALOGS 6,020,473 A 2/2000 Keytet al. 6,057,428 A * 5/2000 Keytet al...... 530.399 (71) Applicant: Trophogen Inc., Rockville, MD (US) 2: R R388 Rita etal (72) Inventors: Mariusz W. Szkudlinski, Rockville, g: R 839; Snowski et al. MD (US); Bruce D. Weintraub, 6,291,740 B1 9, 2001 Bremel et al. Rockville, MD (US) 6,361,992 B1 3/2002 Szkudlinski et al. 6,475,796 B1 1 1/2002 Pollitt et al. (73) Assignee: Trophogen, Inc., Rockville, MD (US) 3. R 1 838, (his et al. 7.005.505 B 2.2006 Keytet al. (*) Notice: Subject to any disclaimer, the term of this 2001, 0046521 A1 11, 2001 E. al. patent is extended or adjusted under 35 2003/0064922 A1 4/2003 Nissen et al. U.S.C. 154(b) by 0 days. 2004/0265972 A1 12/2004 Weintraub et al. 2005.0054036 A1 3/2005 Bates et al. 21) Appl. No.: 14/312,446 (21) App 9 FOREIGN PATENT DOCUMENTS (22) Filed: Jun. 23, 2014 JP 1998-511557 11, 1998 O O JP 2000-507456 6, 2000 (65) Prior Publication Data JP 2001-5.17075 10, 2001 JP 2003-5 17275 5, 2003 US 2014/0308264 A1 Oct. 16, 2014 WO 90.08832 8, 1990 WO 97.08313 3, 1997 Related U.S. Application Data WO 98,07832 2, 1998 WO OOf 17360 3, 2000 (62) Division of application No. 12/089,296, filed as WO OO,25805 5, 2000 application No. PCT/US2006/039181 on Oct. 6, 2006, WO 2005/042575 5, 2005 now Pat. No. 8,759,285. WO 2005, O724.17 8, 2005 (60) Provisional application No. 60/808,106, filed on May OTHER PUBLICATIONS 25, 2006, provisional application No. 60/723,917, Keytet al., “Identification of vascular endothelial growth factor deter filed on Oct. 6, 2005. minants for binding KDR and FLT-1 receptors. Generation of recep tor-selective VEGF variants by site-directed mutagenesis,” Journal of (51) Int. Cl. Biological Chemistry, 271 (10):5638-5646 (1998). A6 IK38/8 (2006.01) Siemeister et al., “An antagonistic vascular endothelial growth factor A6 IK38/6 (2006.01) (VEGF) variant inhibits VEGF-stimulated receptor A6 IK38/17 (2006.01) autophosphorylation and proliferation of human endothelial cells.” C07K I4/475 (2006.01) Proc. Natl Acad Sci USA, 95(8): 4625-4629 (1998). C07K I4/52 (2006.01) Lietal. “Receptor-selective Variants of Human Vascular Endothelial A6 IK 45/06 (2006.01) Growth Factor.” Journal of Biological Chemistry, 275 (38): 29823-8 A61 K38/00 (2006.015 (1998). (52) U.S. Cl. * cited by examiner CPC ...... A61K 38/1866 (2013.01); A61K 45/06 (2013.01); C07K 14/475 (2013.01); C07K Primary Examiner — Christine J Saoud 14/52 (2013.01); A61 K38/00 (2013.01) Assistant Examiner — Jon M Lockard (58) Field of Classification Search (74) Attorney, Agent, or Firm — Morgan Lewis & Bockius None LLP See application file for complete search history. (57) ABSTRACT (56) References Cited Modified VEGF proteins that inhibit VEGF-mediated activa U.S. PATENT DOCUMENTS tion or proliferation of endothelial cells are disclosed. The analogs may be used to inhibit VEGF-mediated activation of 4.902,505 A 2/1990 Pardridge et al. endothelial cells in angiogenesis-associated diseases Such as 5,008,050 A 4, 1991 Cullis et al. 5,017,566 A 5, 1991 Bodor cancer, inflammatory diseases, eye diseases, and skin disor 5,380,531 A 1/1995 Chakrabarti et al. ders. 5,604,198 A 2f1997 Poduslo et al. 5,612,034 A 3/1997 Pouletty et al. 22 Claims, 6 Drawing Sheets U.S. Patent Jul. 14, 2015 Sheet 1 of 6 US 9,078,860 B2

Figure 1A

Binding of VEGF mutants&WT to imobilized KOR-FC 2500 e 83K 2000 WT

1500 5 1000

500

O 10-6 10-6 10-4 10-3 10-2 10-1 100 VEGF(ug/ml)

Figure 1B HUVEC-2 Cell Proliferation Assay-Glo 225 Wt 200 o 83K 175 s S 150 O 125 SS 100 75 HTTTo TTTIn-Tim-Trim-Trim-rrttem-rm 0 10-13 10-12 10-11 10-10 10-0 0-0 0- 0-8 VEGF (M) U.S. Patent Jul. 14, 2015 Sheet 2 of 6 US 9,078,860 B2

Figure 2A Yeast VEGF Mutants Binding Assay

125 e Y-VEGF A3H 100 E44R

O HTTTwo-TTTTwo-rrnu-TTTTTTm-rrm-Trium 0 1013 10-12 10-11 101 100 10 10-7 10-8 VEGF (M)

Figure 2B HUVEC-2 Cell Proliferation Assay-Glo

225 Wt 200 o E44R 175 150 125 100 -

0 10-13 10-1210-11 10-10 0-0 0-0 0-7 10-8 VEGF (M) U.S. Patent Jul. 14, 2015 Sheet 3 of 6 US 9,078,860 B2

Figure 3A

Yeast VEGF Mutants Binding Assay

125 e Y-VEGF A3 100 a EE72/73RR

0 1-S 1012 101 10-10 10 0 107 10 VEGF (M)

Figure 3B HUVEC-2 Cell Proliferation Assay-Glo

225 Wt 200 o EE7273/RR 175 150 125 10-G =c 75 -trator TTTTTTTTTTTTTTTTTTo-Trp-rate 0 10-13 10-12 101110-10 10-0 10-s 10-7 10-8 VEGF (M)

U.S. Patent Jul. 14, 2015 Sheet 5 of 6 US 9,078,860 B2

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0000Z 0000|| (Wido) punoq-93A-l US 9,078,860 B2 1. 2 VEGF ANALOGS KDR is the predominant receptor in angiogenic signaling, whereas Flt-1 is associated with the regulation of blood vessel CROSS-REFERENCE TO RELATED morphogenesis and Flt-4 regulates lymphangiogenesis. APPLICATIONS These receptors are expressed almost exclusively on endot helial cells, with a few exceptions such as the expression of This application claims the benefit of U.S. Provisional Flt-1 in monocytes where it mediates chemotaxis (Barleon et Application No. 60/723,917, filed Oct. 6, 2005, and U.S. al., 1996, Blood. 87: 3336-3343). Provisional Application No. 60/808,106, filed May 25, 2006, VEGF receptors are closely related to Fms, Kit and PDGF which are herein incorporated by reference in their entireties. receptors. They consist of seven extracellular immunoglobu 10 lin (Ig)-like domains, a transmembrane (TM) domain, a regu SEQUENCE LISTING SUBMISSION VIA latory juxtamembrane domain, an intracellular tyrosine EFS-WEB kinase domain interrupted by a short peptide, the kinase insert domain, followed by a sequence carrying several tyrosine A computer readable text file, entitled “056815-5005-01 residues involved in recruiting downstream signaling mol SequenceListing..txt created on or about Jun. 20, 2014, with 15 ecules. Mutation analysis of the extracellular domains of a file size of about 146 kb contains the sequence listing for this Flt-1 and KDR show that the second and third Ig-like domains application and is hereby incorporated by reference in its constitute the high-affinity ligand-binding domain for VEGF entirety. with the first and fourth Ig domains apparently regulating ligand binding and receptor dimerization, respectively FIELD OF INVENTION (Davis-Smyth et al., 1998, J. Biol. Chem. 273: 3216-3222: Fuh et al., 1998, J. Biol. Chem. 273: 11197-11204; and Shin This application relates to the design and use of vascular kai et al., 1998, J. Biol. Chem. 273:31283-31288). Receptor endothelial growth factor (VEGF) analogs as VEGF receptor tyrosine kinases are activated upon ligand-mediated receptor antagonists to inhibitor reduce angiogenesis for the treatment dimerization (Hubbard, 1991, Prog. Biophys. Mol. Biol. 71: of conditions and diseases associated with angiogenesis. The 25 343-358: Jiang and Hunter, 1999, Curr. Biol. 9: R568-R571; application also discloses VEGF analogs that exhibit and Lemmon and Schlessinger, 1998, Methods Mol. Biol. 84: increased receptor binding affinity to native receptors such as 49-71). Signal specificity of VEGF receptors is further modu KDR. lated upon recruitment of coreceptors, such as neuropilins, heparin Sulfate, integrins or cadherins. BACKGROUND OF INVENTION 30 VEGF molecules interact with one or more tyrosine kinase receptors during angiogenesis. For instance, VEGF-A acts Vascular endothelial growth factors (VEGFs) regulate predominantly through KDR and Flt-1. VEGF-C and blood and lymphatic vessel development. They are predomi VEGF-D similarly are specific ligands for KDR and nantly produced by endothelial, hematopoietic and stromal VEGFR3. PIGF and VEGF-B are believed to bind only to cells in response to hypoxia and stimulation with growth 35 Flt-1. Viral VEGF-E variants activate KDR, VEGF-F variants factors such as transforming growth factors, interleukins and interact with either VEGFR3 or KDR. platelet-derived growth factor. In addition to the two classical receptors, there are several In mammals, VEGFs are encoded by a family of genes and membrane or soluble receptors modulating VEGF bioactivity include VEGF-A, VEGF-B, VEGF-C, VEGF-D and Placenta and angiogenesis. For instance, neuropilin-1 and neuropilin-2 like Growth Factor (PIGF). Highly related proteins include 40 interact with both KDR and Flt-1, respectively, stimulating orf virus-encoded VEGF-like proteins referred to as VEGF-E signaling of those receptors. Isoforms of VEGF-A, VEGF-B, and a series of snake venoms referred to as VEGF-F. VEGFs PlGF-2 have been shown to bind to neuropilin-1 (Soker et al., and VEGF-related proteins are members of the Platelet 1998, Cell.92: 735-745; Makinen et al., 1999, J. Biol. Chem. Derived Growth Factor (PDGF) supergene family of cystine 274: 21217-21222; and Migdal et al., 1998, J. Biol. Chem. knot growth factors. All members of the PDGF supergene 45 273:22272-22278). VEGF isoforms capable of interacting of family share a high degree of structural homology with PDGF interacting with neuropilin, i.e., those isoforms with exon 7 or (see U.S. patent application Ser. No. 09/813,398 which is 6 and 7, are also capable of interacting with heparin Sulfate. herein incorporated by reference in its entirety). Although VEGF-A is the best characterized of the VEGF VEGF-A, VEGF-B and PIGF are predominantly required proteins, the molecular basis of the interaction between for blood vessel formation, whereas VEGF-C and VEGF-D 50 VEGF-A and KDR and Flt-1 is not well understood. are essential for the formation of lymphatic vessels. Angio Although VEGFR1 binds VEGF-A with a 50-fold higher genesis is the process by which new blood vessels or lym affinity than KDR, KDR is considered to be the major trans phatic vessels form by developing from pre-existing vessels. ducer of VEGF-A angiogenic effects, i.e., mitogenicity, The process is initiated when VEGFs bind to receptors on chemotaxis and induction of tube formation (Binetruy endothelial cells, signaling activation of endothelial cells. 55 Tourniere et al., Supra). There is, however, growing evidence Activated endothelial cells produce enzymes which dissolve that Flt-1 has a significant role in hematopoiesis and in the tiny holes in the basement membrane Surrounding existing recruitment of monocytes and other bone-marrow derived vessels. Endothelial cells then begin to proliferate and cells that may home in on tumor vasculature and promote migrate out through the dissolved holes of the existing vessel angiogenesis (Hattori et al., 2002, Nature Med. 8: 841-849; to form new vascular tubes (Alberts et al., 1994, Molecular 60 Gerber et al., 2002, Nature. 417: 954-958; and Luttun et al., Biology of the Cell. Garland Publishing, Inc., New York, N.Y. 2002, Nature Med. 8: 831-840). Further, in some cases Flt-1 1294 pp.). is expressed by tumor cells and may mediate a chemotactic Three type III receptor tyrosine kinases are activated by signal, thus potentially extending the role of this receptor in VEGFs during angiogenesis: fims-like tyrosine kinase (Flt-1. cancer growth (Wey et al., 2005, Cancer. 104: 427-438). also known as VEGFR1), kinase domain receptor or kinase 65 A single VEGF-Ahomodimer induces dimerization of two insert domain-containing receptor (KDR, also known as KDR receptors and autophosphorylation of their cytoplas VEGFR2 and Flk-1) and Flt-4 (also known as VEGFR3). matic portions. Previous studies suggested that by analogy to US 9,078,860 B2 3 4 glycoprotein hormones, the charged amino acid residues in KDR interaction and/or block the KDR signal transduction the peripheral loops of VEGF-A are also critical in providing pathway including: peptides that block binding of VEGF to high affinity electrostatic interactions with its respective KDR (Binetruy-Tourniere et al., 2000, EMBO.J. 19(7): 1525 receptors (Szkudlinski et al., 1996, Nat. Biotechnol. 14(10): 33); antibodies to VEGF (Kim et al., 1993, Nature 362, 841 1257-63; Fuh et al., supra; Muller et al., 1997, Proc. Natl. 844; Lanai et al., 1998, J. Cancer 77,933-936; Margolinet al., Acad. Sci. U.S.A. 94(14): 7192-7: Keytet al., 1996, J. Biol. 2001, J. Clin. Oncol. 19, 851-856); antibodies to KDR (Luet Chem. 271 (10): 5638-46). However, it should be noted that al., 2003, supra; Zhu et al., 1998, Cancer Res. 58,3209-3214: many mutations in VEGF-A have no major effect on receptor Zhu et al. 2003, Leukemia 17, 604-611; Prewett et al., 1999, binding affinity. Mutations in the peripheral loops of VEGF Cancer Res. 59,5209-5218); soluble receptors (Holash et al., primarily have resulted in loss-of-function. Further, there 10 appear to be no previous amino acid Substitutions increasing 2002, Proc. Natl. Acad. Sci. USA99, 11393-11398; Clavelet binding affinity to KDR more than 2-fold. al. supra); tyrosine kinase inhibitors (Fong et al., 1999, Can Angiogenesis is responsible for beneficial biological cer Res. 59, 99-106; Wood et al., 2000, Cancer Res. 60, events such as wound healing, myocardial infarction repair, 2178-2189: Grosios et al., 2004, Inflamm Res. 53(4):133-42): and ovulation. On the other hand, angiogenesis is also respon 15 anti-VEGF immunotoxins (Olson et al., 1997, Int. J. Cancer sible for causing or contributing to diseases such as growth 73, 865-870); ribozymes (Pavco et al., 2000, Clin. Cancer and metastasis of Solid tumors (Isayeva et al., 2004, Int. J. Res. 6, 2094-2103); antisense mediated VEGF suppression Oncol. 25(2):335-43: Takeda et al., 2002, Ann Surg. Oncol. (Forster et al., 2004, Cancer Lett. 20; 212(1):95-103); RNA 9(7):610-16); atherosclerosis; abnormal neovascularization interference (Takei et al., 2004, Cancer Res. 64(10):3365-70; of the eye as seen in diseases Such as retinopathy of prema Reichet al., 2003, Mol. Vis. 9:210-6); and undersulfated, low turity, diabetic retinopathy, retinal vein occlusion, and age molecular weight glycol-split heparin (Pisano et al., 2005, related macular degeneration (Yoshida et al., 1999. Histol Glycobiology. 15(2) 1-6). Some of these treatments, however, Histopathol. 14(4): 1287-94: Aiello, 1997, Ophthalmic Res. have resulted in undesirable side effects. For instance, Genen 29(5):354-62); chronic inflammatory conditions such as tech's Avastin, a monoclonal antibody that targets VEGF, has rheumatoid arthritis osteoarthritis, and septic arthritis; neu 25 been reported to cause an increase in serious arterial throm rodegenerative disease (Ferrara, N., 2004, Endocr. Rev. 25: boembolic events in Some colon cancer patients and serious, 581-611); placental insufficiency, i.e., preeclampsia (Ferrara, and in some cases even fatal, hemoptysis in non-Small cell Supra); and skin diseases such as dermatitis, psoriasis, warts, lung cancer patients (Ratner, 2004, Nature Biotechnol. cutaneous malignancy, decubitus ulcers, stasis ulcers, pyo 22(10): 1198). More recently, Genentech has reported that genic granulomas, hemangiomas, Kaposi's sarcoma, hyper 30 gastrointestinal perforations were observed in 11% of ovarian trophic scars, and keloids (Arbiser, 1996, J. Am. Acad. Der cancer patients (5 women out of 44 in trial) treated with matol. 34(3):486-97). During rheumatoid arthritis, for Avastin (Genentech Press Release dated Sep. 23, 2005). example, endothelial cells become activated and express Similarly, the first VEGF-targeting drug, Pfizer's receptor adhesion molecules and chemokines, leading to leukocyte tyrosine kinase inhibitor SU5416, exhibited severe toxicities migration from the blood into the tissue. Endothelial cell 35 that included thromboembolic events, prompting Pfizer to permeability increases, leading to edema formation and discontinue development (Ratner, supra). Given the wide swelling of the joints (Middleton et al., 2004, Arthritis Res. variety of patients that stand to benefit from the development Ther. 6(2):60-72). of effective anti-angiogenic treatments and the drawbacks of VEGF, in particular VEGF-A, has been implicated in many Some known anti-angiogenesis treatments, there remains a of the diseases and conditions associated with increased, 40 need for novel anti-angiogenic therapeutics. decreased, and/or dysregulated angiogenesis (Binetruy Tourniere et al., 2000, EMBO J. 19(7): 1525-33). For SUMMARY OF INVENTION instance, VEGF has been implicated in promoting solid tumor growth and metastasis by stimulating tumor-associated This invention encompasses VEGF analogs and nucleic angiogenesis (Lu et al., 2003, J. Biol. Chem. 278(44): 43496 45 acids encoding the same, which exhibit strong binding affin 43507). VEGF is also a significant mediator of intraocular ity for one or more native VEGF receptors compared to wild neovascularization and permeability. Overexpression of type VEGF. The invention also encompasses VEGF analogs VEGF in transgenic mice results in clinical intraretinal and and nucleic acids encoding same, which exhibit a dissociation Subretinal neovascularization, and the formation of leaky of receptor binding affinity and bioactivity. Specifically, the intraocular blood vessels detectable by angiography, as seen 50 in vivo and in vitro bioactivities of the disclosed analogs are in human eye disease (Miller, 1997, Am. J. Pathol. 151(1): substantially decreased compared to wild-type VEGF, 13-23). Additionally, VEGF has been identified in the perito whereas the binding affinity to one or more native receptors is neal fluid of women with unexplained infertility and about the same or Substantially increased compared to wild endometriosis (Miedzybrodzki et al., 2001, Ginekol. Pol. type VEGF. The VEGF analogs may demonstrate at least 72(5): 427-430), and the overexpression of VEGF in testis 55 about a three to four fold increase in receptor binding affinity and epididymis has been found to cause infertility in trans to a native receptor such as KDR. genic mice (Korpelainen et al., 1998, J. Cell Biol. 143(6): In one embodiment of the invention, the VEGF analogs are 1705-1712). Recently, VEGF-A has been identified in the modified VEGF homodimers or heterodimers. These mol synovial fluid and serum of patients with rheumatoid arthritis ecules contain at least one mutation which can be present in (RA), and its expression is correlated with disease severity 60 one or both subunits of the VEGF molecule. In one embodi (Clavel et al., 2003, Joint Bone Spine. 70(5): 321-6). Given ment of the invention, the VEGF analog containing the one or the involvement of pathogenic angiogenesis in Such a wide more mutations is VEGF-A. The VEGF-A analog can be any variety of disorders and diseases, inhibition of angiogenesis, VEGF-A isoform, for instance, an isoform of 121, 145, 148, and particularly of VEGF signaling, is a desirable therapeutic 165, 183, 189, or 206 amino acids. In one embodiment, the goal. 65 VEGF-A analog of the invention is a VEGFb isoform. In Inhibition of angiogenesis and tumor inhibition has been another embodiment, the VEGF molecule containing one or achieved by using agents that either interrupt VEGF-A and more mutations is VEGF-B, VEGF-C, VEGF-D or PlGF. US 9,078,860 B2 5 6 The present invention includes a VEGF fusion protein con The modified analogs of the invention act as VEGF recep taining one or more mutations in one or more subunits. The tor antagonists and thus provide a long awaited Solution for VEGF fusion protein of the invention includes at least one patients Suffering from a wide spectrum of diseases and con VEGF subunit, i.e., subunit, fused to at least one subunit of a ditions associated with angiogenesis. The VEGF receptor different protein, including, but not limited to, other cystine 5 antagonists can be administered to a patient alone or in con knotgrowth factors or glycoproteins. For instance, the inven junction with another VEGF receptor antagonist, an anti tion includes a chimera VEGF analog in which the VEGF cancer drug, or an anti-angiogenesis drug for the treatment of molecule contains a VEGF-A subunit fused to a VEGF-B, disease associated with angiogenesis, including but not lim VEGF-C, VEGF-D, VEGF-E, VEGF-F, PDGF or PIGF sub ited to, Solid tumor cancers, hemangiomas, rheumatoid unit; a VEGF-B subunit fused to a VEGF-A, VEGF-C, 10 arthritis, osteoarthritis, septic arthritis, asthma, atherosclero VEGF-D, VEGF-E, VEGF-F, PDGF or PIGF subunit; a sis, idiopathic pulmonary fibrosis, vascular restenosis, arte riovenous malformations, meningiomas, neovascular glau VEGF-C subunit fused to a VEGF-A, VEGF-B, VEGF-D, coma, psoriasis, Kaposi's Syndrome, angiofibroma, VEGF-E, VEGF-F, PDGF or PIGF subunit; a VEGF-D sub hemophilic joints, hypertrophic scars, Osler-Weber syn unit fused to a VEGF-A, VEGF-B, VEGF-C, VEGF-E, 15 drome, pyogenic granuloma, retrolental fibroplasias, Sclero VEGF-F, PDGF or PIGF subunit; or a PIGF subunit fused to derma, trachoma, Von Hippel-Lindau disease, vascular adhe a VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF sion pathologies, synovitis, dermatitis, neurological F, or PDGF subunit. The subunits may optionally be separated degenerative diseases, preeclampsia, unexplained female by a linker peptide. The invention also includes different infertility, endometriosis, unexplained male infertility, ptery isoforms of the same VEGF fused together, e.g., VEGFs gium, wounds, Sores, skin ulcers, gastric ulcers, and duodenal subunit fused to VEGFb. ulcers. In one embodiment, the VEGF analog is a single chain molecule. For instance, the VEGF analog of the invention BRIEF DESCRIPTION OF THE DRAWINGS includes two VEGF subunits, i.e., monomers, linked together via a linker peptide. One or both linked subunits can contain 25 FIG. 1A is a graph comparing binding of the I83K mutant one or more basic amino acid Substitutions. Further, the and wild-type VEGF-A to KDR. FIG. 1B is a graph showing linked subunits can be different VEGF protein subunits and a decrease in proliferation of HUVEC-2 endothelial cells in can be different isoforms of the same subunit. For instance, the presence of the I83KVEGF-A mutant compared to wild the present invention includes a wild-type VEGFs subunit type VEGF-A. linked via a GS linker to a VEGFs subunit with a 183K 30 FIG. 2A is a graph comparing binding of the E44R analog amino acid Substitution. and wild-type VEGF-A to KDR. FIG. 2B is a graph compar In another embodiment of the invention, a VEGF-A, ing HUVEC-2 cell proliferation in the presence of the E44R VEGF-B, VEGF-C, VEGF-D, or PlGF subunit or dimer.com VEGF-A analog versus wild-type VEGF-A. prising one or more mutations is fused to a toxin. The peptide FIG. 3A is a graph comparing binding of the E72R--E73R of this embodiment can be useful for the targeting and 35 VEGF mutant and wild-type VEGF-A to KDR. FIG. 3B is a destruction of tumor cells. graph comparing HUVEC-2 cell proliferation in the presence The VEGF analogs of the invention include one or more of the E72R+E73RVEGF mutant versus wild-type VEGF-A. basic amino acid Substitutions, such as lysine or arginine, FIG. 4 is a graph comparing binding of E44R and EE72/ from the group of positions 44, 67, 72,73, 83, and 87. In one 73RR mutants to wild-type VEGF-A. embodiment of the invention, the VEGF analog contains a 40 FIG. 5 is a graph comparing binding of Q87K mutant to basic amino acid substitution at position 83 and optionally wild-type VEGF-A. one or more basic amino acid substitutions at positions 44, 67. FIG. 6 is a graph comparing binding of E67K mutant to 72 and 73. For instance, the invention includes a VEGF ana wild-type VEGF-A. log with a 183K mutation. The invention also includes, for instance, a VEGF analog with basic amino acids at positions 45 DETAILED DESCRIPTION OF THE INVENTION 72, 73 and 83. VEGF analogs with the basic amino acid substitutions The present invention provides modified angiogenic described herein may contain additional amino acid substitu growth factors of the vascular endothelial growth factor tions to further increase receptor binding affinity to KDR (VEGF) family which exhibit surprising activity as VEGF and/or decrease receptor binding affinity to neuropilin-1. For 50 receptorantagonists. AS VEGF receptor antagonists, the com instance, the invention includes mutations at positions 146 pounds of the invention have “anti-angiogenic' properties. and 160 in the which act to disrupt the neuropilin-1 binding Being “modified’ means that, while the protein contains an site. amino acid sequence which differs from a wild-type VEGF of Analogs of the invention can also contain additional amino interest, i.e., human VEGF or animal VEGF, the sequence has acid substitutions which confer enhanced stability and 55 not been changed such that it is identical to the known VEGF increased serum half-life. For instance, the invention includes sequence of other species. The terms “mutated” and “substi amino acids Substitutions which eliminate proteolytic cleav tuted are used interchangeably herein to refer to modified age sites such substitutions at positions 111 and 148. amino acid residues. The terms “modified VEGF molecules', The VEGF receptor antagonists of the present invention “modified VEGF proteins”, “VEGF analogs”, “VEGF recep can exhibit increased plasma half-life as compared to wild 60 tor antagonists”, “VEGF chimeras”, “VEGF fusion proteins’ type VEGF. This may be accomplished by further modifying and “VEGF single chain molecules” are used interchange a VEGF analog by methods known in the art to increase ably hereinto refer to modified VEGF-A, VEGF-B, VEGF-C, half-life or, alternatively, increased plasma half-life may be VEGF-D, and PlGF analog molecules. an inherent characteristic of a VEGF analog. The VEGF Antagonists’ are used interchangeably herein to refer to receptor antagonists of the invention can also exhibit an 65 molecules which act to block, inhibit or reduce the natural, increase in rate of absorption and/or decreased duration of biological activities of VEGF, such as the induction of angio action compared to wild-type VEGF. genesis. The term “anti-angiogenic' as used herein means US 9,078,860 B2 7 8 that the modified VEGF molecules of the invention block, analogs of the invention may display increased or similar inhibit or reduce the process of angiogenesis, or the process receptor binding affinity to KDR compared to wild-type by which new blood or lymphatic vessels form from pre VEGF-A, but may display decreased receptor binding affinity existing vessels. The activities of the VEGF analogs of the to Flt-1, neuropilin-1 or neuropilin-2 compared to wild-type invention disrupt normal VEGF/receptor signaling which VEGF-A. usually occurs when VEGF binds to a receptor. Accordingly, The VEGF analogs of the invention also display a decrease the analogs of the invention are VEGF receptor antagonists. in bioactivity compared to wild-type VEGF. “Bioactivity” Without wishing to be bound by a theory, it is believed that the refers to the natural, biological activities of VEGF in vivo and VEGF analogs of the invention disrupt the dimerization of in vitro, including, but not limited to, the ability of VEGF to KDR necessary for signaling. 10 induce cell proliferation in endothelial cells. A decrease in Inhibition of angiogenesis may be complete or partial. The bioactivity results in a decrease in angiogenesis. In one VEGF receptor antagonist may inhibit angiogenesis at least embodiment of the invention, the VEGF analogs of the inven about 5%, at least about 10%, at least about 20%, at least tion display a decrease in bioactivity compared to wild-type about 25%, at least about 30%, at least about 40%, at least VEGF of the same isoform. For instance, a VEGFs analog about 50%, at least about 60%, at least about 70%, at least 15 of the invention can display a decrease in bioactivity com about 80%, at least about 90%, and at least about 100% in pared to wild-type VEGFs, and a VEGFb analog can vitro and in vivo. Inhibition of angiogenesis can be measured display a decrease in bioactivity compared to wild-type by a skilled artisan by methods known in the art. The deter VEGFb. mination of inhibition of angiogenesis can include the use of Bioactivity can be assessed by several methods known in negative and/or positive controls. For instance, a skilled arti the art, including, but not limited to, in vitro cell viability san can conclude that a VEGF analog of the invention inhibits assays which assay the viability of endothelial cells such as VEGF-induced angiogenesis by comparing angiogenesis in a human umbilical vein endothelial cells (HUVEC) upon expo subject treated with a VEGF analog of the invention to a sure to VEGF. A decrease in endothelial cell viability of at similar subject not treated with a VEGF analog. least about 5%, at least about 10%, at least about 15%, at least The modified VEGF molecules of the invention display 25 about 20%, at least about 25%, at least about 30%, at least increased receptor binding affinity or similar receptor binding about 35%, at least about 40%, at least about 45%, at least affinity to one or more native VEGF receptors compared to about 50%, at least about 60%, at least about 70%, at least that of wild-type VEGF. As used herein, a native VEGF recep about 80%, at least about 90%, or at least about 95% or more tor is an unmodified receptor that specifically interacts with compared to resulting from exposure to wild-type VEGF is VEGF. For instance, an endogenous VEGF receptor is a 30 indicative of a decrease in bioactivity. native VEGF receptor. In one embodiment of the invention, Bioactivity can be assessed in vivo as well. For instance, the native receptor is KDR. KDR is a receptor of VEGF-A, bioactivity can be assessed in vivo in a subject with a tumor by VEGF-C, VEGF-D, VEGF-E and VEGF-F. In another detecting a lack of increase in angiogenesis around a tumor. embodiment, the native receptor is Flt-1. Flt-1 is a receptor of The detection of a lack of increase in angiogenesis can be VEGF-A, VEGF-B and PIGF. 35 accomplished by several methods known in the art including, “Receptor binding affinity” refers to the ability of a ligand but not limited to, an in vivo matrigel migration assay, a disc to bind to a receptor in vivo or in vitro and can be assessed by angiogenesis assay, an assay comprising a dorsal skinfold methods readily available in the art including, but not limited chamber in mice, a corneal transplant and a sponge implant to, competitive binding assays and direct binding assays. As model of angiogenesis. In one embodiment, angiogenesis is used herein, receptor binding affinity refers to the ability of 40 assessed by comparing angiogenesis of and around the tumor VEGF molecules to bind to native VEGF receptors, includ to that of a tumor of similar type, size and location in an ing, but not limited to, Flt-1 (also known as VEGF-R1), KDR untreated Subject. Biopsy methods as known in the art can be (also known as VEGF-R2) and Flt-4 (also known as VEGF used to extract tissue and analyze for vessel formation. R3). For instance, the modified VEGF-A molecules of the “Dissociation of receptor binding affinity and bioactivity invention display increased binding receptor affinity or simi 45 refers to the concept that receptor binding affinity and bioac lar binding affinity to KDR compared to wild-type VEGF-A. tivity are not correlated. In comparison, receptor binding In one embodiment, the increase in receptor binding affinity affinity and bioactivity are correlated for wild-type VEGF of the modified VEGF molecules of the invention is at least proteins such as wild-type VEGF-A. An increase in receptor about 1.25 fold, at least about a 1.5 fold, at least about a 1.75 binding ability, for example, would be expected to result in an fold, at least about 2 fold, at least about 3 fold, at least about 50 increase in bioactivity for wild-type VEGF-A. On the other 4 fold, at least about 5 fold, at least about 6 fold, at least about hand, the modified VEGF molecules of the invention demon 7 fold, at least about 8 fold, at least about 9 fold or at least strate a similar receptor binding affinity or an increase in about 10 fold greater than that of wild-type VEGF. receptor binding affinity as compared to wild-type VEGF but In another embodiment, the modified VEGF exhibits a a decrease in bioactivity as compared to wild-type VEGF. receptor binding affinity to KDR and/or other receptor that is 55 Mammalian VEGFs are produced in multiple isoforms due involved in angiogenesis that is similar or comparable to that to alternative splicing of a family of related genes. The of wild-type VEGF. Similar or comparable receptor binding present invention describes VEGF analogs which correspond affinity is at least about 75%, at least about 80%, at least about to VEGF isoforms involved in angiogenesis. The VEGF ana 85%, at least about 90%, at least about 95%, or at least about logs of the present invention can be created using any VEGF 97% or more of that of wild-type VEGF. For instance, the 60 isoform unless otherwise indicated. invention includes VEGF-A analogs exhibiting about 75% to VEGF-A can existin isoforms including, but not limited to, 85%, about 85% to 95% and about 95% to 100% the receptor 121, 145, 148, 165, 183, 189, and 206 amino acids, respec binding affinity exhibited by wild-type VEGF. tively. The three main mRNA species are VEGF, VEGFs The present invention also includes VEGF analogs which and VEGFs. As used herein, VEGF (SEQ ID NO.: 6), display increased or similar receptor binding affinity to at 65 VEGF (SEQ ID NO.: 8), VEGF (SEQ ID NO.: 10), least one native receptor but display decreased receptor bind VEGF (SEQ ID NO. 4), VEGFb (SEQ ID NO.: 13), ing affinity to another native receptor. For instance, VEGF-A VEGF (SEQID NO.: 15), VEGF (SEQID NO.: 17) and US 9,078,860 B2 10 VEGF (SEQID NO.19) are isoforms of VEGF-Acapable PlGF-1 and PlGF-2 have been reported as being capable of of being modified to possess anti-angiogenic properties. The inducing endothelial cell migration (Migdal et al., 1998, J. amino acid positions described herein are based on a VEGF Biol. Chem. 273:22272-22278). In one embodiment of the molecule lacking a leader sequence Such as the leader invention, the VEGF analog is a PlGF analog. In another sequence of SEQ ID NO. 3. The amino acid sequences of 5 embodiment, the VEGF analog is PIGF-1 or PIGF-2. PIGF VEGF-A isoforms with leader sequence are the sequences of analogs include “modified PlGF proteins”, “PlGF analogs”. SEQ ID NOS.: 2, 5, 7, 9, 12, 14, 16 and 18. “PIGF receptor antagonists”, “PlGF chimeras”, “PlGF fusion The various isoforms of VEGF-A share a common amino proteins’ and “PlGF single chain molecules. PlGF analogs terminal domain consisting of 110 amino acids. VEGF-A are PlGF molecules with at least one modified PIGF subunit. isoforms have a receptor binding domain encoded by exons 10 The VEGF analogs of the invention are modified animal or 2-5. The most notable difference between the isoforms are human VEGF molecules. In one embodiment of the inven found in the neuropilin and heparin binding domains which tion, the VEGF analogs are mammalian VEGF molecules. In are encoded by exons 6a, 6b, 7a and 7b. another embodiment of the invention, the VEGF analogs are The most common VEGF-A isoform is VEGFs. The avian VEGF molecules. The VEGF analogs of the present nucleic acid encoding VEGFs is the sequence of SEQ ID 15 invention include, but are not limited to, modified primate, NO.: 1. Recently, an endogenous splice variant referred to as canine, feline, bovine, equinine, porcine, Ovine, murine, rat VEGFb was described which contains sequences encoded and rabbit VEGF molecules. In one embodiment, the animal by exon 9, instead of exon 8, at the carboxy terminus. The VEGF analog is a VEGF-A analog. For instance, the animal nucleic acid molecule encoding this protein is the sequence of VEGF-A analog of the invention can be an animal VEGFs SEQ ID NO.: 11. VEGFb (SEQ ID NO.: 12 with leader or VEGFb analog. sequence: SEQID NO.: 13 without leader sequence) inhib The modified VEGF molecules of species other than ited VEGF signaling in endothelial cells when added with human have substitutions at positions corresponding to those VEGF (see Woolard et al., 2004, Cancer Research. 64: in the modified human VEGF molecules disclosed herein and 7822-7835; see also U.S. 2005/0054036 which is herein may be identified using any alignment program, including but incorporated by reference in its entirety). 25 not limited to DNASIS, ALIONment, SIM and GCG pro In one embodiment of the invention, the VEGF analogs are grams such as Gap, BestFit, FrameAlign, and Compare. As VEGF-A analogs. VEGF-A analogs include “modified can be appreciated by one of skill in the art, the corresponding VEGF-A proteins”, “VEGF-A receptor antagonists'. amino acid to be replaced with a basic amino acid may not be “VEGF-A chimeras”, “VEGF-A fusion proteins” and identical to the one inhuman VEGF-A. For instance, a skilled “VEGF-A single chain molecules.” A VEGF-A analog is a 30 artisan would appreciate that a glutamate (E) may correspond VEGF-A molecule containing at least one modified VEGF-A to a different acidic amino acid in an animal Such as aspartate subunit. (D). VEGF-B exists in two isoforms, VEGF-B, (SEQ ID In another embodiment, the corresponding amino acid is NO.: 48) and VEGF-Bs (SEQID NO. 50) (Makinen et al., identified as being located in the same general position within 1999, 3. Biol. Chem. 274:21217-21222). In one embodiment 35 a defined structure, for instance, on an outer loop structure. of the invention, the VEGF analog is a VEGF-B analog. The structure of a protein can be predicted using Software VEGF-B analogs include “modified VEGF-B proteins”, based on the amino acids of the protein. Accordingly, one of “VEGF-B analogs”, “VEGF-B receptor antagonists', skill in the art can use Software that predicts protein folding “VEGF-B chimeras”, “VEGF-B fusion proteins” and and loop structure to identify the corresponding position in a “VEGF-B single chain molecules.” A VEGF-B analog is a 40 related protein. VEGF-B molecule containing at least one modified VEGF-B Design of VEGF Receptor Antagonists Subunit. The VEGF receptor antagonists encompassed by the VEGF-C is produced as a propeptide (SEQ ID NO. 51) present invention may be designed by comparing the amino that is proteolytically cleaved to form a 21-kdactive protein acid sequences of the VEGF of interest to that of other species (Nicosia, 1998, Am. J. Path. 153: 11-16). In one embodiment 45 to identify basic residues in the proteins of VEGF of other of the invention, the VEGF analog is a VEGF-C analog. species. For instance, a VEGF-A molecule of instance can be VEGF-C analogs include “modified VEGF-C proteins”, designed by comparing a human VEGF-A to that of another “VEGF-C analogs”, “VEGF-C receptor antagonists'. species. Such methods are disclosed in U.S. Pat. No. 6,361, “VEGF-C chimeras”, “VEGF-C fusion proteins” and 992, which is herein incorporated by reference in its entirety. “VEGF-C single chain molecules.” A VEGF-C analog is a 50 Consideration may also be given to the relative biological VEGF-C molecule containing at least one modified VEGF-C activity of VEGF from various species as to which species to Subunit. choose for comparison and amino acid Substitution. Further VEGF-D is also produced as a propeptide (SEQID NO.: homology modeling based on the structure of related glyco 52) that is proteolytically cleaved to form an active protein. proteins is useful to identify Surface-exposed amino acid VEGF-D is 48% identical to VEGF-C (Nicosia, supra). In one 55 residues. Homology modeling can be performed by methods embodiment of the invention, the VEGF analog is a VEGF-D generally know in the art, including, but not limited to, the use analog. VEGF-D analogs include “modified VEGF-D pro of protein modeling computer Software. teins”, “VEGF-D analogs”, “VEGF-D receptor antagonists'. The present invention also provides a modified VEGF pro “VEGF-D chimeras”, “VEGF-D fusion proteins” and tein, wherein the modified VEGF comprises an amino acid(s) “VEGF-D single chain molecules.” A VEGF-D analog is a 60 Substituted at a position(s) corresponding to the same amino VEGF-D molecule containing at least one modified VEGF-D acid position in a VEGF protein from another species having Subunit. an increased binding affinity and/or decreased bioactivity Placenta growth factor (P1GF) exists in three isoforms, over the wild-type VEGF. For example, snake venom PIGF-1 (SEQ ID NO. 54), PIGF-2 (SEQ ID NO. 56) and VEGF-F binds to KDR with high affinity and strongly stimu PIGF-3 (SEQ ID NO. 58). PIGF-2 contains an exon 6 65 lates proliferation of vascular endothelial cells in vitro. One encoded peptide which bestows heparin and neuropilin-1 can compare human VEGF-A to snake venom VEGF, design binding properties absent in the other two isoforms. Both human VEGF-A proteins with amino acid substitutions at one US 9,078,860 B2 11 12 or more positions where the Snake Venom and human log designed in the VEGFb splice variant. In another sequences differ, construct human VEGF-A proteins with the embodiment, VEGF-B, and PIGF-2 analogs can be selected changes, and administer the modified human designed which exhibit decreased receptor binding affinity to VEGF-A to humans. Although snake venom VEGF-F dem neuropilin-1 and increased binding affinity to Flt-1. onstrates an increase in KDR binding affinity and bioactivity, In one embodiment of the invention, VEGF analogs are i.e., binding affinity and bioactivity are correlated, compared designed to exhibit decreased receptor binding affinity to to human VEGF, one of skill in the art would understand that neuropilin-1 or neuropilin-2 compared to wild-type VEGF by amino acid Substitutions could be empirically tested to iden disrupting the VEGF neuropilin binding site. This can be tify amino acid substitutions which increase receptor binding accomplished by reducing the number of cysteine amino acid affinity but decrease or have no effect on bioactivity. An 10 residues in the neuropilin-1 receptor binding domain. For amino acid substitution which increases receptor binding instance, VEGFs analogs can be designed to disrupt the affinity and/or decreases or has no effect on bioactivity may neuropilin 1 binding site in VEGFs by Substituting the cys then be combined with one or more other amino acid substi teine residues at positions 146 and/or 160 of SEQID NO. 4 tutions known to increase receptor binding affinity and/or with amino acids such as serine which cause a disruption of decrease bioactivity. 15 the disulfide bridge. The substitution of cysteine residues at In another embodiment of the invention, the modified positions 146 and 160 of SEQID NO. 4 disrupts neuropilin-1 VEGF molecule can contain one or more amino acids substi binding but does not disrupt heparin binding. Mutations at tuted at a position(s) corresponding to the same amino acid positions 146 and/or 160 can be coupled with one or more position in a VEGF homolog that naturally exists in arthro mutations to increase, maintain or restore receptor binding pods. In arthropods, a single growth factor performs the tasks affinity to KDR, Flt-1 and/or VEGFR3 as described herein. performed by PDGF and VEGF in higher organisms. One of Similarly, the present invention includes VEGF analogs skill in the art would understand that amino acid substitutions which exhibit decreased receptor binding affinity to neuropi could be empirically tested to identify amino acid substitu lin-2 compared to wild-type VEGF. For instance, the inven tions which increase receptor binding affinity but decrease or tion includes VEGF-C and VEGF-D analogs which exhibit have no effect on bioactivity, or, alternatively, have little 25 reduced binding affinity to neuropilin-2 but increased or simi effect on receptor binding affinity but decrease bioactivity. lar binding affinity to KDR and/or VEGFR3 compared to Further, the present invention provides a modified VEGF, wild-type VEGF-C or VEGF-D, respectively. wherein the modified VEGF comprises a basic amino acid(s) The invention also includes VEGF analogs which exhibit Substituted at a position(s) corresponding to the same amino enhanced stability and resistance to proteases. In one embodi acid in a different VEGF or VEGF isoform or closely related 30 ment, amino acids Substitutions at positions A111 and A148 glycoprotein such as proteins in the PDGF family from the of SEQ ID NO. 4 are incorporated in a VEGF-A analog to same species or different species. For example, VEGFs can improve resistance to proteases. The invention also includes be compared to PDGF from the same species and amino acid VEGF-C and VEGF-D analogs which contain mutations pre substitutions made to the VEGF protein based on any venting the cleavage of the VEGF-C propeptide or VEGF-D sequence divergence. A skilled artisan can compare two or 35 propeptide, respectively. For instance, the present invention more sequences of VEGF proteins or VEGF-related proteins includes VEGF-C and VEGF-D analogs that contain one or using methods known in the art such as the use of alignment more mutations which induce resistance to serine protease software, including but not limited to, DNASIS, ALION plasmin and/or other members of the plasminogen family. ment, SIM and GCG programs such as Gap, BestFit, In another embodiment of the invention, VEGF analogs FrameAlign, and Compare. 40 which exhibit increased receptor binding affinity to one or In another aspect of the invention, the amino acid Substi more VEGF receptors, preferably KDR, can be created in a tutions described herein can be incorporated into closely naturally occurring VEGF molecule which exhibits antago related proteins such as VEGF-E (SEQID NO.: 60), VEGF-F nistic properties. For instance, VEGFb, an isoform iso (SEQID NO.: 62) and PDGF (SEQID NO.: 63 and SEQID lated from kidney tissue, can be modified to incorporate the NO.: 64). For instance, one or more basic amino acid substi 45 amino acid Substitutions associated with an increase in recep tutions selected from the group consisting of E67, E72, E73, tor binding ability and decrease in bioactivity of the protein. I83 and Q87 can be compared to a PDGF isoform from the Similarly, a skilled artisan could incorporate the amino acid same species and amino acid substitutions made to the PDGF substitutions of the present invention in synthetic or new isoform. isoforms of VEGF which contain the properties of VEGFb. The VEGF analogs of the invention may be designed to 50 In particular, the mutations of the invention can be used with display a decreased receptor binding affinity to Flt-1 recep other VEGF proteins which contain the amino acids SLTRKD tors compared to wild-type VEGF-A. Although these analogs (SEQID NO.: 70), i.e., the amino acids coded for by what has display a decreased receptor binding affinity to Flt-1, they been termed exon 9, in addition to or in place of the amino may have an increased or comparable receptor binding affin acids coded for by exon 8 (CDKPRR: SEQID NO.:71). ity to KDR compared to wild-type VEGF-A. 55 Amino Acid Substitutions The VEGF analogs of the invention may be designed to The VEGF analogs of the present invention contain one or display a decreased receptor binding affinity to co-receptors, more basic amino acid substitutions which confer enhanced including, but not limited to, neuropilin-1 or neuropilin-2 receptor binding affinity and decreased bioactivity. In one compared to that of wild-type VEGF. Analogs with decreased embodiment of the invention, the VEGF analogs are VEGF receptor binding affinity to neuropilin-1 or neuropilin-2 may 60 receptor antagonists, including but not limited to VEGF-A have increased or similar receptor binding affinity to KDR, antagonists. Flt-1 or VEGR3 compared to that of wild-type VEGF. For A modified VEGF molecule of the invention may have a instance, VEGF-A analogs can be designed which exhibit basic amino acid Substitution in one or more subunits, i.e., decreased receptor binding affinity to neuropilin-1 and monomers, of VEGF. Basic amino acids comprise the amino increased receptor binding affinity to KDR and/or Flt-1. In 65 acids lysine (K), arginine (R) and histidine (H), and any other one embodiment of the invention, the VEGF-A displaying basic amino acids which may be a modification of any of decreased receptor binding affinity to neuropilin-1 is an ana these three amino acids, synthetic basic amino acids not nor US 9,078,860 B2 13 14 mally found in nature, or any other amino acids which are In another embodiment of the invention, the analog is a positively charged at a neutral pH. Preferred amino acids, VEGFb molecule containing one or more basic amino among others, are selected from the group consisting of lysine acids at positions E44, E67, E72, E73 and Q87 and optionally and arginine. a basic amino acid substitution at position I83. When the In one embodiment, a modified VEGF molecule of the VEGF-A isoform is VEGF165b, it is possible to generate a invention comprises at least one modified subunit, wherein VEGF analog of the invention with increased binding affinity the modified Subunit comprises a basic amino acid substitu and decreased bioactivity compared to wild-type VEGF-A, tion at position I83 of wild-type human VEGFs (SEQ ID including VEGFs, by incorporating a single amino acid NO.: 4), VEGF (SEQID NO.: 6), VEGF (SEQID NO.: modification that would otherwise only result in an increase 8), VEGF (SEQ ID NO.: 10), VEGFb (SEQ ID NO.: 10 in receptor binding affinity in other VEGFs. 13), VEGF (SEQID NO. 15), VEGF (SEQIDNO. 17) As can be appreciated by a skilled artisan, the invention or VEGF (SEQ ID NO.: 19). For instance, the invention includes VEGF proteins and VEGF-related proteins other that includes an I83Kamino acid substitution in SEQID NOS.: 4, VEGF-A that contain basic amino acid modifications corre 6, 8, 10, 13, 15, 17 or 19 corresponding to the amino acid 15 sponding to those of positions E44, E67, E72, E73 and/or Q87 sequences of VEGF-A isoforms. of VEGF-A (SEQ ID NO. 4). For instance, the invention The invention also includes a basic amino acid Substitution includes a modified VEGF-B analog (SEQID NOS.: 48 and in the position corresponding to position 83 in other VEGF 50) containing one or more basic amino acid substitutions at molecules, i.e., VEGF-B, VEGF-C, VEGF-D and PlGF, such positions A44, E67, G72, Q73 and S87 and a modified as position I83 of VEGF-B (SEQ ID NO.: 48) or VEGF VEGF-F analog (SEQ ID NO.: 62) containing one or more Bs (SEQID NO. 50) and position 191 of PlGF-1 (SEQID basic amino acid substitutions at positions E44, E67, E72, NO.:54), PIGF-2 (SEQIDNO.:56) or PIGF-3 (SEQID NO. E73 and Q87. 58). A modified animal, i.e., non-human, VEGF-A molecule of The invention includes modified VEGF molecules in ani the invention can likewise contain additional amino acid mals other than humans, wherein the VEGF molecule con 25 modifications to increase binding affinity or decrease bioac tains, in one or more subunits, a basic amino acid Substitution tivity of the modified animal VEGF molecule compared to in the position corresponding to position 83 inhuman VEGF wild-type animal VEGF. The invention includes the use of A. In one embodiment, the modified animal VEGF is a modi these modifications in conjunction with an amino acid Sub fied VEGF-A molecule. For instance, the present invention stitution that corresponds to I83 of SEQ ID NO. 4 as includes a basic amino acid substitution at position I83 in 30 described above. For instance, the present invention includes primate (SEQID NO.: 22), position I82 in bovine (SEQ ID one or more basic amino acid Substitutions selected from the NO. 25), position I82 in canine (SEQID NO. 28), position group of positions E44, E67, E72, E73, I83 and I87 of primate I83 in chicken (SEQID NO.:31), position I82 inequine (SEQ (long-tailed macaque) VEGF-A (SEQ ID NO.: 22); one or ID NO. I82), position I82 in murine (SEQ ID NO. 37), more basic amino acid substitutions selected from the group position I82 in porcine (SEQID NO.: 40), position I82 of rat 35 of positions E43, E66, E71, E72, I82 and Q86 of bovine (SEQ ID NO.: 43) and position I82 in ovine (SEQ ID NO.: VEGF-A (SEQ ID NO. 25); one or more basic amino acid 46). substitutions selected from the group of positions E43. E66, The invention also envisions a modified VEGF-related pro E71, E72, I82 and Q86 of canine VEGF-A (SEQID NO.: 28); tein, including, but not limited to VEGF-E. VEGF-F and one or more basic amino acid Substitutions selected from the PDGF, containing an amino acid Substitution corresponding 40 group of positions E44, E67, D72, V73, I83 and Q87 of avian to position I83 of SEQ ID NO. 4. For instance, VEGF-F (chicken) VEGF-A (SEQ ID NO. 31); one or more basic (SEQ ID NO.: 62) can be modified to include an I83 amino amino acid substitutions selected from the group of positions acid Substitution. E43, E66, A71, E72, I82 and Q86 of equine VEGF-A (SEQ The modified VEGF molecule of the invention can contain ID NO. 34); one or more basic amino acid substitutions basic amino acid substitutions which further increase the 45 selected from the group of positions E43, E66, S71, E72, I82 binding affinity or decrease bioactivity of VEGF compared to and Q86 of murine VEGF-A (SEQID NO. 37): one or more wild-type VEGF such as wild-type VEGF-A. VEGF mol basic amino acid substitutions selected from the group of ecules with basic amino acid Substitutions at one or more of positions E43, E66, E71, E72, I82 and Q86 of porcine positions 44, 67,72, 73 and/or 87 of VEGF (SEQID NO.: VEGF-A (SEQ ID No.: 40); one or more basic amino acid 4), VEGF (SEQ ID NO.: 6), VEGF (SEQ ID NO.: 8), 50 substitutions selected from the group of positions E43. E66, VEGF (SEQID NO.: 10), VEGFb (SEQID NO.: 13), S71, E72, I82 and Q86 of rat VEGF-A (SEQID NO.:43); and VEGF (SEQID NO. 15), VEGF (SEQID NO.: 17) and one or more basic amino acid Substitutions selected from the VEGF (SEQID NO. 19) can increase binding affinity for group of positions E43. E66, E71, E72, I82 and Q86 of ovine KDR compared to wild-type VEGF. For instance, the inven VEGF-A (SEQID NO.:46). tion includes the basic amino acid modifications E44R, 55 VEGF analogs containing one or more basic amino acid E44K, E72R, E72K, E73R, E73K, Q87R, Q87K and E67K. Substitutions can also be combined with amino acid substitu In one embodiment of the invention, basic amino Substitu tions designed to disrupt a co-receptor binding site. In one tions corresponding to positions 44, 67, 72, 73 and/or 87 of embodiment, the VEGF analogs of the invention contain a SEQID NO. 4 are coupled with the basic amino acid substi disrupted neuropilin-1 binding site. The neuropilin-1 binding tution corresponding to position 83 of SEQ ID NO. 4 to 60 site comprises amino acids 111 to 165 of VEGFs (SEQID produce a VEGF receptorantagonists. For instance, the modi NO.: 04). This domain overlaps the heparin binding domain fied amino acids of the present invention include basic amino encoded by exons 6 and 7. The invention includes any amino acid substitutions at positions 72+73+83, 44+83, 72+83, acid modifications in or near (i.e., within about 5 amino acids) 73+83, 44+72+83, 44+73+83, 44+72+73+83, 44+83+87, that disrupt the neuropilin-1 binding site domain but which do 83+87, 67+72+73+83; 44+67+83, 67+72+83, 67+73+83, 65 not disrupt the ability of the heparin binding domain to bind 44+67+72+83, 44+67+73+83, 44+67+72+73+83, heparin Sulfate. Such amino acid modifications can be deter 44+67+83+87 and 67+83+87. mined empirically by a skilled artisan. US 9,078,860 B2 15 16 In one embodiment of the invention, the neuropilin-1 bind The modified proteins of the invention may also contain ing domain is disrupted by reducing the number of cysteine further substitutions, particularly conservative substitutions amino acid residues in the domain, i.e., by reducing the num that do not alter the enhanced properties of the protein. Typi ber of cysteine amino acid residues between amino acids 111 cally, however, Such modified proteins will contain less than to 165 of VEGF-A. For instance, VEGFs analogs can be 5 five substitutions at positions other than those listed above, designed to disrupt the neuropilin 1 binding site by Substitut and may exhibit complete amino acid sequence identity with ing the cysteine residues at positions 146 and/or 160 of SEQ the corresponding wild-type VEGF subunits in positions ID NO. 4 with amino acids such as serine which cause a other that the positions listed above. disruption of the disulfide bridge. The substitution of cysteine As can be appreciated by a skilled artisan, all amino acid 10 Substitutions and peptide modifications disclosed in the residues at positions 146 and 160 of SEQID NO. 4 disrupts present invention can be incorporated in any VEGF protein or neuropilin-1 binding but does not disrupt heparin binding. related protein, regardless of species, because of the high The neuropilin-1 binding site can also be disrupted by ending degree of homology between VEGF proteins and related pro the amino acid peptide at position 146 or 160. teins. A skilled artisan can correlate the amino acid substitu The invention can also included modifications of amino 15 tions described herein using methods known in the art, acids Surrounding amino acids at positions 146 and 160 of including, but not limited to, the use of amino acid sequence SEQ ID NO. 4 such that the cysteine residues of positions alignment software. 146 and 160 are unable to form a disulfide bridge. For VEGF Analogs with Increased Serum Half-Life instance, the invention includes, but is not limited to, one or The VEGF analogs of the invention may have an increased more amino acid substitutions at positions 136 through 165 plasma half-life as compared to wild-type VEGF. In one which are capable of disrupting the formation of a disulfide embodiment, the modification(s) which increases or main bridge. tains receptor binding affinity and decreases bioactivity as A modified VEGF analog of the invention containing one compared to wild-type VEGF also increases the plasma half or more of the basic amino acid substitutions corresponding life of the VEGF as compared to wild-type VEGF. In another to E44, E67, E72, E73, I83 and Q87 of SEQ ID NO.: 4 25 embodiment, the modified VEGF proteins of the invention described herein. For instance, the invention includes VEGF are further modified such that the plasma half-life is increased as compared to wild type VEGF. There are many modifications known in the art that can be used to increase the half-life of proteins, in particular glyco 30 proteins. For instance, the modified VEGF proteins of the invention may further comprise at least one sequence with a potential glycosylation site including sequences comprising N-glycosylation and/or O-glycosylation sites on either the alpha or beta chain. Sequences providing potential glycosy 35 lation recognition sites may be either an N-terminal or C-ter minal extension on either subunit. Exemplary modified pro teins contain an N-terminal extension on a Subunit that is selected from the group consisting of ANITV (SEQID NO.: 72) and ANITVNITV (SEQID NO.: 73). 40 Increased half-life may also be provided by the use of a peptide extensions such as a carboxyl terminal extension peptide of hCG. See U.S. Ser. No. 09/519,728 which is herein incorporated by reference in its entirety. A subunit of a VEGF analog may be covalently bound by any method known in the 45 art to a CTEP, e.g., by a peptide bond or by a heterobifunc tional reagentable to form a covalent bond between the amino terminus and carboxyl terminus of a protein, including but not limited to a peptide linker. In another embodiment of the invention, the basic amino 50 acid substitutions of the invention are coupled with one or more amino acid Substitutions that enhance stability and increase serum half-life by eliminating one or more pro teolytic cleavage sites. In one embodiment, the additional amino acid Substitutions reduce proteolytic cleavage. In 55 another embodiment, the additional amino acid Substitutions prevent proteolytic cleavage. The invention includes VEGF analogs that contain one or more mutations which induce resistance to plasmin and other members of the plasminogen family. In one embodiment of the invention, at least one 60 subunit of a VEGF molecule contains an amino acid substi tution corresponding to amino acid positions A111 and/or A148 such as A111P and/or A148P of VEGFs (SEQ ID NO. 4) or VEGF165b (SEQID NO.: 13). For instance, the invention includes VEGF, VEGF as VEGF as VEGFs, 65 VEGFs and VEGF containing anamino acid substitution at position A111. The invention includes one or more muta tions in VEGF-B, VEGF-C, VEGF-D and PIGF which inhibit US 9,078,860 B2 17 18 or reduce protease cleavage. For instance, the invention ther, a VEGF analog containing one or more linker peptides includes amino acid Substitutions which prevent the cleavage can link more than one type of VEGF protein or isoform. For of VEGF-C and VEGF-D necessary for bioactivity. instance, the present invention includes, but is not limited to, In another embodiment, half-life can be increased by link a modified VEGF single chain molecule with a wild-type ing VEGF monomers and by constructing fusion proteins. 5 VEGFs monomer linked to a modified VEGFs monomer Increasing the size of a VEGF analog without interfering with containing an I83B substitution; a wild-type VEGFs mono binding sites can increase the half-life of the molecule. mer linked to a modified VEGFb containing an I83B sub stitution; and a modified VEGFs monomer fused to a modi Increased half-life may be provided by crosslinking, fied VEGF-F monomer. including but not limited to pegylation or conjugation of other VEGF Fusion Proteins appropriate chemical groups. Such methods are known in the 10 The present invention also includes fusion proteins, i.e., art, for instance as described in U.S. Pat. No. 5,612,034, U.S. chimeras, containing one or more modified VEGF proteins or Pat. No. 6,225,449, and U.S. Pat. No. 6,555,660, each of fragments. “Fusion protein’ and "chimera’ are used inter which is incorporated by reference in its entirety. Half-life changeably herein. As used herein, a VEGF moiety is a VEGF may also be increased by increasing the number of negatively protein or protein fragment containing one or more of the charged residues within the molecule, for instance, the num 15 basic amino acid substitutions of the invention. A VEGF ber of glutamate and/or aspartate residues. Such alteration fusion protein can have one or more VEGF moieties. may be accomplished by site directed mutagenesis or by an Such a fusion protein may be made by ligating the appro insertion of an amino acid sequence containing one or more priate nucleic acid sequences encoding the desired amino negatively charged residues into said modified VEGF, includ acid sequences to each other by methods known in the art, in ing insertions selected from the group consisting of GEFT the proper coding frame, and expressing the fusion protein by and GEFTT, among others. any of the means described herein. Alternatively, such a The half-life of a protein is a measurement of protein fusion protein may be made by protein synthesis techniques, stability and indicates the time necessary for a one-halfreduc for example, using a peptide synthesizer. tion in the concentration of the protein. The serum half-life of The fusion protein of the invention contains at least one the modified VEGF molecules described herein may be deter 25 VEGF protein or protein fragment containing one or more mined by any method suitable for measuring VEGF levels in basic amino acid Substitutions described herein. In one samples from a subject over time, for example, but not limited embodiment the fusion protein contains a basic amino acid to, immunoassays using anti-VEGF antibodies to measure substitution at position I83 of VEGF (SEQ ID NO. 4), VEGF levels in serum samples taken over a period of time VEGFb (SEQ ID NO. 13), VEGF (SEQ ID NO.: 6), after administration of the modified VEGF, or by detection of 30 VEGF (SEQ ID NO. 8), VEGF (SEQ ID NO.: 10), labeled VEGF molecules, i.e., radiolabeled molecules, in VEGF (SEQID NO. 15), VEGF (SEQID NO.: 17) or samples taken from a subject after administration of the VEGF (SEQ ID NO. 19). In another embodiment, the labeled VEGF. fusion protein contains at least one basic amino acid Substi The rate of absorption of a VEGF analog of the present tution at a position corresponding to I83K of SEQID NO.: 4 invention may result in increased or decreased duration of 35 in another VEGF protein, for instance, an isoform of VEGF action. A VEGF analog with an increased rate of absorption B, VEGF-C, VEGF-D or PIGF. As can be appreciated by a and decreased duration of action may be beneficial for skilled artisan, human or animal VEGF proteins or fragments patients receiving a VEGF analog pharmaceutical composi thereof may be used for the fusion proteins of the invention. tion by way of subcutaneous administration or other route of In one embodiment of the invention, two different VEGF administration generally associated with a slow rate of 40 protein Subunits or fragments thereofare fused. For instance, absorption and/or increased duration of action by counteract the invention includes a VEGF-A subunit or fragment thereof ing the absorption qualities associated with the route of fused to a VEGF-B subunit or fragment thereof, a VEGF-C administration. subunit or fragment thereof, a VEGF-D subunit or fragment Linker thereof, or a PIGF subunit or fragment thereof: a VEGF-B The VEGF analog of the invention can contain two or more 45 subunit or fragment thereof fused to a VEGF-A subunit or monomers separated by a linker peptide. A linker peptide can fragment thereof, a VEGF-C subunit or fragment thereof, a be used to form a VEGF analog in a single chain conforma VEGF-D subunit or fragment thereof, or a PIGF subunit or tion. A skilled artisan can appreciate that various types of fragment thereof: a VEGF-C subunit or fragment thereof linkers can be used in the present invention to form a VEGF fused to a VEGF-A subunit or fragment thereof, a VEGF-B single chain molecule that is capable of binding a VEGF 50 subunit or fragment thereof, a VEGF-D subunit or fragment receptor and which acts as a VEGF receptor antagonist. A thereof, or a PIGF subunit or fragment thereof; a VEGF-D linker peptide should not hinder the ability of the single chain subunit or fragment thereof fused to a VEGF-A subunit or molecule to bind a VEGF receptor. fragment thereof, a VEGF-B subunit or fragment thereof, a The linker peptide can range from about 2 to about 50 or VEGF-C subunit or fragment thereof, or a PIGF subunit or more amino acids in length. For instance, the linker can 55 fragment thereof, and a PIGF subunit or fragment thereof consist of about 2 amino acids, about 3 amino acids, about 4 fused to a VEGF-A subunit or fragment thereof, a VEGF-B amino acids, about 5 amino acids, about 6 amino acids, about subunit or fragment thereof, a VEGF-C subunit or fragment 7 amino acids, about 8 amino acids, about 9 amino acids, thereof, or a VEGF-D subunit or fragment thereof. about 10 amino acids, about 10-15 amino acids, or about The invention includes fusion proteins comprised of two or 15-20 amino acids. In one embodiment of the invention, the 60 more different isoforms of the same VEGF protein or frag linker is Gly-Seror contains Gly-Ser. In another embodiment, ments thereof. For instance, the invention includes a fusion the linker is a glycine-rich polypeptide chain. protein comprised of a VEGFs subunit or fragment thereof VEGF molecules containing a linker can be constructed fused to a VEGF, subunit or fragment thereof, a VEGFs using the methods described herein. A skilled artisan would Subunit or fragment thereof, a VEGF as Subunit or fragment be able to appreciate that VEGF analog molecules of the 65 or subunit thereof, a VEGFb subunit or fragment thereof, a invention containing linker peptides can include any of the VEGFs subunit or fragment thereof, a VEGFs subunit or mutations described herein, in one or more monomers. Fur fragment thereof, or a VEGF subunit or fragment thereof. US 9,078,860 B2 19 20 The invention also includes a VEGFb subunit or fragment In one embodiment, the present invention provides a phar thereof fused to a VEGF, subunit or fragment thereof, a maceutical composition comprising a soluble toxin fused to a VEGF as subunit or fragment thereof, a VEGF as subunit or modified VEGF and a pharmaceutically acceptable carrier. In fragment or subunit thereof, a VEGFs subunit or fragment another embodiment, the present invention provides the use thereof, a VEGFs subunit or fragment thereof, a VEGFs, ofa modified VEGF fusion protein comprising a soluble toxin Subunit or fragment thereof, or a VEGF subunit or frag for the manufacture of a medicament for the treatment or ment thereof. prevention of diseases or conditions associated with angio The basic amino acid substitutions of the invention may be genesis. present in one or more subunits of the protein. For example, a Without wishing to be bound by a theory, it is believed that 10 the VEGF-toxin fusion protein of the invention prevents or fusion protein containing a VEGFs subunit and VEGFsb reduces angiogenesis, the growth of tumors and/or the spread Subunit may only contain an amino acid Substitution in the of cancer by targeting and killing the VEGF receptor and VEGFs subunit. The invention includes a wild-type Surrounding endothelial and tumor cells. VEGFs subunit fused by way of a GS linker to a VEGFs Expression and/or Synthesis of VEGF Receptor Antago containing an I83Kamino acid Substitution. As can be appre 15 nists ciated by one of skill in the art, the fusion proteins of the The present invention includes nucleic acids encoding the present invention containing one mutated Subunit can be cre modified VEGF proteins of the invention, as well as vectors ated in both orientations, i.e., the Subunit containing the muta and host cells for expressing the nucleic acids. tion can beat either the N- or C-terminus of the fusion protein. As used herein, the terms “nucleic acid' or “polynucle In another embodiment of the invention, a VEGF subunitor otide' refer to deoxyribonucleotides or ribonucleotides and fragment thereof is fused to a related protein subunit or frag polymers thereof in either single or double stranded form. ment thereof. For instance, a VEGF subunit or fragment The invention includes a nucleic acid molecule which codes thereof can be fused to a PDGF subunit or other glycoprotein for a modified VEGF molecule of the invention. For instance, subunit or fragment thereof. the invention includes a nucleic acid molecule that codes for As can be appreciated by one of ordinary skill in the art, the 25 a modified VEGFs molecule. The nucleic acid molecule of fusion proteins described herein can be constructed using SEQ ID NO.: 1 which codes for wild-type VEGFs can be human or animal VEGF sequences. Further, a fusion protein mutated by methods known in the art such that the mutated can be constructed using a human VEGF subunit fused to an VEGF, nucleic acid molecule codes for the modified pro animal VEGF subunit. tein. Similarly, the nucleic acid molecule of SEQID NO.: 11 AVEGF fusion protein should be understood to be a VEGF 30 which codes for wild-type VEGFb can be mutated by analog. All modifications disclosed herein, for instance, methods known in the art such that it codes for a VEGFb modifications to further increase receptor binding affinity, molecule of the invention. modifications to increase half-life and stability, modifications Once a nucleic acid encoding a particular modified VEGF to reduce or inhibit protease cleavage, and modifications to of interest, or a region of that nucleic acid encoding a portion disrupt a co-receptor binding site such as a neuropilin-1 bind 35 of the protein containing a basic amino acid Substitution, is ing site can be incorporated in one or more subunits of the constructed, modified, or isolated, that nucleic acid can then VEGF fusion protein. be cloned into an appropriate vector, which can direct the in The fusion proteins of the invention can also contain a vivo or in vitro synthesis of the modified VEGF protein. linker separating the two or more VEGF subunits or VEGF Alternatively, the nucleic acid encoding a VEGF analog of the related protein subunits. The linker can be covalently linked 40 invention may be cloned or modified directly in the expres to and between the peptides of the fusion protein. sion vector of interest. The vector is contemplated to have the VEGF and Toxin Fusion Proteins necessary functional elements that direct and regulate tran The present invention provides fusion proteins comprising Scription of the inserted gene, or hybrid gene. These func a toxin and one or more modified VEGF subunits, i.e., mono tional elements include, but are not limited to, a promoter, mers, containing one or more of the basic amino acid Substi 45 regions upstream or downstream of the promoter, Such as tutions described herein. For instance, the VEGF monomer, enhancers that may regulate the transcriptional activity of the i.e., subunit, of a VEGF-toxin fusion protein can contain a promoter, an origin of replication, appropriate restriction sites basic amino acid at one or more amino acid positions corre to facilitate cloning of inserts adjacent to the promoter, anti sponding to the amino acid positions from the group consist biotic resistance genes or other markers which can serve to ing of 44, 67, 72,73, 83 and 87 (SEQ ID NO. 4 or SEQID 50 select for cells containing the vector or the vector containing NO.: 13). The VEGF and toxin fusion proteins of the inven the insert, RNA splice junctions, a transcription termination tion may optionally contain a linker sequence separating the region, or any other region which may serve to facilitate the toxin and one or more VEGF subunits. expression of the inserted gene or hybridgene. (Seegenerally, As used herein, the term “toxin refers to a poisonous Sambrook et al., Molecular Cloning: A Laboratory Manual, Substance of biological origin. The toxin of the invention may 55 2nd ed. (1989)). Appropriate promoters for the expression of be a soluble toxin as known in the art. The fusion proteins nucleic acids in different host cells are well known in the art, comprising a soluble toxin may be used to target tumors. Such and are readily interchanged depending on the vector-host fusion proteins may also be used for diagnostic purposes. system used for expression. Exemplary vectors and host cells Examples of toxins include, but are not limited to, are described in U.S. Pat. No. 6,361,992, which is herein Pseudomonas exotoxins (PE), Diphtheria toxins (DT), ricin 60 incorporated by reference in its entirety. toxin, abrintoxin, anthrax toxins, shiga toxin, botulism toxin, There are numerous E. coli (Escherichia coli) expression tetanus toxin, cholera toxin, maitotoxin, palytoxin, cigua vectors known to one of ordinary skill in the art which are toxin, textilotoxin, batrachotoxin, alpha conotoxin, taipoxin, useful for the expression of the nucleic acid insert. Other tetrodotoxin, alpha tityustoxin, saxitoxin, anatoxin, micro vectors suitable for use include expression vectors from cystin, aconitine, exfoliatin toxins A and B, enterotoxins, 65 bacilli, such as Bacillus subtilis, and other enterobacteri toxic shock syndrome toxin (TSST-1), Y. pestis toxin, gas aceae. Such as Salmonella, Serratia, and various Pseudomo gangrene toxin, and others. nas species. These expression vectors will typically contain US 9,078,860 B2 21 22 expression control sequences compatible with the host cell expression control sequences, such as an origin of replication, (e.g., an origin of replication). In addition, any number of a a promoter, an enhancer, and necessary information process variety of well-known promoters will be present, such as the ing sites, such as ribosome binding sites, RNA splice sites, lactose promoter system, a tryptophan (Trp) promoter sys polyadenylation sites, and transcriptional terminator tem, a beta-lactamase promoter system, or a promoter system 5 sequences. Exemplary expression control sequences are pro from phage lambda. The promoters will typically control moters derived from immunoglobulin genes, SV40, Adenovi expression, optionally with an operator sequence, and have rus, Bovine Papilloma Virus, etc. The vectors containing the ribosome binding site sequences for example, for initiating nucleic acid segments of interest can be transferred into the and completing transcription and translation. If necessary, an host cell by well-known methods, which vary depending on amino terminal methionine can be provided by insertion of a 10 the type of cellular host. For example, calcium chloride trans Met codon 5' and in-frame with the downstream nucleic acid formation is commonly utilized for prokaryotic cells, insert. Also, the carboxy-terminal extension of the nucleic whereas calcium phosphate, DEAE dextran, or lipofectin acid insert can be removed using standard oligonucleotide mediated transfection or electroporation may be used for mutagenesis procedures. other cellular hosts. Additionally, yeast expression systems can be used. There 15 Expression of the gene or hybridgene can be either in vivo are several advantages to yeast expression systems. First, or in vitro. In vivo synthesis comprises transforming prokary evidence exists that proteins produced in a yeast Secretion otic or eukaryotic cells that can serve as host cells for the systems exhibit correct disulfide pairing. Second, post-trans vector. For instance, techniques for transforming fungi are lational glycosylation is efficiently carried out by yeast secre well known in the literature, and have been described, for tory systems. The Saccharomyces cerevisiae pre-pro-alpha- 20 instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. factor leader region (encoded by the MF"-1 gene) is routinely Sci. USA 75: 1929-1933, 1978), Yelton et al., (Proc. Natl. used to direct protein secretion from yeast. (Brake, et al., Acad. Sci. USA 81: 1740-1747, 1984), and Russell (Nature “varies-Factor-Directed Synthesis and Secretion of Mature 301: 167-169, 1983). Other techniques for introducing cloned Foreign Proteins in Saccharomyces cerevisiae.” Proc. Nat. DNA sequences into fungal cells. Such as electroporation Acad. Sci., 81:4642-4646 (1984)). The leader region of pre- 25 (Becker and Guarente, Methods in Enzymol. 194: 182-187, pro-alpha-factor contains a signal peptide and a pro-segment 1991) may be used. The genotype of the host cell will gener which includes a recognition sequence for a yeast protease ally contain a genetic defect that is complemented by the encoded by the KEX2 gene. This enzyme cleaves the precur selectable marker present on the expression vector. Choice of Sorprotein on the carboxyl side of a Lys-Arg dipeptide cleav a particular host and selectable marker is well within the level age signal sequence. The VEGF coding sequence can be 30 of ordinary skill in the art. fused in-frame to the pre-pro-alpha-factor leader region. This Cloned DNA sequences comprising modified VEGF and construct is then put under the control of a strong transcription VEGF fusion proteins of the invention may beintroduced into promoter. Such as the alcohol dehydrogenase I promoter or a cultured mammalian cells by, for example, calcium phos glycolytic promoter. The nucleic acid coding sequence is phate-mediated transfection (Wigler et al., Cell 14: 725, followed by a translation termination codon which is fol- 35 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, lowed by transcription termination signals. Alternatively, the 1981; Graham and Vander Eb, Virology 52:456, 1973.) Other nucleic acid coding sequences can be fused to a second pro techniques for introducing cloned DNA sequences into mam tein coding sequence, such as S.26 or beta-galactosidase, malian cells, such as electroporation (Neumann et al., EMBO which may be used to facilitate purification of the fusion J. 1: 841-845, 1982), or lipofection may also be used. In order protein by affinity chromatography. The insertion of protease 40 to identify cells that have integrated the cloned DNA, a select cleavage sites to separate the components of the fusion pro able marker is generally introduced into the cells along with tein is applicable to constructs used for expression in yeast. the gene or cDNA of interest. Preferred selectable markers for Efficient post-translational glycosolation and expression of use in cultured mammalian cells include genes that confer recombinant proteins can also be achieved in Baculovirus resistance to drugs, such as neomycin, hygromycin, and systems. 45 methotrexate. The selectable marker may be an amplifiable Mammalian cells permit the expression of proteins in an selectable marker. A preferred amplifiable selectable marker environment that favors important post-translational modifi is the DHFR gene. A particularly preferred amplifiable cations such as folding and cysteine pairing, addition of com marker is the DHFR (see U.S. Pat. No. 6.291,212) cDNA plex carbohydrate structures, and secretion of active protein. (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: Vectors useful for the expression of active proteins in mam- 50 2495-2499, 1983). Selectable markers are reviewed by Thilly malian cells are characterized by insertion of the protein (Mammalian Cell Technology, Butterworth Publishers, coding sequence between a strong viral or other promoter and Stoneham, Mass.) and the choice of selectable markers is well a polyadenylation signal. The vectors can contain genes con within the level of ordinary skill in the art. ferring hygromycin resistance, gentamicin resistance, or Alternatively, expression of the gene can occur in an in other genes or phenotypes Suitable for use as selectable mark- 55 vitro expression system. For example, in vitro transcription ers, or methotrexate resistance for gene amplification. The systems are commercially available which are routinely used chimeric protein coding sequence can be introduced into a to synthesize relatively large amounts of mRNA. In such in Chinese hamster ovary (CHO) cell line using a methotrexate vitro transcription systems, the nucleic acid encoding the resistance-encoding vector, or other cell lines using Suitable modified VEGF would be cloned into an expression vector selection markers. Presence of the vector DNA in trans- 60 adjacent to a transcription promoter. For example, the Blue formed cells can be confirmed by Southern blot analysis. Script II cloning and expression vectors contain multiple clon Production of RNA corresponding to the insert coding ing sites which are flanked by Strong prokaryotic transcrip sequence can be confirmed by Northern blot analysis. A num tion promoters. (Stratagene Cloning Systems, La Jolla, ber of other suitable host cell lines capable of secreting intact Calif.). Kits are available which contain all the necessary human proteins have been developed in the art, and include 65 reagents for in vitro synthesis of an RNA from a DNA tem the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat plate such as the BlueScript vectors. (Stratagene Cloning Sys cells, etc. Expression vectors for these cells can include tems, La Jolla, Calif.). RNA produced in vitro by a system US 9,078,860 B2 23 24 such as this can then be translated in vitro to produce the subunits fused to a toxin. The VEGF analog and VEGF analog desired VEGF analog (Stratagene Cloning Systems, LaJolla, fusion protein of the invention can be isolated and purified by Calif.). means known in the art. Another method of producing a VEGF receptor antagonist All of the VEGF analogs of the invention contain at least is to link two peptides or polypeptides together by protein 5 one basic amino acid substitution in at least one VEGF sub chemistry techniques. Peptides or polypeptides can be chemi unit. In one embodiment of the invention, the VEGF analogs cally synthesized using currently available laboratory equip of the invention contain at least two basic amino acid Substi ment using either Fmoc (9-fluorenylmethyloxycarbonyl) or tutions, at least 3 basic amino acid substitutions, at least 4 Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosys basic amino acid Substitutions or at least 5 basic amino acid 10 substitutions in at least one or at least two VEGF subunits. tems, Inc., Foster City, Calif.). One skilled in the art can The invention includes VEGF analogs containing VEGF readily appreciate that a peptide or polypeptide correspond active fragments, i.e., peptides that are not full length pro ing to a hybrid VEGF protein can be synthesized by standard teins. Active fragments of the modified VEGF of the inven chemical reactions. For example, a peptide or polypeptide can tion can also be synthesized directly or obtained by chemical be synthesized and not cleaved from its synthesis resin 15 or mechanical disruption of larger modified VEGF protein. whereas the other fragment of a hybrid peptide can be syn An active fragment is defined as an amino acid sequence of at thesized and subsequently cleaved from the resin, thereby least about 5 consecutive amino acids, at least 10 consecutive exposing a terminal group which is functionally blocked on amino acids, at least 20 consecutive amino acids, at least 30 the other fragment. By peptide condensation reactions, these consecutive amino acids, at least 40 consecutive amino acids, two fragments can be covalently joined via a peptide bond at at least 50 consecutive amino acids, at least 60 consecutive their carboxyl and amino termini, respectively, to form a amino acids, at least 70 consecutive amino acids, at least 80 hybrid peptide. (Grant, G. A., “Synthetic Peptides: A User consecutive amino acids, at least 90 consecutive amino acids, Guide.” W. H. Freeman and Co., N.Y. (1992) and Bodansky, at least 100 consecutive amino acids, at least 110 consecutive M. and Trost, B., Ed., “Principles of Peptide Synthesis.” amino acids, at least 120 consecutive amino acids, at least 130 Springer-Verlag Inc., N.Y. (1993)). Alternatively, the peptide 25 consecutive amino acids, at least 140 consecutive amino or polypeptide can by independently synthesized in vivo as acids, at least 150 consecutive amino acids, or at least 160 described above. Once isolated, these independent peptides consecutive amino acids derived from the natural amino acid or polypeptides may be linked to form a VEGF via similar sequence, which has the relevant activity, e.g., binding or peptide condensation reactions. For example, enzymatic or regulatory activity. The fragments, whether attached to other chemical ligation of cloned or synthetic peptide segments can 30 sequences or not, can also include insertions, deletions, Sub stitutions, or other selected modifications of particular allow relatively short peptide fragments to be joined to pro regions or specific amino acids residues, provided the activity duce larger peptide fragments, polypeptides or whole protein of the peptide is not significantly altered or impaired com domains (Abrahmsen, L., et al., Biochemistry, 30:4151 pared to the modified VEGF. These modifications can provide (1991); Dawson, at al., “Synthesis of Proteins by Native 35 for some additional property, Such as to remove/add amino Chemical Ligation.” Science, 266:776-779 (1994)). acids capable of disulfide bonding, to increase its biolongev The invention also provides fragments of modified VEGF ity and/or bioactivity, etcetera. In any case, the peptide must which have antagonist activity. The polypeptide fragments of possess a bioactive property, such as binding activity, regula the present invention can be recombinant proteins obtained tion of binding at the binding domain, etcetera. Functional or by cloning nucleic acids encoding the peptides in an expres 40 active regions of the VEGF may be identified by mutagenesis sion system capable of producing the peptides. For example, of a specific region of the hormone, followed by expression amino or carboxy-terminal amino acids can be sequentially and testing of the expressed polypeptide. Such methods are removed from either the native or the VEGF protein and the readily apparent to a skilled practitioner in the art and can respective activity tested in one of many available assays include site-specific mutagenesis of the nucleic acid encoding described above. In another example, the modified proteins of 45 the receptor (Zoller, M. J. et al.). the invention may have a portion of either amino terminal or Methods of Use carboxy terminal amino acids, or even an internal region of The invention encompasses methods for reducing VEGF the protein, replaced with a polypeptide fragment or other mediated angiogenesis, comprising contacting a cell express moiety, Such as biotin, which can facilitate in the purification ing kinase domain receptor (KDR) with the VEGF analogs, of the modified VEGF. For example, a modified VEGF can be 50 including VEGF-Ass and VEGF-Asb analogs, described fused to a maltose binding protein, through either peptide herein such that VEGF-mediated angiogenesis is reduced. chemistry of cloning the respective nucleic acids encoding KDR-expressing cells to be targeted by the methods of the the two polypeptide fragments into an expression vector Such invention can include either or both prokaryotic and eukary that the expression of the coding region results in a hybrid otic cells. Such cells may be maintained in vitro, or they may polypeptide. The hybrid polypeptide can be affinity purified 55 be present in Vivo, for instance in a patient or Subject diag by passing it over an amylose affinity column, and the modi nosed with cancer or another angiogenesis-related disease. fied VEGF can then be separated from the maltose binding The present invention includes methods of treating a region by cleaving the hybrid polypeptide with the specific patient diagnosed with an angiogenesis-related disease or protease factor Xa. (See, for example, New England Biolabs condition with a therapeutically effective amount of any of Product Catalog, 1996, pg. 164). 60 the VEGF receptor antagonists described herein, comprising The VEGF analog of the invention can be a heterodimer or administering said VEGF analog or fusion protein to said a homodimer. In one embodiment, the VEGF analog is a patient Such that said angiogenesis-related disease or condi fusion protein containing one or more VEGF subunits. The tion is reduced or inhibited. In order to measure the reduction VEGF fusion protein of the invention can be a single chain of angiogenesis, the patient’s results may be compared to that protein containing two or more VEGF subunits separated by 65 of a patient administered a placebo. Exemplary angiogenesis linking peptides. In another embodiment, the VEGF analog of related diseases are described throughout this application, the invention is a fusion protein containing one or more VEGF and include but are not limited to diseases selected from the US 9,078,860 B2 25 26 group consisting of tumors and neoplasias, hemangiomas, said patient such that infertility is deemed treated by one of rheumatoid arthritis, osteoarthritis, septic arthritis, asthma, skill in the art. Infertility can be measured by quantitative and atherosclerosis, idiopathic pulmonary fibrosis, vascular rest qualitative parameters known in the art such as quantity of enosis, arteriovenous malformations, meningioma, neovas oocytes, fertilization rate, blastocyst formation rate, and cular glaucoma, psoriasis, Kaposi's Syndrome, angiofi embryo formation rate. Such infertility diseases include any broma, hemophilic joints, hypertrophic scars, Osler-Weber disease associated with the expression of VEGF that compro syndrome, pyogenic granuloma, retrolental fibroplasias, scle mises a patient’s fertility including but not limited to unex roderma, trachoma, Von Hippel-Lindau disease, Vascular plained female infertility, endometriosis, and unexplained adhesion pathologies, synovitis, dermatitis, endometriosis, male infertility. The invention includes but is not limited to pterygium, diabetic retinopathy, neovascularization associ 10 treatment of infertility by administration of a VEGF analog ated with corneal injury or grafts, wounds, Sores, and ulcers alone or in combination with other anti-VEGF treatments, (skin, gastric and duodenal). anti-angiogenesis treatments, and/or infertility treatments. A patient Suffering from a disease caused by or exacerbated The present invention also includes methods of treating a by an increase in angiogenesis, a decrease in angiogenesis, or patient diagnosed with an angiogenesis-associated eye dis otherwise dysregulated angiogenesis can be treated with a 15 ease with a therapeutically effective amount of any of the VEGF analog alone or in combination with a known VEGF VEGF receptor antagonists described herein, comprising receptor antagonist, an anti-angiogenesis therapy, an anti administering saidantagonist to said patient Such that said eye cancer therapy, or other therapy known to treat the disease or disease is reduced or inhibited. Such eye diseases include any condition. As used herein, “therapy” includes but is not lim eye disease associated with abnormal intraocular neovascu ited to a known drug. Known VEGF receptor antagonists or larization, including but not limited to retinopathy of prema anti-angiogenesis therapies include but are not limited to turity, diabetic retinopathy, retinal vein occlusion, and age agents that either interrupt VEGF/KDR interaction and/or related macular degeneration. The invention includes but is block the KDR signal transduction pathway Such as peptides not limited to treatment of angiogenesis-related eye diseases that block binding of VEGF to KDR, antibodies to VEGF, by administration of a VEGF analog alone or in combination antibodies to KDR, soluble receptors, tyrosine kinase inhibi 25 with other anti-VEGF treatments, anti-angiogenesis treat tors, anti-VEGF immunotoxins, ribozymes, antisense medi ments, and/or other eye disease treatments. For example, a ated VEGF suppression, and undersulfated, low molecular VEGF analog of the present invention could be administered weight glycol-split heparin. to a patient in conjunction with Pfizer's Macugen (pegap If a VEGF analog of the invention is used in combination tanib) which is a pegylated anti-VEGFaptamer which acts by with another therapy, the coupling of the therapies results in a 30 binding to and inhibiting the activity of VEGF for the treat synergistic effect. In addition, a VEGF analog of the present ment of diabetic macular edema, retinal vein occlusion, and invention can be combined with a drug associated with an age-related macular degeneration. undesirable side effect. By coupling a VEGF analog with The present invention also includes methods of treating a Such a drug, the effective dosage of the drug with the side patient diagnosed with an angiogenesis-associated inflam effect can be lowered to reduce the probability of the side 35 matory condition or autoimmune disease with a therapeuti effect from occurring. cally effective amount of any of the VEGF receptor antago The invention includes methods of treating a patient diag nists described herein, comprising administering said nosed with cancer with a therapeutically effective amount of antagonist to said patient such that said inflammatory condi any of the VEGF receptor antagonists described herein, com tion is reduced or inhibited. Such inflammatory conditions or prising administering said antagonist to said patient such that 40 diseases include any inflammatory disorder associated with the spread of said cancer is reduced or inhibited, i.e., metasta expression of VEGF and activation of cells by VEGF, includ sis is reduced or inhibited. The invention includes methods of ing but not limited to all types of arthritis and particularly treating a patient diagnosed with cancer with a therapeuti rheumatoid arthritis and osteoarthritis, asthma, pulmonary cally effective amount of any of the VEGF receptor antago fibrosis and dermatitis. The invention includes but is not nists described herein, comprising administering said antago 45 limited to treatment of angiogenesis-related inflammatory nist to said patient Such that the growth of a tumor is reduced conditions or autoimmune disease by administration of a or inhibited. In one embodiment, the VEGF analog functions VEGF analog alone or in combination with other anti-VEGF by inhibiting angiogenesis by reducing or preventing VEGF treatments, anti-angiogenesis treatments, inflammation induced angiogenesis. In another embodiment, the VEGF therapeutics, and/or autoimmune disease therapeutics. analog is a VEGF-toxin fusion protein that prevents or 50 In another embodiment of the present invention, the modi reduces angiogenesis by targeting or killing tumor cells, vas fied VEGF protein of the invention is used as a diagnostic. The cular cells such as endothelial cells and/or VEGF receptors. VEGF analogs of the invention or VEGF receptors can dis Cancers treatable by the methods of the present invention played on a synthetic Surface. Such as in a protein or peptide include all Solid tumor and metastatic cancers, including but array. Such an array is well known in the art and can be used not limited to those selected from the group consisting of 55 to screen for VEGF analogs which bind to KDR and other bladder, breast, liver, bone, kidney, colon, ovarian, prostate, receptors known to be involved in angiogenesis. The VEGF pancreatic, lung, brain and skin cancers. The invention analogs disclosed herein can be used as positive controls to includes but is not limited to treatment of cancer with a VEGF assess the ability of putative VEGF analogs to bind to KDR analog of the present invention, alone, in combination with and other receptors known to be involved in angiogenesis. chemotherapy, or in combination with radiation therapy by 60 The invention also includes an array comprising the VEGF methods known in the art (see U.S. Pat. No. 6,596,712). For analogs of the present invention to screen for putative VEGF instance, a VEGF analog may be used with cesium, iridium, receptors which may be involved in angiogenesis. iodine, or cobalt radiation. Assays Suitable for characterizing the analogs described The present invention includes methods of treating a herein are described in PCT/US/99/05908, which is herein patient diagnosed with infertility with a therapeutically effec 65 incorporated by reference in its entirety. For instance, various tive amount of any of the VEGF receptor antagonists immunoassays may be used including but not limited to com described herein, comprising administering said antagonist to petitive binding assays and non-competitive assay systems US 9,078,860 B2 27 28 using techniques such as radioimmunoassays, ELISA, sand therapies, infertility therapies, autoimmune disease therapies, wich immunoassays, immunoradiometric assays, gel diffu inflammation therapies, ocular disease therapies, and skin sion precipitin reactions, immunodiffusion assays, in situ disease therapies. immunoassays, western blots, precipitation reactions, agglu The present invention thus also provides pharmaceutical tination assays, complement fixation assays, immunofluores compositions suitable for administration to a subject. The cence assays, Protein A assays, and immunoelectrophoresis carrier can be a liquid, so that the composition is adapted for assays, etcetera. parenteral administration, or can be solid, i.e., a tablet or pill Pharmaceutical Formulations formulated for oral administration. Further, the carrier can be The invention provides methods of diagnosis and treatment in the form of a nebulizable liquid or solid so that the com 10 position is adapted for inhalation. When administered by administration to a subject of an effective amount of a parenterally, the composition should be pyrogen free and in therapeutic of the invention. The Subject may be an animal, an acceptable parenteral carrier. Active compounds can alter including but not limited to animals such as cows, pigs, natively be formulated or encapsulated in liposomes, using horses, chickens, cats, dogs, etc., and is preferably a mammal, known methods. Other contemplated formulations include and most preferably human. In a specific embodiment, a 15 projected nanoparticles and immunologically based formula non-human mammal is the Subject. tions. The pharmaceutical compositions of the invention com Liposomes are completely closed lipid bilayer membranes prise an effective amount of one or more modified VEGF which contain entrapped aqueous Volume. Liposomes are proteins of the present invention in combination with the vesicles which may be unilamellar (single membrane) or pharmaceutically acceptable carrier. The compositions may multilamellar (onion-like structures characterized by mul further comprise other known drugs suitable for the treatment tiple membrane bilayers, each separated from the next by an of the particular disease being targeted. An effective amount aqueous layer). The bilayer is composed of two lipid mono of the VEGF receptor antagonist of the present invention is layers having a hydrophobic “tail” region and a hydrophilic that amount that blocks, inhibits or reduces VEGF stimula “head’ region. In the membrane bilayer, the hydrophobic tion of endothelial cells compared to that which would occur 25 (nonpolar) “tails” of the lipid monolayers orient toward the in the absence of the compound; in other words, an amount center of the bilayer, whereas the hydrophilic (polar) “heads' that decreases the angiogenic activity of the endothelium, orient toward the aqueous phase. compared to that which would occur in the absence of the The liposomes of the present invention may be formed by compound. The effective amount (and the manner of admin any of the methods known in the art. Several methods may be istration) will be determined on an individual basis and will 30 used to form the liposomes of the present invention. For be based on the specific therapeutic VEGF receptor antago example, multilamellar vesicles (MLVs), stable plurilamellar nist being used and a consideration of the subject (size, age, vesicles (SPLVs), small unilamellar vesicles (SUV), or general health), the condition being treated (cancer, arthritis, reverse phase evaporation vesicles (REVs) may be used. Pref eye disease, etc.), the severity of the symptoms to be treated, erably, however, MLVs are extruded through filters forming the result sought, the specific carrier or pharmaceutical for 35 large unilamellar vesicles (LUVs) of sizes dependent upon mulation being used, the route of administration, and other the filter size utilized. In general, polycarbonate filters of 30, factors as would be apparent to those skilled in the art. The 50, 60, 100, 200 or 800 nm pores may be used. In this method, effective amount can be determined by one of ordinary skill in disclosed in Cullis et al., U.S. Pat. No. 5,008,050, relevant the art using techniques as are known in the art. Therapeuti portions of which are incorporated by reference herein, the cally effective amounts of the compounds described herein 40 liposome Suspension may be repeatedly passed through the can be determined using in vitro tests, animal models or other extrusion device resulting in a population of liposomes of dose-response studies, as are known in the art. The VEGF homogeneous size distribution. proteins of the present invention can be used alone or in For example, the filtering may be performed through a conjunction with other therapies. The therapeutically effec straight-through membrane filter (a Nuclepore polycarbonate tive amount may be reduced when a VEGF analog is used in 45 filter) or a tortuous path filter (e.g. a Nuclepore Membrafil conjunction with another therapy. filter (mixed cellulose esters) of 0.1 um size), or by alternative The pharmaceutical compositions of the invention may be size reduction techniques such as homogenization. The size prepared, packaged, or sold informulations suitable for intra of the liposomes may vary from about 0.03 to above about 2 dermal, intravenous, Subcutaneous, oral, rectal, vaginal, microns in diameter; preferably about 0.05 to 0.3 microns and parenteral, intraperitoneal, topical, pulmonary, intranasal, 50 most preferably about 0.1 to about 0.2 microns. The size buccal, ophthalmic, intrathecal, epidural or another route of range includes liposomes that are MLVs, SPLVs, or LUVs. administration. The compounds may be administered by any Lipids which can be used in the liposome formulations of convenient route, for example by infusion or bolus injection, the present invention include synthetic or natural phospholip by absorption through epithelial or mucocutaneous linings ids and may include phosphatidylcholine (PC), phosphati (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may 55 dylethanolamine (PE), phosphatidylserine (PS), phosphati be administered together with other biologically active dylglycerol (PG), phosphatidic acid (PA), agents. Administration can be systemic or local. For example, phosphatidylinositol (PI), sphingomyelin (SPM) and cardio the pharmaceutical compositions of the invention can be lipin, among others, either alone or in combination, and also administered locally to a tumor via microinfusion. Further, in combination with cholesterol. The phospholipids useful in administration may be by a single dose or a series of doses. 60 the present invention may also include dimyristoylphosphati For pharmaceutical uses, the VEGF analogs of the present dylcholine (DMPC) and dimyristoylphosphatidylglycerol invention may be used in combination with a pharmaceuti (DMPG). In other embodiments, distearoylphosphatidylcho cally acceptable carrier, and can optionally include a pharma line (DSPC), dipalmitoylphosphatidylcholine (DPPC), or ceutically acceptable diluent or excipient. Further, the VEGF hydrogenated soy phosphatidylcholine (HSPC) may also be analogs of the present invention may be used in combination 65 used. Dimyristoylphosphatidylcholine (DMPC) and diar with other known therapies, including but not limited to anti achidonoylphosphatidylcholine (DAPC) may similarly be VEGF therapies, anti-angiogenesis therapies, anti-cancer used. US 9,078,860 B2 29 30 During preparation of the liposomes, organic solvents may The pharmaceutical compositions of the invention further also be used to Suspend the lipids. Suitable organic solvents comprise a depot formulation of biopolymers such as biode for use in the present invention include those with a variety of gradable microspheres. Biodegradable microspheres are used polarities and dielectric properties, which solubilize the lip to control drug release rates and to target drugs to specific ids, for example, chloroform, methanol, ethanol, dimethyl 5 sites in the body, thereby optimizing their therapeutic sulfoxide (DMSO), methylene chloride, and solvent mixtures response, decreasing toxic side effects, and eliminating the Such as benzene:methanol (70:30), among others. As a result, inconvenience of repeated injections. Biodegradable micro Solutions (mixtures in which the lipids and other components spheres have the advantage over large polymer implants in are uniformly distributed throughout) containing the lipids that they do not require Surgical procedures for implantation are formed. Solvents are generally chosen on the basis of their 10 and removal. biocompatibility, low toxicity, and solubilization abilities. The biodegradable microspheres used in the context of the To encapsulate the VEGF receptor antagonist(s) of the invention are formed with a polymer which delays the release inventions into the liposomes, the methods described in U.S. of the proteins and maintains, at the site of action, a therapeu Pat. No. 5,380.531, relevant portions of which are incorpo tically effective concentration for a prolonged period of time. rated by reference herein, may be used with the analog(s) of 15 The polymer can be chosen from ethylcellulose, polystyrene, the present invention. poly(e-caprolactone), poly(lactic acid) and poly(lactic acid Liposomes containing the VEGF analog(s) of the present co-glycolic acid) (PLGA). PLGA copolymer is one of the invention may be used therapeutically in mammals, espe synthetic biodegradable and biocompatible polymers that has cially humans, in the treatment of a number of disease states reproducible and slow-release characteristics. An advantage or pharmacological conditions which require Sustained of PLGA copolymers is that their degradation rate ranges release formulations as well as repeated administration. The from months to years and is a function of the polymer molecu mode of administration of the liposomes containing the lar weight and the ratio of polylactic acid to polyglycolic acid agents of the present invention may determine the sites and residues. Several products using PLGA for parenteral appli cells in the organism to which the VEGF analog may be cations are currently on the market, including Lupron Depot delivered. 25 and Zoladex in the United States and Enantone Depot, The liposomes of the present invention may be adminis Decapeptil, and Pariodel LA in Europe (see Yonsei, Med J. tered alone but will generally be administered in admixture 2000 December; 41(6):720-34 for review). with a pharmaceutical carrier selected with regard to the The pharmaceutical compositions of the invention may intended route of administration and standard pharmaceutical further be prepared, packaged, or sold in a formulation Suit practice. The preparations may be injected parenterally, for 30 able for nasal administration as increased permeability has example, intravenously. For parenteral administration, they been shown through the tightjunction of the nasal epithelium can be used, for example, in the form of a sterile aqueous (Pietro and Woolley, The Science behind Nastech's intranasal Solution which may contain other solutes, for example, drug delivery technology. Manufacturing Chemist, August, enough salts or glucose to make the Solution isotonic, should 2003). Such formulations may comprise dry particles which isotonicity be necessary or desired. The liposomes of the 35 comprise the active ingredient and which have a diameter in present invention may also be employed Subcutaneously or the range from about 0.5 to about 7 nanometers, and prefer intramuscularly. Other uses, depending upon the particular ably from about 1 to about 6 nanometers. Such compositions properties of the preparation, may be envisioned by those are conveniently in the form of dry powders for administra skilled in the art. tion using a device comprising a dry powder reservoir to For the oral mode of administration, the liposomal formu 40 which a stream of propellant may be directed to disperse the lations of the present invention can be used in the form of powder or using a self-propelling solvent/powder-dispensing tablets, capsules, lozenges, troches, powders, syrups, elixirs, container Such as a device comprising the active ingredient aqueous solutions and Suspensions, and the like. In the case of dissolved or Suspended in a low-boiling propellant in a sealed tablets, carriers which can be used include lactose, sodium container. Preferably, such powders comprise particles citrate and salts of phosphoric acid. Various disintegrants 45 wherein at least 98% of the particles by weight have a diam Such as starch, lubricating agents, and talc are commonly eter greater than 0.5 nanometers and at least 95% of the used in tablets. For oral administration in capsule form, useful particles by number have a diameter less than 7 nanometers. diluents are lactose and high molecular weight polyethylene More preferably, at least 95% of the particles by weight have glycols. When aqueous Suspensions are required for oral use, a diameter greater than 1 nanometer and at least 90% of the the active ingredient is combined with emulsifying and Sus 50 particles by number have a diameter less than 6 nanometers. pending agents. If desired, certain Sweetening and/or flavor Dry powder compositions preferably include a solid fine ing agents can be added. powder diluent Such as Sugar and are conveniently provided For the topical mode of administration, the pharmaceutical in a unit dose form. formulations of the present invention may be incorporated Pharmaceutical compositions of the invention formulated into dosage forms such as a solution, Suspension, gel, oil, 55 for nasal delivery may also provide the active ingredient in the ointment or salve, and the like. Preparation of such topical form of droplets of a solution or Suspension. Such formula formulations are described in the art of pharmaceutical for tions may be prepared, packaged, or sold as aqueous or dilute mulations as exemplified, for example, by Gennaro et al. alcoholic solutions or Suspensions, optionally sterile, com (1995) Remington’s Pharmaceutical Sciences, Mack Pub prising the active ingredient, and may conveniently be admin lishing. For topical application, the compositions could also 60 istered using any nebulization or atomization device. Such be administered as a powder or spray, particularly in aerosol formulations may further comprise one or more additional form. For administration to humans in the treatment of dis ingredients including, but not limited to, a flavoring agent ease states or pharmacological conditions, the prescribing Such as Saccharin Sodium, a Volatile oil, a buffering agent, a physician will ultimately determine the appropriate dosage of Surface active agent, or a preservative such as methylhy the agent for a given human Subject, and this can be expected 65 droxybenzoate. The droplets provided by this route of admin to vary according to the age, weight and response of the istration preferably have an average diameter in the range individual as well as the pharmacokinetics of the agent used. from about 0.1 to about 200 nanometers. US 9,078,860 B2 31 32 Another formulation suitable for intranasal administration ing affinity and optionally antagonistic properties is contem is a coarse powder comprising the active ingredient and hav plated in one embodiment of the present invention. ing an average particle from about 0.2 to 500 micrometers. The Successful production of transgenic, non-human ani Such a formulation is administered in the manner in which mals has been described in a number of patents and publica Snuff is taken i.e. by rapid inhalation through the nasal pas tions, such as, for example U.S. Pat. No. 6,291.740 (issued sage from a container of the powder held close to the nose. Sep. 18, 2001): U.S. Pat. No. 6,281,408 (issued Aug. 28, Formulations suitable for nasal administration may, for 2001); and U.S. Pat. No. 6,271,436 (issued Aug. 7, 2001) the example, comprise from about as little as 0.1% (w/w) and as contents of which are hereby incorporated by reference in much as 100% (w/w) of the active ingredient, and may further their entireties. comprise one or more of the additional ingredients described 10 The ability to alter the genetic make-up of animals, such as herein. domesticated mammals including cows, pigs, goats, horses, In some embodiments, the compositions of the invention cattle, and sheep, allows a number of commercial applica may be administered by inhalation. For inhalation therapy, tions. These applications include the production of animals the active ingredients may be in a solution useful for admin which express large quantities of exogenous proteins in an istration by metered dose inhalers or in a form suitable for a 15 easily harvested form (e.g., expression into the milk or dry powder inhaler. In another embodiment, the compositions blood), the production of animals with increased weight gain, are suitable for administration by bronchial lavage. feed efficiency, carcass composition, milk production or con Suitable formulations for oral administration include hard tent, disease resistance and resistance to infection by specific or softgelatin capsules, pills, tablets, including coated tablets, microorganisms and the production of animals having elixirs, Suspensions, syrups or inhalations and controlled enhanced growth rates or reproductive performance. Animals release forms thereof. which contain exogenous DNA sequences in their genome The VEGF receptor antagonists of the present invention are referred to as transgenic animals. can be administered acutely (i.e., during the onset or shortly The most widely used method for the production of trans after events leading to inflammation), or can be administered genic animals is the microinjection of DNA into the pronuclei during the course of a degenerative disease to reduce orame 25 of fertilized embryos (Wall et al., J. Cell. Biochem. 49:113 liorate the progression of symptoms that would otherwise 1992). Other methods for the production of transgenic ani occur. The timing and interval of administration is varied mals include the infection of embryos with retroviruses or according to the Subjects symptoms, and can be administered with retroviral vectors. Infection of both pre- and post-im at an interval of several hours to several days, over a time plantation mouse embryos with either wild-type or recombi course of hours, days, weeks or longer, as would be deter 30 nant retroviruses has been reported (Janenich, Proc. Natl. mined by one skilled in the art. A typical daily regime can be Acad. Sci. USA 73:1260 (1976; Janenich et al., Cell 24:519 from about 0.01 g/kg body weight per day, from about 1 1981: Stuhlmannet al., Proc. Natl. Acad. Sci. USA81:7151 mg/kg body weight per day, from about 10 mg/kg body 1984; Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927 weight per day, from about 100 mg/kg body weight per day, 1985; Van der Putten et al., Proc. Natl. Acad. Sci. USA and from about 1 g/kg body weight per day. 35 82:6148-6152 (1985; Stewart et al., EMBO J. 6:383-388 The VEGF receptor antagonists of the invention may be 1987). administered intravenously, orally, intranasally, intraocu An alternative means for infecting embryos with retrovi larly, intramuscularly, intrathecally, or by any Suitable route ruses is the injection of virus or virus-producing cells into the in view of the VEGF protein, the protein formulation and the blastocoele of mouse embryos (Jahner, D. et al., Nature 298: disease to be treated. Modified VEGF for the treatment of 40 623 1982). The introduction of transgenes into the germline inflammatory arthritis can be injected directly into the syn of mice has been reported using intrauterine retroviral infec ovial fluid. Modified VEGF for the treatment of solid tumors tion of the midgestation mouse embryo (Jahner et al., Supra may be injected directly into the tumor. Modified VEGF for 1982). Infection of bovine and ovine embryos with retrovi the treatment of skin diseases may be applied topically, for ruses or retroviral vectors to create transgenic animals has instance in the form of a lotion or spray. Intrathecal adminis 45 been reported. These protocols involve the microinjection of tration, i.e. for the treatment of brain tumors, can comprise retroviral particles or growth arrested (i.e., mitomycin injection directly in to the brain. Alternatively, modified C-treated) cells which shed retroviral particles into the perivi VEGF may be coupled or conjugated to a second molecule (a telline space of fertilized eggs or early embryos (PCT Inter “carrier”), which is a peptide or non-proteinaceous moiety national Application WO 90/08832 (1990; and Haskell and selected for its ability to penetrate the blood-brain barrier and 50 Bowen, Mol. Reprod. Dev.,40:3861995). PCT International transport the active agent across the blood-brain barrier. Application WO 90/08832 describes the injection of wild Examples of suitable carriers are disclosed in U.S. Pat. Nos. type feline leukemia virus B into the perivitelline space of 4,902,505; 5,604,198; and 5,017,566, which are herein incor sheep embryos at the 2 to 8 cell stage. Fetuses derived from porated by reference in their entirety. injected embryos were shown to contain multiple sites of An alternative method of administering the VEGF receptor 55 integration. antagonists of the present invention is carried out by admin U.S. Pat. No. 6,291,740 (issued Sep. 18, 2001) describes istering to the Subject a vector carrying a nucleic acid the production of transgenic animals by the introduction of sequence encoding the modified VEGF protein, where the exogenous DNA into pre-maturation oocytes and mature, vector is capable of directing expression and secretion of the unfertilized oocytes (i.e., pre-fertilization oocytes) using ret protein. Suitable vectors are typically viral vectors, including 60 roviral vectors which transduce dividing cells (e.g., vectors DNA viruses, RNA viruses, and retroviruses. Techniques for derived from murine leukemia virus MLVI). This patent also utilizing vector delivery systems and carrying out gene describes methods and compositions for cytomegalovirus therapy are known in the art (see Lundstrom, 2003, Trends promoter-driven, as well as mouse mammary tumor LTR Biotechnol. 21(3): 117-22, for a recent review). expression of various recombinant proteins. Transgenic Animals 65 U.S. Pat. No. 6,281,408 (issued Aug. 28, 2001) describes The production of transgenic non-human animals that con methods for producing transgenic animals using embryonic tain a modified VEGF construct with increased receptor bind stem cells. Briefly, the embryonic stem cells are used in a US 9,078,860 B2 33 34 mixed cell co-culture with a morula to generate transgenic vammin. VEGFs has glutamate residues at positions 72 and animals. Foreign genetic material is introduced into the 73, whereas vammin contains a glycine and lysine residue at embryonic stem cells prior to co-culturing by, for example, these positions, respectively. By modifying VEGF-A to con electroporation, microinjection or retroviral delivery. ES cells tain two basic amino acid residues at positions 72 and 73, the transfected in this manner are selected for integrations of the modified VEGF-A demonstrated a significant increase in gene via a selection marker Such as neomycin. receptor binding affinity compared to wild-type VEGF-A U.S. Pat. No. 6.271,436 (issued Aug. 7, 2001) describes the (FIG. 3A). production of transgenic animals using methods including isolation of primordial germ cells, culturing these cells to Example 2 produce primordial germ cell-derived cell lines, transforming 10 both the primordial germ cells and the cultured cell lines, and Characterization of VEGF Receptor Antagonists using these transformed cells and cell lines to generate trans genic animals. The efficiency at which transgenic animals are VEGF analogs I83K, E44R, E72RE73R, E67K and Q87K generated is greatly increased, thereby allowing the use of were created and assayed for their ability to bind to KDR and homologous recombination in producing transgenic non-ro 15 to decrease cell proliferation compared to wild-type VEGF. dent animal species. Methods Kits Containing Modified VEGF Proteins VEGF analogs expressed by yeast cells were incubated In a further embodiment, the present invention provides with immobilized KDR-Fc and the ability of the analogs to kits containing a VEGF analog and/or VEGF analog fusion bind to KDR-Fc was assayed. The binding assay was per proteins, which can be used, for instance, for therapeutic or formed as follows: non-therapeutic applications. The kit comprises a container 1. Nunc MaxiSorpTM96 microwell plates were coated with with a label. Suitable containers include, for example, bottles, 150 ng?well KDR-Fc (R&D System, Inc.) and 100 ul 50 mM vials, and test tubes. The containers may be formed from a sodium bicarbonate buffer (15 mM NaCO+35 mM variety of materials such as glass or plastic. The container NaHCO) at pH 9.6. A separate plate was used for each VEGF holds a composition which includes a VEGF analog or VEGF 25 analog and wild-type VEGF tested. fusion protein that is effective for therapeutic or non-thera 2. The plates were incubated at 4°C. overnight. peutic applications, such as described above. The label on the 3. The next day, the wells were washed three times in container indicates that the composition is used for a specific washing buffer (0.05% tween in PBS). therapy or non-therapeutic application, and may also indicate 4. The wells were blocked with PBS with 3% BSA, 0.03% directions for either in vivo or in vitro use, such as those 30 tween for 1 hour at room temperature. described above. 5. After blocking, the wells were washed three times in The kit of the invention will typically comprise the con washing buffer (0.05% tween in PBS). tainer described above and one or more other containers 6. VEGF-A (wild-type or mutant) were added at different comprising materials desirable from a commercial and user concentrations to the wells in 50 ul binding buffer (1% BSA standpoint, including buffers, diluents, filters, needles, 35 and 0.03% tween in PBS). Syringes, and package inserts with instructions for use. The 7. 'I-labeled VEGF-A (wild-type or mutant) at 70,000 kit of the invention may also include a control consisting of cpm/well (PerkinElmer) was added to each well in 50 ul wild-type VEGF such as wild-type VEGF, or VEGFb. binding buffer (1% BSA and 0.03% tween in PBS). The following examples are provided to describe and illus 8. The contents of the wells were mixed and incubated for trate the present invention. As such, they should not be con 40 2 hours at room temperature with slow shaking. strued to limit the scope of the invention. Those in the art will 9. The wells were washed three times with washing buffer appreciate that many other embodiments also fall within the (0.05% tween in PBS). scope of the invention, as it is described herein above and in 10. To each well, 120 ul of lysis buffer (0.2 MNaOH+0.5% the claims. SDS) was added. Plate was shaken vigorously for 20 minutes 45 at room temperature. EXAMPLES 11. The lysis buffer from each well was transferred to an individual tube. The wells were washed with lysis buffer two Example 1 times additional times and combined with the lysis solution buffer in the corresponding tube. Design of VEGF Receptor Antagonists 50 12. The measure of binding for wild-type VEGF-A and various VEGF-A mutants was determined by counting with a VEGF-A antagonists of the present invention were gamma counter. designed to increase receptor binding affinity and decrease The ability of HUVEC endothelial cells to proliferate in the bioactivity as compared to wild-type VEGF-A. One method presence of the VEGF analogs was assayed as follows: by which this was done was by adding a positive charge to the 55 1. HUVEC endothelial cells (passage 6) were seeded into loops of VEGF-A. This approach to design Super-antagonists 96 well plates at 3,000 cells/well using Media-200 with involves a combination of different methods known in the art growth factors and incubated overnight. including but not limited to homology modeling, sequence 2. After overnight incubation, the media was removed and comparisons, charge Scanning mutagenesis, and linking Media 199 (Invitrogen) with 2% dialysis FBS (Invitrogen) monomers and introduction of mutations in the context of 60 was added. linked monomers. 3. Cells were incubated for 20 hours. Vammin, or snake venom VEGF, has been shown to bind to 4. Wild-type VEGF-A and VEGF-A analogs were serial KDR-IgG with high affinity and strongly stimulate prolifera diluted in Media 199 with 2% dialysis FBS in the 96-well tion of vascular endothelial cells in vitro (see Yamazaki et al., plates, starting at 200 ng/well. 2003, J. Biol. Chem. 278, 51985-51988, which is herein 65 5. The media was removed from each well and replaced incorporated by reference in its entirety). VEGF-A receptor with 200 ul/well diluted VEGF media. antagonists were designed based on VEGF is homology to 6. Cells were incubated at 37° C. for 72 hours. US 9,078,860 B2 35 36 7. Cell proliferation was analyzed using Promega's Cell (FIGS. 2A, 3A, 4, 5 and 6). However, analogs E44R and Titer-Glo R. Luminescent Cell Viability Assay. Briefly, Cell EE72/73RR demonstrated little to no change in endothelial Titer buffer was thawed, transferred into CellTilter-Glo sub cell proliferation (FIGS. 2B and 3B). These results show that VEGFs analogs comprising I83K can effectively function strate, and mixed well to make substrate mixture. 100 ul as a VEGF-A receptor antagonist. Further, although VEGF-A growth media was removed from each well into a new 96 well analogs E44R and EE72/73RR were unable to decrease plate and mixed well with 100 ul substrate mixture. The plates endothelial cell proliferation alone, when added to I83K, were shaken for 2 minutes and incubated at room temperature these modifications have the potential of further increasing for an additional ten minutes. receptor binding affinity. 8. Plates were read for luminescent signal using a plate All publications, patents and patent applications discussed reader with integration time set at 250 mS (Tecan). 10 in this application are incorporated herein by reference. Analysis While in the foregoing specification this invention has been The receptor binding affinity of the I83K analog to KDR described in relation to certain preferred embodiments, Fc was slightly less than that of wild-type VEGF-A (FIG. thereof, and may details have been set forth for purposes of 1A). However, the I83K analog demonstrated a significant 15 illustration, it will be apparent to those skilled in the art that decrease in endothelial cell proliferation compared to wild- the invention is susceptible to additional embodiments and type VEGF-A (FIG. 1B). VEGF-A analogs E44R, EE72/ that certain of the details described herein may be varied 73RR, E67K and Q87K all demonstrated an increase in considerably without departing from the basic principles of receptor cell binding affinity compared to wild-type VEGF-A the invention.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 73

<21 Os SEQ ID NO 1 &211s LENGTH: 576 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 atgaactitt c togctgtc.ttg ggtgcattgg agcc ttgcct togctgcticta cct c caccat 60 gccaagtggit Cocaggctgc acccatggca galaggaggag ggcagaatca to acgaagtg 12O gtgaagttca tsatgtct a to agcgcagc tact.gc.cat C caatcgaga C cctggtggac 18O

atct tcc agg agtaccctga tigagat.cgag tacatct tca agc.cat cotg tdtgcc cctd 24 O

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcc.cac taggagt cc 3 OO aac at Cacca toagattat gcggatcaaa cct caccalag gc.ca.gcacat aggagagatg 360 agct tcc tac agcacaacaa atgtgaatgc agaccalaaga aagatagagc aaga caagaa 42O aatcc ctgtg ggccttgct C agagcggaga aagcatttgt ttgtacaaga toccagacg 48O

tgtaaatgtt Cotgcaaaaa cacagacitcg cgttgcaagg caggcagot tagttaaac 54 O

gaacgtactt gcagatgtga caa.gc.cgagg cqgtga 576

<21 Os SEQ ID NO 2 &211s LENGTH: 191 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 2 Met Asn. Phe Lieu. Lieu. Ser Trp Val His Trp Ser Lieu Ala Lieu Lleu Lieu. 1. 5 1O 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 3 O Gly Gly Glin Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Glin 35 4 O 45 Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu SO 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Val Pro Leu

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95 US 9,078,860 B2 37 38 - Continued

Thr Glu Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 12 O 125 Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Asn Pro Cys Gly 13 O 135 14 O Pro Cys Ser Glu Arg Arg Llys His Leu Phe Val Glin Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin 1.65 17O 17s Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185 19 O

<210s, SEQ ID NO 3 &211s LENGTH: 26 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Met Asn. Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala 2O 25

<210s, SEQ ID NO 4 &211s LENGTH: 1.65 212. TYPE PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thir Lieu. 2O 25 3O Val Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 Pro Ser Cys Val Pro Lieu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Glin Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Llys His Lieu. Phe 115 12 O 125 Val Glin Asp Pro Glin Thr Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser 13 O 135 14 O

Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg Cys 145 150 155 160

Asp Llys Pro Arg Arg 1.65

<210s, SEQ ID NO 5 &211s LENGTH: 147 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 9,078,860 B2 39 40 - Continued

<4 OOs, SEQUENCE: 5

Met Asn. Phe Lieu. Lell Ser Trp Wall His Trp Ser Lell Ala Luell Luell Luell 1. 1O 15

Tyr Luell His His Ala Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25

Gly Gly Glin Asn His His Glu Wall Val Lys Phe Met Asp Wall Glin 35 4 O 45

Arg Ser His Pro Ile Glu Thir Lieu. Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Glu Ile Glu Tyr Ile Phe Lys Pro Ser Wall Pro Luell 65 70

Met Arg Gly Gly Cys Asn Asp Glu Gly Lell Glu Wall Pro 85 90 95

Thir Glu Glu Ser Asn Ile Thir Met Glin Ile Met Arg Ile Lys Pro His 105 11 O

Glin Gly Glin His Ile Gly Glu Met Ser Phe Luell Glin His Asn 115 12 O 125

Glu Cys Arg Pro Asp Arg Ala Arg Glin Glu Asn Asp 13 O 135 14 O

Pro Arg Arg 145

<210s, SEQ ID NO 6 &211s LENGTH: 121 212. TYPE PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Wall Wall Lys 1. 1O 15

Phe Met Asp Wall Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu Thir Luell 2O 25

Wall Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe 35 4 O 45

Pro Ser Wall Pro Lell Met Arg Gly Cys Asn Asp Glu SO 55 6 O

Gly Luell Glu Wall Pro Thir Glu Glu Ser ASn Ile Thir Met Glin Ile 65 70 8O

Met Arg Ile Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95

Lell Glin His Asn Glu Arg Pro Lys Asp Arg Ala Arg 1OO 105 11 O

Glin Glu Asn Asp Pro Arg Arg 115 12 O

<210s, SEQ ID NO 7 &211s LENGTH: 171 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 7 Met Asn. Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25 US 9,078,860 B2 41 - Continued Gly Gly Glin Asn His His Glu Val Val Llys Phe Met Asp Val Tyr Glin 35 4 O 45 Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu SO 55 6 O Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Val Pro Leu 65 70 7s 8O Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 12 O 125 Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Lys Llys Ser Val 13 O 135 14 O Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg Llys Llys Ser Arg Tyr 145 150 155 160 Llys Ser Trp Ser Val Cys Asp Llys Pro Arg Arg 1.65 17O

<210s, SEQ ID NO 8 &211s LENGTH: 145 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 8 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thir Lieu. 2O 25 3O Val Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 Pro Ser Cys Val Pro Lieu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Glin Glu Lys Llys Ser Val Arg Gly Lys Gly Lys Gly Glin Lys Arg Llys 115 12 O 125 Arg Llys Llys Ser Arg Tyr Lys Ser Trp Ser Val Cys Asp Llys Pro Arg 13 O 135 14 O Arg 145

<210s, SEQ ID NO 9 &211s LENGTH: 174 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 9 Met Asn. Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 3O US 9,078,860 B2 43 44 - Continued Gly Gly Glin Asn His His Glu Val Val Llys Phe Met Asp Val Tyr Glin 35 4 O 45 Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu SO 55 6 O Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Val Pro Leu 65 70 7s 8O Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 12 O 125 Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Asn Pro Cys Gly 13 O 135 14 O Pro Cys Ser Glu Arg Arg Llys His Leu Phe Val Glin Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser Arg Cys Llys Met 1.65 17O

<210s, SEQ ID NO 10 &211s LENGTH: 148 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 10 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thir Lieu. 2O 25 3O Val Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 Pro Ser Cys Val Pro Lieu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Glin Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Llys His Lieu. Phe 115 12 O 125 Val Glin Asp Pro Glin Thr Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser 13 O 135 14 O Arg Cys Llys Met 145

<210s, SEQ ID NO 11 &211s LENGTH: 606 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 atgaactittctgctgtc.ttg ggtgcattgg agccttgcct togctgcticta cct coac cat 6 O gccalagtggit C cc aggctgc acc catggca gaaggaggag ggcagaatca t cacgaagtg 12 O gtgaagttca tatgtcta t cagcgcago tactgcc at C caatcgaga C cctggtggac 18O US 9,078,860 B2 45 46 - Continued atct tccagg agtaccctga tgagat.cgag taCat Cttca agc catcCtg ttgc.ccctg 24 O atgcgatgcg ggggctgctg Caatgacgag ggCCtggagt gtgtgcc cac tgaggagt cc 3OO aaCatCacca tgcagattat gcggatcaaa cct caccaag gcc agcacat aggagagatg 360 agct tcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc alagacaagaa aatc.cc tigtg ggccttgctic agagdggaga aag catttgt ttgtacaaga tcc.gcagacg tgtaaatgtt cctgcaaaaa Cacagactic cgttgcaagg cgaggcagct tgagttaaac 54 O gaacgtactt gcagat ct ct Caccaggaaa gactgataca gaacgat.cga tacagaaacc acgctg 606

<210s, SEQ ID NO 12 &211s LENGTH: 191 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 12

Met Asin Phe Leu Lleu Ser Trp Val His Trp Ser Lell Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25

Gly Gly Glin Asn His His Glu Wall Val Llys Phe Met Asp Wall Tyr Glin 35 4 O 45

Arg Ser Cys His Pro Ile Glu Thir Lieu Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Glu Ile Glu Tyr Ile Phe Llys Pro Ser Wall Pro Luell 65 70 7s 8O

Met Arg Gly Gly Cys Cys Asn Asp Glu Gly Lell Glu Wall Pro 85 90 95

Thir Glu Glu Ser Asn. Ile Thir Met Glin Ile Met Arg Ile Lys Pro His 105 11 O

Glin Gly Glin His Ile Gly Glu Met Ser Phe Lieu. Glin His Asn 115 12 O 125

Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Asn Pro 13 O 135 14 O

Pro Ser Glu Arg Arg Llys His Lieu. Phe Wall Glin Asp Pro Gn. Thir 145 150 155 160

Ser Cys Lys Asn Thr Asp Ser Arg Ala Arg Glin 1.65 17O 17s

Lell Glu Luell Asin Glu Arg Thr Cys Arg Ser Lieu. Thir Arg Lys Asp 185 19 O

<210s, SEQ ID NO 13 &211s LENGTH: 1.65 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13

Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Wall Val Lys 1. 5 1O 15

Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu. 25

Wall Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45

Pro Ser Cys Val Pro Leu Met Arg Cys Asn Asp Glu SO 55 6 O US 9,078,860 B2 47 48 - Continued

Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Glin Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Llys His Lieu. Phe 115 12 O 125 Val Glin Asp Pro Glin Thr Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser 13 O 135 14 O Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg Ser 145 150 155 160 Lieu. Thir Arg Lys Asp 1.65

<210s, SEQ ID NO 14 &211s LENGTH: 209 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 14 Met Asn. Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 3O Gly Gly Glin Asn His His Glu Val Val Llys Phe Met Asp Val Tyr Glin 35 4 O 45 Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu SO 55 6 O Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Val Pro Leu 65 70 7s 8O Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 12 O 125 Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Lys Llys Ser Val 13 O 135 14 O Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg Llys Llys Ser Arg Pro 145 150 155 160 Cys Gly Pro Cys Ser Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro 1.65 17O 17s Glin Thr Cys Lys Cys Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala 18O 185 19 O

Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg 195 2OO 2O5

Arg

<210s, SEQ ID NO 15 &211s LENGTH: 183 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15 US 9,078,860 B2 49 50 - Continued

Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Wal Wall Lys 15

Phe Met Asp Wall Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu Thir Luell 2O 25

Wall Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe 35 4 O 45

Pro Ser Wall Pro Lell Met Arg Gly Gly Cys Asn Asp Glu SO 55 6 O

Gly Luell Glu Wall Pro Thir Glu Glu Ser ASn Ile Thir Met Glin Ile 65 70 8O

Met Arg Ile Pro His Glin Gly Glin His Ile Gly Glu Met Ser Phe 85 90 95

Lell Glin His Asn Glu Arg Pro Asp Arg Ala Arg 1OO 105 11 O

Glin Glu Lys Ser Wall Arg Gly Lys Gly Lys Gly Glin Arg 115 12 O 125

Arg Lys Ser Arg Pro Cys Gly Pro Ser Glu Arg His 13 O 135 14 O

Lell Phe Wall Glin Asp Pro Glin Thir Cys Ser Asn Thir 145 150 155 160

Asp Ser Arg Lys Ala Arg Glin Luell Glu Luell Asn Glu Arg Thir 1.65 17O 17s

Arg Asp Lys Pro Arg Arg 18O

SEQ ID NO 16 LENGTH: 215 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 16

Met Asn. Phe Lieu. Lell Ser Trp Wall His Trp Ser Lell Ala Luell Luell Luell 1. 5 1O 15

Tyr Luell His His Ala Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25

Gly Gly Glin Asn His His Glu Wall Wall Phe Met Asp Wall Glin 35 4 O 45

Arg Ser His Pro Ile Glu Thir Luell Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Glu Ile Glu Tyr Ile Phe Pro Ser Wall Pro Luell 65 70

Met Arg Gly Gly Cys Asn Asp Glu Gly Lell Glu Wall Pro 85 90 95

Thir Glu Glu Ser Asn Ile Thir Met Glin Ile Met Arg Ile Lys Pro His 105 11 O

Glin Gly Glin His Ile Gly Glu Met Ser Phe Luell Glin His Asn 115 12 O 125

Glu Cys Arg Pro Asp Arg Ala Arg Glin Glu Ser Wall 13 O 135 14 O

Arg Gly Gly Gly Glin Arg Arg Ser Arg Tyr 145 150 155 160

Ser Trp Ser Wall Pro Gly Pro Cys Ser Glu Arg Lys His 1.65 17O 17s

Lell Phe Wall Glin Asp Pro Glin Thir Cys Ser Lys Asn Thir 18O 185 19 O US 9,078,860 B2 51 - Continued

Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys 195 2OO 2O5 Arg Cys Asp Llys Pro Arg Arg 21 O 215

<210s, SEQ ID NO 17 &211s LENGTH: 189 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 17 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thir Lieu. 2O 25 3O Val Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 Pro Ser Cys Val Pro Lieu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Gln Glu Lys Llys Ser Val Arg Gly Lys Gly Lys Gly Gln Lys Arg Llys 115 12 O 125 Arg Llys Llys Ser Arg Tyr Lys Ser Trp Ser Val Pro Cys Gly Pro Cys 13 O 135 14 O Ser Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Glin Thir Cys Llys 145 150 155 160 Cys Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu 1.65 17O 17s Lieu. Asn. Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185

<210s, SEQ ID NO 18 &211s LENGTH: 232 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18 Met Asn. Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 1O 15 Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 3O Gly Gly Glin Asn His His Glu Val Val Llys Phe Met Asp Val Tyr Glin 35 4 O 45

Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Val Pro Leu 65 70 7s 8O

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95

Thr Glu Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O US 9,078,860 B2 53 - Continued

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 12 O 125 Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Lys Llys Ser Val 13 O 135 14 O Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg Llys Llys Ser Arg Tyr 145 150 155 160 Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Lieu Met Pro Trp 1.65 17O 17s Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 18O 185 19 O His Leu Phe Val Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195 2OO 2O5 Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr 21 O 215 22O Cys Arg Cys Asp Llys Pro Arg Arg 225 23 O

<210s, SEQ ID NO 19 &211s LENGTH: 2O6 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 1O 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu Thir Lieu 2O 25 3O Val Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 Pro Ser Cys Val Pro Lieu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Glin Ile 65 70 7s 8O Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95 Lieu. Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Lys Asp Arg Ala Arg 1OO 105 11 O Glin Glu Lys Llys Ser Val Arg Gly Lys Gly Lys Gly Glin Lys Arg Llys 115 12 O 125 Arg Llys Llys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg 13 O 135 14 O Cys Cys Lieu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro 145 150 155 160 Cys Ser Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Glin Thr Cys 1.65 17O 17s

Lys Cys Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. 18O 185 19 O

Glu Lieu. Asn. Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 195 2OO 2O5

<210s, SEQ ID NO 2 O &211s LENGTH: 576 &212s. TYPE: DNA <213> ORGANISM: Macaca fascicularis

<4 OOs, SEQUENCE: 2O US 9,078, 860 B2 55 56 - Contin lued atgaacttitc ggtgcattgg agccttgcct tgctgctgta CCt CCaC cat 6 O gccalagtggit CCC aggctgc acccatggca galaggaggag ggcagaatca t cacgaagtg 12 O gtgaagttca tggatgtcta t cagcgcagc tactgccatc Caatcgagac Cctggtggac 18O atct tccagg agtaccctga tgagattgag taCat Cttca agc catcctg tgtgc.ccctg 24 O atgcgatgtg ggggctgctg Caatgacgag ggCCtggagt gtgtgcc cac tgaggagt cc 3OO aaCatCacca tgcagattat gcggatcaaa cct caccaag gcc agcacat aggagagatg 360 agct tcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc alagacaagaa aatc.cc tigtg ggccttgctic agagdggaga aag catttgt ttgtacaaga tcc.gcagacg tgtaaatgtt cctgcaaaaa Cacagactic cgttgcaagg cgaggcagct tgagttaaac 54 O gaacgtactt gcagatgtga caa.gc.cgagg cggtga 576

<210s, SEQ ID NO 21 &211s LENGTH: 191 212. TYPE : PRT &213s ORGANISM: Macaca fascicularis

<4 OOs, SEQUENCE: 21 Met Asin Phe Leu Lleu Ser Trp Val His Ser Lieu Ala Lieu. Lieu. Luell 1. 5 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25 3O

Gly Gly Glin Asn His His Glu Wall Wall Phe Met Asp Wall Tyr Glin 35 4 O 45

Arg Ser Cys His Pro Ile Glu Thir Luell Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Glu Ile Glu Tyr Ile Phe Pro Ser Cys Val Pro Leu 65 70 8O

Met Arg Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Wall Pro 85 90 95

Thir Glu Glu Ser Asn. Ile Thir Met Glin Ile Met Arg Ile Llys Pro His 105 11 O

Glin Gly Glin His Ile Gly Glu Met Ser Phe Luell Glin His Asn 115 12 O 125

Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Asn. Pro 13 O 135 14 O

Pro Ser Glu Arg Arg Llys His Luell Phe Wall Glin Asp Pro Gn. Thir 145 150 155 160

Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Glin 1.65 17O 17s

Lell Glu Luell Asin Glu Arg Thr Cys Arg Asp Llys Pro Arg Arg 185 19 O

<210s, SEQ ID NO 22 &211s LENGTH: 1.65 212. TYPE : PRT <213> ORGANISM: Macaca fascicularis

<4 OOs, SEQUENCE: 22 Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Val Val Lys 1. 5 15 Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thir Lieu. 25 US 9,078,860 B2 57 58 - Continued

Wall Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45

Pro Ser Val Pro Leu Met Arg Cys Cys Asn Asp Glu SO 55 6 O

Gly Luell Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thir Met Glin Ile 65 70 7s 8O

Met Arg Ile Llys Pro His Glin Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95

Lell Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Asp Arg Ala Arg 105 11 O

Glin Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Lieu. Phe 115 12 O 125

Wall Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thir Asp Ser 13 O 135 14 O

Arg Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thir Arg Cys 145 150 155 160

Asp Pro Arg Arg 1.65

<210s, SEQ ID NO 23 &211s LENGTH: 573 &212s. TYPE: DNA &213s ORGANISM: Bos talurus

<4 OOs, SEQUENCE: 23 atgaactittctgct ct cittg ggtacattgg agccttgcct tgctgcticta CCttcac Cat 6 O gccalagtggit CCC aggctgc acccatggca galaggagggc agaaaccc.ca cgaagttggtg 12 O aagttcatgg atgtc.tacca gcgcagottc tgc.cgt.ccca tcq agaccct ggtggaCat C 18O titcCaggagt acccagatga gattgagttc attittcaagc gcc cctgatg 24 O cggtgcgggg gctgctgtaa tgacgaaagt Ctggagtgttg tgcc.cactga ggagttcaac 3OO atcaccatgc agattatgcg gatcaaac ct caccaaag.cc agcacat agg agagatgagc 360 titcc tacago ataacaaatg tgaatgcaga cCaaagaaag ataaa.gcaa.g gcaagaaaat

CCCtgtgggc cittgct caga gcggagaaag catttgtttg tacaagatcc gCagacgtgt aaatgttcct gcaaaaacac agact cqcgt. tgcaaggcga ggcagcttga gttaaacgaa 54 O cg tacttgca gatgtgacaa gcc.gaggcgg tga

<210s, SEQ ID NO 24 &211s LENGTH: 190 212. TYPE : PRT &213s ORGANISM: Bos taurus

<4 OOs, SEQUENCE: 24 Met Asin Phe Leu Lleu Ser Trp Val His Trp Ser Lell Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25 3O

Gly Glin Lys Pro His Glu Wal Wall Llys Phe Met Asp Wall Glin Arg 35 4 O 45

Ser Phe Arg Pro Ile Glu Thr Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Phe Ile Phe Llys Pro Ser Wall Pro Luell Met 65 70

Arg Gly Gly Cys Cys Asn Asp Glu Ser Lieu. Glu Wall Pro Thir US 9,078,860 B2 59 60 - Continued

85 90 95

Glu Glu Phe Asn. Ile Thir Met Glin Ile Met Arg Ile Pro His Glin 105 11 O

Ser Glin His Ile Gly Glu Met Ser Phe Leul Glin His Asn Cys Glu 115 12 O 125

Arg Pro Llys Lys Asp Lys Ala Arg Glin Glu Asn Pro Gly Pro 13 O 135 14 O

Cys Ser Glu Arg Arg Llys His Lieu. Phe Wall Glin Asp Pro Glin Thr Cys 145 150 155 160

Ser Cys Lys Asn. Thir Asp Ser Arg Cys Ala Arg Gln Lieu. 1.65 17O 17s

Glu Luell Asn Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185 19 O

SEO ID NO 25 LENGTH: 164 TYPE : PRT ORGANISM: Bos talurus

< 4 OOs SEQUENCE: 25 Ala Pro Met Ala Glu Gly Gly Glin Llys Pro His Glu Wall Wall Llys Phe 1. 5 1O 15

Met Asp Wall Tyr Glin Arg Ser Phe Cys Arg Pro Ile Glu Thir Lieu Wall 25

Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Phe Ile Phe Llys Pro 35 4 O 45

Ser Cys Wall Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Ser SO 55 6 O

Lell Glu Wall Pro Thr Glu Glu Phe Asn. Ile Thir Met Glin Ile Met 65 70 7s 8O

Arg Ile Pro His Glin Ser Glin His Ile Gly Glu Met Ser Phe Lieu. 85 90 95

Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Llys Asp Ala Arg Glin 105 11 O

Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg His Luell Phe Wall 115 12 O 125

Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Llys Asn Thir Asp Ser Arg 13 O 135 14 O

Cys Ala Arg Glin Lieu. Glu Lieu. Asin Glu Arg Thir Arg 145 150 155 160

Lys Pro Arg Arg

<210s, SEQ ID NO 26 &211s LENGTH: 645 &212s. TYPE: DNA <213> ORGANISM: Canis familiaris

<4 OOs, SEQUENCE: 26 atgaactittctgct ct cittg ggtgcattgg agccttgcct tgctgcticta CCt CCaC cat 6 O gccalagtggit CCC aggctgc gcc tatggca ggaggaga.gc a Calaa CCCC a cgaagttggtg 12 O aagttcatgg acgt.ctacca gcgcagot ac tgc.cgt.ccca ttgagaccct ggtggaCat C 18O titcCaggagt accctgacga gat.cgagtac atc.ttcaagc catcc togcgt. gcc cctgatg 24 O cggtgttgggg gctgctgtaa tgatgagggc Ctagagtgcg tgcc.cactga ggagttcaac 3OO atcaccatgc agattatgcg gatcaaac ct Cat Caaggcc agcacat agg ggagatgagt 360 US 9,078,860 B2 61 62 - Continued titcc togcago atagdaaatg tgaatgcaga cCaaagaaag atagagcaa.g gcaagaaaaa aaat caattic gaggaaaggg galaggggcaa. aaaagaaagc gcaagaaatc ccggtataaa

CCCtggagcg titcCctgtgg gcc ttgctica gag.cggagaa agcatttgtt tgtacaagat 54 O cc.gcagacgt gtaaatgttc ctgcaaaaac acagact cqc gttgcaaggc gaggcagctt gagittaaacg aacgtacttg Cagatgtgac aag.ccgaggc ggtga 645

<210s, SEQ ID NO 27 &211s LENGTH: 214 212. TYPE : PRT &213s ORGANISM: Canis familiaris

<4 OOs, SEQUENCE: 27

Met Asn. Phe Lieu. Lieu. Ser Trp Val His Trp Ser Lell Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Gly Gly 25

Glu His Lys Pro His Glu Wal Wall Llys Phe Met Asp Wall Glin Arg 35 4 O 45

Ser Tyr Arg Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Wall Pro Luell Met 65 70 7s

Arg Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu Wall Pro Thir 85 90 95

Glu Glu Phe Asn Ile Thir Met Glin Ile Met Arg Ile Pro His Glin 105 11 O

Gly Glin His Ile Gly Glu Met Ser Phe Leul Glin His Ser Cys Glu 115 12 O 125

Arg Pro Lys Asp Arg Ala Arg Glin Glu Lys Ser Ile Arg 13 O 135 14 O

Gly Lys Gly Gly Glin Lys Arg Ser Arg 145 150 155 160

Pro Trp Ser Wall Pro Cys Gly Pro Cys Ser Glu Arg His Lieu. 1.65 17O 17s

Phe Wall Glin Asp Pro Gln Thr Cys Lys Cys Ser Asn Thir Asp 18O 185 19 O

Ser Arg Cys Ala Arg Glin Lieu. Glu Lieu. Asn Glu Arg Thir 195 2O5

Asp Pro Arg Arg 21 O

<210s, SEQ ID NO 28 &211s LENGTH: 188 212. TYPE : PRT <213> ORGANISM: Canis familiaris

<4 OOs, SEQUENCE: 28

Ala Pro Met Ala Gly Gly Glu. His Llys Pro His Glu Wall Wall Llys Phe 1. 5 1O 15

Met Asp Val Tyr Glin Arg Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Wall 25

Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro 35 45

Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Asn Asp Glu Gly US 9,078,860 B2 63 64 - Continued

SO 55 6 O

Lieu. Glu. Cys Val Pro Thr Glu Glu Phe Asn. Ile Thir Met Glin Ile Met 65 70 7s 8O

Arg Ile Llys Pro His Glin Gly Glin His Ile Gly Glu Met Ser Phe Lieu. 85 90 95 Glin His Ser Lys Cys Glu. Cys Arg Pro Llys Llys Asp Arg Ala Arg Glin 105 11 O Glu Lys Llys Ser Ile Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg 115 12 O 125

Llys Llys Ser Arg Tyr Llys Pro Trip Ser Wall Pro Cys Gly Pro Cys Ser 13 O 135 14 O Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Gln Thr Cys Lys Cys 145 150 155 160 Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. 1.65 17O 17s Asn Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185

<210s, SEQ ID NO 29 &211s LENGTH: 651 &212s. TYPE: DNA <213> ORGANISM: Gallus gallus <4 OOs, SEQUENCE: 29 atgaactittctgct cacttg gatcc actgg cgctgct ct a tictgcagagc 6 O gC9gagttgtt Caaggctgc tcCggCCCtg ggggatgggg agcggaagcc Caacgaagtt 120 atcaaatticc toggaagttcta cgaacgcagc ttctgcagga Caattgaga C cctggtggac 18O attitt.ccagg agtaccctga tgaggtggag taCat attca ggc catcCtg tdtgcct ctg 24 O atgagatgtg cgggttgctg cgg.cgatgag ggcct agaat gtgtc.cctgt ggatgtgtac 3OO aacgtcacga tiggagat.cgc aagaattaaa c cc catcaga gtcagcacat agcgcacatg 360 agctitc.ttac agcacagtaa atgtgactgc agaccaaaga aagatgtcaa aaataaacaa gaaaaaaaat caaag.cgagg aaaggggalag ggtcaaaaga gaaag.cgcaa gaaaggc.cgg tacaaaccac cca.gctitt ca Ctgtgagc ct tgcticagaga ggagaaagca Cttgtttgta 54 O caagat cocc agacct gtaa atgttcctgc aaatt cacag act cacgttg Caagt cagg

Cagcttgagt taaacgagcg cacttgcaga tgttgaaaaac cgaga.cggtg a 651

<210s, SEQ ID NO 3 O &211s LENGTH: 216 212. TYPE: PRT <213> ORGANISM: Gallus gallus

<4 OOs, SEQUENCE: 30 Met Asin Phe Leu Lleu. Thir Trp Ile His Trp Gly Lieu Ala Ala Lieu. Lieu. 1. 5 1O 15

Tyr Lieu. Glin Ser Ala Glu Lieu. Ser Lys Ala Ala Pro Ala Lieu. Gly Asp 25 3O

Gly Glu Arg Llys Pro Asn. Glu Val Ile Llys Phe Lieu. Glu Val Tyr Glu 35 4 O 45

Arg Ser Phe Cys Arg Thr Ile Glu Thir Lieu Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Asp Glu Val Glu Tyr Ile Phe Arg Pro Ser Cys Val Pro Leu 65 70 7s 8O US 9,078,860 B2 65 66 - Continued

Met Arg Ala Gly Gly Asp Glu Gly Lell Glu Cys Wall Pro 85 90 95

Wall Asp Wall Tyr Asn Wall Thir Met Glu Ile Ala Arg Ile Lys Pro His 1OO 105 11 O

Glin Ser Glin His Ile Ala His Met Ser Phe Luell Glin His Ser 115 12 O 125

Asp Cys Arg Pro Lys Asp Wall Asn Glin Glu Ser 13 O 135 14 O

Lys Arg Gly Gly Lys Gly Glin Arg Lys Arg Gly Arg 145 150 155 160

Pro Pro Ser Phe His Glu Pro Ser Glu Arg Arg 1.65 17O

His Luell Phe Wall Glin Asp Pro Glin Thir Ser Cys Phe 18O 185 19 O

Thir Asp Ser Arg Cys Ser Arg Glin Luell Glu Lell Asn Glu Arg Thir 195 2OO 2O5

Arg Glu Lys Pro Arg Arg 21 O 215

<210s, SEQ ID NO 31 &211s LENGTH: 190 212. TYPE : PRT &213s ORGANISM: Gallus gallus

<4 OOs, SEQUENCE: 31

Ala Pro Ala Lieu. Gly Asp Gly Glu Arg Lys Pro Asn Glu Wall Ile 1. 5 15

Phe Luell Glu Wall Tyr Glu Arg Ser Phe Arg Thir Ile Glu Thir Luell 2O 25

Wall Asp Ile Phe Glin Glu Pro Asp Glu Wall Glu Tyr Ile Phe Arg 35 4 O 45

Pro Ser Wall Pro Lell Met Arg Ala Gly Cys Gly Asp Glu SO 55 6 O

Gly Luell Glu Wall Pro Wall Asp Wall ASn Wall Thir Met Glu Ile 65 70

Ala Arg Ile Pro His Glin Ser Glin His Ile Ala His Met Ser Phe 85 90 95

Lell Glin His Ser Lys Asp Arg Pro Asp Wall Asn 1OO 105 11 O

Glin Glu Lys Ser Arg Gly Gly Gly Glin Arg 115 12 O 125

Arg Gly Arg Tyr Pro Pro Ser Phe His Glu Pro 13 O 135 14 O

Cys Ser Glu Arg Lys His Luell Phe Wall Glin Asp Pro Glin Thir Cys 145 150 155 160

Ser Lys Phe Thir Asp Ser Arg Ser Arg Glin Luell 1.65 17O 17s

Glu Luell Asn Glu Arg Thir Arg Cys Glu Lys Pro Arg Arg 18O 185 19 O

SEQ ID NO 32 LENGTH: TYPE: DNA ORGANISM: Equus caballus

< 4 OOs SEQUENCE: 32 US 9,078,860 B2 67 - Continued atgaacttitc tgct ct cittg ggtgcattgg agccttgcct togctgcticta cct coaccat 6 O gccalagtggit CCC aggctgc acccatggca gaaggagagc ataaaac cca tgaagttggtg 12 O aagttcatgg acgt.ctacca gcgcagot ac tgc.cgt.ccaa to gag accct ggtggaCat C 18O titcCaggagt accc.cgatga gat.cgagtac atc.ttcaagc catcc tdtgt gcc cctgatg 24 O cggtgttgggg gctgctgcaa. cgacgagggc Ctagagtgcg toccactgc ggagttcaac 3OO atcaccatgc agattatgcg gatcaaac ct caccaaagcc aacacatagg agagatgagt 360 titcc tacago at agcaaatg tgaatgcaga cCaaagaaag at aaa.gcaa.g gcaagaaaat

CCCtgtgggc cittgct caga gcggagaaag catttgtttg tacaagatcc gCagacgtgt aaatgttcct gcaaaaacac agact cqcgt. tgcaaggcga ggcagcttga gttaaacgaa 54 O cg tacttgca gatgtgacaa gcc.gaggcgg tga

SEQ ID NO 33 LENGTH: 190 TYPE : PRT ORGANISM: Equus caballus

< 4 OOs SEQUENCE: 33 Met Asin Phe Leu Lleu Ser Trp Val His Trp Ser Lieu. Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25

Glu His Lys Thir His Glu Wal Wall Llys Phe Met Asp Wall Glin Arg 35 4 O 45

Ser Tyr Arg Pro Ile Glu Thr Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Cys Wall Pro Luell Met 65 70 7s 8O

Arg Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu Cys Wall Pro Thir 85 90 95

Ala Glu Phe Asn. Ile Thir Met Glin Ile Met Arg Ile Pro His Glin 105 11 O

Ser Glin His Ile Gly Glu Met Ser Phe Lieu. Glin His Ser Cys Glu 115 12 O 125

Arg Pro Llys Lys Asp Lys Ala Arg Glin Glu Asn Pro Gly Pro 13 O 135 14 O

Cys Ser Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Gln Thr Cys 145 150 155 160

Ser Cys Lys Asn. Thir Asp Ser Arg Cys Llys Ala Arg Gln Lieu. 1.65 17O 17s

Glu Luell Asin Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185 19 O

SEQ ID NO 34 LENGTH: 164 TYPE : PRT ORGANISM: Equus caballus

< 4 OOs SEQUENCE: 34

Ala Pro Met Ala Glu Gly Glu. His Lys Thr His Glu Wall Wall Llys Phe 1. 5 1O 15

Met Asp Val Tyr Glin Arg Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Wall 25 3O

Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro US 9,078,860 B2 69 70 - Continued

35 4 O 45

Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly SO 55 6 O

Lieu. Glu. Cys Val Pro Thr Ala Glu Phe Asin Ile Thir Met Glin Ile Met 65 70 7s 8O

Arg Ile Llys Pro His Glin Ser Gln His Ile Gly Glu Met Ser Phe Lieu. 85 90 95

Glin His Ser Lys Cys Glu. Cys Arg Pro Llys Llys Asp Ala Arg Glin 1OO 105 11 O

Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg His Luell Phe Wall 115 12 O 125

Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thir Asp Ser Arg 13 O 135 14 O

Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thir Arg 145 150 155 160 Llys Pro Arg Arg

<210s, SEQ ID NO 35 &211s LENGTH: 573 &212s. TYPE: DNA <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 35 atgaactitt C totgtcttg ggtgcactgg accctggctt tactgctgta CCt CCaC cat 6 O gccalagtggit C cc aggctgc acccacgaca gaaggagagc agalagtocca tgaagtgat C 12 O aagttcatgg acgt.ctacca gcgaagctac to cqtccala ttgagaccct ggtggaCat C 18O titcCaggagt accc.cgacga gatagagtac atcttcaagc gcc.gctgatg 24 O cgctgtgcag gctgctgtaa catgaagcc ctggagtgcg tgcc.cacgt.c agaga.gcaac 3OO atcaccatgc agat catgcg gatcaaacct caccaaag.cc agcacat agg agagatgagc 360 titcc tacagc acago.cgatg taatgcaga C caaagaaag acaggacaaa gcc agaaaat Cactgtgagc Cttgttcaga gcggagaaag catttgtttg tccaagatcc gCagacgtgt aaatgttcct gcaaaaacac agact cqcgt to aaggcga ggcagcttga gttaaacgaa 54 O cgtacttgca gatgtgacaa gCC gaggcgg ta

<210s, SEQ ID NO 36 &211s LENGTH: 214 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 36

Met Asin Phe Leu Lleu Ser Trp Val His Trp Thr Lell Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Thir Thir Glu Gly 2O 25 3O

Glu Gln Lys Ser His Glu Val Ile Llys Phe Met Asp Wall Glin Arg 35 4 O 45

Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Wall Pro Luell Met 65 70 7s

Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala Lieu. Glu Wall Pro Thir 85 90 95 US 9,078,860 B2 71 - Continued Ser Glu Ser Asn Ile Thr Met Glin Ile Met Arg Ile Llys Pro His Glin 1OO 105 11 O Ser Gln His Ile Gly Glu Met Ser Phe Leu Gln His Ser Arg Cys Glu 115 12 O 125 Cys Arg Pro Llys Lys Asp Arg Thr Llys Pro Glu Lys Llys Ser Val Arg 13 O 135 14 O Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg Llys Llys Ser Arg Phe Lys 145 150 155 160 Ser Trp Ser Val His Cys Glu Pro Cys Ser Glu Arg Arg Llys His Lieu. 1.65 17O 17s Phe Val Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp 18O 185 19 O Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg 195 2OO 2O5 Cys Asp Llys Pro Arg Arg 21 O

<210s, SEQ ID NO 37 &211s LENGTH: 188 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 37 Ala Pro Thir Thr Glu Gly Glu Gln Lys Ser His Glu Val Ile Llys Phe 1. 5 1O 15 Met Asp Val Tyr Glin Arg Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Val 2O 25 30 Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro 35 4 O 45 Ser Cys Val Pro Lieu Met Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala SO 55 6 O Lieu. Glu. Cys Val Pro Thir Ser Glu Ser Asn Ile Thr Met Glin Ile Met 65 70 7s 8O Arg Ile Llys Pro His Glin Ser Gln His Ile Gly Glu Met Ser Phe Leu 85 90 95 Glin His Ser Arg Cys Glu. Cys Arg Pro Llys Lys Asp Arg Thr Llys Pro 1OO 105 11 O Glu Lys Llys Ser Val Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg 115 12 O 125 Lys Llys Ser Arg Phe Lys Ser Trp Ser Val His Cys Glu Pro Cys Ser 13 O 135 14 O Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Glin Thr Cys Lys Cys 145 150 155 160 Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. 1.65 17O 17s Asn Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185

<210s, SEQ ID NO 38 &211s LENGTH: 573 &212s. TYPE: DNA <213s ORGANISM: Sus scrofa

<4 OOs, SEQUENCE: 38 atgaactittctgctgtc.ttg ggtgcattgg agccttgcct togctgcticta cct coac cat 6 O gccalagtggit C cc aggctgc acc catggca gaaggagacic agaalacc cca Caagtggtg 12 O US 9,078,860 B2 73 74 - Continued aagttcatgg acgt.ctacca gcgcagot ac tgc.cgt.ccaa tcgaga.ccct ggtggaCat C 18O titcCaggagt accc.cgatga gat.cgagtac atc.ttcaagc gcc cctgatg 24 O cggtgcgggg gctgctgcaa. cgacgaaggt Ctggagtgttg tgcc.cactga ggagttcaac 3OO atcaccatgc agattatgcg gatcaaac ct Cacca aggcc agcacat agg agagatgagc 360 titcc tacago acaacaaatg tgaatgcaga cCaaagaaag atagagcgag gcaagaaaat

CCCtgtgggc cittgct caga gcggagaaag catttgtttg tacaagatcc gCagacgtgt aaatgttcct gcaaaaacac agact cqcgt. tgcaaggcga ggcagcttga gttaaacgaa 54 O cg tacttgca gatgtgacaa gcc.gaggcgg tga

<210s, SEQ ID NO 39 &211s LENGTH: 190 212. TYPE : PRT <213s ORGANISM: Sus scrofa

<4 OOs, SEQUENCE: 39 Met Asin Phe Leu Lleu Ser Trp Val His Trp Ser Lell Ala Luell Lieu. Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 25

Asp Glin Lys Pro His Glu Wal Wall Llys Phe Met Asp Wall Glin Arg 35 4 O 45

Ser Tyr Arg Pro Ile Glu Thr Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Wall Pro Luell Met 65 70 7s

Arg Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu Wall Pro Thir 85 90 95

Glu Glu Phe Asn. Ile Thir Met Glin Ile Met Arg Ile Pro His Glin 105 11 O

Gly Glin His Ile Gly Glu Met Ser Phe Leul Glin His Asn Cys Glu 115 12 O 125

Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Asn Pro Gly Pro 13 O 135 14 O

Cys Ser Glu Arg Arg Llys His Lieu. Phe Wall Glin Asp Pro Glin Thr Cys 145 150 155 160

Ser Cys Lys Asn. Thir Asp Ser Arg Cys Ala Arg Gln Lieu. 1.65 17O 17s

Glu Luell Asn Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185 19 O

<210s, SEQ ID NO 4 O &211s LENGTH: 164 212. TYPE : PRT &213s ORGANISM: Sus scrofa

<4 OOs, SEQUENCE: 4 O

Ala Pro Met Ala Glu Gly Asp Glin Llys Pro His Glu Wall Wall Llys Phe 1. 5 1O 15

Met Asp Val Tyr Glin Arg Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Wall 25

Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro 35 45

Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Asn Asp Glu Gly US 9,078,860 B2 75 - Continued

SO 55 6 O

Lell Glu Cys Val Pro Thr Glu Glu Phe Asn. Ile Thir Met Glin Ile Met 65 70 7s 8O

Arg Ile Llys Pro His Glin Gly Glin His Ile Gly Glu Met Ser Phe Lieu. 85 90 95

Glin His Asn Lys Cys Glu. Cys Arg Pro Llys Llys Asp Arg Ala Arg Glin 105 11 O

Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg His Lieu. Phe Wall 115 12 O 125

Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Llys Asn Thir Asp Ser Arg 13 O 135 14 O

Cys Ala Arg Glin Lieu. Glu Lieu. Asin Glu Arg Thir Arg Cys Asp 145 150 155 160

Lys Pro Arg Arg

SEQ ID NO 41 LENGTH: 573 TYPE: DNA ORGANISM: Rattus norvegicus

< 4 OOs SEQUENCE: 41 atgaactittctgct ct cittg ggtgcactgg accctggctt tactgctgta cct coaccat 6 O gccalagtggit C cc aggctgc acccacgaca galaggggagc agaaag.ccca talagtggtg 12 O aagttcatgg acgt.ctacca gcgcagot at tgc.cgt.ccga ttgaga.ccct ggtggaCat C 18O titcCaggagt accCCgatga gatagagtat atc.ttcaagc Cgt CCtgttgt gcc cctaatg 24 O cggtgtgcgg gctgctgcaa. tgatgaagcc Ctggagtgcg tgcc.cacgt.c ggaga.gcaac 3OO gtcactatgc agat catgcg gatcaaac ct caccaaag.cc agcacat agg agagatgagc 360 titcCtgcagc at agcagatg tgaatgcaga cCaaagaaag atagaacaaa goc agaaaat

Cactgtgagc Cttgttcaga gcggagaaag catttgtttg tccalagatcc gcagacgtgt aaatgttcct gcaaaaacac agact cqcgt. tgcaaggcga ggcagcttga gttaaacgaa 54 O cgtacttgca gatgtgacaa gccaaggcgg tga

<210s, SEQ ID NO 42 &211s LENGTH: 214 212. TYPE: PRT &213s ORGANISM: Rattus norvegicus

<4 OOs, SEQUENCE: 42

Met Asin Phe Leu Lleu Ser Trp Val His Trp Thr Lell Ala Luell Luell Luell 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Thir Thr Glu Gly 25 3O

Glu Glin Lys Ala His Glu Val Val Llys Phe Met Asp Wall Tyr Glin Arg 35 4 O 45

Ser Tyr Cys Arg Pro Ile Glu Thr Lieu Val Asp Ile Phe Gln Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro Ser Wall Pro Luel Met 65 70

Arg Ala Gly Cys Cys Asn Asp Glu Ala Lieu. Glu Wa Pro Thr 85 90 95

Ser Glu Ser Asn. Wall. Thir Met Glin Ile Met Arg Ile Pro His Glin 105 11 O US 9,078,860 B2 77 - Continued Ser Gln His Ile Gly Glu Met Ser Phe Leu Gln His Ser Arg Cys Glu 115 12 O 125 Cys Arg Pro Llys Lys Asp Arg Thr Llys Pro Glu Lys Llys Ser Val Arg 13 O 135 14 O Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg Llys Llys Ser Arg Phe Lys 145 150 155 160 Ser Trp Ser Val His Cys Glu Pro Cys Ser Glu Arg Arg Llys His Lieu. 1.65 17O 17s Phe Val Glin Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp 18O 185 19 O Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. Asn. Glu Arg Thr Cys Arg 195 2OO 2O5 Cys Asp Llys Pro Arg Arg 21 O

<210s, SEQ ID NO 43 &211s LENGTH: 188 212. TYPE: PRT <213> ORGANISM: Rattus norvegicus

<4 OOs, SEQUENCE: 43 Ala Pro Thir Thr Glu Gly Glu Gln Lys Ala His Glu Val Val Llys Phe 1. 5 1O 15 Met Asp Val Tyr Glin Arg Ser Tyr Cys Arg Pro Ile Glu Thir Lieu Val 2O 25 3O Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Llys Pro 35 4 O 45 Ser Cys Val Pro Lieu Met Arg Cys Ala Gly Cys Cys Asn Asp Glu Ala SO 55 6 O Lieu. Glu. Cys Val Pro Thir Ser Glu Ser Asn Val Thr Met Glin Ile Met 65 70 7s 8O Arg Ile Llys Pro His Glin Ser Gln His Ile Gly Glu Met Ser Phe Leu 85 90 95 Glin His Ser Arg Cys Glu. Cys Arg Pro Llys Lys Asp Arg Thr Llys Pro 1OO 105 11 O Glu Lys Llys Ser Val Arg Gly Lys Gly Lys Gly Glin Lys Arg Lys Arg 115 12 O 125 Lys Llys Ser Arg Phe Lys Ser Trp Ser Val His Cys Glu Pro Cys Ser 13 O 135 14 O Glu Arg Arg Llys His Lieu. Phe Val Glin Asp Pro Glin Thr Cys Lys Cys 145 150 155 160 Ser Cys Lys Asn. Thir Asp Ser Arg Cys Lys Ala Arg Glin Lieu. Glu Lieu. 1.65 17O 17s Asn Glu Arg Thr Cys Arg Cys Asp Llys Pro Arg Arg 18O 185

<210s, SEQ ID NO 44 &211s LENGTH: 441 &212s. TYPE: DNA <213> ORGANISM: Ovis airies

<4 OOs, SEQUENCE: 44 atgaactittctgct ct cittg ggtgcattgg agccttgcct togctgcticta cct tcac cat 6 O gccalagtggit C cc aggctgc acc catggca gaaggagggc agaalacc cca talagtgatg 12 O aagttcatgg atgtctacca gcgcagottctg.ccgt.ccca ttgaga.ccct ggtggacat c 18O US 9,078,860 B2 79 - Continued titcCaggagt acccagatga gattgagttc attittcaagc cgt.cctgttgt gcc cctgatg 24 O cggtgcgggg gctgctgtaa tgacgaaagt Ctggagtgttg tgcc.cactgaggagttcaac 3OO atcaccatgc agattatgcg gatcaaac ct caccaaag.cc agcacat agg agagatgagt 360 titcc tacago ataacaaatg taatgcaga cCaaagaaag ataaa.gcaag gcaagaaaaa tgtgacaa.gc cgaggcggtg a 441

<210s, SEQ ID NO 45 &211s LENGTH: 146 212. TYPE : PRT &213s ORGANISM: Ovis airies

<4 OOs, SEQUENCE: 45 Met Asn. Phe Lieu Lleu Ser Trp Wall His Trp Ser Lieu Ala Lieu. Lieu. Lieu. 1. 5 1O 15

Tyr Luell His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 3O

Gly Glin Lys Pro His Glu. Wall Met Llys Phe Met Asp Val Tyr Glin Arg 35 4 O 45

Ser Phe Arg Pro Ile Glu Thir Lieu Val Asp Ile Phe Glin Glu Tyr SO 55 6 O

Pro Asp Glu Ile Glu Phe Ile Phe Llys Pro Ser Cys Val Pro Leu Met 65 70 7s 8O

Arg Gly Gly Cys Cys Asn Asp Glu Ser Lieu. Glu Cys Val Pro Thr 85 90 95

Glu Glu Phe ASn Ile Thr Met Gln Ile Met Arg Ile Llys Pro His Glin 1OO 105 11 O

Ser Glin His Ile Gly Glu Met Ser Phe Leul Glin His Asn Lys Cys Glu 115 12 O 125

Arg Pro Lys Ala Arg Glin Glu Lys Cys Asp Llys Pro 13 O 135 14 O Arg Arg 145

<210s, SEQ ID NO 46 &211s LENGTH: 120 212. TYPE : PRT <213> ORGANISM: Ovis airies

<4 OOs, SEQUENCE: 46 Ala Pro Met Ala Glu Gly Gly Glin Llys Pro His Glu Val Met Llys Phe 1. 5 1O 15

Met Asp Wall Tyr Glin Arg Ser Phe Cys Arg Pro Ile Glu. Thir Lieu Wall 2O 25 3O

Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Phe Ile Phe Llys Pro 35 4 O 45

Ser Cys Wall Pro Leu Met Arg Gly Gly Cys Cys Asn Asp Glu Ser SO 55 6 O

Lell Glu Wa Pro Thir Glu Glu Phe Asn. Ile Thir Met Glin Ile Met 65 70 8O

Arg Ile Pro His Glin Ser Glin His Ile Gly Glu Met Ser Phe Lieu. 85 90 95

Glin His Asn Lys Cys Glu Arg Pro Llys Llys Asp Lys Ala Arg Glin 1OO 105 11 O

Glu Cys Asp Llys Pro Arg Arg 115 12 O US 9,078,860 B2 81 - Continued

<210s, SEQ ID NO 47 &211s LENGTH: 188 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 47 Met Ser Pro Lieu. Lieu. Arg Arg Lieu. Lieu. Lieu Ala Ala Lieu. Lieu. Glin Lieu. 1. 5 1O 15 Ala Pro Ala Glin Ala Pro Val Ser Glin Pro Asp Ala Pro Gly His Glin 2O 25 3O Arg Llys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Glin 35 4 O 45 Pro Arg Glu Val Val Val Pro Leu. Thr Val Glu Lieu Met Gly Thr Val SO 55 6 O Ala Lys Glin Lieu Val Pro Ser Cys Val Thr Val Glin Arg Cys Gly Gly 65 70 7s 8O Cys Cys Pro Asp Asp Gly Lieu. Glu. Cys Val Pro Thr Gly Glin His Glin 85 90 95 Val Arg Met Glin Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 1OO 105 11 O Glu Met Ser Lieu. Glu Glu. His Ser Glin Cys Glu. Cys Arg Pro Llys Llys 115 12 O 125 Lys Asp Ser Ala Val Llys Pro Asp Ser Pro Arg Pro Lieu. Cys Pro Arg 13 O 135 14 O Cys Thr Gln His His Glin Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 145 150 155 160 Cys Arg Arg Arg Ser Phe Lieu. Arg Cys Glin Gly Arg Gly Lieu. Glu Lieu. 1.65 17O 17s Asn Pro Asp Thr Cys Arg Cys Arg Llys Lieu. Arg Arg 18O 185

<210s, SEQ ID NO 48 &211s LENGTH: 167 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 48 Pro Val Ser Glin Pro Asp Ala Pro Gly His Glin Arg Llys Val Val Ser 1. 5 1O 15 Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val 2O 25 3O Val Pro Leu. Thr Val Glu Lieu Met Gly Thr Val Ala Lys Gln Leu Val 35 4 O 45 Pro Ser Cys Val Thr Val Glin Arg Cys Gly Gly Cys Cys Pro Asp Asp SO 55 6 O Gly Lieu. Glu. Cys Val Pro Thr Gly Gln His Glin Val Arg Met Glin Ile 65 70 7s 8O

Lieu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Lieu. Glu 85 90 95 Glu. His Ser Glin Cys Glu. Cys Arg Pro Llys Llys Lys Asp Ser Ala Val 1OO 105 11 O Llys Pro Asp Ser Pro Arg Pro Leu. Cys Pro Arg Cys Thr Glin His His 115 12 O 125

Glin Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg Cys Arg Arg Arg Ser 13 O 135 14 O US 9,078,860 B2 83 - Continued

Phe Lieu. Arg Cys Glin Gly Arg Gly Lieu. Glu Lieu. Asn Pro Asp Thr Cys 145 150 155 160 Arg Cys Arg Llys Lieu. Arg Arg 1.65

<210s, SEQ ID NO 49 &211s LENGTH: 2O7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 49 Met Ser Pro Lieu. Lieu. Arg Arg Lieu. Lieu. Lieu Ala Ala Lieu. Lieu. Glin Lieu. 1. 5 1O 15 Ala Pro Ala Glin Ala Pro Val Ser Glin Pro Asp Ala Pro Gly His Glin 2O 25 3O Arg Llys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Glin 35 4 O 45 Pro Arg Glu Val Val Val Pro Leu. Thr Val Glu Lieu Met Gly Thr Val SO 55 6 O Ala Lys Glin Lieu Val Pro Ser Cys Val Thr Val Glin Arg Cys Gly Gly 65 70 7s 8O Cys Cys Pro Asp Asp Gly Lieu. Glu. Cys Val Pro Thr Gly Glin His Glin 85 90 95 Val Arg Met Glin Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 1OO 105 11 O Glu Met Ser Lieu. Glu Glu. His Ser Gln Cys Glu. Cys Arg Pro Llys Llys 115 12 O 125 Lys Asp Ser Ala Val Llys Pro Asp Arg Ala Ala Thr Pro His His Arg 13 O 135 14 O Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 145 150 155 160 Ser Pro Ala Asp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 1.65 17O 17s His Ala Ala Pro Ser Thr Thr Ser Ala Lieu. Thr Pro Gly Pro Ala Ala 18O 185 19 O Ala Ala Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 195 2OO 2O5

<210s, SEQ ID NO 50 &211s LENGTH: 186 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 50 Pro Val Ser Glin Pro Asp Ala Pro Gly His Glin Arg Llys Val Val Ser 1. 5 1O 15

Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln Pro Arg Glu Val Val 2O 25 3O

Val Pro Leu. Thr Val Glu Lieu Met Gly Thr Val Ala Lys Gln Leu Val 35 4 O 45

Pro Ser Cys Val Thr Val Glin Arg Cys Gly Gly Cys Cys Pro Asp Asp SO 55 6 O

Gly Lieu. Glu. Cys Val Pro Thr Gly Gln His Glin Val Arg Met Glin Ile 65 70 7s 8O

Lieu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Lieu. Glu 85 90 95 US 9,078,860 B2 85 86 - Continued

Glu His Ser Glin Glu Arg Pro Lys Asp Ser Ala Wall 105 11 O

Pro Asp Arg Ala Ala Thir Pro His His Arg Pro Glin Pro Arg Ser 115 12 O 125

Wall Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro Ser Pro Ala Asp Ile 13 O 135 14 O

Thir His Pro Thir Pro Ala Pro Gly Pro Ser Ala His Ala Ala Pro Ser 145 150 155 160

Thir Thir Ser Ala Lell Thir Pro Gly Pro Ala Ala Ala Ala Ala Asp Ala 1.65 17s

Ala Ala Ser Ser Wall Ala Gly Gly Ala 18O 185

<210s, SEQ ID NO 51 &211s LENGTH: 419 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 51

Met His Lieu. Lieu. Gly Phe Phe Ser Wall Ala Ser Lell Luell Ala Ala 1. 5 15

Ala Luell Luell Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 25

Glu Ser Gly Luell Asp Lell Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 4 O 45

Thr Ala Ala Ser Asp Lieu Glu Glu Gln Lieu Arg Ser Wall Ser SO 55 6 O

Ser Wall Glu Lell Met Thir Wall Luell Tyr Pro Glu Tyr Trp Met 65 70

Glin Lell Arg Gly Gly Trp Glin His Asn Arg Glu Glin 85 90 95

Ala Asn Luell Asn Ser Arg Thir Glu Glu Thir Ile Phe Ala Ala Ala 105 11 O

His Asn Thir Glu Ile Lell Lys Ser Ile Asp Asn Glu Trp Arg 115 12 O 125

Thir Glin Met Pro Arg Glu Wall Ile Asp Wall Gly Glu Phe 13 O 135 14 O

Gly Wall Ala Thir Asn Thir Phe Phe Pro Pro Wall Ser Wall Tyr 145 150 155 160

Arg Gly Gly Cys Asn Ser Glu Gly Luell Glin Met Asn Thir 1.65 17O 17s

Ser Thir Ser Tyr Lell Ser Thir Luell Phe Glu Ile Thir Wall Pro Luell 18O 185 19 O

Ser Glin Gly Pro Pro Wall Thir Ile Ser Phe Ala Asn His Thir Ser 195

Arg Met Ser Lell Asp Wall Tyr Arg Glin Wall His Ser Ile 21 O 215 22O

Ile Arg Ser Lell Pro Ala Thir Luell Pro Glin Glin Ala Ala Asn 225 23 O 235 24 O

Thir Pro Thir Asn Tyr Met Trp Asn ASn His Ile Arg 245 250 255

Lell Ala Glin Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 26 O 265 27 O

Thir Asp Gly Phe His Asp Ile Gly Pro ASn Glu Luell Asp Glu US 9,078,860 B2 87 - Continued

27s 28O 285 Glu Thir Cys Glin Cys Val Cys Arg Ala Gly Lieu. Arg Pro Ala Ser Cys 29 O 295 3 OO Gly Pro His Lys Glu Lieu. Asp Arg Asn. Ser Cys Glin Cys Val Cys Llys 3. OS 310 315 32O Asn Llys Lieu. Phe Pro Ser Glin Cys Gly Ala Asn Arg Glu Phe Asp Glu 3.25 330 335 Asn Thr Cys Glin Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Glin Pro 34 O 345 35. O Lieu. Asn Pro Gly Lys Cys Ala Cys Glu. Cys Thr Glu Ser Pro Glin Lys 355 360 365 Cys Lieu. Leu Lys Gly Lys Llys Phe His His Glin Thr Cys Ser Cys Tyr 37 O 375 38O Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 385 390 395 4 OO Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro 4 OS 41O 415

Gln Met Ser

<210s, SEQ ID NO 52 &211s LENGTH: 354 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 52 Met Tyr Arg Glu Trp Val Val Val ASn Val Phe Met Met Lieu. Tyr Val 1. 5 1O 15 Glin Lieu Val Glin Gly Ser Ser Asn. Glu. His Gly Pro Wall Lys Arg Ser 2O 25 3O Ser Glin Ser Thir Lieu. Glu Arg Ser Glu Glin Glin Ile Arg Ala Ala Ser 35 4 O 45 Ser Lieu. Glu Glu Lieu. Lieu. Arg Ile Thr His Ser Glu Asp Trp Llys Lieu. SO 55 6 O Trp Arg Cys Arg Lieu. Arg Lieu Lys Ser Phe Thir Ser Met Asp Ser Arg 65 70 7s 8O Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95 Glu Thir Lieu Lys Val Ile Asp Glu Glu Trp Glin Arg Thr Glin Cys Ser 1OO 105 11 O Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Lieu. Gly Lys Ser Thr 115 12 O 125 Asn Thr Phe Phe Llys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly 13 O 135 14 O Cys Cys Asn Glu Glu Ser Lieu. Ile Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155 160

Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu. Thir Ser Val Pro 1.65 17O 17s Glu Lieu Val Pro Val Llys Val Ala Asn His Thr Gly Cys Lys Cys Lieu. 18O 185 19 O

Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Glin 195 2OO 2O5 Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Llys Lieu. Cys Pro Ile 21 O 215 22O

Asp Met Lieu. Trp Asp Ser Asn Lys Cys Lys Cys Val Lieu. Glin Glu Glu US 9,078,860 B2 89 - Continued

225 23 O 235 24 O Asn Pro Lieu Ala Gly. Thr Glu Asp His Ser His Lieu. Glin Glu Pro Ala 245 250 255 Lieu. Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu. Cys Val 26 O 265 27 O Cys Llys Thr Pro Cys Pro Lys Asp Lieu. Ile Gln His Pro Lys Asn. Cys 27s 28O 285 Ser Cys Phe Glu. Cys Lys Glu Ser Lieu. Glu Thr Cys Cys Glin Llys His 29 O 295 3 OO Lys Lieu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 3. OS 310 315 32O His Thr Arg Pro Cys Ala Ser Gly Llys Thir Ala Cys Ala Lys His Cys 3.25 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Glin Gly Pro His Ser Arg Llys 34 O 345 35. O

Asn. Pro

<210s, SEQ ID NO 53 &211s LENGTH: 149 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 53 Met Pro Val Met Arg Lieu Phe Pro Cys Phe Leu Gln Lieu. Leu Ala Gly 1. 5 1O 15 Lieu Ala Lieu Pro Ala Val Pro Pro Gln Gln Trp Ala Lieu Ser Ala Gly 2O 25 3O Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly 35 4 O 45 Arg Ser Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu SO 55 6 O Tyr Pro Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. 65 70 7s 8O Lieu. Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro 85 90 95 Val Glu Thir Ala Asn Val Thr Met Glin Lieu. Lieu Lys Ile Arg Ser Gly 1OO 105 11 O Asp Arg Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Gln His Val Arg Cys 115 12 O 125 Glu Cys Arg Pro Lieu. Arg Glu Lys Met Llys Pro Glu Arg Cys Gly Asp 13 O 135 14 O Ala Val Pro Arg Arg 145

<210s, SEQ ID NO 54 &211s LENGTH: 131 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 54 Lieu Pro Ala Val Pro Pro Glin Glin Trp Ala Lieu. Ser Ala Gly Asn Gly 1. 5 1O 15

Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly Arg Ser 2O 25 3O

Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu Tyr Pro 35 4 O 45 US 9,078,860 B2 91 - Continued

Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. Leu Arg SO 55 6 O Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro Val Glu 65 70 7s 8O Thir Ala Asn Val Thr Met Gln Lieu. Lieu Lys Ile Arg Ser Gly Asp Arg 85 90 95 Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Glin His Val Arg Cys Glu. Cys 1OO 105 11 O Arg Pro Lieu. Arg Glu Lys Met Llys Pro Glu Arg Cys Gly Asp Ala Val 115 12 O 125 Pro Arg Arg 13 O

<210s, SEQ ID NO 55 &211s LENGTH: 170 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 55 Met Pro Val Met Arg Lieu Phe Pro Cys Phe Leu Gln Lieu. Leu Ala Gly 1. 5 1O 15 Lieu Ala Lieu Pro Ala Val Pro Pro Glin Gln Trp Ala Lieu. Ser Ala Gly 2O 25 3O Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly 35 4 O 45 Arg Ser Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu SO 55 6 O Tyr Pro Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. 65 70 7s 8O Lieu. Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro 85 90 95 Val Glu Thir Ala Asn Val Thr Met Glin Lieu. Lieu Lys Ile Arg Ser Gly 1OO 105 11 O Asp Arg Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Gln His Val Arg Cys 115 12 O 125 Glu Cys Arg Pro Lieu. Arg Glu Lys Met Llys Pro Glu Arg Arg Arg Pro 13 O 135 14 O Lys Gly Arg Gly Lys Arg Arg Arg Glu Lys Glin Arg Pro Thir Asp Cys 145 150 155 160 His Lieu. Cys Gly Asp Ala Val Pro Arg Arg 1.65 17O

<210s, SEQ ID NO 56 &211s LENGTH: 152 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 56 Lieu Pro Ala Val Pro Pro Glin Glin Trp Ala Lieu. Ser Ala Gly Asn Gly 1. 5 1O 15

Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly Arg Ser 2O 25 3O

Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu Tyr Pro 35 4 O 45

Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. Leu Arg SO 55 6 O US 9,078,860 B2 93 - Continued

Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro Val Glu 65 70 7s 8O Thir Ala Asn Val Thr Met Gln Lieu. Lieu Lys Ile Arg Ser Gly Asp Arg 85 90 95 Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Glin His Val Arg Cys Glu. Cys 1OO 105 11 O Arg Pro Lieu. Arg Glu Lys Met Llys Pro Glu Arg Arg Arg Pro Lys Gly 115 12 O 125 Arg Gly Lys Arg Arg Arg Glu Lys Glin Arg Pro Thr Asp Cys His Lieu 13 O 135 14 O Cys Gly Asp Ala Val Pro Arg Arg 145 150

<210s, SEQ ID NO 57 &211s LENGTH: 221 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 57 Met Pro Val Met Arg Lieu Phe Pro Cys Phe Leu Gln Lieu. Leu Ala Gly 1. 5 1O 15 Lieu Ala Lieu Pro Ala Val Pro Pro Glin Gln Trp Ala Lieu. Ser Ala Gly 2O 25 3O Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly 35 4 O 45 Arg Ser Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu SO 55 6 O Tyr Pro Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. 65 70 7s 8O Lieu. Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro 85 90 95 Val Glu Thir Ala Asn Val Thr Met Glin Lieu. Lieu Lys Ile Arg Ser Gly 1OO 105 11 O Asp Arg Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Gln His Val Arg Cys 115 12 O 125 Glu Cys Arg His Ser Pro Gly Arg Glin Ser Pro Asp Met Pro Gly Asp 13 O 135 14 O Phe Arg Ala Asp Ala Pro Ser Phe Lieu Pro Pro Arg Arg Ser Lieu Pro 145 150 155 160 Met Leu Phe Arg Met Glu Trp Gly Cys Ala Lieu. Thr Gly Ser Glin Ser 1.65 17O 17s Ala Val Trp Pro Ser Ser Pro Val Pro Glu Glu Ile Pro Arg Met His 18O 185 19 O Pro Gly Arg Asin Gly Llys Lys Glin Glin Arg Llys Pro Lieu. Arg Glu Lys 195 2OO 2O5

Met Llys Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg 21 O 215 22O

<210s, SEQ ID NO 58 &211s LENGTH: 2O3 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 58 Lieu Pro Ala Val Pro Pro Glin Glin Trp Ala Lieu. Ser Ala Gly Asn Gly 1. 5 1O 15 US 9,078,860 B2 95 - Continued

Ser Ser Glu Val Glu Val Val Pro Phe Glin Glu Val Trp Gly Arg Ser 2O 25 3O Tyr Cys Arg Ala Lieu. Glu Arg Lieu Val Asp Val Val Ser Glu Tyr Pro 35 4 O 45 Ser Glu Val Glu. His Met Phe Ser Pro Ser Cys Val Ser Lieu. Leu Arg SO 55 6 O Cys Thr Gly Cys Cys Gly Asp Glu Asn Lieu. His Cys Val Pro Val Glu 65 70 7s 8O Thir Ala Asn Val Thr Met Gln Lieu. Lieu Lys Ile Arg Ser Gly Asp Arg 85 90 95 Pro Ser Tyr Val Glu Lieu. Thr Phe Ser Glin His Val Arg Cys Glu. Cys 1OO 105 11 O Arg His Ser Pro Gly Arg Glin Ser Pro Asp Met Pro Gly Asp Phe Arg 115 12 O 125 Ala Asp Ala Pro Ser Phe Lieu Pro Pro Arg Arg Ser Lieu Pro Met Lieu 13 O 135 14 O Phe Arg Met Glu Trp Gly Cys Ala Lieu. Thr Gly Ser Glin Ser Ala Val 145 150 155 160 Trp Pro Ser Ser Pro Val Pro Glu Glu Ile Pro Arg Met His Pro Gly 1.65 17O 17s Arg Asn Gly Lys Lys Glin Glin Arg Llys Pro Lieu. Arg Glu Lys Met Lys 18O 185 19 O Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg 195 2OO

<210s, SEQ ID NO 59 &211s LENGTH: 345 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 59 Met Ser Lieu. Phe Gly Lieu Lleu Lieu. Lieu. Thir Ser Ala Lieu Ala Gly Glin 1. 5 1O 15 Arg Glin Gly Thr Glin Ala Glu Ser Asn Lieu Ser Ser Lys Phe Glin Phe 2O 25 3O Ser Ser Asn Lys Glu Glin Asn Gly Val Glin Asp Pro Gln His Glu Arg 35 4 O 45 Ile Ile Thr Val Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro SO 55 6 O His Thr Tyr Pro Arg Asn Thr Val Lieu Val Trp Arg Lieu Val Ala Val 65 70 7s 8O Glu Glu Asn Val Trp Ile Glin Lieu. Thir Phe Asp Glu Arg Phe Gly Lieu. 85 90 95 Glu Asp Pro Glu Asp Asp Ile Cys Llys Tyr Asp Phe Val Glu Val Glu 1OO 105 11 O

Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr 115 12 O 125

Val Pro Gly Lys Glin Ile Ser Lys Gly Asin Glin Ile Arg Ile Arg Phe 13 O 135 14 O Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile His Tyr 145 150 155 160

Asn. Ile Wal Met Pro Glin Phe Thr Glu Ala Wal Ser Pro Ser Wall Lieu. 1.65 17O 17s

Pro Pro Ser Ala Lieu Pro Lieu. Asp Lieu. Lieu. Asn. Asn Ala Ile Thr Ala US 9,078,860 B2 97 - Continued

18O 185 19 O Phe Ser Thr Lieu. Glu Asp Lieu. Ile Arg Tyr Lieu. Glu Pro Glu Arg Trp 195 2OO 2O5 Glin Lieu. Asp Lieu. Glu Asp Lieu. Tyr Arg Pro Thir Trp Gln Lieu. Lieu. Gly 21 O 215 22O Lys Ala Phe Val Phe Gly Arg Llys Ser Arg Val Val Asp Lieu. Asn Lieu. 225 23 O 235 24 O Lieu. Thr Glu Glu Val Arg Lieu. Tyr Ser Cys Thr Pro Arg Asin Phe Ser 245 250 255 Val Ser Ile Arg Glu Glu Lieu Lys Arg Thr Asp Thr Ile Phe Trp Pro 26 O 265 27 O Gly Cys Lieu. Lieu Val Lys Arg Cys Gly Gly Asn. Cys Ala Cys Cys Lieu. 27s 28O 285 His Asn. Cys Asn. Glu. Cys Glin Cys Val Pro Ser Llys Val Thir Lys Llys 29 O 295 3 OO Tyr His Glu Val Lieu. Glin Lieu. Arg Pro Llys Thr Gly Val Arg Gly Lieu. 3. OS 310 315 32O His Llys Ser Lieu. Thir Asp Wall Ala Lieu. Glu. His His Glu Glu. Cys Asp 3.25 330 335 Cys Val Cys Arg Gly Ser Thr Gly Gly 34 O 345

<210s, SEQ ID NO 60 &211s LENGTH: 326 212. TYPE: PRT <213> ORGANISM; Homo sapiens <4 OOs, SEQUENCE: 60 Thr Glin Ala Glu Ser Asn Lieu Ser Ser Llys Phe Glin Phe Ser Ser Asn 1. 5 1O 15 Lys Glu Glin Asn Gly Val Glin Asp Pro Gln His Glu Arg Ile Ile Thr 2O 25 3O Val Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro His Thr Tyr 35 4 O 45 Pro Arg Asn Thr Val Lieu Val Trp Arg Lieu Val Ala Val Glu Glu Asn SO 55 6 O Val Trp Ile Glin Lieu. Thir Phe Asp Glu Arg Phe Gly Lieu. Glu Asp Pro 65 70 7s 8O Glu Asp Asp Ile Cys Llys Tyr Asp Phe Val Glu Val Glu Glu Pro Ser 85 90 95 Asp Gly. Thir Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr Val Pro Gly 1OO 105 11 O Lys Glin Ile Ser Lys Gly Asn Glin Ile Arg Ile Arg Phe Val Ser Asp 115 12 O 125 Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile His Tyr Asn Ile Val 13 O 135 14 O

Met Pro Glin Phe Thr Glu Ala Wal Ser Pro Ser Wall Leu Pro Pro Ser 145 150 155 160

Ala Lieu Pro Lieu. Asp Lieu. Lieu. Asn. Asn Ala Ile Thr Ala Phe Ser Thr 1.65 17O 17s

Lieu. Glu Asp Lieu. Ile Arg Tyr Lieu. Glu Pro Glu Arg Trp Gln Lieu. Asp 18O 185 19 O

Lieu. Glu Asp Lieu. Tyr Arg Pro Thir Trp Gln Lieu. Lieu. Gly Lys Ala Phe 195 2OO 2O5 US 9,078,860 B2 99 100 - Continued

Wall Phe Gly Arg Llys Ser Arg Val Wall Asp Luell Asn Lieu. Lieu. Thr Glu 21 O 215 22O

Glu Val Arg Lieu. Tyr Ser Cys Thr Pro Arg ASn Phe Ser Wal Ser Ile 225 23 O 235 24 O

Arg Glu Glu Lieu Lys Arg Thr Asp Thir Ile Phe Trp Pro Gly Cys Luell 245 250 255

Lell Val Lys Arg Cys Gly Gly Asn Cys Ala Cys Lieu. His Asn Cys 26 O 265 27 O

Asn Glu Cys Glin Cys Val Pro Ser Wall Thir Llys Llys Tyr His Glu 27s 28O 285

Wall Lieu. Glin Lieu. Arg Pro Llys Thr Gly Wall Arg Gly Lieu. His Lys Ser 29 O 295 3 OO

Lell Thir Asp Wall Ala Lieu. Glu. His His Glu Glu Cys Asp Cys Val Cys 3. OS 310 315 32O Arg Gly Ser Thr Gly Gly 3.25

<210s, SEQ ID NO 61 &211s LENGTH: 147 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 61

Met Asin Phe Lieu Lleu Ser Trp Val His Trp Ser Lieu Ala Lieu. Lieu Luell 1. 5 15

Tyr Lieu. His His Ala Lys Trp Ser Glin Ala Ala Pro Met Ala Glu Gly 2O 25 30

Gly Gly Glin Asn His His Glu Val Wall Lys Phe Met Asp Val Tyr Glin 35 4 O 45

Arg Ser Tyr Cys His Pro Ile Glu Thir Luell Wall Asp Ile Phe Glin Glu SO 55 6 O

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Luell 65 70

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Lieu. Glu. Cys Val Pro 85 90 95

Thir Glu Glu Ser Asn. Ile Thir Met Glin Ile Met Arg Ile Llys Pro His 1OO 105 11 O

Glin Gly Glin His Ile Gly Glu Met Ser Phe Luell Glin His Asn Lys Cys 115 12 O 125

Glu Cys Arg Pro Llys Lys Asp Arg Ala Arg Glin Glu Lys Cys Asp Lys 13 O 135 14 O

Pro Arg Arg 145

<210s, SEQ ID NO 62 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 62

Ala Pro Met Ala Glu Gly Gly Gly Glin Asn His His Glu Wal Wall Lys 1. 5 15

Phe Met Asp Val Tyr Glin Arg Ser Tyr Cys His Pro Ile Glu. Thr Luell 2O 25

Wall Asp Ile Phe Glin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 4 O 45 US 9,078,860 B2 101 102 - Continued

Pro Ser Wall Pro Leu Met Arg Gly Gly Cys Cys Asn Asp Glu SO 55 6 O

Gly Luell Glu Cys Wall Pro Thr Glu Glu Ser ASn Ile Thir Met Glin Ile 65 70 7s 8O

Met Arg Ile Lys Pro His Glin Gly Glin His Ile Gly Glu Met Ser Phe 85 90 95

Lell Glin His Asn Lys Cys Glu. Cys Arg Pro Lys Asp Arg Ala Arg 1OO 105 11 O

Glin Glu Lys Asp Llys Pro Arg Arg 115 12 O

<210s, SEQ ID NO 63 &211s LENGTH: 211 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 63

Met Arg Thr Lieu. Ala Cys Lieu. Lieu. Luell Luell Gly Gly Tyr Luell Ala 1. 5 15

His Wall Luell Ala Glu Glu Ala Glu Ile Pro Arg Glu Wall Ile Glu Arg 2O 25

Lell Ala Arg Ser Glin Ile His Ser Ile Arg Asp Lell Glin Arg Luell Luell 35 4 O 45

Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Luell Asp Thir Ser Luell Arg SO 55 6 O

Ala His Gly Wall His Ala Thr Lys His Wall Pro Glu Arg Pro Luell 65 70 75

Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Wall Pro Ala Wall Cys 85 90 95

Thir Arg Thir Val Ile Tyr Glu Ile Pro Arg Ser Glin Wall Asp Pro 105 11 O

Thir Ser Ala Asn Phe Lieu. Ile Trp Pro Pro Wall Glu Wall 115 12 O 125

Thir Gly Cys Asn Thr Ser Ser Wall Cys Glin Pro Ser Arg 13 O 135 14 O

Wall His His Arg Ser Val Llys Val Ala Wall Glu Tyr Wall Arg Lys 145 150 155 160

Pro Luell Lys Glu Val Glin Wall Arg Luell Glu Glu His Luell Glu 1.65 17O 17s

Ala Ala Thir Thir Ser Lieu. Asn Pro Asp Arg Glu Glu Asp 18O 185 19 O

Thir Gly Arg Pro Arg Glu Ser Gly Lys Arg Arg Arg Luell 195 2OO 2O5

Pro Thir 21 O

<210s, SEQ ID NO 64 &211s LENGTH: 196 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 64 Met Arg Thr Lieu Ala Cys Lieu. Lieu. Lieu. Lieu. Gly Cys Gly Tyr Lieu Ala 1. 5 15 His Val Lieu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg 25 US 9,078,860 B2 103 104 - Continued

Lell Ala Arg Ser Glin Ile His Ser Ile Arg Asp Lell Glin Arg Lieu. Luell 35 4 O 45

Glu Ile Asp Ser Wall Gly Ser Glu Asp Ser Luell Asp Thir Ser Luell Arg SO 55 6 O

Ala His Gly Wall His Ala Thir His Wall Pro Glu Lys Arg Pro Luell 65 70

Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Wall Pro Ala Wall 85 90 95

Thir Arg Thir Wall Ile Glu Ile Pro Arg Ser Glin Wall Asp Pro 105 11 O

Thir Ser Ala Asn Phe Lell Ile Trp Pro Pro Wall Glu Wall Arg 115 12 O 125

Thir Gly Cys Asn Thir Ser Ser Wall Cys Glin Pro Ser Arg 13 O 135 14 O

Wall His His Arg Ser Wall Wall Ala Wall Glu Wall Arg Lys 145 150 155 160

Pro Luell Lys Glu Wall Glin Wall Arg Luell Glu Glu His Luell Glu 1.65 17O 17s

Ala Ala Thir Thir Ser Luell Asn Pro Asp Arg Glu Glu Asp 18O 185 19 O

Thir Asp Wall Arg 195

<210s, SEQ ID NO 65 &211s LENGTH: 1338 212. TYPE PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 65

Met Val Ser Tyr Trp Asp Thir Gly Wall Luell Luell Ala Luell Luell Ser 1. 5 15

Cys Luell Luell Luell Thir Gly Ser Ser Ser Gly Ser Lell Lys Asp Pro 25 3O

Glu Luell Ser Luell Gly Thir Glin His Ile Met Glin Ala Gly Glin Thir 35 4 O 45

Lell His Luell Glin Arg Gly Glu Ala Ala His Lys Trp Ser Luell Pro SO 55 6 O

Glu Met Wall Ser Glu Ser Glu Arg Luell Ser Ile Thir Ser Ala 65 70

Gly Arg Asn Gly Glin Phe Ser Thir Lell Thir Luell Asn Thir 85 90 95

Ala Glin Ala Asn His Thir Gly Phe Tyr Ser Luell Ala Wall 1OO 105 11 O

Pro Thir Ser Lys Glu Thir Glu Ser Ala Ile Tyr Ile Phe Ile 115 12 O 125

Ser Asp Thir Gly Arg Pro Phe Wall Glu Met Ser Glu Ile Pro Glu 13 O 135 14 O

Ile Ile His Met Thir Glu Gly Arg Glu Luell Wall Ile Pro Arg Wall 145 150 155 160

Thir Ser Pro Asn Ile Thir Wall Thir Luell Lys Phe Pro Luell Asp Thir 1.65 17O 17s

Lell Ile Pro Asp Gly Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 18O 185 19 O

Ile Ile Ser Asn Ala Thir Lys Glu Ile Gly Lell Lell Thir Glu 195 2OO 2O5 US 9,078,860 B2 105 106 - Continued

Ala Thir Wall Asn Gly His Lell Thir ASn Tyr Lell Thir His Arg 21 O 215 22O

Glin Thir Asn Thir Ile Ile Asp Wall Glin Ile Ser Thir Pro Arg Pro Wall 225 23 O 235 24 O

Luell Luell Arg Gly His Thir Luell Wall Luell ASn Thir Ala Thir Thir 245 250 255

Pro Luell Asn Thir Wall Glin Met Thir Trp Ser Pro Asp Glu 26 O 265 27 O

Asn Arg Ala Ser Wall Arg Arg Arg Ile Asp Glin Ser Asn Ser His 285

Ala Asn Ile Phe Tyr Ser Wall Luell Thir Ile Asp Lys Met Glin Asn 29 O 295 3 OO

Asp Gly Luell Tyr Thir Arg Wall Arg Ser Gly Pro Ser Phe Lys 3. OS 310 315

Ser Wall Asn Thir Ser Wall His Ile Asp Ala Phe Ile Thir Wall 3.25 330 335

His Arg Lys Glin Glin Wall Luell Glu Thir Wall Ala Gly Lys Arg Ser 34 O 345 35. O

Arg Luell Ser Met Wall Lys Ala Phe Pro Ser Pro Glu Wall Wall 355 360 365

Trp Luell Asp Gly Lel Pro Ala Thir Glu Ser Ala Arg Luell 37 O 375

Thir Arg Ser Lel Ile Ile Asp Wall Thir Glu Glu Asp Ala 385 390 395 4 OO

Gly Asn Thir Ile Lel Lell Ser Ile Lys Glin Ser Asn Wall Phe 4 OS 415

Asn Luell Thir Ala Thir Lel Ile Wall Asn Wall Pro Glin Ile Glu 425 43 O

Ala Wall Ser Ser Phe Pro Asp Pro Ala Luell Pro Luell Gly Ser 435 44 O 445

Arg Glin Ile Luell Thir Thir Ala Gly Ile Pro Glin Pro Thir Ile 450 45.5 460

Lys Trp Phe Trp His Pro Asn His Asn His Ser Glu Ala Arg Cys 465 470

Asp Phe Ser Asn Asn Glu Glu Ser Phe Ile Lell Asp Ala Asp Ser 485 490 495

Asn Met Gly Asn Arg Ile Glu Ser Ile Thir Glin Arg Met Ala Ile Ile SOO 505

Glu Gly Lys Asn Lys Met Ala Ser Thir Luell Wall Wall Ala Asp Ser Arg 515 525

Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser ASn Lys Wall Gly Thir Wall 53 O 535 54 O

Gly Arg Asn Ile Ser Phe Ile Thir Asp Wall Pro Asn Gly Phe His 5.45 550 555 560

Wall Asn Luell Glu Lys Met Pro Thir Glu Gly Glu Asp Lell Luell Ser 565 st O sts

Thir Wall Asn Lys Phe Lell Arg Asp Wall Thir Trp Ile Luell Luell 585 59 O

Arg Thir Wall Asn Asn Arg Thir Met His Ser Ile Ser Glin Lys 595 6OO 605

Met Ala Ile Thir Lys Glu His Ser Ile Thir Luell Asn Lell Thir Ile Met 610 615 62O US 9,078,860 B2 107 108 - Continued

Asn Wall Ser Luell Glin Asp Ser Gly Thir Tyr Ala Cys Arg Ala Arg Asn 625 630 635 64 O

Wall Thir Gly Glu Glu Ile Luell Glin Lys Glu Ile Thir Ile Arg 645 650 655

Asp Glin Glu Ala Pro Lell Luell Arg Asn Luell Ser Asp His Thir Wall 660 665 67 O

Ala Ile Ser Ser Ser Thir Thir Luell Asp His Ala Asn Gly Wall Pro 675 685

Glu Pro Glin Ile Thir Trp Phe Asn Asn His Lys Ile Glin Glin Glu 69 O. 695 7 OO

Pro Gly Ile Ile Lell Gly Pro Gly Ser Ser Thir Lell Phe Ile Glu Arg 7 Os

Wall Thir Glu Glu Asp Glu Gly Wall His Ala Thir Asn Glin 72 73 O 73

Gly Ser Wall Glu Ser Ser Ala Tyr Luell Thir Wall Glin Gly Thir Ser 740 74. 7 O

Asp Ser Asn Lell Glu Lell Ile Thir Luell Thir Thir Wall Ala 760 765

Ala Thir Luell Phe Trp Lell Lell Luell Thir Luell Luell Ile Arg Met Lys 770 775

Arg Ser Ser Ser Glu Ile Thir Asp Luell Ser Ile Ile Met Asp 79 O 79.

Pro Asp Glu Wall Pro Lell Asp Glu Glin Cys Glu Arg Lell Pro Tyr Asp 805 810 815

Ala Ser Trp Glu Phe Ala Arg Glu Arg Lieu. Lys Lieu Gly Lys Ser 825 83 O

Lell Gly Arg Gly Ala Phe Gly Lys Wall Wall Glin Ala Ser Ala Phe Gly 835 84 O 845

Ile Lys Ser Pro Thir Cys Arg Thir Wall Ala Wall Met Luell 850 855 860

Glu Gly Ala Thir Ala Ser Glu Ala Luell Met Thir Glu Luell Lys 865 88O

Ile Luell Thir His Ile Gly His His Lel Asn Wall Wall Asn Luell Luell Gly 885 890 895

Ala Thir Lys Glin Gly Gly Pro Lel Met Wall Ile Wall Glu Tyr 9 OO 905 91 O

Gly Asn Lell Ser Asn Tyr Lel Ser Arg Asp Luell Phe 915 92 O 925

Phe Luell Asn Asp Ala Ala Luell His Met Glu Pro Glu 93 O 935 94 O

Met Glu Pro Gly Lell Glu Glin Gly Pro Arg Lell Asp Ser Wall 945 950 955 96.O

Thir Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Glin Glu Asp Lys Ser 965 97O 97.

Lell Ser Asp Wall Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 98O 985 99 O

Pro Ile Thir Met Glu Asp Lell Ile Ser Tyr Ser Phe Glin Val Ala Arg 995 1OOO 1005

Gly Met Glu Phe Lieu. Ser Ser Arg Lys Cys Ile His Arg Asp Lieu. 1010 5 1 O2O

Ala Ala Arg Asn. Ile Lieu. Lieu. Ser Glu Asn. Asn. Wall Val Lys Ile 1025 103 O 1035

Asp Phe Gly Lieu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr US 9,078,860 B2 110 - Continued

O4 O OSO

Val Arg Lys Gly Asp Thir Arg Lieu. Pro Luell Met Ala Pro O55

Glu Ser Ile Phe Asp Ser Thir Asp Wall Trp Of O

Ser Tyr Gly Val Lieu Luell rp Glu Ile Phe Ser Lell Gly Gly Ser O85 O9 O O95

Pro Tyr Pro Gly Val Glin Met Asp Glu Asp Phe Ser Arg Lell OO O5

Arg Glu Gly Met Arg Met Arg Ala Pro Glu Thir Pro Glu

Ile Tyr Glin Ile Met Luell Trp His Arg Pro Glu

Arg Pro Arg Phe Ala Glu Lieu Wall Glu Lell Asp Lell Lell

Glin Ala Asn. Wall Glin Glin Asp Gly Asp Pro Ile Asn

Ala e Lieu. Thr Gly Asn Ser Gly Phe Thir Thir Pro Ala

Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Pro Phe

Asn Ser Gly Ser Ser Asp Asp Val Arg Wall Asn Ala Phe 2O5 215

Phe Met Ser Lieu. Glu Arg Thir Phe Glu Glu Luell Lell Pro 220 23 O

Asn Ala Thir Ser Met Phe Glin Gly Ser Ser Thir 235 245

Lieu. Lieu Ala Ser Pro Met Arg Phe Thir rp Thir Asp Ser 250 26 O

Llys Pro Lys Ala Ser Luell Lys Ile Asp Luell Arg Wall Thir Ser 265 27s

Ser Lys Glu Ser Gly Luell Ser Asp Wall Ser Arg Pro Ser Phe 28O 29 O

His Ser Ser Cys Gly His Wall Ser Glu Gly Arg Phe Thir 295

Tyr Asp His Ala Glu Luell Glu Arg Ile Ala Ser Pro 310

Pro Pro Asp Tyr Asn Ser Wall Wall Luell Ser Pro Pro Ile 3.25 335

<210s, SEQ ID NO 66 &211s LENGTH: 1356 212. TYPE: PRT &213s ORGANISM: Homo Sap iens

<4 OOs, SEQUENCE: 66

Met Glin Ser Llys Val Lell Lieu Ala Wa l Ala Lieu. Trp Lieu. Cys Wall Glu 1. 5 15

Thir Arg Ala Ala Ser Val Gly Leu Pr o Ser Wal Ser Lieu. Asp Leul Pro 25

Arg Lieu. Ser Ile Glin Lys Asp Ile Le u. Thir Ile Lys Ala Asn Th Thr 35 4 O 45

Lieu. Glin Ile Thir Cys Arg Gly Gln Air g Asp Lieu. As p Trp Luell Trp Pro SO 55 6 O US 9,078,860 B2 111 112 - Continued

Asn Asn Glin Ser Gly Ser Glu Glin Arg Wall Glu Wall Thir Glu Cys Ser 65 70 8O

Asp Gly Luell Phe Cys Thir Luell Thir Ile Pro Lys Wall Ile Gly Asn 85 90 95

Asp Thir Gly Ala Tyr Phe Tyr Arg Glu Thir Asp Luell Ala Ser 105 11 O

Wall Ile Tyr Wall Tyr Wall Glin Asp Arg Ser Pro Phe Ile Ala Ser 115 12 O 125

Wall Ser Asp Glin His Gly Wall Wall Ile Thir Glu Asn Asn 13 O 135 14 O

Thir Wall Wall Ile Pro Cys Lell Gly Ser Ile Ser Asn Lell Asn Wall Ser 145 150 155 160

Lell Ala Arg Tyr Pro Glu Arg Phe Wall Pro Asp Gly Asn Arg 1.65 17O 17s

Ile Ser Trp Asp Ser Gly Phe Thir Ile Pro Ser Tyr Met Ile 18O 185 19 O

Ser Ala Gly Met Wall Phe Cys Glu Ala Ile Asn Asp Glu Ser 195

Glin Ser Ile Met Ile Wall Wall Wall Wall Gly Arg Ile Tyr 21 O 215 22O

Asp Wall Wall Luell Ser Pro Ser His Gly Ile Glu Lell Ser Wall Gly Glu 225 23 O 235 24 O

Luell Wall Luell Asn Thir Ala Arg Thir Glu Lell Asn Wall Gly Ile 245 250 255

Asp Phe Asn Trp Glu Pro Ser Ser His Gln His Lys Lieu 26 O 265 27 O

Wall Asn Arg Asp Lell Thir Glin Ser Gly Ser Glu Met Phe 285

Lell Ser Thir Luell Thir Ile Asp Gly Wall Thir Arg Ser Asp Glin Gly Luell 29 O 295 3 OO

Tyr Thir Ala Ala Ser Ser Gly Luell Met Thir Asn Ser Thir 3. OS 310 315

Phe Wall Arg Wall His Glu Pro Phe Wall Ala Phe Gly Ser Gly Met 3.25 330 335

Glu Ser Luell Wall Glu Ala Thir Wall Gly Glu Arg Wall Arg Ile Pro Ala 34 O 345 35. O

Luell Gly Tyr Pro Pro Pro Glu Ile Trp Tyr Asn Gly 355 360 365

Ile Pro Luell Glu Ser Asn His Thir Ile Ala Gly His Wall Luell Thir 37 O 375

Ile Met Glu Wall Ser Glu Arg Asp Thir Gly ASn Tyr Thir Wall Ile Luell 385 390 395 4 OO

Thir Asn Pro Ile Ser Glu Glin Ser His Wall Wall Ser Luell Wall 4 OS 41O 415

Wall Wall Pro Pro Glin Ile Gly Glu Ser Lell Ile Ser Pro Wall 425 43 O

Asp Ser Tyr Glin Tyr Gly Thir Thir Glin Thir Luell Thir Cys Thir Wall Tyr 435 44 O 445

Ala Ile Pro Pro Pro His His Ile His Trp Trp Glin Luell Glu Glu 450 45.5 460

Glu Ala Asn Glu Pro Ser Glin Ala Wall Ser Wall Thir Asn Pro Tyr 465 470 47s 48O

Pro Glu Glu Trp Arg Ser Wall Glu Asp Phe Glin Gly Gly Asn US 9,078,860 B2 113 114 - Continued

485 490 495

Ile Glu Wall Asn Lys Asn Glin Phe Ala Luell Ile Glu Gly Lys Asn SOO 505

Thir Wall Ser Thir Lell Wall Ile Glin Ala Ala ASn Wall Ser Ala Luell 515 525

Cys Glu Ala Wall Asn Lys Wall Gly Arg Gly Glu Arg Wall Ile Ser 53 O 535 54 O

Phe His Wall Thir Gly Pro Glu Ile Thir Luell Glin Pro Asp Met Glin 5.45 550 555 560

Pro Thir Glu Glin Glu Ser Wall Ser Luell Trp Thir Ala Asp Arg Ser 565 st O sts

Thir Phe Glu Asn Lell Thir Trp Lys Luell Pro Glin Pro Luell Pro 585 59 O

Ile His Wall Gly Glu Lell Pro Thir Pro Wall Asn Luell Asp Thir 595 6OO 605

Lell Trp Luell Asn Ala Thir Met Phe Ser ASn Ser Thir Asn Asp Ile 610 615

Lell Ile Met Glu Lell Lys Asn Ala Ser Luell Glin Asp Glin Gly Asp Tyr 625 630 635 64 O

Wall Luell Ala Glin Asp Arg Thir Lys Arg His Wall Wall 645 650 655

Arg Glin Luell Thir Wall Lell Glu Arg Wall Ala Pro Thir Ile Thir Gly Asn 660 665 67 O

Lell Glu Asn Glin Thir Thir Ser Ile Gly Glu Ser Ile Glu Wall Ser 675 685

Thir Ala Ser Gly Asn Pro Pro Pro Glin Ile Met Trp Phe Asp Asn 69 O. 695 7 OO

Glu Thir Luell Wall Glu Asp Ser Gly Ile Wall Luell Asp Gly Asn Arg 7 Os

Asn Luell Thir Ile Arg Arg Wall Arg Glu Asp Glu Gly Luell Tyr Thir 72 73 O 73

Glin Ala Cys Ser Wall Lell Gly Cys Ala Wall Glu Ala Phe Phe 740 74. 7 O

Ile Ile Glu Gly Ala Glin Glu Lys Thir Asn Luell Glu Ile Ile Ile Luell 760 765

Wall Gly Thir Ala Wall Ile Ala Met Phe Phe Trp Lell Lell Luell Wall Ile 770 775

Ile Luell Arg Thir Wall Lys Arg Ala Asn Gly Gly Glu Lell Thir Gly 78s 79 O 79.

Luell Ser Ile Wall Met Asp Pro Asp Glu Luell Pro Lell Asp Glu His 805 810 815

Glu Arg Luell Pro Asp Ala Ser Trp Glu Phe Pro Arg Asp 825 83 O

Arg Luell Lys Luell Gly Pro Luell Gly Gly Ala Phe Gly Glin Wall 835 84 O 845

Ile Glu Ala Asp Ala Phe Gly Ile Asp Thir Ala Thir Arg Thir 850 855 860

Wall Ala Wall Lys Met Lell Glu Gly Ala Thir His Ser Glu His Arg 865 87O 87s

Ala Luell Met Ser Glu Lell Ile Luell Ile His Ile Gly His His Luell 885 890 895

Asn Wall Wall Asn Lell Lell Gly Ala Cys Thir Pro Gly Gly Pro Luell 9 OO 905 91 O US 9,078,860 B2 115 116 - Continued

Met Wall Ile Wall Glu Phe Cys Llys Phe Gly Asn Lieu Ser Thr Tyr Lieu 915 92 O 925

Arg Ser Arg Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg 93 O 935 94 O

Phe Arg Glin Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Lieu Lys 945 950 955 96.O

Arg Arg Luell Asp Ser Ile Thir Ser Ser Glin Ser Ser Ala Ser Ser Gly 965 97O 97.

Phe Wall Glu Glu Lys Ser Lieu. Ser Asp Val Glu Glu Glu Glu Ala Pro 98O 985 99 O

Glu Asp Luell Asp Phe Lieu. Thir Lieu. Glu. His Lieu. Ile Cys Tyr 995 1OOO 1005

Ser Phe Glin Wall Ala Lys Gly Met Glu Phe Lieu Ala Ser Arg Llys O1O O15 O2O

His Arg Asp Luell Ala Ala Arg Asn. Ile Lieu. Lieu. Ser Glu O3 O O35

Wall Val Lys Ile Cys Asp Phe Gly Lieu Ala Arg Asp Ile O45 OSO Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Lieu Pro O6 O O65

Lell Trp Met Ala Pro Glu Thir Ile Phe Asp Arg Val Tyr Thr O7 O8O

Ile Ser Asp Wall Trp Ser Phe Gly Val Lieu Lleu Trp Glu Ile O9 O O95

Phe Lell Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu O5 10

Glu Arg Arg Luell Lys Glu Gly Thr Arg Met Arg Ala Pro 2O 25

Asp Thir Thir Pro Glu Met Tyr Glin Thr Met Lieu. Asp Cys Trp 35 4 O

His Glu Pro Ser Glin Arg Pro Thr Phe Ser Glu Lieu Val Glu SO 55

His Gly Asn Lieu. Luell Glin Ala Asn Ala Glin Glin Asp Gly Lys 65 70

Asp Ile Wall Leu Pro e Ser Glu. Thir Lieu. Ser Met Glu Glu 8O 85

Asp Gly Luell Ser Luell Pro Thir Ser Pro Val Ser Cys Met Glu 95 2OO

Glu Glu Val Cys Asp Pro Llys Phe His Tyr Asp Asn Thr Ala 21 O 215

Gly Ser Gln Tyr Luell Glin Asn. Ser Lys Arg Llys Ser Arg Pro 225 23 O

Wall Wall Lys Thr Phe Glu Asp Ile Pro Lieu. Glu Glu Pro Glu 24 O 245

Wall Wall Ile Pro Asp Asp Asin Glin Thr Asp Ser Gly Met Val 255 26 O

Lell Ser Glu Glu Lieu. Llys Thr Lieu. Glu Asp Arg Thr Llys Lieu 27 O 27s

Ser Ser Phe Gly Gly Met Val Pro Ser Lys Ser Arg Glu Ser 285 29 O

Wall Ser Glu Gly Ser Asin Glin Thr Ser Gly Tyr Glin Ser Gly 3OO 305 US 9,078,860 B2 117 118 - Continued

Tyr His Ser Asp Asp Thr Asp Thir Thr Val Tyr Ser Ser Glu Glu 1310 131 5 132O

Ala Glu Lieu Lleu Lys Lieu. Ile Glu Ile Gly Val Glin Thr Gly Ser 1325 133 O 1335

Thir Ala Glin Ile Leul Glin Pro Asp Ser Gly Thr Thr Luel Ser Ser 134 O 134 5 1350

Pro Pro Wall 1355

<210s, SEQ ID NO 67 &211s LENGTH : 1298 212. TYPE : PRT &213s ORGANI SM: Homo sapiens

<4 OOs, SEQUEN CE: 67

Met Glin Arg Gly Ala Ala Lell Luell Arg Luell Trp Lell Luell Gly 1. 5 15

Lieu. Lieu. Asp Gly Lell Wall Ser Gly Tyr Ser Met Thir Pro Pro Thir Luell 25 3O

Asn. Ile Thr Glu Glu Ser His Wall Ile Asp Thir Gly Asp Ser Luell Ser 35 4 O 45

Ile Ser Cys Arg Gly Glin His Pro Luell Glu Trp Ala Trp Pro Gly Ala SO 55 6 O

Glin Glu Ala Pro Ala Thir Gly Asp Asp Ser Glu Asp Thir Gly Wall 65 70

Val Arg Asp Glu Gly Thir Asp Ala Arg Pro Wall Luell 85 90 95

Lieu. Lieu. His Glu Wall His Ala Asn Asp Thir Gly Ser Wall Tyr 105 11 O

Ile Ala Arg Ile Glu Gly Thir Thir Ala Ala Ser Ser 115 12 O 125

Tyr Val Phe Wall Arg Asp Phe Glu Glin Pro Phe Ile Asn Pro Asp 13 O 135 14 O

Thir Lieu. Lieu. Wall Asn Arg Asp Ala Met Trp Wall Pro Luell Wall 145 150 155 160

Ser Ile Pro Gly Lell Asn Wall Thir Luell Arg Ser Glin Ser Ser Wall Luell 1.65 17s

Trp Pro Asp Gly Glin Glu Wall Wall Trp Asp Asp Arg Arg Gly Met Luell 18O 185 19 O

Wall Ser Thr Pro Lell Lell His Asp Ala Luell Tyr Lell Glin Glu Thir 195

Thir Trp Gly Asp Glin Asp Phe Luell Ser Asn Pro Phe Lell Wall His Ile 21 O 215 22O

Thr Gly Asn Glu Lell Tyr Asp Ile Glin Luell Luell Pro Arg Ser Luell 225 23 O 235 24 O

Glu Lieu. Lieu. Wall Gly Glu Luell Wall Luell ASn Thir Wall Trp Ala 245 250 255

Glu Phe Asn Ser Gly Wall Thir Phe Asp Trp Asp Pro Gly Lys Glin 26 O 265 27 O

Ala Glu Arg Gly Trp Wall Pro Glu Arg Arg Ser Glin Glin Thir His 27s 285

Thir Glu Lieu. Ser Ser Ile Lell Thir Ile His ASn Wall Ser Glin His Asp 29 O 295 3 OO

Lieu. Gly Ser Wall Cys Ala Asn Asn Gly Ile Glin Arg Phe Arg 3. OS 310 315 32O US 9,078,860 B2 119 120 - Continued

Glu Ser Thir Glu Wall Ile Wall His Glu Asn Pro Phe Ile Ser Wall Glu 3.25 330 335

Trp Luell Gly Pro Ile Lell Glu Ala Thir Ala Gly Asp Glu Luell Wall 34 O 345 35. O

Luell Pro Wall Lys Lell Ala Ala Pro Pro Pro Glu Phe Glin Trp 355 360 365

Lys Asp Gly Ala Lell Ser Gly Arg His Ser Pro His Ala Luell 37 O 375

Wall Luell Glu Wall Thir Glu Ala Ser Thir Gly Thir Thir Luell Ala 385 390 395 4 OO

Lell Trp Asn Ser Ala Ala Gly Luell Arg Arg ASn Ile Ser Luell Glu Luell 4 OS 415

Wall Wall Asn Wall Pro Pro Glin Ile His Glu Lys Glu Ala Ser Ser Pro 425 43 O

Ser Ile Tyr Ser Arg His Ser Arg Glin Ala Luell Thir Cys Thir Ala Tyr 435 44 O 445

Gly Wall Pro Luell Pro Lell Ser Ile Glin Trp His Trp Arg Pro Trp Thir 450 45.5 460

Pro Met Phe Ala Glin Arg Ser Luell Arg Arg Arg Glin Glin Glin 465 470

Asp Luell Met Pro Glin Arg Asp Trp Arg Ala Wall Thir Thir Glin Asp 485 490 495

Ala Wall Asn Pro Ile Glu Ser Luell Asp Thir Trp Thir Glu Phe Wall Glu SOO 505

Gly Asn Thir Wall Ser Lys Luell Wall Ile Glin Asn Ala Asn Wall 515 525

Ser Ala Met Lys Wall Wall Ser Asn Lys Wall Gly Glin Asp Glu 53 O 535 54 O

Arg Luell Ile Phe Tyr Wall Thir Thir Ile Pro Asp Gly Phe Thir Ile 5.45 550 555 560

Glu Ser Pro Ser Glu Glu Luell Luell Glu Gly Glin Pro Wall Luell Luell 565 st O sts

Ser Glin Ala Asp Ser Tyr Glu His Lell Arg Trp 585 59 O

Lell Asn Luell Ser Thir Lell His Asp Ala His Gly Asn Pro Luell Luell Luell 595 605

Asp Cys Asn Wall His Lell Phe Ala Thir Pro Lell Ala Ala Ser Luell 610 615

Glu Glu Wall Ala Pro Gly Ala Arg His Ala Thir Lell Ser Luell Ser Ile 625 630 635 64 O

Pro Arg Wall Ala Pro Glu His Glu Gly His Wall Glu Wall Glin 645 650 655

Asp Arg Arg Ser His Asp His Cys His Luell Ser Wall 660 665 67 O

Glin Ala Luell Glu Ala Pro Arg Luell Thir Glin ASn Lell Thir Asp Luell Luell 675 68O 685

Wall Asn Wall Ser Asp Ser Lell Glu Met Glin Cys Lell Wall Ala Gly Ala 69 O. 695 7 OO

His Ala Pro Ser Ile Wall Trp Asp Glu Arg Lell Luell Glu Glu 7 Os

Ser Gly Wall Asp Lell Ala Asp Ser Asn Glin Lell Ser Ile Glin 72 73 O 73 US 9,078,860 B2 121 122 - Continued

Arg Wall Arg Glu Glu Asp Ala Gly Arg Luell Cys Ser Wall Cys Asn 740 74. 7 O

Ala Gly Cys Val Asn Ser Ser Ala Ser Wall Ala Wall Glu Gly Ser 760 765

Glu Asp Gly Ser Met Glu Ile Wall Ile Luell Wall Gly Thir Gly Wall 770 775

Ile Ala Wall Phe Phe Trp Wall Luell Luell Luell Luell Ile Phe Asn Met 79 O 79.

Arg Arg Pro Ala His Ala Asp Ile Thir Gly Lell Ser Ile Ile 805 810 815

Met Asp Pro Gly Glu Wall Pro Luell Glu Glu Glin Glu Tyr Luell Ser 82O 825 83 O

Asp Ala Ser Glin Trp Glu Phe Pro Arg Glu Arg Lell His Luell Gly 835 84 O 845

Arg Wall Luell Gly Tyr Gly Ala Phe Gly Wall Wall Glu Ala Ser Ala 850 855 860

Phe Gly Ile His Llys Gly Ser Ser Asp Thir Wall Ala Wall Met 865

Lell Glu Gly Ala Thir Ala Ser Glu His Arg Ala Lell Met Ser Glu 885 890 895

Lell Ile Lieu. Ile His Ile Gly Asn His Luell Asn Wall Wall Asn Luell 9 OO 905 91 O

Lell Gly Ala Cys Thr Pro Glin Gly Pro Luell Met Wall Ile Wall Glu 915 92 O 925

Phe Cys Asn Lieu Ser Asn Phe Lieu. Arg Ala Arg Asp 93 O 935 94 O

Ala Phe Ser Pro Cys Ala Ser Pro Glu Glin Arg Gly Arg Phe 945 950 955 96.O

Arg Ala Met Wall Glu Lell Arg Luell Asp Arg Arg Arg Pro Gly Ser 965 97.

Ser Asp Arg Wall Lieu. Phe Arg Phe Ser Lys Thir Glu Gly Gly Ala 98O 985 99 O

Arg Arg Ala Ser Pro Asp Glu Ala Glu Asp Lieu. Trp Lieu. Ser Pro 995

Lell Thir Met Glu Asp Lieu Val Cys Ser Phe Glin Wall Ala Arg O1O 5

Gly Met Glu Phe Lieu Ala Ser Arg Ile H is Arg Asp Lell O25 O O35

Ala Arg ASn Ile Lieu. Luell Ser Glu Ser Asp V al Wall Ile 5 OSO

Phe Gly Lieu Ala Arg Asp Ile Lys A Sp Pro Asp O65

Wall Gly Ser Ala Arg Luell Pro Luell Met Ala Pro

Glu Ser Ile Phe Asp Llys Val Tyr Thir Thir Glin S Asp Wall Trp O85

Ser Phe Gly Val Lieu. Lieu. Trp Glu Ile Phe Ser L. el Gly Ala Ser 1 OO 11 O

Pro yr Pro Gly Val Glin Ile Asn Glu Glu Phe Cys Glin Arg Lell 115 125

Arg Gly Thr Arg Met Arg Ala Pro Glu Lieu. A la Thir Pro Ala 13 O 14 O

Ile Arg Arg Ile Met Lieu. Asn. Cys Trp Ser Gly A Sp Pro Ala US 9,078,860 B2 123 124 - Continued

145 15 O 155 Arg Pro Ala Phe Ser Glu Lieu Val Glu Ile Lieu. Gly Asp Lieu. Lieu. 16 O 1.65 17 O Glin Gly Arg Gly Lieu. Glin Glu Glu Glu Glu Val Cys Met Ala Pro 17s 18O 185 Arg Ser Ser Glin Ser Ser Glu Glu Gly Ser Phe Ser Glin Val Ser 190 195 2OO Thir Met Ala Lieu. His Ile Ala Glin Ala Asp Ala Glu Asp Ser Pro 2O5 21 O 215 Pro Ser Lieu. Glin Arg His Ser Lieu Ala Ala Arg Tyr Tyr Asn Trp 22O 225 23 O Val Ser Phe Pro Gly Cys Lieu Ala Arg Gly Ala Glu Thir Arg Gly 235 24 O 245 Ser Ser Arg Met Lys Thr Phe Glu Glu Phe Pro Met Thr Pro Thr 250 255 26 O Thr Tyr Lys Gly Ser Val Asp Asn Glin Thr Asp Ser Gly Met Val 265 27 O 27s Lieu Ala Ser Glu Glu Phe Glu Glin Ile Glu Ser Arg His Arg Glin 28O 285 29 O Glu Ser Gly Phe Arg 295

<210s, SEQ ID NO 68 &211s LENGTH: 923 212. TYPE: PRT <213> ORGANISM; Homo sapiens <4 OOs, SEQUENCE: 68 Met Glu Arg Gly Lieu Pro Lieu. Lieu. Cys Ala Val Lieu Ala Lieu Val Lieu. 1. 5 1O 15 Ala Pro Ala Gly Ala Phe Arg Asn Asp Llys Cys Gly Asp Thir Ile Llys 2O 25 3O Ile Glu Ser Pro Gly Tyr Lieu. Thir Ser Pro Gly Tyr Pro His Ser Tyr 35 4 O 45 His Pro Ser Glu Lys Cys Glu Trp Lieu. Ile Glin Ala Pro Asp Pro Tyr SO 55 6 O Glin Arg Ile Met Ile Asn. Phe Asn Pro His Phe Asp Lieu. Glu Asp Arg 65 70 7s 8O Asp Cys Llys Tyr Asp Tyr Val Glu Val Phe Asp Gly Glu Asn. Glu Asn 85 90 95 Gly His Phe Arg Gly Lys Phe Cys Gly Lys Ile Ala Pro Pro Pro Val 1OO 105 11 O Val Ser Ser Gly Pro Phe Leu Phe Ile Llys Phe Val Ser Asp Tyr Glu 115 12 O 125 Thr His Gly Ala Gly Phe Ser Ile Arg Tyr Glu Ile Phe Lys Arg Gly 13 O 135 14 O

Pro Glu. Cys Ser Glin Asn Tyr Thr Thr Pro Ser Gly Val Ile Llys Ser 145 150 155 160

Pro Gly Phe Pro Glu Lys Tyr Pro Asn Ser Lieu. Glu. Cys Thr Tyr Ile 1.65 17O 17s

Val Phe Ala Pro Llys Met Ser Glu Ile Ile Leu Glu Phe Glu Ser Phe 18O 185 19 O

Asp Leu Glu Pro Asp Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr 195 2OO 2O5 US 9,078,860 B2 125 126 - Continued

Asp Arg Luell Glu Ile Trp Asp Gly Phe Pro Asp Wall Gly Pro His Ile 21 O 215 22O

Gly Arg Gly Glin Thir Pro Gly Arg Ile Arg Ser Ser Ser 225 23 O 235 24 O

Gly Ile Luell Ser Met Wall Phe Thir Asp Ser Ala Ile Ala Lys Glu 245 250 255

Gly Phe Ser Ala Asn Tyr Ser Wall Luell Glin Ser Ser Wall Ser Glu Asp 26 O 265 27 O

Phe Cys Met Glu Ala Lell Gly Met Glu Ser Gly Glu Ile His Ser 27s 285

Asp Glin Ile Thir Ala Ser Ser Glin Tyr Ser Thir Asn Trp Ser Ala Glu 29 O 295 3 OO

Arg Ser Arg Luell Asn Tyr Pro Glu Asn Gly Trp Thir Pro Gly Glu Asp 3. OS 310 315

Ser Arg Glu Trp Ile Glin Wall Asp Luell Gly Lell Lell Arg Phe Wall 3.25 330 335

Thir Ala Wall Gly Thir Glin Gly Ala Ile Ser Glu Thir Lys 34 O 345 35. O

Wall Thir Ile Asp Wall Ser Ser Asn Gly Glu Asp 355 360 365

Trp Ile Thir Ile Glu Gly Asn Pro Wall Lell Phe Glin Gly Asn 37 O 375

Thir Asn Pro Thir Asp Wall Wall Wall Ala Wall Phe Pro Pro Luell Ile 385 390 395 4 OO

Thr Arg Phe Wall Arg Ile Pro Ala Thr Trp Glu Gly Ile Ser 4 OS 415

Met Arg Phe Glu Wall Gly Lys Ile Thir Asp Pro Ser 425 43 O

Gly Met Luell Gly Met Wall Ser Gly Luell Ile Ser Asp Ser Glin Ile Thir 435 44 O 445

Ser Ser Asn Glin Gly Asp Arg Asn Trp Met Pro Glu Asn Ile Arg Luell 450 45.5 460

Wall Thir Ser Arg Ser Gly Trp Ala Luell Pro Pro Ala Pro His Ser Tyr 465 470

Ile Asn Glu Trp Lell Glin Ile Asp Luell Gly Glu Glu Ile Wall Arg 485 490 495

Gly Ile Ile Ile Glin Gly Gly His Arg Glu Asn Wall Phe Met SOO 505

Arg Phe Ile Gly Ser Asn Asn Gly Ser Asp Trp Met 515 52O 525

Ile Met Asp Asp Ser Arg Ala Ser Phe Glu Gly Asn Asn 53 O 535 54 O

Asn Tyr Asp Thir Pro Glu Lell Arg Thir Phe Pro Ala Lell Ser Thir Arg 5.45 550 555 560

Phe Ile Arg Ile Tyr Pro Glu Arg Ala Thir His Gly Gly Luell Gly Luell 565 st O sts

Arg Met Glu Luell Lell Gly Glu Wall Glu Ala Pro Thir Ala Gly Pro 585 59 O

Thir Thir Pro Asn Gly Asn Lell Wall Asp Glu Cys Asp Asp Asp Glin Ala 595 605

Asn Cys His Ser Gly Thir Gly Asp Asp Phe Glin Lell Thir Gly Gly Thir 610 615

Thir Wall Luell Ala Thir Glu Pro Thir Wall Ile Asp Ser Thir Ile Glin US 9,078,860 B2 127 128 - Continued

625 630 635 64 O

Ser Glu Phe Thr Tyr Gly Phe Asn Cys Glu Phe Gly Trp Gly Ser 645 650 655

His Thir Phe Cys His Trp Glu His Asp ASn His Wall Glin Luell 660 665 67 O

Trp Ser Wall Lel Thir Ser Lys Thr Gly Pro Ile Glin Asp His Thir 675 68O 685

Asp Gly Asn Phe Ile Tyr Ser Glin Ala Asp Glu Asn Glin Gly 69 O. 695 7 OO

Wall Ala Arg Lel Wall Ser Pro Wall Wall Ser Glin Asn Ser Ala His 7 Os 71O

Met Thir Phe Trp Tyr His Met Ser Gly Ser His Wall Gly Thir Luell 72 73 O 73

Arg Wall Lel Arg Tyr Gln Lys Pro Glu Glu Asp Glin Luell Wall 740 74. 7 O

Trp Met Ala Ile Gly His Glin Gly Asp His Trp Glu Gly Arg Wall 7ss 760 765

Lell Luell His Ser Lieu Lys Lieu. Glin Wall Ile Phe Glu Gly Glu 770 775

Ile Gly Gly Asn Lieu. Gly Gly Ile Ala Wall Asp Asp Ile Ser Ile 79 O 79.

Asn Asn His Ile Ser Glin Glu Asp Ala Pro Ala Asp Luell Asp 805 810 815

Asn Pro Glu Ile Lys Ile Asp Glu Thir Gly Ser Thir Pro Gly 825 83 O

Glu Gly Glu Gly Glu Gly Asp Asn Ile Ser Arg Pro Gly 835 84 O 845

Asn Wall Luell Thir Lieu. Glu Pro Ile Luell Ile Thir Ile Ile Ala Met 850 855 860

Ser Ala Luell Gly Val Lieu. Lieu. Gly Ala Wall Cys Gly Wall Wall Luell Tyr 865 87O

Ala Trp His Asn Gly Met Ser Glu Arg Asn Lell Ser Ala Luell 885 890 895

Glu Asn Asn Phe Glu Lieu Wall Asp Gly Wall Lell Lys Asp 9 OO 905 91 O

Luell Asn Thir Gln Ser Thr Tyr Ser Glu Ala 915 92 O

<210s, SEQ ID NO 69 &211s LENGTH: 931 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 69

Met Asp Met Phe Pro Leu. Thir Trp Wall Phe Luell Ala Lell Phe Ser 1. 5 15

Arg His Glin Wall Arg Gly Glin Pro Asp Pro Pro Gly Gly Arg Luell 2O 25 3O

Asn Ser Lys Asp Ala Gly Tyr Ile Thir Ser Pro Gly Tyr Pro Glin Asp 35 4 O 45

Pro Ser His Glin Asn. Cys Glu Trp Ile Wall Tyr Ala Pro Glu Pro SO 55 6 O

Asn Glin Ile Wall Lieu. Asn. Phe Asn Pro His Phe Glu Ile Glu 65 70 7s US 9,078,860 B2 129 130 - Continued

His Asp Tyr Asp Phe Ile Glu Ile Arg Asp Gly Asp Ser Glu 85 90 95

Ser Ala Asp Luell Lell Gly His Cys Gly ASn Ile Ala Pro Pro Thir 105 11 O

Ile Ile Ser Ser Gly Ser Met Luell Ile Phe Thir Ser Asp 115 12 O 125

Ala Arg Glin Gly Ala Gly Phe Ser Luell Arg Glu Ile Phe Thir 13 O 135 14 O

Gly Ser Glu Asp Ser Asn Phe Thir Ser Pro Asn Gly Thir Ile 145 150 155 160

Glu Ser Pro Gly Phe Pro Glu Pro His Asn Lell Asp Cys Thir 1.65 17O 17s

Phe Thir Ile Luell Ala Pro Met Glu Ile Ile Lell Glin Phe Luell 18O 185 19 O

Ile Phe Asp Luell Glu His Asp Pro Luell Glin Wall Gly Glu Gly Asp 195 2OO

Tyr Asp Trp Lell Asp Ile Trp Asp Gly Ile Pro His Wall Gly Pro 21 O 215 22O

Lell Ile Gly Tyr Cys Gly Thir Thir Pro Ser Glu Luell Arg Ser 225 23 O 235 24 O

Ser Thir Gly Ile Lell Ser Lell Thir Phe His Thir Asp Met Ala Wall Ala 245 250 255

Asp Gly Phe Ser Ala Arg Tyr Luell Wall His Glin Glu Pro Luell 26 O 265 27 O

Glu Asn Phe Gln Cys Asn Wall Pro Lieu Gly Met Glu Ser Gly Arg Ile 27s 285

Ala Asn Glu Glin Ile Ser Ala Ser Ser Thir Ser Asp Gly Arg Trp 29 O 295 3 OO

Thir Pro Glin Glin Ser Arg Lell His Gly Asp Asp Asn Gly Trp Thir Pro 3. OS 310 315

Asn Luell Asp Ser Asn Glu Tyr Luell Glin Wall Asp Lell Arg Phe Luell 3.25 330 335

Thir Met Luell Thir Ala Ile Ala Thir Glin Gly Ala Ile Ser Arg Glu Thir 34 O 345 35. O

Glin Asn Gly Tyr Tyr Wall Ser Luell Glu Wall Ser Thir Asn 355 360 365

Gly Glu Asp Trp Met Wall Tyr Arg His Gly Lys Asn His Wall Phe 37 O 375

Glin Ala Asn Asn Asp Ala Thir Glu Wall Wall Luell Asn Luell His Ala 385 390 395 4 OO

Pro Luell Luell Thir Arg Phe Wall Arg Ile Arg Pro Glin Thir Trp His Ser 4 OS 415

Gly Ile Ala Luell Arg Lell Glu Luell Phe Gly Cys Arg Wall Thir Asp Ala 425 43 O

Pro Ser Asn Met Lell Gly Met Luell Ser Gly Lell Ile Ala Asp Ser 435 44 O 445

Glin Ile Ser Ala Ser Ser Thir Glin Glu Tyr Luell Trp Ser Pro Ser Ala 450 45.5 460

Ala Arg Luell Wall Ser Ser Arg Ser Gly Trp Phe Pro Arg Ile Pro Glin 465 470 47s

Ala Glin Pro Gly Glu Glu Trp Luell Glin Wall Asp Lell Gly Thir Pro 485 490 495

Thir Wall Gly Wall Ile Ile Glin Gly Ala Arg Gly Gly Asp Ser Ile US 9,078,860 B2 131 132 - Continued

SOO 505

Thir Ala Wall Glu Ala Arg Ala Phe Wall Arg Phe Lys Wall Ser 515 525

Ser Luell Asn Gly Lys Asp Trp Glu Ile Glin Asp Pro Arg Thir Glin 53 O 535 54 O

Glin Pro Luell Phe Glu Gly Asn Met His Tyr Asp Thir Pro Asp Ile 5.45 550 555 560

Arg Arg Phe Asp Pro Ile Pro Ala Glin Tyr Wall Arg Wall Pro Glu 565 st O sts

Arg Trp Ser Pro Ala Gly Ile Gly Met Arg Luell Glu Wall Luell Gly 585 59 O

Asp Trp Thir Asp Ser Pro Thir Wall Glu Thir Lell Gly Pro Thir Wall 595 605

Ser Glu Glu Thir Thir Thir Pro Pro Thir Glu Glu Glu Ala Thir 610 615

Glu Gly Glu Asn Cys Ser Phe Glu Asp Asp Asp Luell Glin Luell 625 630 635 64 O

Pro Ser Gly Phe Asn Asn Phe Asp Phe Luell Glu Glu Pro Cys Gly 645 650 655

Trp Met Asp His Ala Trp Luell Arg Thir Thir Trp Ala Ser Ser 660 665 67 O

Ser Ser Pro Asn Asp Arg Thir Phe Pro Asp Asp Arg Asn Phe Luell Arg 675 685

Lell Glin Ser Asp Ser Glin Arg Glu Gly Glin Tyr Ala Arg Luell Ser 690 695 7 OO

Pro Pro Wall His Lell Pro Arg Ser Pro Wall Cys Met Glu Phe Tyr 7 Os

Glin Ala Thir Gly Gly Arg Gly Wall Ala Luell Glin Wall Wall Arg Ala 72 73 O

Ser Glin Glu Ser Lys Lell Lell Trp Wall Ile Arg Glu Asp Glin Gly 740 74. 7 O

Glu Trp Lys His Gly Arg Ile Ile Luell Pro Ser Asp Met 760 765

Glin Ile Wall Phe Glu Gly Wall Ile Gly Gly Arg Ser Gly Ile 770 775

Ala Ile Asp Asp Ile Arg Ile Ser Thir Asp Wall Pro Lell Glu Asn Cys 79 O 79.

Met Glu Pro Ile Ser Ala Phe Ala Gly Glu ASn Phe Wall Asp Ile 805 810 815

Pro Glu Ile His Glu Arg Glu Gly Tyr Glu Asp Glu Ile Asp Asp Glu 825 83 O

Glu Wall Asp Trp Ser Asn Ser Ser Ser Ala Thir Ser Gly Ser Gly 835 84 O 845

Ala Pro Ser Thir Asp Glu Ser Trp Luell Tyr Thir Luell Asp Pro 850 855 860

Ile Luell Ile Thir Ile Ile Ala Met Ser Ser Luell Gly Wall Luell Luell Gly 865 87O 88O

Ala Thir Ala Gly Lell Lell Luell Cys Thir Ser Ser Gly 885 890 895

Lell Ser Ser Arg Ser Thir Thir Luell Glu ASn Asn Phe Glu Luell 9 OO 905 91 O

Asp Gly Luell Lys His Wall Met ASn His Glin Cys 915 92 O 925 US 9,078,860 B2 133 134 - Continued

Ser Glu Ala 93 O

<210s, SEQ ID NO 70 &211s LENGTH: 6 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 70 Ser Lieu. Thir Arg Lys Asp 1.

<210s, SEQ ID NO 71. &211s LENGTH: 6 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 71. Cys Asp Llys Pro Arg Arg 1.

<210s, SEQ ID NO 72 &211s LENGTH: 5 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 72

Ala Asn. Ile Thr Wall 1. 5

<210s, SEQ ID NO 73 &211s LENGTH: 9 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 73

Ala Asn. Ile Thir Wall Asn. Ile Thir Wall 1. 5

What is claimed: 7. The VEGF-A receptor antagonist of claim 5, wherein the 1. A human vascular endothelial growth factor A (VEGF additional Substitutions are selected from the group consist A) receptor antagonist comprising a Substitution at an isoleu 45 ing of E72K and E73K of SEQID NO:4. cine residue corresponding to I83 in SEQID NO:4 or SEQID 8. The VEGF-A receptor antagonist of claim 5, wherein the NO:13, wherein the substitution is a lysine, arginine or histi dine, and the Substitution results in a decrease in bioactivity as additional Substitution is at a position corresponding to E44R compared to wild-type VEGF. or E44K of SEQ ID NO:4. 2. The VEGF-A receptor antagonist of claim 1, wherein the 9. The VEGF-A receptor antagonist of claim 5, wherein the VEGF-A receptor antagonist comprises an amino acid 50 additional Substitution is at a position corresponding to Q87K sequence of SEQID NO: 4, and I83 is substituted by a lysine, or Q87L of SEQ ID NO:4. arginine or histidine. 10. The VEGF-A receptor antagonist of claim 5, wherein 3. The VEGF-A receptor antagonist of claim 1, wherein the the additional substitution corresponds to E67K of SEQ ID VEGF-A receptor antagonist comprises an amino acid NO:4. sequence of SEQ ID NO: 13, and I83 is substituted by a 55 11. The VEGF-A receptor antagonist of claim 1, wherein lysine, arginine or histidine. 4. The VEGF-A receptor antagonist of claim 1, which is interaction of the VEGF-A receptor antagonist and a native expressed as at least one of Subunits of a homodimer or VEGF-A receptor results in inhibition of angiogenesis. heterodiner having two subunits. 12. The VEGF-A receptor antagonist of claim 11, wherein 5. The VEGF-A receptor antagonist of claim 1, wherein the 60 the native VEGF-A receptor is kinase insert domain receptor antagonist contains one or more additional basic amino acid (KDR). Substitutions at the position(s) corresponding to the residues 13. The VEGF-A receptor antagonist of claim 5, wherein selected from the group consisting of positions E44, E67. the antagonist contains the amino acid substitutions corre E72, E73 and Q87 of SEQID NO: 4. sponding to E72R, E73R and 183K of SEQID NO:4. 6. The VEGF-A receptor antagonist of claim 5, wherein the 65 14. The VEGF-A receptor antagonist of claim 5, wherein additional Substitutions are selected from the group consist the antagonist contains the amino acid substitutions corre ing of E72R and E73R of SEQID NO:4. sponding to E44R, E72R, E73R and I83K of SEQID NO:4. US 9,078,860 B2 135 136 15. The VEGF-A receptor antagonist of claim 1, further toxin, cholera toxin, maitotoxin, palytoxin, ciguatoxin, tex comprising an amino acid Substitution at a position corre itilotoxin, batrachotoxin, alpha conotoxin, taipoxin, tetrodot sponding to C146 or C160 of SEQID NO:4. oxin, alpha tityustoxin, saxitoxin, anatoxin, microcystin, 16. The VEGF-A receptor antagonist of claim 1, wherein aconitine, exfoliatin toxins A, exfoliatin B, an enterotoxin, the amino acid Substitution is at a position corresponding to toxic shock syndrome toxin (TSST-1), Y. pestis toxin and a C146S or C160S of SEQ ID NO:4. gas gangrene toxin. 17. The VEGF-A receptor antagonist of claim 1, further 21. The VEGF-A receptor antagonist of claim 1, compris comprising an amino acid Substitution at a position corre sponding to A111 and/or A148 of SEQID NO: 4. ing a VEGF-A selected from the group consisting of 18. The VEGF-A receptor antagonist of claim 1, wherein 10 VEGF (SEQ ID NO:4), VEGF (SEQ ID NO:13), the amino acid Substitution is at a position corresponding to VEGF (SEQ ID NO:6), VEGF (SEQ ID NO:8), A111P and/or A148P of SEQID NO:4. VEGF (SEQ ID NO:10), VEGF (SEQ ID NO:15), 19. The VEGF-A receptor antagonist of claim 1, further VEGF (SEQID NO:17), and VEGF (SEQ ID NO:19), comprising a toxin. wherein I83 is substituted by lysine, arginine or histidine. 20. The VEGF-A receptor antagonist of claim 19, wherein 15 22. A pharmaceutical composition comprising the the toxin is selected from the group consisting of a Pseudomo VEGF-A receptor antagonist of claim 1 and at least one nas exotoxin (PE), a Diphtheria toxin (DT), ricintoxin, abrin excipient. toxin, anthrax toxins, Shiga toxin, botulism toxin, tetanus