AU2013213745B2

AU2013213745B2 - VEGF analogs and methods of use

Info

Publication number: AU2013213745B2
Application number: AU2013213745A
Authority: AU
Inventors: Mariusz W. Szkudlinski; Bruce D. Weintraub
Original assignee: Trophogen Inc
Current assignee: Trophogen Inc
Priority date: 2005-10-06
Filing date: 2013-08-09
Publication date: 2015-10-29
Anticipated expiration: 2026-10-06
Also published as: AU2013213745A1

Abstract

VEGF Analogs and Methods of Use Abstract Modified VEGF proteins that inhibit VEGF-mediated activation or proliferation of endothelial cells are disclosed. The analogs may be used to inhibit VEGF-mediated activation of endothelial cells in angiogenesis-associated diseases such as cancer, inflammatory diseases, eye diseases, and skin disorders.

Description

AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention title: VEGF analogs and methods of use This application is a divisional of Australian Patent Application No 2006302345 which is the Australian national phase entry of PCT/US2006/039181, which claims priority to US provisional patent application No 60/808,106 filed 25 May 2006 and US provisional patent application No 60/723,917 filed 6 October 2005 and. Each of these applications is herein incorporated by reference in their entireties. The following statement is a full description of this invention, including the best method of performing it known to us: 1 VEGF ANALOGS AND METHODS OF USE INVENTORS: MARIUSZ W. SZKUDLINSKI AND BRUCE WEINTRAUB FIELD OF INVENTION [0001] This application relates to the design and use of vascular endothelial growth factor (VEGF) analogs as VEGF receptor antagonists to inhibit or reduce angiogenesis for the treatment of conditions and diseases associated with angiogenesis. The application also discloses VEGF analogs that exhibit increased receptor binding affinity to native receptors such as KDR. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of U.S. Provisional Application No. 60/723,917, filed October 6, 2005, and U.S. Provisional Application No. 60/808,106, filed May 25, 2006, which are herein incorporated by reference in their entireties. BACKGROUND OF INVENTION [0003] Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development. They are predominantly produced by endothelial, hematopoietic and stromal cells in response to hypoxia and stimulation with growth factors such as transforming growth factors, interleukins and platelet-derived growth factor. [0004] In mammals, VEGFs are encoded by a family of genes and include VEGF-A, VEGF-B, VEGF-C, VEGF-D and Placenta like Growth Factor (PlGF). Highly related proteins include oif virus-encoded VEGF-like proteins referred to as VEGF-E and a series of snake venoms referred to as VEGF-F. VEGFs and VEGF-related proteins are members of 42723 vl/DC 1a the Platelet Derived Growth Factor (PDGF) supergene family of cystine kmot growth factors. All members of the PDGF supergene family share a high degree of structural homology with PDGF (see U.S. Patent Application 09/813,398 which is herein incorporated by reference in its entirety). [0005] VEGF-A, VEGF-B and PlGF are predominantly required for blood vessel fonnation, whereas VEGF-C and VEGF-D are essential for the formation of lymphatic vessels. Angiogenesis is the process by which new blood vessels or lymphatic vessels form by developing from pre-existing vessels. The process is initiated when VEGFs bind to receptors on endothelial cells, signaling activation of endothelial cells. Activated endothelial cells produce enzymes which dissolve tiny holes in the basement membrane surrounding existing vessels. Endothelial cells then begin to proliferate and migrate out through the dissolved holes of the existing vessel to form new vascular tubes (Alberts et al., 1994, Molecular Biology of the Cell. Garland Publishing, Inc., New York, N.Y. 1294 pp.). [0006] Three type III receptor tyrosine kinases are activated by VEGFs during angiogenesis:frns-like tyrosine kinase (Flt-1, also known as VEGFRl), kinase domain receptor or kinase insert domain-containing receptor (KDR, also known as VEGFR2 and Flk-1) and Flt-4 (also known as VEGFR3). KDR is the predominant receptor in angiogenic signaling, whereas Flt-1 is associated with the regulation of blood vessel morphogenesis and Flt-4 regulates lymphangiogenesis. These receptors are expressed almost exclusively on endothelial cells, with a few exceptions such as the expression of Flt- 1 in monocytes where it mediates chemotaxis (Barleon et al., 1996, Blood. 87: 3336-3343). [0007] VEGF receptors are closely related to Fms, Kit and PDGF receptors. They consist of seven extracellular immunoglobulin (Ig)-like domains, a transmembrane (TM) domain, a regulatory juxtamembrane domain, an intracellular tyrosine kinase domain interrupted by a short peptide, the kinase insert domain, followed by a sequence carrying several tyrosine residues involved in recruiting downstream signaling molecules. Mutation analysis of the 42723 vl/DC 2 extracellular domains of Flt-I and KDR show that the second and third Ig-like domains constitute the high-affinity ligand-binding domain for VEGF with the first and fourth Ig domains apparently regulating ligand binding and receptor dimerization, respectively (Davis-Smyth et al., 1998, J. Biol. Chem. 273: 3216-3222; Fuh et al., 1998, J. Biol. Chem. 273: 11197-11204; and Shinkai et al., 1998, J. Biol. Chem. 273: 31283-31288). Receptor tyrosine kinases are activated upon ligand-mediated receptor dimerization (Hubbard, 1991, Prog. Biophys. Mol. Biol. 71: 343-358; Jiang and Hunter, 1999, Curr. Biol. 9: R568-R571; and Lemmon and Schlessinger, 1998, Methods Mol. Biol. 84: 49-71). Signal specificity of VEGF receptors is further modulated upon recruitment of coreceptors, such as neuropilins, heparin sulfate, integrins or cadherins. [0008] VEGF molecules interact with one or more tyrosine kinase receptors during angiogenesis. For instance, VEGF-A acts predominantly through KDR and Flt-1. VEGF-C and VEGF-D similarly are specific ligands for KDR and VEGFR3. PIGF and VEGF-B are believed to bind only to Flt-1. Viral VEGF-E variants activate KDR. VEGF-F variants interact with either VEGFR3 or KDR. [0009] In addition to the two classical receptors, there are several membrane or soluble receptors modulating VEGF bioactivity and angiogenesis. For instance, neuropilin- 1 and neuropilin-2 interact with both KDR and Flt-1, respectively, stimulating signaling of those receptors. Isoforms of VEGF-A, VEGF-B, PlGF-2 have been shown to bind to neuropilin-I (Soker et al., 1998, Cell. 92: 735-745; Makinen et al., 1999, J. Biol. Chem. 274: 21217 21222; and Migdal et al., 1998, J. Biol. Chem. 273: 22272-22278). VEGF isoforms capable of interacting of interacting with neuropilin, i.e., those isoforms with exon 7 or 6 and 7, are also capable of interacting with heparin sulfate. [00101 Although VEGF-A is the best characterized of the VEGF proteins, the molecular basis of the interaction between VEGF-A and KDR and Flt-I is not well understood. Although VEGFR1 binds VEGF-A with a 50-fold higher affinity than KDR, KDR is 42723 vl/DC 3 considered to be the major transducer of VEGF-A angiogenic effects, i.e., mitogenicity, chemotaxis and induction of tube fonnation (Binetruy-Tourniere et al., supra). There is, however, growing evidence that Flt-I has a significant role in hematopoiesis and in the recruitment of monocytes and other bone-marrow derived cells that may home in on tumor vasculature and promote angiogenesis (Hattori et al., 2002, Nature Med. 8: 841-849; Gerber et al., 2002, Nature. 417: 954-958; and Luttun et al., 2002, Nature Med. 8: 831-840). Further, in some cases Flt-i is expressed by tumor cells and may mediate a chemotactic signal, thus potentially extending the role of this receptor in cancer growth (Wey et al., 2005, Cancer. 104: 427-438). [0011] A single VEGF-A homodimer induces dimerization of two KDR receptors and autophosphorylation of their cytoplasmatic portions. Previous studies suggested that by analogy to glycoprotein hormones, the charged amino acid residues in the peripheral loops of VEGF-A are also critical in providing high affinity electrostatic interactions with its respective receptors (Szkudlinski et al., 1996, Nat. Biotechnol. 14(10): 1257-63; Fuh et al., supra; Muller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94(14): 7192-7; Keyt et al., 1996, J. Biol. Chem. 271(10): 563 8-46). However, it should be noted that many mutations in VEGF A have no major effect on receptor binding affinity. Mutations in the peripheral loops of VEGF primarily have resulted in loss-of-function. Further, there appear to be no previous amino acid substitutions increasing binding affinity to KDR more than 2-fold. [0012] Angiogenesis is responsible for beneficial biological events such as wound healing, myocardial infarction repair, and ovulation. On the other hand, angiogenesis is also responsible for causing or contributing to diseases such as growth and metastasis of solid tumors (Isayeva et al., 2004, Int. J. Oncol. 25(2):335-43; Takeda et al., 2002, Ann Surg. Oncol. 9(7):610-16); atherosclerosis; abnormal neovascularization of the eye as seen in diseases such as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration (Yoshida et al., 1999, Histol Histopathol. 14(4):1287-94; 42723 v1/DC 4 Aiello, 1997, Ophthalmic Res. 29(5):354-62); chronic inflammatory conditions such as rheumatoid arthritis osteoarthritis, and septic arthritis; neurodegenerative disease (Ferrara, N., 2004, Endocr. Rev. 25: 581-611); placental insufficiency, i.e., preeclampsia (Ferrara, supra); and skin diseases such as dermititis, psoriasis, warts, cutaneous malignancy, decubitus ulcers, stasis ulcers, pyogenic granulomas, hemangiomas, Kaposi's sarcoma, hypertrophic scars, and keloids (Arbiser, 1996, J. Am. Acad. Dermatol. 34(3):486-97). During rheumatoid arthritis, for example, endothelial cells become activated and express adhesion molecules and chemokines, leading to leukocyte migration from the blood into the tissue. Endothelial cell permeability increases, leading to edema formation and swelling of the joints (Middleton et al., 2004, Arthritis Res. Ther. 6(2):60-72). [0013] VEGF, in particular VEGF-A, has been implicated in many of the diseases and conditions associated with increased, decreased, and/or dysregulated angiogenesis (Binetruy Tourniere et al., 2000, EMBO J. 19(7): 1525-33). For instance, VEGF has been implicated in promoting solid tumor growth and metasta-sis by stimulating tumor-associated angiogenesis (Lu et al., 2003, J. Biol. Chem. 278(44): 43496-43507). VEGF is also a signficant mediator of intraocular neovascularization and permeability. Overexpression of VEGF in transgenic mice results in clinical intraretinal and subretinal neovascularization, and the formation of leaky intraocular blood vessels detectable by angiography, as seen in human eye disease (Miller, 1997, Am. J. Pathol. 151(1): 13-23). Additionally, VEGF has been identified in the peritoneal fluid of women with unexplained infertility and endometriosis (Miedzybrodzki et al., 2001, Ginekol. Pol. 72(5): 427-430), and the overexpression of VEGF in testis and epididymis has been found to cause infertility in transgenic mice (Korpelainen et al., 1998, J. Cell Biol. 143(6): 1705-1712). Recently, VEGF-A has been identified in the synovial fluid and serum of patients with rheumatoid arthritis (RA), and its expression is correlated with disease severity (Clavel et al., 2003, Joint Bone Spine. 70(5): 321-6). Given the involvement of pathogenic angiogenesis in such a 42723 v1/DC 5 wide variety of disorders and diseases, inhibition of angiogenesis, and particularly of VEGF signaling, is a desirable therapeutic goal. [00141 Inhibition of angiogenesis and tumor inhibition has been achieved by using agents that either interrupt VEGF-A and KDR interaction and/or block the KDR signal transduction pathway including: peptides that block binding of VEGF to KDR (Binetruy-Toumiere et al., 2000, EMBO J. 19(7): 1525-33); antibodies to VEGF (Kim et al., 1993, Nature 362, 841 844; Kanai et al., 1998, J. Cancer 77, 933-936; Margolin et al., 2001, J. Clin. Oncol. 19, 851-856); antibodies to KDR (Lu et al., 2003, supra; Zhu et al., 1998, Cancer Res. 58, 3209-3214; Zhu et al. 2003, Leukemia 17, 604-611; Prewett et al., 1999, Cancer Res. 59, 5209-5218); soluble receptors (Holash et al., 2002, Proc. Natl. Acad. Sci. USA 99, 11393 11398; Clavel et al. supra); tyrosine kinase inhibitors (Fong et al., 1999, Cancer Res. 59, 99 106; Wood et al., 2000, Cancer Res. 60, 2178-2189; Grosios et al., 2004, Inflamm Res. 53(4):133-42); anti-VEGF immunotoxins (Olson et al., 1997, Int. J. Cancer 73, 865-870); ribozymes (Pavco et al., 2000, Clin. Cancer Res. 6, 2094-2103); antisense mediated VEGF suppression (Forster et al., 2004, Cancer Lett. 20;212(1):95-103); RNA interference (Takei et al., 2004, Cancer Res. 64(10):3365-70; Reich et al., 2003, Mol Vis. 9:210-6); and undersulfated, low molecular weight glycol-split heparin (Pisano et al., 2005, Glycobiology. 15(2) 1-6). Some of these treatments, however, have resulted in undesirable side effects. For instance, Genentech's Avastin, a monoclonal antibody that targets VEGF, has been reported to cause an increase in serious arterial thromboembolic events in some colon cancer patients and serious, and in some cases even fatal, hemoptysis in non-small cell lung cancer patients (Ratner, 2004, Nature Biotechnol. 22(10):1198). More recently, Genentech has reported that gastrointestinal perforations were observed in 11% of ovarian cancer patients (5 women out of 44 in trial) treated with Avastin (Genentech Press Release dated September 23, 2005). Similarly, the first VEGF-targeting drug, Pfizer's receptor tyrosine kinase inhibitor SU5416, exhibited severe toxicities that included thromboembolic events, 42723 v1/DC 6 prompting Pfizer to discontinue development (Ratner, supra). Given the wide variety of patients that stand to benefit from the development of effective anti-angiogenic treatments and the drawbacks of some known anti-angiogenesis treatments, there remains a need for novel anti-angiogenic therapeutics. SUMMARY OF INVENTION [00151 This invention encompasses VEGF analogs and nucleic acids encoding the same, which exhibit strong binding affinity for one or more native VEGF receptors compared to wild-type VEGF. The invention also encompasses VEGF analogs and nucleic acids encoding same, which exhibit a dissociation of receptor binding affinity and bioactivity. Specifically, the in vivo and in vitro bioactivities of the disclosed analogs are substantially decreased compared to wild-type VEGF, whereas the binding affinity to one or more native receptors is about the same or substantially increased compared to wild-type VEGF. The VEGF analogs may demonstrate at least about a three to four fold increase in receptor binding affinity to a native receptor such as KDR. [0016] In one embodiment of the invention, the VEGF analogs are modified VEGF homodimers or heterodimers. These molecules contain at least one mutation which can be present in one or both subunits of the VEGF molecule. In one embodiment of the invention, the VEGF analog containing the one or more mutations is VEGF-A. The VEGF-A analog can be any VEGF-A isoform, for instance, an isoform of 121, 145, 148, 165, 183, 189, or 206 amino acids. In one embodiment, the VEGF-A analog of the invention is a VEGF 16 5 b isoform. In another embodiment, the VEGF molecule containing one or more mutations is VEGF-B, VEGF-C, VEGF-D or PlGF. [0017] The present invention includes a VEGF fusion protein containing one or more mutations in one or more subunits. The VEGF fusion protein of the invention includes at 42723 vl/DC 7 least one VEGF subunit, i.e., subunit, fused to at least one subunit of a different protein, including, but not limited to, other cystine knot growth factors or glycoproteins. For instance, the invention includes a chimera VEGF analog in which the VEGF molecule contains a VEGF-A subunit fused to a VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, PDGF or PIGF subunit; a VEGF-B subunit fused to a VEGF-A, VEGF-C, VEGF-D, VEGF E, VEGF-F, PDGF or PlGF subunit; a VEGF-C subunit fused to a VEGF-A, VEGF-B, VEGF-D, VEGF-E, VEGF-F, PDGF or PIGF subunit; a VEGF-D subunit fused to a VEGF A, VEGF-B, VEGF-C, VEGF-E, VEGF-F, PDGF or PlGF subunit; or a P1GF subunit fused to a VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or PDGF subunit. The subunits may optionally be separated by a linker peptide. The invention also includes different isoforms of the same VEGF fused together, e.g., VEGF 165 subunit fused to VEGFi 65 b. [0018] In one embodiment, the VEGF analog is a single chain molecule. For instance, the VEGF analog of the invention includes two VEGF subunits, i.e., monomers, linked -together via a linker peptide. One or both linked subunits can contain one or more basic amino acid substitutions. Further, the linked subunits can be different VEGF protein subunits and can be different isoforms of the same subunit. For instance, the present invention includes a wild-type VEGFi 65 subunit linked via a GS linker to a VEGF 1 65 subunit with a 183K amino acid substitution. [0019] In another embodiment of the invention, a VEGF-A, VEGF-B, VEGF-C, VEGF-D, or P1GF subunit or dimer comprising one or more mutations is fused to a toxin. The peptide of this embodiment can be useful for the targeting and destruction of tumor cells. [0020] The VEGF analogs of the invention include one or more basic amino acid substitutions, such as lysine or arginine, from the group of positions 44, 67, 72, 73, 83, and 87. In one embodiment of the invention, the VEGF analog contains a basic amino acid substitution at position 83 and optionally one or more basic amino acid substitutions at 42723 vl/DC 8 positions 44, 67, 72 and 73. For instance, the invention includes a VEGF analog with a 183K mutation. The invention also includes, for instance, a VEGF analog with basic amino acids at positions 72, 73 and 83. [0021] VEGF analogs with the basic amino acid substitutions described herein may contain additional amino acid substitutions to further increase receptor binding affinity to KDR and/or decrease receptor binding affinity to neuropilin-1. For instance, the invention includes mutations at positions 146 and 160 in the which act to disrupt the neuropilin-1 binding site. [0022] Analogs of the invention can also contain additional amino acid substitutions which confer enhanced stability and increased serum half-life. For instance, the invention includes amino acids substitutions which eliminate proteolytic cleavage sites such substitutions at positions 111 and 148. [0023] The VEGF receptor antagonists of the present invention can exhibit increased plasma half-life as compared to wild-type VEGF. This may be accomplished by further modifying a VEGF analog by methods known in the art to increase half-life or, alternatively, increased plasma half-life may be an inherent characteristic of a VEGF analog. The VEGF receptor antagonists of the invention can also exhibit an increase in rate of absorption and/or decreased duration of action compared to wild-type VEGF. [0024] The modified analogs of the invention act as VEGF receptor antagonists and thus provide a long awaited solution for patients suffering from a wide spectrum of diseases and conditions associated with angiogenesis. The VEGF receptor antagonists can be administered to a patient alone or in conjunction with another VEGF receptor antagonist, an anti-cancer drug, or an anti-angiogenesis drug for the treatment of disease associated with angiogenesis, including but not limited to, solid tumor cancers, hemangiomas, rheumatoid arthritis, osteoarthritis, septic arthritis, asthma, atherosclerosis, idiopathic pulmonary fibrosis, vascular restenosis, arteriovenous malformations, meningiomas, neovascular 42723 v1/DC 9 glaucoma, psoriasis, Kaposi's Syndrome, angiofibroma, hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasias, scleroderma, trachoma, von Hippel-Lindau disease, vascular adhesion pathologies, synovitis, dermatitis, neurological degenerative diseases, preeclampsia, unexplained female infertility, endometriosis, unexplained male infertility, pterygium, wounds, sores, skin ulcers, gastric ulcers, and duodenal ulcers. BRIEF DESCRIPTION OF THE DRAWINGS [0025] Figure 1A is a graph comparing binding of the 183K mutant and wild-type VEGF A to KDR. Figure lB is a graph showing a decrease in proliferation of HUVEC-2 endothelial cells in the presence of the 183K VEGF-A mutant compared to wild-type VEGF A. [0026] . Figure 2A is a graph comparing binding of the E44R analog and wild-type VEGF A to KDR. Figure 2B is a graph comparing HUVEC-2 cell proliferation in the presence of the E44R VEGF-A analog versus wild-type VEGF-A. [0027] Figure 3A is a graph comparing binding of the E72R+E73R VEGF mutant and wild-type VEGF-A to KDR. Figure 3B is a graph comparing HUVEC-2 cell proliferation in the presence of the E72R+E73R VEGF mutant versus wild-type VEGF-A. [00281 Figure 4 is a graph comparing binding of E44R and EE72/73RR mutants to wild type VEGF-A. [00291 Figure 5 is a graph comparing binding of Q87K mutant to wild-type VEGF-A. [00301 Figure 6 is a graph comparing binding of E67K mutant to wild-type VEGF-A. 42723 v1/DC 10 DETAILED DESCRIPTION OF THE INVENTION [0031] The present invention provides modified angiogenic growth factors of the vascular endothelial growth factor (VEGF) family which exhibit surprising activity as VEGF receptor antagonists. As VEGF receptor antagonists, the compounds of the invention have "anti angiogenic" properties. Being "modified" means that, while the protein contains an amino acid sequence which differs from a wild-type VEGF of interest, i.e., human VEGF or animal VEGF, the sequence has not been changed such that it is identical to the known VEGF sequence of other species. The terms "mutated" and "substituted" are used interchangeably herein to refer to modified amino acid residues. The terms "modified VEGF molecules", "modified VEGF proteins", "VEGF analogs", "VEGF receptor antagonists", "VEGF chimeras", "VEGF fusion proteins" and "VEGF single chain molecules" are used interchangeably herein to refer to modified VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF analog molecules. [0032] "Antagonists" are used interchangeably herein to refer to molecules which act to block, inhibit or reduce the natural, biological activities of VEGF, such as the induction of angiogenesis. The term "anti-angiogenic" as used herein means that the modified VEGF molecules of the invention block, inhibit or reduce the process of angiogenesis, or the process by which new blood or lymphatic vessels form from pre-existing vessels. The activities of the VEGF analogs of the invention disrupt normal VEGF/receptor signaling which usually occurs when VEGF binds to a receptor. Accordingly, the analogs of the invention are VEGF receptor antagonists. Without wishing to be bound by a theory, it is believed that the VEGF analogs of the invention disrupt the dimerization of KDR necessary for signaling. [00331 Inhibition of angiogenesis may be complete or partial. The VEGF receptor antagonist may inhibit angiogenesis at least about 5%, at least about 10%, at least about 42723 vl/DC 11 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, and at least about 100% in vitro and in vivo. Inhibition of angiogenesis can be measured by a skilled artisan by methods known in the art. The detennination of inhibition of angiogenesis can include the use of negative and/or positive controls. For instance, a skilled artisan can conclude that a VEGF analog of the invention inhibits VEGF-induced angiogenesis by comparing angiogenesis in a subject treated with a VEGF analog of the invention to a similar subject not treated with a VEGF analog. [00341 The modified VEGF molecules of the invention display increased receptor binding affinity or similar receptor binding affinity to one or more native VEGF receptors compared to that of wild-type VEGF. As used herein, a native VEGF receptor is an unmodified receptor that specifically interacts with VEGF. For instance, an endogenous VEGF receptor is a native VEGF receptor. In one embodiment of the invention, the native receptor is KDR. KDR is a receptor of VEGF-A, VEGF-C, VEGF-D, VEGF-E and VEGF-F. In another embodiment, the native receptor is Flt-1. Flt-1 is a receptor of VEGF-A, VEGF-B and PlGF. [00351 "Receptor binding affinity" refers to the ability of a ligand to bind to a receptor in vivo or in vitro and can be assessed by methods readily available in the art including, but not limited to, competitive binding assays and direct binding assays. As used herein, receptor binding affinity refers to the ability of VEGF molecules to bind to native VEGF receptors, including, but not limited to, Flt-1 (also known as VEGF-R1), KDR (also known as VEGF R2) and Flt-4 (also known as VEGF-R3). For instance, the modified VEGF-A molecules of the invention display increased binding receptor affinity or similar binding affinity to KDR compared to wild-type VEGF-A. In one embodiment, the increase in receptor binding affinity of the modified VEGF molecules of the invention is at least about 1.25 fold, at least about a 1.5 fold, at least about a 1.75 fold, at least about 2 fold, at least about 3 fold, at least 42723 v1/DC 12 about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold or at least about 10 fold greater than that of wild-type VEGF. [0036] In another embodiment, the modified VEGF exhibits a receptor binding affinity to KDR and/or other receptor that is involved in angiogenesis that is similar or comparable to that of wild-type VEGF. Similar or comparable receptor binding affinity is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% or more of that of wild-type VEGF. For instance, the invention includes VEGF-A analogs exhibiting about 75% to 85%, about 85% to 95% and about 95% to 100% the receptor binding affinity exhibited by wild-type VEGF. [0037] The present invention also includes VEGF analogs which display increased or similar receptor binding affinity to at least one native receptor but display decreased receptor binding affinity to another native receptor. For instance, VEGF-A analogs of the invention may display increased or similar receptor binding affinity to KDR compared to wild-type VEGF-A, but may display decreased receptor binding affinity to Flt-1, neuropilin- 1 or neuropilin-2 compared to wild-type VEGF-A. [0038] The VEGF analogs of the invention also display a decrease in bioactivity compared to wild-type VEGF. "Bioactivity" refers to the natural, biological activities of VEGF in vivo and in vitro, including, but not limited to, the ability of VEGF to induce cell proliferation in endothelial cells. A decrease in bioactivity results in a decrease in angiogenesis. In one embodiment of the invention, the VEGF analogs of the invention display a decrease in bioactivity compared to wild-type VEGF of the same isoform. For instance, a VEGF165 analog of the invention can display a decrease in bioactivity compared to wild-type VEGF 165 , and a VEGF 165 b analog can display a decrease in bioactivity compared to wild-type VEGF 165 b. [0039] Bioactivity can be assessed by several methods known in the art, including, but not limited to, in vitro cell viability assays which assay the viability of endothelial cells such as 42723 vl/DC 13 human umbilical vein endothelial cells (HUVEC) upon exposure to VEGF. A decrease in endothelial cell viability of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or more compared to resulting from exposure to wild-type VEGF is indicative of a decrease in bioactivity. [00401 Bioactivity can be assessed in vivo as well. For instance, bioactivity can be assessed in vivo in a subject with a tumor by detecting a lack of increase in angiogenesis around a tumor. The detection of a lack of increase in angiogenesis can be accomplished by several methods known in the art including, but not limited to, an in vivo matrigel migration assay, a disc angiogenesis assay, an assay comprising a dorsal skinfold chamber in mice, a corneal transplant and a sponge implant model of angiogenesis. In one embodiment, angiogenesis is assessed by comparing angiogenesis of and around the tumor to that of a tumor of similar type, size and location in an untreated subject. Biopsy methods as known in the art can be used to extract tissue and analyze for vessel formation. [00411 "Dissociation" of receptor binding affinity and bioactivity refers to the concept that receptor binding affinity and bioactivity are not correlated. In comparison, receptor binding affinity and bioactivity are correlated for wild-type VEGF proteins such as wild-type VEGF A. An increase in receptor binding ability, for example, would be expected to result in an increase in bioactivity for wild-type VEGF-A. On the other hand, the modified VEGF molecules of the invention demonstrate a similar receptor binding affinity or an increase in receptor binding affinity as compared to wild-type VEGF but a decrease in bioactivity as compared to wild-type VEGF. [0042] Mammalian VEGFs are produced in multiple isoforms due to alternative splicing of a family of related genes. The present invention describes VEGF analogs which 42723 v1/DC 14 correspond to VEGF isoforms involved in angiogenesis. The VEGF analogs of the present invention can be created using any VEGF isoforn unless otherwise indicated. [00431 VEGF-A can exist in isoforms including, but not limited to, 121, 145, 148, 165, 183, 189, and 206 amino acids, respectively. The three main mRNA species are VEGF1 21 , VEGF1 65 and VEGF 18 9 . As used herein, VEGF1 21 (SEQ ID NO.: 6), VEGF 1 45 (SEQ ID NO.: 8), VEGF 1 48 (SEQ ID NO.: 10), VEGF 1 65 (SEQ ID NO.: 4), VEGF 1 6 5 b (SEQ ID NO.: 13),

VEGF

1 83 (SEQ ID NO.: 15), VEGF1 89 (SEQ ID NO.: 17) and VEGF 06 (SEQ ID NO.: 19) are isofonns of VEGF-A capable of being modified to possess anti-angiogenic properties. The amino acid positions described herein are based on a VEGF molecule lacking a leader sequence such as the leader sequence of SEQ ID NO.: 3. The amino acid sequences of VEGF-A isoforms with leader sequence are the sequences of SEQ ID NOs.: 2, 5, 7, 9, 12, 14, 16 and 18. [0044] The various isoforms of VEGF-A share a common amino-terminal domain consisting of 110 amino acids. VEGF-A isofonns have a receptor binding domain encoded by exons 2-5. The most notable difference between the isoforms are found in the neuropilin and heparin binding domains which are encoded by exons 6a, 6b, 7a and 7b. [0045] The most common VEGF-A isoform is VEGF 165 . The nucleic acid encoding

VEGF

1 6 5 is the sequence of SEQ ID NO.: 1. Recently, an endogenous splice variant referred to as VEGF 165 b was described which contains sequences encoded by exon 9, instead of exon 8, at the carboxy terminus. The nucleic acid molecule encoding this protein is the sequence of SEQ ID NO.: 11. VEGF 1 65 b (SEQ ID NO.: 12 with leader sequence; SEQ ID NO.: 13 without leader sequence) inhibited VEGF signaling in endothelial cells when added with

VEGF

1 65 (see Woolard et al., 2004, Cancer Research. 64: 7822-7835; see also U.S. 2005/0054036 which is herein incorporated by reference in its entirety). [0046] In one embodiment of the invention, the VEGF analogs are VEGF-A analogs. VEGF-A analogs include "modified VEGF-A proteins", "VEGF-A receptor antagonists", 42723 vl/DC 15 "VEGF-A chimeras", "VEGF-A fusion proteins" and "VEGF-A single chain molecules." A VEGF-A analog is a VEGF-A molecule containing at least one modified VEGF-A subunit. [00471 VEGF-B exists in two isofonns, VEGF-B 167 (SEQ ID NO.: 48) and VEGF-BI 86 (SEQ ID NO.: 50) (Makinen et al., 1999, J. Biol. Chem. 274: 21217-21222). In one embodiment of the invention, the VEGF analog is a VEGF-B analog. VEGF-B analogs include "modified VEGF-B proteins", "VEGF-B analogs", "VEGF-B receptor antagonists", "VEGF-B chimeras", "VEGF-B fusion proteins" and "VEGF-B single chain molecules." A VEGF-B analog is a VEGF-B molecule containing at least one modified VEGF-B subunit. [0048] VEGF-C is produced as a propeptide (SEQ ID NO.: 51) that is proteolytically cleaved to form a 21-kd active protein (Nicosia, 1998, Am. J. Path. 153: 11-16). In one embodiment of the invention, the VEGF analog is a VEGF-C analog. VEGF-C analogs include "modified VEGF-C proteins", "VEGF-C analogs", "VEGF-C receptor antagonists", "VEGF-C chimeras", "VEGF-C fusion proteins" and "VEGF-C single chain molecules." A VEGF-C analog is a VEGF-C molecule containing at least one modified VEGF-C subunit. [00491 VEGF-D is also produced as a propeptide (SEQ ID NO.: 52) that is proteolytically cleaved to form an active protein. VEGF-D is 48% identical to VEGF-C (Nicosia, supra). In one embodiment of the invention, the VEGF analog is a VEGF-D analog. VEGF-D analogs include "modified VEGF-D proteins", "VEGF-D analogs", "VEGF-D receptor antagonists", "VEGF-D chimeras", "VEGF-D fusion proteins" and "VEGF-D single chain molecules." A VEGF-D analog is a VEGF-D molecule containing at least one modified VEGF-D subunit. [0050] Placenta growth factor (PlGF) exists in three isoforms, PlGF-I (SEQ ID NO.: 54), PlGF-2 (SEQ ID NO.: 56) and PlGF-3 (SEQ ID NO.: 58). PlGF-2 contains an exon 6 encoded peptide which bestows heparin and neuropilin-1 binding properties absent in the other two isoforms. Both PlGF-l and PlGF-2 have been reported as being capable of inducing endothelial cell migration (Migdal et al., 1998, J. Biol. Chem. 273: 22272-22278). 42723 vl/DC 16 In one embodiment of the invention, the VEGF analog is a PlGF analog. In another embodiment, the VEGF analog is PlGF-l or PlGF-2. PlGF analogs include "modified PlGF proteins", "PIGF analogs", "PlGF receptor antagonists", "PIGF chimeras", "PlGF fusion proteins" and " PlGF single chain molecules." P1GF analogs are PlGF molecules with at least one modified PlGF subunit. [00511 The VEGF analogs of the invention are modified animal or human VEGF molecules. In one embodiment of the invention, the VEGF analogs are mammalian VEGF molecules. In another embodiment of the invention, the VEGF analogs are avian VEGF molecules. The VEGF analogs of the present invention include, but are not limited to, modified primate, canine, feline, bovine, equinine, porcine, ovine, murine, rat and rabbit VEGF molecules. In one embodiment, the animal VEGF analog is a VEGF-A analog. For instance, the animal VEGF-A analog of the invention can be an animal VEGF 165 or

VEGF

165 b analog. [0052] The modified VEGF molecules of species other than human have substitutions at positions corresponding to those in the modified human VEGF molecules disclosed herein and may be identified using any alignment program, including but not limited to DNASIS, ALIONment, SIM and GCG programs such as Gap, BestFit, FrameAlign, and Compare. As can be appreciated by one of skill in the art, the corresponding amino acid to be replaced with a basic amino acid may not be identical to the one in human VEGF-A. For instance, a skilled artisan would appreciate that a glutamate (E) may correspond to a different acidic amino acid in an animal such as aspartate (D). 100531 In another embodiment, the corresponding amino acid is identified as being located in the same general position within a defined structure, for instance, on an outer loop structure. The structure of a protein can be predicted using software based on the amino acids of the protein. Accordingly, one of skill in the art can use software that predicts protein folding and loop structure to identify the corresponding position in a related protein. 42723 vl/DC 17 [00541 Design of VEGF Receptor Antagonists [00551 The VEGF receptor antagonists encompassed by the present invention may be designed by comparing the amino acid sequences of the VEGF of interest to that of other species to identify basic residues in the proteins of VEGF of other species. For instance, a VEGF-A molecule of instance can be designed by comparing a human VEGF-A to that of another species. Such methods are disclosed in U.S. 6,361,992, which is herein incorporated by reference in its entirety. Consideration may also be given to the relative biological activity of VEGF from various species as to which species to choose for comparison and amino acid substitution. Further homology modeling based on the structure of related glycoproteins is useful to identify surface-exposed amino acid residues. Homology modeling can be performed by methods generally know in the art, including, but not limited to, the use of protein modeling computer software. [0056] The present invention also provides a modified VEGF protein, wherein the modified VEG.F comprises an amino acid(s) substituted at a position(s) corresponding to the same amino acid position in a VEGF protein from another species having an increased binding affinity and/or decreased bioactivity over the wild-type VEGF. For example, snake venom VEGF-F binds to KDR with high affinity and strongly stimulates proliferation of vascular endothelial cells in vitro. One can compare human VEGF-A to snake venom VEGF, design human VEGF-A proteins with amino acid substitutions at one or more positions where the snake venom and human sequences differ, construct human VEGF-A proteins with the selected changes, and administer the modified human VEGF-A to humans. Although snake venom VEGF-F demonstrates an increase in KDR binding affinity and bioactivity, i.e., binding affinity and bioactivity are correlated, compared to human VEGF, one of skill in the art would understand that amino acid substitutions could be empirically tested to identify amino acid substitutions which increase receptor binding affinity but decrease or have no effect on bioactivity. An amino acid substitution which increases 42723 v1/DC 18 receptor binding affinity and/or decreases or has no effect on bioactivity may then be combined with one or more other amino acid substitutions known to increase receptor binding affinity and/or decrease bioactivity. [00571 In another embodiment of the invention, the modified VEGF molecule can contain one or more amino acids substituted at a position(s) corresponding to the same amino acid position in a VEGF homolog that naturally exists in arthropods. In arthropods, a single growth factor performs the tasks performed by PDGF and VEGF in higher organisms. One of skill in the art would understand that amino acid substitutions could be empirically tested to identify amino acid substitutions which increase receptor binding affinity but decrease or have no effect on bioactivity, or, alternatively, have little effect on receptor binding affinity but decrease bioactivity. [0058] Further, the present invention provides a modified VEGF, wherein the modified VEGF comprises a basic amino acid(s) substituted at a position(s) corresponding to the same amino acid in a different VEGF or VEGF isoform or closely related glycoprotein such as proteins in the PDGF family from the same species or different species. For example, VEGFi 65 can be compared to PDGF from the same species and amino acid substitutions made to the VEGF protein based on any sequence divergence. A skilled artisan can compare two or more sequences of VEGF proteins or VEGF-related proteins using methods known in the art such as the use of alignment software, including but not limited to, DNASIS, ALIONment, SIM and GCG programs such as Gap, BestFit, FrameAlign, and Compare. [0059] In another aspect of the invention, the amino acid substitutions described herein can be incorporated into closely related proteins such as VEGF-E (SEQ ID NO.: 60), VEGF F (SEQ ID NO.: 62) and PDGF (SEQ ID NO.: 63 and SEQ ID NO.: 64). For instance, one or more basic amino acid substitutions selected from the group consisting of E67, E72, E73, 183 and Q87 can be compared to a PDGF isoform from the same species and amino acid substitutions made to the PDGF isoform. 42723 v1/DC 19 [0060] The VEGF analogs of the invention may be designed to display a decreased receptor binding affinity to Flt-i receptors compared to wild-type VEGF-A. Although these analogs display a decreased receptor binding affinity to Flt-1, they may have an increased or comparable receptor binding affinity to KDR compared to wild-type VEGF-A. [0061] The VEGF analogs of the invention may be designed to display a decreased receptor binding affinity to co-receptors, including, but not limited to, neuropilin- 1 or neuropilin-2 compared to that of wild-type VEGF. Analogs with decreased receptor binding affinity to neuropilin- 1 or neuropilin-2 may have increased or similar receptor binding affinity to KDR, Flt-I or VEGR3 compared to that of wild-type VEGF. For instance, VEGF-A analogs can be designed which exhibit decreased receptor binding affinity to neuropilin- 1 and increased receptor binding affinity to KDR and/or Flt- 1. In one embodiment of the invention, the VEGF-A displaying decreased receptor binding affinity to neuropilin-1 is an analog designed in the VEGF 1 65 b splice variant. In another embodiment,

VEGF-B

1 67 and PlGF-2 analogs can be designed which exhibit decreased receptor binding affinity to neuropilin-1 and increased binding affinity to Flt-1. [00621 In one embodiment of the invention, VEGF analogs are designed to exhibit decreased receptor binding affinity to neuropilin- 1 or neuropilin-2 compared to wild-type VEGF by disrupting the VEGF neuropilin binding site. This can be accomplished by reducing the number of cysteine amino acid residues in the neuropilin-1 receptor binding domain. For instance, VEGF 1 65 analogs can be designed to disrupt the neuropilin 1 binding site in VEGF 1 65 by substituting the cysteine residues at positions 146 and/or 160 of SEQ ID NO.: 4 with amino acids such as serine which cause a disruption of the disulfide bridge. The substitution of cysteine residues at positions 146 and 160 of SEQ ID NO.: 4 disrupts neuropilin-1 binding but does not disrupt heparin binding. Mutations at positions 146 and/or 160 can be coupled with one or more mutations to increase, maintain or restore receptor binding affinity to KDR, Flt-I and/or VEGFR3 as described herein. 42723 vl/DC 20 [0063] Similarly, the present invention includes VEGF analogs which exhibit decreased receptor binding affinity to neuropilin-2 compared to wild-type VEGF. For instance, the invention includes VEGF-C and VEGF-D analogs which exhibit reduced binding affinity to neuropilin-2 but increased or similar binding affinity to KDR and/or VEGFR3 compared to wild-type VEGF-C or VEGF-D, respectively. [0064] The invention also includes VEGF analogs which exhibit enhanced stability and resistance to proteases. In one embodiment, amino acids substitutions at positions Al 11 and A148 of SEQ ID NO.: 4 are incorporated in a VEGF-A analog to improve resistance to proteases. The invention also includes VEGF-C and VEGF-D analogs which contain mutations preventing the cleavage of the VEGF-C propeptide or VEGF-D propeptide, respectively. For instance, the present invention includes VEGF-C and VEGF-D analogs that contain one or more mutations which induce resistance to serine protease plasmin and/or other members of the plasminogen family. [0065] - In another embodiment of the invention, VFGF analogs which exhibit increased receptor binding affinity to one or more VEGF receptors, preferably KDR, can be created in a naturally occurring VEGF molecule which exhibits antagonistic properties. For instance,

VEGF

1 6 5 b, an isoform isolated from kidney tissue, can be modified to incorporate the amino acid substitutions associated with an increase in receptor binding ability and decrease in bioactivity of the protein. Similarly, a skilled artisan could incorporate the amino acid substitutions of the present invention in synthetic or new isofonns of VEGF which contain the properties of VEGF 16 5 b. In particular, the mutations of the invention can be used with other VEGF proteins which contain the amino acids SLTRKD (SEQ ID NO.: 70), i.e., the amino acids coded for by what has been termed exon 9, in addition to or in place of the amino acids coded for by exon 8 (CDKIPRR; SEQ ID NO.: 71). [0066] Amino Acid Substitutions 42723 v1/DC 21 [0067] The VEGF analogs of the present invention contain one or more basic amino acid substitutions which confer enhanced receptor binding affinity and decreased bioactivity. In one embodiment of the invention, the VEGF analogs are VEGF receptor antagonists, including but not limited to VEGF-A antagonists. [00681 A modified VEGF molecule of the invention may have a basic amino acid substitution in one or more subunits, i.e., monomers, of VEGF. Basic amino acids comprise the amino acids lysine (K), arginine (R) and histidine (H), and any other basic amino acids which may be a modification of any of these three amino acids, synthetic basic amino acids not normally found in nature, or any other amino acids which are positively charged at a neutral pH. Preferred amino acids, among others, are selected from the group consisting of lysine and arginine. [0069] In one embodiment, a modified VEGF molecule of the invention comprises at least one modified subunit, wherein the modified subunit comprises a basic amino acid substitution at pQsition 183.of wild-type human VEGF 1 6 5 (SEQ ID NO.: 4), VEGF121 (SEQ ID NO.: 6), VEGF 1 45 (SEQ ID NO.: 8), VEGF1 48 (SEQ ID NO.: 10), VEGF 165 b (SEQ ID NO.: 13), VEGF 1 83 (SEQ ID NO.: 15), VEGFi 8 9 (SEQ ID NO.: 17) or VEGF 20 6 (SEQ ID NO.: 19). For instance, the invention includes an 183K amino acid substitution in SEQ ID NOs.: 4, 6, 8, 10, 13, 15, 17 or 19 corresponding to the amino acid sequences of VEGF-A isoforms. [0070] The invention also includes a basic amino acid substitution in the position corresponding to position 83 in other VEGF molecules, i.e., VEGF-B, VEGF-C, VEGF-D and P1GF, such as position 183 of VEGF-B 1 67 (SEQ ID NO.: 48) or VEGF-B 186 (SEQ ID NO.: 50) and position 191 of PlGF-1 (SEQ ID NO.: 54), PIGF-2 (SEQ ID NO.: 56) or PlGF 3 (SEQ ID NO.: 58). [0071] The invention includes modified VEGF molecules in animals other than humans, wherein the VEGF molecule contains, in one or more subunits, a basic amino acid 42723 v1/DC 22 substitution in the position corresponding to position 83 in human VEGF-A. In one embodiment, the modified animal VEGF is a modified VEGF-A molecule. For instance, the present invention includes a basic amino acid substitution at position 183 in primate (SEQ ID NO.: 22), position 182 in bovine (SEQ ID NO.: 25), position 182 in canine (SEQ ID NO.: 28), position 183 in chicken (SEQ ID NO.: 31), position 182 in equine (SEQ ID NO.: 182), position 182 in murine (SEQ ID NO.: 37), position 182 in porcine (SEQ ID NO.: 40), position 182 of rat (SEQ ID NO.: 43) and position 182 in ovine (SEQ ID NO.: 46). [00721 The invention also envisions a modified VEGF-related protein, including, but not limited to VEGF-E, VEGF-F and PDGF, containing an amino acid substitution corresponding to position 183 of SEQ ID NO.: 4. For instance, VEGF-F (SEQ ID NO.: 62) can be modified to include an 183 amino acid substitution. [0073] The modified VEGF molecule of the invention can contain basic amino acid substitutions which further increase the binding affinity or decrease bioactivity of VEGF compared to wild-type VEGF such as wild-type VEGF-A. VEGF molecules with basic amino acid substitutions at one or more of positions 44, 67, 72, 73 and/or 87 of VEGF165 (SEQ ID NO.: 4), VEGF121 (SEQ ID NO.: 6), VEGF1 45 (SEQ ID NO.: 8), VEGF1 48 (SEQ ID NO.: 10), VEGF 165 b (SEQ ID NO.: 13), VEGF1 8 3 (SEQ ID NO.: 15), VEGF1 89 (SEQ ID NO.: 17) and VEGF 2 06 (SEQ ID NO.: 19) can increase binding affinity for KDR compared to wild-type VEGF. For instance, the invention includes the basic amino acid modifications E44R, E44K, E72R, E72K, E73R, E73K, Q87R, Q87K and E67K. [0074] In one embodiment of the invention, basic amino substitutions corresponding to positions 44, 67, 72, 73 and/or 87 of SEQ ID NO.: 4 are coupled with the basic amino acid substitution corresponding to position 83 of SEQ ID NO.: 4 to produce a VEGF receptor antagonists. For instance, the modified amino acids of the present invention include basic amino acid substitutions at positions 72+73+83, 44+83, 72+83, 73+83, 44+72+83, 42723 v1/DC 23 44+73+83, 44+72+73+83, 44+83+87, 83+87, 67+72+73+83; 44+67+83, 67+72+83, 67+73+83, 44+67+72+83, 44+67+73+83, 44+67+72+73+83, 44+67+83+87 and 67+83+87. [0075] In another embodiment of the invention, the analog is a VEGF 165 b molecule containing one or more basic amino acids at positions E44, E67, E72, E73 and Q87 and optionally a basic amino acid substitution at position 183. When the VEGF-A isoform is VEGFl65b, it is possible to generate a VEGF analog of the invention with increased binding affinity and decreased bioactivity compared to wild-type VEGF-A, including VEGF 165 , by incorporating a single amino acid modification that would otherwise only result in an increase in receptor binding affinity in other VEGFI 65. [0076] As can be appreciated by a skilled artisan, the invention includes VEGF proteins and VEGF-related proteins other that VEGF-A that contain basic amino acid modifications corresponding to those of positions E44, E67, E72, E73 and/or Q87 of VEGF-A (SEQ ID NO.: 4). For instance, the invention includes a modified VEGF-B analog (SEQ ID NOs.: 48 and 50) containing one or more basic amino acid substitutions at positions A44, E67, G72, Q73 and S87 and a modified VEGF-F analog (SEQ ID NO.: 62) containing one or more basic amino acid substitutions at positions E44, E67, E72, E73 and Q87. [00771 A modified animal, i.e., non-human, VEGF-A molecule of the invention can likewise contain additional amino acid modifications to increase binding affinity or decrease bioactivity of the modified animal VEGF molecule compared to wild-type animal VEGF. The invention includes the use of these modifications in conjunction with an amino acid substitution that corresponds to 183 of SEQ ID NO.: 4 as described above. For instance, the present invention includes one or more basic amino acid substitutions selected from the group of positions E44, E67, E72, E73, 183 and 187 of primate (long-tailed macaque) VEGF A (SEQ ID NO.: 22); one or more basic amino acid substitutions selected from the group of positions E43, E66, E71, E72, 182 and Q86 of bovine VEGF-A (SEQ ID NO.: 25); one or more basic amino acid substitutions selected from the group of positions E43, E66, E71, 42723 vl/DC 24 E72, 182 and Q86 of canine VEGF-A (SEQ ID NO.: 28); one or more basic amino acid substitutions selected from the group of positions E44, E67, D72, V73, 183 and Q87 of avian (chicken) VEGF-A (SEQ ID NO.: 31); one or more basic amino acid substitutions selected from the group of positions E43, E66, A71, E72, 182 and Q86 of equine VEGF-A (SEQ ID NO.: 34); one or more basic amino acid substitutions selected from the group of positions E43, E66, S71, E72, 182 and Q86 of murine VEGF-A (SEQ ID NO.: 37); one or more basic amino acid substitutions selected from the group of positions E43, E66, E71, E72, 182 and Q86 of porcine VEGF-A (SEQ ID No.: 40); one or more basic amino acid substitutions selected from the group of positions E43, E66, S71, E72, 182 and Q86 of rat VEGF-A (SEQ ID NO.: 43); and one or more basic amino acid substitutions selected from the group of positions E43, E66, E71, E72, 182 and Q86 of ovine VEGF-A (SEQ ID NO.: 46). [0078] VEGF analogs containing containing one or more basic amino acid substitutions can also be combined with amino acid substitutions designed to disrupt a co-receptor binding site. In one embodiment, the VEGF analogs of the invention contain a disrupted neuropilin 1 binding site. The neuropilin-1 binding site comprises amino acids 111 to 165 of VEGFi 65 (SEQ ID NO.: 04). This domain overlaps the heparin binding domain encoded by exons 6 and 7. The invention includes any amino acid modifications in or near (i.e., within about 5 amino acids) that disrupt the neuropilin- 1 binding site domain but which do not disrupt the ability of the heparin binding domain to bind heparin sulfate. Such amino acid modifications can be determined empirically by a skilled artisan. [00791 In one embodiment of the invention, the neuropilin-1 binding domain is disrupted by reducing the number of cysteine amino acid residues in the domain, i.e., by reducing the number of cysteine amino acid residues between amino acids 111 to 165 of VEGF-A. For instance, VEGF 165 analogs can be designed to disrupt the neuropilin 1 binding site by substituting the cysteine residues at positions 146 and/or 160 of SEQ ID NO.: 4 with amino acids such as seine which cause a disruption of the disulfide bridge. The substitution of 42723 vl/DC 25 cysteine residues at positions 146 and 160 of SEQ ID NO.: 4 disrupts neuropilin-1 binding but does not disrupt heparin binding. The neuropilin-1 binding site can also be disrupted by ending the amino acid peptide at position 146 or 160. [00801 The invention can also included modifications of amino acids surrounding amino acids at positions 146 and 160 of SEQ ID NO.: 4 such that the cysteine residues of positions 146 and 160 are unable to fonn a disulfide bridge. For instance, the invention includes, but is not limited to, one or more amino acid substitutions at positions 136 through 165 which are capable of disrupting the formation of a disulfide bridge. [0081] A modified VEGF analog of the invention containing one or more of the basic amino acid substitutions corresponding to E44, E67, E72, E73, 183 and Q87 of SEQ ID NO.: 4 described herein. For instance, the invention includes VEGF analogs with amino acid substitutions at positions E44B+C 1 46X, E44B+C 1 60X, E44B+C 1 46X+C 1 60X, E67B+C146X, E67B+C160X, E67B+C146X+C160X, E44B+E67B+C146X, E44B+E67B+C160X, E44B+E67B+CI46X+C160X, E72B+C146X, E72B+C160X, E72B+C146X+C160X, E73B+C146X, E73B+C160X, E73B+C146X+C160X, E72B+E73B+C146X, E72B+E73B+C160X, E72B+E73B+C146X+C160X, I83B+C146X, 183B+C160X, 183B+C146X+C160X, Q87B+C146X, Q87B+C160X, Q87B+C146X+C160X, E44B+E67B+E72B+C146X, E44B+E67B+E72B+C160X, E44B+E67B+E72+CI46X+C160X, E44B+E67B+E73B+C146X, E44B+E67B+E73B+C160X, E44B+E67B+E73B+C146X+C160X, E44B+E67B+E72B+E73B+C146X, E44B+E67B+E72B+E73B+C160X, E44B+E67B+E72B+E73B+C146X+C160X, E67B+E72B+E73B+C146X, E67B+E72B+E73B+C160X, E67B+E72B+E73B+C146X+C160X, E44B+E72B+E73B+C146X, E44B+E72B+E73B+C160X, E44B+E72B+E73B+C146X+C160X, E44B+183B+C146X, E44B+183B+CI60X, E44B+183B+CI46X+C160X, E67B+183B+C146X, E67B+183B+C160X, 42723 v1/DC 26 E67B+I83B+C146X+C160X, E44B+E67B+183B+C146X, E44B+E67B+183B+C160X, E44B+E67B+183B+C146X+C160X, E72B+183B+C146X, E72B+183B+C160X, E72B+183B+C146X+C160X, E73B+183B+C146X, E73B+183B+C160X, E73B+183B+C146X+C160X, E72B+E73B+I83B+C146X, E72B+E73B+I83B+C160X, E72B+E73B+I83B+C146X+C160X, 183B+Q87B+C146X, 183B+Q87B+C160X, 183B+Q87B+C146X+C160X, E44B+E67B+E72B+183B+C146X, E44B+E67B+E72B+183B+C160X, E44B+E67B+E72+183B+C146X+C160X, E44B+E67B+E73B+183B+C146X, E44B+E67B+E73B+183B+C160X, E44B+E67B+E73B+I83B+C146X+C160X, E44B+E67B+E72B+E73B+183B+C146X, E44B+E67B+E72B+E73B+I83B+C160X, E44B+E67B+E72B+E73B+I83B+CI46X+C160X, E67B+E72B+E73B+183B+C146X, E67B+E72B+E73B+183B+C160X, E67B+E72B+E73B+183B+C146X+C160X, E44B+E72B+E73B+183B+C146X, E44B+E72B+E73B+183B+CI60X and E44B+E72B+E73B+183B+C146X+C16OX, wherein B is a basic amino acid and X is any amino acid other than cysteine. In one embodiment, X is serine. [00821 The modified proteins of the invention may also contain further substitutions, particularly conservative substitutions that do not alter the enhanced properties of the protein. Typically, however, such modified proteins will contain less than five substitutions at positions other than those listed above, and may exhibit complete amino acid sequence identity with the corresponding wild-type VEGF subunits in positions other that the positions listed above. [0083] As can be appreciated by a skilled artisan, all amino acid substitutions and peptide modifications disclosed in the present invention can be incorporated in any VEGF protein or related protein, regardless of species, because of the high degree of homology between VEGF proteins and related proteins. A skilled artisan can correlate the amino acid 42723 vl/DC 27 substitutions described herein using methods known in the art, including, but not limited to, the use of amino acid sequence alignment software. [0084] VEGF Analogs with Increased Serum Half-Life [0085] The VEGF analogs of the invention may have an increased plasma half-life as compared to wild-type VEGF. In one embodiment, the modification(s) which increases or maintains receptor binding affinity and decreases bioactivity as compared to wild-type VEGF also increases the plasma half-life of the VEGF as compared to wild-type VEGF. In another embodiment, the modified VEGF proteins of the invention are further modified such that the plasma half-life is increased as compared to wild type VEGF. [00861 There are many modifications known in the art that can be used to increase the half-life of proteins, in particular glycoproteins. 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 glycosylation recognition sites may be either an N-terminal or C-terminal extension on either subunit. Exemplary modified proteins contain an N-terminal extension on a subunit that is selected from the group consisting of ANITV (SEQ ID NO.: 72) and ANITVNITV (SEQ ID NO.: 73). [00871 Increased half-life may also be provided by the use of a peptide extensions such as a carboxyl terminal extension peptide of hCG. See US 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 art to a CTEP, e.g., by a peptide bond or by a heterobifunctional reagent able to form a covalent bond between the amino terminus and carboxyl terminus of a protein, including but not limited to a peptide linker. [0088] In another embodiment of the invention, the basic amino acid substitutions of the invention are coupled with one or more amino acid substitutions that enhance stability and 42723 vI/DC 28 increase serum half-life by eliminating one or more proteolytic cleavage sites. In one embodiment, the additional amino acid substitutions reduce proteolytic cleavage. In 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 subunit of a VEGF molecule contains an amino acid substitution corresponding to amino acid positions Al 11 and/or A148 such as Al 11P and/or Al48Pof

VEGF

1 65 (SEQ ID NO.: 4) or VEGF165b (SEQ ID NO.: 13). For instance, the invention includes VEGF 1 2 1, VEGF1 45 , VEGF 1 48 , VEGF1 83 , VEGF 18 9 and VEGF 2 0 6 containing an amino acid substitution at position Al 11. The invention includes one or more mutations in VEGF-B, VEGF-C, VEGF-D and PlGF which inhibit or reduce protease cleavage. For instance, the invention includes amino acid substitutions which prevent the cleavage of VEGF-C and VEGF-D necessary for bioactivity. [0089] In another embodiment, half-life can be increased-by linking VEGF monomers and by constructing fusion proteins. Increasing the size of a VEGF analog without interfering with binding sites can increase the half-life of the molecule. [0090] Increased half-life may be provided by crosslinking, including but not limited to pegylation or conjugation of other appropriate chemical groups. Such methods are known in the art, for instance as described in U.S. Patent 5,612,034, U.S. Patent 6,225,449, and U.S. Patent 6,555,660, each of which is incorporated by reference in its entirety. Half-life may also be increased by increasing the number of negatively charged residues within the molecule, for instance, the number of glutamate and/or aspartate residues. Such alteration may be accomplished by site directed mutagenesis or by an insertion of an amino acid sequence containing one or more negatively charged residues into said modified VEGF, including insertions selected from the group consisting of GEFT and GEFTT, among others. 42723 vl/DC 29 [0091] The half-life of a protein is a measurement of protein stability and indicates the time necessary for a one-half reduction in the concentration of the protein. The serum half-life of the modified VEGF molecules described herein may be detennined by any method suitable for measuring VEGF levels in samples from a subject over time, for example, but not limited to, immunoassays using anti-VEGF antibodies to measure VEGF levels in serum samples taken over a period of time after administration of the modified VEGF, or by detection of labeled VEGF molecules, i.e., radiolabeled molecules, in samples taken fiom a subject after administration of the labeled VEGF. [00921 The rate of absorption of a VEGF analog of the present invention may result in increased or decreased duration of action. A VEGF analog with an increased rate of absorption and decreased duration of action may be beneficial for patients receiving a VEGF analog pharmaceutical composition by way of subcutaneous administration or other route of administration generally associated with a slow rate of absorption and/or increased duration of action by counteracting the absorption qualities associated with the route of administration. [00931 Linker [00941 The VEGF analog of the invention can contain two or more monomers separated by a linker peptide. A linker peptide can be used to form a VEGF analog in a single chain conformation. A skilled artisan can appreciate that various types of linkers can be used in the present invention to form a VEGF single chain molecule that is capable of binding a VEGF receptor and which acts as a VEGF receptor antagonist. A linker peptide should not hinder the ability of the single chain molecule to bind a VEGF receptor. [00951 The linker peptide can range from about 2 to about 50 or more amino acids in length. For instance, the linker can consist of about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 42723 vl/DC 30 amino acids, about 9 amino acids, about 10 amino acids, about 10-15 amino acids, or about 15-20 amino acids. In one embodiment of the invention, the linker is Gly-Ser or contains Gly-Ser. In another embodiment, the linker is a glycine-rich polypeptide chain. [0096] VEGF molecules containing a linker can be constructed using the methods described herein. A skilled artisan would be able to appreciate that VEGF analog molecules of the invention containing linker peptides can include any of the mutations described herein, in one or more monomers. Further, a VEGF analog containing one or more linker peptides can link more than one type of VEGF protein or isoform. For instance, the present invention includes, but is not limited to, a modified VEGF single chain molecule with a wild-type VEGF 16 5 monomer linked to a modified VEGF 16 5 monomer containing an 183B substitution; a wild-type VEGF 1 65 monomer linked to a modified VEGF 16 5 b containing an 183B substitution; and a modified VEGF 165 monomer fused to a modified VEGF-F monomer. [0097] VEGF Fusion Proteins [00981 The present invention also includes fusion proteins, i.e., chimeras, containing one or more modified VEGF proteins or fragments. "Fusion protein" and "chimera" are used interchangeably herein. As used herein, a VEGF moiety is a VEGF protein or protein fragment containing one or more of the basic amino acid substitutions of the invention. A VEGF fusion protein can have one or more VEGF moieties. [00991 Such a fusion protein may be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the fusion protein by any of the means described herein. Alternatively, such a fusion protein may be made by protein synthesis techniques, for example, using a peptide synthesizer. 42723 vl/DC 31 [00100] The fusion protein of the invention contains at least one VEGF protein or protein fragment containing one or more basic amino acid substitutions described herein. In one embodiment the fusion protein contains a basic amino acid substitution at position 183 of VEGF1 65 (SEQ ID NO. 4), VEGF 1 6 5 b (SEQ ID NO. 13), VEGF121 (SEQ ID NO.: 6),

VEGF

145 (SEQ ID NO.: 8), VEGF 1 48 (SEQ ID NO.: 10), VEGF 1 83 (SEQ ID NO.: 15), VEGFis 9 (SEQ ID NO.: 17) or VEGF 206 (SEQ ID NO.: 19). In another embodiment, the fusion protein contains at least one basic amino acid substitution at a position corresponding to 183K of SEQ ID NO.: 4 in another VEGF protein, for instance, an isoform of VEGF-B, VEGF-C, VEGF-D or P1GF. As can be appreciated by a skilled artisan, human or animal VEGF proteins or fragments thereof may be used for the fusion proteins of the invention. [001011 In one embodiment of the invention, two different VEGF protein subunits or fragments thereof are fused. For instance, the invention includes a VEGF-A subunit or fragment thereof fused to a VEGF-B subunit or fragment thereof, a VEGF-C subunit or fragment thereof, a VEGF-D subunit or fragment thereof, or a PlGF subunit or fragment thereof; a VEGF-B subunit or fragment thereof fused to a VEGF-A subunit or fragment thereof, a VEGF-C subunit or fragment thereof, a VEGF-D subunit or fragment thereof, or a PlGF subunit or fragment thereof, a VEGF-C subunit or fragment thereof fused to a VEGF A subunit or fragment thereof, a VEGF-B subunit or fragment thereof, a VEGF-D subunit or fragment thereof, or a PlGF subunit or fragment thereof, a VEGF-D subunit or fragment thereof fused to a VEGF-A subunit or fragment thereof, a VEGF-B subunit or fragment thereof, a VEGF-C subunit or fragment thereof, or a P1GF subunit or fragment thereof; and a PlGF subunit or fragment thereof fused to a VEGF-A subunit or fragment thereof, a VEGF B subunit or fragment thereof, a VEGF-C subunit or fragment thereof, or a VEGF-D subunit or fragment thereof. [001021 The invention includes fusion proteins comprised of two or more different isoforms of the same VEGF protein or fragments thereof. For instance, the invention 42723 vI/DC 32 includes a fusion protein comprised of a VEGF 16 5 subunit or fragment thereof fused to a VEGF1 21 subunit or fragment thereof, a VEGF 1 4 5 subunit or fragment thereof, a VEGF148 subunit or fragment or subunit thereof, a VEGF 165 b subunit or fragment thereof, a VEGF 183 subunit or fragment thereof, a VEGF1 89 subunit or fragment thereof, or a VEGF 2 0 6 subunit or fragment thereof The invention also includes a a VEGF16 5 b subunit or fragment thereof fused to a VEGF121 subunit or fragment thereof, a VEGF 145 subunit or fragment thereof, a

VEGF

148 subunit or fi-agment or subunit thereof, a VEGF 165 subunit or fragment thereof, a VEGF1 83 subunit or fragment thereof, a VEGFi 8 9 subunit or fragment thereof, or a VEGF 206 subunit or fragment thereof [00103] The basic amino acid substitutions of the invention may be present in one or more subunits of the protein. For example, a fusion protein containing a VEGF165 subunit and

VEGF

1 6 5 b subunit may only contain an amino acid substitution in the VEGF 16 5 subunit. The invention includes a wild-type VEGF 1 65 subunit fused by way of a GS linker to a VEGF 165 containing an 183K amino acid substitution. As can be appreciated by one of skill in the art, the fusion proteins of the present invention containing one mutated subunit can be created in both orientations, i.e., the subunit containing the mutation can be at either the N- or C terminus of the fusion protein. [001041 In another embodiment of the invention, a VEGF subunit or fragment thereof is fused to a related protein subunit or fragment thereof. For instance, a VEGF subunit or fragment thereof can be fused to a PDGF subunit or other glycoprotein subunit or fragment thereof. [00105] As can be appreciated by one of ordinary skill in the art, the fusion proteins described herein can be constructed using human or animal VEGF sequences. Further, a fusion protein can be constructed using a human VEGF subunit fused to an animal VEGF subunit. 42723 vl/DC 33 [00106] A VEGF fusion protein should be understood to be a VEGF analog. All modifications disclosed herein, for instance, modifications to further increase receptor binding affinity, modifications to increase half-life and stability, modifications to reduce or inhibit protease cleavage, and modifications to disrupt a co-receptor binding site such as a neuropilin- I binding site can be incorporated in one or more subunits of the VEGF fusion protein. [00107] The fusion proteins of the invention can also contain a linker separating the two or more VEGF subunits or VEGF-related protein subunits. The linker can be covalently linked to and between the peptides of the fusion protein. [00108] VEGF and Toxin Fusion Proteins [00109] The present invention provides fusion proteins comprising a toxin and one or more modified VEGF subunits, i.e., monomers, containing one or more of the basic amino acid substitutions described herein. For instance, the VEGF monomer, i.e., subunit, of a VEGF toxin fusion protein can contain a basic amino acid at one or more amino acid positions corresponding to the amino acid positions from the group consisting of 44, 67, 72, 73, 83 and 87 (SEQ ID NO.: 4 or SEQ ID NO.: 13). The VEGF and toxin fusion proteins of the invention may optionally contain a linker sequence separating the toxin and one or more VEGF subunits. [00110] As used herein, the term "toxin" refers to a poisonous substance of biological origin. The toxin of the invention may be a soluble toxin as known in the art. The fusion proteins comprising a soluble toxin may be used to target tumors. Such fusion proteins may also be used for diagnostic purposes. [001111 Examples of toxins include, but are not limited to, Pseudomonas exotoxins (PE), Diphtheria toxins (DT), ricin toxin, abrin toxin, anthrax toxins, shiga toxin, botulism toxin, tetanus toxin, cholera toxin, maitotoxin, palytoxin, ciguatoxin, textilotoxin, batrachotoxin, 42723 vl/DC 34 alpha conotoxin, taipoxin, tetrodotoxin, alpha tityustoxin, saxitoxin, anatoxin, microcystin, aconitine, exfoliatin toxins A and B, enterotoxins, toxic shock syndrome toxin (TSST-1), Y. pestis toxin, gas gangrene toxin, and others. [001121 In one embodiment, the present invention provides a pharmaceutical composition comprising a soluble toxin fused to a modified VEGF and a pharmaceutically acceptable carrier. In another embodiment, the present invention provides the use of a modified VEGF fusion protein comprising a soluble toxin for the manufacture of a medicament for the treatment or prevention of diseases or conditions associated with angiogenesis. [00113] Without wishing to be bound by a theory, it is believed that the VEGF-toxin fusion protein of the invention prevents or reduces angiogenesis, the growth of tumors and/or the spread of cancer by targeting and killing the VEGF receptor and surrounding endothelial and tumor cells. [00114] Expression and/or Synthesis of VEGF Receptor Antagonists [00115] The present invention includes nucleic acids encoding the modified VEGF proteins of the invention, as well as vectors and host cells for expressing the nucleic acids. [00116] As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double stranded form. The invention includes a nucleic acid molecule which codes for a modified VEGF molecule of the invention. For instance, the invention includes a nucleic acid molecule that codes for a modified VEGF 1 6 5 molecule. The nucleic acid molecule of SEQ ID NO.: 1 which codes for wild-type VEGF 1 65 can be mutated by methods known in the art such that the mutated VEGF 1 6 5 nucleic acid molecule codes for the modified protein. Similarly, the nucleic acid molecule of SEQ ID NO.: 11 which codes for wild-type

VEGF

1 6 5 b can be mutated by methods known in the art such that it codes for a VEGF 16 5 b molecule of the invention. 42723 v1I/DC 35 [001171 Once a nucleic acid encoding a particular modified VEGF of interest, or a region of that nucleic acid encoding a portion of the protein containing a basic amino acid substitution, is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of the modified VEGF protein. Alternatively, the nucleic acid encoding a VEGF analog of the invention may be cloned or modified directly in the expression vector of interest. The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or hybrid gene. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989)). Appropriate promoters for the expression of nucleic acids in different host cells are well known in the art, and are readily interchanged depending on the vector-host system used for expression. Exemplary vectors and host cells are described in U.S. 6,361,992, which is herein incorporated by reference in its entirety. [00118] There are numerous E. coli (Escherichia coli) expression vectors known to one of ordinary skill in the art which are useful for the expression of the nucleic acid insert. Other vectors suitable for use include expression vectors from bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. These expression vectors will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan 42723 vl/DC 36 (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the downstream nucleic acid insert. Also, the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures. [00119] Additionally, yeast expression systems can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomnyces cerevisiae pre-pro alpha-factor leader region (encoded by the MF"-1 gene) is routinely used to direct protein secretion from yeast. (Brake, et al., " varies-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae." Proc..Nat. Acad. Sci., 81:4642-4646 (1984)). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene. This enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. The VEGF coding sequence can be fused in-frame to the pre-pro alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or beta galactosidase, which may be used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post 42723 vl/DC 37 translational glycosolation and expression of recombinant proteins can also be achieved in Baculovirus systems. [00120] Mammalian cells pennit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral or other promoter and a polyadenylation signal. The vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Exemplary expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other cellular hosts. 42723 vl/DC 38 [00121] Expression of the gene or hybrid gene can be either in vivo or in vitro. In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector. For instance, techniques for transfonring fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81: 1740 1747, 1984), and Russell (Nature 301: 167-169, 1983). Other techniques for introducing cloned DNA sequences into fungal cells, such as electroporation (Becker and Guarente, Methods in Enzymol. 194: 182-187, 1991) may be used. The genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art. [00122] Cloned DNA sequences comprising modified VEGF and VEGF fusion proteins of the invention may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniques for introducing cloned DNA sequences into mammalian cells, such as electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), or lipofection may also be used. In order to identify cells that have integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is the DHFR gene. A particularly preferred amplifiable marker is the DHFR (see U.S. Patent 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth 42723 vl/DC 39 Publishers, Stoneham, Mass.) and the choice of selectable markers is well within the level of ordinary skill in the art. [00123] Alternatively, expression of the gene can occur in an in vitro expression system. For example, in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA. In such in vitro transcription systems, the nucleic acid encoding the modified VEGF would be cloned into an expression vector adjacent to a transcription promoter. For example, the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters. (Stratagene Cloning Systems, La Jolla, Cailf.). Kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, Cailf.). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired VEGF analog (Stratagene Cloning Systems, La Jolla, Cailf.). [001241 Another method of producing a VEGF receptor antagonist is to link two peptides or polypeptides together by protein chemistry techniques. Peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9 fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, Cailf.). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to a hybrid VEGF protein can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a hybrid peptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a hybrid peptide. (Grant, G. A., "Synthetic Peptides: A User Guide," W. H. Freeman and Co., N.Y. (1992) and Bodansky, M and Trost, B., Ed., 42723 vl/DC 40 "Principles of Peptide Synthesis," Springer-Verlag Inc., N.Y. (1993)). Alternatively, the peptide or polypeptide can by independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form a VEGF via similar peptide condensation reactions. For example, enzymatic or chemical ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen, L., et al., Biochemistry, 30:4151 (1991); Dawson, et al., "Synthesis of Proteins by Native Chemical Ligation," Science, 266:776-779 (1994)). [00125] The invention also provides fragments of modified VEGF which have antagonist activity. The polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding the peptides in an expression system capable of producing the peptides. For example, amino or carboxy-terminal amino acids can be sequentially removed from either the native or the VEGF protein and the respective activity tested in one of many available assays described above. In another example, the modified proteins of the invention may have a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the protein, replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified VEGF. For example, a modified VEGF can be fused to a maltose binding protein, through either peptide chemistry of cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide. The hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified VEGF can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164). [00126] The VEGF analog of the invention can be a heterodimer or a homodimer. In one embodiment, the VEGF analog is a fusion protein containing one or more VEGF subunits. 42723 vl/DC 41 The VEGF fusion protein of the invention can be a single chain protein containing two or more VEGF subunits separated by linking peptides. In another embodiment, the VEGF analog of the invention is a fusion protein containing one or more VEGF subunits fused to a toxin. The VEGF analog and VEGF analog fusion protein of the invention can be isolated and purified by means known in the art. [001271 All of the VEGF analogs of the invention contain at least one basic amino acid substitution in at least one VEGF subunit. In one embodiment of the invention, the VEGF analogs of the invention contain at least two basic amino acid substitutions, at least 3 basic amino acid substitutions, at least 4 basic amino acid substitutions or at least 5 basic amino acid substitutions in at least one or at least two VEGF subunits. [00128] The invention includes VEGF analogs containing VEGF active fragments, i.e., peptides that are not full length proteins. Active fragments of the modified VEGF of the invention can also be synthesized directly or obtained by chemical or mechanical disruption of larger modified VEGF protein. An active fragment is defined as an amino acid sequence of at least about 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 60 consecutive amino acids, at least 70 consecutive amino acids, at least 80 consecutive amino acids, at least 90 consecutive amino acids, at least 100 consecutive amino acids, at least 110 consecutive amino acids, at least 120 consecutive amino acids, at least 130 consecutive amino acids, at least 140 consecutive amino acids, at least 150 consecutive amino acids, or at least 160 consecutive amino acids derived from the natural amino acid sequence, which has the relevant activity, e.g., binding or regulatory activity. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the peptide is not significantly altered or impaired compared to the modified VEGF. These modifications can 42723 vl/DC 42 provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its biolongevity and/or bioactivity, etcetera. In any case, the peptide must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etcetera. Functional or active regions of the VEGF may be identified by mutagenesis of a specific region of the honnone, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the receptor (Zoller, M. J. et al.). [001291 Methods of Use [00130] The invention encompasses methods for reducing VEGF-mediated angiogenesis, comprising contacting a cell expressing kinase domain receptor (KDR) with the VEGF analogs, including VEGF-A 165 and VEGF-A 16 5 b analogs, described herein such that VEGF mediated angiogenesis is reduced. KDR-expressing cells to be targeted by the methods of the invention can include either or both prokaryotic and eukaryotic cells. Such cells may be maintained in vitro, or they may be present in vivo, for instance in a patient or subject diagnosed with cancer or another angiogenesis-related disease. [00131] The present invention includes methods of treating a patient diagnosed with an angiogenesis-related disease or condition with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said VEGF analog or fusion protein to said patient such that said angiogenesis-related disease or condition is reduced or inhibited. In order to measure the reduction of angiogenesis, the patient's results may be compared to that of a patient administered a placebo. Exemplary angiogenesis-related diseases are described throughout this application, and include but are not limited to diseases selected from the group consisting of tumors and neoplasias, hemangiomas, rheumatoid arthritis, osteoarthritis, septic arthritis, asthma, atherosclerosis, 42723 v1/DC 43 idiopathic pulmonary fibrosis, vascular restenosis, arteriovenous malfonnations, meningioma, neovascular glaucoma, psoriasis, Kaposi's Syndrome, angiofibroma, hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasias, sclerodenna, trachoma, von Hippel-Lindau disease, vascular adhesion pathologies, synovitis, dermatitis, endometriosis, pterygium, diabetic retinopathy, neovascularization associated with corneal injury or grafts, wounds, sores, and ulcers (skin, gastric and duodenal). [001321 A patient suffering from a disease caused by or exacerbated by an increase in angiogenesis, a decrease in angiogenesis, or otherwise dysregulated angiogenesis can be treated with a VEGF analog alone or in combination with a known VEGF receptor antagonist, an anti-angiogenesis therapy, an anti-cancer therapy, or other therapy known to treat the disease or condition. As used herein, "therapy" includes but is not limited to a known drug. Known VEGF receptor antagonists or anti-angiogenesis therapies include but are not limited to agents that either interrupt VEGF/KDR interaction and/or block the KDR signal transduction pathway such as peptides that block binding of VEGF to KDR, antibodies to VEGF, antibodies to KDR, soluble receptors, tyrosine kinase inhibitors, anti VEGF immunotoxins, ribozymes, antisense mediated VEGF suppression, and undersulfated, low molecular weight glycol-split heparin. [00133] If a VEGF analog of the invention is used in combination with another therapy, the coupling of the therapies results in a synergistic effect. In addition, a VEGF analog of the present invention can be combined with a drug associated with an undesirable side effect. By coupling a VEGF analog with such a drug, the effective dosage of the drug with the side effect can be lowered to reduce the probability of the side effect from occurring. [00134] The invention includes methods of treating a patient diagnosed with cancer with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said antagonist to said patient such that the spread of said cancer is 42723 vl/DC 44 reduced or inhibited, i.e., metastasis is reduced or inhibited. The invention includes methods of treating a patient diagnosed with cancer with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said antagonist to said patient such that the growth of a tumor is reduced or inhibited. In one embodiment, the VEGF analog functions by inhibiting angiogenesis by reducing or preventing VEGF-induced angiogenesis. In another embodiment, the VEGF analog is a VEGF-toxin fusion protein that prevents or reduces angiogenesis by targeting or killing tumor cells, vascular cells such as endothelial cells and/or VEGF receptors. [001351 Cancers treatable by the methods of the present invention include all solid tumor and metastatic cancers, including but not limited to those selected from the group consisting of bladder, breast, liver, bone, kidney, colon, ovarian, prostate, pancreatic, lung, brain and skin cancers. The invention includes but is not limited to treatment of cancer with a VEGF analog of the present invention, alone, in combination with chemotherapy, or in combination with radiation therapy by methods known in the art (see U.S. Patent 6,596,712). For instance, a VEGF analog may be used with cesium, iridium, iodine, or cobalt radiation. [001361 The present invention includes methods of treating a patient diagnosed with infertility with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said antagonist to said patient such that infertility is deemed treated by one of skill in the art. Infertility can be measured by quantitative and qualitative parameters known in the art such as quantity of oocytes, fertilization rate, blastocyst formation rate, and embryo formation rate. Such infertility diseases include any disease associated with the expression of VEGF that compromises a patient's fertility including but not limited to unexplained female infertility, endometriosis, and unexplained male infertility. The invention includes but is not limited to treatment of infertility by administration of a VEGF analog alone or in combination with other anti-VEGF treatments, anti-angiogenesis treatments, and/or infertility treatments. 42723 vl/DC 45 [00137] The present invention also includes methods of treating a patient diagnosed with an angiogenesis-associated eye disease with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said antagonist to said patient such that said eye disease is reduced or inhibited. Such eye diseases include any eye disease associated with abnormal intraocular neovascularization, including but not limited to retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age related macular degeneration. The invention includes but is not limited to treatment of angiogenesis-related eye diseases by administration of a VEGF analog alone or in combination with other anti-VEGF treatments, anti-angiogenesis treatments, and/or other eye disease treatments. For example, a VEGF analog of the present invention could be administered to a patient in conjunction with Pfizer's Macugen (pegaptanib) which is a pegylated anti-VEGF aptamer which acts by binding to and inhibiting the activity of VEGF for the treatment of diabetic macular edema, retinal vein occlusion, and age-related macular degeneration. [00138] The present invention also includes methods of treating a patient diagnosed with an angiogenesis-associated inflammatory condition or autoimmune disease with a therapeutically effective amount of any of the VEGF receptor antagonists described herein, comprising administering said antagonist to said patient such that said inflammatory condition is reduced or inhibited. Such inflammatory conditions or diseases include any inflammatory disorder associated with expression of VEGF and activation of cells by VEGF, including but not limited to all types of arthritis and particularly rheumatoid arthritis and osteoarthritis, asthma, pulmonary fibrosis and dermatitis. The invention includes but is not limited to treatment of angiogenesis-related inflammatory conditions or autoimmune disease by administration of a VEGF analog alone or in combination with other anti-VEGF treatments, anti-angiogenesis treatments, inflammation therapeutics, and/or autoimmune disease therapeutics. 42723 vl/DC 46 [00139] In another embodiment of the present invention, the modified VEGF protein of the invention is used as a diagnostic. The VEGF analogs of the invention or VEGF receptors can displayed on a synthetic surface, such as in a protein or peptide array. Such an array is well known in the art and can be used to screen for VEGF analogs which bind to KDR and other receptors known to be involved in angiogenesis. The VEGF analogs disclosed herein can be used as positive controls to assess the ability of putative VEGF analogs to bind to KDR and other receptors known to be involved in angiogenesis. The invention also includes an array comprising the VEGF analogs of the present invention to screen for putative VEGF receptors which may be involved in angiogenesis. [00140] Assays suitable for characterizing the analogs described herein are described in PCT/US/99/05908, which is herein incorporated by reference in its entirety. For instance, various immunoassays may be used including but not limited to competitive binding assays and non-competitive assay systems using techniques such as radioimmunoassays, ELISA, sandwich immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays, western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, Protein A assays, and immunoelectrophoresis assays, etcetera. [00141] Pharmaceutical Formulations [00142] The invention provides methods of diagnosis and treatment by administration to a subject of an effective amount of a therapeutic of the invention. The subject may be an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject. [00143] The pharmaceutical compositions of the invention comprise an effective amount of one or more modified VEGF proteins of the present invention in combination with the 42723 vl/DC 47 pharmaceutically acceptable carrier. The compositions may further comprise other known drugs suitable for the treatment of the particular disease being targeted. An effective amount of the VEGF receptor antagonist of the present invention is that amount that blocks, inhibits or reduces VEGF stimulation of endothelial cells compared to that which would occur in the absence of the compound; in other words, an amount that decreases the angiogenic activity of the endothelium, compared to that which would occur in the absence of the compound. The effective amount (and the manner of administration) will be determined on an individual basis and will be based on the specific therapeutic VEGF receptor antagonist being used and a consideration of the subject (size, age, general health), the condition being treated (cancer, arthritis, eye disease, etc.), the severity of the symptoms to be treated, the result sought, the specific carrier or pharmaceutical formulation being used, the route of administration, and other factors as would be apparent to those skilled in the art. The effective amount can be determined by one of ordinary skill in the art using techniques as are known in the art. Therapeutically effective amounts of the compounds described herein can be determined using in vitro tests, animal models or other dose-response studies, as are known in the art. The VEGF proteins of the. present invention can be used alone or in conjunction with other therapies. The therapeutically effective amount may be reduced when a VEGF analog is used in conjunction with another therapy. [00144] The pharmaceutical compositions of the invention may be prepared, packaged, or sold in formulations suitable for intradermal, intravenous, subcutaneous, oral, rectal, vaginal, parenteral, intraperitoneal, topical, pulmonary, intranasal, buccal, ophthalmic, intrathecal, epidural or another route of administration. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. For example, the pharmaceutical compositions of the invention can be 42723 vl/DC 48 administered locally to a tumor via microinfusion. Further, administration may be by a single dose or a series of doses. [00145] For pharmaceutical uses, the VEGF analogs of the present invention may be used in combination with a phannaceutically acceptable carrier, and can optionally include a pharnaceutically acceptable diluent or excipient. Further, the VEGF analogs of the present invention may be used in combination with other known therapies, including but not limited to anti-VEGF therapies, anti-angiogenesis therapies, anti-cancer therapies, infertility therapies, autoimmune disease therapies, inflammation therapies, ocular disease therapies, and skin disease therapies. [001461 The present invention thus also provides pharmaceutical compositions suitable for administration to a subject. The carrier can be a liquid, so that the composition is adapted for parenteral administration, or can be solid, i.e., a tablet or pill formulated for oral administration. Further, the carrier can be in the form of a nebulizable liquid or solid so that the composition is adapted for inhalation. When administered parenterally, the composition should be pyrogen free and in an acceptable parenteral carrier. Active compounds can alternatively be formulated or encapsulated in liposomes, using known methods. Other contemplated formulations include projected nanoparticles and immunologically based formulations. [00147] Liposomes are completely closed lipid bilayer membranes which contain entrapped aqueous volume. Liposomes are vesicles which may be unilamellar (single membrane) or multilamellar (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads" orient toward the aqueous phase. 42723 vl/DC 49 [00148] The liposomes of the present invention may be formed by any of the methods known in the art. Several methods may be used to form the liposomes of the present invention. For example, multilamellar vesicles (MLVs), stable plurilamellar vesicles (SPLVs), small unilamellar vesicles (SUV), or reverse phase evaporation vesicles (REVs) may be used. Preferably, however, MLVs are extruded through filters fonning large unilamellar vesicles (LUVs) of sizes dependent upon the filter size utilized. In general, polycarbonate filters of 30, 50, 60, 100, 200 or 800 nm pores may be used. In this method, disclosed in Cullis et al., U.S. Pat. No. 5,008,050, relevant portions of which are incorporated by reference herein, the liposome suspension may be repeatedly passed through the extrusion device resulting in a population of liposomes of homogeneous size distribution. [001491 For example, the filtering may be performed through a straight-through membrane filter (a Nuclepore polycarbonate filter) or a tortuous path filter (e.g. a Nuclepore Membrafil filter (mixed cellulose esters) of 0.1 tm size), or by alternative size reduction techniques such as homogenization. The size of the liposomes may vary from about 0.03 to above about 2 microns in diameter; preferably about 0.05 to 0.3 microns and most preferably about 0.1 to about 0.2 microns. The size range includes liposomes that are MLVs, SPLVs, or LUVs. [00150] Lipids which can be used in the liposome formulations of the present invention include synthetic or natural phospholipids and may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM) and cardiolipin, among others, either alone or in combination, and also in combination with cholesterol. The phospholipids useful in the present invention may also include dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). In other embodiments, distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), or hydrogenated soy phosphatidylcholine (HSPC) may also be used. 42723 vl/DC 50 Dimyristoylphosphatidylcholine (DMPC) and diarachidonoylphosphatidylcholine (DAPC) may similarly be used. [00151] During preparation of the liposomes, organic solvents may also be used to suspend the lipids. Suitable organic solvents for use in the present invention include those with a variety of polarities and dielectric properties, which solubilize the lipids, for example, chloroform, methanol, ethanol, dimethylsulfoxide (DMSO), methylene chloride, and solvent mixtures such as benzene:methanol (70:30), among others. As a result, solutions (mixtures in which the lipids and other components are unifonnly distributed throughout) containing the lipids are formed. Solvents are generally chosen on the basis of their biocompatability, low toxicity, and solubilization abilities. [00152] To encapsulate the VEGF receptor antagonist(s) of the inventions into the liposomes, the methods described in U.S. Patent No. 5,380,531, relevant portions of which are incorporated by reference herein, may be used with the analog(s) of the present invention. [00153] Liposomes containing the VEGF analog(s) of the present invention may be used therapeutically in mammals, especially humans, in the treatment of a number of disease states or pharmacological conditions which require sustained release formulations as well as repeated administration. The mode of administration of the liposomes containing the agents of the present invention may determine the sites and cells in the organism to which the VEGF analog may be delivered. [00154] The liposomes of the present invention may be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The preparations may be injected parenterally, for example, intravenously. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic, should 42723 v1/DC 51 isotonicity be necessary or desired. The liposomes of the present invention may also be employed subcutaneously or intramuscularly. Other uses, depending upon the particular properties of the preparation, may be envisioned by those skilled in the art. [00155] For the oral mode of administration, the liposomal formulations of the present invention can be used in the fonn of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers which can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, lubricating agents, and talc are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added. [00156] For the topical mode of administration, the pharmaceutical formulations of the present invention may be incorporated into dosage forms such as a solution, suspension, gel, oil, ointment or salve, and the like. Preparation of such topical formulations are described in the art of pharmaceutical formulations as exemplified, for example, by Gennaro et al. (1995) Remington's Pharmaceutical Sciences, Mack Publishing. For topical application, the compositions could also be administered as a powder or spray, particularly in aerosol form. For administration to humans in the treatment of disease states or pharmacological conditions, the prescribing physician will ultimately determine the appropriate dosage of the agent for a given human subject, and this can be expected to vary according to the age, weight and response of the individual as well as the pharmacokinetics of the agent used. [00157] The pharmaceutical compositions of the invention further comprise a depot formulation of biopolymers such as biodegradable microspheres. Biodegradable microspheres are used to control drug release rates and to target drugs to specific sites in the body, thereby optimizing their therapeutic response, decreasing toxic side effects, and 42723 vl/DC 52 eliminating the inconvenience of repeated injections. Biodegradable microspheres have the advantage over large polymer implants in that they do not require surgical procedures for implantation and removal. [00158] The biodegradable microspheres used in the context of the invention are formed with a polymer which delays the release of the proteins and maintains, at the site of action, a therapeutically effective concentration for a prolonged period of time. The polymer can be chosen from ethylcellulose, polystyrene, poly(s-caprolactone), poly(lactic acid) and poly(lactic acid-co-glycolic acid) (PLGA). PLGA copolymer is one of the synthetic biodegradable and biocompatible polymers that has reproducible and slow-release characteristics. An advantage of PLGA copolymers is that their degradation rate ranges from months to years and is a function of the polymer molecular weight and the ratio of polylactic acid to polyglycolic acid residues. Several products using PLGA for parenteral applications are currently on the market, including Lupron Depot and Zoladex in the United States and Enantone Depot, Decapeptil, and Pariodel LA in Europe (see Yonsei, Med J. 2000 Dec;41(6):720-34 for review). [00159] The phannaceutical compositions of the invention may further be prepared, packaged, or sold in a formulation suitable for nasal administration as increased permeability has been shown through the tight junction of the nasal epithelium (Pietro and Woolley, The Science behind Nastech's intranasal drug delivery technology. Manufacturing Chemist, August, 2003). Such formulations may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. 42723 v1/DC 53 Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose fonn. [001601 Pharmaceutical compositions of the invention formulated for nasal delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. Such fonnulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers. [001611 Another fonnulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose. [00162] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein. [001631 In some embodiments, the compositions of the invention may be administered by inhalation. For inhalation therapy, the active ingredients may be in a solution useful for 42723 vl/DC 54 administration by metered dose inhalers or in a form suitable for a dry powder inhaler. In another embodiment, the compositions are suitable for administration by bronchial lavage. [001641 Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. [00165] The VEGF receptor antagonists of the present invention can be administered acutely (i.e., during the onset or shortly after events leading to inflammation), or can be administered during the course of a degenerative disease to reduce or ameliorate the progression of symptoms that would otherwise occur. The timing and interval of administration is varied according to the subject's symptoms, and can be administered at an interval of several hours to several days, over a time course of hours, days, weeks or longer, as would be determined by one skilled in the art. A typical daily regime can be from about 0.01 ptg/kg body weight per day, from about 1 mg/kg body weight per day, from about 10 mg/kg body weight per day, from about 100 mg/kg body weight per day, and from about 1 g/kg body weight per day. [001661 The VEGF receptor antagonists of the invention may be administered intravenously, orally, intranasally, intraocularly, intramuscularly, intrathecally, or by any suitable route in view of the VEGF protein, the protein formulation and the disease to be treated. Modified VEGF for the treatment of inflammatory arthritis can be injected directly into the synovial fluid. Modified VEGF for the treatment of solid tumors may be injected directly into the tumor. Modified VEGF for the treatment of skin diseases may be applied topically, for instance in the fonn of a lotion or spray. Intrathecal administration, i.e. for the treatment of brain tumors, can comprise injection directly into the brain. Alternatively, modified VEGF may be coupled or conjugated to a second molecule (a "carrier"), which is a peptide or non-proteinaceous moiety selected for its ability to penetrate the blood-brain barrier and transport the active agent across the blood-brain barrier. Examples of suitable 42723 vl/DC 55 carriers are disclosed in U.S. Patent Nos. 4,902,505; 5,604,198; and 5,017,566, which are herein incorporated by reference in their entirety. [00167] An alternative method of administering the VEGF receptor antagonists of the present invention is carried out by administering to the subject a vector carrying a nucleic acid sequence encoding the modified VEGF protein, where the vector is capable of directing expression and secretion of the protein. Suitable vectors are typically viral vectors, including DNA viruses, RNA viruses, and retroviruses. Techniques for utilizing vector delivery systems and carrying out gene therapy are known in the art (see Lundstrom, 2003, Trends Biotechnol. 21(3):117-22, for a recent review). [00168] Transgenic Animals [00169] The production of transgenic non-human animals that contain a modified VEGF construct with increased receptor binding affinity and optionally antagonistic properties is contemplated in one embodiment of the present invention. [00170] The successful production of transgenic, non-human animals has been described in a number of patents and publications, such as, for example U.S. Patent 6,291,740 (issued September 18, 2001); U.S. Patent 6,281,408 (issued August 28, 2001); and U.S. Patent 6,271,436 (issued August 7, 2001) the contents of which are hereby incorporated by reference in their entireties. [001711 The ability to alter the genetic make-up of animals, such as domesticated mammals including cows, pigs, goats, horses, cattle, and sheep, allows a number of commercial applications. These applications include the production of animals which express large quantities of exogenous proteins in an easily harvested form (e.g., expression into the milk or blood), the production of animals with increased weight gain, feed efficiency, carcass composition, milk production or content, disease resistance and resistance to infection by specific microorganisms and the production of animals having enhanced growth rates or 42723 v1/DC 56 reproductive perfonnance. Animals which contain exogenous DNA sequences in their genome are referred to as transgenic animals. [001721 The most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos (Wall et al., J. Cell. Biochem. 49:113 [1992]). Other methods for the production of transgenic animals include the infection of embryos with retroviruses or with retroviral vectors. Infection of both pre and post-implantation mouse embryos with either wild-type or recombinant retroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]; Janenich et al., Cell 24:519 [1981]; Stuhlmann et al., Proc. Natl. Acad. Sci. USA 81:7151 [1984]; Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]; Van der Putten et al., Proc. Natl. Acad Sci. USA 82:6148-6152 [1985]; Stewart et al., EMBO J. 6:383-388 [1987]). [00173] An alternative means for infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoele of mouse embryos (Jahner, D. et al., Nature 298:623 [1982]). The introduction of transgenes into the germline of mice has been reported using intrauterine retroviral infection of the midgestation mouse embryo (Jahner et al., supra [1982]). Infection of bovine and ovine embryos with retroviruses or retroviral vectors to create transgenic animals has been reported. These protocols involve the micro injection of retroviral particles or growth arrested (i.e., mitomycin C-treated) cells which shed retroviral particles into the perivitelline space of fertilized eggs or early embryos (PCT International Application WO 90/08832 [1990]; and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]. PCT International Application WO 90/08832 describes the injection of wild type feline leukemia virus B into the perivitelline space of sheep embryos at the 2 to 8 cell stage. Fetuses derived from injected embryos were shown to contain multiple sites of integration. [00174] U.S. Patent 6,291,740 (issued September 18, 2001) describes the production of transgenic animals by the introduction of exogenous DNA into pre-maturation oocytes and 42723 vl/DC 57 mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]). This patent also describes methods and compositions for cytomegalovirus promoter-driven, as well as mouse mammary tumor LTR expression of various recombinant proteins. [001751 U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for producing transgenic animals using embryonic stem cells. Briefly, the embryonic stem cells are used in a mixed cell co-culture with a morula to generate transgenic animals. Foreign genetic material is introduced into the embryonic stem cells prior to co-culturing by, for example, electroporation, microinjection or retroviral delivery. ES cells transfected in this manner are selected for integrations of the gene via a selection marker such as neomycin. [001761 U.S. Patent 6,271,436 (issued August 7, 2001) describes the production of transgenic animals using methods including isolation of primordial germ cells, culturing these cells to produce primordial gern cell-derived cell lines, transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species. [001771 Kits Containing Modified VEGF Proteins [001781 In a further embodiment, the present invention provides kits containing a VEGF analog and/or VEGF analog fusion proteins, which can be used, for instance, for therapeutic or non-therapeutic applications. The kit comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which includes a VEGF analog or VEGF fusion protein that is effective for therapeutic or non therapeutic applications, such as described above. The label on the container indicates that 42723 v1/DC 58 the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. [001791 The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The kit of the invention may also include a control consisting of wild type VEGF such as wild-type VEGFs65 or VEGF 165 b. [001801 The following examples are provided to describe and illustrate the present invention. As such, they should not be construed to limit the scope of the invention. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims. .001811 Examples [001821 Example 1: Design of VEGF Receptor Antagonists [001831 VEGF-A antagonists of the present invention were designed to increase receptor binding affinity and decrease bioactivity as compared to wild-type VEGF-A. One method by which this was done was by adding a positive charge to the loops of VEGF-A. This approach to design super-antagonists involves a combination of different methods known in the art including but not limited to homology modeling, sequence comparisons, charge scanning mutagenesis, and linking monomers and introduction of mutations in the context of linked monomers. [001841 Vammin, or snake venom VEGF, has been shown to bind to KDR-IgG with high affinity and strongly stimulate proliferation of vascular endothelial cells in vitro (see Yamazaki et al., 2003, J. Biol. Chem. 278, 51985-51988, which is herein incorporated by reference in its entirety). VEGF-A receptor antagonists were designed based on VEGF 1 65 42723 v1/DC 59 homology to vammin. VEGF165 has glutamate residues at positions 72 and 73, whereas vammin contains a glycine and lysine residue at these positions, respectively. By modifying VEGF-A to contain two basic amino acid residues at positions 72 and 73, the modified VEGF-A demonstrated a significant increase in receptor binding affinity compared to wild type VEGF-A (Figure 3A). [001851 Example 2: Characterization of VEGF receptor antagonists [00186] VEGF analogs I83K, E44R, E72RE73R, E67K andQ87K were created and assayed for their ability to bind to KDR and to decrease cell proliferation compared to wild-type VEGF. [00187] Methods [00188] VEGF analogs expressed by yeast cells were incubated with immobilized KDR-Fc and the ability of the analogs to bind to KDR-Fc was assayed. The binding assay was performed as follows: [00189] 1. Nunc MaxiSorpTM 96 microwell plates were coated with 150 ng/well KDR-Fc (R & D System, Inc.) and 100 pl 50 mM sodium bicarbonate buffer (15 mM Na 2

CO

3 + 35 mM NaHCO 3 ) at pH 9.6. A separate plate was used for each VEGF analog and wild-type VEGF tested. [00190] 2. The plates were incubated at 4'C overnight. [00191] 3. The next day, the wells were washed three times in washing buffer (0.05% tween in PBS). [00192] 4. The wells were blocked with PBS with 3% BSA, 0.03% tween for 1 hour at room temperature. 42723 v1/DC 60 [001931 5. After blocking, the wells were washed three times in washing buffer (0.05% tween in PBS). [00194] 6. VEGF-A (wild-type or mutant) were added at different concentrations to the wells in 50 ptl binding buffer (1% BSA and 0.03% tween in PBS). [00195] 7. 125 I- labeled VEGF-A (wild-type or mutant) at 70,000 cpm/well (PerkinElmer) was added to each well in 50 pl binding buffer (1% BSA and 0.03% tween in PBS). [00196] 8. The contents of the wells were mixed and incubated for 2 hours at room temperature with slow shaking. [001971 9. The wells were washed three times with washing buffer (0.05% tween in PBS). [00198] 10. To each well, 120 pl of lysis buffer (0.2 M NaOH + 0.5% SDS) was added. Plate was shaken vigorously for 20 minutes at room temperature. [00199] 11. The lysis buffer from each well was transferred to an individual tube. The wells were washed with lysis buffer two times additional times and combined with the lysis solution buffer in the corresponding tube. [00200] 12. The measure of binding for wild-type VEGF-A and various VEGF-A mutants was determined by counting with a gamma counter. [00201] The ability of HUVEC endothelial cells to proliferate in the presence of the VEGF analogs was assayed as follows: [00202] 1. HUVEC endothelial cells (passage 6) were seeded into 96 well plates at 3,000 cells/well using Media-200 with growth factors and incubated overnight. [00203] 2. After overnight incubation, the media was removed and Media 199 (Invitrogen) with 2% dialysis FBS (Invitrogen) was added. [002041 3. Cells were incubated for 20 hours. 42723 vl/DC 61 [00205] 4. Wild-type VEGF-A and VEGF-A analogs were serial diluted in Media 199 with 2% dialysis FBS in the 96-well plates, starting at 200 ng/well. [00206] 5. The media was removed from each well and replaced with 200 pl/well diluted VEGF media. [00207] 6. Cells were incubated at 370 C for 72 hours. [00208] 7. Cell proliferation was analyzed using Promega's CellTiter-Glo@ Luminescent Cell Viability Assay. Briefly, CellTiter buffer was thawed, transferred into CellTiter-Glo substrate, and mixed well to make substrate mixture. 100 pl growth media was removed from each well into a new 96 well plate and mixed well with 100 1d substrate mixture. The plates were shaken for 2 minutes and incubated at room temperature for an additional ten minutes. [00209] 8. Plates were read for luminescent signal using a plate reader with integration time set at 250mS (Tecan). [00210] Analysis [002111 The receptor binding affinity of the 183K analog to KDR-Fc was slightly less than that of wild-type VEGF-A (Figure 1A). However, the 183K analog demonstrated a significant decrease in endothelial cell proliferation compared to wild-type VEGF-A (Figure 1B). VEGF-A analogs E44R, EE72/73RR, E67K and Q87K all demonstrated an increase in receptor cell binding affinity compared to wild-type VEGF-A (Figures 2A, 3A, 4, 5 and 6). However, analogs E44R and EE72/73RR demonstrated little to no change in endothelial cell proliferation (Figures 2B and 3B). These results show that VEGF165 analogs comprising 183K can effectively function as a VEGF-A receptor antagonist. Further, although VEGF-A analogs E44R and EE72/73RR were unable to decrease endothelial cell proliferation alone, when added to 183K, these modifications have the potential of further increasing receptor binding affinity. 42723 vl/DC 62 [00212] All publications, patents and patent applications discussed in this application are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments, thereof, and may details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. 42723 v1/DC 63

Claims

1. A vascular endothelial growth factor (VEGF) receptor antagonist which is a human VEGF-A having a substitution at an isoleucine residue corresponding to 183 in SEQ ID NO:4 or SEQ ID NO:13, wherein the substitution is a positively charged amino acid.

2. The VEGF receptor antagonist of claim 1, wherein the VEGF receptor antagonist comprises an amino acid sequence of SEQ ID NO: 4, wherein 183 is substituted by a positively charged amino acid.

3. The VEGF receptor antagonist of claim 1, wherein the VEGF receptor antagonist comprises an amino acid sequence of SEQ ID NO: 13, wherein 183 is substituted by a positively charged amino acid.

4. The VEGF receptor antagonist of any one of claims 1 to 3, wherein the positively charged amino acid is lysine, arginine or histidine.

5. The VEGF receptor antagonist of claim 1, wherein the human VEGF-A is expressed as at least one subunit of a homodimer or heterodimer.

6. The VEGF receptor antagonist of claim 1, wherein the antagonist contains one or more additional basic amino acid substitutions at the position(s) corresponding to the residue(s) selected from the group consisting of positions E44, E67, E72, E73 and Q87 of SEQ ID NO.: 4.

7. The VEGF receptor antagonist of claim 6, wherein the additional substitutions are selected from those corresponding to the group consisting of E72R, E72K, E73R, and E73K of SEQ ID NO:4.

8. The VEGF receptor antagonist of claim 6, wherein the additional substitution is one corresponding to E44R, E44K, E67K, Q87K or Q87L of SEQ ID NO:4.

9. The VEGF receptor antagonist of claim 1, wherein interaction of the VEGF receptor antagonist and a native VEGF receptor results in inhibition of angiogenesis.

10. The VEGF receptor antagonist of claim 9, wherein the native VEGF receptor is KDR. 64

11. The VEGF receptor antagonist of claim 6, wherein the antagonist contains the amino acid substitutions corresponding to E72R, E73R, and 183K of SEQ ID NO:4.

12. The VEGF receptor antagonist of claim 6, wherein the antagonist contains the amino acid substitutions corresponding to E44R, E72R, E73R and 183K of SEQ ID NO:4.

13. The VEGF receptor antagonist of any one of claims 1, 2, 4, 5 or 6, further comprising an amino acid substitution at the position corresponding to C146 or C160 of SEQ ID NO.: 4.

14. The VEGF receptor antagonist of claim 13, wherein the amino acid substitution is one corresponding to C146S or C160S of SEQ ID NO:4.

15. The VEGF receptor antagonist of claims 1, 6 or 13, further comprising an amino acid substitution at the position corresponding to A 111 and/or A148 of SEQ ID NO.: 4.

16. The VEGF receptor antagonist of claim 15, wherein the amino acid substitution is one corresponding to Al IP and/or A148P of SEQ ID NO:4.

17. The VEGF receptor antagonist of claim 1, further comprising a toxin.

18. The VEGF receptor antagonist of claim 17, wherein the toxin is selected from the group consisting of a Pseudomonas exotoxin (PE), a Diphtheria toxin (DT), ricin toxin, abrin toxin, anthrax toxins, shiga toxin, botulism toxin, tetanus toxin, cholera toxin, maitotoxin, palytoxin, ciguatoxin, texitilotoxin, batrachotoxin, alpha conotoxin, taipoxin, tetrodotoxin, alpha tityustoxin, saxitoxin, anatoxin, microcystin, aconitine, exfoliatin toxins A, exfoliatin B, an enterotoxin, toxic shock syndrome toxin (TSST-I), Y.pestis toxin and a gas gangrene toxin.

19. The VEGF receptor antagonist of claim 1, wherein said VEGF-A is selected from the group consisting of VEGF 165 .(SE ID NO:4), VEGFi 65 b (SEQ ID NO:13), VEGF 1 2 1 (SEQ ID NO: 6), VEGF 145 (SEQ ID NO: 8), VEGF 14 8 (SEQ ID NO:10), VEGFi 8 3 (SEQ ID NO: 15), VEGF 18 9 (SEQ ID NO: 17) and VEGF 2 5 (SEQ ID NO: 19).

20. A pharmaceutical composition comprising the VEGF receptor antagonist of claims 1, 6, 13 or 17. 65