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.

What is claimed: 
     
         1 . A modified vascular endothelial growth factor (VEGF) comprising at least one mutation causing the modified VEGF to act as a receptor antagonist, wherein the mutation results in a dissociation of receptor binding affinity and bio activity. 
     
     
         2 . The VEGF receptor antagonist of  claim 1 , wherein the receptor binding affinity of the antagonist to a native VEGF receptor is greater than the receptor binding affinity of wild-type VEGF to the native VEGF receptor. 
     
     
         3 . The VEGF receptor antagonist of  claim 2 , wherein the native VEGF receptor is Flt-1 or KDR. 
     
     
         4 . The VEGF receptor antagonist of  claim 2 , wherein there is at least about a 3 to 4 fold increase in receptor binding affinity. 
     
     
         5 . The VEGF receptor antagonist of  claim 2 , wherein there is at least about a 2 fold increase in receptor binding affinity. 
     
     
         6 . The VEGF receptor antagonist of  claim 1 , wherein the bioactivity for the antagonist is decreased compared to the bioactivity for a wild-type VEGF. 
     
     
         7 . The VEGF receptor antagonist of  claim 1 , wherein the antagonist is expressed as a homodimer or heterodimer. 
     
     
         8 . The VEGF receptor antagonist of  claim 7 , wherein each subunit of the homodimer or heterodimer contains the at least one mutation. 
     
     
         9 . The VEGF receptor antagonist of  claim 8 , wherein one subunit of the homodimer or heterodimer contains the at least one mutation. 
     
     
         10 . The VEGF receptor antagonist of  claim 1 , wherein the antagonist is expressed as a fusion protein comprising one or more VEGF subunits. 
     
     
         11 . The VEGF receptor antagonist of  claim 10 , wherein the one or more VEGF subunits each contain the at least one mutation. 
     
     
         12 . The VEGF receptor antagonist of  claim 10 , wherein at least one VEGF subunit contains the at least one mutation. 
     
     
         13 . The VEGF receptor antagonist of  claim 10  further comprising a linker peptide. 
     
     
         14 . The VEGF receptor antagonist of  claim 10  further comprising a toxin. 
     
     
         15 . The VEGF receptor antagonist of  claim 1 , wherein the mutation is a basic amino acid substitution at a position corresponding to position 83 of SEQ ID NO.: 4. 
     
     
         16 . The VEGF receptor antagonist of  claim 15 , wherein said basic amino acid is I83K. 
     
     
         17 . The VEGF receptor antagonist of  claim 15 , wherein said basic amino acid is I83R. 
     
     
         18 . The VEGF of  claim 15 , wherein the antagonist contains one or more additional basic amino acid substitutions corresponding to the group consisting of positions 44, 67, 72, 73 and 87 of SEQ ID NO.: 4. 
     
     
         19 . The VEGF receptor antagonist of  claim 18 , wherein the additional substitutions are selected from the group consisting of E72R and E73R. 
     
     
         20 . The VEGF receptor antagonist of  claim 18 , wherein the additional substitutions are selected from the group consisting of E72K and E73K. 
     
     
         21 . The VEGF receptor antagonist of  claim 18 , wherein the additional substitution is E44R or E44K. 
     
     
         22 . The VEGF receptor antagonist of  claim 18 , wherein the additional substitution is Q87K or Q87L. 
     
     
         23 . The VEGF receptor antagonist of  claim 18 , wherein the additional substitution is E67K. 
     
     
         24 . The VEGF receptor antagonist of  claim 1 , wherein interaction of the VEGF-A receptor antagonist and a native VEGF receptor results in inhibition of angiogenesis. 
     
     
         25 . The VEGF receptor antagonist of  claim 24 , wherein the native VEGF receptor is KDR. 
     
     
         26 . The VEGF receptor antagonist of  claim 18 , wherein the antagonist contains the amino acids E72R+E73R+I83K. 
     
     
         27 . The VEGF receptor antagonist of  claim 18 , wherein the antagonist contains the amino acids E44R+E72R+E73R+I83K. 
     
     
         28 . The VEGF antagonist of  claims 15  and  18 , wherein the antagonist contains one or more additional amino acid substitutions in amino acids corresponding to positions 111-165 of SEQ ID NO.: 4 which disrupt neuropilin-1 binding. 
     
     
         29 . The VEGF antagonist of  claim 28 , wherein the additional amino acid substitutions do not disrupt heparin sulfate binding. 
     
     
         30 . The VEGF antagonist of  claim 28 , wherein the antagonist contains an amino acid substitution at the position corresponding to C146 or C160 of SEQ ID NO.: 4. 
     
     
         31 . The VEGF antagonist of  claim 30 , wherein the amino acid substitution is C146S or C160S. 
     
     
         32 . The VEGF antagonist of  claim 28 , wherein the antagonist contains amino acid substitutions at the positions corresponding to C146 and C160 of SEQ ID NO.: 4. 
     
     
         33 . The VEGF antagonist of  claim 32 , wherein the amino acid substitution is C146S and C160S. 
     
     
         34 . The VEGF antagonist of  claims 15  and  18 , wherein the antagonist contains one or more additional amino acid substitutions which reduce or prevent protease cleavage of the antagonist. 
     
     
         35 . The VEGF antagonist of  claim 34 , wherein the protease is plasmin. 
     
     
         36 . The VEGF antagonist of  claim 34 , wherein the one or more additional amino acid substitutions are selected from the group of positions corresponding to positions A111 and A148 of SEQ ID NO.: 4. 
     
     
         37 . The VEGF antagonist of  claim 36 , wherein the amino acid substitution is A111P or A148P. 
     
     
         38 . The VEGF antagonist of  claim 36 , wherein the amino acid substitutions are A111P and A148P. 
     
     
         39 . The VEGF antagonist of  claim 14 , 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, textilotoxin, batrachotoxin, alpha conotoxin, taipoxin, tetrodotoxin, alpha tityustoxin, saxitoxin, anatoxin, microcystin, aconitine, exfoliatin toxins A, exfoliatin B, an enterotoxin, toxic shock syndrome toxin (TSST-1),  Y. pestis  toxin and a gas gangrene toxin. 
     
     
         40 . The VEGF antagonist of  claims 8 - 12 , wherein at least one of said subunits is a VEGF-A subunit. 
     
     
         41 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is a VEGF 165  subunit. 
     
     
         42 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is VEGF 165 b. 
     
     
         43 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is a VEGF 121 . subunit. 
     
     
         44 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is VEGF 145 . 
     
     
         45 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is a VEGF 148 . subunit. 
     
     
         46 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is VEGF 183 . 
     
     
         47 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is a VEGF 189 . subunit. 
     
     
         48 . The VEGF antagonist of  claim 40 , wherein the said VEGF-A subunit is VEGF 206 . 
     
     
         49 . The VEGF antagonist of  claims 8 - 12 , wherein at least one of said subunits is a VEGF-B subunit. 
     
     
         50 . The VEGF antagonist of  claim 49 , wherein the said VEGF-B subunit is a VEGF-B 167  subunit. 
     
     
         51 . The VEGF antagonist of  claim 49 , wherein the said VEGF-B subunit is VEGF-B 186 . 
     
     
         52 . The VEGF antagonist of  claims 8 - 12 , wherein at least one of said subunits is a VEGF-C subunit. 
     
     
         53 . The VEGF antagonist of  claims 8 - 12 , wherein at least one of said subunits is a VEGF-D subunit. 
     
     
         54 . The VEGF antagonist of  claims 8 - 12 , wherein at least one of said subunits is a PlGF subunit. 
     
     
         55 . The VEGF antagonist of  claim 54 , wherein the said PlGF subunit is PlGF-1. 
     
     
         56 . The VEGF antagonist of  claim 54 , wherein the said PlGF subunit is PlGF-2. 
     
     
         57 . The VEGF antagonist of  claims 1 - 56 , wherein angiogenesis is partially inhibited. 
     
     
         58 . The VEGF antagonist of  claims 1 - 56 , wherein angiogenesis is about almost completely inhibited. 
     
     
         59 . A pharmaceutical composition comprising the VEGF receptor antagonist of any of  claims 1 - 58 . 
     
     
         60 . The composition of  claim 59  further comprising a pharmaceutically acceptable carrier. 
     
     
         61 . The composition of  claim 60 , wherein said composition is formulated for aerosol deliver. 
     
     
         62 . The composition of  claim 61 , wherein said composition is formulated as a nasal spray. 
     
     
         63 . The composition of  claim 60 , wherein the composition is formulated for oral administration. 
     
     
         64 . The composition of  claim 63 , wherein the composition is formulated as a tablet, pill, or capsule. 
     
     
         65 . The composition of  claim 60 , wherein the composition is formulated as a depot or suppository. 
     
     
         66 . The composition of  claim 60  further comprising one or more additional drugs from the group consisting of anti-VEGF drug, anti-angiogenic drug, anti-cancer drug, infertility drug, autoimmune drug, inflammation drug, ocular disease drug, and skin disease drug. 
     
     
         67 . A method of treating a patient diagnosed with cancer with a therapeutically effective amount of the VEGF receptor antagonist of any of  claims 1 - 58 , comprising administering said antagonist to said patient such that spread of said cancer is reduced or inhibited. 
     
     
         68 . The method of  claim 67  wherein said cancer is a solid tumor cancer selected from the group consisting of bladder, breast, liver, bone, kidney, colon, ovarian, prostate, pancreatic, lung, brain, breast, and skin. 
     
     
         69 . A method of treating a patient diagnosed with an angiogenesis-associated eye disease with a therapeutically effective amount of the VEGF receptor antagonist of any  claims 1 - 58 , comprising administering said antagonist to said patient such that said eye disease is reduced or inhibited. 
     
     
         70 . The method of  claim 69 , wherein said eye disease is selected from the group consisting of retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, macular degeneration, and neovascularization associated with corneal injury or grafts. 
     
     
         71 . A method of treating a patient diagnosed with an angiogenesis-related disease or condition with a therapeutically effective amount of the VEGF receptor antagonist of any  claims 1 - 58 , comprising administering said antagonist to said patient such that said angiogenesis-related disease is reduced or inhibited. 
     
     
         72 . The method of  claim 71 , wherein said disease or condition is selected from the group consisting of hemangiomas, rheumatoid arthritis, osteoarthritis, septic arthritis, asthma, atherosclerosis, idiopathic pulmonary fibrosis, vascular restenosis, arteriovenous malformations, meningiomas, neovascular 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, unexplained female infertility, endometriosis, unexplained male infertility, pterygium, wounds, sores, skin ulcers, gastric ulcers, and duodenal ulcers. 
     
     
         73 . The VEGF receptor antagonist of  claim 1 , wherein one or both subunits of the VEGF comprise a modification to prolong half-life. 
     
     
         74 . The VEGF receptor antagonist of  claim 73 , wherein the modification to prolong half-life is a N terminal or C terminal extension. 
     
     
         75 . The VEGF receptor antagonist of  claim 73 , wherein the modification to prolong half-life is pegylation. 
     
     
         76 . The VEGF receptor antagonist of  claim 10 , wherein the fusion protein contains two VEGF or VEGF-related protein subunits fused together. 
     
     
         77 . The VEGF receptor antagonist of  claim 76 , wherein the two VEGF or VEGF-related protein subunits are selected from the group consisting of a VEGF-A subunit fused to a VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, PDGF or PlGF 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 PlGF subunit; a VEGF-D subunit fused to a VEGF-A, VEGF-B, VEGF-C, VEGF-E, VEGF-F, PDGF or PlGF subunit; and a PlGF subunit fused to a VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, or PDGF subunit. 
     
     
         78 . A VEGF 165 b receptor antagonist comprising one or more mutations selected from the group consisting of E44B, E67B, E72B, E73B, I83B and Q87B of SEQ ID NO.: 13, wherein B is a basic amino acid. 
     
     
         79 . The VEGF 165 b receptor antagonist of  claim 78 , wherein the antagonist exhibits an increased receptor binding affinity compared to wild-type VEGF 165  or wild-type VEGF 165 b. 
     
     
         80 . A VEGF receptor antagonist comprising one or more mutations disrupting neuropilin-1 binding and comprising one or more basic amino acid substitutions from the group consisting of E44, E67, E72, E73, I83 and Q87 of SEQ ID NO.:4. 
     
     
         81 . The VEGF receptor antagonist of  claim 80 , wherein said one or more mutations disrupting neuropilin-1 binding are selected from the group consisting of C146S and C160S.