Nova Patents
CA2184584A1

Vascular endothelial growth factor 2

Abstract

Disclosed is a human VEGF2 polypeptide and DNA (RNA) encoding such VEGF2 pol ypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and anta gonist against such polypeptide. Such polypeptides may be combined with a suitable pharmaceutical carrier or diluent to provide dia gnostic, therapeutic and or prophylactic effects against various diseases. Also provided are methods of using the antibodies and antagonists to inhibit the action of VEGF2 for therapeutic purposes. Disclosed is a human VEGF2 polypeptide and DNA (RNA) encoding such VEGF2 polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonist against such polypeptide. Such polypeptides may be combined with a suitable pharmaceutical carrier or diluent to provide diagnostic, therapeutic and or prophylactic effects against various diseases. Also provided are methods of using the antibodies and antagonists to inhibit the action of VEGF2 for therapeutic purposes.

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Projected expiry passed 12 May 2014, 12.4 years ago.

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14 claims: 11 independent, 3 dependent

  1. 1
    CLAIMED ARE DEFINED AS FOLLOWS:1. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a proprotein portion of a protein comprising SEQ ID NO:2.
  2. 2
    An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a mature portion of a protein comprising SEQ ID NO:2.
  3. 3
    An isolated polynucleotide, or one which is substantially identical to said polynucleotide, which encodes a VEGF-2 polypeptide comprising amino acids +61 to +74 of SEQ ID NO:2.
  4. 6
    An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a VEGF-2 polypeptide that is a fragment of SEQ ID NO:2 comprising amino acids +38 to +118.
  5. 7
    An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a VEGF-2 polypeptide, which hybridizes to a polynucleotide consisting of the complement of SEQ ID NO:1 under the following conditions: hybridization followed by a final wash using: (a) 0.5 x SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C;or (b) 0.2 x SSC at 65°C. CA 02184584 2001-03-14 -358. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a VEGF-2 polypeptide, which hybridizes to the cDNA contained in ATCC Deposit No. 75698 under the following conditions: hybridization followed by a final wash using: (a) 0.5 x SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C;or (b) 0.2 x SSC at 65°C.
  6. 8
    9. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment of SEQ ID NO:2, wherein said polypeptide fragment has angiogenic activity.
  7. 9
    10. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment encoded by the cDNA contained in ATCC Deposit No. 75698, wherein said fragment has angiogenic activity.
  8. 10
    11. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment of SEQ ID NO:2, wherein said polypeptide fragment has endothelial cell proliferative activity.
  9. 11
    12. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, comprising a polynucleotide that encodes a polypeptide fragment encoded by the cDNA contained in ATCC Deposit No. 75698, wherein said fragment has endothelial cell proliferative activity.
  10. 12
    13. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a protein encoded by the cDNA contained in ATCC Deposit No. 75698,
  11. 13
    14. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a proprotein portion of a protein encoded by the cDNA contained in ATCC Deposit No.75698. CA 02184584 2002-01-23 -3615. An isolated polynucleotide, or one which is substantially identical to said polynucleotide, encoding a mature portion of a protein encoded by the cDNA contained in ATCC Deposit No. 75698. 16. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, fused to a heterologous polynucleotide. 17. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, fused to a polynucleotide which encodes a heterologous polypeptide. 18. A vector comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15. 19. A vector comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, operatively associated with a regulatory sequence that controls gene expression. 20. The polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, fused, in frame, to a polynucleotide sequence encoding a polypeptide which facilitates purification of the resultant fusion polypeptide. 21. A recombinant host cell transformed, transfected or transduced with the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to
  12. 14
    15. 22. A recombinant host cell comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, operatively associated with a heterologous regulatory sequence that controls gene expression. CA 02184584 2002-01-23 -3723. A method for producing a VEGF-2 polypeptide, comprising:(a) culturing the host cell of claim 22, under conditions suitable to produce the polypeptide;and (b) recovering the polypeptide from the cell culture. 24. The isolated polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, wherein the polynucleotide is DNA. 25. The isolated polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15, wherein the polynucleotide is RNA. 26. A method for producing a VEGF-2 polypeptide, comprising: (a) culturing a genetically engineered host cell comprising an isolated mammalian RNA or cDNA which hybridizes to a polynucleotide, or one which is substantially identical to said polynucleotide, consisting of the complement of SEQ ID NO:1 under the following conditions: hybridization followed by a final wash using: (i) 0.5 x SSC, 0.1% sodium dodecyl sulfate (SDS) at 60°C;or (ii) 0.2 x SSC at 65°C.;and (b) recovering the polypeptide from the cell culture. 27. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a proprotein portion of a protein comprising the amino acid sequence of SEQ ID NO:2. 28. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a mature portion of a protein comprising the amino acid sequence of SEQ ID NO:2. 29. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a protein encoded by the cDNA contained in ATCC Deposit No. 75698. CA 02184584 2002-01-23 -3830. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a proprotein portion of a protein encoded by the cDNA contained in ATCC Deposit No. 75698. 31. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a mature portion of a protein encoded by the cDNA contained in ATCC Deposit No. 75698. 32. An isolated VEGF-2 polypeptide, or one which is substantially identical to said isolated polypeptide and exhibits the activity of said isolated polypeptide, comprising amino acids +61 to +74 of SEQ ID NO:2. 33. The isolated polypeptide, or one which is substantially identical to said polypeptide, of claim 32, comprising amino acids +1 to +326 of SEQ ID NO:2. 34. The isolated polypeptide, or one which is substantially identical to said polypeptide, of claim 32, comprising amino acids -24 to +326 of SEQ ID NO:2. 35. An isolated VEGF-2 polypeptide, or one which is substantially identical to said isolated polypeptide and exhibits the activity of said isolated polypeptide, that is a fragment of SEQ ID NO: 2 comprising amino acids +38 to +118. 36. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of SEQ ID NO:2, wherein said fragment has angiogenic activity. 37. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment encoded by the cDNA contained in ATCC Deposit No. 75698, wherein said fragment has angiogenic activity. CA 02184584 2001-03-14 -3938. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment of SEQ ID NO:2, wherein said fragment has endothelial cell proliferative activity. 39. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising a polypeptide fragment encoded by the cDNA contained in ATCC Deposit No. 75698, wherein said fragment has endothelial cell proliferative activity. 40. The polypeptide, or one which is substantially identical to said polypeptide, produced by the method of claim 23 or 26. 41. A method for producing a VEGF-2 polypeptide, comprising: (a) performing cell-free translation of mRNA encoding the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27 to 40;and (b) recovering the polypeptide, or one which is substantially identical to said polypeptide, from the translation mixture. 42. The polypeptide, or one which is substantially identical to said polypeptide, produced by the method of claim 41. 43. A fusion protein comprising the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, fused to a heterologous polypeptide. 44. The polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, within a homodimer or a heterodimer. 45. The polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, which is glycosylated. CA 02184584 2001-03-14 -4046. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, in a conditioned medium for culturing vascular endothelial cells in vitro. 47. The use according to claim 46, wherein said polypeptide, or one which is substantially identical to said polypeptide, is added to said conditioned medium in a concentration from 10 pg/ml to 10 ng/ml. 48. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, in a conditioned medium for culturing lymphatic endothelial cells in vitro. 49. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, to stimulate the endothelial cell proliferation in a patient in need of such therapy. 50. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, to stimulate angiogenesis in a patient in need of such therapy. 51. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, in vascular graft surgery wherein the polypeptide stimulates the proliferation of endothelial cells in the patient. 52. The use according to claim 51, wherein said proliferation of endothelial cells stimulates vascularization. 53. The use of the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, to stimulate the proliferation of lymphatic endothelial cells in a patient in need of such therapy. CA 02184584 2001-03-14 -4154. The use of the polynucleotide, or one which is substantially identical to said polynucleotide, of any one of claims 1 to 15 to express a polypeptide, in vivo, which when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy. 55. A retroviral particle comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of claim 25. 56. A method for preparing a producer cell wherein the retroviral particle of claim 55 is used to transduce a packaging cell. 57. The method of claim 56, wherein said packaging cell is chosen from the group of cell lines comprising: PE501, PA317, ψ-2, ^-AM, PA12, T19-14X, VT-19-17-H2, ^GRE, ^CRIP, GP+E-86, GP+envAml2, and DAN. 58. A producer cell prepared by the method of claim 56, for the production of the retroviral particle of claim 55. 59. An adenovirus comprising the polynucleotide, or one which is substantially identical to said polynucleotide, of claim 24. 60. The use of the retroviral particle of claim 55 to transduce cells ex vivo, wherein said cells become capable of in vivo expression of a polypeptide with VEGF-2 activity. 61. The use of the retroviral particle of claim 55 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy. CA 02184584 2001-03-14 -4262. The use of the retroviral particle of claim 55 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy. 63. The use of the producer cell of claim 58 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy. 64. The use of the producer cell of claim 58 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy. 65. The use of the adenovirus of claim 59 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate endothelial cell proliferation in a patient in need of such therapy. 66. The use of the adenovirus of claim 59 to express in vivo a polypeptide, or one which is substantially identical to said polypeptide, which, when expressed will stimulate angiogenesis in a patient in need of such therapy. 67. The use according to any one of claims 49, 54, 61, 63 or 65, wherein said endothelial cell proliferation leads to angiogenesis. 68. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has a wound. 69. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has tissue damage. 70. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has bone damage. CA 02184584 2001-03-14 -4371. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has ischemia. 72. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has had a myocardial infarction. 73. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient has had coronary bypass surgery. 74. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient requires revascularization of damaged tissue. 75. The use according to any one of claims 49, 50, 54, 61, 62, 63, 64, 65, 66 or 67, wherein said patient requires growth of one or more chosen from the group comprising: bone;peridontium;and ligament tissue. 76. The use according to any one of claims 53, 54, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, wherein said patient is human. 77. A composition comprising a pharmaceutically acceptable earner and at least one polypeptide according to any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, or at least one polypeptide which is substantially identical to a polypeptide according to any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42. 78. A composition comprising a pharmaceutically acceptable carrier and at least one polynucleotide according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least one polynucleotide which is substantially identical to a polynucleotide according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. CA 02184584 2001-03-14 -4479, A composition according to claim 77 or 78, wherein said carrier is chosen from the group comprising: saline, buffered saline, dextrose, water, glycerol, ethanol and any combination thereof. 80. A composition according to claim 77 or 78, designed for administration by a convenient method chosen from the group comprising: topical, intravenous, intraperitoneal, intramuscular, intratumor, subcutaneous, intranasal and intradermal routes. 81. A pharmaceutical kit comprising: (a) one or more containers containing a composition according to claim 77 or 78;and optionally (b) a notice, in the form prescribed by a governmental agency regulating the manufacture, sale or use of pharmaceutical or biological products, which reflects approval by the agency of manufacture, use or sale for human administration. 82. A diagnostic kit for use with an antibody with specificity for the polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42, including more than one element selected from the list comprising: (a) a receptacle for sample collection;(b) a solid support for immobilization of any polypeptides present within said sample;(c) a contained amount of a blocking solution comprising a protein, a buffer and a preservative, wherein said protein will bind in any unoccupied sites of the solid support;(d) a contained amount of a wash solution comprising a buffer and a preservative;(e) a contained amount of a signal developing reagent whereby said reagent will react with said antibody to generate a signal as a positive result;(f) a contained amount of a standard solution of a polypeptide, or one which is substantially identical to said polypeptide, of any one of claims 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 42 in a buffer;and (g) instructions for use. CA 02184584 2001-03-14 -4583. An isolated polypeptide, or one which is substantially identical to said polypeptide, comprising amino acids +61 to +74 of SEQ ID NO:2, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies. 84. An isolated polypeptide, or one which is substantially identical to said polypeptide, that is a fragment of SEQ ID NO:2 comprising amino acids +38 to +118, wherein said polypeptide is used as an immunogen for the production of anti-VEGF-2 antibodies. WO 95/24473 PCT/US94/05291
Independent claims12