VEGFR-3 ligand binding molecules and uses thereof
View Patent ↗The present invention is directed to VEGFR-3 ligand binding molecules and uses thereof to modulate angiogenesis and/or lymphangiogenesis. A glycosylation sequon of wildtype VEGFR-3 has been modified to eliminate glycosylation in the ligand binding molecules.
1. A purified or isolated, soluble, ligand binding polypeptide comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 47-115 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T,
wherein the polypeptide binds to at least one ligand polypeptide selected from human VEGF-C, VEGF-D, and PIGF.
2. The purified or isolated, soluble, ligand binding polypeptide according to claim 1 , comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 47-210 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T.
3. The purified or isolated, soluble, ligand binding polypeptide according to claim 1 , comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 47-314 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T.
4. The purified or isolated, soluble, ligand binding polypeptide according to claim 1 , comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 25-314 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T.
5. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 that retains four N-glycosylation sequon sites corresponding to positions 33-35 of SEQ ID NO: 2, positions 166-168 of SEQ ID NO: 2, positions 251-253 of SEQ ID NO: 2, and positions 299-301 of SEQ ID NO: 2.
6. The purified or isolated, soluble, ligand binding polypeptide according to claim 5 , that is glycosylated at said four N-glycosylation sequon sites.
7. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 that binds human VEGF-C with a K d or 1 nM or less.
8. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 , that binds human VEGF-D with a K d of 5 nM or less.
9. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 , wherein the amino acid in the polypeptide corresponding to position 104 of SEQ ID NO: 2 is deleted or replaced with another amino acid.
10. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 , wherein the amino acid corresponding to position 104 of SEQ ID NO: 2 is deleted or replaced with another amino acid selected from the group consisting of glutamine, aspartate, glutamate, arginine, and lysine.
11. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 , further comprising a signal peptide.
12. The purified or isolated, soluble, ligand binding polypeptide according to claim 4 , further comprising at least one polyethylene glycol moiety attached to the polypeptide.
13. The purified or isolated, soluble, ligand binding polypeptide according to claim 12 , comprising polyethylene glycol of about 20-40 kDa attached to the amino terminus of the polypeptide.
14. A soluble, ligand binding molecule comprising the soluble, ligand binding polypeptide according to claim 4 connected to a heterologous peptide.
15. The soluble, ligand binding molecule according to claim 14 , wherein the heterologous peptide comprises an immunoglobulin constant domain fragment.
16. The soluble, ligand binding molecule according to claim 15 , wherein the immunoglobulin constant fragment comprises amino acids 306-537 of SEQ ID NO: 3.
17. The soluble, ligand binding molecule according to claim 15 , wherein the immunoglobulin constant domain fragment is an IgG constant domain fragment.
18. The soluble, ligand binding molecule according to claim 14 , optionally comprising a linker connecting the heterologous peptide to the soluble, ligand binding polypeptide.
19. The soluble, ligand binding molecule according to claim 14 that comprises a polypeptide in which a C-terminal amino acid of the soluble, ligand binding polypeptide is directly attached to an N-terminal amino acid of the heterologous peptide by a peptide bond.
20. The soluble, ligand binding molecule according to claim 14 , further comprising a signal peptide that directs secretion of the molecule from a cell that expresses the molecule.
21. The soluble, ligand binding molecule according to claim 14 , wherein the molecule comprises the amino acid sequence set forth in SEQ ID NO: 3.
22. The soluble, ligand binding molecule according to claim 14 , wherein the ligand binding polypeptide and the heterologous peptide are linked by amide bonding to form a single polypeptide chain.
23. The soluble, ligand binding molecule according to claim 14 further comprising a detectable label.
24. A conjugate comprising the soluble, ligand binding molecule according to claim 14 and a chemotherapeutic agent.
25. The soluble, ligand binding polypeptide according to claim 4 further comprising a detectable label.
26. A conjugate comprising the soluble, ligand binding polypeptide according to claim 4 and a chemotherapeutic agent.
27. A composition comprising a purified or isolated, soluble, ligand binding polypeptide or ligand binding molecule according to claim 4 and a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier.
28. The composition according to claim 27 , that is formulated for topical administration.
29. The composition according to claim 28 , that is in the form of a solid, a paste, an ointment, a gel, a liquid, an aerosol, a mist, a polymer, a film, an emulsion, or a suspension.
30. The composition according to claim 27 , that is formulated for intravitreal administration.
31. A method of inhibiting neovascularization in a subject, the method comprising administering to the subject a composition according to claim 27 in an amount effective to inhibit neovascularization in the subject.
32. The method according to claim 31 , wherein the subject has been diagnosed with a tumor, and wherein the composition is administered in an amount effective to inhibit neovascularization in the tumor.
33. The method according to claim 32 , wherein the composition is administered locally to the tumor or to the organ or tissue from which the tumor has been surgically removed.
34. The method according to claim 32 , wherein the composition is administered in an amount effective to inhibit VEGF-C and/or VEGF-D in the tumor of the subject from binding to or stimulating VEGFR-2 and/or VEGFR-3 expressed in tumor cells.
35. A method of inhibiting choroidal or retinal neovascularization in a subject, the method comprising administering to the subject a composition according to claim 27 , in an amount effective to inhibit retinal neovascularization in the subject.
36. The method according to claim 35 , wherein the composition is administered locally to the eye of the subject.
37. The method according to claim 36 , wherein the composition is administered by intravitreal injection.
38. The method according to claim 36 , wherein the composition is administered by intravitreal implant.
39. The method according to claim 36 , wherein the composition is administered by topical administration.
40. The method according to claim 35 , wherein the composition is administered in an amount effective to inhibit VEGF-C and/or VEGF-D in the eye of the subject from binding to or stimulating VEGFR-2 and/or VEGFR-3 expressed in cells of the eye or vessels of the eye.
41. The method according to claim 35 , further comprising administering an antibiotic to the subject.
42. The method according to claim 41 , wherein the antibiotic is selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, clozacillin, dicloxacillin, flucoxacillin, mezlocillin, nafcillin, penicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, soxycycline, minocycline, oxytetracycline, and tetracycline.
43. A method of treating a subject having an ocular disorder associated with retinal neovascularization, the method comprising administering to the subject a composition according to claim 27 , in an amount effective to inhibit retinal neovascularization in the subject.
44. The method of claim 43 , wherein the ocular disorder is selected from the group consisting of macular degeneration, diabetic retinopathy and macular telangiectasia.
45. The purified or isolated, soluble, ligand binding polypeptide according to claim 1 , comprising an amino acid sequence that is identical to the sequence of amino acids defined by positions 47-115 of SEQ ID NO: 2, positions 47-210 of SEQ ID NO: 2, positions 47-314 of SEQ ID NO: 2, or positions 47-752 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T.
46. The purified or isolated, soluble, ligand polypeptide according to claim 1 , wherein the polypeptide comprises amino acids 23-290 of SEQ ID NO: 3.
47. The purified or isolated, soluble, ligand binding polypeptide according to claim 1 , comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 25-752 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T.
48. A purified or isolated, ligand binding polypeptide comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 25-314 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T,
wherein the polypeptide lacks VEGFR-3 Ig-like domains 4-7, lacks a VEGFR-3 transmembrane domain, and lacks a VEGFR-3 intracellular domain, and
wherein the polypeptide binds human VEGF-C or human VEGF-D.
49. A purified or isolated, soluble, ligand binding polypeptide comprising an amino acid sequence having at least 95% identity to the sequence of amino acids defined by positions 25-314 of SEQ ID NO: 2, with the proviso that positions of the polypeptide corresponding to positions 104-106 of SEQ ID NO: 2 are not identical to N-X-S or N-X-T,
wherein the polypeptide binds human VEGF-C or human VEGF-D and inhibits VEGF-C- or VEGF-D-binding to VEGFR-3 or inhibits VEGF-C- or VEGF-D-mediated stimulation of VEGFR-3 in a cell expressing VEGFR-3 on its surface.