Receptors as novel inhibitors of vascular endothelial growth factor activity and processes for their production

• US 6,100,071 A
• Filed: 05/07/1996
• Issued: 08/08/2000
• Est. Priority Date: 05/07/1996
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Ferrara, Napoleone, Davis-Smyth, Terri Lynn, Presta, Leonard, Chen, Helen Hsifei
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US08/643,839
• Time in Patent Office: 1,554 Days
• Field of Search: 530/350, 530/387.3, 536/23.4, 536/23.1, 514/12, 435/69.1, 435/320.1, 435/325, 435/252.3, 435/254.11, 435/366, 435/69.7, 424/178.1
• US Class Current: 435/69.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

The present invention is directed to novel chimeric VEGF receptor proteins comprising amino acid sequences derived from the vascular endothelial growth factor (VEGF) receptors flt-1 and KDR, including the murine homologue to the human KDR receptor FLK-1, wherein said chimeric VEGF receptor proteins bind to VEGF and antagonize the endothelial cell proliferative and angiogenic activity thereof. The present invention is also directed to nucleic acids and expression vectors encoding these chimeric VEGF receptor proteins, host cells harboring such expression vectors, pharmaceutically acceptable compositions comprising such proteins, methods of preparing such proteins and to methods utilizing such proteins for the treatment of conditions associated with undesired vascularization.

Claims

1. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein comprising tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises an amino acid sequence selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) an amino acid sequence consisting essentially of the Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) an amino acid sequence consisting of the Ig-like domain 1 of the KDR tyrosine kinase receptor;

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises an amino acid sequence selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) an amino acid sequence consisting essentially of the Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) an amino acid sequence consisting essentially of the Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises an amino acid sequence selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) an amino acid sequence consisting essentially of the Ig-like domain 3 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) an amino acid sequence consisting essentially of the Ig-like domain 3 of the KDR tyrosine kinase receptor; and

wherein said VEGF receptor protein possesses at least one of (a), (b), (e), (f), (i) or (j) and at least one of (c), (d), (g), (h), (k) or (l).

2. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

1), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2).

3. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

1), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2).

4. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

1), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1).

5. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

2), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1).

6. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

2), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1).

7. The VEGF receptor protein according to claim 1, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO:

2), said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1) and said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2).

23. A composition of matter comprising a VEGF receptor protein according to any one of claims 1, 8, 15, 20, 21, or 22 compounded with a pharmaceutically acceptable carrier.

24. A nucleic acid encoding the VEGF receptor protein of any one of claims, 1, 8, 15, 20, 21, or 22.

25. A replicable expression vector capable in a transformed host cell of expressing the VEGF receptor protein of any one of claims 1, 8, 15, 20, 21, or 22.

26. Host cells transformed with the replicable expression vector according to claim 25.

27. Host cells according to claim 26 which are CEN4 cells.

28. A method of producing a VEGF receptor protein comprising the steps of introducing into a suitable expression system the expression vector of claim 25 and effecting the expression of said VEGF receptor protein.

29. A VEGF receptor protein of any one of claims 1, 8, 15, 20, 21, or 22 which is soluble.

30. A VEGF receptor protein of any one of claims 1, 8, 15 or 21 which is membrane bound.

8. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein consisting of tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 fused to a heterologous polypeptide,

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 is selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) an amino acid sequence consisting essentially of the Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) an amino acid sequence consisting essentially of the Ig-like domain 1 of the KDR tyrosine kinase receptor;

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) an amino acid sequence consisting essentially of the Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) an amino acid sequence consisting essentially of the Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 is selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) an amino acid sequence consisting essentially of the Ig-like domain 3 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) an amino acid sequence consisting of the Ig-like domain 3 of the KDR tyrosine kinase receptor.

9. The VEGF receptor protein according to claim 8, wherein said heterologous polypeptide is an immunoglobulin or fragment thereof.

10. The VEGF receptor protein according to claim 8, wherein said heterologous polypeptide is an immunoglobulin constant region.

11. The VEGF receptor protein according to claim 8, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 are all derived from the flt-1 tyrosine kinase receptor.

12. The VEGF receptor protein according to claim 8, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 are all derived from the KDR tyrosine kinase receptor.

13. The VEGF receptor protein according to claim 8, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1 and 3 are derived from the flt-1 tyrosine kinase receptor and said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is derived from the KDR tyrosine kinase receptor.

14. The VEGF receptor protein according to claim 8, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1 and 3 are derived from the KDR tyrosine kinase receptor and said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is derived from the flt-1 tyrosine kinase receptor.

15. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein consisting of tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3,

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 is selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) an amino acid sequence consisting essentially of the Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) an amino acid sequence consisting essentially of the Ig-like domain 1 of the KDR tyrosine kinase receptor);

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) an amino acid sequence consisting essentially of the Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) an amino acid sequence consisting essentially of the Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 is selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) an amino acid sequence consisting essentially of the Ig-like domain 3 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) an amino acid sequence consisting essentially of the Ig-like domain 3 of the KDR tyrosine kinase receptor.

16. The VEGF receptor protein according to claim 15, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 are all derived from the flt-1 tyrosine kinase receptor.

17. The VEGF receptor protein according to claim 15, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 are all derived from the KDR tyrosine kinase receptor.

18. The VEGF receptor protein according to claim 15, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1 and 3 are derived from the flt-1 tyrosine kinase receptor and said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is derived from the KDR tyrosine kinase receptor.

19. The VEGF receptor protein according to claim 15, wherein said tyrosine kinase receptor-derived immunoglobulin-like domains 1 and 3 are derived from the KDR tyrosine kinase receptor and said tyrosine kinase receptor-derived immunoglobulin-like domain 2 is derived from the flt-1 tyrosine kinase receptor.

20. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein comprising tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3, wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises an amino acid sequence selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the KDR tyrosine kinase receptor;

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises an amino acid sequence selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises an amino acid sequence selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) a functionally equivalent amino acid sequence variant of Ig-like domain 3 of the KDR tyrosine kinase receptor; and

wherein said VEGF receptor protein possesses at least one of (a), (b), (e), (f), (i) or (j) and at least one of (c), (d), (g), (h), (k) or (l), wherein said VEGF receptor protein possesses no more than one of (b), (d), (f), (h), (j) and (l), and wherein said functionally equivalent amino acid sequence variant is obtained by a single modification selected from the group consisting of a deletion of 1 to 7 residues, an insertion of 1 to 5 residues, and a substitution of 1 residue.

21. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein consisting of tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3 fused to a heterologous polypeptide,

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 comprises an amino acid sequence selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the KDR tyrosine kinase receptor;

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises an amino acid sequence selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises an amino acid sequence selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) a functionally equivalent amino acid sequence variant of Ig-like domain 3 of the KDR tyrosine kinase receptor; and

wherein said VEGF receptor protein possesses no more than one of (b), (d), (f), (h), (j) and (l), and wherein said functionally equivalent amino acid sequence variant is obtained by a single modification selected from the group consisting of a deletion of 1 to 7 residues, an insertion of 1 to 5 residues, and a substitution of 1 residue.

22. A VEGF receptor protein that is capable of binding to VEGF and thereby exerting an inhibitory effect thereon, said VEGF receptor protein consisting of tyrosine kinase receptor-derived immunoglobulin-like domains 1, 2 and 3,

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 1 is selected from the group consisting of:

(a) amino acids 32-128 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(b) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(c) amino acids 32-118 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(d) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the KDR tyrosine kinase receptor;

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 2 comprises an amino acid sequence selected from the group consisting of;

(e) amino acids 134-226 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(f) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the flt-1 tyrosine kinase receptor;

(g) amino acids 124-220 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(h) a functionally equivalent amino acid sequence variant of Ig-like domain 2 of the KDR tyrosine kinase receptor; and

wherein said tyrosine kinase receptor-derived immunoglobulin-like domain 3 comprises an amino acid sequence selected from the group consisting of;

(i) amino acids 232-331 of the flt-1 tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

1);

(j) a functionally equivalent amino acid sequence variant of Ig-like domain 1 of the flt-1 tyrosine kinase receptor;

(k) amino acids 226-327 of the KDR tyrosine kinase receptor shown in FIG. 1 (SEQ ID NO;

2); and

(l) a functionally equivalent amino acid sequence variant of Ig-like domain 3 of the KDR tyrosine kinase receptor; and

wherein said VEGF receptor protein possesses no more than one of (b), (d), (f), (h), (j) and (l), and wherein said functionally equivalent amino acid sequence variant is obtained by a single modification selected from the group consisting of a deletion of 1 to 7 residues, an insertion of 1 to 5 residues, and a substitution of 1 residue.