Fusion protein capable of binding VEGF

• US 7,087,411 B2
• Filed: 06/30/2003
• Issued: 08/08/2006
• Est. Priority Date: 06/08/1999
• Status: Expired due to Term

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Papadopoulos, Nicholas J., Daly, Thomas J., Fandl, James P.
• Primary Examiner(s): Andres, Janet L.
• Assistant Examiner(s): Howard, Zachary
• Application Number:
  US10/609,775
• Publication Number:
  US 20040014667A1
• Time in Patent Office: 1,135 Days
• Field of Search: None
• 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 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Nucleic acid molecules and multimeric proteins capable of binding vascular endothelial growth factor (VEGF). VEGF mini-traps are disclosed which are therapeutically useful for treating VEGF-associated conditions and diseases, and are specifically designed for local administration to specific organs, tissues, and/or cells.

Claims

1. An isolated nucleic acid molecule encoding a fusion polypeptide capable of binding vascular endothelial growth factor (VEGF) consisting of components (R1R2)_X, and a multimerizing component (MC) capable of interacting with another MC to form a multimeric structure, wherein X≧

1, R1 is VEGF receptor component Ig domain 2 of Flt-1 consisting of amino acids 27–

126 of SEQ ID NO;

8 or 27–

129 of SEQ ID NO;

10, R2 is Ig domain 3 of Flk-1 consisting of amino acids 127–

228 of SEQ ID NO;

8 or 130–

231 of SEQ ID NO;

10, and MC is an amino acid sequence between 1 to about 200 amino acids in length having at least one cysteine residue.

2. The nucleic acid molecule of claim 1, encoding an amino acid sequence selected from the group consisting of SEQ ID NO:

2, 5, 23 and 25.

3. A replicable expression vector comprising a nucleic acid molecule encoding a fusion protein which binds vascular endothelial growth factor (VEGF), wherein the fusion protein consists of a first receptor component, a second receptor component, and a multimerizing component, wherein the first receptor component is amino acids 27–

126 of SEQ ID NO;

8 or 27–

129 of SEQ ID NO;

10, the second receptor component is amino acids 127–

228 of SEQ ID NO;

8 or 130–

231 of SEQ ID NO;

10, and the multimerizing component is an amino acid sequence between 1 to about 200 amino acids having at least one cysteine residue.

4. A method of producing a VEGF fusion protein comprising the step of introducing the expression vector of claim 3 into an isolated host cell, growing the cell under conditions permitting production of the fusion protein and recovering the fusion protein so produced.

5. An isolated nucleic acid molecule encoding a fusion polypeptide which binds vascular endothelial growth factor (VEGF), consisting of a first receptor component, a second receptor component, and a multimerizing component, wherein the first receptor component is amino acids 27–

126 of SEQ ID NO;

8 or 27–

129 of SEQ ID NO;

10, the second receptor component is amino acids 127–

228 of SEQ ID NO;

8 or 130–

231 of SEQ ID NO;

10, and the multimerizing component is an amino acid sequence between 1 to about 200 amino acids having at least one cysteine residue.

6. The isolated nucleic acid molecule of claim 5, wherein the fusion polypeptide is selected from the group consisting of SEQ ID NO:

2, 5, 23 and 25.

7. A fusion polypeptide encoded by the nucleic acid molecule of claim 5.

8. The fusion polypeptide of claim 7, wherein the components are connected directly to each other or via one or more spacer sequences.

9. A vascular endothelial cell growth factor (VEGF) trap, comprising a multimer of two or more fusion polypeptides of claim 7.

10. The VEGF trap of claim 9 which is a dimer.

11. The VEGF trap of claim 10, wherein the MC is a cysteine residue and the cysteine residue of a first fusion polypeptide forms a covalent disulfide bond with the cysteine residue of a second fusion polypeptide.

12. A composition comprising the VEGF trap of claim 11 and a pharmaceutically acceptable carrier.

13. The VEGF trap of claim 10, wherein the MC is four amino acids and comprises at least one cysteine residue.

14. A vascular endothelial growth factor (VEGF) trap, comprising a dimer which binds VEGF consisting of two fusion polypeptides, each fusion polypeptide consisting of a first receptor component, a second receptor component, and a multimerizing component (MC), wherein the first receptor component is amino acids 27–

126 of SEQ ID NO;

8 or 27–

129 of SEQ ID NO;

10, the second receptor component is amino acids 127–

228 of SEQ ID NO;

8 or 130–

231 of SEQ ID NO;

10, and wherein the MC is XCXC (SEQ ID NO;

3).

15. A vascular endothelial growth factor (VEGF) trap, comprising a dimer which binds VEGF consisting of two fusion polypeptides, each fusion polypeptide consisting of a first receptor component, a second receptor component, and a multimerizing component (MC), wherein the first receptor component is amino acids 27–

126 of SEQ ID NO;

8 or 27–

129 of SEQ ID NO;

10, the second receptor component is amino acids 127–

228 of SEQ ID NO;

8 or 130–

231 of SEQ ID NO;

10, and wherein the MC is ACGC (SEQ ID NO;

4).

16. A composition comprising the VEGF trap of claim 15 and a pharmaceutically acceptable carrier.