Recombinant proteins containing Shiga-like toxin and vascular endothelial growth factor fragments

• US 20040166565A1
• Filed: 01/27/2004
• Published: 08/26/2004
• Est. Priority Date: 03/22/2000
• Status: Active Grant

• Current Assignee: Sibtech Incorporated
• Original Assignee: Sibtech Incorporated
• Inventors: Backer, Marina V., Backer, Joseph M.
• Granted Patent: US 7,267,973 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• 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 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention is directed to an isolated nucleic acid encoding a fusion protein comprising (1) the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof; and (2) human vascular endothelial growth factor, or a truncated or mutated version thereof; wherein the fusion protein possesses ribosome inactivating activity and ability to bind to cellular VEGF receptors. The present invention is also directed to polypeptides the above combination of toxin and growth factor, as well as expression vectors and transformed cells incorporating the above nucleic acid. The invention is also directed to pharmaceutical compositions and methods for treating patients suffering from diseases relating to angiogenesis.

Claims

1. An isolated nucleic acid encoding a fusion protein comprising:

(1) the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof, said subunit having the nucleic acid sequence of SEQ ID NO;

9, or a truncated or mutated version thereof; and

(2) human vascular endothelial growth factor, or a truncated or mutated version thereof, said growth factor having the nucleic acid sequence of SEQ ID NO;

10, or a truncated or mutated version thereof;

wherein said fusion protein possesses ribosome inactivating activity.

2. The isolated nucleic acid of claim 1, wherein said fusion protein specifically binds to vascular endothelial growth factor receptors.

3. The isolated nucleic acid of claim 2, wherein said fusion protein is capable of being internalized by a cell which expresses said receptors.

4. The isolated nucleic acid of claim 3, wherein said internalization occurs by endocytosis.

5. The isolated nucleic acid of claim 1, wherein said isolated nucleic acid has the nucleic acid sequence of SEQ ID NO:

11.

6. An isolated polypeptide comprising:

(1) the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof, wherein said subunit has the nucleic acid sequence of SEQ ID NO;

9 or a truncated or mutated version thereof; and

(2) human vascular endothelial growth factor, or a truncated or mutated version thereof, wherein said growth factor has the nucleic acid sequence of SEQ ID NO;

10, or a truncated or mutated version thereof;

wherein said isolated polypeptide possesses ribosome inactivating activity.

7. The isolated polypeptide of claim 6, wherein said isolated polypeptide specifically binds vascular endothelial growth factor receptors.

8. The isolated polypeptide of claim 7, wherein said isolated polypeptide is internalized by a cell which expresses said receptors.

9. The isolated polypeptide of claim 6, wherein said internalization occurs by endocytosis.

10. The isolated polypeptide of claim 6, wherein said isolated polypeptide is encoded by the nucleic acid of SEQ ID NO:

11.

11. An expression vector, comprising:

(1) a nucleic acid encoding a fusion protein comprising the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof; and

human vascular endothelial growth factor, or a truncated or mutated version thereof; and

(2) a promoter sequence operably linked to said nucleic acid to allow expression of said nucleic acid;

said expression vector comprising the nucleic acid sequence of SEQ ID NO;

11.

12. The expression vector of claim 11, wherein said fusion protein is capable of specifically binding to vascular endothelial growth factor receptors.

13. The expression vector of claim 12, wherein said fusion protein is internalized by a cell which expresses said receptors.

14. A bacterial cell transformed with the expression vector of claim 11.

15. A method of inactivating ribosomes in a cell, comprising the steps of:

(a) contacting a cell with a polypeptide comprising;

(1) the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof, said subunit having the nucleic acid sequence of SEQ ID NO;

9, or a truncated or mutated version thereof; and

(2) human vascular endothelial growth factor, or a truncated or mutated version thereof, said growth factor having the nucleic acid sequence of SEQ ID NO;

10, or a truncated or mutated version thereof;

under conditions which permit said polypeptide to be internalized into said cell and inactivate ribosomes in said cell.

16. The method of claim 15, wherein said fusion protein specifically binds vascular endothelial growth factor receptors.

17. The method of claim 16, wherein said fusion protein is internalized by a cell which expresses said receptors.

18. A composition for inhibiting endothelial cell growth in a patient, comprising:

(A) a fusion protein comprising the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof; and

human vascular endothelial growth factor, or a truncated or mutated version thereof, said fusion protein possessing ribosome inactivating activity; and

(B) a pharmaceutically acceptable carrier;

wherein said A subunit of said Shiga-like bacterial toxin, or a truncated or mutated version thereof, is encoded by the nucleic acid sequence of SEQ ID NO;

9, and wherein said human vascular endothelial growth factor, or a truncated or mutated version thereof, is encoded by the nucleic acid sequence of SEQ ID NO;

11.

19. A method of treating a patient suffering from a pathophysiological condition that depends on angiogenesis, comprising:

providing to said patient an effective amount of a composition comprising a fusion protein comprising the A subunit of Shiga-like bacterial toxin, or a truncated or mutated version thereof; and

human vascular endothelial growth factor, or a truncated or mutated version thereof, said fusion protein possessing ribosome inactivating activity; and

a pharmaceutically acceptable carrier;

wherein said A subunit of said Shiga-like bacterial toxin, or a truncated or mutated version thereof, is encoded by the nucleic acid sequence of SEQ ID NO;

9, and wherein said human vascular endothelial growth factor, or a truncated or mutated version thereof, is encoded by the nucleic acid sequence of SEQ ID NO;

11.

20. The method of claim 19, wherein said method is used in combination with other treatments for said pathophysiological condition.

21. The method of claim 19, wherein the efficacy of said treatment is enhanced by decrease in oxygen or nutrient supplies that would arise from damage to endothelium caused by said protein and pharmaceutical compositions.

22. An isolated nucleic acid comprising SEQ ID NO:

9 and SEQ ID NO;

11.

23. A fusion protein encoded by a nucleic acid sequence comprising SEQ ID NO:

9 and SEQ ID NO;

11.

24. An isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO:

11.

25. A fusion protein encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO:

11.