Biologically active B-chain homodimers

• US 5,516,896 A
• Filed: 07/13/1989
• Issued: 05/14/1996
• Est. Priority Date: 02/25/1985
• Status: Expired due to Term

• Current Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Inventors: Kelly, James D., Murray, Mark J.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/381,096
• Time in Patent Office: 2,497 Days
• Field of Search: 530/350, 530/324, 530/399, 530/397, 530/380, 530/356, 514/12, 435/69.4
• US Class Current: 530/399
• CPC Class Codes: A61K 35/16 Blood plasma; Blood serum umbilical cord blood A61K35/51
• 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: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 2740/11022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: Y10S 930/12 Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides
• CPC Class Codes: Y10S 930/221 Retrovirus related, or human immunodeficiency virus related, or simian immunodeficiency virus related

Abstract

Dimeric proteins having substantially the same biological activity as PDGF are disclosed. More specifically, the protein may have two substantially identical polypeptide chains, each of the chains being substantially homologous to the B-chain of PDGF. Alternatively, the protein may have two polypeptide chains that are substantially identical to the B-chain of PDGF. In addition, proteins comprising polypeptides that are variants or derivatives of the B-chain of PDGF are also disclosed. Therapeutic compositions containing these proteins and methods for enhancing the wound-healing process in warm-blooded animals are also disclosed.

Claims

1. A protein having two PDGF-B polypeptide chains, said chains having one or more of the following modifications:

(a) one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain;

(b) a deletion of 1 to 14 amino acids at the N-terminus of the protein; and

(c) a deletion of 1 to 9 amino acids at the C-terminus of the protein.

2. The protein of claim 1 wherein the cysteine residue corresponding to residue 43 is changed to serine.

3. The protein of claim 1 wherein the cysteine residue corresponding to residue 52 is changed to serine.

4. The protein of claim 1 wherein the cysteine residue corresponding to residue 53 is changed to serine.

5. The protein of claim 1 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 109, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

6. The protein of claim 1 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

7. The protein of claim 1 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 1 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

8. The protein according to claim 5, 6, or 7, further comprising one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

9. A therapeutic composition comprising a protein having two PDGF-B polypeptide chains, said chains having one or more of the following modifications:

(a) one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain;

(b) a deletion of 1 to 14 amino acids at the N-terminus of the protein; and

(c) a deletion of 1 to 9 amino acids at the C-terminus of the protein, said protein being chemotactic or mitogenic for fibroblasts, and a physiologically acceptable carrier or diluent.

10. The therapeutic composition of claim 9 wherein the cysteine residue corresponding to residue 43 is changed to serine.

11. The therapeutic composition of claim 9 wherein the cysteine residue corresponding to residue 52 is changed to serine.

12. The therapeutic composition of claim 9 wherein the cysteine residue corresponding to residue 53 is changed to serine.

13. The therapeutic composition of claim 9 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 109, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

14. The therapeutic composition of claim 9 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

15. The therapeutic composition of claim 9 wherein polypeptide chains comprise the B-chain of PDGF from amino acid 1 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

16. The therapeutic composition according to claim 13, 14, or 15, further comprising one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

17. The therapeutic composition of claim 9 wherein said protein is present in a concentration of from about 1 to 50 micrograms per milliliter of total volume.

18. The therapeutic composition of claim 9 wherein said carrier or diluent is selected from the group consisting of albumin, sterile water, and saline.

19. The therapeutic composition of claim 18, including an adjuvant.

20. The therapeutic composition of claim 19 wherein said adjuvant is selected from the group consisting of collagen, hyaluronic acid, fibronectin, factor XIII, and an antibiotic.

21. A method for enhancing the wound-healing process in warm-blooded animals, comprising administering to the animal a therapeutically effective amount of a protein having two PDGF-B polypeptide chains, said chains having one or more of the following modifications:

(a) one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain;

(b) a deletion of 1 to 14 amino acids at the N-terminus of the protein; and

(c) a deletion of 1 to 9 amino acids at the C-terminus of the protein, said protein being chemotactic and mitogenic for fibroblasts, and a physiologically acceptable carrier or diluent.

22. The method of claim 21 wherein the cysteine residue corresponding to residue 43 is changed to serine.

23. The method of claim 21 wherein the cysteine residue corresponding to residue 52 is changed to serine.

24. The method of claim 21 wherein the cysteine residue corresponding to residue 53 is changed to serine.

25. The method of claim 21 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 109, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

26. The method of claim 21 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 15 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

27. The method of claim 21 wherein said polypeptide chains comprise the B-chain of PDGF from amino acid 1 to amino acid 101, optionally having one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

28. The method according to claim 25, 26, or 27, further comprising one or more amino acid substitutions of cysteine residues corresponding to residues 43, 49, 52, and 53 of the PDGF B chain.

29. The method of claim 21 wherein said protein is present in a concentration of from about 1 to 50 micrograms per milliliter of total volume.

30. The method of claim 21 wherein said carrier or diluent is selected from the group consisting of albumin, sterile water, and saline.

31. The method of claim 21 wherein said protein and physiologically acceptable carrier or diluent is administered topically.

32. The method of claim 21, including administering an adjuvant.

33. The method of claim 32 wherein said adjuvant is selected from the group consisting of collagen, hyaluronic acid, fibronectin, factor XIII, and antibiotics.