Chelating polymers for labeling of proteins

• US 6,203,775 B1
• Filed: 03/19/1993
• Issued: 03/20/2001
• Est. Priority Date: 03/19/1993
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Trubetskoy, Vladimir S., Torchilin, Vladimir P.
• Primary Examiner(s): Dees, José G.
• Assistant Examiner(s): JONES, DAMERON LEVEST
• Application Number:
  US08/034,912
• Time in Patent Office: 2,923 Days
• Field of Search: 424/1.11, 424/9, 424/85.8, 424/85.91, 424/1.41, 424/1.45, 424/1.53, 424/1.57, 424/1.69, 424/9.34, 534/10, 534/14, 534/15, 534/16, 530/391.3, 530/391.5, 530/391.7, 530/391.9
• US Class Current: 424/1.69
• CPC Class Codes: C07K 1/13 Labelling of peptides

Abstract

A method of labelling a protein with a metal involving chelating a metal to a compound, with a molecular weight of at least 1,000, and a reactive group capable of forming a covalent bond with a target protein, and conjugating the metallically-labelled compound to a protein.

Claims

1. A method for labelling a protein with a metal comprising:

(a) providing a synthetic polypeptide to which a metal is chelated, said polypeptide having a molecular weight of at least 1,000 daltons, being purified from unchelated metal, and comprising a reactive group;

(b) providing a protein comprising a sulfhydryl group which is either naturally occurring in said protein or the product of chemical modification; and

(c) reacting said polypeptide with said protein to form a covalent bond at a single site therebetween via said reactive group and said sulfhydryl group to yield a metallically-labelled protein substantially free of unchelated metal.

2. The method of claim 1, wherein said metal is a radioisotope.

3. The method of claim 1, wherein said metal is paramagnetic.

4. The method of claim 1, wherein said metal is chelated at multiple sites to said polypeptide.

5. The method of claim 1, wherein said polypeptide is poly-L-lysine.

6. The method of claim 1, wherein said reactive group is a thiol-reactive group.

7. The method of claim 6, wherein said thiol-reactive group is a maleimido group.

8. The method of claim 1, wherein said protein is an antibody.

9. The method of claim 8, wherein said antibody is an monoclonal antibody.

10. The method of claim 9, wherein said monoclonal antibody is an Fab′

fragment.

11. A method of making a metallically-labelled complex having a molecular weight of at least 1,000 daltons, said method comprising:

(a) providing a synthetic polypeptide, comprising a reactive group, which polypeptide covalently bonds to a protein at a single site via said reactive group, and further comprising a chelating group; and

(b) contacting the polypeptide of (a) with a metal to cause chelation therewith.

12. The method of claim 11, wherein said metal is a radioisotope.

13. The method of claim 11, wherein said polypeptide is a poly-L-lysine.

14. The method of claim 11, wherein said reactive group is a thiol-reactive group.

15. The method of claim 14, wherein said thiol-reactive group is a maleimido group.

16. The method of claim 11, wherein said chelating group is at multiple sites.

17. The method of claim 16, wherein said chelating group is DTPA.

18. A synthetic polypeptide to which a metal is chelated, having a molecular weight of at least 1,000 daltons, being purified from unchelated metal, comprising a reactive group, which polypeptide covalently bonds to a protein at a single site via said reactive group.

19. The polypeptide of claim 18, wherein said metal is a radioisotope.

20. The polypeptide of claim 18, wherein said protein is an antibody.

21. The polypeptide of claim 20, wherein said antibody is an monoclonal antibody.

22. The polypeptide of claim 21, wherein said monoclonal antibody is an Fab′

fragment.

23. A metal-labelled complex, comprising a synthetic polypeptide to which a metal is chelated, having a molecular weight of at least 1,000 daltons, being purified from unchelated metal, and being covalently bonded to a protein at a single site, said protein having substantially no unchelated metal associated therewith.

24. The complex of claim 23, wherein said metal is a radioisotope.

25. The complex of claim 23, wherein said protein is an antibody.

26. The complex of claim 25, wherein said antibody is an monoclonal antibody.

27. The complex of claim 26, wherein said monoclonal antibody is an Fab′

fragment.

28. A method of labelling cells in a mammal, comprising administering to a mammal the compound of claim 23, wherein said protein specifically binds to said cells.

29. The method of claim 28, wherein said metal is Gd.

30. The method of claim 28, wherein said metal is a radioisotope.

31. The method of claim 30, wherein said radioisotope is ¹¹¹-In.

32. The method of claim 30, wherein said radioisotope is ^99m-Tc.

33. A method of killing unwanted cells in a mammal, said method comprising administering to a mammal the compound of claim 24, wherein said radioisotope is cytotoxic, and wherein said protein specifically binds to said cells.

34. The method of claim 33, wherein said tissue is a tumor.

35. The method of claim 33, wherein said radioisotope is ¹⁸⁶-Re.

36. The method of claim 33, wherein said radioisotope is ¹⁸⁸-Re.