Preparation of a polymer/interleukin-2 conjugate

• US 4,902,502 A
• Filed: 01/23/1989
• Issued: 02/20/1990
• Est. Priority Date: 01/23/1989
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Aldwin, Lois, Katre, Nandini, Nitecki, Danute E., Goodson, Robert J.
• Primary Examiner(s): Schofer, Joseph L.
• Assistant Examiner(s): Pili-Curtis, Carmen B.
• Application Number:
  US07/299,235
• Time in Patent Office: 393 Days
• Field of Search: 424/78, 424/83, 424/85, 530/351, 514/2
• US Class Current: 530/351
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Abstract

The present invention is a process for preparing a pharmaceutical composition comprising a biologically active conjugated protein. It comprises a polyethylene glycol or a polyoxyethylated polyol conjugated to IL-2. This protein is conjugated to reduce its immunogenicity, and increase it solubility, and increase its circulating in vivo half-life.

Claims

1. A method for producing a PEG/IL-2 conjugate or a polyoxyethylated polyol/IL-2 conjugate, comprising:

a. contacting a PEG or a polyoxyethylated polyol, which has at least one hydroxyl group, with a chloroformate under the appropriate reaction conditions to form a PEG or a polyoxyethylated polyol active ester; and

b. contacting the PEG or the polyoxyethylated polyol active ester with IL-2 under the appropriate reaction conditions to form a PEG/IL-2 conjugate or a polyoxyethylated polyol/IL-2 conjugate.

2. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol active ester is contacted with the IL-2 in the molar ratio of 1 to 20 moles active esters to one mole IL-2.

3. A method in accordance with claim 1, wherein the active ester is contacted with the IL-2 in the molar ratio of 6 to 16 moles active esters to one mole IL-2.

4. A method in accordance with claim 1, wherein the active ester is contacted with the IL-2 in the molar ratio of 10 to 14 moles active esters to one mole IL-2.

5. A method in accordance with claim 1 wherein the conjugate has a molar ratio of from 1 to 3 PEGs or polyoxyethylated polyols per one IL-2.

6. A method in accordance with claim 1, wherein the conjugate has a molar ratio of from 2 to 3 PEGs or polyoxyethylated per one IL-2.

7. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 1000 and 40,000.

8. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol has a average molecular weight between 2000 and 20,000.

9. A method in accordance with claim 1, wherein the PEG or the polyoxyethylated polyol has an averaage molecular weight between 3,000 and 12,000.

10. A method in accordance with claim 4 wherein the polymer is PEG.

11. A method in accordance with claim 4 wherein the polymer is POG.

12. A method in accordance with claim 1, wherein the IL-2 is a mutein.

13. A method in accordance with claim 12, wherein the IL-2 mutein which has up to six N-terminal deletions and the cysteine at position 125 is replaced by serine.

14. A method in accordance with claim 1, wherein the chloroformate is para-nitrophenyl chloroformate or, ortho-nitrophenyl chloroformate.

15. A method in accordance with claim 1, wherein the PEG or polyoxyethylated polyol is covalently conjugated to the IL-2 via a urethane bond.

16. A method for producing a PEG/IL-2 conjugate, comprising:

a. contacting PEG, which has at least one hydroxyl group, with a chloroformate under the appropriate reaction conditions to form a PEG active ester; and

b. contacting the PEG active ester with IL-2 under the appropriate reaction conditions to form a PEG/IL-2 conjugate.

17. A method in accordance with claim 16, wherein the PEG active ester is contacted with the IL-2 in the molar ratio of 10 to 14 moles PEG esters to one mole IL-2.

18. A method in accordance with claim 16, wherein the conjugate has a molar ratio of between 1 and 3 PEGs per IL-2.

19. A method in accordance with claim 16, wherein the PEG has a average molecular weight between 2000 and 20,000.

20. A method in accordance with claim 16, wherein the PEG has a molecular weight between 3,000 to 12,000.

21. A method in accordance with claim 16, wherein the IL-2 is a mutein.

22. A method in accordance with claim 21, wherein IL-2 mutein which has up to six N-terminal deletions and the cysteine at position 125 is replaced by serine.

23. A method in accordance with claim 16, wherein the chloroformate is para-nitrophenyl chloroformate or, ortho-nitrophenyl chloroformate.

24. A method in accordance with claim 16, wherein the PEG is covalently conjugated to the IL-2 via a urethane bond.

25. A method for producing a PEG/IL-2 conjugate, comprising contacting PEG, having a terminal hydroxy group, with para-nitrophenyl chloroformate under the appropriate reaction conditions to form a PEG active ester;

and contacting the PEG active ester with des-ala ser₁₂₅, IL-2 to form a PEG/IL-2 conjugate.