Preparation and use of sialylated glycoforms of soluble complement receptor 1 (CR1)

• US 6,057,131 A
• Filed: 11/05/1998
• Issued: 05/02/2000
• Est. Priority Date: 08/07/1992
• Status: Expired due to Fees

• Current Assignee: AVANT Immunotherapeutics, Inc. (Celldex Therapeutics Incorporated)
• Original Assignee: AVANT Immunotherapeutics, Inc. (Celldex Therapeutics Incorporated)
• Inventors: Freeman, Anne Mary, Lifter, John, Gosselin, Michael L., Smith, Richard A. G., Yeh, Chang-Jing Grace, Marsh, Henry C. Jr.
• Primary Examiner(s): Fitzgerald, David L.
• Application Number:
  US09/186,827
• Time in Patent Office: 544 Days
• Field of Search: 514/8, 530/412, 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/166 Streptokinase
• CPC Class Codes: A61K 38/49 Urokinase; Tissue plasminogen activator
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• 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 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

Provided are methods of making preparations of recombinant soluble Complement Receptor type 1 (sCR1) defined with respect to the distribution of sCR1 glycoforms and therapeutic methods using them. The preparations are suitable for treatment of allograft or xenograft rejection, diseases involving inflammation or inappropriate complement activation, and thrombotic or shock state conditions. Preferred methods employ sCR1 glycoforms that are sialylated, have a pI of ≦5.1, or have a sialic acid: mannose molar ratio of ≧0.25.

Claims

1. A method of delaying transplant rejection in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of sCR1 glycoprotein molecules, in which said composition exhibits dominant isoforms of sCR1 glycoprotein molecules which have an isoelectric point, pI, less than or equal to 5.1 as determined by chromatofocusing, and wherein the pI of said dominant isoforms increases after neuraminidase treatment.

9. The method according to claim 1, 2, 3, 4, or 5 in which the transplant is a xenograft.

10. The method according to claim 1, 2, 3, 4, or 5 which the transplant is an allograft.

11. The method of claim 1, 2, 3, 4, or 5 wherein said sCR1 glycoprotein molecules comprise the 30 extracellular short consensus repeats of human CR1.

2. A method of delaying transplant rejection in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of sCR1 glycoprotein molecules, wherein substantially all of the sCR1 glycoprotein molecules in said composition contain one or more complex oligosaccharide structures, wherein at least about 40% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

3. The method of claim 2, wherein at least about 70% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

4. A method of delaying transplant rejection in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of sCR1 glycoprotein molecules, in which 70% of all of said sCR1 glycoprotein molecules have an isoelectric point, pI, less than or equal to 5.1 as determined by chromatofocusing, and wherein the pI of said dominant isoforms increases after neuraminidase treatment.

5. A method of delaying transplant rejection in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of sCR1 glycoprotein molecules having an average molar ratio of sialic acid to mannose greater than or equal to 0.25.

6. A method according to claim 5, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.38.

7. A method according to claim 5, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.42.

8. A method according to claim 5, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.53.

12. A method for making an sCR1 glycoprotein preparation comprising:

(a) expressing DNA encoding an sCR1 polypeptide in a mammalian host cell in culture under conditions wherein cell growth is not limited by nutrient supply and wherein said host cell is capable of sialylation of oligosaccharide structures;

(b) recovering a composition comprising a plurality of sCR1 glycoprotein molecules from the culture;

(c) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation which exhibits dominant sCR1 glycoprotein isoforms having an isolelectric point, pI, less than or equal to 5.1 as determined by chromatofocusing, wherein the pI of said dominant isoforms increases after neuraminidase treatment.

19. The method of claim 12, 13, 14, or 15, further comprising:

(d) isolating sialic acid-containing sCR1 glycoprotein molecules from the preparation obtained in step (c).

20. The method of claim 12, 13, 14, or 15 wherein said sCR1 polypeptide comprises the 30 extracellular short consensus repeats (SCRs) of human CR1.

13. A method for making an sCR1 glycoprotein preparation comprising:

(a) expressing DNA encoding an sCR1 polypeptide in a mammalian host cell in culture under conditions wherein cell growth is not limited by nutrient supply and wherein said host cell is capable of sialylation of oligosaccharide structures;

(b) recovering a composition comprising a plurality of sCR1 glycoprotein molecules from the culture;

(c) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation wherein substantially all of the sCR1 glycoprotein molecules contain one or more complex oligosaccharide structures, wherein at least about 40% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

14. The method according to claim 13, wherein at least about 70% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

15. A method for making an sCR1 glycoprotein preparation comprising:

(a) expressing DNA encoding an sCR1 polypeptide in a mammalian host cell in culture under conditions wherein cell growth is not limited by nutrient supply and wherein said host cell is capable of sialylation of oligosaccharide structures;

(b) recovering a composition comprising a plurality of sCR1 glycoprotein molecules from the culture;

(c) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation of sCR1 glycoprotein molecules having an average molar ratio of sialic acid to mannose greater than or equal to 0.25.

16. A method according to claim 15, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.38.

17. A method according to claim 15, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.42.

18. A method according to claim 15, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.53.

21. A method for making an sCR1 glycoprotein preparation comprising:

(a) providing a composition comprising a plurality of sCR1 glycoprotein molecules produced by a mammalian cell; and

(b) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation which exhibits dominant sCR1 glycoprotein isoforms having an isolelectric point, pI, less than or equal to 5.1 as determined by chromatofocusing, wherein the pI of said dominant isoforms increases after neuraminidase treatment.

28. The method of claim 21, 22, 23, or 24 wherein said sCR1 glycoprotein molecules comprise the 30 extracellular short consensus repeats (SCRs) of human CR1.

22. A method for making an sCR1 glycoprotein preparation comprising:

(a) providing a composition comprising a plurality of sCR1 glycoprotein molecules produced by a mammalian cell; and

(b) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation wherein substantially all of the sCR1 glycoprotein molecules contain one or more complex oligosaccharide structures, wherein at least about 40% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

23. The method according to claim 22, wherein at least about 70% of said oligosaccharide structures are terminated with one or more sialic acid residues per oligosaccharide structure.

24. A method for making an sCR1 glycoprotein preparation comprising:

(a) providing a composition comprising a plurality of sCR1 glycoprotein molecules produced by a mammalian cell; and

(b) recovering sCR1 glycoprotein isoforms from the composition to afford a preparation of sCR1 glycoprotein molecules having an average molar ratio of sialic acid to mannose greater than or equal to 0.25.

25. A method according to claim 24, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.38.

26. A method according to claim 24, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.42.

27. A method according to claim 24, wherein the sCR1 glycoprotein molecules have an average molar ratio of sialic acid to mannose greater than or equal to 0.53.