Product and process for T lymphocyte immunosuppression

• US 6,264,950 B1
• Filed: 08/17/1999
• Issued: 07/24/2001
• Est. Priority Date: 04/10/1996
• Status: Expired due to Fees

• Current Assignee: National Jewish Medical and Research Center
• Original Assignee: National Jewish Medical and Research Center
• Inventors: Staerz, Uwe D.
• Primary Examiner(s): Saunders, David
• Assistant Examiner(s): DECLOUX, AMY M
• Application Number:
  US09/375,419
• Time in Patent Office: 707 Days
• Field of Search: 424/134.1, 424/192.1, 424/193.1, 424/194.1, 435/69.1, 435/455, 435/471, 435/325, 435/328, 435/425, 530/350, 530/387.3, 530/391.1, 530/6, 530/868, 530/413, 530/416, 530/417, 530/412, 514/8, 514/885
• US Class Current: 424/134.1
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C07K 14/70507 CD2
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70532 B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 14/70553 Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 14/70589 CD45
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/2833 against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: Y10S 530/868 involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance

Abstract

The present invention relates to a product and process for suppressing an immune response using a T lymphocyte veto molecule capable of blocking cell surface molecules responsible for T cell activation. Disclosed is a CD4 or CD2 molecule, associated with an immunoglobulin molecule capable of binding to a major histocompatibility antigen. Also disclosed is a method to produce a T lymphocyte veto molecule, a therapeutic composition comprising a T lymphocyte veto molecule and methods to use T lymphocyte veto molecules in therapeutic processes requiring suppression of an immune response.

Claims

1. A T lymphocyte immunosuppression molecule comprising a chimeric molecule having a CD4 protein, wherein said protein is linked to a targeting immunoglobulin molecule that binds by its variable region to a molecule on the surface of a tissue graft cell that differentiates a host cell from said tissue graft cell.

2. The immunosuppression molecule of claim 1, wherein said CD4 protein comprises at least one V and at least one C2 domain of a CD4 protein.

3. The immunosuppression molecule of claim 1, wherein said protein comprises an amino acid sequence SEQ ID NO:

1.

4. The immunosuppression molecule of claim 1, wherein said protein is linked by a peptide bond to the constant region domain of said immunoglobulin molecule, to form a protein:

Ig conjugated molecule.

5. The immunosuppression molecule of claim 4, wherein said immunoglobulin molecule of said protein:

Ig conjugated molecule comprises a constant region domain of an IgG2a molecule.

6. The immunosuppression molecule of claim 4, wherein said protein:

Ig conjugated molecule comprises an amino acid sequence comprising SEQ ID NO;

17.

7. The immunosuppression molecule of claim 1, wherein said targeting immunoglobulin binds to a major histocompatibility molecule.

8. The immunosuppression molecule of claim 1, wherein said protein is linked to said targeting immunoglobulin by a chemically produced di-sulfide bond.

9. The immunosuppression molecule of claim 1, wherein said chimeric molecule can be secreted from a cell that produces said molecule.

15. The immunosuppression molecule of claim 1, wherein said CD4 protein comprises first and second V domains and first and second C2 domains of a CD4 protein.

16. The immunosuppression molecule of claim 1, wherein said CD4 protein comprises a leader sequence.

17. The immunosuppression molecule of claim 16, wherein said leader sequence co an amino acid sequence SEQ ID NO:

29.

10. A recombinant cell that expresses a chimeric T lymphocyte immunosuppression molecule, said recombinant cell having:

(1) a first recombinant molecule comprising a first nucleic acid molecule operatively linked to an expression vector, said first nucleic acid molecule having a nucleic acid sequence encoding a first CD4 protein; and

(2) a second recombinant molecule comprising a second nucleic acid molecule operatively linked to an expression vector, said second nucleic acid molecule having a nucleic acid sequence encoding a second protein comprising a targeting immunoglobulin molecule selected from the group consisting of a targeting immunoglobulin molecule having a variable region that binds to a tissue graft cell surface molecule that differentiates a host cell from a tissue graft cell and a targeting immunoglobulin molecule that selectively targets a molecule on the surface of a cell involved in an autoimmune response.

11. The recombinant cell of claim 10, wherein said recombinant cell is not capable of stimulating a T cell response.

12. The recombinant cell of claim 10, wherein said recombinant cell is derived from a host cell selected from the group consisting of a fibroblast, a pluripotent progenitor cell, an epithelial cell, a neural cell, a T cell line and a B cell line.

13. The recombinant cell of claim 10, wherein said first nucleic acid molecule comprises a nucleic acid sequence that encodes an amino acid sequence SEQ ID NO:

1.

14. The recombinant cell of claim 10, wherein said first nucleic acid molecule comprises a nucleic acid sequence that encodes an amino acid sequence SEQ ID NO:

29.

18. A composition comprising:

a T lymphocyte immunosuppression molecule comprising;

a chimeric molecule having a targeting immunoglobulin molecule selected from the group consisting of a targeting immunoglobulin molecule having a variable region that binds to a tissue graft cell surface molecule that differentiates a host cell from a tissue graft cell and a targeting immunoglobulin molecule having a variable region that selectively targets a molecule on the surface of a cell involved in an autoimmune response, wherein said targeting molecule is linked to a CD4 protein; and

a pharmaceutically acceptable carrier.

19. A method for producing a T lymphocyte veto molecule, comprising:

(a) providing a first protein comprising a CD4protein;

(b) providing a second protein comprising a targeting immunoglobulin molecule selected from the group consisting of a targeting immunoglobulin molecule that binds to a tissue graft cell surface molecule that differentiates a host cell from a tissue graft cell and a targeting immunoglobulin molecule having a variable region that selectively targets a molecule on the surface of a cell involved in an autoimmune response; and

(c) linking said first protein to said second protein to form a chimeric molecule.

20. The method of claim 19, wherein said first protein is produced by culturing a cell transformed with a recombinant molecule comprising a nucleic acid molecule operatively linked to an expression vector, said nucleic acid molecule comprising a nucleic acid sequence that encodes an amino acid sequence SEQ ID NO:

1.

21. The method of claim 19, wherein said second protein is produced by culturing a cell transformed with a recombinant molecule comprising a nucleic acid molecule operatively linked to an expression vector, said nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence derived from said immunoglobulin molecule.

22. The method of claim 19, wherein said step of linking comprises combining said first and second proteins and contacting said proteins with a reagent capable of creating di-sulfide bonds between said first and second proteins.

23. The method of claim 19, wherein said step of linking is performed using N-succinimidyl-3-1-(1-pyridyldthio)-propionate.

24. The method of claim 19, wherein said chimeric molecule is recovered from said first and second proteins that have not been linked.

25. The method of claim 19, wherein said chimeric molecule is recovered from said first and second proteins that have not been linked by a method selected from the group consisting of size separation chromatography, affinity chromatography, ion-exchange chromatography, reverse phase chromatography, chromatofocussing, hydroxyapatite adsorption and electrophoresis systems.

26. A T lymphocyte immunosuppression molecule comprising a chimeric molecule having a CD4 protein;

wherein said protein is linked to a targeting immunoglobulin that binds by its variable region to a molecule which selectively targets a cell involved in an autoimmune response.

27. The immunosuppression molecule of claim 26, wherein said protein comprises an amino acid sequence SEQ ID NO:

1.

28. The immunosuppression molecule of claim 26, wherein said protein is linked by a peptide bond to the constant region domain of said immunoglobulin molecule, to form a protein:

Ig conjugated molecule.

29. The immunosuppression molecule of claim 28, wherein said immunoglobulin molecule of said protein:

Ig conjugated molecule comprises a constant region domain of an IgG2a molecule.

30. The immunosuppression molecule of claim 28, wherein said protein:

Ig conjugated molecule comprises an amino acid sequence comprising SEQ ID NO;

17.

31. The immunosuppression molecule of claim 26, wherein said targeting immunoglobulin molecule comprises an immunoglobulin that binds to a protein selected from the group consisting of a major histocompatibility molecule, asialoglycoprotein receptor, bile acid receptor, LMA surface target molecules, heart cell surface proteins, islets of Langerhans cell surface proteins, c-kit receptor and blood proteins.

32. The immunosuppression molecule of claim 26, wherein said protein is linked to said targeting immunoglobulin by a chemically produced di-sulfide bond.

33. The immunosuppression molecule of claim 26, wherein said chimeric molecule can be secreted from a cell that produces said molecule.

34. A T lymphocyte immunosuppression molecule comprising a chimeric molecule having a CD4 protein, wherein said protein is linked to a monovalent targeting immunoglobulin molecule that binds by its variable region to a molecule on the surface of a cell selected from the group consisting of a tissue graft cell that differentiates a host cell from said tissue graft cell, and a cell involved in an autoimmune response.