Reducing the immunogenicity of fusion proteins

• US 20030166877A1
• Filed: 03/29/2002
• Published: 09/04/2003
• Est. Priority Date: 03/30/2001
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Lexigen Pharmaceuticals Corporation
• Inventors: Hamilton, Anita A., Gillies, Stephen D., Way, Jeffrey
• Granted Patent: US 6,992,174 B2
• Time in Patent Office: Days
• US Class Current: 530/395
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/5434 IL-12
• CPC Class Codes: C07K 14/55 IL-2
• 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 16/30 from tumour cells
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• 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 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: Y10S 424/809 involving immunoglobulin or antibody fragment, e.g. fab', fv, fc, heavy chain or light chain
• CPC Class Codes: Y10S 530/868 involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, or immunotolerance

Abstract

Disclosed are compositions and methods for producing fusion proteins with reduced immunogenicity. Fusion proteins of the invention include a junction region having an amino acid change that reduces the ability of a junctional epitope to bind to MHC Class II, thereby reducing its interaction with a T-cell receptor. Methods of the invention involve analyzing, changing, or modifying one or more amino acids in the junction region of a fusion protein in order to identify a T-cell epitope and reduce its ability to interact with a T cell receptor. Compositions and methods of the invention are useful in therapy.

Claims

1. A method for reducing the immunogenicity of a fusion protein, the method comprising:

i. identifying a candidate T-cell epitope within a junction region spanning a fusion junction of a fusion protein; and

,

ii. changing an amino acid within the junction region to reduce the ability of the candidate T-cell epitope to interact with a T cell receptor.

2. A fusion protein produced by the method of claim 1.

13. A fusion protein of claim 2, 4, or 9, wherein the protein comprises an Ig region.

14. A fusion protein of claim 2, 4, or 9, wherein the protein comprises a serum albumin region.

15. A fusion protein of claim 2, 4, or 9, wherein the protein comprises a cytokine activity.

16. A fusion protein of claim 2, 4, or 9, wherein the protein comprises a hormone activity.

17. A fusion protein of claim 13, wherein the Ig region, the Ig region comprises sequences of more than one antibody isotype.

3. A method for reducing the immunogenicity of a fusion protein, the method comprising changing a candidate T-cell epitope within a junction region spanning a fusion junction of a fusion protein to reduce the ability of the candidate T-cell epitope to interact with a T cell receptor:

i. T-cell epitope

4. A fusion protein produced by the method of claim 3.

5. A method for reducing the immunogenicity of a fusion protein, the method comprising introducing a glycosylation site within the junction region spanning the fusion junction.

7. A method of claim 5, the method comprising introducing a glycosylation site within 5 amino acids of the fusion junction.

8. A method of claim 5, the method comprising introducing a glycosylation site within 2 amino acids of the fusion junction.

10. A fusion protein produced by the method of claim 5, 6, 7, 8, or 9.

11. A method of claim 5-8, wherein the glycosylation is an N-linked glycosylation.

12. A method of claim 5-8, wherein the glycosylation is an O-linked glycosylation.

6. A method for reducing the immunogenicity of a fusion protein, the method comprising introducing a glycosylation site within 10 amino acids of the fusion junction.

9. A method for reducing the immunogenicity of a fusion protein, the method comprising the steps of:

i. providing a fusion protein with a junction region comprising a substituted amino acid; and

ii. assaying said fusion protein in an immunogenicity assay.

18. A fusion protein with reduced immunogenicity comprising

a first protein;

and,

a second protein linked to said first protein via a fusion junction,

wherein the amino acid sequence of a junction region surrounding the fusion junction is modified to remove a non-self T-cell epitope.

19. The fusion protein of claim 18, wherein the junction region comprises between 1 and 25 amino acids.

20. The fusion protein of claim 18, wherein the junction region comprises between 1 and 15 amino acids.

21. The fusion protein of claim 18, wherein the junction region comprises between 1 and 9 amino acids.

22. The fusion protein of claim 18, wherein the junction region comprises an N-linked or an O-linked glycosylation site.

23. The fusion protein of claim 18, wherein the junction region comprises a spacer or linker.

24. The fusion protein of claim 18, wherein the junction region comprises an Asn-X-Ser/Thr-Gly-amino acid sequence, wherein X is any amino acid.

25. The fusion protein of claim 18, wherein the first protein comprises an Ig molecule or a fragment thereof.

26. The fusion protein of claim 25, wherein the C-terminus of said Ig molecule or fragment thereof is linked to the N-terminus of said second protein.

27. The fusion protein of claim 18, wherein the junction region comprises an IgG sequence having an ATAT amino acid sequence instead of an LSLS amino acid sequence.

28. The fusion protein of claim 25, wherein the Ig molecule or fragment thereof comprises an Fc molecule.

29. The fusion protein of claim 25, wherein the Ig molecule or fragment thereof comprises amino acid sequences from two antibody isotypes.

30. The fusion protein of claim 29, wherein the Ig molecule or fragment thereof comprises IgG1 and IgG2 amino acid sequences.

31. The fusion protein of claim 18, wherein the second protein has cytokine activity.

32. The fusion protein of claim 18, wherein the second protein has hormone activity.

33. The fusion protein of claim 18, wherein the first protein is an albumin protein.

34. A method of reducing the immunogenicity of a fusion protein, the method comprising:

i. identifying an amino acid in a peptide in a junction region, wherein the amino acid is selected from the group consisting of a leucine, a valine, an isoleucine, a methionine, a phenylalanine, a tryptophan and a tyrosine; and

ii. changing the amino acid in the peptide, such that the ability of the peptide to bind to MHC Class II is reduced.

35. A fusion protein produced by the method of claim 34.

36. A nucleic acid encoding a fusion protein with reduced immunogenicity, the fusion protein comprising:

i. a first protein;

ii. a second protein linked to the first protein via a fusion junction, wherein the amino acid sequence of a junction region spanning the fusion junction is modified to remove a non-self T-cell epitope.

iii.

37. A nucleic acid of claim 36, wherein the junction region comprises a linker.