REDUCING THE IMMUNOGENICITY OF FUSION PROTEINS

• US 20100016562A1
• Filed: 09/01/2009
• Published: 01/21/2010
• Est. Priority Date: 03/30/2001
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Hamilton, Anita A., Gillies, Stephen D., Way, Jeffrey
• Granted Patent: US 7,973,150 B2
• Time in Patent Office: Days
• US Class Current: 530/402
• 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.

2-17. -17. (canceled)

18. A nucleic acid encoding a fusion protein with reduced immunogenicity comprising;

a non-immunoglobulin protein and

an immunoglobulin protein fused to said non-immunoglobulin protein via a fusion junction,

wherein the amino acid sequence of a junction region surrounding the fusion junction is modified by substitution or deletion of one or more amino acids to remove a non-self T-cell epitope.

26. The nucleic acid of claim 18, wherein the C-terminus of the immunoglobulin protein is fused to the N-terminus of the non-immunoglobulin protein.

27. The nucleic acid of claim 18, wherein the junction region comprises an IgG sequence having an ATAT amino acid sequence (amino acids 3-6 of SEQ ID NO:

6) instead of an LSLS amino acid sequence (amino acids 3-6 of SEQ ID NO;

5).

31. The nucleic acid of claim 18, wherein the non-immunoglobulin protein has cytokine activity.

32. The nucleic acid of claim 18, wherein the non-immunoglobulin protein has hormone activity.

33. The nucleic acid of claim 18, wherein the non-immunoglobulin protein is an albumin protein.

38. The nucleic acid of claim 18, wherein the junction region comprises an IgG region wherein an LSLS amino acid sequence (amino acids 3-6 of SEQ ID NO:

5) is mutated without generating a T-cell epitope.

39. The nucleic acid of claim 18, wherein the immunoglobulin protein comprises two immunoglobulin isotypes.

40. The nucleic acid of claim 18, wherein the substitution or deletion comprises a substitution of a leucine to a threonine, alanine or proline.

41. The nucleic acid of claim 18, wherein the substitution or deletion comprises an amino acid substitution to glycine or proline.

19-25. -25. (canceled)

28-30. -30. (canceled)

34-37. -37. (canceled)