Enhancing the circulating half-life of antibody-based fusion proteins

• US 7,790,415 B2
• Filed: 01/27/2009
• Issued: 09/07/2010
• Est. Priority Date: 02/11/2000
• Status: Expired due to Fees

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Gillies, Stephen D., Lo, Kin-Ming, Burger, Christa
• Primary Examiner(s): Saunders; David A
• Application Number:
  US12/360,682
• Publication Number:
  US 20090264627A1
• Time in Patent Office: 588 Days
• Field of Search: None
• US Class Current: 435/69.7
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 5/1013 the side chain containing O or S as heteroatoms, e.g. Cys, Ser

Abstract

Disclosed are compositions and methods for enhancing the circulating half-life of antibody-based fusion proteins. Disclosed methods and compositions rely on altering the amino acid sequence of the junction region between the antibody moiety and the fused protein moiety in an antibody-based fusion protein. An antibody-based fusion protein with an altered amino acid sequence in the junction region has a greater circulating half-life when administered to a mammal. Disclosed methods and compositions are particularly useful for reducing tumor size and metastasis in a mammal.

Claims

1. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal Ig chain linked to a C-terminal non-Ig protein, the Ig chain comprising an IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE constant domain and an amino acid substitution introducing a hydrophobic or non-polar amino acid within 10 amino acids from the C-terminus of the Ig chain.

2. The method of claim 1, wherein the constant domain comprises at least one of a CH1, CH2, or CH3 domain.

3. The method of claim 1, wherein the constant domain comprises at least one of a CH2 or CH3 domain.

4. The method of claim 1, wherein said amino acid substitution increases the hydrophobicity of said antibody-based fusion protein.

5. The method of claim 1, wherein said substitution replaces a charged amino acid with a hydrophobic or nonpolar amino acid.

6. The method of claim 1, wherein said substitution changes the C-terminal amino acid of the Ig chain.

7. The method of claim 6, wherein the Ig chain comprises the CH2 domain of the IgG2 constant region and a C-terminal lysine is substituted with a non-polar or hydrophobic amino acid.

8. The method of claim 7, wherein the C-terminal lysine is substituted with an alanine.

9. The method of claim 7, wherein the non-Ig protein is a cytokine.

10. The method of claim 1, wherein the Ig chain comprises part of an Ig heavy chain.

11. The method of claim 10, wherein said part of an Ig heavy chain further has binding affinity for an immunoglobulin protection receptor.

12. The method of claim 10, wherein said Ig chain has substantially reduced binding affinity for a Fc receptor selected from the group consisting of Fcγ

RI, Fcγ

RII and Fcγ

RIII, when compared to the binding affinity of an unsubstituted Ig chain for said Fc receptor.

13. The method of claim 1, wherein the Ig chain comprises at least the CH2 domain of an IgG2 or an IgG4 constant region.

14. The method of claim 1, further comprising a linker between said Ig chain and said non-Ig protein.

15. The method of claim 1, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Leu, Trp, Ala, and Gly.

16. The method of claim 15, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Trp, Ala, and Gly.

17. The method of claim 1, wherein said non-Ig protein is a secreted protein.

18. The method of claim 17, wherein said non-Ig protein is a mature form of said secreted protein.

19. The method of claim 1, wherein said non-Ig protein is interleukin-2.

20. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal Ig chain linked to a C-terminal non-Ig protein, the Ig chain comprising:

at least one of a CH2 and CH3 domain; and

an amino acid sequence that is non-natural within 10 amino acids from its C-terminus, the non-natural amino acid sequence comprising an amino acid substitution introducing a hydrophobic or non-polar amino acid.

21. The method of claim 20, wherein the Ig chain is an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE chain.

22. The method of claim 20, wherein said amino acid substitution increases the hydrophobicity of said antibody-based fusion protein.

23. The method of claim 20, wherein said amino acid substitution replaces a charged amino acid with a hydrophobic or non-polar amino acid.

24. The method of claim 20, wherein said substitution changes the C-terminal amino acid of the Ig chain.

25. The method of claim 24, wherein the Ig chain comprises the CH2 domain of the IgG2 constant region and a C-terminal lysine is substituted with a nonpolar or hydrophobic amino acid.

26. The method of claim 25, wherein the C-terminal lysine is substituted with an alanine.

27. The method of claim 25, wherein the non-Ig protein is a cytokine.

28. The method of claim 20, wherein the Ig chain comprises at least the CH2 domain of an IgG2 or an IgG4 constant region.

29. The method of claim 20, further comprising a linker between said Ig chain and said non-Ig protein.

30. The method of claim 20, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Leu, Trp, Ala, and Gly.

31. The method of claim 30, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Trp, Ala, and Gly.

32. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal immunoglobulin (Ig) chain linked to a C-terminal non-Ig protein, the Ig chain comprising an amino acid substitution within 10 amino acids from the C-terminus, the substitution replacing a charged amino acid with a hydrophobic or non-polar amino acid.

33. The method of claim 32, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Leu, Trp, Ala, and Gly.

34. The method of claim 33, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Trp, Ala, and Gly.

35. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal immunoglobulin (Ig) chain linked to a C-terminal non-Ig protein, the N-terminal Ig chain comprising an amino acid substitution within 10 amino acids from the C-terminus of the Ig chain, the substitution introducing a hydrophobic or non-polar amino acid selected from the group consisting of Ala, Gly and Trp.

36. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal Ig chain linked to a C-terminal non-Ig protein, the C-terminal non-Ig protein comprising an amino acid substitution introducing a hydrophobic or non-polar amino acid within 10 amino acids of the N-terminus of the C-terminal non-Ig protein.

37. The method of claim 36, wherein said amino acid substitution increases the hydrophobicity of said antibody-based fusion protein.

38. The method of claim 36, wherein the Ig chain comprises (i) a deletion of a charged an amino acid within 10 amino acids of the C-terminus of the Ig chain, or (ii) an amino acid alteration introducing a hydrophobic or non-polar amino acid within 10 amino acids of the C-terminus of the Ig chain.

39. The method of claim 36, wherein the hydrophobic or non-polar amino acid is Ala.

40. The method of claim 36, further comprising a linker between said Ig chain and said non-Ig protein.

41. The method of claim 36, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Leu, Trp, Ala, and Gly.

42. A method for increasing the circulating half-life of an antibody-based fusion protein having an immunoglobulin (Ig) chain linked to a non-Ig protein via a junction point, the method comprising expressing the antibody-based fusion protein as an N-terminal immunoglobulin (Ig) chain linked to a C-terminal non-Ig protein, the Ig chain comprising at least one of a CH2 and a CH3 domain, and the C-terminal non-Ig protein comprising an amino acid alteration within 10 amino acids of the N-terminus of the C-terminal non-Ig protein, the alteration introducing an amino acid selected from the group consisting of Leu and Trp.