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

• US 7,091,321 B2
• Filed: 02/09/2001
• Issued: 08/15/2006
• Est. Priority Date: 02/11/2000
• Status: Expired due to Term

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: EMD Lexigen Research Center Corp.
• Inventors: Gillies, Stephen D., Lo, Kin-Ming, Burger, Christa
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US09/780,668
• Publication Number:
  US 20020147311A1
• Time in Patent Office: 2,013 Days
• Field of Search: 530/387.3, 424/134.1
• US Class Current: 530/387.3
• 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. An antibody-based fusion protein comprising an N-terminal immunoglobulin (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, wherein said antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid substitution.

2. The antibody-based fusion protein of claim 1, wherein said non-Ig protein is a secreted protein.

3. The antibody-based fusion protein of claim 2, wherein said non-Ig protein is a mature form of said secreted protein.

4. The antibody-based fusion protein of claim 1, wherein said non-Ig protein is selected from the group consisting of a cytokine, a ligand-binding protein, and a protein toxin.

5. The antibody-based fusion protein of claim 4, wherein said cytokine is selected from the group consisting of a tumor necrosis factor, an interleukin, and a lymphokine.

6. The antibody-based fusion protein of claim 5, wherein said tumor necrosis factor is tumor necrosis factor alpha.

7. The antibody-based fusion protein of claim 5, wherein said interleukin is interleukin-2.

8. The antibody-based fusion protein of claim 5, wherein said lymphokine is a lymphotoxin or a colony stimulating factor.

9. The antibody-based fusion protein of claim 8, wherein said colony stimulating factor is a granulocyte-macrophage colony stimulating factor.

10. The antibody-based fusion protein of claim 4, wherein said ligand-binding protein is selected from the group consisting of CD₄, CTLA-4, TNF receptor, and an interleukin receptor.

11. The fusion protein of claim 1 further comprising an amino acid substitution introducing a hydrophobic or non-polar amino acid within the Ig chain, wherein said antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without the amino acid substitutions.

12. The fusion protein of claim 1, wherein said hydrophobic or non-polar amino acid is Ala.

17. The antibody-based fusion protein of claim 1, 13, or 15, wherein said amino acid substitution increases the hydrophobicity of said antibody-based fusion protein.

27. The fusion protein of claim 1, 13 or 15, further comprising a linker between said Ig chain and said non-Ig protein.

29. The fusion protein of claim 1, 13, 15, or 28, wherein said hydrophobic or non-polar amino acid is selected from the group consisting of Leu, Trp, Ala, and Gly.

13. An antibody-based fusion protein comprising an N-terminal immunoglobulin (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, wherein said antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid substitution.

14. The antibody-based fusion protein of claim 13 wherein the constant domain comprises at least one of a CH1, CH2, or CH3 domain.

18. The antibody-based fusion protein of claim 13 or 15, wherein said substitution changes the C-terminal amino acid of the Ig chain.

19. The fusion protein of claim 18 wherein the C-terminal amino acid of said N-terminal Ig chain is substituted.

20. The antibody-based fusion protein of claim 18 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.

21. The antibody-based fusion protein of claim 20, wherein the C-terminal lysine is substituted with an alanine.

22. The antibody-based fusion protein of claim 20, wherein the non-Ig protein is a cytokine.

23. The antibody-based fusion protein of claim 13 or 15, wherein the Ig chain comprises part of an Ig heavy chain.

24. The antibody-based fusion protein of claim 23, wherein said part of an Ig heavy chain further has binding affinity for an immunoglobulin protection receptor.

25. The antibody-based fusion protein of claim 23, 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.

26. The antibody-based fusion protein of claim 13 or 15, wherein the Ig chain comprises at least the CH2 domain of an IgG2 or an IgG4 constant region.

15. An antibody-based fusion protein comprising 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 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, wherein the antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid substitution.

16. The antibody-based fusion protein of claim 15 wherein the Ig chain is an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE chain.

28. An antibody-based fusion protein comprising 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, wherein the antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid substitution.

30. An antibody-based fusion protein comprising 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, wherein said antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid alteration.

31. An antibody-based fusion protein comprising 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, wherein the antibody-based fusion protein has a longer circulating half-life in vivo than a corresponding antibody-based fusion protein without said amino acid substitution.