ENHANCING THE CIRCULATING HALF LIFE OF ANTIBODY-BASED FUSION PROTEINS

• US 20030105294A1
• Filed: 02/24/1999
• Published: 06/05/2003
• Est. Priority Date: 02/25/1998
• Status: Abandoned Application

• Current Assignee: EMD Lexigen Research Center Corp.
• Original Assignee: EMD Lexigen Research Center Corp.
• Inventors: WESOLOWSKI, JOHN, GILLIES, STEPHEN, LAN, YAN, LO, KIN-MING
• Application Number:
  US09/256,156
• Publication Number:
  US 20030105294A1
• Time in Patent Office: Days
• US Class Current: 530/351
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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

Abstract

Disclosed are methods for the genetic construction and expression of antibody-based fusion proteins with enhanced circulating half-lives. The fusion proteins of the present invention lack the ability to bind to immunoglobulin Fc receptors, either as a consequence of the antibody isotype used for fusion protein construction, or through directed mutagenesis of antibody isotypes that normally bind Fc receptors. The fusion proteins of the present invention may also contain a functional domain capable of binding an immunoglobulin protection receptor.

Claims

1. An antibody-based fusion protein with an enhanced circulating half-life, comprising at least a portion of an immunoglobulin (Ig) heavy chain having substantially reduced binding affinity for an Fc receptor, said portion of heavy chain being linked to a second non-Ig protein, said antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein.

2. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain comprises at least the CH2 domain of an IgG2 or IgG4 constant region.

3. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain comprises at least a portion of an IgG1 constant region having a mutation or a deletion at one or more amino acid selected from the group consisting of Leu₂₃₄, Leu₂₃₅, Gly₂₃₆, Gly₂₃₇, Asn₂₉₇, and Pro₃₃₁.

4. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain comprises at least a portion of an IgG3 constant region having a mutation or a deletion at one or more amino acid selected from the group consisting of Leu₂₈₁, Leu₂₈₂, Gly₂₈₃, Gly₂₈₄, Asn₃₄₄, and Pro₃₇₈.

6. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain has substantially reduced binding affinity for a Fc receptor selected from the group consisting of Fcγ

RI, Fcγ

RII and Fcγ

RIII.

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

8. The antibody-based fusion protein of claim 1, wherein said cytokine is selected from the group consisting of a tumor necrosis factor, an interleukin, and a lymphokine.

9. The antibody-based fusion protein of claim 8, wherein said tumor necrosis factor is tumor necrosis factor alpha.

10. The antibody-based fusion protein of claim 8, wherein said interleukin is interleukin-2.

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

12. The antibody-based fusion protein of claim 11, wherein said colony stimulating factor is a granulocyte-macrophage colony stimulating factor.

13. The antibody-based fusion protein of claim 1, wherein said ligand-binding protein is selected from the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin receptor.

5. The antibody-based fusion protein of claim , wherein said portion of heavy chain further has binding affinity for an immunoglobulin protection receptor.

14. A method of increasing the circulating half-life of an antibody-based fusion protein, comprising the step of linking at least a portion of an Ig heavy chain to a second non-Ig protein, said portion of heavy chain having substantially reduced binding affinity for an Fc receptor, thereby forming an antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein.

15. The method of claim 14, wherein said portion of heavy chain comprises at least the CH2 domain of an IgG2 or IgG4 constant region.

18. The method of claim 14, 16 or 17, wherein said portion of heavy chain further has binding affinity for an immunoglobulin protection receptor.

19. The method of claim 14, 16 or 17, wherein said portion of heavy chain has substantially reduced binding affinity for a Fc receptor selected from the group consisting of Fcγ

RI, Fcγ

RII and Fcγ

RIII.

20. The method of claim 14, 16 or 17, wherein said second non-Ig protein is selected from the group consisting of a cytokine, a ligand-binding protein, and a protein toxin.

21. The method of claim 14, 16 or 17, wherein said cytokine is selected from the group consisting of a tumor necrosis factor, an interleukin, and a lymphokine.

22. The method of claim 21, wherein said tumor necrosis factor is tumor necrosis factor alpha.

23. The method of claim 21, wherein said interleukin is interleukin-2.

24. The method of claim 21, wherein said lymphokine is a lymphotoxin or a colony stimulating factor.

25. The antibody-based fusion protein of claim 24, wherein said colony stimulating factor is a granulocyte-macrophage colony stimulating factor.

26. The method of claim 14, 16 or 17, wherein said ligand-binding protein is selected from the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin receptor.

16. A method of increasing the circulating half-life of an antibody-based fusion protein, comprising the steps of:

(a) introducing a mutation or a deletion at one or more amino acid of an IgG1 constant region, said amino acid selected from the group consisting of Leu₂₃₄, Leu₂₃₅, Gly₂₃₆, Gly₂₃₇, Asn₂₉₇, and Pro₃₃₁, thereby producing an Ig heavy chain having substantially reduced binding affinity for an Fc receptor; and

(b) linking at least a portion of the heavy chain of step (a) to a second non-Ig protein,

thereby forming an antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein.

17. A method of increasing the circulating half-life of an antibody-based fusion protein, comprising the steps of:

(a) introducing a mutation or a deletion at one or more amino acid of an IgG3 constant region, said amino acid selected from the group consisting of Leu₂₈₁, Leu₂₈₂, Gly₂₈₃, Gly₂₈₄, Asn₃₄₄, and Pro₃₇₈, thereby producing an Ig heavy chain having substantially reduced binding affinity for an Fc receptor; and

(b) linking at least a portion of the Ig heavy chain of step (a) to a second non-Ig protein,

thereby forming an antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein.