Compositions and methods for generating chimeric heteromultimers

• US 7,429,652 B2
• Filed: 08/03/2004
• Issued: 09/30/2008
• Est. Priority Date: 08/01/2001
• Status: Expired due to Term

• Current Assignee: Abmaxis, Inc. (Merck & Co., Inc.)
• Original Assignee: Abmaxis, Inc. (Merck & Co., Inc.)
• Inventors: Zhong, Pingyu, Wang, Caili, Wang, Xinwei, Luo, Peizhi, Liu, Shengjiang, Li, Shengfeng
• Primary Examiner(s): Blanchard; David J.
• Application Number:
  US10/911,127
• Publication Number:
  US 20050009139A1
• Time in Patent Office: 1,519 Days
• Field of Search: None
• US Class Current: 536/23.4
• 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 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention provides a technique for specific assembly of monomeric polypeptides to form a heteromultimer. This technique is particularly useful for generating a genetically diverse repertoire of heteromultimers such as antigen-binding units. The invention also provides both non-single-chain and single-chain antigen-binding units that are assembled by the technique described herein. The present invention also provides recombinant polynucleotides, vectors, host cells, and kits for producing the subject antigen-binding units. Further provided by the invention are methods of using the subject antigen-binding units.

Claims

1. A recombinant polynucleotide comprising a coding sequence that encodes a light (L) chain polypeptide of a non-single-chain antigen-binding unit, wherein the non-single-chain antigen-binding unit comprises:

(a) the encoded light (L) chain polypeptide having a light (L) chain variable region fused in-frame to a first heterodimerization polypeptide and (b) a heavy (H) chain polypeptide having a heavy (H) chain variable region fused in-frame to a second heterodimerization polypeptide wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization polypeptides, said first and second heterodimerization polypeptides comprising heterodimeric receptor polypeptides that mediate heterodimerization.

3. A recombinant polynucleotide comprising a first coding sequence that encodes the L chain polypeptide of claim 1, and a second coding sequence that encodes the H chain polypeptide of claim 2, wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization polypeptides;

and further wherein at least one of the heterodimerization polypeptides is essentially incapable of forming a homodimer under physiological buffer conditions and/or at physiological body temperatures.

4. A vector comprising the recombinant polynucleotide of claim 1, or 2.

5. The vector of claim 4, wherein the vector is an expression vector.

6. The vector of claim 4, wherein the vector is a phage display vector.

7. A selectable library of expression vectors encoding a repertoire of antigen binding units, comprising more than one vector of claim 4.

8. The selectable library of claim 7, wherein the vector is a phage display vector.

9. An isolated host cell comprising the vector of claim 4.

10. The isolated host cell of claim 9, wherein the recombinant polynucleotide encoding the L chain polypeptide and the polynucleotide encoding the H chain polypeptide, are present in a single vector.

11. The isolated host cell of claim 9, wherein the recombinant polynucleotide encoding the L chain polypeptide and the polynucleotide encoding the H chain polypeptide, are present in separate vectors.

12. The isolated host cell of claim 9, wherein the host cell is a eukaryotic cell.

13. The isolated host cell of claim 9, wherein the host cell is a prokaryotic cell.

31. A kit comprising a vector of claim 4 in suitable packaging.

2. A recombinant polynucleotide comprising a coding sequence that encodes a heavy (H) chain polypeptide of a non-single-chain antigen-binding unit, wherein the non-single-chain antigen-binding unit comprises:

(a) a light (L) chain polypeptide comprising a light (L) chain variable region fused in-frame to a first heterodimerization polypeptide and (b) the encoded heavy (H) chain polypeptide comprising a heavy (H) chain variable region fused in-frame to a second heterodimerization polypeptide wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization polypeptides, said first and second heterodimerization polypeptides comprising heterodimeric receptor sequences that mediate heterodimerization.

14. A method of producing a non-single-chain antigen-binding unit, comprising:

(a) expressing in a host cell a first recombinant polynucleotide encoding a light (L) chain polypeptide comprising a light (L) chain variable region fused in-frame to a first heterodimerization polypeptide, and a second recombinant polynucleotide encoding a heavy (H) chain polypeptide comprising a heavy (H) chain variable region fused in-frame to a second heterodimerization polypeptide;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization polypeptides and optionally (b) isolating the antigen-binding unit expressed in the host cell.

15. The method of claim 14, wherein the non-single-chain antigen-binding expressed in step (a) is displayed on surface of the host cell.

16. The method of claim 14, wherein the non-single-chain antigen-binding expressed in step (a) is displayed on a phage particle.

17. The method of claim 14, wherein the host cell is a eukaryotic cell.

18. The method of claim 14, wherein the host cell is a prokaryotic cell.

20. The method of claim 14, wherein the L chain and the H chain polypeptides dimerize via non-covalent pairwise affinity.

28. The method of claim 14, wherein the light (L) chain variable region is human.

29. The method of claim 14, wherein the heavy (H) chain variable region is human.

30. The method of claim 14, wherein the non-single-chain antigen-binding unit exhibits an apparent binding affinity at least one order of magnitude higher than that of a single-chain antigen-binding unit (scFv) that is stabilized by a peptide linker.

19. The method of 14, wherein the first and second heterodimerization polypeptides form a coiled-coil dimer.

21. The method of claim 19, wherein the L chain polypeptide further comprises a flexon that is flanked by the L chain variable region and the first heterodimerization polypeptide.

22. The method of claim 19, wherein the H chain polypeptide further comprises a flexon that is flanked by the H chain variable region and the second heterodimerization polypeptide.

23. The method of claim 19, wherein both the first and the second heterodimerization polypeptides are linked to at least one cysteine residue.

24. The method of claim 19, wherein the non-single-chain antigen-binding unit is multivalent.

25. The method of claim 19, wherein the non-single-chain antigen-binding unit is multispecific.

26. The method of claim 25, wherein the non-single-chain antigen-binding unit is bispecific.

27. The method of claim 25, wherein the non-single-chain antigen-binding unit is trispecific.