Compositions and methods for generating chimeric heteromultimers

• US 20050009139A1
• Filed: 08/03/2004
• Published: 01/13/2005
• Est. Priority Date: 08/01/2001
• Status: Active Grant

• Current Assignee: Caili Wang, Peizhi Luo, Pingyu Zhong, Shengfeng Li, Shengjiang Liu, Xinwei Wang
• Original Assignee: Caili Wang, Peizhi Luo, Pingyu Zhong, Shengfeng Li, Shengjiang Liu, Xinwei Wang
• Inventors: Zhong, Pingyu, Wang, Caili, Wang, Xinwei, Luo, Peizhi, Liu, Shengjiang, Li, Shengfeng
• Granted Patent: US 7,429,652 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 light (L) chain polypeptide having a light (L) chain variable region fused in-frame to a first heterodimerization sequence;

(b) a heavy (H) chain polypeptide having a heavy (H) chain variable region fused in-frame to a second heterodimerization sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences; and

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

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.

9. A vector comprising the recombinant polynucleotide of any one of claims 1, 2, 4, 5, 7, and 8.

10. The vector of claim 9, wherein the vector is an expression vector.

11. The vector of claim 9, wherein the vector is a phage display vector.

12. A selectable library of expression vectors encoding a repertoire of antigen binding units, comprising more than one vector of claim 9.

13. The selectable library of claim 12, wherein the vector is a phage display vector.

14. A host cell comprising the recombinant polynucleotides of any one of claims 1, 2, 4, 5, 7, and 8.

15. The host cell of claim 14, wherein the recombinant polynucleotide encoding the L chain polypeptide and the polynucleotide encoding the H chain polypeptide, are present in a single vector.

16. The host cell of claim 14, wherein the recombinant polynucleotide encoding the L chain polypeptide and the polynucleotide encoding the H chain polypeptide, are present in separate vectors.

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

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

43. A kit comprising a vector of claim 9 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 having a light (L) chain variable region fused in-frame to a first heterodimerization sequence;

(b) the heavy (H) chain polypeptide having a heavy (H) chain variable region fused in-frame to a second heterodimerization sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences; and

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

4. 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 light (L) chain polypeptide having a light (L) chain variable region fused to a first heterodimerization sequence;

(b) a heavy (H) chain polypeptide having a heavy (H) chain variable region fused to a second heterodimerization sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences, said first and second heterodimerization sequences comprising heterodimeric receptor sequences that mediate heterodimerization of the receptors.

6. A recombinant polynucleotide comprising a first coding sequence that encodes the L chain polypeptide of claim 4, and a second coding sequence that encodes the H chain polypeptide of claim 5.

5. 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 to a first heterodimerization sequence;

(b) the heavy (H) chain polypeptide comprising a heavy (H) chain variable region fused to a second heterodimerization sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences, said first and second heterodimerization sequences comprising heterodimeric receptor sequences that mediate heterodimerization of the receptors.

7. A recombinant polynucleotide comprising a coding sequence that encodes the single-chain antigen-binding unit, wherein the single-chain antigen-binding unit comprises:

a light (L) chain variable region and a heavy (H) chain variable region connected by a first and a second heterodimerization sequence spanning the distance between the C-terminus of one of the region to the N-terminus of the other region, wherein the two regions form an intra-molecular dimer via pairwise affinity of the first and second heterodimerization sequences; and

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

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

(a) expressing in a host cell a recombinant polynucleotide of claim 7 or 8; and

optionally

(b) isolating the single-chain antigen-binding unit expressed in the host cell.

40. The method of claim 39, wherein the polynucleotide is contained in a phage display vector.

8. A recombinant polynucleotide comprising a coding sequence that encodes the single-chain antigen-binding unit, wherein the single-chain antigen-binding unit comprises:

a light (L) chain variable region and a heavy (H) chain variable region connected by a first and a second heterodimerization sequence spanning the distance between the C-terminus of one of the region to the N-terminus of the other region, wherein the two regions form an intra-molecular dimer via pairwise affinity of the first and second heterodimerization sequences, said first and second heterodimerization sequences comprising heterodimeric receptor sequences that mediate heterodimerization of the receptors.

19. 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 sequence, 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 sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences; and

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

optionally

(b) isolating the antigen-binding unit expressed in the host cell.

20. A method of claim 19, wherein both of the first and second heterodimerization sequences are essentially incapable of forming homodimers under physiological buffer conditions and at physiological body temperatures.

22. The method of claim 19 or 21, wherein the non-single-chain antigen-binding expressed in step (a) is displayed on surface of the host cell.

23. The method of claim 19 or 21, wherein the non-single-chain antigen-binding expressed in step (a) is displayed on a phage particle.

24. The method of claim 19 or 21, wherein the host cell is a eukaryotic cell.

25. The method of claim 19 or 21, wherein the host cell is a prokaryotic cell.

27. The method of claim 19 or 21, wherein the L chain and the H chain polypeptides dimerize via non-covalent pairwise affinity.

35. The method of claim 19 or 21, wherein the L chain polypeptide comprises sequences from a human light chain.

36. The method of claim 19 or 21, wherein the H chain polypeptide comprises sequences from a human heavy chain.

41. The method of claim 19 or 21, wherein the non-single-chain antigen-binding 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.

42. The method of claim 19, wherein said first and second heterodimerization polypeptides are essentially incapable of forming homodimers when mixed in equimolar.

21. 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 sequence, 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 sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences, said first and second heterodimerization sequences comprising heterodimeric receptor sequences that mediate heterodimerization of the receptors; and

optionally

(b) isolating the antigen-binding unit expressed in the host cell.

26. The method of 19 or 21, wherein the first and second heterodimerization sequences form a coiled-coil dimer.

28. The method of claim 26, wherein the L chain polypeptide further comprises a flexon that is flanked by the L chain variable region and the first heterodimerization sequence.

29. The method of claim 26, wherein the H chain polypeptide further comprises a flexon sequence that is flanked by the H chain variable region and the second heterodimerization sequence.

30. The method of claim 26, wherein both the first and the second heterodimerization sequences are linked to at least one cysteine residue.

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

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

33. The method of claim 32, wherein the non-single-chain antigen-binding unit is bispecific.

34. The method of claim 32, wherein the non-single-chain antigen-binding unit is trispecific.

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

(a) preparing a first recombinant polynucleotide encoding a light (L) chain polypeptide comprising a light (L) chain variable region fused in-frame to a first heterodimerization sequence, 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 sequence;

wherein the L chain and the H chain polypeptides dimerize via pairwise affinity of the first and second heterodimerization sequences; and

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

(b) allowing the first and second polypeptides to dimerize via pairwise affinity of the first and second heterodimerization sequences.

38. The method of claim 37, wherein step (b) comprises dimerizing the first and the second polypeptides in vitro.