Compositions and methods for generating antigen-binding units

• US 20040265901A1
• Filed: 08/22/2002
• Published: 12/30/2004
• Est. Priority Date: 08/22/2001
• Status: Active Grant

• Current Assignee: Bio-Thera Solutions, Ltd.
• Original Assignee: Shengfeng Li
• Inventors: Li, Shengfeng
• Granted Patent: US 7,179,595 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2318/10 Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2565/207 Three hybrid system

Abstract

The present invention provides non-single-chain antigen-binding units that is stabilized by leucine zipper sequences. The experimental design is particularly useful for generating and screening for Nsc Abus that remain the binding capabilities to their respective antigens within a cell. The present invention also provides recombinant polynucleotides, host cells and kits comprising the vectors. Further provided by the invention are methods of using the subject vectors.

Claims

1. A non-single-chain antigen-binding unit comprising:

(a) a light (L) chain polypeptide comprising a light (L) chain variable region fused to a first leucine zipper sequence;

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

wherein the L chain and the H chain polypeptides dimerize to form an antigen-binding site through an interaction between the first and second leucine zipper sequences.

2. The non-single-chain antigen-binding unit of claim 1, wherein the first leucine zipper sequence is Fos leucine zipper and the second leucine zipper sequence is Jun leucine zipper.

3. The non-single-chain antigen-binding unit of claim 1, wherein the first leucine zipper sequence is Jun leucine zipper and the second leucine zipper sequence is Fos leucine zipper.

4. The non-single-chain antigen-binding unit of claim 1, wherein either the L chain or the H chain polypeptide is further fused to a gene activation moiety region.

5. The non-single-chain antigen-binding unit of claim 1, wherein the L chain polypeptide further comprises a flexon that is flanked by the L chain variable region and the first leucine zipper sequence.

6. The non-single-chain antigen-binding unit of claim 1, wherein the H chain polypeptide further comprises a flexon that is flanked by the H chain variable region and the second leucine zipper sequence.

7. The non-single-chain antigen-binding unit of claim 1, wherein the L chain variable region comprises variable region sequences of a human antibody.

8. The non-single-chain antigen-binding unit of claim 1, wherein the H chain variable region comprises variable region sequences of a human antibody.

9. A recombinant polynucleotide comprising a coding sequence that encodes the L chain polypeptide of claim 1.

10. A recombinant polynucleotide comprising a coding sequence that encodes the H chain polypeptide of claim 1.

11. 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 of polypeptide of claim 1.

13. A vector comprising the recombinant polynucleotide of any one of the claims 9 to 12.

14. The vector of claim 13, wherein the vector is an expression vector.

15. A selectable library of expression vector encoding a repertoire of antigen-binding units, comprising more than one vector of claim 13.

16. A host cell expressing the recombinant polynucleotides of any one of the claims 9 to 12.

19. The host cell of claim 16, wherein the cell is prokaryotic.

42. A kit comprising the vector of claim 13 in suitable packaging.

12. A recombinant polynucleotide comprising a coding sequence that encodes either a L or H chain polypeptide that is fused to a gene activation moiety region.

17. The host cell of 16, wherein the cell is eukaryotic.

18. The host cell of claim 17, wherein the eukaryotic cell is yeast cell.

20. A method of generating a non-single-chain antigen-binding unit in a yeast cell, comprising co-expressing

(a) a light (L) chain polypeptide comprising a light (L) chain variable region fused to a first leucine zipper sequence;

and

(b) a heavy (H) chain polypeptide comprising a heavy (H) chain variable region fused to a second leucine zipper sequence,

wherein the L and H chain polypeptides dimerize to form an antigen-binding site through an interaction between the first and second leucine zipper sequences.

21. The method of claim 20, wherein the first leucine zipper sequence is Fos leucine zipper and the second leucine zipper sequence is Jun leucine zipper.

22. The method of claim 20, wherein the first leucine zipper sequence is Jun leucine zipper and the second leucine zipper sequence is Fos leucine zipper.

23. The method of claim 20, wherein the L chain polypeptide further comprises a flexon that is flanked by the L chain variable region and the first leucine zipper sequence.

24. The method of claim 20, wherein the H chain polypeptide further comprises a flexon that is flanked by the H chain variable region and the second leucine zipper sequence.

25. The method of claim 20, wherein the L chain variable region comprises variable region sequences of a human antibody.

26. The method of claim 20, wherein the H chain variable region comprises variable region sequences of a human antibody.

27. A method of identifying a non-single-chain antigen-binding unit that is immunoreactive with a desired antigen in a yeast cell, comprising:

(a) recombinantly co-expressing within a population of yeast cells

(i) a reporter gene operably linked to a first DNA-binding-protein recognition site (DNA-BPRS);

(ii) a first antigen fusion gene encoding the desired antigen fused in-frame with a first DNA-binding moiety which specifically binds to said first DNA-BPRS;

(iii) a plurality of expression vectors that encodes a genetically diverse repertoire of antigen-binding units, each antigen-binding unit comprising a variable region of a first antibody chain fused to a first dimerization sequence, and a variable region of a second antibody chain fused to a second dimerization sequence and a gene activation moiety;

wherein the variable regions of the first and second antibody chains dimerize to form an antigen-binding site through an interaction between the first and second dimerization sequences; and

(b) detecting expression of said reporter gene, wherein an increase in the expression indicates a specific binding between an antigen binding fragment and the desired antigen, thereby identifying an antigen binding unit that is immunoreactive with the desired antigen.

28. The method of claim 27, wherein the first antibody chain is light chain, and the second antibody chain is heavy chain.

29. The method of claim 27, wherein the first antibody chain is heavy chain, and the second antibody chain is light chain.

30. The method of claim 27, wherein the first dimerization sequence is Fos leucine zipper and the second dimerization sequence is Jun leucine zipper.

31. The method of claim 27, wherein the first dimerization sequence is Jun leucine zipper and the second dimerization sequence is Fos leucine zipper.

32. The method of claim 27, wherein co-expression of a plurality of expression vectors comprises mating a first population of yeast cells that carries expression vectors encoding a repertoire comprising variable regions of a first antibody chain, with a second population of yeast cells that carries expression vectors encoding a repertoire comprising variable regions of a second antibody chain.

33. The method of claim 27, further comprising the step of counter selecting yeast cells that express the reporter gene independent of the specific interaction between an antigen binding fragment and the desired antigen.

34. The method of claim 27 wherein the step of counter selecting further comprising:

(a) recombinantly co-expressing within the population of yeast cells

(i) a counterselectable gene operably linked to a second DNA-binding protein recognition site (DNA-BPRS);

(ii) a second antigen fusion gene encoding a second antigen fused in-frame with a second DNA-binding moiety which specifically binds to the second DNA-BPRS, wherein the second antigen differs structurally from the first antigen;

(b) culturing the yeast cells under condition suitable for expression for the reporter gene and the counterselectable gene; and

(c) detecting growth of yeast cells and expression of the reporter gene, wherein the growth of the yeast cells and an increase in the reporter gene expression indicate that a specific binding between an antigen-binding unit and the desired antigen has occurred.

35. The method of claim 27, wherein the reporter gene is selected from the group consisting of LEU2, TRP1, HIS3, LacZ, URA3, and MEL.

36. The method of claim 27, wherein said DNA-binding-protein recognition site comprises at least one binding site for a protein selected from the group consisting of GAL4, LexA, and Ace1.

37. The method of claim 27, wherein said DNA-binding moiety comprises the DNA-binding domain of a protein selected from the group consisting of GAL4, LexA, and Ace1.

38. The method of claim 27, wherein said gene activating moiety comprises the transcription activation domain selected from the group consisting of GAL4 and VP16.

39. The method of claim 34, wherein said counterselectable gene is selected from the group consisting of URA3, LYS5, GAL1, CYH2, and CAN1.

40. The method of claim 39, wherein said counterselectable gene is integrated into the genome of the population of mating or mated yeast cells.

41. The method of claim 39, wherein said expression of said counterselectable reporter gene is lethal to a yeast cell.