METHODS AND COMPOSITIONS FOR GENERATION OF GERMLINE HUMAN ANTIBODY GENES

• US 20150183853A1
• Filed: 07/09/2014
• Published: 07/02/2015
• Est. Priority Date: 09/09/2003
• Status: Abandoned Application

• Current Assignee: Integrigen, Inc.
• Original Assignee: Integrigen, Inc.
• Inventors: SHARMA, Vikram, LEONARD, Lindsay, SMIDER, Vaughn
• Application Number:
  US14/327,486
• Publication Number:
  US 20150183853A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 16/005 constructed by phage libraries
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Abstract

The present invention relates to a method for in vitro producing polynucleotides encoding human germline antibody V-regions. Also disclosed is a library of human germline antibody V-region genes.

Claims

1. A method for producing a polynucleotide encoding a human germline antibody V-region, comprising the steps of:

(a) obtaining a V minigene or a J minigene; and

(b) joining the V minigene with at least one J minigene, or joining the J minigene with a V minigene, wherein the J minigene is located at the 3′

end of the V minigene.

2. The method of claim 1, wherein a D minigene is further joined to the 3′

end of the V minigene and the 5′

end of the J minigene.

3. The method of claim 1, wherein the V minigene or the J minigene in step (a) is obtained by chemical synthesis.

4. The method of claim 1, wherein the V minigene or the J minigene in step (a) is obtained by amplification from a germline DNA library.

5. The method of claim 1, wherein step (b) is performed by primer extension using at least two oligonucleotide primers.

6. The method of claim 2, wherein step (b) is performed by primer extension using at least three oligonucleotide primers.

7. The method of claim 5, wherein one of the primers comprises homology to both the V minigene and the J minigene.

8. The method of claim 6, wherein one of the primers comprises homology to both the V minigene and the D minigene.

9. The method of claim 6, wherein one of the primers comprises homology to both the D minigene and the J minigene.

10. The method of claim 6, wherein at least one of the oligonucleotide primers comprises degeneracy at one nucleotide position.

11. The method of claim 1, wherein the V minigene is derived from human immunoglobulin kappa locus.

12. The method of claim 1, wherein the V minigene is derived from human immunoglobulin lambda locus.

13. The method of claim 1, wherein the V minigene is derived from human immunoglobulin heavy chain locus.

14. The method of claim 1, wherein the V-region comprises a serine protease triad.

15. A library comprising member polynucleotides encoding exogenously rearranged human germline antibody V-regions.

16. The library of claim 15, wherein the germline V-regions are light chain V-regions.

17. The library of claim 16, wherein each of the light chain V-regions is operably linked to an endogenously rearranged heavy chain V-region.

18. The library of claim 15, wherein the germline V-regions are heavy chain V-regions.

19. The library of claim 18, wherein each of the heavy chain V-regions is operably linked to an endogenously rearranged light chain V-region.

20. The library of claim 15, wherein the germline V-regions comprise operably linked heavy chain and light chain V-regions.

21. The library of claim 15, which is a phage library.

22. The library of claim 15, which resides in a eukaryotic cell.

23. The library of claim 15, which is a ribosome display library.

24. The library of claim 15, which is an RNA display library.

25. The library of claim 15, which is a plasmid display library.