Immunoglobulin libraries

Patent metadata

Field	Value
Publication number	US-9244070-B2
Application number	US-201313856291-A
Country	US
Kind code	B2
Filing date	Apr 3, 2013
Priority date	Nov 30, 2006
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

Methods and compositions for the screening and isolation of ligand-binding polypeptides, such as antibodies. In some aspects, methods of the invention enable the isolation of intact soluble antibodies comprising a constant domain. Screening methods that employ genetic packages such as bacteria and bacteriophages enable high through-put identification of ligand binding molecules.

First claim

What is claimed is:

1. A method for isolating a genetic package comprising a DNA encoding a ligand-binding polypeptide comprising the steps of:
a) obtaining a population of genetic packages comprising a plurality of nucleic acid sequences encoding distinct ligand-binding polypeptides and encoding a second polypeptide that binds non-covalently to such ligand-binding polypeptides, wherein individual packages of said population further comprise a ligand-binding polypeptide encoded by the nucleic acid sequence of that package in complex with the encoded second polypeptide that binds non-covalently to the ligand-binding polypeptide;
b) contacting said population with a target ligand; and
c) selecting at least one genetic package based on binding of a distinct ligand-binding polypeptide encoded by said nucleic acid sequence to the ligand.

2. The method of claim 1 , wherein the ligand-binding polypeptide is an antibody.

3. The method of claim 2 , wherein the antibody is a single chain antibody, a Fab fragment or scFv.

4. The method of claim 2 , wherein the antibody comprises a heavy chain.

5. The method of claim 1 , wherein the antibody is an intact antibody.

6. The method of claim 1 , wherein the antibody does not comprise a light chain polypeptide.

7. The method of claim 1 , whereby the antibody comprises the VL and Ck or CL domains of an antibody.

8. The method of claim 1 , wherein the antibody comprises VH, CH1 and CH2 domains of an antibody.

9. The method of claim 1 , wherein the genetic package is a bacteriophage.

10. The method of claim 9 , wherein the bacteriophage is a filamentous phage.

11. The method of claim 10 , wherein the filamentous phage is bacteriophage M13.

12. The method of claim 1 , where the genetic package is a bacterial cell.

13. The method of claim 12 , where the bacterial cell is a Gram negative bacterial cell.

14. The method of claim 13 , where the Gram negative bacterial cell is an E coli cell.

15. The method of claim 13 , wherein the distinct ligand-binding polypeptide is comprised in the periplasm of the Gram negative bacterial cell.

16. The method of claim 13 , further comprising (ii) disrupting the outer membrane of the bacterial cells before contacting the bacterial cells with a target ligand.

17. The method of claim 16 , wherein disrupting the outer membrane of the bacterial cell further comprises removing the outer membrane of said bacterium.

18. The method of claim 1 , wherein target ligand is labeled.

19. The method of claim 18 , wherein target ligand is labeled with a fluorophore, a radioisotope or an enzyme.

20. The method of claim 1 , wherein target ligand is immobilized.

21. The method of claim 1 , wherein the selecting of step (iv) is further defined as comprising at least two rounds of selection wherein the sub-population of genetic packages obtained in the first round of selection is subjected to at least a second round of selection based on the binding of the distinct ligand-binding polypeptide to the target ligand.

22. The method of claim 21 , comprising two to ten rounds of selection.

23. The method of claim 1 , wherein the selecting is carried out by FACS or magnetic separation.

24. The method of claim 1 , further comprising contacting the genetic packages with at least two target ligands.

25. The method of claim 24 , wherein the at least two target ligands comprise distinct labels.

26. The method of claim 24 , further comprising selecting genetic packages based on binding of the distinct antibody to at least two target ligands.

27. The method of claim 24 , further comprising selecting genetic packages based on binding of the distinct ligand-binding polypeptide to at least a first target ligand and based on the distinct ligand-binding polypeptide not binding to at least a second target ligand.

28. The method of claim 1 , further comprising removing target ligand not bound to the distinct ligand-binding polypeptide.

29. The method of claim 1 , wherein the second polypeptide that binds to the ligand-binding polypeptide comprises an antibody-binding domain.

30. The method of claim 29 , wherein the antibody-binding domain binds to the constant region (Fc) of an antibody.

31. The method of claim 30 , wherein the antibody-binding domain binds to a Fc of an IgG antibody.

32. The method of claim 29 , wherein the antibody-binding domain comprises a mammalian, bacterial or synthetic Fc binding domain.

33. The method of claim 32 , wherein the bacterial Fc binding domain is an S aureus protein A or protein G domain.

34. The method of claim 32 , wherein the synthetic Fc binding domain is ZZ polypeptide.

35. The method of claim 13 , wherein the second polypeptide that binds to the ligand-binding polypeptide is anchored to the inner membrane of the Gram negative bacterial cell.

36. The method of claim 35 , wherein the second polypeptide that binds to the ligand-binding polypeptide comprises a membrane anchoring polypeptide fused to the N or C terminus of the polypeptide.

37. The method of claim 36 , wherein the membrane anchoring polypeptide is the first six amino acids encoded by the E coli NlpA gene, the gene III protein of filamentous phage or a fragment thereof, an inner membrane lipoprotein or fragment thereof.

38. The method of claim 37 , wherein the membrane anchoring polypeptide is an inner membrane lipoprotein or fragment thereof selected from the group consisting of: AraH, MglC, MalF, MalG, MalC, MalD, RbsC, RbsC, ArtM, ArtQ, GlnP, ProW, HisM, HisQ, LivH, LivM, LivA, LivE, DppB, DppC, OppB, AmiC, AmiD, BtuC, ThuD, FecC, FecD, FecR, FepD, NikB, NikC, CysT, CysW, UgpA, UgpE, PstA, PstC, PotB, PotC, PotH, Pod, ModB, NosY, PhnM, LacY, SecY, To 1 C, Dsb, B, DsbD, TouB, TatC, CheY, TraB, ExbD, ExbB and Aas.

39. The method of claim 1 , wherein the target ligand is a cancer-associated protein, a cell surface protein, an enzyme, a virus, a glycoprotein or a cell receptor ligand.

40. The method of claim 13 , wherein the population of Gram negative bacterial cells is produced by a method comprising the steps of: (a) preparing a plurality of nucleic acid sequences encoding a plurality of ligand-binding polypeptides; and (b) transforming a population of Gram negative bacteria with said nucleic acid sequences wherein the Gram negative bacterial cells comprise the ligand-binding polypeptide in the periplasm along with a second polypeptide that binds to the ligand-binding polypeptide.

41. The method of claim 1 , further defined as a method of producing a nucleic acid sequence encoding a ligand-binding polypeptide and further comprising the step of:
v) cloning a nucleic acid sequence encoding the ligand-binding polypeptide from the genetic package to produce a nucleic acid sequence encoding a ligand-binding polypeptide.

42. The method of claim 41 , wherein cloning comprises amplification of the nucleic acid sequence.

43. The method of claim 41 , further defined as a method of producing ligand-binding polypeptide having a specific affinity for a target ligand further comprising the step of:
vi) expressing the nucleic acid sequence enc

Assignees

• Res Dev Foundation

Inventors

• Georgiou George
• Mazor Yariv

Classifications

• C07K2317/565

  Complementarity determining region [CDR] · CPC title

• C07K16/1278

  Bacillus (G) · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C07K16/16

  against material from plants · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title