Antibody affinity engineering by serial epitope-guided complementarity replacement

• US 20050008625A1
• Filed: 02/13/2004
• Published: 01/13/2005
• Est. Priority Date: 02/13/2003
• Status: Abandoned Application

• Current Assignee: Kalobios Incorporated
• Original Assignee: Kalobios Incorporated
• Inventors: Her, Jeng-Horng, Balint, Robert F.
• Application Number:
  US10/778,726
• Publication Number:
  US 20050008625A1
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/55 Fab or Fab'

Abstract

This invention provides for a novel means for obtaining human idiologs for any non-human antibody to any target by epitope guided replacement of variable regions using competitive cell-based methods in which the competitor can be either the reference antibody or a ligand that binds to the same epitope on the target as the reference antibody.

Claims

1. An in vivo method for replacing a variable region in a reference antibody with a variable region from an antibody library, said method comprising the steps of:

a) ecombinantly altering a population of host cells by;

introducing into the host cells both a gene encoding a competitor having the binding properties of the reference antibody and a gene encoding a target molecule recognized by the competitor and reference antibody; and

,

introducing into the host cells, members of a library of genes encoding hybrid antibodies, wherein the antibodies comprising the library have a variable region from the reference antibody and a variable region from an antibody library, so that cells of the population contain different hybrid antibodies; and

,

b) etecting cells that both express the genes of step (a) and permit the binding of the hybrid antibody to the target in the presence of the competitor.

2. A method of claim 1 wherein the competitor is a reference antibody.

3. A method of claim 1 wherein the competitor is a natural ligand of the target molecule able to compete with the reference antibody for binding to the target molecule.

4. A method of claim 1 wherein the competitor is an artificial non-antibody ligand of the target molecule able to compete with the reference antibody for binding to the target binding partner.

5. A method of claim 1 wherein the competitor has a non-human Vh and a V1 region that is either human or non-human.

6. A method of claim 1 wherein the competitor is a hybrid antibody comprising human and nonhuman variable regions and the library encodes fully human antibodies.

7. A method of claim 1 where the competitor is a single chain antibody Fv fragment.

8. A method of claim 1 where the competitor is an antibody Fab fragment.

9. A method of claim 1 wherein the detection is by a self-inhibited reactivation system.

10. A method of claim 1 wherein the detection is by a competitive activation system.

11. A method of claim 1 where the test library members are bound to an activator or a detection system.

12. A method of claim 1 wherein the detection is by an enzyme fragment or subunit complementation system.

13. A method of claim 1 wherein a non-human variable region has been replaced by a human variable region.

14. A method of claim 1 wherein the selected antibody has a greater affinity for the target than does the reference antibody.

15. A method of claim 1 wherein the selected antibody is more stable than the reference antibody.

16. A method of claim 1 wherein the selected antibody has a greater specificity for the target than does the reference antibody.

17. A in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody said method comprising the steps of:

a) ecombinantly altering a population of host cells by;

i. ntroducing into the host cells both a gene encoding a reference antibody having a nonhuman Vh or V1 and a gene encoding a target binding partner recognized by the reference antibody, and

ii. introducing into the host cells, members of a library of genes encoding antibody members have a variable region of the reference antibody and a human variable region from an antibody library so that cells of the population contain different hybrid binding partners, and;

b) detecting cells that both express the genes of step (a) and permit the binding of the hybrid binding partner to the target in the presence of the reference antibody.

18. A method of claim 17 wherein the reference antibody has a non-human Vh and a V1 region that is either human or non-human.

19. A method of claim 17 wherein the reference antibody comprises a human and nonhuman variable region and the library encodes fully human antibodies.

20. A method of claim 17 wherein the reference antibody comprises a non-human V1 and a human Vh.

21. A method of claim 17 where the reference antibody is a single chain antibody.

22. A method of claim 17 where the reference antibody is a Fab.

23. A method of claim 17 wherein the detection is by a self-inhibited reactivation system.

24. A method of claim 17 wherein the detection is by a competitive reactivation system.

25. A method of claim 17 where the library members are bound to an activator or a detection system.

26. A method of claim 17 wherein the detection is by a complementation system.

27. A method of claim 17 wherein the humanized antibody has a greater affinity for the target than the competitor binding pair partner.

28. A population of cells comprising:

i. recombinantly introduced gene encoding a competitor with the binding properties of a reference antibody;

ii. recombinantly introduced gene encoding a target molecule recognized by the competitor; and

,

iii. library of genes encoding hybrid antibodies, wherein the antibodies comprising the library have a variable region from the reference antibody and a variable region from a natural antibody repertoire, so that cells of the population contain different hybrid antibodies.

29. A population of recombinantly altered cells of claim 28 comprising:

i. gene encoding a competitor that is a reference antibody having a nonhuman Vh or V1;

ii. gene encoding a target by the reference antibody; and

,

ii. alibrary of genes encoding antibodies having a variable region of the reference antibody and a human variable region from an antibody library so that cells of the population contain different hybrid binding partners;

30. A population of claim 28 wherein the library members are attached to a part of multipart reporter system.

31. A population of claim 28 wherein the target molecule has a self-inhibited reporter attached to it.