Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule

• US 20050255527A1
• Filed: 05/13/2005
• Published: 11/17/2005
• Est. Priority Date: 05/15/2004
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Yang, Jihong, Quarmby, Valerie Elizabeth
• Granted Patent: US 7,514,223 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/4241 against anti-human or anti-animal Ig
• CPC Class Codes: C07K 16/4258 against anti-receptor Ig
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

The invention is directed to a cross-screening system and methods of the invention utilizing a combination of an immunoassay (IA) and electrochemiluminescence assay (ECLA) to identify molecules that have binding affinities for a target molecule. The cross-screening system and methods of the invention can detect molecules that have binding affinities for the target molecule below the detection limits of the individual immunoassay or ECLA. The cross-screening system and methods of the invention are useful for generating a pool of candidate analyte molecules enriched in a desired characteristic, such as low binding affinity for a target molecule. Low affinity antibodies identified by the cross-screening system and methods of the invention are useful, for example, in assessing the safety and efficacy of biological therapeutics.

Claims

1. A method of enriching a pool of analyte molecules with candidate analyte molecules that selectively bind a target molecule, comprising:

(a) determining ECLA responses for individual members of a pool of analyte molecules binding a target molecule;

(b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule; and

(c) generating a pool of candidate analyte molecules comprising;

i) IA⁻

/ECLA⁺, and enriched for low affinity analyte molecules;

ii) IA⁺/ECLA⁺ or IA⁺/ECLA, and enriched for high affinity analyte molecules;

or

iii) IA⁺/ECLA⁻

, and enriched for analyte molecules that bind the target molecule at a binding site not recognized by ECLA.

2. The method of claim 1, wherein the pool of candidate analyte molecules is IA⁻

/ECLA⁺, and enriched for low affinity analyte molecules.

3. The method of claim 2, wherein the pool of candidate analyte molecule is IA⁺/ECLA⁺ or IA⁺/ECLA⁻

, and enriched for high affinity analyte molecules.

4. The method of claim 2, wherein the pool of candidate analyte molecules is IA⁺/ECLA⁻

, and enriched for analyte molecules that bind the target molecule at a binding site not recognized by ECLA.

6. The method of claim 1, further comprising:

(a) applying a detection limit to the analysis of the ECLA response, wherein an ECLA response equal to or greater than the ECLA detection limit identifies an ECLA⁺ analyte molecule and an ECLA response less than the ECLA detection identifies an ECLA⁻

analyte molecule; and

(b) applying a detection limit to the analysis of the ELISA response, wherein an ELISA response equal to or greater than the ELISA detection limit is ELISA⁺ and an ELISA response less than the ELISA detection limit is ELISA⁻

.

7. The method of claim 6, wherein the ELISA detection limit is 0.5 O.D. at 650 nm.

8. The method of claim 6, wherein the detection limit for the ECLA response is 250 ECLU.

9. The method of claim 1, further comprising confirming specific binding affinity of an analyte molecule selected from the enriched pool of candidate analyte molecules.

10. The method of claim 9, wherein a K_dissoc of about 10⁻

6 1/sec or less identifies a high affinity analyte molecule.

11. The method of claim 9, wherein a K_dissoc greater than about 10⁻

6 1/sec identifies a low affinity analyte molecule.

12. The method of claim 9, wherein a K_dissoc greater or equal to about 10⁻

5 1/sec identifies a low affinity analyte molecule.

13. The method of claim 9, wherein a K_dissoc greater or equal to about 10⁻

3 1/sec identifies a low affinity analyte molecule.

14. The method of claim 9, wherein a K_D equal to or greater than about 10⁻

8 M identifies a low affinity analyte molecule.

15. The method of claim 9, wherein a K_D of about 10⁻

6 M to about 10<sup>−

8</sup> M identifies a low affinity analyte molecule.

16. The method of claim 1, wherein the analyte molecules are antibodies or antigen binding portions thereof.

17. The method of claim 16, wherein the antibodies are anti-therapeutic antibodies.

18. The method of claim 1, wherein the target molecule is an antigen.

19. The method of claim 18, wherein the antigen is an antibody or antigen binding portion thereof.

20. The method of of claim 1, wherein the target molecule is an antibody or antigen binding fragment thereof.

21. The method of claim 20, wherein the antibody is a therapeutic antibody.

22. The method of claim 21, wherein the antibody binds CD20.

23. The method of claim 21, wherein the antibody binds VEGF.

24. The method of of claims 16, wherein the antibodies are monoclonal.

25. The method of claim 16, further comprising isotyping the antibodies.

26. The method of claim 16, wherein the antibodies are IgG.

27. An antibody having a K_dissoc in the range of 10⁻

2 to 10<sup>−

6</sup> prepared by the method of claim 1.

28. An antibody having a K_D in the range of 10⁻

6 M to 10<sup>−

8</sup> M prepared by the method of claim 1.

29. The antibody of claim 27, comprising an anti-therapeutic antibody.

30. The antibody of claim 28, comprising an anti-therapeutic antibody.

5. A method of identifying candidate low affinity analyte molecules from a pool of analyte molecules, comprising:

(a) determining ECLA responses for individual members of the pool of analyte molecules binding a target molecule; and

(b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule;

wherein analyte molecules that are IA⁻

/ECLA⁺ are identified as candidate low affinity molecules.