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

• US 7,514,223 B2
• Filed: 05/13/2005
• Issued: 04/07/2009
• 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
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Cheu; Changhwa J
• Application Number:
  US11/128,981
• Publication Number:
  US 20050255527A1
• Time in Patent Office: 1,425 Days
• Field of Search: 435/7.1, 435/7.2, 435/7.91, 435/7.92
• US Class Current: 435/7.1
• 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 electrochemiluminescence assay (ECLA) responses for individual members of a pool of analyte molecules binding a target molecule;

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

) analyte molecule;

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

(d) applying a detection limit to the analysis of the IA responses, wherein an IA response equal to or greater than the IA detection limit is immunoassay positive (IA⁺) and an IA response less than the IA detection limit is immunoassay negative (IA⁻

);

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

(i) immunoassay negative and electrochemiluminescence assay positive molecules (IA⁻

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

(ii) immunoassay positive and electrochemiluminescence assay positive molecules (IA⁺/ECLA⁺) or immunoassay positive and electrochemiluminescence assay negative molecules (IA⁺/ECLA⁻

), and enriched for high affinity analyte molecules;

or

(iii) immunoassay positive and electrochemiluminescence as say negative molecules (IA⁺/ECLA⁻

), and enriched for analyte molecules that bind the target molecule at a binding site not recognized by electrochemiluminescence assay (ECLA); and

(f) confirming specific binding affinity of an analyte molecule selected from the enriched pool of candidate analyte molecules.

2. The method of claim 1, wherein the pool of candidate molecules is immunoassay negative and electrochemiluminescence assay positive (IA⁻

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

3. The method of claim 1, wherein the pool of candidate analyte molecules is immunoassay positive and electrochemiluminescence assay positive (IA⁺/ECLA⁺) or immunoassay positive and electrochemiluminescence assay negative (IA⁺/ECLA⁻

), and enriched for high affinity analyte molecules.

4. The method of claim 2, wherein the pool of candidate analyte molecules is immunoassay positive and electrochemiluminescence assay negative (IA⁺/ECLA⁻

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

8. The method of claim 1, wherein a K_dissoc of about 10⁻

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

9. The method of claim 1, wherein a K_dissoc greater than about 10⁻

6 1/sec identifies a low affinity analyte molecule.

10. The method of claim 1, wherein a K_dissoc greater or equal to about 10⁻

5 1/sec identifies a low affinity analyte molecule.

11. The method of claim 1, wherein a K_dissoc greater or equal to about 10⁻

3 1/sec identifies a low affinity analyte molecule.

12. The method of claim 1, wherein a K_D equal to or greater than about 10⁻

8 M identifies a low affinity analyte molecule.

13. The method of claim 1, wherein a K_D of about 10⁻

6 M to about 10<sup>−

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

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

15. The method of claim 14, wherein the antibodies are anti-therapeutic antibodies.

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

17. The method of claim 16, wherein the antigen is an antibody or antigen binding portion thereof.

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

19. The method of claim 18, wherein the antibody is a therapeutic antibody.

20. The method of claim 19, wherein the antibody binds CD20.

21. The method of claim 19, wherein the antibody binds vascular endothelial growth factor (VEGF).

22. The method of claims 14, wherein the antibodies are monoclonal.

23. The method of claim 14, further comprising isotyping the antibodies.

24. The method of claim 14, wherein the antibodies are IgG.

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

2 1/sec to 10<sup>−

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

26. 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.

27. The antibody of claim 25, comprising an anti-therapeutic antibody.

28. The antibody of claim 26, 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;

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

) analyte molecule;

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

(d) applying a detection limit to the analysis of the IA responses, wherein an IA response equal to or greater than the IA detection limit is immunoassay positive (IA⁺) and an IA response less than the IA detection limit is immunoassay negative (IA⁻

);

wherein analyte molecules that are immunoassay negative and electrochemilunilnescence assay positive (IA⁻

/ECLA⁺); and

(e) confirming specific binding affinity of an analyte molecule selected from the candidate low affinity molecules are identified as candidate low affinity molecules.

6. The method of claim 5, wherein the IA is ELISA and the ELISA detection limit is 0.5 O.D. at 650 nm.

7. The method of claim 5, wherein the detection limit for the ECLA response is 250 electrochemiluminescence units (ECLU).