Methods for screening antibodies

Patent metadata

Field	Value
Publication number	US-9725500-B2
Application number	US-201113581236-A
Country	US
Kind code	B2
Filing date	Feb 28, 2011
Priority date	Mar 2, 2010
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

Abstract

The invention provides methods for making antibody conjugates for use in antibody screening assays and antibody conjugates produced by the claimed methods.

First claim

What is claimed:

1. A method for making antibody conjugates for use in high throughput screening assays comprising the steps of:
providing a plurality of antibody-containing samples that vary with respect to antibody quantity and antibody sequence provided that, in a majority of the antibody-containing samples, substantially all of the antibody present in each of the samples is of the same sequence;
immobilizing the antibodies of the antibody-containing samples on a solid support to provide a plurality of antibody-containing samples comprising immobilized antibodies;
fully reducing the reducible disulfide bonds of the immobilized antibodies to provide a plurality of samples comprising reduced immobilized antibodies; wherein the reduction is selective for reducible disulfide bonds;
reacting the reduced immobilized antibodies with capping agent, drug or drug-linker, and optionally a detection agent to provide immobilized antibody conjugates; wherein the capping agent, drug or drug-linker, and optional detection agent selectively react with reactive thiols, the capping agent, drug or drug-linker, and optional detection agent are provided in molar excess, and the ratio of capping agent, drug or drug linker, and optional detection agent is selected so as to achieve a desired level of drug loading; and
eluting the immobilized antibody conjugates to provide a plurality of antibody conjugate samples.

2. The method of claim 1 wherein the reducible disulfide bonds are naturally occurring.

3. The method of claim 1 wherein the antibodies have substantially the same number of reducible disulfide bonds.

4. The method of claim 1 wherein the quantity of antibody present in the antibody-containing samples prior to immobilizing, reduction, conjugation, and elution is not known.

5. The method claim 1 wherein the antibody-containing samples have from 1 μg to 100 μg of antibody present in the sample.

6. The method of claim 1 wherein the antibody containing samples have from 1 μg to 20 μg of antibody present in the sample.

7. The method claim 1 wherein the capping agent, drug or drug-linker and detection agent comprise a maleimide group.

8. The method of claim 1 wherein the drug-linker loading between samples is substantially uniform.

9. The method of claim 1 wherein the antibody conjugate samples have an average drug-linker loading of about 4 drug-linkers per antibody.

10. The method of claim 1 wherein the antibody-containing samples are unpurified CHO cell culture supernatant.

11. The method of claim 1 wherein the antibody-containing samples are cell culture supernatant samples and_substantially all of the cell culture media used for antibody production was IgG depleted media.

12. The method of claim 1 further comprising the step of:
assaying for an activity of the antibody conjugates and making a comparison between the antibodies that constitute the antibody conjugates based on an activity of the antibody conjugates.

13. The method claim 12 wherein the activity is cytotoxicity.

14. The method of claim 1 wherein the antibody-containing samples are unpurified hybridoma supernatant samples comprising unquantified antibody produced from hybridoma clones and wherein substantially all of the antibody present in each sample is from a single hybridoma clone.

15. The method of claim 14 wherein substantially all of the cell culture media used for antibody production was IgG depleted media.

16. The method of claim 14 wherein there is from 1 μg to 100 μg of antibody present in each sample of hybridoma supernatant.

17. The method of claim 14 wherein there is from 1 μg to 50 μg of antibody present in each sample of hybridoma supernatant.

18. The method of claim 14 further comprising the steps of (i) determining the actual or relative quantity of antibody present in the antibody conjugate samples; (ii) assaying an activity of the antibody conjugates; (iii) selecting an antibody based on the results of the assay and the actual or relative quantity of antibody present in the antibody conjugate samples.

19. The method of claim 14 wherein the detection agent is a fluorescent label.

20. The method of claim 14 wherein the capping agent, drug or drug-linker and detection agent comprise a maleimide group.

21. The method of claim 1 further comprising the steps of:
performing a cytotoxicity assay on the_antibody conjugates; and selecting an antibody of the basis of the outcome of the assay.

22. A method for making antibody conjugates for use in antibody screening assays comprising the steps of:
providing a plurality of antibody containing samples that vary with respect to antibody quantity and antibody sequence provided that, in a majority of the plurality of the antibody containing samples, substantially all of the antibody present in each of the samples is of the same sequence;
immobilizing the antibodies of the antibody containing samples_on a solid support to provide a plurality of samples comprising immobilized antibodies;
fully reducing the reducible disulfide bonds of the immobilized antibodies to provide a plurality of samples comprising reduced immobilized antibodies, wherein the reduction is selective for reducible disulfide bonds;
reacting the reduced immobilized antibodies with capping agent, and a detection agent to provide a plurality of samples comprising immobilized antibody conjugates, wherein the capping and detection agent selectively react with reactive thiols, the capping agent, and detection agent are provided in molar excess, and the ratio of capping agent and detection agent is selected so as to achieve a desired level of capping agent and/or detection agent loading; and
eluting the antibody conjugates to provide a plurality of antibody conjugate samples comprising free antibody conjugates.

23. The method of claim 22 further comprising the steps of:
performing a cytotoxicity assay on the antibody conjugates; and selecting an antibody of the basis of the outcome of the assay.

Assignees

• Lyon Robert
• Benjamin Dennis
• Ryan Maureen
• Seattle Genetics Inc

Inventors

• Lyon Robert
• Benjamin Dennis
• Ryan Maureen

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K1/04

  on carriers {(C07K1/003, C07K1/006 take precedence)} · CPC title

• G01N33/533

  with fluorescent label · CPC title