Methods for screening antibodies

Abstract

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

What is claimed: 
     
         1 . A method for making antibody conjugates for use in antibody screening assays, the method comprising the steps of:
 (a) providing a first and second antibody-containing sample, wherein the first and second antibody-containing samples vary with respect to antibody quantity and antibody sequence provided that substantially all of the antibody present in the first sample is of the same sequence and substantially all of the antibody present in the second sample is of the same sequence, wherein the antibodies of the first and second samples have sulfhydryl groups introduced into said antibodies;   (b) immobilizing the antibodies on solid support to provide a first and second sample comprising immobilized antibodies;   (c) fully reducing the interchain disulfide bonds and introduced sulfhydryl groups of the immobilized antibodies to provide a first sample comprising reduced immobilized antibodies and second sample comprising reduced immobilized antibodies;   (d) 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   (e) eluting the antibody conjugates to provide a first and second antibody drug conjugate composition.   
     
     
         2 . The method of  claim 1  wherein the antibody in the first and second antibody-containing samples have the same number of introduced sulfhydryl groups and the reduced immobilized antibodies are contacted with the same ratio of capping agent, drug or drug-linker, and optional detection agent. 
     
     
         3 . The method of  claim 1  wherein the antibody in the first and second antibody-containing samples have different number of introduced sulfhydryl groups and the reduced immobilized antibodies are contacted with a different ratio of capping agent, drug or drug-linker, and optional detection agent. 
     
     
         4 . The method of  claim 1  wherein the reduced immobilized antibodies are reacted with a detection agent. 
     
     
         5 . The method of  claim 4  wherein the detection agent is a fluorophore and the resultant antibody conjugates compositions have an average fluorophore loading of about 3 fluorophores per antibody. 
     
     
         6 . The method of  claim 1  wherein following elution of the antibody conjugates, the actual or relative quantity of antibody present in the antibody conjugate compositions is determined. 
     
     
         7 . The method of  claim 1  wherein the antibody present in the antibody-containing samples prior to immobilization is impure. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 1  wherein the antibody-containing samples independently have from 1 μg to 100 μg of antibody present in each of the sample. 
     
     
         10 . The method of  claim 9  wherein the antibody-containing samples independently have from 1 μg to 50 μg of antibody present in each of the samples. 
     
     
         11 . The method of  claim 10  wherein the antibody containing samples independently have from 1 μg to 20 μg of antibody present in each of the samples. 
     
     
         12 . The method of  claim 1  wherein the antibody in the first antibody-containing sample and the antibody in the second antibody-containing sample are of the same species. 
     
     
         13 . The method of  claim 1  wherein the capping agent, drug or drug-linker and detection agent comprise a maleimide group. 
     
     
         14 . The method of  claim 13  wherein the drug-linker loading between samples is substantially uniform. 
     
     
         15 . The method of  claim 14  wherein the antibody conjugate compositions have an average drug-linker loading of about 2 drug-linkers per antibody. 
     
     
         16 . The method of  claim 1  wherein the antibody-containing samples are cell culture supernatant samples. 
     
     
         17 . The method of  claim 16  wherein the cell culture supernatant is unpurified hybridoma cell culture supernatant. 
     
     
         18 . The method of  claim 16  wherein the cell culture supernatant is unpurified CHO cell culture supernatant. 
     
     
         19 . The method of  claim 16  wherein the cell culture media used for providing the antibody-containing samples was substantially IgG depleted. 
     
     
         20 . The method of  claim 16  wherein the solid support is added to the cell culture supernatant. 
     
     
         21 . A method for making antibody conjugates for use in high throughput screening assays comprising the steps of:
 (a) providing a plurality of samples of unpurified hybridoma supernatant comprising unquantified antibody produced from a plurality of hybridoma clones, wherein the plurality of samples vary with respect to antibody quantity and antibody sequence provided that, in a majority of the plurality of the samples, substantially all of the antibody present in each sample is from a single hybridoma clone wherein the antibodies of the plurality of samples have sulfhydryl groups introduced into said antibodies;   (b) immobilizing the unquantified antibodies on solid support to provide a plurality of samples comprising immobilized antibodies;   (c) fully reducing the intrachain disulfide and introduced sulfhydryl groups of the immobilized antibodies to provide a plurality of samples comprising reduced immobilized antibodies;   (d) reacting the reduced immobilized antibodies with capping agent, drug or drug-linker, and a detection agent to provide immobilized antibody conjugates, wherein the capping agent, drug or drug-linker, and 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 detection agent is selected so as to achieve a desired level of drug loading; and   (e) eluting the immobilized antibody conjugates from the solid supports to provide a plurality of antibody conjugate compositions.   
     
     
         22 . The method of  claim 21  wherein the cell culture media used for production of the antibody-containing samples was substantially IgG depleted. 
     
     
         23 . The method  claim 21  wherein there is from 1 μg to 100 μg of antibody present in each sample of hybridoma supernatant. 
     
     
         24 . The method  claim 23  wherein there is from 1 μg to 50 μg of antibody present in each sample of hybridoma supernatant. 
     
     
         25 . The method of  claim 24  wherein there is from 1 μg to 20 μg of antibody present in each sample of hybridoma supernatant. 
     
     
         26 . The method of  claim 21  wherein the detection agent is a fluorescent label. 
     
     
         27 . The method of  claim 21  wherein the capping agent, drug or drug-linker and detection agent comprise a maleimide group. 
     
     
         28 . The method of  claim 21  wherein the drug-linker loading between samples is substantially uniform. 
     
     
         29 . A method for preparing antibody conjugates, the method comprising the steps of:
 (a) 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 a single sample is of the same sequence;   (b) immobilizing the antibodies on solid support to provide a plurality of samples comprising immobilized antibodies;   (c) fully reducing the interchain disulfide bonds and introduced sulfhydryl groups of the immobilized antibodies to provide a plurality of samples comprising reduced immobilized antibodies,   (d) reacting the reduced immobilized antibodies with capping agent, drug or drug-linker, and optionally a detection agent to provide a plurality of samples comprising 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   (e) eluting the antibody conjugates to provide a plurality of antibody conjugate compositions.   
     
     
         30 . A method for selecting an antibody for use in an antibody drug conjugate, the method comprising the steps of:
 (a) 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 a single sample is of the same sequence;   (b) immobilizing the antibodies on a solid support to provide a plurality of samples comprising immobilized antibodies;   (c) fully reducing the intrachain disulfide bonds and introduced sulfhydryl groups of the immobilized antibodies to provide a plurality of samples comprising reduced immobilized antibodies;   (d) reacting the reduced immobilized antibodies with capping agent, drug or drug-linker, and optionally a detection agent to provide a plurality of samples comprising 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;   (e) eluting the antibody conjugates to provide a plurality of antibody conjugate compositions comprising free antibody conjugates;   (f) assaying for an activity of the antibody conjugates;   (g) determining the actual or relative quantity of antibody present in the antibody conjugate compositions;   (h) selecting the antibody providing antibody drug conjugates with the greatest activity based on the results of the assay and the actual or relative quantity of antibody present in the antibody conjugate compositions.   
     
     
         31 . The method of  claim 30 , wherein the activity is cytotoxicity.