Comparative analysis of monoclonal antibody-drug conjugate binding by flow cytometry

Flow cytometric methods for the evaluation of the cell surface binding properties of monoclonal antibody (MoAb)-drug/toxin conjugates defining tumor-associated antigens are presented. In these techniques, suspension cultures of solid human tumor cell lines are incubated with either varying dilutions of MoAb or MoAb-drug conjugates followed by FITC-conjugated anti-mouse immunoglobulin antibodies in an indirect assay or with FITC-conjugated MoAbs specific for the tumor target cell line in a competition assay. The amount of fluorescent probe bound is measured by flow cytometry and the mean fluorescence intensity determined. The relative binding capacity is quantified by linear regression of the mean fluorescence versus the concentration of primary antibody or antibody conjugate. The application of these techniques to several drug and toxin conjugates of MoAb KS1/4, which defines a human adenocarcinoma-associated antigen, demonstrates that these assays can be effectively utilized to monitor the effects of covalent chemical modification on a MoAb's antigen binding reactivity.