Flow Cytometry As A Single Platform Tool To Evaluate Multiple Mechanisms Of Actions Of Therapeutic Antibodies

The unique ability of flow cytometry to simultaneously examine intricate details of multiple cell subsets is unparalleled and the platform is now a dominant tool for measuring antibody mediated effector functions on cancer cells. The objective of this study is to illustrate the utility of flow cytometry as a single platform for concurrently measuring the multiple mechanisms of actions of therapeutic antibodies such as ADCC, ADCP, Apoptosis, CDC and Trogocytosis on human cancer cells and immune functioning cells. Rituximab® was tested with the Burkett9s Lymphoma cell line (Daudi) for all assays between the ranges of 0.01 and 100ug/mL. Apoptosis was evaluated by measuring Caspase 3/7 staining. ADCC assays were established with Rituximab®/CFSE labeled Daudi cells and human PBMC’s. ADCP was assessed with Rituximab®/ CFSE labeled Daudi cells and CD14+/CD11b+ in vitro generated phagocytes using a 4 hour exposure. CDC assays were established with Daudi cells/ 10% pooled human serum using 7-AAD as the cytotoxic indicator in a 4 hour assay. Trogocytosis was measured up to 4 hours with Rituximab®/ Daudi and PBMC9s and CD19/20 and CD14. All assays were analyzed using a FACS ARIA III flow cytometer and replicates of 3 were evaluated for statistical relevance. Results demonstrated that Rituximab® induced effects were observed in all 5 assays using flow cytometry. Apoptosis was induced when 0.01ug/mL Rituximab® was present. ADCP occurred at levels as low as 0.01ug/mL Rituximab®. ADCC and CDC occurred at levels as low as 0.1ug/mL Rituximab®. Trogocytosis, indicated by the transfer of CD19+ B cells to CD14+ monocytes, occurred at 0.1ug/ml Rituximab®. In summary, we demonstrated the clinical applicability of flow cytometry as a single platform to simultaneously evaluate five different mechanisms of actions that can occur when therapeutic antibodies are used to treat cancer cells. Citation Format: Antony R. Chadderton, Shane Harvey, Brandy Strake, Jill Giles-Komar, Renold J. Capocasale, T. Shantha Raju. Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3518.