Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro

Introduction CD19 directed chimeric antigen receptor T cell (CART19) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia (ALL). With the emergence of therapeutic anti CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CART19 to exert their antitumor effect in a subsequent therapy. Tafasitamab is an Fc enhanced humanized anti CD19 monoclonal antibody which mediates antibody dependent cellular toxicity (ADCC), antibody dependent cellular phagocytosis and direct cytotoxicity. It is currently being studied in phase 2/3 clinical trials in diffuse large B cell lymphoma (DLBCL) in combination with lenalidomide and bendamustine. Objectives To investigate the potential for functional interference between tafasitamab and CART19. Methods CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ). In vitro functional activity of tafasitamab was assessed through killing assays against CD19+ cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM6 (ALL). To study whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition we incubated NALM6 or JEKO with up to 100 μg/ml tafasitamab, to saturate the receptors, and then performed flow cytometry using FMC63 anti CD19 antibody (it carries the same CD19 binding domain as CAR19). To investigate the potential impact of such binding competition on CART19 cell effector functions, we cocultured tafasitamab with JEKO at increasing concentrations of up to 100 μg/ml, and then added CART19 at different effector to target (E:T) ratios to the cell culture. Results In a 24 hours ADCC (Fig 1A) and T cell cytotoxicity assays (CART19, E:T titrations) distinct activity was observed for both therapies on all tested cell lines. Secondly, after testing the CD19 binding competition, FMC63 antibody failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Fig 1B). Next, after coculturing tafasitamab and CART19 in JEKO, it was evident that the presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Fig 1C), degranulation (Fig 1D), cytokine production or proliferation of CART19 (Fig 1E). Conclusions Our studies indicate that CART19 cells continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. The question of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models.