RT Journal Article SR Electronic T1 Src/lck inhibitor dasatinib reversibly switches off cytokine release and T cell cytotoxicity following stimulation with T cell bispecific antibodies JF Journal for ImmunoTherapy of Cancer JO J Immunother Cancer FD BMJ Publishing Group Ltd SP e002582 DO 10.1136/jitc-2021-002582 VO 9 IS 7 A1 Gabrielle Leclercq A1 Hélène Haegel A1 Anneliese Schneider A1 Anna Maria Giusti A1 Estelle Marrer-Berger A1 Christophe Boetsch A1 Antje-Christine Walz A1 Vesna Pulko A1 Johannes Sam A1 John Challier A1 Cristiano Ferlini A1 Alex Odermatt A1 Pablo Umaña A1 Marina Bacac A1 Christian Klein YR 2021 UL http://jitc.bmj.com/content/9/7/e002582.abstract AB Background T cell engagers are bispecific antibodies recognizing, with one moiety, the CD3ε chain of the T cell receptor and, with the other moiety, specific tumor surface antigens. Crosslinking of CD3 upon simultaneous binding to tumor antigens triggers T cell activation, proliferation and cytokine release, leading to tumor cell killing. Treatment with T cell engagers can be associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release syndrome (CRS). Tyrosine kinases such as SRC, LCK or ZAP70 are involved in downstream signaling pathways after engagement of the T cell receptor and blocking these kinases might serve to abrogate T cell activation when required (online supplemental material 1). Dasatinib was previously identified as a potent kinase inhibitor that switches off CAR T cell functionality.Methods Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of dasatinib combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB, CD19-TCB or HLA-A2 WT1-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. To determine the effective dose of dasatinib, the Incucyte system was used to monitor the kinetics of TCB-mediated target cell killing in the presence of escalating concentrations of dasatinib. Last, the effects of dasatinib were evaluated in vivo in humanized NSG mice co-treated with CD19-TCB. The count of CD20+ blood B cells was used as a readout of efficacy of TCB-mediated killing and cytokine levels were measured in the serum.Results Dasatinib concentrations above 50 nM prevented cytokine release and switched off-target cell killing, which were subsequently restored on removal of dasatinib. In addition, dasatinib prevented CD19-TCB-mediated B cell depletion in humanized NSG mice. These data confirm that dasatinib can act as a rapid and reversible on/off switch for activated T cells at pharmacologically relevant doses as they are applied in patients according to the label.Conclusion Taken together, we provide evidence for the use of dasatinib as a pharmacological on/off switch to mitigate off-tumor toxicities or CRS by T cell bispecific antibodies.The authors declare that all relevant data to the study are included in the article or uploaded as online supplemental information.