P09.01 The use of FDA approved JAK, mTOR and Src inhibitors to regulate T cell-bispecific antibody-induced cytokine release while not preventing T cell cytotoxicity (2021)

<h3>Background</h3> T cell bispecific antibodies (TCBs) are potent T cell engagers, harboring a 2+1 format with one binder to the CD3ε chain and two binders to specific tumor antigens. Crosslinking of CD3 with tumor antigens triggers T cell activation and proliferation, cytokine release and tumor cell killing. TCB treatment is sometimes associated with safety liabilities due to on-target on-tumor or on-target off-tumor cytotoxicity and cytokine release. Off-tumor activity of the TCB may occur if the targeted tumor antigens are expressed on healthy cells, which may potentially result in tissue damages and compromise the patient’s safety. Patients treated with TCBs may also experience a Cytokine Release Syndrome (CRS), characterized by fever, hypotension and respiratory deficiency and associated with the release of pro-inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and IL-1β. Tyrosine kinases such as Src, mTOR and JAK1/2 are involved in downstream signaling pathways after engagement of the T cell receptor. <h3>Materials and Methods</h3> 52 FDA approved kinase inhibitors were screened in the presence of T cells activated on CD3 coated plates, mimicking TCB stimulation. Src, mTOR and JAK inhibitors were selected based on their capacity to prevent both, cytokine release and T cell proliferation. Using an <i>in vitro</i> model of target cell killing by human peripheral blood mononuclear cells stimulated with TCBs, we validated the effects of mTOR, JAK and Src kinase inhibitors on TCB-induced T cell activation, tumor cell killing and cytokine release. <i>In vivo</i>, the effect of mTOR, JAK and Src kinase inhibitors on TCB-induced cytokine release was confirmed in humanized NOD scid gamma (NSG) mice engrafted with human hematopoietic stem cells and treated with CD19-TCB. <h3>Results</h3> In line with previous reports for CAR-T cells, dasatinib (a src inhibitor) was found to fully switch off TCB-induced T cell functionality as well as the other src inhibitors bosutinib and ponatinib. In contrast, temsirolimus, sirolimus and everolimus (mTOR inhibitors) and ruxolitinib, baricitinib, tofacitinib, and fedratinib (JAK1/2 inhibitors) were found to more potently prevent TCB-induced cytokine release without blocking TCB-mediated target cell killing. <h3>Conclusions</h3> These results provide evidence that the mechanisms of TCB-dependent cytokine release and tumor cell killing can be uncoupled. The FDA-approved mTOR and JAK1/2 inhibitors could potentially be used to mitigate CRS whereas the Src inhibitor dasatinib could rather stand as a potential antidote for on-target off-tumor activity or high-grade CRS. <h3>Disclosure Information</h3> <b>G. Leclercq:</b> A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. <b>H. Haegel:</b> A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. <b>A. Schneider:</b> A. Employment (full or part-time); Modest; Roche. <b>A. Giusti:</b> A. Employment (full or part-time); Modest; Roche. <b>V. Pulko:</b> A. Employment (full or part-time); Modest; Roche. <b>A. Toso:</b> A. Employment (full or part-time); Modest; Roche. <b>T. Zimmermann:</b> A. Employment (full or part-time); Modest; Roche. <b>L. Green:</b> A. Employment (full or part-time); Modest; Roche. <b>N. Steinhoff:</b> A. Employment (full or part-time); Modest; Roche. <b>J. Sam:</b> A. Employment (full or part-time); Modest; Roche. <b>M. Bacac:</b> A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. <b>P. Umaña:</b> A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche. <b>C. Klein:</b> A. Employment (full or part-time); Modest; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Roche.