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Background T cell bispecific antibodies (TCBs) are extremely potent T cell engagers, harboring a 2+1 format with one binder to the CD3e chain and two binders to specific tumor antigens. Crosslinking of CD3 with tumor antigens triggers T cell activation, proliferation and cytokine release, leading to tumor cell killing.1 2 TCB treatment is sometimes associated with safety liabilities due to on-target on-tumor, on-target off-tumor cytotoxic activity and cytokine release. Patients treated with TCBs may 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.3 Off-tumor toxicity may occur if target antigens are expressed in healthy cells, which may potentially result in tissue damages and compromise the patient‘s safety. Rapid pharmacological blockade of T cell activation and proliferation is a promising approach to mitigate these life-threatening toxicities. 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. Dasatinib was identified as a potent candidate that switches off CAR T cell functionality.4 5 Methods Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the reversible effects of dasatinib combined with CEA-TCB or HLA-A2-WT1-TCB on T cell activation and proliferation, target cell killing and cytokine release. At assay endpoints, T cell phenotype and target cell killing were measured by flow cytometry and supernatants were analyzed by Luminex to assess cytokine release. To determine the effective dose of dasatinib, the Incucyte system was used to follow kinetics of target cells killing by TCB in the presence of a dose response of dasatinib concentrations. Results 100 nM dasatinib prevented TCB-mediated target cell killing when added in the system upon restimulation of activated T cells (figure 1). Dasatinib concentrations above 50 nM fully switched off target cell killing (figure 2) which was restored upon removal of dasatinib. These data confirm that dasatinib act as a potent and reversible on/off switch for activated T cells at pharmacologically relevant doses as they are applied in patients according to the label.6 Conclusions 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 engaging therapies. These data are being validated in vivo. References Bacac M, Fauti T, Sam J, Colombetti S, Weinzierl T, Ouaret D, et al. A novel carcinoembryonic antigen T-Cell Bispecific Antibody [CEA TCB] for the treatment of solid tumors. Clin Cancer Res 2016;22(13):3286–97. Bacac M, Klein C, Umana P. CEA TCB: A novel head-to-tail 2:1 T cell bispecific antibody for treatment of CEA-positive solid tumors. Oncoimmunology 2016;5(8):e1203498. Shimabukuro-Vornhagen A, Godel P, Subklewe M, Stemmler HJ, Schloser HA, Schlaak M, et al. Cytokine release syndrome. J Immunother Cancer 2018;6(1):56. Weber EW, Lynn RC, Sotillo E, Lattin J, Xu P, Mackall CL. Pharmacologic control of CAR-T cell function using dasatinib. Blood Advances 2019;3(5):711–7. Mestermann K, Giavridis T, Weber J, Rydzek J, Frenz S, Nerreter T, et al. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Science Translational Medicine 2019;11(499):eaau5907. Wang X, Roy A, Hochhaus A, Kantarjian HM, Chen TT, Shah NP. Differential effects of dosing regimen on the safety and efficacy of dasatinib: retrospective exposure-response analysis of a Phase III study. Clinical pharmacology : advances and applications 2013;5:85–97.

Background75% of diagnosed bladder tumors are non-muscle-invasive (NMIBC)[1, 2]. Most require intravesical instillation of M.bovis Bacillus Calmette-Guérin (BCG). Recurrence after immunotherapy occurs in ~50% patients. Development of treatments for BCG-resistant disease has lagged partly because few studies have attempted to understand the relationship between timing of tumor recurrence, reasoning for the recurrence, and the state of immune system at the time of recurrence.Immune exhaustion is observed following microbial infections, cancers and chronic inflammation [3–5]. Natural Killer (NK) cells are among the earliest responders[6–8] and undergo a similar program of exhaustion as T cells[9]. HLA-E strongly inhibits NKG2A-expressing NK and CD8+T cells and is commonly upregulated on tumors[10]. We evaluated the potential restorative capacity of NKG2A and PD-L1-blockade for reinvigorating NK and CD8+T cell antitumor functions in in BCG-resistant bladder cancer.Methods mRNA analysis of 2,892 genes was performed on tumor tissue of NMIBC patients before and after BCG therapy (n=35). Immunostaining (serial-IHC,immunofluorescence,imaging-mass cytometry) was performed on consecutive tissue sections. Single-cell-RNA-sequencing (scRNAseq) was performed on fresh bladder tumors (NMIBC,n=4; MIBC,n=9). OLink Proteomics (”Inflammation” panel) was performed longitudinally on plasma/urine from a prospective cohort of NMIBC patients. Patient tumors (n=3) were expanded as organoids and co-cultured with autologous tumor-derived NK and CD8+T cells in presence/absence of anti-PD-L1/NKG2A antibodies.ResultsWe demonstrate a robust local TME and systemic response to BCG that correlates with chronic inflammation and adaptive resistance rather than with preventing tumor recurrence. This resistance is mediated through IFN-γ-production by tumor-infiltrating NKG2A+NK and NKG2A+PD-1+CD8+T cells and results in increased HLA-E and PD-L1 on recurring tumors. Co-culture of treatment-naïve NMIBC tumors with recombinant IFN-gamma directly enhanced expression of PD-L1 and HLA-E. Longitudinal analysis of plasma before and during BCG immunotherapy revealed an inflammatory signature, including but not limited to IFN-gamma, that is maintained throughout treatment.Immunostaining and scRNAseq of NMIBC specimens revealed highly enriched infiltration by NKG2A+NK and NKG2A+CD8+T cells in HLA-EBrightPD-L1+ tumors and were spatially organized relative to tumors in a manner suggesting direct inhibition. Tumor-derived NK and CD8+T cells from BCG-resistant patients were co-cultured with autologous tumor organoids. Preliminary analyses demonstrated an improved anti-tumor response in presence of NKG2A/PD-L1-blockade.ConclusionsOur data support a model of BCG-resistance that points to a novel checkpoint axis that contributes to BCG-resistance: HLA-E/NKG2A. New insights into this axis in NMIBC and how it is altered with repeated BCG exposure will enable us to explore combination therapies (PD-L1/NKG2A-blockade) that may reduce BCG-resistance and provide durable response.ReferencesEidinger D, Morales A: Discussion paper: treatment of superficial bladder cancer in man. Ann N Y Acad Sci 1976, 277:239–240.Morales A, Eidinger D, Bruce AW: Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Urol 1976, 116:180–183.Blank CU, Haining WN, Held W, Hogan PG, Kallies A, Lugli E, Lynn RC, Philip M, Rao A, Restifo NP et al: Defining ‘T cell exhaustion’. Nat Rev Immunol 2019, 19:665–674.Hashimoto M, Kamphorst AO, Im SJ, Kissick HT, Pillai RN, Ramalingam SS, Araki K, Ahmed R: CD8 T Cell Exhaustion in Chronic Infection and Cancer: Opportunities for Interventions. Annu Rev Med 2018, 69:301–318.McLane LM, Abdel-Hakeem MS, Wherry EJ: CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu Rev Immunol 2019, 37:457–495.Lanier LL: NK cell receptors. Annu Rev Immunol 1998, 16:359–393.Biron CA, Gazzinelli RT: Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr Opin Immunol 1995, 7:485–496.Welsh RM, Jr.: Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction. J Exp Med 1978, 148:163–181.da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, Kuchroo VK, Osman I, Bhardwaj N: Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol Res 2014, 2:410–422.van Hall T, Andre P, Horowitz A, Ruan DF, Borst L, Zerbib R, Narni-Mancinelli E, van der Burg SH, Vivier E: Monalizumab: inhibiting the novel immune checkpoint NKG2A. J Immunother Cancer 2019, 7:263.Ethics ApprovalPrimary urothelial bladder cancer tumor tissue was obtained after obtaining informed consent in the context of an Institutional Review Board (IRB)-approved genitourinary cancer clinical database and specimen collection protocol (IRB #10-1180) at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai (New York, NY).