A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy

Abstract

T cell-based therapies have induced cancer remissions, though most tumors ultimately progress, reflecting inherent or acquired resistance including antigen escape. Better understanding of how T cells eliminate tumors will help decipher resistance mechanisms. We used a CRISPR/Cas9 screen and identified a necessary role for Fas-FasL in antigen-specific T-cell killing. We also found that Fas-FasL mediated off-target "bystander" killing of antigen-negative tumor cells. This localized bystander cytotoxicity enhanced clearance of antigen-heterogeneous tumors in vivo, a finding that has not been shown previously. Fas-mediated on-target and bystander killing was reproduced in chimeric antigen receptor (CAR-T) and bispecific antibody T-cell models and was augmented by inhibiting regulators of Fas signaling. Tumoral FAS expression alone predicted survival of CAR-T-treated patients in a large clinical trial (NCT02348216). These data suggest strategies to prevent immune escape by targeting both the antigen expression of most tumor cells and the geography of antigen-loss variants.SigNifiCANCe: This study demonstrates the first report of in vivo Fas-dependent bystander killing of antigen-negative tumors by T cells, a phenomenon that may be contributing to the high response rates of antigen-directed immunotherapies despite tumoral heterogeneity. Small molecules that target the Fas pathway may potentiate this mechanism to prevent cancer relapse. intRoductionT cell-based immunotherapies-including adoptive transfer of engineered T cells, bispecific antibodies, and checkpoint blockade-have revolutionized cancer treatment. However, even with the remarkably high response rates of chimeric antigen receptor (CAR)-T-treated patients, most either progress or relapse within one year (1-3). Microenvironmental factors contributing to T-cell priming (4-6) and T cell-intrinsic factors (7, 8) both influence antitumor immunity, but tumor cell-intrinsic factors have the most abundant clinical evidence for contributing to treatment potency and failures.The clearest such mechanism is target antigen (Ag) modulation-expression downregulation, lineage switching, or emergence of splice variants-which is the most common cause of relapse following CAR-T therapy for B-cell acute

Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma

“…26,41 Cytokine-mediated cell death in the presence of IAP antagonists may include cell-to-cell contact-dependent engagement of the TNF superfamily (TNF/TRAIL/FAS) and their receptors, a mechanism implicated in T cell-dependent bystander killing. 42 Third, we show that tolinapant treatment can promote T-cell infiltration into the tumor. In preclinical models, a proinflammatory tumor microenvironment with an increased CD8 1 T-cell infiltration was detected after tolinapant treatment.…”

CD19 target evasion as a mechanism of relapse in large B-cell lymphoma treated with axicabtagene ciloleucel

“…Our findings align with the ability of CD28-based CARs to control low antigen tumors. 11-13 Alternate mechanisms underlying target-related evasion reported in the literature are disrupted CD19 cell membrane transport after endoplasmic reticulum, 11,14 accidental insertion of CARs in tumor cells, 15 or possible suboptimal bystander FAS-mediated killing of CD19 − cells, 16 further driving their selection. This mechanism of relapse complements other recently described mechanisms consisting of suboptimal product T-cell fitness and CAR expansion in vivo, 3 or the role of immune TME-related factors that may influence outcomes.…”

Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis

“…Ectopic FAS expression at the tumor site was recently shown to improve CAR T cell efficacy against embryonal carcinomas by enabling antigen-independent FASLdependent tumor cell lysis [40]. Evidence for FAS-dependent bystander killing of antigennegative tumors by T cells was also delivered by Upadhyay et al, who postulated that combining CAR T cells with small molecules targeting the FAS pathway may potentiate this mechanism [57]. Monoclonal antibodies that act as a FAS agonist have also been developed as an alternative strategy to activate tumor cell killing via the FAS-FASL axis.…”