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Abstract
Background T-cell based immunotherapies such as CAR-T, bispecific mAb, transgenic T cells and checkpoint blockade have profound efficacy in multiple tumor types but share a common limitation – target antigen (Ag) escape.1 2 One approach to address this limitation has been therapy directed at a ‘parallel’ target (e.g. CD22 after CD19 loss), however, these lineage markers are frequently lost together.3 Here, we describe an alternate, broadly applicable, approach: potentiating fasL/fas-signaling to increase localized bystander killing of Ag-tumor cells and thereby prevent Ag escape.
Methods We used a CRISPR/Cas9 library to screen for tumor expressed molecules that inhibit or facilitate T-cell killing. We then evaluated one candidate -fas- using murine transgenic T cells, murine and human CAR-T cells, bispecific mAb redirected PBMC, and tumoral RNAseq data from a large CAR-T clinical trial.
Results GFP-specific (JEDI) CD8 T cells were co-cultured with on-target (GFP+) and bystander (mCherry+) lymphoma cells that had been transfected with a CRISPR/Cas9 library; this screen revealed several tumor-expressed candidate molecules inhibiting or facilitating T-cell killing. Notably, we observed a marked dependence on fas for on-target tumor killing and then, surprisingly, an exquisite dependence on fas for localized bystander tumor killing. (figure 1).Because bystander tumor killing appeared critically fas-dependent, we hypothesized that potentiating fas-signaling might increase bystander killing. An in vitro screen of small molecules that modulate fas-pathway revealed several candidates, including inhibitors of histone deacetylases (HDAC), inhibitors of apoptosis proteins (IAP) and Bcl-2 family members in murine and human systems (figure 2). To validate these candidates, we demonstrated that HDACi increased GFP-specific T cell killing of both on-target and bystander lymphoma cells, in a completely fas-dependent manner (figure 3). Similarly, using a bispecific antibody-based system, we demonstrated increased, fas-dependent, T cell killing of both on-target and bystander human lymphoma cells with inhibitors of IAP and bcl-2 family members (e.g. MCL1).
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Conclusions T-cell mediated tumor killing can be potentiated with fas pathway modulators. This augmentation improves both fas-dependent Ag+ and Ag-tumor cell death. Further studies of modulating the fas pathway alongside T-cell based immunotherapies are needed as potential treatments to prevent antigen escape and improve patient outcomes.
Acknowledgements We thank the flow cytometry core facility, microscopy core facility, and the CCMS animal facility at ISMMS.
Ethics Approval The studies were approved by The Mount Sinai Institutional Review Board.
References
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Majzner R, Mackall C. Tumor antigen escape from CAR T-cell therapy. Cancer Discov 2018;8(10):1219–1226.
Jacoby E, Nguyen S, Fountaine T, et al. CD19 CAR immune pressure induces B-precusor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat Commun 2016; 7:12320.
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