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bioRxiv · 10.1101/2025.09.22.677496

Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

Lin, M. J.·Chorazeczewski, J. K.·Pantsulaia, G.·Coooper, A.·Sohn, M.·Reci, S.·Mateus-Tique, J.·Hirsh, N. H.·Xie, X.·Odak, I.·Dutta, R.·Charytonowicz, D.·Upadhyay, R.·Brown, B. D.·Huse, M.·Kline, J.·Brody, J. D.
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Abstract

Antigen (Ag) escape is a frequent mechanism of relapse after CAR-T therapy, even though only [~]1% of leukemic and [~]0.1% of lymphoma cells are Ag- at baseline. In this study, we modeled extreme Ag heterogeneity (>20%) to define how Fas/FasL-dependent bystander killing contributes to tumor clearance. Across patient cohorts, Fas expression predicted survival after CD19 CAR-T therapy, particularly in CD19-low disease. In both murine and human systems, Fas-dependent bystander killing required Ag stimulation and cell contact, operated within a defined therapeutic window, and could eradicate large fractions of Ag- tumors in vivo. Pharmacologic potentiation with inhibitor of apoptosis protein antagonists or genetic stabilization of CAR-T membrane-bound FasL enhanced bystander killing but simultaneously induced CD4 T cell fratricide, which was rescued by CAR-T Fas knockout. Importantly, Fas sensitization also enabled bispecific antibody-redirected T cells to mediate bystander killing in resistant tumors. Finally, targeting tumor-associated macrophages triggered Fas-dependent clearance of neighboring tumor cells. These findings establish Fas-mediated bystander killing as a generalizable and therapeutically actionable axis to prevent Ag escape and broaden the scope of targeted T cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/677496v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@11bedorg.highwire.dtl.DTLVardef@d09d07org.highwire.dtl.DTLVardef@1f53c1dorg.highwire.dtl.DTLVardef@13d96f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Fas-mediated bystander killing can be potentiated by IAP inhibition and FasL stabilization, and further improved by decoupling CD4 T cell fratricide via Fas knockout. Schematic representation of CAR-T bystander killing across three mechanistic states. Left (Baseline): Antigen-positive (CD19) tumor cells activate CAR-T cells, leading to FasL-mediated cytotoxicity of neighboring antigen-negative (CD19-) tumor cells via membrane-bound FasL. Limited FasL expression and ADAM10-mediated shedding constrain bystander killing, and CD4 T cells are largely spared. Middle (Potentiated): Enhancing Fas signaling through IAP inhibition (IAPi), increasing FasL expression, and reducing FasL shedding result in improved bystander killing but also increased CD4 T cell fratricide. Right (Potentiated & Decoupled): Combining IAPi with FasL stabilization and CAR-T Fas knockout (FasKO) augments bystander killing while eliminating CD4 T cell fratricide.

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Lin, M. J., Chorazeczewski, J. K., Pantsulaia, G., Coooper, A., Sohn, M., Reci, S., Mateus-Tique, J., Hirsh, N. H., Xie, X., Odak, I., Dutta, R., Charytonowicz, D., Upadhyay, R., Brown, B. D., Huse, M., Kline, J., Brody, J. D.. 2025-09-24. Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors. https://doi.org/10.1101/2025.09.22.677496

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