Mateus-Tique, J. | Search bioRxiv

bioRxiv2025

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

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.

immunology↗

bioRxiv2021

Targeting macrophages with CAR-T cells delays solid tumor progression and enhances anti-tumor immunity

Tumor-associated macrophages (TAMs) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably to PD1 blockade, and significantly extended mouse survival. Anti-tumor effects were mediated by F4.CAR-T-produced IFN-{gamma}, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigens and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof-of-principle evidence to support CAR-T targeting of TAMs as a means to enhance antitumor immunity.

immunology↗