Abstract 40: Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit
Authors: Yi Fei , Cohen Tal , Zumbo Paul , Eltilib Razan , Arkin Hannah , Gormally Michael , Hackett Christopher S. , Kropp Korbinian , Chandran Smita , Park Jae H. , Betel Doron , Klebanoff Christopher A. 
Published: March 2024

Abstract
Adoptive cell transfer of chimeric antigen receptor (CAR) engineered T and NK cells has revolutionized the treatment of many B-lymphoid malignancies; however, limited persistence of infused lymphocytes can restrain the full potential of this approach. Apoptosis induction through the FAS ligand/FAS pathway is a key negative regulator of lymphocyte survival. To define which cellular subsets express FASLG, the gene encoding FAS ligand, we generated a single-cell transcriptomic atlas from 37 patients with diverse cancer types. We discovered that FASLG expression is highly constrained and limited primarily to T and NK cells while stromal and tumor cells express minimal to no FASLG. Multiplex RNA in situ hybridization of clinical samples from patients treated with anti-CD19 CAR-T cells confirmed these results and revealed significantly higher FASLG content in CAR-expressing compared with non-CAR expressing T cells. Based on these findings, we hypothesized that FAS ligand expression by CAR+ T and NK cells will engage FAS+CAR+ cells to induce apoptosis and limit cellular persistence. To test this hypothesis, we performed competitive fitness assays using two phenotypically discernable populations of human CAR-T and CAK-NK cells that are either responsive or unresponsive to FAS-signaling through expression of a FAS dominant negative receptor (ΔFAS). ΔFAS+ and ΔFAS− CAR-expressing cells were co-infused 1:1 into tumor bearing hosts and persistence of each population was quantified. For both CAR-T and CAR-NK, we measured significant enrichment in ΔFAS-expressing cells across multiple tissues, demonstrating that FAS-signaling regulates CAR+ cell survival in a cell-intrinsic manner. Knock out (KO) of FASLG using CRISPR/Cas9 prevented population skewing while KO of the AAVS1 control locus had no effect, indicating that CAR-derived FASLG is sufficient to restrain the persistence of transferred cells. By contrast, we discovered that FASLG was dispensable for CAR-T and CAR-NK mediated tumor killing both in vitro and in vivo. Finally, using two different tumor models, we found that adoptive transfer of CAR-T or CAR-NK cells that co-express ΔFAS resulted in superior antitumor efficacy compared with control cells that express the CAR alone. Taken together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS/FAS ligand auto-regulatory circuit. Further, they indicate that the therapeutic benefit of a FAS-dominant negative receptor is broadly applicable and agnostic of whether FASLG is expressed by cells in the tumor microenvironment.
Citation Format: Fei Yi, Tal Cohen, Paul Zumbo, Razan Eltilib, Hannah Arkin, Michael Gormally, Christopher S. Hackett, Korbinian Kropp, Smita Chandran, Jae H. Park, Doron Betel, Christopher A. Klebanoff. Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 40.