Work

166 CARD8- and SRCAP- deficient CAR T-cells promote durable responses and limit functional exhaustion

Year: 2026

Type: conference-abstract

Abstract: Background Chimeric antigen receptor (CAR) T-cell therapy can induce deep and durable remissions in patients with B-cell malignancies; however, most ultimately relapse due to insufficient CAR T-cell persistence and development of exhaustion following prolonged antigen exposure. 1 Patients achieving >8-year remissions in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) retain CAR T-cells with remarkable proliferative capacity and functional durability despite chronic stimulation.2 3 However, the molecular programs supporting this exceptional clinical benefit remain incompletely understood. We previously reported a CLL patient in whom the CAR lentivector disrupted TET2 in a dominant CD8 CAR T-cell clone, conferring marked proliferative advantage and long-term persistence, with this expanded clone believed to be the principal driver of the durable response.4 Subsequent studies identified integration sites associated with clinical response, although their functional relevance remained unclear.5 Using a patient-informed CRISPR-Cas9 screening strategy, we identified CARD8 and SRCAP as negative regulators of CAR T-cell fitness and evaluated their functional contribution to CAR T-cell activity.Methods A focused CRISPR-Cas9 library targeting 180 genes enriched at lentiviral vector integration sites was incorporated into a one-shot anti-CD19 BBζ-CAR construct. CAR T-cells were generated from healthy donor T-cells, enriched, electroporated with Cas9 ribonucleoproteins (RNPs) or mock treated, expanded, and evaluated in chronic stimulation assays and in an immunocompromised (NSG) NALM6-GFP-luciferase xenograft model. Candidate genes were ranked using the drugZ algorithm and validated individually using Cas9-RNP editing (>80% knockout efficiency). Mice received doses associated with robust tumor control of unedited CAR T-cells or suboptimal doses of CARD8- or SRCAP-knockout CAR T-cells. Tumor burden, CAR T-cell persistence, phenotype, and transcriptional profiles were assessed longitudinally.Results Edited CAR T-cells demonstrated enhanced proliferation following repeated antigen stimulation in vitro and improved tumor control in vivo. Enriched hits converged on pathways involved in MAPK signaling, DNA damage, apoptosis, migration, and cell-cycle. Individual validation confirmed that disruption of CARD8 and SRCAP enhanced tumor clearance and survival. CARD8-deficient CAR T-cells displayed reduced frequencies of PD-1+ TIGIT+ CD39+ exhausted CD8 T-cells, whereas SRCAP-deficient CAR T-cells exhibited increased frequencies of PD-1/TIGIT/CD39 triple-negative CD8 T-cells and enrichment of effector-memory populations at endpoint. RNA-sequencing from in vivo samples demonstrated activation of T-cell receptor signaling and immune activation programs in CARD8-deficient cells and enhanced cell-cycle programs in SRCAP-deficient cells.Conclusions CARD8 and SRCAP regulate CAR T-cell persistence and function. Disruption of either gene enhanced antitumor activity, improved persistence, and reduced features of T-cell dysfunction, supporting their development as targets for next-generation CAR T-cell therapies.References Cappell,KM, Sherry RM, Yang JC, Goff SL, Vanasse DA, McIntyre L, Rosenberg SA, Kochenderfer JN. Long-term follow-up of anti-CD19 chimeric antigen receptor T-cell therapy. J Clin Oncol. 2020;38:3805–3815.Bai Z, Feng B, McClory SE, de Oliveira BC, Diorio C, Gregoire C, Tao B, Yang L, Zhao Z, Peng L, et al. Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission. Nature 2024;634:702–711.Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MA, Gao P, Bandyopadhyay S, Sun H, Zhao Z, et al. Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature 2022;602:503–509.Fraietta JA, Nobles CL, Sammons MA, Lundh S, Carty SA, Reich TJ, Cogdill AP, Morrissette JJD, DeNizio JE, Reddy S, et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature 2018;558:307–312.Nobles CL, Sherrill-Mix S, Everett JK, Reddy S, Fraietta JA, Porter DL, Frey N, Gill SI, Grupp SA, Maude SL, et al. CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration. J Clin Invest. 2020;130:673–685.Ethics Approval Human T cells were isolated from commercially purchased, de-identified leukopaks from healthy donors. The use of these materials is exempt from IRB review. All lentiviral and gene-editing procedures were conducted in accordance with protocols approved by the Cleveland Clinic Institutional Biosafety Committee (Protocol # 2210). Animal experiments were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Protocol # 3023).

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0166