Abstract C006: In vivo CRISPR-screen identifies determinants of early CAR-T cell dysfunction in solid tumors

Abstract CAR-T cell therapy has achieved remarkable clinical success for the treatment of hematologic malignancies; however, durable and sustained regressions in patients with solid tumors remain uncommon. A major cause of resistance is CAR-T cell dysfunction driven by chronic antigen exposure within the tumor site. To uncover molecular drivers of this dysfunction, we developed an in vivo CRISPR-based screen using a xenograft model that recapitulates tumor escape due to loss of CAR-T cell activity. We hypothesized that targeted ablation of the identified candidate genes would enhance CAR-T cell efficacy against solid tumors. NSG mice bearing ovarian xenograft tumors were treated with a single dose of anti-HER2 CD28-based CAR-T cells. Tumor-infiltrating T cells (TILs) were isolated short after infusion (effective phase) and after therapy failure (dysfunctional phase). Bulk RNA-seq and single-cell RNA-seq analysis were performed on dysfunctional CAR-TILs compared to effective CAR-TILs and preinfusion product. Effective CAR-TILs retained functional activity but were already transcriptionally committed to dysfunction. In contrast, dysfunctional CAR-TILs displayed phenotypic and functional hallmarks of T-cell exhaustion and a transcriptional program resembling dysfunctional TILs from cancer patients. To uncover mediators of CAR-T cell dysfunction we selected 300 significantly upregulated genes in dysfunctional CAR-TILs, generated a CRISPR/Cas9 library, and performed an in vivo loss-of-function screen in mice bearing ovarian tumors. Using the MAGeCK algorithm, we identified 14 significantly enriched candidate genes, 10 of which were upregulated in CAR-T cells at early stages after treatment. Three of the candidates, including ZC3H12C, TG, and ITGB8 were selected for individual validation. Genetic ablation of all validated candidates enhanced CAR-T cell persistence and antitumor efficacy. Single-cell RNA-seq and ATAC-seq confirmed preferential expression of these genes in exhausted T cells in our model. Analysis of previously published single-cell datasets from human TILs validated the relevance of these candidate genes in TILs undergoing exhaustion in cancer patients. Top hit in our screen ZC3H12C (Regnase-3) was further characterized under chronic antigen stimulation. Genetic ablation of ZC3H12C enhanced HER2-CD28z CAR-T cell proliferation, cytotoxicity, and cytokine production in vitro, and improved antitumor responses and persistence in vivo. CAR-TILs from ZC3H12C-KO-treated mice showed superior functionality and reduced expression of exhaustion makers. Comparable results were observed in lung and pancreatic tumor models with Mesothelin-CD28z and CD19-BBz CARs. In conclusion, by using an in vivo custom CRISPR screen, guided by transcriptional data on dysfunctional CAR-TILs, we identified novel and promising gene targets for engineering CAR-T cells with enhanced efficacy against solid tumors. Citation Format: Paula Barbao, Alba Rodriguez-Garcia, Ariel Galindo-Albarrán, Marta Giménez-Alejandre, Pablo Clavero, Marta Botas, Teresa Lobo-Jarne, Guillem Colell, Joan Castellsagué, Guim Cascalló, Irene Andreu-Saumell, Marta Soria-Castellano, Berta Marzal, Beatriz Martin-Mur, Anna Esteve-Codina, Alvaro Urbano-Ispizua, Aleix Prat, Luca Gattinoni, Marco Antonio. Mendoza-Parra, Sonia Guedan. In vivo CRISPR-screen identifies determinants of early CAR-T cell dysfunction in solid tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C006.