Gattinoni, L. | Search bioRxiv

bioRxiv2026

In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

CAR-T cell therapy has shown limited efficacy in solid tumors, largely due to T cell dysfunction driven by chronic antigen exposure. To uncover mediators of this dysfunction, we developed an in vivo screening platform using an ovarian xenograft tumor model in which CD28-based CAR-T cells undergo exhaustion leading to tumor escape. Transcriptomic profiling of tumor-infiltrating CAR-T cells at different stages revealed dynamic upregulation of exhaustion-associated genes. We used this data to design a focused CRISPR/Cas9 library and performed an in vivo screen. We identified 14 significantly enriched candidate genes, among which ZC3H12C emerged as the top hit. Single-cell RNA and ATAC-seq confirmed ZC3H12C expression in CAR-T cells undergoing early exhaustion in vivo. ZC3H12C disruption enhanced CAR-T cell persistence and antitumor efficacy while reducing exhaustion, across both CD28- and 4-1BB-based CARs targeting distinct antigens. These results highlight ZC3H12C as a promising target to improve CAR-T therapy in solid tumors.

immunology↗

bioRxiv2026

Targeting ZC3H12C improves T cell persistence and antitumor function in adoptive T cell therapy

Adoptive T cell therapy (ACT) has achieved remarkable clinical responses in hematologic malignancies but remains limited by progressive T cell dysfunction under chronic antigen stimulation. Here, we identify ZC3H12C as a conserved feature of dysfunctional T cells and show that its disruption enhances the durability and antitumor activity of engineered T cells. By integrating single-cell chromatin accessibility and transcriptomic profiling of human tumor-infiltrating lymphocytes (TILs), we identified the ZC3H12C locus as selectively remodeled in exhausted T cells. ZC3H12C induction is largely absent across acute T cell activation contexts, indicating regulation that is specific to chronic antigen-driven dysfunction. Genetic disruption of ZC3H12C improves T cell expansion, cytotoxicity, and expression of effector molecules during repeated in vitro stimulation, translating into enhanced tumor control in vivo across both T cell receptor (TCR) and chimeric antigen receptor (CAR) T cell therapy platforms. Improved efficacy is observed in hematologic, solid, and metastatic tumor models and is accompanied by increased T cell persistence. Further, ZC3H12C is enriched in clinical pre-infusion CAR T cell products associated with non-response. Together, these findings identify ZC3H12C as a T cell dysfunction-specific target to improve ACT performance.

immunology↗