Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia.

BACKGROUND: Chimeric Antigen Receptor (CAR)-T cell therapy holds considerable promise for the treatment of CD7+ T cell malignancies. However, a major challenge limiting clinical development of CD7-targeted CARs has been fratricide, a process of self-cytotoxicity caused by the shared expression of CD7 on malignant and healthy cells. Current solutions, including CD7 gene editing, intracellular retention or cell-sorting strategies, add significant complexity and cost, thereby limiting the feasibility and cost-effectiveness of this therapy. In this study, we evaluated whether fratricide can instead be overcome by tailoring standard manufacturing protocols to generate effective CAR-T cell products. Specifically, we report the GMP-compliant development of UMCG-001, a non-proprietary, academically generated, third-generation ligand-based CD7-targeting autologous CAR-T product. METHODS: Production of UMCG-001 was explored using standard manufacturing protocols with various cytokine mixtures and GMP-grade human plate lysate (HPL). A GMP-compliant process was optimized with both healthy donor and patient material and subsequently transferred to our GMP facility. The anti-leukemic activity of UMCG-001 was assessed in vitro and in vivo, followed by in-depth multi-omics characterization to evaluate the impact of the fratricide phase on product quality. RESULTS: Supplementation with human platelet lysate during the ex vivo expansion phase effectively restored expansion and improved viability of fratricide-driven CAR⁺CD7Low/Neg cells. These unedited cells exhibited robust CD7-specific antileukemic activity and achieved ~ 90% prolonged survival in a T-ALL xenograft model. Notably, spectral flow and single-cell RNA sequencing revealed enrichment of a central memory phenotype and a high TCR diversity, with increased activation/proliferation profile. Importantly, the exhaustion profile of UMCG-001 remained comparable to benchmark CD19 CAR-T. These results confirmed that the fratricide selection process maintained a diverse and functionally competent T cell repertoire. Finally, UMCG-001 batches were successfully manufactured from autologous patient-derived material with no detectable contamination through malignant blasts. CONCLUSIONS: These data support the feasibility of adapting standard manufacturing protocols to overcome fratricide and demonstrate the potential for clinical translation of scalable, unedited UMCG-001 cells for the treatment of T-cell malignancies.