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

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. 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. 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. 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.