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Background PRAME-targeted T cell receptor (TCR) T cell therapy induces deep and durable responses in melanoma and sarcoma but has shown limited efficacy in other solid tumors. To tackle suboptimal T cell engraftment, fitness and immune suppression by the tumor microenvironment (TME), TK-6302 was developed as a best-in-class candidate. It incorporates a high-affinity TCR, a chimeric CD8 co-receptor that engages CD4 T cells and provides co-stimulation (co-stim CD8CoR), and a FAS switch receptor (SwR) to enhance T cell fitness in the periphery and resist TME-induced apoptosis. A non-viral genome-editing manufacturing process was developed to generate large numbers of fit and potent PRAME TCR-T cells with characteristics associated with clinical responses.Methods Six TK-6302 products were generated at-scale using leukaphereses from five healthy donors and one melanoma patient. CD4 and CD8 T cells were non-virally genome edited to express PRAME-TCR, co-stim CD8CoR, and a FAS SwR with simultaneous knock-out (KO) of endogenous TCR. Therapeutic cell numbers were calculated from cell count, viability, and knock-in (KI) percentage. T cell purity, memory phenotype and activation/exhaustion were assessed by flow cytometry. Bulk RNA-sequencing enabled deep characterization of TCR-T cells. Functional activity was assessed by cytokine secretion and serial killing in co-culture with 2D and 3D multi-cellular cancer spheroid models reflecting the immunosuppressive TME.Results All six TK-6302 drug products (DPs) were successfully manufactured at the highest target dose, with average viability of 94%, negligible cellular impurities, and high KI efficiency (24-42%), as measured by flow cytometry. TK-6302 DPs were highly enriched for memory cells, with ≥45% of cells exhibiting stem cell/central memory-like phenotype, and minimal PD-1 and LAG-3 expression (<10%). High and specific cytotoxicity and IFN-gamma production were observed after co-culture with cancer cells. All six DPs showed serial killing capacity, completing 3 to 8 rounds of killing after repetitive challenge with new cancer cells. Importantly, TK-6302 cells were able to efficiently eliminate multi-cellular cancer spheroids mimicking the challenges of the immunosuppressive TME. Significant differences were not observed for the analyses between runs using donor-derived or patient-derived starting material. Gene expression analysis using bulk RNA-Sequencing is ongoing, with early data indicating T cell fitness-associated gene signatures.Conclusions The non-viral genome-editing manufacturing process of TK-6302 consistently yields TCR-T product at therapeutically relevant doses. Comprehensive characterization of healthy donor- and patient derived DP showed characteristics associated with improved clinical outcomes, supporting the potential for broad, deep and durable responses in hard-to-treat indications.