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Background Adoptive cell therapy (ACT) using T cell receptor-engineered T cells (TCR-T cell) represents a promising strategy for treating solid tumors by redirecting autologous T cells to recognize tumor-specific neoantigens. While TCR-T cell therapies have demonstrated clinical activity, tumor heterogeneity and HLA loss remain key barriers to durable responses. Consequently, targeting multiple neoantigens simultaneously may enhance therapeutic efficacy. However, current manufacturing processes, which require parallel production and testing of multiple single-TCR-transduced cell products, are time- and resource-intensive, limiting the feasibility of administering multi-neoantigen-specific TCR-T (multi-TCR-T) cell therapy in clinical settings.Methods We developed two novel manufacturing approaches to streamline the generation of TCR-T cell products that target multiple neoantigens simultaneously: (1) pooled plasmid (ppl), where plasmids encoding distinct neoantigen-specific TCRs are combined before retroviral packaging; and (2) pooled supernatant (psup), where individually generated viral supernatants are mixed prior to T cell transduction. These were compared with the currently clinically used pooled-cell (pcell) method where individual TCR-T cell populations are generated separately and pooled post-expansion. Functional activity, neoantigen specificity, and TCR representation were assessed using peptide-loaded antigen presenting cell (APC) co-cultures, tumor killing assays and high-dimensional flow cytometry analysis.Results Multi-TCR-T cells generated via ppl and psup methods expressed multiple functional TCRs and recognized their cognate neoantigens in both CD8 + and CD4+ T cell compartments. The pooled TCR-T products showed neoantigen-specific activation (measured by 4-1BB upregulation) and interferon gamma (IFNg) production comparable to that of single TCR-transduced cells when co-cultured against all target peptides simultaneously. The ppl and psup products demonstrated robust activation and cytotoxicity against heterogeneous tumor cells. TCR specific HLA-tetramer staining assay confirmed the co-expression of multiple TCRs within individual T cells in the pooled products. Compared to the pooled cell method, ppl and psup strategies offered comparable functionality with reduced production complexity.Conclusions Our findings demonstrate that multifunctional TCR-T cell products can be manufactured via simplified retroviral engineering approaches for simultaneous targeting of multiple patient-specific neoantigens in a single product. We aim to apply these manufacturing strategies to enhance the therapeutic efficacy in our upcoming TCR-T cell clinical trials.