Simultaneous gene engineering of T cells with multiple neoantigen-specific T cell receptors for adoptive cell transfer.

T cell receptor (TCR) T cell therapy targeting tumor-specific antigens and neoantigens can mediate impressive tumor regressions in solid tumors. However, solid epithelial tumors are characterized by inherent tumor heterogeneity that can lead to tumor escape and thwart TCR T cell therapy. Targeting multiple cancer antigens in a single TCR T product may help overcome the antigen heterogeneity among metastatic deposits. However, the time and expense of preparing multiple TCR-transduced T cell populations under good manufacturing practice (GMP) for patient treatment has limited this approach to target 1 or 2 antigens. Here, we design strategies to enhance TCR T cell therapy efficacy by simultaneously targeting multiple neoantigens. We developed two novel manufacturing processes to introduce multiple neoantigen-specific TCRs into T cells, using gamma-retroviral delivery. These strategies resulted in multipotent neoantigen-reactive TCR T cell products, which demonstrated functionality against multiple tumor neoantigens and displayed cytotoxicity against heterogeneous tumor cells in vitro. High-dimensional single-cell and single-clone T cell analysis confirmed the presence of multiple TCR-expressing T cells in the products and demonstrated functional recognition of multiple neoantigens. This study, thus, addresses the challenges of tumor heterogeneity and immune escape mechanisms in solid tumors, paving the way for more effective TCR T cell therapies in cancer treatment.