Riboflavin biosynthesis byproducts increase the vulnerability of cancer to adoptive MAIT TCR-engineered T-cell transfer

Introduction: Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes expressing a semi-invariant T-cell receptor (TCR) alpha-chain (TRAV1-2 joined to TRAJ33/12/20) paired with a restricted TCR beta repertoire. Unlike conventional T cells that respond to peptide antigens, MAIT cells recognize and kill infected or transformed cells presenting non-polymorphic MHC class-I (MR1) molecule covalently loaded with microbial riboflavin (vitamin B2) biosynthetic intermediates. A key riboflavin precursor, 5-amino-6-D-ribitylaminouracil (5-A-RU), undergoes non-enzymatic condensation to form potent MR1 ligands such as 5-OP-RU, which can prime tumor cells to express MR1. These features make MAIT TCRs an attractive platform for adoptive T-cell therapy across cancer types, independent of HLA molecules or tumor genetics. Methods: Human CD3+ T cells isolated from healthy donor peripheral blood mononuclear cells (PBMC) were engineered to express MAIT TCRs (TRAV1-2/TRAJ12 or TRAV1-2/TRAJ33) using a CRISPR-Cas12-mediated homology-directed repair (HDR) strategy. TCR knockout ( TRAC / TRBC double-knockout) T cells lacking any introduced TCR served as controls. Antitumor activity was assessed in vitro and in vivo. Cytotoxicity against the human B-cell acute lymphoblastic leukemia line NALM6 was measured by flow cytometry. For in vivo evaluation, luciferase-expressing NALM6 cells were injected intravenously into NOD/SCID/ Il2rg −/− (NSG) mice, followed by infusion of engineered T cells. A 5-A-RU prodrug was administered intraperitoneally. Tumor burden was monitored by bioluminescent imaging. Results: In vitro, MAIT TCR-engineered human T cells exhibited distinct baseline cytotoxicity against NALM6 cells (approximately 60-70% for J12 and ~10% for J33 in the absence of ligand). Addition of 5-A-RU resulted in near-complete tumor cell elimination for both MAIT TCR variants. In xenograft models, neither J12 nor J33 MAIT TCR T cells showed significant antitumor activity without exogenous 5-A-RU. However, intraperitoneal delivery of the 5-A-RU prodrug had no effect in TCR-knockout controls but markedly enhanced the antitumor activity of both MAIT TCR-engineered T-cell products. Mice receiving MAIT TCR T cells plus 5-A-RU showed delayed tumor progression and significantly improved survival compared with controls. Conclusion: MAIT TCR engineering confers potent antitumor activity to human T cells, which is strongly augmented by riboflavin pathway metabolites such as 5-A-RU. These findings support a model in which metabolite supplementation prime tumor cells to express MR1 and redirect MAIT TCR-engineered T cells cytotoxic activity towards tumor. Further studies are underway to define the therapeutic window, evaluate potential toxicities, and identify the breadth of tumor types responsive to MAIT TCR-based adoptive cell therapy.