Dual-antigen recognition iPSC-derived CAR-T cells for B-cell malignancies: establishment of a COVID-19 vaccine synergy strategy.

INTRODUCTION: CD19-directed chimeric antigen receptor (CD19-CAR) T-cell therapy has markedly improved outcomes in relapsed and refractory B-cell malignancies, but its efficacy remains limited by insufficient in vivo persistence and functional exhaustion. We have generated functionally rejuvenated T-cells (rejTs) by reprogramming antigen-specific cytotoxic T lymphocytes (CTLs) into induced pluripotent stem cells (iPSCs) and redifferentiating them into CTLs with restored proliferative capacity. In this study, we explored a vaccine synergy strategy to enhance the persistence of CAR-rejuvenated CTLs (CARrejTs) through T-cell receptor (TCR) restimulation. METHODS: SARS-CoV-2 spike protein-specific rejTs (COVID19-rejTs) were established from iPSCs derived from spike protein-specific CTLs. A CD19-CAR was introduced into these iPSCs to generate dual-antigen recognition CARrejTs targeting CD19 and COVID-19 spike protein (1919-CARrejTs). Subsequently, 1919-CARrejTs were assessed for cytotoxicity, proliferative capacity, and exhaustion phenotype using (51)Cr release assays, sequential rechallenge assays, and CFSE-based proliferation analysis with CAR- or TCR-dependent stimulation. RESULTS: 1919-CARrejTs uniformly expressed both CD19-CAR and spike protein-specific TCRs, retained antigen-specific cytotoxicity, and exhibited a rejuvenated phenotype with higher expression of granzyme B and perforin and lower expression of exhaustion markers compared with conventional CD19-CAR-T cells. Dual-antigen recognition enhanced cytotoxicity under matched antigen presentation, and 1919-CARrejTs maintained durable tumor control in sequential rechallenge assays. CFSE dilution analysis revealed that TCR-mediated stimulation by spike protein-specific peptide provided strong proliferative capacity of 1919-CARrejTs in an HLA-dependent manner. CONCLUSION: The combination of iPSC-mediated rejuvenation and dual-antigen recognition via CAR and native TCR confers superior cytotoxicity, persistence, and proliferative potential compared to conventional CD19-CAR-T cells. These findings provide a proof-of-concept for a vaccine-synergy strategy in which in vivo TCR restimulation supports selective expansion and sustained antitumor effect of dual-antigen recognition T-cells that can be a promising treatment approach for B-cell malignancies.