ABSTRACT: Adoptive T cell therapy has emerged as a promising strategy for treating solid tumors. However, its clinical application remains constrained by major limitations, including manufacturing complexity in autologous settings and risks of graft-versus-host disease (GvHD), HLA restriction and donor variability in allogeneic contexts. To address these challenges, we present a scalable platform for the generation of hematopoietic stem and progenitor cell (HSPC)-derived, mono-specific cytotoxic T cells targeting the NY-ESO-1 antigen. Using an ex vivo, feeder-free, serum-free culture system, we differentiate gene-engineered HSPCs into allogeneic T cells (AlloESO-T) with a uniform cytotoxic phenotype and robust antitumor activity. These cells employ dual tumor-targeting strategies by antigen-specific killing via a transgenic TCR, and antigen-independent cytotoxicity via natural killer receptors. Compared to conventional PBMC-derived TCR-T cells, Allogeneic ESO-T cells display superior cytotoxic potency, selective solid tumor homing, durable killing persistence, and resilience against immune evasion. Notably, they exhibit a favorable safety profile, with low risks of graft-versus-host disease (GvHD) and cytokine release syndrome (CRS), alongside stable hypoimmunogenic features. Collectively, our findings demonstrate the feasibility, universality, and therapeutic potential of HSPC-engineered, off-the-shelf mono-specific cytotoxic T cells. This work lays a foundation for next-generation T cell immunotherapies that are scalable, safe, and broadly applicable to diverse solid tumors.
