iPSC-derived, multiplex-engineered CD8αβT cells deliver antigen-specific, potent, and durable tumor cytotoxicity without CD4αβT support – powering scalable, off-the-shelf allogeneic αβT cell therapy in hematological malignancies

Abstract Autologous T cell therapies have reshaped cancer treatment paradigms, yet persistent challenges in scalability, product consistency, and manufacturing cost continue to restrict their accessibility. This therapeutic gap underscores the urgent need for robust, off-the-shelf alternatives capable of delivering broad, reliable, and cost-effective patient coverage. Here, we introduce a next-generation, induced pluripotent stem cells (iPSC)-derived allogeneic CD8αβT cell (iCD8αβT) platform—engineered to eliminate the need for CD4αβT cells for streamlined manufacturing, while achieving ultra-purity and >10⁹-fold lineage-directed expansion from a single iPSC cell. This platform delivers durable, tumor-specific cytotoxicity alongside intrinsic mechanisms of immune evasion, offering a transformative solution for scalable, truly affordable, and effective allogeneic cell therapy in hematologic malignancies. To drive functional persistence and lower activation thresholds, we implemented a multiplex knockout (KO) strategy targeting CISH and a key SOCS family member, both negative regulators of the activation cytokine signaling. In parallel, we modulated apoptotic pathways to alleviate activation-induced cell death. This combinatorial engineering dramatically reduced cytokine dependency and enabled vigorous expansion and effector function even at physiological—or subphysiological—IL-2/IL-15 levels. Notably, these benefits were achieved without constitutive cytokine receptor or cytokine overexpression, sidestepping the risks of tonic signaling and premature exhaustion. To further amplify tumor-driven proliferation and activation, we deployed an innovative signal converter (SC) module. Upon engagement with tumor targets, a secreted ligand activates the SC, unleashing a synthetic costimulatory signal through its intracellular domain. This module selectively intensifies iCD8αβT cell expansion in the tumor microenvironment, fueling a powerful and sustained anti-tumor response. When armed with CARs (e.g., CD19) or a KRAS G12V-specific TCR, engineered iCD8αβT cells consistently achieved antigen specific, complete and durable tumor clearance across multiple rounds of serial tumor rechallenges over the duration of >50 days—a remarkable feat of persistence and potency. Even under immunosuppressive conditions (e.g., high TGF-β1), iCD8αβT cells retained robust and durable cytotoxicity while producing minimal cytokines associated with cytokine release syndrome (e.g., IL-6, IL-10, TNF-α), highlighting both their functional resilience and promising safety profile. Furthermore, leveraging a proprietary, feeder-free differentiation and expansion system, we generated ultra-pure (~95%) CD8αβT cells from a single, multiplex-edited iPSC clone, achieving >10⁹-fold lineage-directed expansion—yielding over one billion fully engineered CD8αβT cells per starting iPSC. The process is fully feeder-free, enrichment-free, and GMP-compliant, enabling clinical development and scalable manufacturing at commercial levels. In conclusion, this next-generation iPSC-derived CD8αβT cell platform redefines the paradigm for off-the-shelf, allogeneic cell therapy. By eliminating the requirement for CD4αβT cell to streamline the manufacturing process and engineering for precision activation, reduced threshold of the activation cytokine required, and tumor-selective persistence, it delivers a powerful combination of scalability, safety, and anti-tumor durability—poised to unlock broader and more equitable access to cell-based cancer therapies.