Beyond ex vivo and in vivo CAR T: antigen-driven CAR T (adCAR-T) expansion method enables rapid, physiological CAR T cells programming.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated transformative efficacy in hematologic malignancies, but its broader use remains constrained by complex ex vivo manufacturing, prolonged production timelines, high cost, and dependence on lymphodepleting chemotherapy. Emerging in vivo CAR-T generation strategies aim to address these limitations, but they introduce additional safety concerns associated with systemic delivery of gene-modifying vectors, including off-target transduction and insertional mutagenesis. This paper describes a novel antigen-driven CAR T-cell expansion platform (adCAR-T) based on co-culture of CAR T cells with engineered target cells expressing defined antigen density and lacking the inhibitory checkpoint ligand PD-L1. This system induces immediate activation, rapid proliferation, and sustained cytotoxic differentiation of CAR T cells without reliance on artificial CD3/CD28 bead stimulation or exogenous cytokine-driven expansion. In contrast to conventional methods, the platform eliminates the lag phase of CAR T-cell expansion and enables rapid scaling to clinically relevant doses (108-109 cells) within several days, depending on the initial cell input. Mechanistically, antigen-driven CAR engagement and target-cell lysis trigger cytokine release and amplification of CAR T cells in a physiologically relevant manner. This process promotes coordinated expansion of both directly antigen-engaged and non-engaged CAR T cells. The platform preserves "functional fitness", minimizes exhaustion, and avoids systemic exposure to gene-delivery vectors. Taken together, this strategy defines a hybrid manufacturing paradigm that bridges the control of ex vivo production with the physiological logic of in vivo activation. Proposed method has a potential to reduce manufacturing complexity, improve safety, and possibly decrease or eliminate the need for lymphodepleting conditioning. This work presents a potential alternative to both standard ex vivo manufacturing and emerging in vivo CAR-T generation approaches, with important implications for improving the accessibility, safety, and cost-effectiveness of CAR T-cell therapies.