PROJECT SUMMARY Despite unprecedented clinical success of chimeric antigen receptor (CAR)-T cell therapy against tumors, widespread application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) very high costs of therapy of up to half of a million dollars; (ii) delays of weeks or months to infuse CAR-T cells to patients with rapidly progressing disease; and (iii) heterogeneous composition and terminal differentiation of infused CAR-T cells as a result of ex vivo culture that limit CAR-T cell engraftment and persistence. Effort to overcome these limitations have focused on closed and automatic manufacturing devices to contain the labor needed to manufacture CAR-T cells ex vivo, and allogeneic off-the-shelf CAR-T cells have been proposed to overcome the need of CAR-T cell manufacturing for each single patient. Despite significant achievements in this space, reducing the time, costs and regulatory burden remains a deep unmet need in CAR-T cell therapy and significant reducing or eliminating ex vivo procedures remains a critical unmet need. In vivo generation of CAR-T cells would eliminate the need for ex vivo procedures, prevent the terminal differentiation of ex vivo expanded CAR-T cells and ensure the potency and longevity of autologous T cells as compared to allogeneic CAR-T cell products that are extensively manipulated to prevent rejection and graft- versus-host disease The research outlined in this proposal develops new biomaterials approaches to reduce the time and effort to produce CAR-T cells in vitro, to enhance CAR-T cell efficacy and persistence in vivo and, finally, to eliminate ex vivo manipulation entirely by generating CAR-T cells entirely within the patient. We propose that biomaterial scaffolds displaying anti-CD3/CD28 antibodies and releasing pro-proliferative interleukins will mediate simultaneous activation and viral transduction of T cells without centrifugation (spinoculation) or transduction agents (retronectin, polybrene) and will facilitate ex vivo genetic reprogramming of T cells by reducing the time and expense of activating naive T-cells and transducing them with viral vectors. We next propose that directly implanting scaffolds seeded with peripheral blood mononuclear cells and CAR- encoding viral vectors will promote release of CAR-T cells into circulation, eliminating ex vivo CAR-T isolation and proliferation protocols to promote a less differentiated cell phenotype associated with longer in vivo persistence. Finally, we propose that, through the inclusion of encapsulated T-cell attracting cytokines, implanted biomaterial scaffolds will generate CAR-T cells entirely in situ through recruitment of host T cells to the scaffold, in-scaffold reprogramming of recruited T cells with resident CAR-encoding viral vectors, and release of reprogrammed CAR-T cells. We expect that our results will provide a basis for a general cellular therapeutic strategy and promote widespread patient ...