PROJECT SUMMARY CAR-T cell therapy has revolutionized the treatment of liquid tumors, including leukemia and lymphoma, and hold enormous promise for treatment of solid cancers as well. However, despite their unprecedented clinical success, widespread utilization of this therapy is hampered by the lengthy and labor-intensive manufacturing procedures. CAR-T cell manufacturing takes weeks, results in very high costs of therapy (~$500,000). The long manufacturing time creates delays of weeks or months to infuse CAR-T cells to patients with rapidly progressing disease. Finally, the extensive ex vivo manipulation creates cell products with heterogeneous composition and terminal differentiation 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. These technologies are promising, but reducing the time, costs and regulatory burden of manufacturing or eliminating ex vivo procedures entirely remains a critical unmet need. In vivo generation of CAR-T cells would eliminate the need for ex vivo procedures, prevent the terminal differenti- ation 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. This proposal outlines the first steps in a highly innovative high-risk/high-reward effort to develop bioin- structive biomaterials scaffolds that generate CAR-T cells entirely within the patient and produce CAR-T cells with improved efficacy and persistence. Our endeavor is built on significant published and preliminary data demonstrating that our biomaterial scaffolds already efficiently activate and mediate CAR-T cell transduction in vitro and efficiently recruit and release CAR-T cells in vivo and reduce CAR-T manufacturing times from weeks to a single day. We propose that the biocompatible alginate biomaterial scaffolds can be modified to encapsulate T cell-attracting chemokines to recruit T cells to the scaffold. After recruitment, the biomaterial scaffolds will provide αCD3/CD28 signaling to activate the T cells. After activation, T cell-specific viral particles either already present in the biomaterial or administered to the biomaterial as a separate step will transduce the T cells, gen- erating tumor specific CAR-T cells in situ in manner compatible with irradiative lymphodepletion. Finally, inter- leukin signaling in the scaffold will expand and promote release of formed CAR-T cells for systemic function. If successful, this approach could have enormous clinical impact by significantly reducing therapy costs and dra- matically expanding the patient population benefiting from CAR-T-cell therapy. We expect...