Project Summary Adoptive T cell therapy (ACT) is a T cell based cancer therapy in which autologous T cells are isolated from the patient, activated and expanded ex vivo, then reinfused into the patient. While ACT has shown great clinical success, the success has been limited to melanoma, and complex manufacturing considerations create a large price tag. Innovations in in scalable, acellular systems for T cell activation, such as artificial antigen presenting cells (aAPCs), has improved the ex vivo expansion of CD8+ T cells by shortening culture times and providing tighter control of the resulting T cell phenotype and function. However, T cell culture with aAPCs still takes several weeks and requires manufacturing labor and cost. Platforms for in vivo activation of antigen-specific T cells would decrease the cost and complexity of T cell therapy. The goal of the proposed project is to create the first biomaterial scaffold for direct, in vivo, antigen-specific activation of CD8+ T cells for cancer immunotherapy. The platform, termed the artificial lymph node (aLN), is a hyaluronic acid hydrogel conjugated with signals 1 (peptide-MHC), 2 (anti-CD28) and 3 (cytokine support) that can be injected subcutaneously to create a T cell activating microenvironment. We will investigate the effects of 3D scaffold parameters on T cell activation as well as gain insight into the dynamics of in vivo antigen- specific T cell activation in an immune competent host. We will develop injectable aLN microparticles (MPs) that will compact in vivo to form a T cell activating scaffold. We will first investigate physical properties of the aLN MPs such as signal density, stiffness, and size. We will also investigate the addition of cell adhesion proteins to facilitate migration of the T cells within the scaffold. Second, we will incorporate a local and sustained signal 3 (cytokine) signaling component. We will test a variety of cytokines, including IL-2, IL-7, IL-15, and IL-21, for their ability to generate both effector and memory cells. With the lead cytokine cocktail, we will test two methods of integration, antibody presentation and encapsulation. These aLN parameters will be optimized for both murine and human antigen- specific T cell expansion. Finally, we will test the efficacy of the aLN MPs for in vivo activation and expansion of CD8+ T cells and their anti-cancer efficacy, using B16-OVA, B16-SIY, and MC38-OVA for mouse T cells, and the human SK-MEL-37 (A2+/NY-ESO-1+) melanoma cell line for human T cells. If successful, this proposal will produce a novel acellular approach for the in situ generation of an antigen-specific T cell response, expanding the access of immunotherapy to more patients.