Project Summary/Abstract 30 lines or less: Adoptive cell therapy (ACT) with chimeric antigen receptor (CAR) T cells has shown dramatic clinical responses in hematologic cancers, with a high proportion of durable complete remissions elicited in leukemias and lymphomas. This success has led to a strong commercial investment establishing adoptive cell therapy as a viable clinical therapy and the first licensure of CAR-T therapy by the FDA in 2017. However, achieving the full promise of CAR-T ACT, especially in solid tumors, will require further advances in this form of cellular therapy. A key challenge is maintaining a sufficient pool of functional CAR-T cells in vivo. In addition, even in hematologic tumors treated effectively with CAR T cells, chemotherapeutic lymphodepletion regimens with high toxicity are often required to ensure the engraftment and initial expansion of the donor cells. We recently discovered an efficient strategy for molecularly targeting peptide vaccines and vaccine adjuvants to lymph nodes, through the use of albumin-binding phospholipid-polymer linkers conjugated to antigens/molecular adjuvants. Albumin constitutively traffics from blood to lymph, and serves as an effective chaperone to concentrate these “amphiphile-vaccine” (amph-vax) components in lymph nodes that would otherwise be rapidly dispersed in the bloodstream following parenteral injection. However, these lipid-polymer conjugates also exhibit the property that they insert in cell membranes on arrival in lymph nodes. We propose here to exploit these dual lymph node targeting and membrane-decorating properties of amph-vax molecules to create a booster vaccine for CAR T cells, which can be used to repeatedly expand and rejuvenate CAR-T directly in vivo- in native lymph nodes and/or tumors. To evaluate this approach in the presence of a complete host immune system, we will test this concept both with human T cells and with an immunocompetent syngeneic mouse model of melanoma recently developed by our lab. Our specific aims are to (1) Characterize the biology of synthetic antigen presentation of amphiphile-ligands from the surface of antigen presenting cells to CAR T cells in vivo, (2) to demonstrate an amph-vax design generalizable to any CAR, (3) to evaluate the capacity of a CAR-T vaccine to expand T cells with enhanced functionality and persistence in vivo, and (4) to test the utility of intratumoral amph-vax delivery to enhance CAR-T and endogenous T cell priming in tandem. These studies will establish a robust technology platform to transform multiple aspects of adoptive cell therapy and address key limitations in existing ACT therapeutic strategies.