Project Summary/Abstract Chimeric antigen receptor (CAR) T cells are engineered ex- vivo to localize to tumors and kill antigen positive cancer cells. This project has a direct relationship to health because CAR T cell therapy has shown promise for treating B cell malignancies, but many barriers limit their efficacy in solid tumors, such as antigen heterogeneity and an immunosuppressive tumor microenvironment. The long-term objectives of this project is to engineer a CAR T cell that can overcome these obstacles. To overcome these obstacles, we combined enzyme-prodrug systems with CAR T cells, generating Synthetic Enzyme Armed KillER (SEAKER) CAR T cells. We engineered human CAR T cells to secrete an enzyme, β-Lactamase, which cleaves cephalothin chemical motifs. We also synthesized cephalothin-linked prodrugs of cytotoxic agents. SEAKER cells deliver β-Lactamase to the tumor, to selectively unmask systemically administered, nontoxic cephalothin prodrugs in the tumor microenvironment. Our lab is the first to demonstrate that cellular therapeutics can generate small molecule drugs at the tumor site in xenograft studies (paper submitted). However, mice used in xenograft studies lack a complete immune system. Therefore, how SEAKER cells interact with a complete, endogenous immune system are not understood. Thus, we will transition our SEAKER platform to fully immunocompetent, syngeneic mouse models to inform clinical application of our technology. Our lab has already characterized murine SEAKER cells, in vitro, and has demonstrated the compatibility of the SEAKER platform with murine cells. The specific aims are: Aim 1: Characterize SEAKER cell kinetics and biodistribution in syngeneic in vivo models. Aim 2: Demonstrate improved anti-tumor efficacy of our SEAKER platform over traditional CAR-T cells in a fully immunocompetent host. The research environment is exceptionally well suited for my goals.This research will be completed at Memorial Sloan-Kettering Cancer Center (MSKCC) and Weill Cornell Medicine (WCM), two prominent institutions with track-records in training successful scientists. MSKCC is a world leader in CAR T cell therapy. The training plan of myself, the applicant, will maximize my potential to achieve the goals for this fellowship and prepare me for an academic career. I have experience in many necessary techniques to complete the proposed research. I have an extensive network of collaborators and peers who can aid the development of new techniques to complete this proposal. I am a PhD candidate, and have completed my required coursework. I have a strong commitment to diversity and will continue to promote equality in biomedical research.