In adoptive immunotherapy, immune cells isolated from patients can be engrafted with synthetic proteins that enable the cells to perform therapeutic functions, such as destroying tumor cells. One class of these proteins is the chimeric antigen receptors (CARs), which are fusions of extracellular single chain antibodies and intracellular signaling domains. The customizable antibody portion can bind to a specific antigen marker on a target cell surface. The modular intracellular signaling portion of the CAR then initiates a response in the immune cell. In cancer immunotherapy, CAR-mediated recognition and killing of cancer cells based on a single marker on the cancer cell has been shown to be clinically useful. However, recent studies also reveal a significant pitfall in safety: markers that are highly abundant on tumor cells can also be present at lower abundance on some normal cells. Unintentional killing of such normal cells by CAR-carrying immune cells has led to severe toxicity or even death of the patient. This project proposes to achieve the following goals: (1) Develop CAR systems that mediate immune cell functions based on combinations of markers instead of single markers. The intended result is that only cancer cells with a specific combination of surface markers will be killed by the immune cell. This proof-of-principle project is envisioned to generate valuable insights into the designs of more sophisticated CAR systems and to improve clinical utility of synthetic protein receptors in cell- based immunotherapy. (2) Compare the properties of the developed CAR systems in both T cells and Natural Killer cells, two major types of immune cells that are therapeutically useful in adoptive immunotherapy. Studies have shown that due to innate differences within the immune cells, a CAR can manifest different functional properties in different cell types. Characterizing CARs and CAR systems in different immune cell types is therefore important for enriching the current understanding of molecular and cellular immunology while defining the range of clinical utility of CARs. By pursuing the proposed research goals, I expect to gain expertise in bio-engineering, cancer biology and immunology. The acquired knowledge and skills will prepare me for an independent career in synthetic biology and cancer immunology. PUBLIC HEALTH RELEVANCE: In adoptive immunotherapy for cancer, a patient's own immune cells can be engrafted with synthetic proteins so that the cells are capable of destroying tumors in the patient. However recent clinical trials revealed a pitfall - insufficient discriminaton between cancer and normal cells in the body, leading to undesirable killing of normal cells by the immune cells and severe toxicity. The goal of this project is to design synthetic protein-based systems that will improve immune cells' recognition of cancer cells and to enrich the current understanding of molecular signaling mechanisms in immune cells.