Fellow: James Cockey

Mentor: Cynthia Leifer

DESCRIPTION (provided by applicant): 

Feline infectious peritonitis (FIP) is an invariably fatal disease in cats caused by a coronaviral infection, to which there are currently no FDA approved treatments. The goal of my thesis project is to engineer immune cells to express a chimeric antigen receptor (CAR) that will detect and eliminate FIP virus (FIPV)-infected cells. CAR immunotherapy has been successful in treating some human cancers but has not yet been developed for acute viral infections like FIPV, nor used at all in cats to date. CARs are comprised of two main components: a single chain antibody fragment (ScFv) and signaling domain(s) from immune costimulatory receptor(s). With generous support from the Feline Health Center, and a pre-A exam Liz Hanson Fellowship, I collected a significant amount of preliminary data and developed key reagents to complete my thesis studies. I cloned the antigen binding region of an anti-spike monoclonal antibody (18A7.4) generated at Cornell by Dr. Fred Scott. Using that sequence, I designed an ScFv specific for FIPV spike protein and engineered the anti-spike CAR (FIPV-S-CAR). I made three different FIPV-S-CAR constructs with three different signaling domains. All three novel FIPV-S-CARs were stably expressed following transduction. Moreover, I obtained hybridomas for feline CD3 and CD56, and with the help of the Wagner lab, I purified the antibodies, labeled them, and demonstrated they can be used to enrich T and natural killer (NK) cells, respectively. I also developed a rapid and robust cytotoxicity assay, and paired cell lines expressing and not expressing target antigens. With these tools in hand, and having recently passed my A exam, I will work on two specific aims. First, I will determine the optimal CARexpressing primary feline lymphocyte to kill specific target cells. Although some studies have investigated feline T or NK cytotoxicity, no study has performed a head-to head comparison, and no study has simultaneously measured cytokine secretion. Cytotoxicity data will inform us of the therapeutic potential of each cell type and cytokine data will provide important information about the potential of T and NK cells to induce cytokine release syndrome, an important limiting clinical outcome of CAR therapy. Second, I will engineer an optimized CAR targeting FIPV spike protein. I will compare my FIPV-S-CAR constructs containing different immune-activating domains for the ability of these CARs to specifically direct immune cell killing of spike expressing target cells. Completion of this study will provide proof-of-principle data using an FIPV model to support the development of FIPV CAR-cell therapy for this devastating disease in cats and will also determine the feasibility of developing a CAR-based immunotherapy for the potential treatment of acute coronaviral infections in cats and humans.