Development of a Tunable Protein Release Hydrogel for the Enhancement of CAR T cell Activity - Project Summary and Abstract Chimeric antigen receptor (CAR) T cells are genetically engineered T lymphocytes designed to sense antigens and mount an immune response. Though CAR T cells have received FDA approval for the treatment of several hematologic malignancies, success in solid tumors is limited by the immunosuppressive tumor microenvironment, and treatment-limiting adverse effects such as on-target, off-tumor toxicity and cytokine release syndrome. Though investigators report strategies for mitigating these limitations such as biomaterials for reshaping the tumor microenvironment, and logic-gated CAR T cells to prevent non-specific toxicity, no proposed strategy has overcome each of these barriers. To surmount these limitations, I propose the use of a novel self- assembled hydrogel for implantation into tumor. This hydrogel will be used in conjunction with a split CAR T cell called a zipCAR, which uses a split adaptor protein (zipFv) to sense antigens. The hydrogel is composed of a 4 arm PEG linker decorated with leucine zippers that can be loaded with zipCargo proteins (payload proteins that are modified with a leucine zipper, including zipFv). The hydrogel supplies the zipFv adaptor protein, cytokines (IL-7), and chemokines (CXCL9). I hypothesize that the use of this in situ hydrogel will overcome the barriers to CAR T cell therapy in solid tumors by: 1) opposing T cell anergy and promoting proliferation in the resection cavity; 2) preventing antigen escape via encapsulation of zipFvs targeting multiple antigens; and, 3) imparting spatiotemporal control over CAR T cell activity. Aim 1 will demonstrate the advantage of the hydrogel by monitoring CAR T cell migration and proliferation in a murine model of ROR1+ lung cancer. Aim 2 will document the efficacy and safety advantages of the hydrogels in a toxicity model of lung cancer. To demonstrate prevention of antigen escape, ROR1- and MSLN-deficient lung cancer cell lines will be created using CRISPR-Cas9 knockouts. In a murine model of antigen escape, these cells will be used to demonstrate superior efficacy in mice treated with zipCAR T cells and hydrogels loaded with zipFvs against both antigens. This proposal builds around five key components of critical research and clinical skills to support my development into an independent engineer/scientist: (1) an interdisciplinary research project focusing on novel surgical biomaterials for enhancement of CAR T cell activity; (2) multi-disciplinary mentoring from Drs. Grinstaff (biomaterials), Wong (immunotherapy); and, Colson (clinical medicine, animal models, and immunology), (3) academic engineer-scientist in research conduct and communication skills, (4) clinical awareness program, overseen by Dr. Yolonda Colson a nationally recognized surgeon, and (5) professional development activities to guide my training goals.
