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 a lack of specific antigens, 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 surgical mesh for implantation into the tumor resection cavity. This mesh will be used in conjunction with a split CAR T cell called a zipCAR, which uses a detached adaptor protein (a “zipFv”) to sense antigens. The mesh is composed of polymeric nanofibers with a matrix of chitosan deposited within the pores. The mesh supplies the zipFv adaptor protein, cytokines (IL-15), and T cell stimulatory antibodies (α- CD3/28). I hypothesize that the use of this surgical mesh 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 of this proposal demonstrates the proliferation advantage of the mesh by monitoring CAR T cell proliferation in a murine model of HER2+ breast cancer. Aim 2 of this proposal demonstrates the efficacy and safety advantages of the meshes in a model of operative debulking of ovarian cancer. To demonstrate prevention of antigen escape, ROR1- and HER2-deficient OVCAR3 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 meshes loaded with zipFvs against both antigens. To demonstrate superior safety, meshes will be utilized in the same model of ovarian cancer with mice that are irradiated to upregulate ROR1 expression in non-hematopoietic stem cells in the bone marrow and spleen, allowing observation of on-target, off-tumor toxicity. This proposal builds around four key components of critical research and clinical skills to support my development into an independent physician 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 physician scientist training in research conduct and communication skills, (4) commitment to an individual development plan (IDP) to guide my training goals.

Project Narrative This proposal describes the training and mentoring of Mr. Eric Bressler and his research project to treat solid tumors using a novel biomaterial in conjunction with a split and programmable chimeric antigen receptor (CAR) T cell system. Using this system, he describes a method for overcoming barriers to efficacy of CAR T cells in solid tumors, specifically poor T cell persistence in the tumor microenvironment, lack of specific antigens, and treatment-limiting adverse effects. The capability of this novel therapeutic system to promote CAR T cell proliferation, prevent antigen escape, and mitigate adverse events will be evaluated.