Abstract Immunotherapies for cancer, including Adoptive Cell Transfer (ACT), chimeric antigen receptor (CAR) T cell therapy, and Immune Checkpoint Blockade (ICB), have seen great clinical success. However, they are still limited by cost, variable efficacy and resistance, and lack of quality targets. Technologies to track immune responses, and in particular tumor-specific T cell responses, would help researchers and clinicians to better understand variable responses in patients, design better targets for therapies, and track patient health outcomes. In this proposal, we aim to adapt our novel hydrogel-based immune cell expansion system to detect and characterize rare, neo-epitope specific T cells in the peripheral blood. This platform, termed the artificial T cell stimulation matrix (aTM), is a hyaluronic acid hydrogel conjugated with signals 1 (peptide-MHC), 2 (anti-CD28) and 3 (cytokine support). In order to create an aTM-based high- throughput detection system, we will first investigate the effects of physical (i.e. stiffness) and biochemical (i.e. activation molecules and cytokines) cues of the aTM on the expansion of neo- epitope specific anti-tumor T cells in mice. We will test the optimized aTM for batched expansion from bulk murine splenocytes in order to create a high-throughput system. Next, we will develop a process to detect and expand tumor-associated antigen-specific T cells from peripheral blood of healthy donors and compare this to current gold standards of antigen-specific T cell detection. Finally, because disease status may impact our ability to detect rare T cells, we will verify that the system can be used for detection of neoepitope-specific T cells from the peripheral blood of melanoma patients. Access to blood from patients will also allow us to determine how ICB treatment affects the immune response to the tumor by investigation of expanded neo-epitope specific T cell phenotype and function before and after therapy. The development of aTM as a high-throughput detection system for neo-epitope, patient-specific T cell responses will allow pre-clinical and clinical researchers to study immune responses to cancer and to improve cancer immunotherapies by bringing the power of these therapies to more patients.

Characterization of the immune response before, during, and after administration of immunotherapy would allow researchers and clinicians to better understand how patients respond to these therapies and how resistance develops, as well as facilitate more informed decisions about therapy choice, dose, and target. One important marker of the immune response against cancer is the presence of mutated neoantigens in tumors, but a major unmet challenge is the ability to detect immune responses to neoantigens within tumors or in the peripheral blood. The proposed research seeks to apply a novel hydrogel-based platform for high throughput detection and expansion of neoantigen-specific immune responses in tumor- bearing mice and humans.