PROJECT SUMMARY Adoptive T cell therapy (ACT) is a form of immunotherapy that involves the repopulation of the host immune system with a selective population of ex vivo expanded, tumor reactive T cells. Tumor reactive T cells can be isolated from the tumor (tumor infiltrating lymphocytes, TIL) or created through genetic engineering (TCR or CAR T cells). By expanding and activating the cells ex vivo, outside of the suppressive tumor microenvironment, and infusing them back into the host, successful response rates can be induced. However, many tumors have high genomic heterogeneity, which can result in decreased neoantigen expression by some cells within the tumor, allowing for immune-escape, limiting the efficacy of checkpoint inhibitors or T cell-based therapies that target a single antigen (CAR-T, TCR). A more robust T cell response that targets a diverse and greater number of tumor neoantigens may lead to more effective immunotherapies. Proponents of TIL therapy contend that these T cells may be a more effective approach to ACT due their ability to recognize a greater number of tumor neoantigens as they are found within a patient’s tumor. Response rates can be induced in up to 72% of patients. However, some patients are unable to undergo surgery to obtain TIL, others are unable to wait the 4-6 weeks required to expand TIL to numbers sufficient for therapy, and other patients may not have sufficient TIL at all. Lastly, while tumor reactive T cells are found in peripheral blood, previous attempts to isolate, increase tumor antigen recognition, and expand these T cells to numbers sufficient for therapy have been futile. However, peripheral blood could serve as a potentially limitless source of T cells, if one can enrich them for tumor antigen recognition. To overcome these limitations, we will employ a bioengineered immune-enhanced tumor-on-a-chip platform (iTOC) to enrich tumor antigen recognition and expand tumor-reactive T cells from peripheral blood. In the iTOC microfluidic platform, tumor cells and autologous lymph node-derived immune cells are combined to form immune-enhanced patient tumor organoids whereby the heterogeneity of and the interplay between the patient’s tumor, stroma, and immune cells remain intact. We hypothesize that circulation of T cells from peripheral blood through the iTOC will result in an enriched population of tumor reactive T cells that are capable of mounting a more robust immune response than TIL or uncirculated peripheral blood T cells. Aim 1 will determine the effect of iTOC perfusion on T cell viability, proliferation, and phenotype. Aim 2 will determine how iTOC perfusion impacts T cell receptor diversity and tumor-specific effector function compared to TIL and uncirculated peripheral blood T cells. These studies will establish the efficacy of the iTOC to improve a patient’s tumor-specific adaptive immune response through on demand generation of a product suitable for adoptive T cell transfer. While our test v...