Abstract The goal of cancer immunotherapy is to build long-lasting tumor-specific immunologic ‘memory’ in patients that enables the lifelong rejection of tumors. The two prominent types of antigen-specific cancer immunotherapy, adoptive T cell therapy and APC-based vaccination, both require expansion of anti-tumor T cells via APCs. However, for the purpose of effective adoptive T cell therapy, the critical question is how to generate, within a short period of time, large numbers of antitumor T cells. Furthermore, in vitro-expanded T cells must also possess the capacity to engraft, proliferate, and persist in vivo with sufficient antitumor function to induce sustained antitumor activity. Autologous antigen-presenting cells (APCs) such as DCs also have several serious limitations. The necessity to access large amounts of cancer patients’ blood to prepare autologous APC from each patient in a timely manner is cumbersome. To overcome these problems, we developed the microfluidic process to generate cell-sized unilamellar vesicles (CUVs) and decorated them with antigen presenting ligands for artificial APCs (or aAPCs). Preliminary results show that aAPCs are able to bind and interact with T cells and cause their expansion. The objective of the present proposal is to further optimize the aAPCs preparation and test its capacity to induce tumor specific responses in vitro and in vivo. The hypothesis is that the optimized aAPC functionalization will result in enhanced expansion of cytotoxic CD8 T cells and a reduction in tumor progression over the present one (original). The Specific Aims are- 1) Bioinspired optimization of artificial antigen presenting cell (aAPC) production via microfluidic engineering. We will insert the antigen presenting ligands in the membrane to mimic cells. The aAPCs will also be produced with hydrogel cytoskeletons to optimize its mechanical properties for maximum T cell expansion. 2) Evaluation of the capacity of aAPCs to induce tumor specific T cell responses in vitro. Using PBMCs from healthy donors and breast cancer patients we will evaluate the capacity of aAPCs to induce cytotoxic T cells. 3) Evaluation of the capacity of aAPCs to induce T cell responses and tumor killing with an in vivo mice tumor model. Methods to scale up the production of aAPCs for in vivo use will be developed. The capacity of aAPCs to kill tumor in vivo in mice will also be determined using a melanoma model. The goal is to produce an aAPC preparation that mimics cells, is stable, easy to produce in large quantities and capable of expanding tumor specific CD8 T cells for immunotherapy of cancer.