PROJECT SUMMARY Current cell expansion procedures for antigen-specific CAR T cells and CAR NK cells result in cells with a broad repertoire of chemokine receptors. Upon infusion, they migrate to off-tumor healthy tissues and exert toxicities by targeting antigens expressed at basal levels. The ability to produce therapeutic cells with affinity to migrate into the tumor microenvironment (TME) is a critical unmet need. The investigating team’s mission is to harness the immune system to treat solid tumors. In line with this mission, the objectives of this R61 are to 1) develop a prototype of the bioreactor to selectively culture chemotactically competent cell-based therapies (CAR T, CAR NK-92), and 2) demonstrate use of this subset for migrating through a tissue-mimicking barrier and kill epithelial ovarian cancer cells in an antigen-specific manner. The rationale for this effort is that it will disrupt the status quo of current cell manufacturing technology, which generates therapeutic cells that traffic not only to tumors but also to healthy tissue. The team will conduct the R61 project under two milestone-driven aims: 1) Develop bioreactor for generating chemotactically competent CAR NK-92 cells; 2) Demonstrate utility of the bioreactor for generating chemotactically competent CAR T cells. In a follow-on R33, the team will use the bioreactor to scale up the production of these chemotactically competent cell therapies for preclinical mouse studies. The impact of bioreactor will be in making treatments safer (by mitigating off-tumor migration of therapeutic cells) and affordable (by reducing manual intervention in the closed-system bioreactor). Mitigating off-tumor migration will reduce the burden of manufacturing because most of the therapeutic cells will reach the target TME site. The significance of this project is also supported by its applicability to other immune cells (CAR NK cells, CAR macrophages) that are currently in development for targeting solid tumors.