PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are innate immune effector cells that play an immediate role in host defense. The activation of NK cells is mediated by receptor-ligand interactions and downstream intracellular signaling path- ways. One type of immunotherapy that has achieved great success in recent years is based on chimeric antigen receptors (CARs). These are engineered receptors composed of both target recognition and cell activation func- tions that can direct immune cells to mediate killing of cancer cells. Although T cells are the predominant immune cell type used for CAR-based immunotherapy, NK cells provide significant advantages over CAR-engineered T cells because they can be derived from non-autologous sources. However, most studies testing CAR-NK cells have used CAR constructs based on T cell signaling pathways that are not optimized for NK cell signaling. In addition, the development of CAR constructs is largely achieved using a trial-and-error experimental approach, and there is no systematic understanding of how altering the CAR signaling domains influences cell activation. The main objective of this proposal is to identify effective NK based-CAR designs using systems biology tools. Our approach combines computational modeling and quantitative phospho-proteomics to generate a de- tailed understanding of CAR-mediated NK cell signaling and cytotoxicity. The outcome of our work will be a set of validated NK CARs that target and kill BCMA-positive multiple myeloma (MM) cancer cells. We will also test the optimized CARs against CS1-expressing MM cells. The approach builds on our team’s extensive experience in modeling and characterizing cell signaling and studying NK cell biology. Guided by strong preliminary data, we propose to pursue three Specific Aims: (1) Characterize intracellular and cellular-level responses of CAR- expressing NK cells; (2) Develop computational models to predict the dynamic responses of CAR-expressing NK cells; (3) Identify novel CAR constructs that effectively activate NK cells. Collectively, our proposed research will generate a quantitative understanding of how CAR signaling encodes NK cell-mediated cytotoxicity and how NK-CAR constructs can be optimized for cancer immunotherapy. Our research will broadly impact the field of cancer immunotherapy by providing insight into how intracellular NK cell signaling and CAR structure influence NK cell activation. Ultimately, this research will expand our knowledge of NK cell signaling and the design criteria for NK-CAR-based immunotherapy for other tumor types and cancer antigens. Our work has the potential to unlock the transformative power of NK-CAR cells for cancer immuno- therapy.

NARRATIVE Natural killer (NK) cells are innate immune effector cells that may be engineered for cancer immunotherapy by expression of chimeric antigen receptors (CARs), engineered proteins that direct NK cells to kill cancer cells. Our proposed studies will generate a fundamental understanding of intracellular signaling mediated by NK-CARs to enable efficient design of optimal CARs for multiple myeloma. This research has the potential to unlock the transformative power of NK-CAR cells for cancer immunotherapy.