Modulation of the tumor microenvironment by CAR-engineered NK cells

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Natural killer (NK) cells play a critical role in antitumor immunity by directly eliminating malignant cells and by regulating tumor-specific adaptive immune responses. NK-cell-based cancer immunotherapies are typically based on adoptive transfer of donor-derived allogeneic NK cells. To enhance their antitumor activity, NK cells can be further engineered to express chimeric antigen receptors (CARs) that facilitate selective recognition and killing of tumor cells, with early stage clinical development of several such approaches ongoing. Nevertheless, in addition to pro-inflammatory cytokines such as interferon (IFN)-gamma, activated NK and CAR NK cells secrete high levels of immuno¬regulatory interleukin (IL)-10, which may counteract pro-inflammatory factors and dampen tumor-suppressive activities of bystander immune cells in the tumor microenvironment. With the proposed project, we aim to better understand the role of NK-cell-derived IL-10 in the interaction of CAR-engineered NK cells with host immune cells within solid tumors, and to enhance endogenous tumor-suppressive mechanisms by modulation of the CAR effector cells' cytokine profile. Using continuously expanding ErbB2 (HER2)-specific CAR NK cells derived from the human NK cell line NK-92 as a clinically relevant model, we will investigate the consequences of IL-10 depletion by antibody-mediated inhibition and CRISPR/Cas9-mediated gene knockout. Cell-intrinsic effects of IL-10 downregulation on growth, cytotoxicity and cytokine production of the CAR NK cells will be analyzed. In addition, in co-culture assays the influence of IL-10-depleted CAR NK cells on polarization of macrophages and maturation of dendritic cells will be studied, since these two cell types appear most crucial for overcoming an immunosuppressive tumor microenvironment and induction of adaptive antitumor immunity. Similarly, the potential enhancement of bystander immune cell activation by CAR NK cells through ectopically expressed pro-inflammatory cytokines such as IL-12 and IL-15 will be investigated. Subsequently, IL-10-depleted CAR NK cells and variants modified to express pro-inflammatory factors will be tested in immunocompetent mouse models with syngeneic, ErbB2-positive tumors in comparison to unmodified CAR NK cells. Similar experiments will be performed in tumor xenograft models in immunodeficient mice transplanted with human peripheral blood mononuclear cells to also address the influence of NK-derived factors on bystander immune cells of human origin in vivo. We expect that insights from this project will not only be relevant for improvement of adoptive cancer immunotherapy with CAR NK-92 cells, but may lead to a generally applicable approach to enhance endogenous antitumor immunity through the activity of genetically engineered primary NK and T cells.