Tumor-specific CAR-NK cells for adoptive cancer immunotherapy

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Natural killer (NK) cells hold promise for adoptive cancer immunotherapy. Similar to T cells, the antitumor activity of NK cells can be enhanced by expression of chimeric antigen receptors (CARs) that facilitate selective recognition and killing of malignant cells. Thereby, characteristics unique to NK cells such as their endogenous natural cytotoxicity complementing CAR-mediated antitumor activity, and the low risk of graft versus host disease upon application of donor-derived cells favor the use of NK cells as CAR-engineered effectors. Nevertheless, challenges such as the limited expansion potential of NK cells and reduced effectiveness of CAR gene transfer are slowing clinical development of CAR-engineered primary NK cells. With the proposed project, we aim to investigate directed ex vivo differentiation of hematopoietic stem cells (HSC) as a strategy for the generation of fully functional NK cells, which will be further expanded using specific cytokine cocktails and gene-modified feeder cells to reliably yield high effector cell numbers. This approach will then be employed to generate CAR-engineered NK cells with enhanced antitumor activity, based on lentiviral CAR gene transfer into HSCs followed by ex vivo differentiation into CAR-NK cells. Thereby, a CAR specific for the tumor-associated surface antigen ErbB2 (HER2) will be employed as a clinically relevant model, with CAR expression restricted to the NK cell lineage through the use of an NK-specific promoter. The resulting ex vivo generated NK and CAR-NK cells will be phenotypically and functionally characterized, and their antitumor activity will be tested against cancer cells in vitro and human tumor xenografts in immunodeficient mice in vivo. Subsequently, the principles established during ex vivo differentiation of CAR-NK cells will be applied for in vivo CAR-NK cell differentiation in humanized mouse model systems. Transplantation of HSCs carrying a CAR sequence expressed in NK cells in a lineage-specific manner may facilitate continuous in vivo repopulation with polyclonal CAR-NK cells and allow durable disease control. We expect these studies to establish CAR gene transduced HSCs as a useful source for the generation of tumor-specific CAR-NK cells in therapeutically relevant numbers, which could serve as a promising alternative to currently applied CAR-T cells for the development of effective cancer immunotherapies.