Strategies to optimize natural killer cell functions in the tumor microenvironment

Sammanfattning: Immune cell-based therapies are currently changing the oncological treatment landscape worldwide. While genetically modified T cells have already been approved for clinical use in few hematological cancers, natural killer (NK) cell-based therapies are employed in early phase clinical trials. NK cells are generally regarded as an alternative to T cells as they possess the advantage of intrinsic killing capacity of malignant cells, cause less side effects post transplantation and can be used in an allogeneic setting. In the context of multiple myeloma (MM), the second most common blood cancer, two chimeric antigen receptor (CAR) T cell products are clinically approved and NK cell-based therapies show promising results in early phase clinical studies. However, relapses occur after CAR-T cell therapy and although NK cells show a good safety profile, it is evident that genetic modifications and combination therapies are needed to improve their efficacy. The research provided in this thesis aims to elaborate strategies to improve NK cell functionality in the tumor microenvironment (TME) through genetic modification and combination with an oncolytic virus, with a special focus on MM. It is well established that immune cell dysfunctionality can occur through chronic stimulation via the programmed death protein 1 (PD1) pathway. Blocking the interaction of PD1 with its ligands PD-L1 and PD-L2 restores immune cell effector functions and is successfully applied in several hematological and solid malignancies. However, in MM, immune checkpoint inhibition has failed due to lack of efficacy in monotherapies and severe side effects in combination therapies. Therefore, paper I explores the feasibility of novel NK cell specific chimeric switch receptors (CSR) that re-direct inhibitory signaling into activating signaling in NK cells upon binding of PD1 ligands. The designed CSR are based on the extracellular domain of PD1 that is linked to the transmembrane and/or intracellular domains of DAP10, DAP12 and CD3z which are known to provide NK cell activation. In paper I, we show that primary PD1-CSR+ NK cells exert superior cytotoxicity and cytokine release against PD-L1+ cancer cell lines and primary autologous MM samples, establishing them as a promising strategy towards PD-L1+ cancers. Targeting CD38 with the monoclonal antibody (mAb) daratumumab has improved treatment outcomes for patients with both newly diagnosed and relapsed or refractory MM, but is also met with resistance to therapy. Moreover, the majority of peripheral blood NK cells express CD38 and are depleted during daratumumab treatment. In paper II, we have introduced an affinity-optimized CD38-CAR construct into CD38dim primary NK cells that naturally occur during a long-term cytokine-based feeder cell-free expansion protocol. This approach leads to a functional NK cell product with superior cytotoxicity against CD38+ MM cell lines and autologous MM samples with minimal risk for fratricide. The data provided here pave the way for advancing this approach as an alternative treatment option for MM. Oncolytic viruses (OV) present an attractive immunotherapeutic platform for combination therapies and are currently tested in pre-clinical and early phase clinical trials for several malignancies. Herpes simplex virus 1 (HSV-1) based OV belong to the most utilized viruses and were shown to effectively infect and lyse malignant MM cells. However, their clinical applicability is met with several challenges, one being a rapid clearance by the immune system. NK cells naturally recognize virally infected cells and have been implicated in both promoting and hampering OV therapy. However, the exact molecular mechanism remain elusive. In paper III we have studied the interaction of primary NK cells with target cells that have been infected with the HSV-1 based OV HSV1716. The data provided in paper III show that primary NK cells increase their degranulation ability against HSV1716-infected target cells, despite the downregulation of important ligands for activating NK cell receptors. Primary NK cells become infected with HSV1716 through direct contact with target cells and increase their activation status. A better understanding of the molecular mechanisms behind NK cell recognition of HSV-1 OV infected target cells might improve immunotherapeutic combination approaches. All in all, the constituting studies provide unique and novel data on how to improve NK cell-based immunotherapies for cancer. Although the main focus is laid on MM, the presented approaches can be expanded to other malignancies in order to improve NK cell therapies and increase NK cell fitness in an immunosuppressive TME.