Ex vivo modification of natural killer cells: The Role of Lipid Nanoparticle-Mediated mRNA Delivery

Cellular immunotherapy is an increasingly prominent field in oncology that prolongs patient survival by enhancing tumor surveillance and clearance. In addition, immunotherapy boosts the suppressed and weakened immune system either by targeting immune cells with monoclonal antibodies (mAbs), engagers, and immune checkpoint inhibitors (ICIs), or through adoptive cell transfer via intravenous infusion of autologous or allogeneic effector cells. Over the past decade, major pharmaceutical companies have been focusing on T cell-based therapies, including genetically modified chimeric antigen receptor (CAR) T cells, which have demonstrated substantial clinical benefit. Nevertheless, extensive research has highlighted associated toxicities, such as cytokine release syndrome (CRS), graft-versus-host disease (GvHD), and neurotoxicity linked to CAR-T cells. Given these risks, along with the significant costs of manufacturing, research efforts are increasingly shifting towards alternative effector cell sources, including natural killer (NK) cells. Unlike T cells, NK cells offer the advantage of not targeting healthy self-tissue, thereby diminishing the risk of triggering a GvHD, making them a promising alternative for the development of allogeneic (“off-the-shelf”) cell products. Their efficacy in treating hematological malignancies has been demonstrated in numerous clinical studies. However, challenges remain in enhancing their effectiveness in solid tumors, improving their penetration in tumor sites, and increasing their proliferation and persistence in the body. To address these limitations, researchers are now focusing on engineering NK cells to support tumor infiltration and to overcome resistance to immunosuppression by the tumor microenvironment (TME). Strategies involve the overexpression of activating and chemotactic signals, as well as downregulation of inhibitory signals. These engineering approaches are often applied in combination with mAbs and engagers to enhance their therapeutic efficacy. Delivery of genetic material is essential for successful modification of NK cells, however, NK cells are hard-to-transfect cells and existing gene delivery methods present significant limitations. While viral vectors are highly effective in transducing NK cells, their large-scale production under good manufacturing practice (GMP) standards is associated with considerable costs and genotoxic risks. Moreover, electroporation has been linked to cellular toxicity and phenotypic alterations of NK cells. Consequently, to advance the field of adoptive cell therapy and generate modified NK cells with enhanced cytotoxicity against both hematological malignancies and solid tumors, the development of a scalable, non-toxic gene delivery system is of high importance. The aim of this thesis was to contribute to the field of NK cell therapeutics by developing a non-viral nanoparticle-based delivery system to efficiently and safely deliver mRNA into NK cells. Additionally, this work investigated the simultaneous delivery of two mRNA molecules for dual NK cell targeting and examined how the incorporation of cell penetrating peptides (CPPs) within the nanoparticles can enhance endosomal escape and mRNA transfection efficiency. Finally, this thesis explored the potential of nanoparticle-mediated overexpression of an activating receptor in NK cells as a synergistic approach with mAbs to enhance the antibody-dependent cellular cytotoxicity (ADCC) function of NK cells.