Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are immune cells known for their lethal capabilities. CTLs develop from naïve CD8+ T cells, acquiring antigen peptides from dendritic cells (DCs), enabling potent, antigen-specific killing, and forming immunological memory. CTLs' specificity and memory play vital roles in viral infection vaccinations. In cancer immunity, CTLs are key players in eliminating cancer cells, and current cancer immunotherapy strategies heavily rely on their effectiveness. We've explored innovative approaches to induce CTLs and recently reported successful antitumor activity induction using these methods. Nevertheless, the complex CTL generation mechanism allows cancer cells to evade killing by impairing neoantigen expression and antigen presentation processes
On the other hand, NK cells exhibit an inherent cytotoxic capability, unlike CTL which necessitates a complex generation process. NK cells can eliminate cancer cells that manage to escape CTL-induced killing due to the presence of both activating and inhibitory receptors. The regulation of NK cells' cytotoxic function relies on maintaining an equilibrium between these receptor signals.
Hence, NK cells are poised to serve as a strategic asset against cancers resistant to CTL. The pursuit of NK cell-targeting therapeutics encompasses two main approaches: bolstering the activation or mobilization of existing NK cells and utilizing NK cell-centered therapies. Various cytokine treatments like interleukin 12 (IL-12), IL-15, and IL-18 have been shown to enhance NK cell functions. Supplementary treatments further amplify the anti-tumor impact through NK cell activation. Effective therapeutic outcomes have also been realized by blocking inhibitory receptors.
Regarding NK cell-based therapy, allogeneic NK cells derived from blood sources and human NK cell lines are commonly employed. In contrast to T cell-based therapy, NK cell-based approaches offer advantages such as diminished graft-versus-host reactions and readily available implementation. Among human NK cell lines, clinical trials have been conducted using NK-92 cells. Notably, NK-92 cells exhibit minimal expression of inhibitory receptors, rendering them receptive to activating signals. As a result, NK-92 cells hold promise as a foundational platform for advancing NK cell-based therapies.
Genetic engineering techniques have been harnessed to enhance the efficacy of NK-92-based therapy. While viral vectors like retroviruses and lentiviruses have been employed for transduction, the potential for insertional mutations remains a concern. In contrast, non-viral vectors offer a safe, cost-effective, and user-friendly alternative. Moreover, these non-viral vectors are well-suited for delivering nucleic acids like small interfering RNA (siRNA) and messenger RNA (mRNA). Despite challenges associated with transfection via non-viral vectors, a breakthrough was achieved through the development of lipid nanoparticles (LNPs) to deliver siRNA into NK-92 cells. Notably, one LNP formulation, CL1H6-LNP, composed of an ionizable lipid, CL1H6, exhibited robust gene silencing capabilities alongside high cell viability. These findings establish CL1H6-LNP as a potent non-viral vector for modifying NK-92 cell function. The remarkable success of mRNA vaccines in combating coronavirus disease 2019 (COVID-19) has propelled mRNA to the forefront of drug modalities. This recognition has extended to the importance of LNPs for mRNA delivery. Although mRNA's gene expression is transient, it aligns well with NK-92-based therapy. NK-92 cells possess limited proliferation and a short lifespan within our bodies, often administered post-irradiation. Thus, even transient mRNA expression can effectively exert its function. Notably, NK-92-based therapy is distinct as living drug therapy, diverging from conventional cell therapies. For the delivery of mRNA into NK-92 cells, a method involving the transfection of chimeric antigen receptor (CAR)-coding mRNA via electroporation has been reported, resulting in the potent killing activity of CAR-NK-92 against cancer cells (Boissel et al., 2009; Boissel et al., 2012). Conversely, utilizing LNPs for this purpose has yet to be explored. This study delves into the potential of CL1H6-LNP for delivering mRNA to NK-92 cells. The mRNA-loaded CL1H6-LNP exhibited superior gene expression compared to DLin-MC3-DMA (MC3)-based LNPs (MC3-LNP) and Lipofectamine MessengerMAX (LF). A comparison of intracellular trafficking between CL1H6-LNP and MC3-LNP revealed the former's high efficiencies in cellular uptake and fusion with endosomal membranes, attributed to its elevated mRNA expression. These findings highlight CL1H6-LNPs as a promising platform for mRNA delivery to NK-92 cells, offering valuable insights for the development of LNPs tailored for NK cell mRNA delivery.
