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Natural killer cells are a promising platform for cancer immunotherapy. Natural killer cells have high intrinsic killing capability, and the insertion of a chimeric antigen receptor can further enhance their antitumor potential. In first-in-human trials, chimeric antigen receptor-natural killer cells demonstrated strong clinical activity without therapy-induced side effects. The applicability of natural killer cells as an "off-the-shelf product makes them highly attractive for gene-engineered cell therapies. Traditionally, viral transduction has been used for gene editing; however, the use of viral vectors remains a safety concern and is associated with high costs and regulatory requirements. Here, we review the current landscape of nonviral approaches for chimeric antigen receptor- natural killer cell generation. This includes transfection of vector particles and electroporation of mRNA and DNA vectors, resulting in transient modification and chimeric antigen receptor expression. In addition, using nonviral transposon technologies, natural killer cells can be stably modified ensuring long-lasting chimeric antigen receptor expression. Finally, we discuss CRISPR/Cas9 tools to edit key genes for natural killer cell functionality. [ABSTRACT FROM AUTHOR]

Although the overall survival in pediatric rhabdomyosarcoma (RMS) has increased over the last decades, the most aggressive subtype of alveolar RMS is in dire need of novel treatment strategies. RMS cells evade cell death induction and immune control by increasing the expression of inhibitors of apoptosis proteins (IAPs), which can be exploited and targeted with stimulation with Smac mimetics. Here, we used the Smac mimetic BV6 to re-sensitize RMS spheroids to cell death, which increased killing induced by natural killer (NK) cells. Single BV6 treatment of RMS spheroids did not reduce spheroidal growth. However, we observed significant spheroidal decomposition upon BV6 pre-treatment combined with NK cell co-cultivation. Molecularly, IAPs s are rapidly degraded by BV6, which activates NF-κB signal transduction pathways in RMS spheroids. RNA sequencing analysis validated NF-κB activation and identified a plethora of BV6-regulated genes. Additionally, BV6 released caspases from IAP-mediated inhibition. Here, caspase-8 might play a major role, as knockdown experiments resulted in decreased NK cell-mediated attack. Taken together, we improved the understanding of the BV6 mechanism of RMS spheroid sensitization to cytotoxic immune cells, which could be suitable for the development of novel combinatory cellular immunotherapy with Smac mimetics. [ABSTRACT FROM AUTHOR]

Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)-T cell therapies have achieved remarkable success. However, application-related toxicities, such as cytokine release syndrome or neurotoxicity, moved natural killer (NK) cells into focus as novel players in immunotherapy. CAR-NK cells provide an advantageous dual killing-capacity by CAR-dependent and -independent mechanisms and induce few side effects. While the majority of trials still use CAR-T cells, CAR-NK cell trials are on the rise with 19 ongoing studies worldwide. This review illuminates the current state of research and clinical application of CAR-NK cells, as well as future developmental potential. [ABSTRACT FROM AUTHOR]

Simple Summary: This review is intended to provide an overview on the history and recent advances of T cell and natural killer (NK) cell-based immunotherapy. While the thymus was discovered as the origin of T cells in the 1960s, and NK cells were first described in 1975, the clinical application of adoptive cell therapies (ACT) only began in the early 1980s with the first lymphokine activated killer (LAK) cell product for the treatment of cancer patients. Over the past decades, further immunotherapies have been developed, including ACT using cytokine-induced killer (CIK) cells, products based on the NK cell line NK-92 as well as specific T and NK cell preparations. Recent advances have successfully improved the effectiveness of T, NK, CIK or NK-92 cells towards tumor-targeting antigens generated by genetic engineering of the immune cells. Herein, we summarize the promising development of ACT over the past decades in the fight against cancer. The promising development of adoptive immunotherapy over the last four decades has revealed numerous therapeutic approaches in which dedicated immune cells are modified and administered to eliminate malignant cells. Starting in the early 1980s, lymphokine activated killer (LAK) cells were the first ex vivo generated NK cell-enriched products utilized for adoptive immunotherapy. Over the past decades, various immunotherapies have been developed, including cytokine-induced killer (CIK) cells, as a peripheral blood mononuclear cells (PBMCs)-based therapeutic product, the adoptive transfer of specific T and NK cell products, and the NK cell line NK-92. In addition to allogeneic NK cells, NK-92 cell products represent a possible "off-the-shelf" therapeutic concept. Recent approaches have successfully enhanced the specificity and cytotoxicity of T, NK, CIK or NK-92 cells towards tumor-specific or associated target antigens generated by genetic engineering of the immune cells, e.g., to express a chimeric antigen receptor (CAR). Here, we will look into the history and recent developments of T and NK cell-based immunotherapy. [ABSTRACT FROM AUTHOR]