Natural killer (NK) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in tumor surveillance, they are fast becoming a next-generation tool for adoptive immunotherapy. Many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. These include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune-function checkpoints, addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic ablation of inhibitory molecules. The next generation of NK cell products will ideally be readily available as an “off-the-shelf” product and stem cell derived to enable potentially unlimited supply. However, several considerations regarding NK cell source, genetic modification and scale up first need addressing. Understanding NK cell biology and interaction within specific tumor contexts will help identify necessary NK cell modifications and relevant choice of NK cell source. Further enhancement of manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer.

Manufacture of chimeric antigen receptor (CAR)‐T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked‐in an anti‐tumor single chain variable fragment (scFv) into the N‐terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR‐T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti‐tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral‐based methods, this method serves as an alternative and versatile way of generating T cells with tumor‐targeting receptors for cancer immunotherapy.

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