Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38^dim phenotype occurring during long-term cytokine stimulation of primary NK cells. Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38^dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. We verified the functionality of the αCD38-CAR-NK cells against CD38⁺ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.