A chimeric antigen receptor tailored to integrate complementary activation signals potentiates the antitumor activity...

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram the target specificity and functions of CAR-expressing effector cells. The design of CAR constructs typically includes an extracellular antigen-binding moiety, hinge (H), transmembrane (TM), and intracellular signaling domains. Conventional CAR constructs are primarily designed for T cells but have been directly adopted for other effector cells, including natural killer (NK) cells, without tailored optimization. Given the benefits of CAR-NK cells over CAR-T cells in terms of safety, off-the-shelf utility, and antigen escape, there is an increasing emphasis on tailoring them to NK cell activation mechanisms.

We first have taken a stepwise approach to modifying CAR components such as the combination and order of the H, TM, and signaling domains to achieve such tailoring in NK cells. Functionality of NK-tailored CARs were evaluated in vitro and in vivo in a model of CD19-expressing lymphoma, along with their expression and signaling properties in NK cells.

We found that NK-CAR driven by the synergistic combination of NK receptors NKG2D and 2B4 rather than DNAM-1 and 2B4 induces potent activation in NK cells. Further, more effective CAR-mediated cytotoxicity was observed following the sequential combination of DAP10, but not NKG2D TM, with 2B4 signaling domain despite the capacity of NKG2D TM to recruit endogenous DAP10 for signaling. Accordingly, an NK-CAR incorporating DAP10, 2B4, and CD3ζ signaling domains coupled to CD8α H and CD28 TM domains was identified as the most promising candidate to improve CAR-mediated cytotoxicity. This NK-tailored CAR provided more potent antitumor activity than a conventional T-CAR when delivered to NK cells both in vitro and in vivo.

Hence, NK receptor-based domains hold great promise for the future of NK-CAR design with potentially significant therapeutic benefits.