Enhancing the Anti-Tumor Activity of Natural Killer (NK) Cells through NK-Biology Tailored Chimeric Antigen Receptors

Abstract

In recent years, chimeric antigen receptor (CAR) T-cell therapy against cancer has shown remarkable efficacy against B-cell malignancies. However, CAR T-cell therapy bears lethal toxicities and requires autologous use, rendering it a high-cost treatment that requires access to well-resourced medical centers. Contrastingly, natural killer (NK) cell-based CAR therapies are safe to use in the allogeneic setting, presenting the opportunity for an “off-the-shelf” therapy with low toxicity. However, there is a lack of literature to tailor CARs to NK-cell biology. In this study, we address this need through testing a panel of next-generation anti-CD19 CARs that contained domains derived from NK-cell receptors (NKRs) in an immortalized human NK-cell line, NK-92. Using flow cytometry and real-time, live-cell imaging (IncuCyte SX5), we found that use of the transmembrane domain (TMD) of NKp46 alone resulted in ~2-fold increase of NK-cell killing of CD19+ B lymphoblastoid cells, compared to a third-generation T-cell–optimized CAR that contained domains from CD28, CD127, and CD3ζ. In addition, NK-cell killing could be further enhanced using co-stimulatory domains DAP10 and CD27. Additionally, considering that TMDs from activating NKRs (e.g., NKp46) recruit adaptor proteins that mediate cell surface expression and signaling, we tested whether this was true for our CARs. Indeed, when using a CAR containing the TMD of CD16 or KIR3DS1, which normally recruits FcRγ or DAP12 respectively, flow cytometry revealed that surface expression could not be achieved in adaptor-deficient cell lines unless cells were co-transduced with corresponding adaptors. With successful CAR testing in NK-92 cells and thorough understanding of NK adaptors, we will expand our study into primary human NK cells and in-vivo xenograft mouse models to translate our findings into a next-generation cellular therapy against cancer that may revolutionize the standard of care for cancer patients.