CAR-NK cells: A promising cellular immunotherapy for cancer

Abstract

Natural Killer (NK) cells and CD8⁺ cytotoxic T cells are two types of immune cells that can kill target cells through similar cytotoxic mechanisms. With the remarkable success of chimeric antigen receptor (CAR)-engineered T (CAR-T) cells for treating haematological malignancies, there is a rapid growing interest in developing CAR-engineered NK (CAR-NK) cells for cancer therapy. Compared to CAR-T cells, CAR-NK cells could offer some significant advantages, including: (1) better safety, such as a lack or minimal cytokine release syndrome and neurotoxicity in autologous setting and graft-versus-host disease in allogenic setting, (2) multiple mechanisms for activating cytotoxic activity, and (3) high feasibility for 'off-the-shelf' manufacturing. CAR-NK cells could be engineered to target diverse antigens, enhance proliferation and persistence in vivo, increase infiltration into solid tumours, overcome resistant tumour microenvironment, and ultimately achieve an effective anti-tumour response. In this review, we focus on recent progress in genetic engineering and clinical application of CAR-NK cells, and discuss current challenges and future promise of CAR-NK cells as a novel cellular immunotherapy in cancer.

Keywords: Cancer; Chimeric antigen receptor; Immunotherapy; NK cells.

Fig. 1

Germline-encoded activating and inhibitory receptors control NK cell activation. One of the best-studied activating receptors is NKG2D, a homodimeric receptor that recognises stress-induced ligands MICA, MICB, and ULBP1-6 expressed on damaged, transformed, or otherwise abnormal cells. Upon engaging with its ligand, NKG2D signals through adapter molecule DAP10 to trigger NK activation. NK cells can also be activated through a variety of natural cytotoxicity receptors (NCRs) – such as NKp30, NKp44, and NKp46 - in response to a diverse set of viral, oncogenic, or stress-induced ligands. Upon binding to their respective ligands, NCRs recruit immunoreceptor tyrosine-based activation motif (ITAM)-bearing adapter molecules DAP12, CD3ζ or FcεRIγ, which in turn initiate activation signalling cascades. Immunoglobulin (Ig) superfamily receptor DNAM-1 can activate NK cells via interaction with CD112. Additionally, NK cells can eliminate tumour cells through CD16-mediated ADCC, in which CD16 expressed on NK cells recognises the Fc portion of the IgG bound to the tumour cell surface. Conversely, inhibitory receptors like NKG2A and inhibitory KIRs carry immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains. They provide inhibitory signalling for NK cells when interacting with their ligands (e.g., HLA-E for CD94/NKG2A and certain HLA-A, B, or C allotypes for inhibitor KIRs) expressed by the target cells. Some Ig superfamily receptors, such as CD96 and TIGIT, interact with CD155 or CD112 and transmit inhibitory signals via additional motifs.

Fig. 2

Sources and manufacturing of CAR-NK cell products. NK92 cell line has been extensively used as a source of CAR-NK cells because CAR-NK92 cells can expand indefinitely in vitro and have reduced sensitivity to repeated freeze/thaw cycles. As malignant cells from a NK cell lymphoma, NK92 cells engineered with CAR must receive lethal irradiation before their infusion into patients. Primary NK cells can be isolated directly from peripheral blood mononuclear cells (PBMCs) of healthy donors or umbilical cord blood (UCB) using NK cell isolation kit. Isolated primary NK cells can be activated, genetically engineered with CAR-expressing vectors (e.g., lentivirus [LVs] or retrovirus [RVs]), and then expanded in NK cell-specific expansion media with cytokines for GMP-grade clinical application. NK cells also can be differentiated from CD34⁺ hematopoietic progenitor cells (HPCs) using a cocktail of cytokines in culture; the resulting NK cells are engineered with CAR and then expanded in vitro before infusion. Recently, induced pluripotent stem cells (iPSCs) have become an attractive source of CAR-NK cells for “off-the-shelf” CAR-NK cell products attributing to their unlimited proliferative capacity. iPSCs can differentiate into CD34⁺ HPCs and then NK cells. Importantly, iPSCs can be genetically engineered with CAR-expressing vectors and the resulting CAR-iPSCs then can be differentiated into CAR-HPCs and finally into CAR-NK cells.