Designs of NKG2D-based immunotherapeutics for cancer

Abstract

Natural killer group 2 D (NKG2D) receptor, one of the activation receptors on NK cells, has gained increasing attention in recent years because its ligands are widely expressed in most cancers. Naturally, NKG2D reacts to 8 different stress-induced ligands, MICA/B, and ULBP1-6. Despite being genomically conserved between human and mouse, NKG2D transcripts have splice variants that can differentiate the two. hNKG2D or mNKG2D (both long and short transcripts) interacts with DAP10 only in human but DAP10/12 in mouse, switching on different effector functions such as IFN-γ production and cytotoxicity. Full-length, extracellular or cytoplasmic domains have been used to construct chimeric antigen receptors (CAR) or implement into the antibody structures including bispecific antibodies. Interestingly, most of the NKG2D CARs, either on T cells or NK cells are investigated in preclinical models of solid tumors. In this article, we reviewed the majority of published NKG2D-based CAR and antibody designs, comparing their respective advantages and disadvantages. We also elaborated how these CARs and antibodies were tested in preclinical cancer models and clinical trials in this review article.

Keywords: CAR-T therapy; NK cell therapy; NKG2D; cancer; immunotherapy.

Figure 1

Human NKG2D receptor and its cognate ligands. NKG2D receptor is a C-type lectin-like molecule expressed primarily on NK cells and subsets of T cells. It has a disulfide-linked homodimer that is associated with four DNAX-activating protein 10 (DAP10) forming a hexameric complex. Upon phosphorylation, the YINM motifs of DAP10 recruit and activate phosphatidyl-inositol 3-kinase (PI3K)/Akt and Grb2/Vav1 molecules leading to cell survival, expansion, cytotoxicity and differentiation. NKG2D ligands include proteins in MHC class-I polypeptide-related sequence (MIC) and UL16-binding protein (ULBP) families. MICA and B (other names PERB11.1/11.2) have three MHC-class I related α1, α2 and α3 domains anchored on cell surface by a transmembrane domain. ULBP 1-6 (other name RAET1I/H/N/E/G/L) consists of only α1 and α2 domains and are attached to the cell membrane via either a glycosylphosphatidylinositol (GPI)-anchor (ULBP1-3, 5 and 6) or transmembrane domains (ULBP 2, 4, and 5). Created in BioRender. Chan, W. (2025) https://BioRender.com/n95p848.

Figure 2

Family of NKG2D-based CAR designs. The antigen-binding domains of the NKG2D-based CARs include extracellular domains of NKG2D receptor molecule (NKG2D ECD) and scFv targeting tumor antigens. The hinge region of the CARs is mostly derived from CD8α molecule, and others are from NKG2D, IgG1/4, or PD1. Except full-length NKG2D or truncated 2B4 is employed in the CAR design, the transmembrane domain of the CARs is CD8α, CD28, or IgG1. For the co-stimulation domains, almost all the NKG2D-based CARs contain CD3ζ, with 4-1BB, DAP12, CD28, DAP10, or CD27 as the second/third co-stimulation domain. Created in BioRender. Chan, W. (2025) https://BioRender.com/777ditl.