A Soluble NK-CAR Mediates the Specific Cytotoxicity of NK Cells toward the Target CD20 Lymphoma Cells.

The structures of chimeric antigen receptors (CARs) currently designed for natural killer (NK) cells are mostly based on knowledge gained about CAR-T cells. Although these CAR-NK cells have shown promising effects, there are still many limitations to their application. In this study, we designed a soluble NK-CAR since the membrane protein NKG2D expressed by NK cells can directly trigger NK cell cytotoxicity by binding with the ligand MICA. This CAR is composed of three segments: the extracellular domain of MICA, an anti-CD20 single-chain variable fragment (anti-CD20 ScFv), and a human IgG Fc component. The nucleotide sequence of the soluble NK-CAR was cloned into a eukaryotic expression vector and expressed in suspension HEK293 cells, and the recombinant NK-CAR protein was then purified in a protein A column. The novel NK-CAR exhibited bifunctional activity, recognizing both the CD20 antigen of target cells and the NKG2D receptor of NKL cells. The NK-CAR activated the NKG2D receptor signaling pathway, causing NKL cells to express CD107a and secrete interferon-gamma. The soluble NK-CAR mediated the NKL cell killing of CD20 Daudi cells in vitro, with a 1 µg/mL concentration inducing the maximum killing effect. Moreover, 51.7% (p < 0.01) of Daudi cells were killed at the effector-to-target ratio of 10:1. In the presence of recombinant rMICA and NKG2D-Ig proteins, this killing effect was reduced to 30% (P < 0.01) owing to competitive interference. Our results highlight the clinical application potential of this novel immunotherapy for killing target tumor cells.