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

Abstract

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 <i>Staphylococcus aureus</i> 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.