A Soluble NK-CAR Mediates the Specific Cytotoxicity of NK Cells toward the Target CD20(+) Lymphoma Cells.
A Soluble NK-CAR Mediates the Specific Cytotoxicity of NK Cells toward the Target CD20(+) Lymphoma Cells.
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可溶性 NK-CAR 介导 NK 细胞对目标 CD20 淋巴瘤细胞的特异性细胞毒性
DOI：
10.14336/ad.2022.0415
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发表时间：
2022-10-01
影响因子：
7.4
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中科院分区：
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目前为自然杀伤（NK）细胞设计的嵌合抗原受体（汽车）的结构主要基于对CAR-T细胞的了解。尽管这些CAR-NK细胞已经显示出有希望的效果，但它们的应用仍然存在许多限制。在这项研究中，我们设计了一种可溶性NK-CAR，因为NK细胞表达的膜蛋白NKG 2D可以通过与配体云母结合直接触发NK细胞的细胞毒性。该CAR由三个片段组成：云母的胞外结构域、抗CD 20单链可变片段（抗CD 20 ScFv）和人IgG Fc组分。将可溶性NK-CAR的核苷酸序列克隆到真核表达载体中，并在悬浮HEK 293细胞中表达，然后在金黄色葡萄球菌蛋白A柱中纯化重组NK-CAR蛋白。新型NK-CAR表现出双功能活性，识别靶细胞的CD 20抗原和NKL细胞的NKG 2D受体。NK-CAR激活NKG 2D受体信号通路，导致NKL细胞表达CD 107 a并分泌干扰素-γ。可溶性NK-CAR在体外介导CD 20 + Daudi细胞的NKL细胞杀伤，其中1 μg/mL浓度诱导最大杀伤作用。此外，在10：1的效靶比下，51.7%（p < 0.01）的Daudi细胞被杀死。在重组rMICA和NKG 2D-Ig蛋白存在下，由于竞争性干扰，这种杀伤作用降低至30%（P < 0.01）。我们的研究结果突出了这种新型免疫疗法在杀死靶肿瘤细胞方面的临床应用潜力。
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 Staphylococcus aureus 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.