Long Jing, Zheng Rong, Ye Sishi, Ke Shanwen, Duan Deming, Wei Cheng, Gao Jimin
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035; Department of Clinical Laboratory, Dujiangyan Traditional Chinese Medicine Hospital, Chengdu 611830, China.
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Jul;39(7):577-585.
Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
目的 本研究旨在构建并鉴定靶向NKG2D配体（NKG2DL）（分泌IL-15Ra-IL-15）的嵌合抗原受体NK92（CAR-NK92）细胞，并验证NKG2D CAR-NK92细胞对多发性骨髓瘤细胞的杀伤活性。方法 采用NKG2D的胞外段连接4-1BB和CD3ζ，以及IL-15Ra-IL-15序列，获得CAR表达框架。包装慢病毒并转导至NK92细胞中，获得NKG2D CAR-NK92细胞。采用CCK-8法检测NKG2D CAR-NK92细胞的增殖情况，ELISA法检测IL-15Ra分泌情况，乳酸脱氢酶（LDH）法检测杀伤效率。采用流式细胞术检测NKp30、NKp44、NKp46分子标志物、凋亡细胞群体比例、CD107a以及颗粒酶B和穿孔素的分泌水平。此外，通过检测脱颗粒能力验证NKG2D CAR-NK92细胞对肿瘤的细胞毒性机制。此外，在NKG2D抗体抑制效应细胞和组胺抑制肿瘤细胞后，利用LDH法检测对细胞杀伤效率的影响。最后，构建多发性骨髓瘤肿瘤异种移植模型，验证其体内抗肿瘤活性。结果 慢病毒转导显著增加了NK92细胞中NKG2D的表达。与NK92细胞相比，NKG2D CAR-NK92细胞的增殖能力较弱。NKG2D CAR-NK92细胞的早期凋亡细胞群体较少，且NKG2D CAR-NK92细胞对多发性骨髓瘤细胞具有更强的细胞毒性。此外，在其培养上清中可检测到IL-15Ra分泌。NKG2D CAR-NK92细胞中NKp44蛋白表达明显增加，表明激活水平增强。抑制试验表明，CAR-NK92细胞对MHC-I链相关蛋白A（MICA）和MICB阳性肿瘤细胞的细胞毒性更依赖于NKG2D CAR与NKG2DL之间的相互作用。用肿瘤细胞刺激NKG2D CAR-NK92细胞后，颗粒酶B和穿孔素表达增加，NK细胞明显上调CD107α。此外，多发性骨髓瘤肿瘤异种移植模型显示，用NKG2D CAR-NK92细胞治疗的小鼠肿瘤明显缩小，且细胞治疗对小鼠体重无明显影响。结论 成功构建了一种靶向NKG2DL（分泌IL-15Ra-IL-15）的CAR-NK92细胞，表明其对多发性骨髓瘤细胞具有有效杀伤作用。
