南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (12): 1877-1884.doi: 10.12122/j.issn.1673-4254.2021.12.18

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表达高亲和PD-1突变体和嵌合抗原受体的双效CBNK细胞的构建及其生物学特性评价

钟慧霖，邹庆剑，刘海霞，王晓民，杜少茵，梁海燕，吴志君，叶俊杰，邹清雁   

  1. 广州熙帝生物科技有限公司，广东 广州 510633；五邑大学生物科技与大健康学院，广东 江门 529020；西湖大学生命科学学院，浙江 杭州 310024
  • 出版日期:2021-12-20 发布日期:2022-01-05

Construction and evaluation of dual-effect cord blood natural killer cells expressing high-affinity PD-1 and chimeric antigen CD19 receptor

ZHONG Huilin, ZOU Qingjian, LIU Haixia, WANG Xiaomin, DU Shaoyin, LIANG Haiyan, WU Zhijun, YE Junjie, ZOU Qingyan   

  1. Guangzhou Cedicine Biotech Co.Ltd, Guangzhou 510633, China; School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China; School of Life Sciences, Westlake University, Hangzhou 310024, China
  • Online:2021-12-20 Published:2022-01-05

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摘要： 目的 开发一种共表达PD-L1高亲和受体PD-1（HAPD1）和嵌合抗原受体（CAR）的新型双效 CBNK（HAPD1-CAR-CBNK）细胞，进一步提高肿瘤免疫治疗的疗效。方法 首先构建可同时表达高亲和PD-1（HAPD1）的靶向CD19的CAR的双效慢病毒载体，并包装慢病毒感染从脐血中分离扩增的单个核细胞获得双效脐血CAR-NK细胞。检测分析感染效率、细胞扩增能力、表面标志物表达率；然后通过体外共培养检测细胞在不同效靶比条件下对靶细胞杀伤效率以及不同扩增时期细胞的杀伤效率。最后在免疫缺陷小鼠（24只）体内验证双效CBNK细胞在体内对靶细胞的杀伤能力。结果 HAPD1和CAR基因利用慢病毒载体可高效转导至CBNK细胞（[18.63±1.88）%]，病毒感染对细胞扩增倍数有一定影响（10.97±2.77 vs 24.84±3.17，P<0.05），但对细胞表面标志物表达没有显著影响（P?0.05）；双效NK细胞在效靶比为5:1和10:1时的靶细胞杀伤效力高于普通CAR-CBNK细胞（[ 68.38±8.08)% ,（79.11±7.42）% vs（49.65±13.60）%，（59.78±9.32）%，P<0.05）。病毒感染后第9~12天双效NK细胞具有最强靶细胞杀伤能力。体内实验进一步证实，双效NK细胞移植28 d后动物白细胞中肿瘤细胞比例低于普通CAR-CBNK细胞移植组（[ 19.21±3.07）% vs（29.08±3.15）%，P<0.05]。结论 本研究成功构建了一种共表达HAPD1和CAR双效CBNK细胞，并在体外和体内实验证实其高效的靶细胞杀伤能力，为肿瘤免疫细胞治疗提供了一个新的有效方案。

关键词: 嵌合抗原受体；CBNK细胞；高亲和PD-1；免疫表型；抗肿瘤活性

Abstract: Objective To obtain novel dual-effect cord blood natural killer cells (CBNKCs) expressing high-affinity PD-1 (HAPD1) and chimeric antigen CD19 receptor (CAR) to improve the effect of CAR-based immunotherapy. Methods A dual-effect lentiviral vector expressing both HAPD1 and CAR targeting CD19 was constructed. CBNKCs were infected withthe vector to obtain HAPD1 CAR19 CBNKCs. The surface markers of the cells including CD3-/CD16+CD56+, CD3+/CD16+CD56+,CD3+/CD4+, and CD3+/CD8+ were tested during cell proliferation. The cytotoxicity of CBNKCs, CAR19 CBNKCs and HAPD1 CAR19 CBNKCs incubated with CD19-positive target cells at the effector-target ratios of 5∶1, 10∶1 and 20∶1 was tested on days 7, 9, 12, and 15 of cell culture. The cytotoxicity of the cells against the target cells was also tested in NPG mice. Results CBNKCs were successfully transduced with T-cell designed CAR19 and HAPD1 CAR19 with an efficiency of (18.63±1.88)%. Infection with the lentiviral vector significantly reduced the cell expansion efficiency of the CBNKCs (10.97±2.77 vs 24.84±3.17, P<0.05) but did not significantly affect the expressions of the surface markers (P>0.05). HAPD1 CAR CBNKCs showed stronger anti-tumor effect than CAR19 CBNKCs [(68.38±8.08)% vs (49.65±13.60)% at the effector-target ratios of 5∶1 and (79.11±7.42)% vs (59.78 ± 9.32)% at 10∶1; P<0.05]. The infected CBNKCs showed the strongest cytotoxicity at 9 and 12 days after lentivirus infection. In the mouse models, transplantation of the dual-effect cells resulted in a significantly lower percentage of tumor cells in white blood cells than transplantation CAR-CBNKCs [(19.21 ± 3.07% ) vs (29.08 ± 3.15)% , P<0.05]. Conclusion We obtained a novel dual-effect CBNKC co-expressing HAPD1 and CAR. The cells show strong cytotoxicity against the target tumor cells both in vitro and in vivo, which sheds light on a new strategy of immunotherapy against tumor cells.

Key words: chimeric antigen receptor; cord blood NK cells; high-affinity PD-1; immunophenotypes; antitumor efficacy