摘要
目的:构建基于靶点人源化小鼠的程序性死亡受体-1(PD-1)/细胞毒性T淋巴细胞抗原-4(CTLA-4)双特异性抗体(BsAb)并对BsAb及其IgG1亚型进行抗癌活性评价和探讨其潜在的作用机制。方法:构建和扩增并纯化不同结构及抗体亚型的PD-1/CTLA-4抗体BsAb1、BsAb2和BsAb3,对纯化BsAb进行靶点亲和力检测,采用荧光素酶报告基因实验和FCM检测抗体的生物学活性。基于B-hPD-1-hPD-L1-h CTLA-4人源化小鼠的MC38-hPD-L1结肠癌细胞移植瘤模型对BsAb进行体内药效评估,并通过移植瘤组织中肿瘤浸润淋巴细胞(TIL)分析PD-1/CTLA-4抗体的作用机制。结果:成功制备的BsAb1、BsAb2及BsAb3对靶点PD-1和CTLA-4均有较强的特异性亲和力、对靶点通路均有不同程度的阻滞活性,均明显抑制移植瘤的生长(P<0.05或P<0.01)。IgG1亚型BsAb体内药效更优(P<0.01),TIL分析发现BsAb2-IgG1明显增加了CTL百分率(P<0.05),显著降低了肿瘤浸润Treg细胞百分率(P<0.01),使肿瘤免疫微环境更有利于杀伤肿瘤细胞;增强ADCC活性的Fc突变体亚型BsAb2-SI则不能进一步提高抗肿瘤活性。结论:具有Fc效应功能的IgG1亚型的PD-1/CTLA-4抗体体内抗癌药效更优,因其可以更好地清除TIL中的Treg细胞。
Objective:To construct programmed death-1(PD-1)/cytotoxic T lymphocyte antigen-4(CTLA-4)bispecific antibody(BsAb)based on the target humanized mice,evaluate the anticancer activity of BsAb and its IgG1 isotype and investigate its potential working mechanism.Methods:Different formats and isotypes of PD-1/CTLA-4 antibodies BsAb1,BsAb2 and BsAb3 were constructed,expanded and purified.The target affinity of purified BsAbs were tested by surface plasmon resonance(SPR),and the biological activity of antibodies were tested by luciferase reporter gene assay and FCM.The efficacy of BsAbs was evaluated in vivo based on the MC38-hPD-L1 colon carcinoma cell transplant tumor model in humanized B-hPD-1-hPD-L1-hCTLA-4 mice.The working mechanism of PD-1/CTLA-4 BsAbs in transplant tumor tissues was investigated by tumor-infiltrating lymphocyte(TIL)analysis.Results:Successfully prepared BsAb1,BsAb2,and BsAb3 all demonstrated relatively strong specific affinity to checkpoint targets PD-1 and CTLA-4,showed different levels of blocker efficacy to target pathways,and significantly suppressed the growth of transplant tumors(P<0.05 or P<0.01).BsAb with IgG1 isotype was more potent in vivo than other isotypes(P<0.01).Tumor-infiltrating lymphocyte analysis revealed that BsAb2-IgG1 significantly increased the percentage of cytotoxic T lymphocytes(CTLs)(P<0.05)and significantly decreased the percentage of tumor-infiltrating regulatory T(Treg)cells(P<0.01),thus making the tumor immune microenvironment more conducive in killing tumor cells.However,the ADCC-enhanced Fc mutation isotype of BsAb2-SI can not further improve anti-tumor activity.Conclusion:The IgG1 PD-1/CTLA-4 antibody with Fc effector function has more potent in vivo anticancer efficacy,because it can better remove Treg cells in tumor-infiltrating lymphocytes.
作者
胡红梅
于秋红
杨毅
沈月雷
刘柏宏
HU Hongmei;YU Qiuhong;YANG Yi;SHEN Yuelie;LIU Baihong(School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004,Jiangxi,China;Yangtze Delta Drug Advanced Research Institute,Nantong 226133,Jiangsu,China;BiocytogenPharmaceutical Technology Co.,Ltd.,Beijing 102609,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2023年第5期380-386,共7页
Chinese Journal of Cancer Biotherapy
基金
山东省抗体药物创新创业共同体项目(No.2021CXCYGTT16)。
