整合素αvβ3放射性核素靶向治疗联合PD-L1免疫治疗的实验研究  

Enhancement of therapeutic efficacy by combination of integrinαvβ3-targeted radiotherapy and anti-PD-L1 immunotherapy:a preclinical study

作　　者：赵亮 富凯丽 姚兰琳 林秋明 郭志德[3] 文雪君 陈小元[4] 吴华[2] 孙龙[2] 林勤[1] 陈皓鋆[2] Zhao Liang;Fu Kaili;Yao Lanlin;Lin Qiuming;Guo Zhide;Wen Xuejun;Chen Xiaoyuan;Wu Hua;Sun Long;Lin Qin;Chen Haojun(Department of Radiation Oncology,the First Affiliated Hospital of Xiamen University,Xiamen 361003,China;Department of Nuclear Medicine&Minnan PET Center,the First Affiliated Hospital of Xiamen University,Xiamen 361003,China;Center for Molecular Imaging and Translational Medicine,School of Public Health,Xiamen University,Xiamen 361102,China;Laboratory of Molecular Imaging and Nanomedicine,National Institute of Biomedical Imaging and Bioengineering,National Institutes of Health,Bethesda,Maryland MD 20892,USA)

机构地区：[1]厦门大学附属第一医院放射治疗科,361003 [2]厦门大学附属第一医院核医学科、闽南PET中心,361003 [3]厦门大学分子影像暨转化医学研究中心,361102 [4]美国国立卫生研究院、生物医学影像与生物工程研究所、分子影像和纳米医学研究中心,美国马里兰州贝塞斯达20892

出　　处：《中华核医学与分子影像杂志》2020年第5期268-274,共7页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家自然科学基金(81701736,81772893)。

摘　　要：目的探讨基于靶向整合素αvβ3的放射性核素靶向治疗(TRT)联合基于程序性死亡受体蛋白配体1(PD-L1)的免疫治疗的抗肿瘤疗效及其潜在机制。方法制备可特异性靶向整合素αvβ3的分子探针177Lu-伊文思蓝(EB)-精氨酸-甘氨酸-天冬氨酸(RGD),并测定其放射性比活度与放化纯。建立结肠癌MC38荷瘤鼠模型,进行生物分布及microSPECT显像研究。通过监测小鼠肿瘤体积和体质量变化来评估疗效与安全性(各组n=9);采用流式细胞术分析A组(对照组;生理盐水治疗)、B组(177Lu-EB-RGD单药治疗,18.5 MBq)、C组(PD-L1抗体单药治疗,10 mg/kg)和D组(联合治疗,18.5 MBq 177Lu-EB-RGD与10 mg/kg PD-L1)荷瘤鼠经治疗后肿瘤微环境(PD-L1+免疫细胞、CD8+T细胞和调节性T细胞)的改变(各组n=3)。采用重复测量方差分析、两独立样本t检验分析数据。结果177Lu-EB-RGD放射性比活度为(55.85±14.00)GBq/μmol,放化纯大于95%。MicroSPECT显像中,177Lu-EB-RGD在荷瘤鼠肿瘤中清晰可见,且摄取率高、滞留时间长,注射后24 h肿瘤/肌肉比值达14.87±0.88,而在正常组织摄取及滞留较少;生物分布结果显示,注射后4 h 177Lu-EB-RGD较177Lu-RGD表现出明显增高的肿瘤摄取[(12.00±1.60)和(3.69±0.37)每克组织百分注射剂量率(%ID/g);t=8.63,P<0.01]。在治疗开始后第6天,A^D各组小鼠肿瘤体积差异有统计学意义(F=7.32,P=0.03),在监测时间内,D组平均肿瘤体积最小,治疗效果最好,7/9的荷瘤鼠表现为完全缓解,且未出现肿瘤复发。流式细胞术结果显示,TRT可导致肿瘤微环境中PD-L1表达上调,B组与A组PD-L1+免疫细胞数差异有统计学意义(CD45+/PD-L1:2.34%与0.95%,CD11b+/PD-L1:2.41%与0.66%;t值:11.17和8.70,均P<0.01),而免疫治疗及联合治疗使C、D组微环境中的CD8+T细胞浸润较A组急剧增加(2.07%与0.26%,2.71%与0.26%;t值:4.10和6.03,均P<0.05)。结论TRT联合免疫治疗可协同增强抗肿瘤疗效,有望应用于可接受TRT的转移性肿�Objective To investigate the therapeutic efficacy and potential mechanisms of integrinαvβ3-targeted radionuclide therapy(TRT)in combination with anti-programmed cell death protein ligand 1(PD-L1)immunotherapy.Methods Integrinαvβ3-targeted molecule Arg-Gly-Asp(RGD)was conjugated with Evans blue(EB)and then labeled with 177Lu to obtain 177Lu-EB-RGD.The radioactivity and radiochemical purity were determined.MicroSPECT imaging,biodistribution,and in vivo therapeutic efficacy were subsequently performed in MC38 murine colon cancer models.Volume of tumor and body mass of mice were observed to assess the therapeutic efficacy and safety(n=9 in each group).Flow cytometry was used to evaluate therapy response of saline-treated(control,group A),18.5 MBq 177Lu-EB-RGD-treated(group B),10 mg/kg PD-L1 antibody-treated(group C),TRT combined with immunotherapy-treated(group D,18.5 MBq 177Lu-EB-RGD and 10 mg/kg PD-L1 antibody)mice and alterations in tumor microenvironment(PD-L1+immune cells,CD8+T cells and regulatory T cells).Independent-sample t test and repeated measures analysis of variance were used for data analysis.Results The radioactivity of 177Lu-EB-RGD was(55.85±14.00)GBq/μmol.SPECT imaging clearly visualized the MC38 tumors in mice models with high uptake and long retention time,the tumor/muscle ratio reached 14.87±0.88 at 24 h postinjection,while less uptake and retention in normal tissues.Tumor uptake of 177Lu-EB-RGD was significantly higher than that of 177Lu-RGD 4 h post-injection((12.00±1.60)vs(3.69±0.37)%ID/g;t=8.63,P<0.01).The efficacy results between each treatment group was significantly different(F=7.32,P=0.03)at day 6 post-treatment.The combination therapy showed the most outstanding anti-tumor efficacy with 7/9 mice showed complete response.Flow cytometry results showed that TRT up-regulated the PD-L1 expression significantly,namely,PD-L1+immune cells in group B and group A were significantly different(CD45+/PD-L1:2.34%vs 0.95%,CD11b+/PD-L1:2.41%vs 0.66%;t values:11.17 and 8.70,both P<0.01);immunoth

关 键 词：结肠肿瘤 伊文思蓝 精氨酸-甘氨酸-天冬氨酸 镥 程序性细胞死亡受体1 小鼠 

分 类 号：R730[医药卫生—肿瘤]