<sup>131</sup>I标记PD-L1抗体在人乳腺癌荷瘤小鼠模型中分子成像的研究

doi:10.3760/cma.j.cn101202-20221028-00328

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (1695 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要目的探讨放射性核素¹³¹I标记的程序性死亡受体配体1单克隆抗体（PD-L1 McAb）的物理性质及其在人乳腺癌荷瘤小鼠模型中进行分子成像的可行性。方法采用氯胺T法以放射性核素¹³¹I标记PD-L1 McAb,采用纸层析法测定¹³¹I标记PD-L1 McAb的标记率,并计算放射性比活度;标记产物¹³¹I-PD-L1 McAb采用葡聚糖凝胶G-25层析柱分离纯化,采用纸层析法测定¹³¹I-PD-L1 McAb的放射性化学纯度,以及¹³¹I-PD-L1 McAb在生理盐水和健康成人血浆中的稳定性。培养人乳腺癌PD-L1阳性细胞株MDA-MB-231细胞,随机分为总结合实验（TB）组和非特异性结合实验（NSB）组,每组又分为6个亚组,各亚组在PH 7.4的磷酸盐缓冲液中分别加入不同体积的¹³¹I-PD-L1 McAb溶液和PD-L1 McAb溶液,配制总量500 μL,使用GC-300型免疫γ-计数器测量各组细胞中¹³¹I-PD-L1 McAb的每分钟计数（cpm）值。根据TB组与NSB组各亚组之间cpm值的差值,使用Scatchard作图分析方法计算¹³¹I-PD-L1 McAb的平衡解离常数KD值,评估¹³¹I-PD-L1 McAb与MDA-MB-231细胞亲和力。取裸鼠3只,于右前肢前臂皮下接种MDA-MB-231细胞悬液制备人乳腺癌荷瘤小鼠模型,观察接种部位成瘤情况并计算肿瘤体积。裸鼠肿瘤接种后第15天按200 MBq/kg尾静脉注射¹³¹I-PD-L1 McAb溶液,分别于注射后5 min、30 min、60 min、24 h、48 h、72 h使用小动物活体成像仪进行放射性核素成像检查,观察¹³¹I-PD-L1 McAb在荷瘤鼠体内浓聚情况;在小动物活体成像检查图像上选择肿瘤部位、对侧前肢相应部位为感兴趣区,计算相应的放射性计数靶（T）值和放射性计数非靶（NT）值,比较不同时间点肿瘤部位与非肿瘤部位的放射性活度比值（T/NT）的变化情况。结果 ¹³¹I标记PD-L1 McAb的标记率为80.10%~82.20%（81.07%±1.06%）,标记产物¹³¹I-PD-L1 McAb的放射性比活度为（2.78±0.23） MBq/μg,放射性化学纯度为99.10%~99.60%（99.37%±0.25%）。¹³¹I-PD-L1 McAb在生理盐水与人新鲜血浆溶液中6 h内放射性化学纯度均大于80%。¹³¹I-PD-L1 McAb与MDA-MB-231细胞KD值为60~64（62±2） nmol/L。3只裸鼠均成功建立乳腺癌荷瘤小鼠模型,接种乳腺癌MDA-MB-231细胞后第6天触及肿块,第15天测量肿瘤体积为0.92~1.12（1.00±0.11） cm³。尾静脉注射¹³¹I-PD-L1 McAb后,不同时间点小动物活体放射性核素成像显示,¹³¹I-PD-L1 McAb早期主要在荷瘤鼠腹部浓聚,24 h时荷瘤鼠右前肢肿瘤部位开始显影,48 h时肿瘤显影最为清晰,72 h时肿瘤部位显影开始模糊。不同时间点放射性计数T/NT值随着注射时间的延长逐渐增加,48 h时T/NT值最大（6.66±1.10）,72 h时开始下降,不同时间点T/NT值比较,差异有统计学意义（F=38.04,P=0.011）。结论放射性核素¹³¹I标记PD-L1 McAb,具有较高的标记率。标记产物¹³¹I-PD-L1 McAb具有较高的放射性化学纯度及适宜的放射性比活度,在体外有良好的稳定性,且与MDA-MB-231细胞亲和力较高,可用于人乳腺癌荷瘤小鼠模型的分子成像研究。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	严小平
	邵晨旭
	年娣
	任丽
	袁超
	李辉
	孙俊杰

关键词 ：乳腺肿瘤, 模型, 动物, 小鼠, 裸, ¹³¹碘, 程序性死亡受体配体1, 分子显像

Abstract：Objective This study aims to explore the physical properties of programmed death-ligand 1 (PD-L1) monoclonal antibody (McAb) labeled with radionuclide ¹³¹I and the feasibility of molecular imaging in mice bearing human breast cancer tumor. Methods Radionuclide ¹³¹I was labeled with PD-L1 McAb by chloramine T method. The labeling rate of ¹³¹I-labeled PD-L1 McAb was determined by paper chromatography, and specific radioactivity was calculated. The labeled product ¹³¹I-PD-L1 McAb was separated and purified by dextran gel chromatography 25 column. The radiochemical purity of ¹³¹I-PD-L1 McAb and its stability in normal saline and healthy adult plasma were determined by paper chromatography. Human breast cancer PD-L1-positive MDA-MB-231 cells were randomly divided into total binding test (TB) and non-specific binding test (NSB) groups. Each group was subdivided into six subgroups. In each subgroup, different volumes of ¹³¹I-PD-L1 McAb and PD-L1 McAb solutions were added to phosphate buffer solution with PH 7.4, respectively, with a total of 500 μL. The count per minute (cpm) of ¹³¹I-PD-L1 McAb in cells of each group was measured by GC-300 immune γ-counter. According to the difference in the cpm value between TB and NSB groups, the equilibrium dissociation constant (KD) of ¹³¹I-PD-L1 McAb was calculated by Scatchard mapping analysis, and the affinity of ¹³¹I-PD-L1 McAb with MDA-MB-231 cells was evaluated. Three nude mice were subcutaneously inoculated with MDA-MB-231 cell suspension into the forearm of the right forelimb to establish a mouse model of human breast cancer. The tumor formation at the inoculation site was observed, and tumor volume was calculated. On the 15th day after tumor inoculation, nude mice were injected with ¹³¹I-PD-L1 McAb solution according to 200 MBq/kg tail vein. At 5 min, 30 min, 60 min, 24 h, 48 h, and 72 h after injection, the mice were examined by living animal imager to observe the concentration of ¹³¹I-PD-L1 McAb. The tumor site and the corresponding part of the contralateral forelimb were selected as the region of interest in the in vivo imaging images of small animals. The corresponding radioactivity target (T) and radioactivity count non-target (NT) values were calculated. Changes in the radioactivity ratio (T/NT) between tumor and non-tumor sites at different time points were compared. Results The labeling efficiency of ¹³¹I-labeled PD-L1 McAb was 80.10%-82.20% (81.07%±1.06%), the specific activity of the labeled product ¹³¹I-PD-L1 McAb was (2.78±0.23) MBq/kg, and the radiochemical purity was 99.10%-99.60%(99.37%±0.25%). The radiochemical purity of ¹³¹I-PD-L1 McAb in normal saline and human fresh plasma was more than 80% within 6 hours, and the difference was statistically significant. The KD value of ¹³¹I-PD-L1 McAb and MDA-MB-231 cells was 60-64 (62±2) nmol/L. The model of breast cancer was established successfully in three nude mice. The tumor was touched on the 6th day after the inoculation of breast cancer MDA-MB-231 cells, and the tumor volume was 0.92-1.12 (1.00±0.11) cm³ on the 15th day. After injection of ¹³¹I-PD-L1 McAb into caudal vein, radionuclide imaging at different time points showed that ¹³¹I-PD-L1 McAb was mainly concentrated in the abdomen of tumor-bearing mice in the early stage. The tumor in the right forelimb of tumor-bearing mice began to develop at 24 hours. The tumor was most clearly developed at 48 hours and began to blur at 72 hours. The value of T/NT increased gradually with prolonged injection time at different time points, reached the maximum at 48 hours, and began to decrease at 72 hours. The T/NT value significantly differed at different time points (F=38.04, P=0.011). Conclusion Radionuclide ¹³¹I labeling PD-L1 McAb has a high labeling rate. The labeled product ¹³¹I-PD-L1 McAb has high radiochemical purity, suitable specific radioactivity, good stability in vitro, and high affinity with MDA-MB-231 cells. Hence, it can be used for molecular imaging of human breast cancer tumor-bearing mice.

Key words： Breast neoplasms Models animal Mice nude ¹³¹I Programmed death-ligand 1 Molecular imaging

收稿日期: 2022-10-28

基金资助:蚌埠医学院自然科学基金（BYKY18110）; 蚌埠医学院研究生科研创新项目（Byycx22032）; 安徽省大学生创新训练项目（S202110367092）

通讯作者: 孙俊杰,Email：391566371@qq.com

引用本文:

严小平, 邵晨旭, 年娣, 任丽, 袁超, 李辉, 孙俊杰. ¹³¹I标记PD-L1抗体在人乳腺癌荷瘤小鼠模型中分子成像的研究[J]. 中华解剖与临床杂志, 2023, 28(10): 679-685.
Yan Xiaoping, Shao Chenxu, Nian Di, Ren Li, Yuan Chao, Li Hui, Sun Junjie. Molecular imaging of ¹³¹I-labeled PD-L1 antibody in mice bearing human breast cancer tumor. Chinese Journal of Anatomy and Clinics, 2023, 28(10): 679-685.

链接本文:

http://www.cjac.com.cn/CN/10.3760/cma.j.cn101202-20221028-00328 或 http://www.cjac.com.cn/CN/Y2023/V28/I10/679

[1]	袁蕙芸, 蒋宇飞, 谭玉婷, 等. 全球癌症发病与死亡流行现状和变化趋势[J].肿瘤防治研究, 2021, 48(6): 642-646. DOI: 10.3971/j.issn.1000-8578.2021.20.1533Yuan HY,Jiang YF,Tan YT,et al.Current status and time trends of cancer incidence and mortality worldwide[J]. Cancer Res Prev Treat, 2021,48(6):642-646. DOI: 10.3971/j.issn.1000-8578.2021.20.1533
[2]	孙莉,赵毅.三阴性乳腺癌治疗的研究进展[J].现代肿瘤医学,2019,27(13):2420-2424. DOI:10.3969/j.issn.1672-4992.2019.13.044Sun L, Zhao Y.Research progress in the treatment of triple negative breast cancer[J]. Modern Oncology, 2019, 27(13): 2420-2424. DOI: 10.3969/j.issn.1672-4992.2019.13.044
[3]	Wang W, Gao Z, Wang L, et al.Application and prospects of molecular imaging in immunotherapy[J]. Cancer Manag Res, 2020,12: 9389-9403.DOI: 10.2147/CMAR.S269773
[4]	Heskamp S, Wierstra PJ, Molkenboer-Kuenen J, et al.PD-L1 micro SPECT/CT imaging for longitudinal monitoring of PD-L1 expression in syngeneic and humanized mouse models for cancer[J]. Cancer Immunol Res, 2019,7(1):150-161. DOI: 10.1158/2326-6066.CIR-18-0280
[5]	Hamid O, Carvajal RD.Anti-programmed death-1 and anti-programmed death-ligand 1 antibodies in cancer therapy[J]. Expert Opin Biol Ther, 2013,13(6):847-861. DOI: 10.1517/14712598.2013.770836
[6]	Saresella M, Rainone V, Al-Daghri NM, et al.The PD-1/PD-L1 pathway in human pathology[J]. Curr Mol Med, 2012,12(3):259-267. DOI: 10.2174/156652412799218903
[7]	Ostrand-Rosenberg S, Horn LA, Haile ST.The programmed death-1 immune-suppressive pathway: barrier to antitumor immunity[J]. J Immunol, 2014,193(8):3835-3841. DOI: 10.4049/jimmunol.1401572
[8]	Krutzek F, Kopka K, Stadlbauer S.Development of radiotracers for imaging of the PD-1/ PD-L1 axis[J]. Pharmaceuticals (Basel), 2022,15(6):747. DOI: 10.3390/ph15060747
[9]	Gao H, Wu Y, Shi J, et al.Nuclear imaging-guided PD-L1 blockade therapy increases effectiveness of cancer immunotherapy[J]. J Immunother Cancer, 2020,8(2):e001156. DOI: 10.1136/jitc-2020-001156
[10]	庞小溪,廖栩鹤,杜毓菁,等.放射免疫分子探针131I-PD-L1 mAb的制备及体内显像实验[J].标记免疫分析与临床, 2018, 25(10): 1536-1541. DOI: 10.11748/bjmy.issn.1006-1703.2018.10.026Pang XX, Liao XH, Du YJ, et al.The preparation and characterization in vivo of a novel molecular probe targeting PD-L1 radiolabeled with iodine-131[J]. Labeled Immunoassays and Clinical Medicine, 2018, 25(10): 1536-1541. DOI: 10.11748/bjmy.issn.1006-1703.2018.10.026
[11]	魏凯,李悦,张苗苗,等.131I标记槲皮素的方法[J].中国老年学杂志, 2014 (17):4874-4877. DOI:10.3969/j.issn.1005-9202.2014.17.075Wei K, Li Y, Zhang MM.The experiment of 131I-labeled quercetin in vitro[J]. Chinese Journal of Gerontology, 2014 (17): 4874-4877. DOI: 10.3969/j.issn.1005-9202.2014.17.075
[12]	许杰华, 张桂雄, 杨敏, 等. 131I标记适配子的优化研究及其稳定性的评价[J].新医学, 2019,50(12):894-897. DOI:10.3969/j.issn.0253-9802.2019.12.005Xu JH, Zhang GX, Yang M, et al.Optimization and evaluation of the stability of 131I labeling aptamer[J]. New Medicine, 2019, 50(12): 894-897. DOI: 10.3969/j.issn.0253-9802.2019.12.005
[13]	Moreno MJ, Loura L, Martins J, et al.Analysis of the equilibrium distribution of ligands in heterogeneous media-approaches and pitfalls[J]. Int J Mol Sci, 2022,23(17):9757. DOI: 10.3390/ijms23179757
[14]	Sung H, Ferlay J, Siegel RL, et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249. DOI: 10.3322/caac.21660
[15]	Sarhangi N, Hajjari S, Heydari SF, et al.Breast cancer in the era of precision medicine[J]. Mol Biol Rep, 2022,49(10):10023-10037. DOI: 10.1007/s11033-022-07571-2
[16]	程曦, 李珂, 魏雪. PD-1/PD-L1在乳腺癌治疗中的应用[J].中国妇幼保健, 2018, 33(12): 2863-2865. DOI: 10.7620/zgfybj.j.issn.1001-4411.2018.12.77
[17]	李阳, 默峰, 王涛, 等. PD-1/PD-L1免疫检查点抑制剂抗肿瘤疗效预测中肿瘤相关生物标记物和HPD预测因子的应用[J].山东医药,2021,61(30):88-92. DOI: 10.3969/j.issn.1002-266X.2021.30.023
[18]	Wen X, Shi C, Zhao L, et al. Immuno-SPECT/PET imaging with radioiodinated anti-PD-L1 antibody to evaluate PD-L1 expression in immune-competent murine models and PDX model of lung adenocarcinoma[J]. Nucl Med Biol, 2020,86-87:44-51. DOI: 10.1016/j.nucmedbio.2020.05.006
[19]	Heskamp S, Hobo W, Molkenboer-Kuenen JD, et al.Noninvasive imaging of tumor PD-L1 expression using radiolabeled anti-PD-L1 antibodies[J]. Cancer Res, 2015,75(14):2928-2936. DOI: 10.1158/0008-5472.CAN-14-3477
[20]	Liang Z, Hu X, Hu H, et al.Novel small 99mTc-labeled affibody molecular probe for PD-L1 receptor imaging[J]. Front Oncol, 2022,12:1017737. DOI: 10.3389/fonc.2022.1017737
[21]	Zhang Y, Ding Y, Li N, et al.Noninvasive imaging of tumor PD-L1 expression using [99mTc]Tc-labeled KN035 with SPECT/CT[J]. Mol Pharm, 2023, 20(1):690-700. DOI: 10.1021/acs.molpharmaceut.2c00874
[22]	Bensch F, van der Veen EL, Lub-de Hooge MN, et al. 89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer[J]. Nat Med, 2018,24(12):1852-1858. DOI: 10.1038/s41591-018-0255-8.