靶向4-1BBL和CD3双信号的协同抗肿瘤作用机制研究  

The Anti-Tumor Mechanism Involved when 4-1BBL and CD3 are Targeted

作　　者：姜文国[1] 张树平[1] 刘荣[2] 李崴[2] 张研君 邵晓枫[2] 王金宏[2] 

机构地区：[1]滨州医学院药理学教研室,药物研究所,山东省滨州市256603 [2]中国医学科学院北京协和医学院血液学研究所、实验血液学国家重点实验室

出　　处：《中国肿瘤临床》2010年第21期1201-1204,共4页Chinese Journal of Clinical Oncology

基　　金：山东省自然科学基金(编号:Q2006C02);山东省高校科技计划项目资助(编号:J10LC82)~~

摘　　要：目的:探讨人4-1BBL胞外区/抗CD20融合蛋白增强抗CD3/抗CD20 diabody介导的靶向杀伤作用及其机制。方法:通过表达纯化人4-1BBL胞外区/抗CD20融合蛋白及抗CD3/抗CD20 diabody,利用台盼蓝计数观察联合应用人4-1BBL胞外区/抗CD20融合蛋白及抗CD3/抗CD20 diabody对淋巴细胞增殖的影响;采用ELISA检测白介素-2(IL-2)水平。RT-PCR检测穿孔素和颗粒酶mRNA的表达。Calcein检测其联合应用抗CD3/抗CD20双功能抗体及PBL对靶细胞Raji细胞的杀伤作用。结果:人4-1BBL胞外区/抗CD20融合蛋白增强抗CD3/抗CD20 diabody对靶细胞Raji细胞的杀伤作用,其机制可能是促进淋巴细胞增殖,减少细胞死亡,促进IL-2分泌及上调穿孔素和颗粒酶mRNA表达。结论:人4-1BBL胞外区/抗CD20融合蛋白与抗CD3/抗CD20diabody联合应用,分别靶向4-1BBL和CD3双信号发挥协同抗肿瘤作用,为肿瘤免疫治疗提供了新的思路。Objective： To examine the anti-tumor mechanism involved when 4-1BBL/CD20 and diabody are used in combination. Methods： Human peripheral blood lymphocytes （PBLs） were isolated by Ficoll - Hypaque density-gradient centrifugation. Cell proliferation was assessed by measuring the conversion of the trazolium salt WST-8. Concentrations of IL-2 were measured by Enzyme-linked immunosorbent assay （ELISA）. Transcript expression of perforin and Grb was investigated by reverse transcription- polymerase chain reaction （RT-PCR）. Cytotoxicity analysis was performed using calcein-AM retention assays. Results： The proliferation of PBLs in the group that was treated with 4-1BBL/CD20 plus the diabody was significantly higher than that of cells treated with either 4-1BBL/CD20 or diabody alone. Combination treatment with the 4-1BBL/anti-CD20 fusion protein and the anti-CD3/anti-CD20 diabody led to significantly increased T cell cytotoxicity to human B lymphoma cells in vitro. Mechanistic studies revealed that significantly higher levels of granzyme B and perforin were detected in PBLs treated with the diabody plus 4-1BBL/CD20, compared to those levels in PBLs treated with the diabody alone. The level of IL-2 expression in PBLs treated with the diabody plus 4-1BBL/CD20 was significantly higher than that in cells treated with either the diabody or 4-1BBL/CD20 alone. Conclusion： Diabody plus 4-1BBL/CD20 is a more effective combination for lymphocyte activation than diabody alone. The combined administration of 4-1BBL/CD20 and diabody augments the antitumor activity of the diabody. Such a reaction may have significance for clinical application in the treatment of human CD20-positive B cell malignancies.

关 键 词：4—1BBL CD20 B淋巴细 胞靶向治疗 

分 类 号：R73-36[医药卫生—肿瘤]