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目的:比较鼻咽癌双价双特异性抗独特型抗体G22-I50与单价的抗独特型抗体G22,I50在体外抗肿瘤免疫情况,并对其可能的机制作初步探讨。方法:诱导表达G22-I50,G22,I50 3种蛋白并用Western印迹及ELISA方法进行活性鉴定。常规分离人外周血单个核细胞(PBMC),分别用G22-I50,G22,I50抗独特型抗体刺激并培养,采用MTT法、LDH释放法分别观察淋巴细胞增殖情况和细胞毒作用,ELISA法测定各抗独特型抗体刺激后培养上清液中IFN-γ,IL-2,IL-4的水平,流式细胞术检测刺激后淋巴细胞表型的改变。结果:Western印迹分析表明表达的G22-I50,G22,I50蛋白均可与FC2(Ab1)特异性地结合;直接ELISA结果表明复性后的蛋白已恢复活性,可用于体外实验研究。与G22、I50组相比,G22-I50刺激后的PBMC增殖明显,且对鼻咽癌细胞HNE2有特异的杀伤作用。G22-I50刺激后的PBMC培养上清液中IFN-γ,IL-2水平均比G22,I50组有所增加,而对IL-4水平没有明显影响。流式细胞术显示刺激后的PBMC中CD4+,CD8+T细胞比例增加,CD4+/CD8+比率增加,而CD4+CD25+T细胞的比例有所减少,其中以G22-I50组最为明显。结论:G22-I50具有更强的免疫原性并能增强PBMC的特异性杀瘤作用,其机制可能与促进PBMC的增殖,诱导具有抗肿瘤作用的Th1细胞因子的分泌并活化CD8+T细胞,抑制CD4+CD25+T细胞的表达有关。
OBJECTIVE: To compare the anti-tumor immunity of bivalent dual anti-idiotypic antibody G22-I50 with monovalent anti-idiotypic antibodies G22 and I50 in nasopharyngeal carcinoma and to explore its possible mechanism. Methods: Three proteins, G22-I50, G22 and I50, were induced and identified by Western blotting and ELISA. The peripheral blood mononuclear cells (PBMCs) were routinely isolated and stimulated with anti-idiotypic antibodies G22-I50, G22 and I50 respectively. The proliferation and cytotoxicity of lymphocytes were observed by MTT assay and LDH release assay respectively. The levels of IFN-γ, IL-2 and IL-4 in culture supernatants after stimulation with each anti-idiotypic antibody were measured. The changes of lymphocyte phenotype after stimulation were detected by flow cytometry. Results: Western blot analysis showed that the expressed G22-I50, G22 and I50 proteins could specifically bind to FC2 (Ab1). The result of direct ELISA showed that the renatured protein was restored and could be used in vitro. Compared with G22 and I50 groups, PBMC proliferation induced by G22-I50 was significantly increased and had specific cytotoxicity on HNE2 cells. The levels of IFN-γ and IL-2 in the supernatant of PBMC stimulated by G22-I50 were higher than those in G22 and I50 groups, but had no obvious effect on the level of IL-4. Flow cytometry showed that the proportion of CD4 + and CD8 + T cells in PBMC increased and the ratio of CD4 + / CD8 + increased, while the proportion of CD4 + CD25 + T cells decreased, especially in G22-I50 group. Conclusion: G22-I50 has stronger immunogenicity and can enhance the specific killing effect of PBMC. The mechanism may be related to the promotion of PBMC proliferation, inducing the secretion of Th1 cytokines with anti-tumor effect and activating CD8 + T cells, Inhibit the expression of CD4 + CD25 + T cells.