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目的:制备真核细胞CHO表达的CD80单链抗体(CD80-scFv)并研究其对不同肿瘤细胞膜型分子的识别及增殖的影响。方法:将表达CD80-scFv的CHO细胞大规模无血清培养收集上清,采用IMAC亲和层析法纯化获取抗人抗体。采用免疫荧光及流式细胞术分析其对人恶性B系淋巴瘤细胞株Daudi、人黑色素瘤细胞株A375、神经胶质瘤细胞株U251及人多发性骨髓瘤细胞株8266及U266细胞膜型CD80分子的识别。在上述肿瘤细胞的培养体系中加入不同浓度的CD80-scFv,MTT法分析其对肿瘤细胞增殖的影响。以天然高表达CD80分子的8266细胞经丝裂霉素处理后作为APC,人PBMC作为效应细胞,分析抗体通过阻断共刺激信号对PBMC增殖的影响。结果:从CHO细胞培养上清中获取的抗人CD80-scFv纯化品的量为6.67 mg/L;该抗体与Daudi、U251、A375、8266及U266的阳性结合率分别为96.8%、39.6%、20.5%、99.9%及3.8%。抗体对高表达CD80分子的Daudi及8266细胞具有增殖抑制作用,加入抗体(终浓度为25μg/mL)培养72 h时的抑制率分别为24.04%及24.16%。同时,抗体通过阻断CD80-CD28介导的共刺激信号,抑制PBMC的增殖。结论:CD80-scFv能够特异识别细胞膜型CD80分子,并对高表达细胞的体外增殖具有抑制作用。
OBJECTIVE: To prepare CD80 single chain antibody (CD80-scFv) expressed in CHO cells by eukaryotic cells and study its effect on the recognition and proliferation of membrane molecules in different tumor cells. Methods: The CHO cells expressing CD80-scFv were cultured on a large-scale serum-free medium and purified by IMAC affinity chromatography to obtain anti-human antibodies. Immunofluorescence and flow cytometry analysis of human malignant B line lymphoma cell line Daudi, human melanoma cell line A375, glioma cell line U251 and human multiple myeloma cell line 8266 and U266 cell membrane type CD80 molecules Recognition. Different concentrations of CD80-scFv were added to the culture system of the tumor cells, and MTT assay was used to analyze the effect on tumor cell proliferation. 8266 cells, which naturally express CD80 molecules, were treated with mitomycin C and APCs were treated with human PBMCs as effector cells. The effects of antibodies on the proliferation of PBMCs were analyzed by blocking co-stimulatory signals. Results: The amount of anti-human CD80-scFv purified from CHO cell culture supernatant was 6.67 mg / L. The positive binding rates of this antibody to Daudi, U251, A375, 8266 and U266 were 96.8%, 39.6% 20.5%, 99.9% and 3.8% respectively. Antibodies could inhibit the proliferation of Daudi and 8266 cells with high expression of CD80, and the inhibition rate of antibody was 24.04% and 24.16% when added with antibody (final concentration 25μg / mL) for 72h. At the same time, antibodies inhibit PBMC proliferation by blocking CD80-CD28-mediated costimulation signals. Conclusion: CD80-scFv can specifically recognize cell membrane CD80 molecules and inhibit the proliferation of highly expressed cells in vitro.