抗相思子毒素抗体的嵌合改造与功能评价

Chimeric transformation and functional evaluation of anti-Abrin antibody

目的:制备并评价针对相思子毒素(Abrin)的具有中和活性的特异性人源化嵌合抗体。方法:从鼠源单抗中钓取抗体可变区轻重链序列4条,两两配对载入人源化IgG1表达载体pFRT-KIgG1,经表达、纯化等步骤得到4株人源化嵌合抗体;间接ELISA测定嵌合抗体与Abrin的结合力;ForteBio测定嵌合抗体与Abrin的亲和力;体外细胞学实验鉴定抗体的中和功能;无细胞体系评价抗体是否阻碍Abrin抑制荧光酶合成;选择6~8周雌性BALB/c小鼠,腹腔注射Abrin和不同浓度抗体,观察抗体的体内保护效应;流式细胞术分析抗体是否阻断Abrin与细胞的结合。结果:ELISA和ForteBio结果显示,4株嵌合抗体中仅H1L1嵌合抗体与Abrin的结合[EC_(50)=(0.1212±0.0040)μg/ml和亲和力(亲和常数3.97×10^(-10)M)]与母本抗体[EC_(50)=(0.15825±0.00235)μg/ml,亲和常数5.59×10^(-10)M]相似;体外细胞保护试验显示,H1L1[EC_(50)=(3.7455±0.0575)ng/ml]约为母本抗体[EC_(50)=(1.5825±0.5335)ng/ml]中和作用的42%;无细胞体系荧光素酶合成试验显示,H1L1[EC_(50)=(0.6729±0.0755)μg/ml]与母本抗体[EC_(50)=(1.0365±0.0045)μg/ml]接近,均能逆转Abrin抑制蛋白合成的作用;BALB/c小鼠体内中和保护实验结果显示,H1L1与10D8均能提供良好的体内保护作用;流式细胞术分析发现,H1L1并不能通过抑制Abrin黏附或进入细胞抑制其毒性。结论:4株嵌合抗体中,仅H1L1是轻、重链正确配对且拥有良好中和活性的候选抗体,有潜在的用于Abrin中毒救治的开发价值,应用前景良好。

Objective:To prepare and evaluate humanized chimeric antibody with neutralizing activity against Abrin toxin.Methods:Four light and heavy chain sequences of antibody variable region were fished from mouse monoclonal antibodies and loaded into humanized IgG1 expression vector pFRT-KIgG1 in pairs.Four humanized chimeric antibodies were obtained by expression and purification.Indirect ELISA was used to determine binding force between chimeric antibodies and Abrin.Fortebio was used to determine affinity between chimeric antibody and Abrin.Antibody neutralization was measured in cell level in vitro.Cell-free system was used to evaluate whether Abrin inhibited synthesis of luciferase.Female BALB/c mice of 6~8 weeks were selected,and Abrin and different concentrations of antibodies were injected intraperitoneally and observed.Flow cytometry was used to analyze whether antibody blocked binding of Abrin to cells.Results:ELISA results and ForteBio showed that binding[EC_(50)=(0.1212±0.0040)μg/ml and affinity=3.97×10^(-10)M]of H1L1 chimeric antibody to Abrin were similar to those of maternal antibody[EC_(50)=(0.15825±0.00235)μg/ml,affinity constant=5.59×10^(-10)M],which is the only one among four chimeric antibodies.In vitro cytoprotection test showed that neutra-lization protection function of H1L1[EC_(50)=(3.7455±0.0575)ng/ml]was about 42%of neutralization effect of maternal antibody[EC_(50)=(1.5825±0.5335)ng/ml];H1L1[EC_(50)=(0.6729±0.0755)μg/ml]was similar to maternal antibody[EC_(50)=(1.0365±0.0045)μg/ml]in luciferase synthesis of cell-free system,which could hinder inhibitory effect of Abrin on protein syinthesis.Protection experiment in vivo results in BALB/c mice showed that H1L1 and 10D8 could provide good protection in vivo;flow cytometry analysis showed that H1L1 could not inhibit toxicity of Abrin by inhibiting its adhesion or entry into cells.Conclusion:Only H1L1 is a neutralizing antibody with correct pairing among four chimeric antibodies,which has a good binding activity and potential value in research and development of treating for Abrin poisoning,and is a candidate antibody drug against Abrin.