卵巢癌抗独特型微抗体主动免疫治疗卵巢癌动物实验

Immuno-therapeutic Study of Anti-idiotype Minibody(Single Chain Fv -CH3)on Ovarian Carcinoma Bearing Mice

目的用模拟人卵巢癌抗原并有满意免疫原性的抗独特型微抗体进行临床前动物实验研究。方法将已构建的抗独特型微抗体融合基因在大肠杆菌进行表达,用Westernblot、竞争抑制ELISA进行微抗体活性测定。20只人淋巴细胞免疫功能重建的荷卵巢癌腹腔瘤SCID小鼠分2组,10只用微抗体免疫后取血采用间接ELISA检测Ab3。观察小鼠腹腔积液生成时间、生存期,取脾做流式细胞分析CD4+、CD8+。结果在宿主大肠杆菌BL21(DE3)成功诱导表达出微抗体,Westernblot结果表明微抗体可同时与COC166-9(6B11的一抗)及羊抗人的IgG1反应。竞争抑制ELISA表明微抗体可模拟原始抗原与一抗结合。微抗体免疫小鼠后可诱导产生Ab3,在末次免疫14d时最高,持续6周降至对照组水平;CD4+/CD8+在末次免疫13d达最高值。对照组和微抗体治疗组小鼠分别在(37.7±5.5)d和(48.6±14.3)d长出血性腹腔积液(P=0.04);两组小鼠生存期分别为(42.5±1.8)d和(59.4±16.8)d(P=0.011)。结论微抗体保留了6B11scFv和人IgG的双重免疫学活性,达到了部分人源化的目的,并有满意的免疫原性,可诱导小鼠产生特异性体液免疫反应,抑制荷瘤小鼠腹腔积液的生成,延长生存期,或许将来可作为卵巢癌疫苗用于临床。

An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer.Methods Using gene engineering technique,prokaryotic expression vector was constructed by genetic fusion of6B11scFv to human IgG1hinge and CH3region.The fusion protein named minibody was induced with IPTG in E.coli and analyzed with Western blot and inhibition ELISA tests respectively.Twenty human-PBL-SCID mice bearing ip Skov3.ip1cells were divided into two groups (10per-group),10mice were immunized repeatedly by minibody every two weeks for three times.Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv(Ab3).The latent period of ascites growth and the mean survival time were observed respectively.CD4 + and CD8 + T cells from the spleen of immunized mice were assayed by flow cytometry.Results SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of50000appeared as the expected size after transformation and induction the host bacteria BL21(DE3).The expressed minibody could be reacted with COC166-9(Ab1of6B11)and binding goat anti-human IgG1antibody in Western blot.Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9instead of primal antigen.Ab3could be detected in the sera of immunized mice with minibody by ELISA test.Ab3reached the highest at the14th day after last vaccination and lasted for6weeks.The ratio of CD4 + /CD8 + was the highest at the13th day after last vaccination.The latent period of ascites growth were(37.7±5.5)days and(48.6±14.3)days(P=0.04)respectively;while the mean survival time were(42.5±1.8)days and (59.4±16.8)days(P=0.011)in the control and minibody group respectively.Conclusions These results demonstrate the successful construction and expression minibody with good immune activities of6B11scFv and human IgG1molecules function.Antigenicity is increased without adjuvants and partial humanization is realized.Minibody can induce humoral anti - idiotypic immunity responses against ovarian carcinoma in vivo.When ascites formation was delayed or prevented and the survival was prolonged in minibody group.We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.