Development of an early detection method and specific immunotherapy of ovarian cancers

为了开发一种新的早期卵巢癌诊断方法，我们制备了抗卵巢癌衍生分子的鼠单克隆抗体（MoAbs），这些分子在患者体内具有免疫原性。尝试了五种不同的免疫抗原/筛选抗原组合策略。在所有的策略中，从30例各种卵巢癌患者的腹水中纯化的免疫复合物（IC）被用作免疫抗原和/或ELISA的筛选抗原。Sephacryl S300凝胶过滤和蛋白A亲和层析。作为免疫抗原，还使用浆液性囊腺癌细胞系（HOC/BR）或卵巢癌的肿瘤组织的无血清条件培养基。从正常健康对照的合并血清纯化的IC用作阴性筛选抗原。我们从8,000多株杂交瘤细胞上清中筛选出7株与卵巢癌腹水LC特异性结合的单克隆抗体，而与正常人IgG和IC均不结合。其中2株单克隆抗体（2Flt和7 E2克隆）也能用蛋白A预包被的微孔板进行夹心ELISA检测卵巢癌患者外周血中的循环IC。HOC/BR细胞系的条件培养基抑制3种MoAb（2F 11、7 E2和H5克隆）的反应性，表明这些抗体检测参与患者IC的卵巢癌衍生分子。因此，认为这些分子表达与患者IgG反应的肿瘤相关表位。

To develop a new diagnostic method for ovarian cancers at an carly stage, we produced murine monoclonal antibodies (MoAbs) against ovarian cancer-derived molecules which are lmmunogenic in patients. Five different strategies of immunizing antigen/screening antigen combination were tried. In all strategies, immune complexes (ICs) purified from ascites of 30 patients with various ovarian cancers were used as immunizing antigens and/or screening antigens in ELISA.The purification of ICs includes ammonium sulphate precipitation. Sephacryl S300 gel filtration, and protein A affinity chromatography. As immunizing antigens, serum-free conditioned medium of a serous cystadenocarcinoma cell line (HOC/BR) or tumor tissue of ovarian cancers were also used. ICs purified from pooled sera of normal healthy controls were used as negative screening antigens. We screened more than 8,000 supernatants of hybridomas in to fusion experiments, and established 7 MoAbs which react with ascites LCs of ovarian cancers, but not with IgG or ICs of normal controls. Among them, 2 MoAbs (2Flt and 7E2 clones) can also detect circulating ICs in peripheral blood from patients with ovarian cancers by sandwich ELISA using protein A precoated micropiates. Conditioned medium of HOC/BR cell line inhibited the reactivity of 3 MoAbs (2Fll, 7E2, and H5 clones), indicating that these antibodies detect ovarian cancer-derived molecules involved in the patients' ICs. These molecules are, therefore, considered to express tumor-associated epitopes reacted by patients' IgG.