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，我们生产了针对卵巢癌衍生分子的鼠源性单抗(MoAbbs)，这些分子在患者中是免疫的。尝试了免疫抗原/筛选抗原组合的五种不同策略。在所有策略中，从30例卵巢癌患者腹水中纯化的免疫复合物(IC)作为免疫抗原和/或筛选抗原用于ELISA。IC的纯化包括硫酸铵沉淀。Sephacryl S300凝胶过滤，蛋白A亲和层析。用浆液性囊腺癌细胞系无血清条件培养液(HOC/BR)或卵巢癌肿瘤组织作为免疫抗原。用从正常健康人混合血清中提纯的ICS作为阴性筛选抗原。在融合实验中，我们筛选了8000多株杂交瘤细胞上清液，建立了7株能与卵巢癌腹水LC反应，但不与正常对照的免疫球蛋白或免疫球蛋白反应的抗体。其中，2株MoAb2Flt和7E2克隆也能用蛋白A包被微孔板的夹心ELISA法检测卵巢癌患者外周血中的ICs。HOC/BR细胞株的条件培养液抑制了3个MoAb2F11、7E2和H5克隆的反应性，表明这些抗体可以检测到与患者ICs有关的卵巢癌衍生分子。因此，这些分子被认为表达与患者的免疫球蛋白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.