Development of human monoclonal antibodies using TC(Trans Chromosomic) mice

目前的研究是从开发针对女性生殖系统癌症的人源化单克隆抗体的策略开始的。使用表达人抗体（重链和轻链K）的新建立的TC（反式染色体）小鼠开发这些抗体。本课题组的主要研究成果如下：以子宫内膜癌细胞株（SNG-S）为抗原，研制出人源性单克隆抗体。根据标准小鼠单克隆抗体方案用PEG 4000进行细胞融合;小鼠脾细胞和骨髓瘤细胞以1：5的比例融合，通过子宫内膜癌组织切片的免疫组织化学染色（ABC法）进行杂交瘤筛选。用人单克隆抗体1-1C的染色结果显示，该抗体染色约70%的 关于我们 在子宫内膜癌组织标本中，它几乎不显示对正常子宫内膜组织标本的染色。除了子宫内膜癌的阳性染色外，该抗体还显示出与正常子宫颈腺体以及宫颈腺癌的强反应性，以及与女性生殖系统无关的几种其他癌症的阳性染色。初步调查，以阐明该抗体识别的抗原表位显示，胰蛋白酶处理的标本没有减少抗体染色，而高碘酸处理完全废除阳性染色的组织标本，因此碳水化合物部分牵连作为该抗体的可能表位。此外，SNG-S细胞的细胞裂解物的SDS-PAGE电泳和蛋白质印迹显示具有膜部分的阳性条带，表明1-1C识别的抗原可能是细胞表面膜糖蛋白。初步研究表明，建立了一个单克隆抗体，显示与卵巢透明细胞癌的强反应性。少

The current study was initiated with the strategy to develop humanized monoclonal antibodies towards cancers of the female genital system. The newly established TC (Trans Chromo) mice which express human antibodies (heavy chain and light chain K) were used to develop these antibodies. Basic investigations have been launched with implications towards future clinical applications; the following results have been obtained.A human monoclonal antibody has been developed using as antigen an endometrial carcinoma cell line culture (SNG-S), originally established by the current research group leaders. Cell fusion was performed with PEG 4000 according to standard mouse monoclonal antibody protocols; mouse splenocytes and myeloma cells were fused at a ratio of 1:5, Hybridoma screening was carried out by immunohistochemical staining (ABC method) of endometrial carcinoma tissue sections. Staining results with the human monoclonal antibody 1-1C showed that the antibody stained approximately 70% of … More endometrial carcinoma tissue specimens while it showed almost no staining towards normal endometrial tissue specimens. In addition to positive staining of endometrial carcinoma, the antibody also showed strong reactivity with normal uterine cervical glands as well as cervical adenocarcinoma in addition to positive staining of several other cancers not related to the female genital system. Preliminary investigations to elucidate the antigenic epitope recognized by this antibody revealed that trypsin treatment of the specimens did not diminish the antibody staining while periodic acid treatment completely abolished positive staining of tissue specimens; thus a carbohydrate moiety was implicated as the possible epitope of this antibody. Furthermore, SDS-PAGE electrophoresis and western blotting of the cell lysates of the SNG-S cells revealed a positive band with the membrane fraction, denoting that the antigen recognized by 1-1C is possibly a cell surface membrane glycoprotein.In addition to the above studies, investigations have been instigated to develop a human monoclonal antibody using as antigen an ovarian clear cell carcinoma cell line culture (RMG-1). Initial studies have revealed establishment of a monoclonal antibody which shows strong reactivity with ovarian clear cell carcinoma. Less