Dharma Rao Thapi, Park Kay J, Smith-Jones Peter, Iasonos Alexia, Linkov Irina, Soslow Robert A, Spriggs David R
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Appl Immunohistochem Mol Morphol. 2010 Oct;18(5):462-72. doi: 10.1097/PAI.0b013e3181dbfcd2.
The CA125 antigen, recognized by the OC125 antibody, is a tissue-specific circulating antigen expressed in ovarian cancer. The CA125 antigen is encoded by the MUC16 gene cloned by Yin and Lloyd. The full-length gene describes a complex tethered mucin protein present primarily in a variety of gynecologic tissues, especially neoplasms. OC125 and other related antibodies react with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. These antibodies are not useful as screening tools, nor can they detect the proximal residual MUC16 protein fragment after cleavage. This has limited its diagnostic and therapeutic applications. Using synthetic peptides, we raised novel-specific antibodies to the carboxy-terminal portion of MUC16 retained by the cell proximal to the putative cleavage site. These antibodies were characterized using fluorescence-activated cell-sorting analysis, enzyme-linked immunoassay, Western blot analysis, and immunohistochemistry. Each of the selected monoclonal antibodies was reactive against recombinant GST-ΔMUC16 protein and the MUC16-transfected SKOV3 cell line. Three antibodies, 4H11, 9C9, and 4A5 antibodies showed high affinities by Western blot analysis and saturation-binding studies of transfected-SKOV3 cells and displayed antibody internalization. Immunohistochemical positivity with novel antibody 4H11 was similar to OC125 but with important differences, including diffuse positivity in lobular breast cancer and a small percentage of OC125-negative ovarian carcinomas that showed intense and diffuse 4H11. Development of such antibodies may be useful for the characterization of MUC16 biology and allow for future studies in targeted therapy and diagnostics.
由OC125抗体识别的CA125抗原是一种在卵巢癌中表达的组织特异性循环抗原。CA125抗原由Yin和Lloyd克隆的MUC16基因编码。全长基因描述了一种主要存在于多种妇科组织尤其是肿瘤中的复杂的连接黏蛋白。OC125及其他相关抗体与仅存在于分子裂解部分的糖基化依赖性抗原发生反应。这些抗体既不能用作筛查工具，也无法检测裂解后近端残留的MUC16蛋白片段。这限制了其诊断和治疗应用。我们使用合成肽制备了针对假定裂解位点近端细胞保留的MUC16羧基末端部分的新型特异性抗体。使用荧光激活细胞分选分析、酶联免疫吸附测定、蛋白质印迹分析和免疫组织化学对这些抗体进行了表征。每种选定的单克隆抗体均与重组GST-ΔMUC16蛋白和MUC16转染的SKOV3细胞系发生反应。通过蛋白质印迹分析以及对转染的SKOV3细胞的饱和结合研究，三种抗体4H11、9C9和4A5抗体显示出高亲和力，并表现出抗体内化。新型抗体4H11的免疫组织化学阳性与OC125相似，但存在重要差异，包括小叶乳腺癌中的弥漫性阳性以及一小部分OC125阴性卵巢癌中显示出强烈且弥漫性的4H11阳性。此类抗体的开发可能有助于MUC16生物学特性的表征，并为未来的靶向治疗和诊断研究提供支持。
