Li Guanjie, Suzuki Hiroyuki, Asano Teizo, Tanaka Tomohiro, Suzuki Hiroyoshi, Kaneko Mika K, Kato Yukinari
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Antibodies (Basel). 2022 Jun 8;11(2):41. doi: 10.3390/antib11020041.
The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10 M and 3.2 × 10 M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications.
上皮细胞粘附分子（EpCAM）是一种细胞表面糖蛋白，在正常细胞和癌细胞上广泛表达。EpCAM参与细胞粘附、增殖、存活、干性维持和肿瘤发生。因此，EpCAM被认为是癌症诊断和治疗的一个有前景的靶点。在本研究中，我们使用基于细胞的免疫接种和筛选（CBIS）方法建立了抗EpCAM单克隆抗体（mAb）。我们通过流式细胞术、蛋白质免疫印迹法和免疫组织化学对它们进行了表征。所建立的重组抗EpCAM mAb之一，recEpMab-37（小鼠IgG，κ链），与过表达EpCAM的中国仓鼠卵巢-K1细胞（CHO/EpCAM）或结肠癌细胞系（Caco-2）发生反应。相比之下，recEpMab-37不与敲除EpCAM的Caco-2细胞发生反应。recEpMab-37对CHO/EpCAM和Caco-2的亲和力分别为2.0×10⁻⁸ M和3.2×10⁻⁸ M。我们观察到144至164位的EpCAM氨基酸参与recEpMab-37的结合。在蛋白质免疫印迹分析中，recEpMab-37检测到了CHO/EpCAM和Caco-2细胞的EpCAM。此外，recEpMab-37可通过免疫组织化学对福尔马林固定石蜡包埋的结肠癌细胞组织进行染色。综上所述，通过CBIS方法建立的recEpMab-37可用于在各种应用中检测EpCAM。
