Ando Hiroshi, Suzuki Masayo, Kato-Nakano Mariko, Kawamoto Shinobu, Misaka Hirofumi, Kimoto Naoya, Furuya Akiko, Nakamura Kazuyasu
a R&D Division, Tokyo Research Park , Kyowa Hakko Kirin Co., Ltd , Machida-shi , Japan.
Biosci Biotechnol Biochem. 2015;79(8):1272-9. doi: 10.1080/09168451.2015.1018124. Epub 2015 Mar 6.
Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.
人紧密连接蛋白3（CLDN3）是一种存在于紧密连接结构中的四次跨膜蛋白，已知在某些恶性肿瘤中过度表达。尽管针对CLDN3细胞外结构域的特异性单克隆抗体（MAb）将是一种有价值的工具，但由于CLDN3在不同物种间的保守序列同源性，使用传统杂交瘤技术生成此类单克隆抗体一直被认为很困难。此外，紧密连接蛋白家族成员之间具有高度的序列相似性，因此应仔细评估单克隆抗体的潜在交叉反应性。为克服这些困难，我们构建了表达CLDN3的中国仓鼠卵巢细胞和Sf9细胞作为免疫原，并进行基于细胞的筛选以消除交叉反应性抗体。结果，我们获得了能识别CLDN3细胞外环但不识别CLDN4、5、6或9细胞外环的单克隆抗体。进一步的体外研究表明，分离出的单克隆抗体具有检测或靶向CLDN3所需的结合特性。
