Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C<sub>44</sub>Mab-46) Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance
Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C<sub>44</sub>Mab-46) Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance
复制标题
使用丙氨酸扫描诱变和表面等离子共振进行抗 CD44 单克隆抗体 (C<sub>44</sub>Mab-46) 的表位作图
DOI：
10.1089/mab.2021.0028
复制
发表时间：
2021
影响因子：
--
通讯作者：
Kato Yukinari
中科院分区：
文献类型：
--
作者：
Takei Junko;Asano Teizo;Suzuki Hiroyuki;Kaneko Mika K.;Kato Yukinari
CD 44是一种I型跨膜蛋白，在各种正常癌细胞中表达，包括胰腺癌、乳腺癌和口腔癌。CD 44与癌症进展、转移和治疗抗性相关。CD 44由20个外显子组成，并且由于中央10个外显子的选择性剪接而存在各种亚型。一些剪接变异体显示出癌症特异性表达模式，并与癌症患者的预后相关。因此，CD 44靶向治疗一直备受关注。在以前的研究中，我们建立了一个抗CD 44单克隆抗体，C44单克隆抗体-46（IgG 1，κ），用于流式细胞术，蛋白质印迹，免疫组化与CD 44 v3 -10胞外域免疫小鼠。本研究采用酶联免疫吸附试验（ELISA）和表面等离子体共振（SPR）技术对C44 Mab-46的结合表位进行了研究。使用缺失突变体的ELISA结果显示，C44 Mab-46与CD 44的161-180 aa的氨基酸（aa）反应。使用ELISA用CD 44的161-180 aa中的点突变体进一步检查C44 Mab-46表位，证明C44 Mab-46表位包含Thr 174、Asp 177和Val 178。在CD 44的161-180 aa的点突变的SPR表明，C44 Mab-46表位包括Thr 174，Asp 175，Asp 176，Asp 177和Val 178。总之，确定C44 Mab-46表位位于CD 44的外显子5中。
CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted with the amino acids (aa) of 161–180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA with point mutants in 161–180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161–180 aa of CD44 demonstrated that the C44Mab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to be located in exon 5 of CD44.