Takei Junko, Asano Teizo, Suzuki Hiroyuki, Kaneko Mika K, Kato Yukinari
Department of Antibody Drug Development, and Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Monoclon Antib Immunodiagn Immunother. 2021 Oct;40(5):219-226. doi: 10.1089/mab.2021.0028.
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, CMab-46 (IgG, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of CMab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that CMab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the CMab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the CMab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the CMab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the CMab-46 epitope was determined to be located in exon 5 of CD44.
CD44是一种I型跨膜蛋白，在包括胰腺癌、乳腺癌和口腔癌在内的各种正常癌细胞中均有表达。CD44与癌症进展、转移及治疗耐药性相关。CD44由20个外显子组成，由于中间10个外显子的可变剪接而存在多种异构体。一些剪接变体呈现癌症特异性表达模式，并与癌症患者的预后相关。因此，靶向CD44的治疗一直备受关注。在之前的一项研究中，我们通过用CD44v3 - 10胞外域免疫小鼠，制备了一种抗CD单克隆抗体CMab - 46（IgG，κ），可用于流式细胞术、蛋白质印迹法和免疫组织化学。本研究使用酶联免疫吸附测定（ELISA）和表面等离子体共振（SPR）结合合成肽来研究CMab - 46的结合表位。使用缺失突变体的ELISA结果表明，CMab - 46与CD44的161 - 180位氨基酸发生反应。使用CD44的161 - 180位氨基酸点突变体的ELISA对CMab - 46表位进行进一步检测，结果表明CMab - 46表位由苏氨酸174、天冬氨酸177和缬氨酸178组成。使用CD44的161 - 180位氨基酸点突变体的SPR结果表明，CMab - 46表位由苏氨酸174、天冬氨酸175、天冬氨酸176、天冬氨酸177和缬氨酸178组成。综合来看，CMab - 46表位被确定位于CD44的第5外显子中。
