Ishikawa Kenichiro, Suzuki Hiroyuki, 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.
Curr Issues Mol Biol. 2023 Jun 21;45(7):5248-5262. doi: 10.3390/cimb45070333.
Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed PANC-1 cells. Among the established clones, CMab-18 (IgM, kappa) reacted with CHO/CD44v3-10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that CMab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that CMab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, CMab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant () of CMab-18 for CHO/CD44v3-10 and HSC-3 was 1.6 × 10 M and 1.7 × 10 M, respectively. Furthermore, CMab-18 detected CD44v3-10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that CMab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.
头颈部鳞状细胞癌（HNSCC）是头颈部最常见的癌症类型，并且已被揭示为癌症中CD44表达第二高的类型。CD44已被作为HNSCC的癌症干细胞标志物进行研究，并在肿瘤恶性进展中起关键作用。特别是，CD44的剪接变异体亚型（CD44v）在癌症中过度表达，并被认为是癌症诊断和治疗的一个有前景的靶点。我们通过用CD44v3-10过表达的胰腺癌细胞系PANC-1免疫小鼠来制备抗CD44的单克隆抗体（mAb）。在已建立的克隆中，CMab-18（IgM，κ）通过流式细胞术与CHO/CD44v3-10反应，但不与CHO/CD44s和亲本CHO-K1反应。使用覆盖变异外显子编码区域的肽进行表位作图显示，CMab-18识别变异体10与恒定外显子16编码序列之间的边界序列。这些结果表明，CMab-18识别含变异体10的CD44v，但不识别CD44s。此外，CMab-18在流式细胞术中能够识别人类口腔鳞状细胞癌（OSCC）细胞系HSC-3。CMab-18对CHO/CD44v3-10和HSC-3的表观解离常数（）分别为1.6×10 M和1.7×10 M。此外，CMab-18在蛋白质印迹法中检测到CD44v3-10，但未检测到CHO/CD44s，并且在使用OSCC组织的免疫组织化学中检测到内源性CD44v10。这些结果表明，CMab-18可用于在流式细胞术和免疫组织化学中检测CD44v10。
