Epitope Mapping of a Cancer-Specific Anti-Podocalyxin Monoclonal Antibody (PcMab-60) Using Enzyme-Linked Immunosorbent Assay and Surface Plasmon Resonance
Epitope Mapping of a Cancer-Specific Anti-Podocalyxin Monoclonal Antibody (PcMab-60) Using Enzyme-Linked Immunosorbent Assay and Surface Plasmon Resonance
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DOI：
10.1089/mab.2021.0030
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发表时间：
2021-10-01
影响因子：
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通讯作者：
Kato, Yukinari
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文献类型：
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作者：
Asano, Teizo;Suzuki, Hiroyuki;Kato, Yukinari
足糖萼蛋白（Podocalyxin，PODXL）是一种I型跨膜唾液酸糖蛋白，其在人类癌症（包括乳腺癌、口腔癌和肺癌）中过表达。PODXL促进肿瘤进展，其表达与不良预后相关。由于PODXL在正常细胞（包括肾足细胞和血管内皮细胞（VEC））中表达，因此癌症特异性单克隆抗体（mAb）对于减少抗体疗法对表达PODXL的癌症的不利影响是必要的。之前，我们通过用LN 229胶质母细胞瘤细胞产生的可溶性PODXL免疫小鼠，建立了针对PODXL的癌症特异性mAb，PcMab-60（小鼠IgM，κ）。PcMab-60与表达PODXL的癌细胞反应，但不与VEC反应。在这项研究中，我们研究了PcMab-60的表位，使用流式细胞术，表面等离子体共振（SPR），酶联免疫吸附测定（ELISA）。SPR结果显示PcMab-60表位由Thr 105、Arg 109、Gly 110、Gly 111、Gly 112、Ser 113、Gly 114、Asn 115、Pro 116和Thr 117组成。ELISA结果显示PcMab-60抗原表位由Arg 109、Gly 110、Gly 111、Gly 112、Ser 113、Gly 114、Asn 115和Pro 116组成。这些结果证明了癌症特异性表位，其被PcMab-60识别。
Podocalyxin (PODXL) is a type I transmembrane sialoglycoprotein that is overexpressed in human cancers, including breast, oral, and lung. PODXL promotes tumor progression, and its expression is associated with poor prognosis. Since PODXL is expressed in normal cells, including kidney podocytes and vascular endothelial cells (VECs), cancer-specific monoclonal antibodies (mAbs) are necessary to reduce the adverse effects of antibody therapy on PODXL-expressing cancers. Previously, we established a cancer-specific mAb against PODXL, PcMab-60 (mouse IgM, kappa), by immunizing mice with soluble PODXL produced by LN229 glioblastoma cells. PcMab-60 reacted with PODXL-expressing cancer cells, but did not react with VECs. In this study, we investigated an epitope of PcMab-60 using flow cytometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). The results of SPR revealed that the PcMab-60 epitope consisted of Thr105, Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117. In contrast, the results of ELISA revealed that the PcMab-60 epitope consisted of Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116. These results demonstrate the cancer-specific epitope, which was recognized by PcMab-60.