鉴定针对肝癌标志物磷脂酰聚糖-3 的纳米抗体。

Identification of nanobodies against hepatocellular carcinoma marker glypican-3.

机构信息

Institute for Medical Biology and Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Sciences, South-Central University for Nationalities, Wuhan, Hubei, 430074, China; Precision Medicine R&D Center, Zhuhai Institute of Advanced Technology, Chinese Academy of Sciences, Zhuhai, Guangdong, 519080, China; Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, 518055, China.

Precision Medicine R&D Center, Zhuhai Institute of Advanced Technology, Chinese Academy of Sciences, Zhuhai, Guangdong, 519080, China.

出版信息

Mol Immunol. 2021 Mar;131:13-22. doi: 10.1016/j.molimm.2021.01.010. Epub 2021 Jan 13.

DOI:10.1016/j.molimm.2021.01.010

PMID:33453658

Abstract

Glypican-3 (GPC3) is a highly specific diagnostic marker for hepatocellular carcinoma (HCC) diagnosis and a potential target in HCC therapy. Nanobodies (Nbs) are promising targeting molecules due to their high specificity and strong affinities to antigens, high stability, deep tissue penetration, and low immunogenicity. In this study, we isolated Nbs against GPC3 marker protein from a synthetic Nb library by phage display. To characterize these Nbs, we performed enzyme-linked immunosorbent assay, immunoprecipitation assay, and immunofluorescent assay to demonstrate that four (G8, G10, G11, and G64) of them bound specifically to recombinant as well as endogenous GPC3, and epitope mapping showed they all bound to N-terminal subunit of GPC3. Furthermore, we found that G64 exhibited high protein stability and GPC3 binding activity in serum at 37℃ for at least 96 h, and G64 did not affect the proliferation of HEK293T cells and HCC cell line HepG2. Our study provides four anti-GPC3 Nbs as promising targeting molecules for HCC diagnostic and therapeutic drugs.

摘要

Glypican-3（GPC3）是肝细胞癌（HCC）诊断的高度特异性诊断标志物，也是 HCC 治疗的潜在靶点。纳米抗体（Nbs）由于其对抗原的高特异性和强亲和力、高稳定性、深组织穿透力和低免疫原性，是很有前途的靶向分子。在这项研究中，我们通过噬菌体展示从合成的 Nb 文库中分离出针对 GPC3 标记蛋白的 Nb。为了表征这些 Nb，我们进行了酶联免疫吸附试验、免疫沉淀试验和免疫荧光试验，证明其中的 4 种（G8、G10、G11 和 G64）能够特异性结合重组和内源性 GPC3，表位作图表明它们都结合 GPC3 的 N 端亚基。此外，我们发现 G64 在 37℃的血清中至少 96 小时具有高的蛋白稳定性和 GPC3 结合活性，并且 G64 不影响 HEK293T 细胞和 HCC 细胞系 HepG2 的增殖。我们的研究提供了 4 种抗 GPC3 Nb，作为 HCC 诊断和治疗药物的有前途的靶向分子。