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.

Glypican-3（GPC3）是肝细胞癌（HCC）诊断的高度特异性诊断标记，是HCC治疗的潜在靶标。纳米抗体（Nbs）由于其高特异性和对抗原的强亲和力，高稳定性，深层组织穿透性和低免疫原性而成为有希望的靶向分子。在这项研究中，我们通过噬菌体展示从合成的Nb库中分离了针对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测定，免疫沉淀测定和免疫荧光测定，以证明其中的四个（G8，G10，G11和G64）特异性结合重组体和内源性GPC3，并且通过表位作图显示它们全部结合到GPC3的N-末端亚基。此外，我们发现，G64在37℃下至少96小时内表现出高蛋白稳定性和血清中的GPC3结合活性，而G64没有影响HEK293T细胞和HCC细胞HepG2的增殖。我们的研究提供了四种抗GPC3 Nbs作为有希望的HCC诊断和治疗药物靶向分子。