Preparation and Characterization of Anti-GPC3 Nanobody Against Hepatocellular Carcinoma

Abstract

Background: Glypican-3 (GPC3) is a newly identified target molecule for the early diagnosis of hepatocellular carcinoma (HCC), while targeted inhibition of GPC3 signaling may help to control the proliferation and metastasis of HCC cells. The purpose of this study was to prepare the anti-GPC3 nanobody and to investigate the affinity of the anti-GPC3 nanobodies in vitro and the anticancer effects on hepatocellular carcinoma in vivo.

Methods: To screen for unknown anti-GPC3 antibodies, we constructed an antibody phage display library. After three rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was expressed in E. coli BL21 cells and purified by affinity chromatography. Competitive ELISA and flow cytometry were conducted to confirm the affinity of the anti-GPC3 nanobodies in vitro. The antitumor effects of VHH_GPC3 were assessed in vivo.

Results: The results showed that the nanobody VHH_GPC3 had specific high-affinity binding to His-GPC3 antigen. Moreover, VHH_GPC3 exhibited specific binding to commercial human GPC3 and recognized the surface GPC3 protein of the hepatoma cell line HepG2. Importantly, in vivo study showed that GPC3 nanobody suppresses the growth of HepG2 and improves the survival rate of tumor mice.

Discussion: In summary, our new anti-GPC3 nanobody suggests a strong application potential for targeted therapy of liver cancer.

Keywords: GPC3; nanobody; phage display library; selection.

Figure 1

Schematic procedure for the production of phage nano-antibody library.

Figure 2

Construction of a phage display library. (A) Anti-GPC3 serum titer of Bactrian Camel. (B) Identification of VHH gene amplification. M: DNA Marker DL 2000; Lanes 1–8: Positive clones.

Figure 3

VHH_GPC3 positive clones were identified by PCR. M: DNA Marker DL2000. -: Negative control. Lanes 1–15: Positive clones.

Figure 4

Expression and purification of VHH_GPC3 antibody. (A) Expression of pET28a-VHH_GPC3 fusion protein analyzed by SDS-PAGE. M: protein marker (10–170 kDa); Lane 1: Transformed bacterium carrying pET28a empty vector without induction, Lane 2: Transformed bacterium carrying pET28a empty vector induced with IPTG; Lane 3: pET28a-VHH_GPC3 recombinant plasmid without induction; Lane4: pET28a-VHH_GPC3 recombinant plasmid induced with 0.5mmol/L IPTG; Lanes5–6: pET28a-VHH_GPC3 recombinant plasmid expressed in the supernatant and precipitation, respectively. (B) Expression of pET28a-VHH_GPC3 fusion protein analyzed by Western blot. Lane1: pET28a-VHH_GPC3 recombinant plasmid without induction; Lane 2:pET28a-VHH_GPC3 recombinant plasmid induced with 0.5mmol/L IPTG; Lanes 3–4: pET28a-VHH_GPC3 recombinant plasmid expressed in the supernatant and precipitation, respectively. (C) Purification of VHH_GPC3 antibody. Lane 1: Purified his-VHH_GPC3 fusion protein in the supernatant.

Figure 5

Determination of affinity of VHH_GPC3 and antigen protein. The 96-well plates were coated with 2μg/mL antigen, incubated with 5μg/mL VHH_GPC3. Skim milk was used as negative control. Commercial anti-human GPC3 mAb was used as positive control.

Figure 6

Detection of VHH_GPC3 binding with GPC3 expressed on HepG2 by FCM. (A) Not incubated by VHH_GPC3 was used as negative group with black line. Commercial anti-human GPC3 mAbs was used as positive with green line. 35 μg and 45μg of VHH_GPC3 were red and blue lines, respectively. (B) Statistical analysis showed a FITC intensity significant increase in HepG2 as compared to the negative control.

Figure 7

GPC3 nanobody inhibits tumor growth in nude BALB/c mice. Nude mice (n=6) were inoculated with HepG2 cells subcutaneously on the back of each nude mouse. After 7 days, tumor mice were treated with 10 mg/kg GPC3 nanobody, BSA (control) and cisplatin. Body weight of mice (A), tumor sizes (B), and the survival rates (C) were monitored at the indicated time points.