Preparation of anti-GPC3 single chain antibody for targeted detection of hepatocellular carcinoma / 生物工程学报

Glypican-3 (GPC3) is a key member of Glypican family and plays an important role in the development, angiogenesis and metastasis of hepatocellular carcinoma (HCC). Most HCC overexpresses GPC3, but GPC3 is hardly detected in normal adult liver and benign liver lesions, so it is regarded as a highly specific diagnostic marker and an ideal therapeutic target for HCC. In this study, we cloned the heavy and light chain variable region gene from the monoclonal antibody targeted to GPC3 screened in the previous stage, and linked it with a segment of flexible peptide (Linker) to obtain the single chain antibody against GPC3. The single chain antibody gene was cloned into vector for prokaryotic expression and purified to obtain high purity protein. Detection shows that the single-chain antibody produced by us has the same binding activity with the full-length antibody, and can accurately target the tumor site of Huh7 tumor-bearing model mice after coupling Cy5.5 fluorescence, suggesting that the single-chain antibody has the potential to realize multi-directional liver cancer precise surgical navigation under the guidance of a probe.

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT

Hepatocellular carcinoma [HCC] is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation [LT] holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein [AFP] messenger RNA [mRNA], Glypican-3 [GPC3] mRNA-expressing cells in the peripheral blood [PB] for prediction of HCC recurrence following orthotopic liver transplantation [OLT]. 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected APF mRNA, follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction [RT-PCR], pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 [285], 15 [52%], and 9 [31%] patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 [24%] patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA - expressing cells in PB seem to have no diagnostic value