Screening And Anti-Carcinoma Activity Of Novel GPC3 Antibody On Hepatoma Carcinoma Cells

Primary liver cancer is one of the common malignant tumors in China, with an increasing incidence recent years. Hepatocellular carcinoma (HCC) is one of the primary liver cancer types. Glypican-3 (GPC3) protein is highly expressed in HCC but not in other liver-related diseases, such as chronic superficial gastritis, tissue adjacent to carcinoma, bile duct carcinoma, benign liver disease and normal liver tissue. Therefore, GPC3 is a new potential targets for clinical diagnosis and therapy of HCC. Compared with the prokaryotic cells display antibody library, mammalian cell membranes display antibody library has its advantages. Single Chain Fragment Variable (scFv) antibody screened from mammalian cell membranes display antibody library is has better specificity and low immunogenicity. Compared with single-chain antibody, full-length antibody is of high stability and long half-life. Full-length antibody with Fc (fragment crystallizable) segment can combine with the target cell that contains Fc receptor and then mediates cellular immunity. Meanwhile it can also activate complement-mediated immunity. Therefore, the full-length and fully humanized GPC3 antibodies have great potential in the diagnosis and treatment of HCC.Part I:Construction of scFv antibody library derived from Peripheral Blood Mononuclear Cell (PBMC) of hepatocellular carcinomaOn the basis of mammalian cell membranes display scFv antibody library constructed previously in our lab, hunman PBMCs of nine cases of liver cancer patients were included to enlarge scFv antibody libraries. The main process is:1) Isolate PBMCs from Peripheral Blood lymphocytes and extract the total RNA; 2) Perform reverse transcription polymerase chain reaction (RT-PCR) with the isolated RNA as template and make complementary DNA (cDNA)available; 3) Variable region of heavy chain (VH) and variable region of light chain (VL) were available by PCR with the cDNAs as template using the multiple primers reported by the reference (42 pairs of heavy chain type primers,16 pairs of Kappa light chain type primers,18 pairs of lambda light chain primers). Use cDNA as template to get VH and VL; 4) Overlapping PCR was conducted to obtain scFv fragments with VH and VL as templates; 5) scFv and pDisplay plasmid are digested with Sfi Ⅰ and Sal Ⅰ and then linked together to form the pDisplay scFv antibody library. We transformed pDisplay-scFv plasmid library derived from liver carcinomas into E.Coli DH5a and sequencing determination. Identify the activity, diversity and capacity of liver cancer scFv antibody library by scFv sequence analysis. Flow cytometry and Western Blotting assay were performed to identify the the corretc expression of pDisplay-scFv antibody library. To sum up, we built a pDisplay-scFv antibody library with activity and diversity. The capacity is about 6.4×104 and it can efficiently express on mammalian cell membrane surface.Part II:Candidate GPC3 scFv obtain by flow cytometry sortingTwo types of pDisplay-scFv plasmid library (HCC and non-HCC) were transfetced into 293T cells with the decreased FITC GPC3 protein concentration (10 nM,5 nM,1 nM) as antigen to using flow cytometry sorting. Results showed that scFv antibody library derived from HCC is beneficial to GPC3 scFv antibody screening. After three rounds of flow cytometry sorting, candidate GPC3 scFv antibody were obtained. The sequence homology of the candidate sequences is 85%-95% homology with the published positive GPC3 antibody.Part III:Construction of full-length GPC3 antibodyThe light and heavy chain sequences are cloned into HXT1 and HXT2 plasmid and named HXT1-VH and HXT2-VL plasmid. Then we transfected HXT1 VH and HXT2 VL plasmids into 293E cell together. Cell supernatant were harvested to identify antibody size by the SDS-PAGE and coomassie brilliant blue staining. The GPC3 antibody is IgG type determined by Western Blotting. We use Protein A affinity column chromatography assay to purify antibodies.Part IV:In vitro activity evaluation of GPC3 antibodyEnzyme-linked immunosorbent assay (ELISA) experiment was performed to identify the affinity of antibody with GPC3 antigen. Fortebio was performend to measure the Kd value of antibody using NTA sensor. The value of Kd is about 1.4×106 M. Huh-7 and HepG2 cells were treated with different concentration of GPC3 antibody (0μg/mL,40μg/mL and 200μg/mL) in proliferation and wound-healing experiment. Huh-7 and HepG2 cells were treated with 0 μg/mL and 100 μg/mL GPC3 antibody in apoptosis experiment. Results show that the antibody has different degree of proliferation inhibition on Huh-7 and HepG2 cells, promotes Huh-7 and HepG2 cell apoptosis and inhibit Huh-7 and HepG2 cell migration and adhesion.Related phenotype and target genes mRNA expression were observed by co-incubation of Huh-7 and HepG2 cells with 150μg/mL GPC3 antibody. RNA was extracted and RT-QPCR was performed. Using cDNA as template and the key molecular primers of proliferation, apoptosis, migration and adhesion, the activity of cellular level is validated by the mRNA level experiment.Part Ⅴ:Preliminary mechanism study of anti-tumor action of GPC3 antibodyFirstly, antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the anti-tumor mechanisms of GPC3 antibody. Human PBMCs were incubated with Huh-7 and HepG2 cells in the addition of GPC3 antibody at the concentration of 200 μg/mL for 12 hours. Results showed that GPC3 antibody mediated an obvious killing effect of PBMCs on Huh-7 and HepG2 cells.Secondly, Wnt signaling pathway is definitely involved with the anti-tumor. Huh-7 and HepG2 cells were treated with 150 μg/mL GPC3 antibody and total RNAs were isolated from cells. RT-QPCR was performed and mRNA level of the key molecular-involved with Wnt signaling pathway was determined. Results showed that GPC3 antibody showed no inhibitory effect on the GPC3 and β-catenin expression. However, obvious inhibitory effect on the mRNA expression of Wnt3a and Wntl was observed. The above conclusion is verified by the Western Blotting experiments of GPC3, Wnt3α and β-catenin. In summary, we presumed that GPC3 antibody plays a role on Wnt signaling pathways by competitive recognition of GPC3 with Wnt molecular or non-classic Wnt signaling pathways. Further experiments need to done to confirm the point.