HC6R1, A Monoclonal Antibody Against IL6R, Has Potent Antitumor Activity Of Hepatocellular Carcinoma

Objective:1. To identify the specificity of HC6R1 and find out its antigen recognition epitope.2. To investigate the therapeutic activity of HC6R1 and its antitumor mechanisms for hepatocellular carcinoma. Materials and Methods:HC6R1 antibodies were produced by hybridoma technique. Flow cytometric and immunohistochemical were used to test whether HC6R1 could specific recognize the cells or tissues of hepatocellular carcinoma. HC6R1 epitope was detected by the immunoprecipitation and mass spectrometry. RNA interference was used to retardant the expression of IL6 R. Use the HC6R1 and anti-IL6 R as the primary antibodies, western blotting and flow cytometric assay were further used to identify the HC6R1 epitope. CCK8 assay, wound heealing, adhesion assy and transwell were employed to find out the effect of HC6R1 in hepatocellular carcinoma cellular proliferation migration, adhesion and invasion. And we also generated xenografted tumor models by substaneously injecting HepG2 or Huh7 cells into the back of mice to research the inhibiting effect of HC6R1 for the carcinoma. Cell apoptosis and cell-cyle was detected by Annexin V/PI double staining and flow cytometric assay. We used gene microarray to detect the target gene of HC6R1. Use the HC6R1 and anti-IL6 R as the primary antibodies, western blotting analysis was carried out to measure the protein expression levels of STAT3、p-STAT3、CyclinD1 and CDK4. Results:1. To produce and identify the HC6R11.1 We produced the monoclonal antibody(HC6R1) by hybridoma technique1.2 Flow cytometric and immunohistochemistry analysis showed that HC6R1 could specific recognize the cells or tissues of hepatocellular carcinoma, but not in other tumor cell lines or normal tissues.1.3 The results of the immunoprecipitation and mass spectrometry showed that IL6 R might be the antigen of HC6R1. The FACS analysis showed that, HC6R1 and anti-IL6 R can not stain the IL6 R silenced hepatoma cells, which might further implied that IL6 R is the antigen recognition epitope of HC6R1.2. The antitumor activity and mechanisms of HC6R1 in the hepatocellular carcinoma2.1 The CCK8 analysis showed that HC6R1 can markedly reduce the proliferation of hepatic carcinoma cell lines after 24 h incubation. Wound healing assay showed that HC6R1 reduced the migration of hepatoma cells significantly. Adhension and transwell analysis showed that adhesion and invasion ability was obviously suppressed after HC6R1 treatment.2.2 We generated xenografted tumor models and found out that, compared with control mIgG, HC6R1 treatment could significantly inhibit the tumor growth. Compared with control mIgG, we also observed that intravescical administration of HC6R1 administration was able to suppress hepatic tumor growth and prolong the survival rate of tumor-bearing mice.2.3 Flow cytometric analysis suggested that HC6R1 can promote cell apoptosis and arrests cell-cycle progression in G1/S phase, which could suppress the cell proliferation.We used gene microarray to detect the target gene of HC6R1, and found Bcl-2, Bcl-xL, Survivin, Myc, Cyclin D1, VEFG, HIF-1, MMP-2, IL-6 and IL-10 genes were significantly activated. Most of them were the downstream target genes of STAT3 signaling pathway. Western blotting assay showed that HC6R1 could suppress the expression levels of p-STAT3, CyclinD1 and CDK4. Conclusions:1. HC6R1 can specific recognized IL6 R on hepatic carcinoma.2. HC6R1 can significantly suppresses cancerous proliferation, migration, adhesion and invasion ability in vitro.3. HC6R1 is able to suppress the hepatic tumor growth and prolong the survival rate of tumor-bearing mice.4. HC6R1 can block the activation of the JAK-STAT3 signaling pathway mediated by IL6, which can inhibit the phosphorylation of STAT3 and then suppress the expression of CyclinD1 or CDK4, further to arrest cell-cycle progression in G1/S phase and promote the cell apoptosis.