Targeted Human Cadherin-17 Monoclonal Antibody In Hepatocellular Carcinoma Therapy

PartIPreparation, purification and identification of monoclonal antibodies against human cadherin 17(CDH17)Purpose:Prepare Lic5 and Lic3 monoclonal antibodies (mAbs) from nude mouse ascites induced by Lic5 and Lic3 hybridoma cells and identify the purity and specificity of mAbs for the study of monoclonal antibody therapy in human hepatocellular carcinoma (HCC).Materials and Methods:Lic5 and Lic3 hybridoma cells were injected into abdominal cavity of BALB/c nu/nu mice and collected the ascites containing the mAbs. Purify Lic5 and Lic3 mAbs from mouse ascites by Protein A affinity chromatography and identify mAbs'purity and specificity by Western Blot and silver staining. Measure mAbs'concentration by Lowry method.Results:1. Lic5 and Lic3 hybridoma cells grew well in high activity. Ascites was collected 7-10 days after Lic5 hybridoma cell injection and 15 days after Lic3 hybridoma cell injection. The average yield of ascites with Lic5 and Lic3 hybridoma cells were 7.25ml and 5.5ml per nude mouse respectively.2. From the silver staining of Lic5 and Lic3 mAbs, we only found two pieces of clear and independent bands at 50kD and 25kD, which represent the mAbs' heavy chain and light chain respectively. Western Blot detected CDH17 in cell lysate of MHCC97L and AGS with a single band at 120kD. Isotypes of Lic5 and Lic3 were identified by test paper and mAbs were IgG2b and IgG2a respectively.3. Lic5 and Lic3 mAbs output of hybridoma ascites were 2.75mg/ml and 11.8mg/ml respectively. Conclusion:1. Mouse ascites containing Lic5 and Lic3 mAbs were harvested with high quality and high titer.2. Lic5 and Lic3 mAbs against human CDH17 were prepared successfully with high purity and specificity.3. Protein A affinity chromatography is suitable for purification of mAbs in the laboratory.Part IICharacterization of changes in biological properties of human hepatocellualr carcinoma cell lines under the anti-CDH17 mAbsPurpose:To study the changes in cell proliferation, migration, invasion, colony formation and related drug sensitivity of human hepatoma cell lines MHCC97L, MHCC97H, PLC/PRF/5 and immortalized human hepatocyte MIHA under the Lic5 and Lic3 mAbs'treatment. To explore the possible underlying mechanisms.Materials and Methods:Detect the CDH17 protein and mRNA level in four cell lines such as MHCC97L, MHCC97H, PLC/PRF/5 and MIHA by the way of Western Blot and quantitative real-time PCR. Examine the binding capacity between Lic5 and Lic3 mAbs and CDH17 in the living cell membrane by the way of immunofluorescence staining. With the PBS and mouse IgG as the control, cell proliferation was detected by the way of MTT assay, cell migration was checked by wound healing assay, cell invasion assay was calculated by transwell method, cell colony formation was detected by plate colony assay. Drug sensitivity assay was monitored by MTT assay. Signaling pathways was explored by Western Blot.Results:1. CDH17 protein and mRNA were detected with a strongly high level in metastatic HCC cell lines MHCC97L, MHCC97H, but very little or undetectable level in primary HCC cell line PLC/PRF/5 and immortalized normal human hepatocyte cell line MIHA.2. Lic5 and Lic3 mAbs can bind with the CDH17 in the membrane of living cell lines MHCC9L and MHCC97H.3. Cell proliferation ability of MHCC97L and MHCC97H in mAbs'groups was significantly weakened. The statistic analysis showed significant difference between mAbs'groups and controls (p<0.05). There was no difference between mAbs'groups and controls in PLC/PRF/5, and MIHA cell lines.4. The number of clone formation, migration and invasion in Lic5 and Lic3 mAbs' groups of MHCC97L and MHCC97H cells was significantly less than that in PBS and mouse Ig groups. Statistic analysis showed significant difference between mAbs'and control groups.5. The drug sensitivity and inhibition rate of cisplatin to MHCC97L and MHCC97H cells was significantly elevated as compared to PBS and mouse Ig controls.6. Lic5 and Lic3 mAbs can downregulate the CDH17 protein expression level in MHCC97L and MHCC97H cells and had no any effect on CDH17 mRNA level.7. Lic5 and Lic3 mAbs can downregulate theβ-catenin protein expression level in MHCC97L and MHCC97H cells, which was the key molecule in Wnt/p-catenin signaling pathway.Conclusion:1. Lic5 and Lic3 mAbs can bind with CDH17 in cell membrane in living MHCC97L and MHCC97H cells.2. The MHCC97L and MHCC97H cells in mAs'groups showed great changes in the cell biological properties as compared to PBS and mouse Ig controls including retarded proliferation; decreased ability in cell clone formation, cell migration and invasion.3. Lic5 and Lic3 mAbs increased the drug sensititvity of MHCC97L and MHCC97H to cisplatin significantly.4. The CDH17 andβ-catenin protein level were significantly reduced by Lic5 and Lic3 mAbs in MHCC97L and MHCC97H cells. Part IIIThe inhibition role of anti-CDH17 monoclonal antibody and combination with cisplatin on hepatocellular carcinoma in vivo and the exploration of the anti-tumor mechanisms.Purpose:To studay the anti-tumor effect of Lic5 and the combination between Lic5 and cisplatin in subcutaneous xenograft model and orthotopic transplantation model of MHCC97L cell. To explore the underlying signaling pathway related to the mAbs' therapy.Materials and Methods:Use MHCC97L or with luciferase transfection to establish the subcutaneous tumor model and orthotopic tansplantation tumor model of human HCC in nude mouse. The subcutaneous tumor models in nude mice were divided into six groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg), cisplatin group(lmg/kg), the combination group of Lic5 mAb and cisplatin(Lic55mg/kg, cisplatin lmg/kg). Observe the impact of Lic5 and the combination with cisplatin on subcutaneous tumor growth. Detect the change of expression level of CDH17 and key molecules in related signaling pathway by the way of Western Blot. The orthotopic transplantation tumor models in nude mice were divided into four groups, PBS empty control group, Mouse IgG parallel control group, Lic5 mAb low dosage group(2.5mg/kg), Lic5 mAb high dosage group(5mg/kg). Use the bioluminescence to montitor the tumr growth in different groups.Results:1. Compared with PBS and mouse Ig control in subcutaneous model, the tumor growth was remarkably inhibited in mAbs'groups. Among the former three groups, the tumor growth in Lic5 and cisplatin combination group showed the strongest inhibition. Statistics analysis showed significant difference between the mAbs'and control gourps. 2. In the subcutaneous tumor models, the lung metastasis rate in PBS and mouse Ig groups were all 83.3%, Lic5 low dosage, high dosage and combination groups were 50%,33%and 0.3. The number of Ki67 positive cells were significantly decreased in mAbs'groups compared with PBS and mouse Ig groups in subcutaneous tumor models. Statistics analysis showed significant difference between mAbs'and control groups.4. Western Blot showed the expression of CDH17 was significantly downregulated in mAbs'treatment groups whereas high expression of CDH17 in control groups. The key molecules in Wnt/β-catenin, (3-catenin and Cyclin D1 were also downregulated in mAbs'treatment groups, but Rb was upregulated in the same groups.5. There was no obvious tissue abnormalities in liver, kidney, lung and spleen organs in mAbs'treatment groups.6. Compared with the PBS and moue Ig controls in orthotopic tumor models, bioluminescence signals and tumor volume were reduced significantly in mAs' treatment groups. Statistics analysis showed significant difference between mAbs' groups and control groups. The tumor lung metastasis rate in PBS and mouse Ig groups were all 100%and 50%,33.3%in Lic5 low and high dosage groups.Conclusion:1. Anti-CDH17 monoclonal antibody Lic5 can significantly inhibit the MHCC97L tumor growth and lung metastasis in nude mouse model and is a promising candidate in HCC targeted therapy.2. Lic5 mAb inhibited MHCC97L tumor growth by downregulation of CDH17 and inactivation of Wnt/β-catenin signaling pathway.3. Lic5 mAb can improve the drug sensitivity to cisplatin in MHCC97L in vivo and provide an effective experimental basis for the multi-drug combination therapy in HCC.4. No risk in biological safety had been observed during the period of mAb administration.