Screening And Identification Of Monoclonal Antibodies Against Murine Hepa 1-6 Hepatoma Cell And Evaluation Of Its Therapeutic Effect In Cancer Therapy

Liver cancer is one of the most common digestive system malignant tumor in clinical practice.At present,the incidence and mortality of liver cancer are still high,mainly due to the low rate of early diagnosis and the lack of a very effective treatment.Traditional treatment(hepatectomy,local ablation and liver transplantation)is the most important way for patients with liver cancer to obtain long-term survival,but there is still the risk of incomplete treatment and easy relapse.However,most patients with liver cancer are often diagnosed at the middle and advanced stages,so they missed the best time for treatment.At present,the treatment for advanced liver cancer is limited.Sorafenib is the first small molec?Lar targeted drug approved by the FDA for the treatment of liver cancer,but the efficacy is still not satisfactory,and the median survival time of patients can only be extended by 3 months.Over the past few years,immune checkpoint inhibitors has made a great breakthrough in the treatment of some advanced cancer,but the objective response rate of PD-1 inhibitors for advanced liver cancer is only 18%,most patients can not benefit from this type of immunotherapy.Although a small number of other anti-liver cancer drugs are testing in the clinical trials,there is still a long way to go to achieve the goal of curing liver cancer.Therefore,the development of a novel,effective target or drug for liver cancer is still urgent and with important clinical significance.The purpose of this study was to screen monoclonal antibodies specific for murine Hepa 1-6 hepatoma,which possess therapeutic potential,and it is expected to identify the precise targets which recognized by these therapeutic monoclonal antibodies.For this purpose,the following work was carried out.First,using the ELISPOT assay we established a cell platform for in vitro screening of monoclonal antibodies specific for murine Hepa 1-6 hepatoma.In this study,murine Hepa 1-6 hepatoma cells were used as immunogens.Two different immunization strategies,including direct immunization and subtractive immunization by cyclophosphamide,were used to screen monoclonal antibodies specific for murine Hepa 1-6 hepatoma.Through the direct immunization method,we obtained a total of 8 monoclonal antibodies with IgM subtype.Through the direct immunization method,we obtained a total of 24 monoclonal antibodies with IgG subtype.By flow cytometry and immunofluorescence,we evaluated the reactivity and specificity of antibodies with either IgM or IgG subtype,respectively,and we also confirmed that these antibodies bound to the proteins on the cell membrane.In order to evaluate the anti-tumor activity of these antibodies,a syngeneic mouse model was developed with subcutaneously implanted Hepa 1-6 cells in C57BL/6 mice.The res?Lts showed that only antibodies with IgG subtype(2G2 and 12D9)had antitumor activity.2G2 and 12D9 can not only prevent the formation of Hepa 1-6 hepatoma,but also treat and completely eliminate Hepa 1-6 hepatoma in mice.In addition,we also demonstrated the therapeutic ability of 2G2 and 12D9 in Hepa 1-6 hepatoma bearing nude mice and NOD-SCID mice.Finally,by using co-immunoprecipitation and time-of-flight mass spectrometry,we identified the target of 2G2 and 12D9 on the cell membrane,named chloride ion channel 1(CLIC1).It showed that the anti-tumor activity of these antibodies may play an important role in interfering the CLIC1 signaling pathway.However,the underlying mechanism still need to be further investigated.In summary,32 monoclonal antibodies specifically against murine Hepa 1-6 hepatoma cells were screened in this study.Among them,monoclonal antibodies with IgG subtype had significantly superior anti-tumor activity in mouse Hepa 1-6 hepatoma-bearing models.In addition,we also conducted a preliminary exploration of the targets of these monoclonal antibodies,and initially identified as CLIC1.This study has important guiding value for the improvement of the screening strategy for novel liver cancer targets,and provides a basis for exploring the new intervention by targeting the CLIC1 for the treatment of liver cancer.