Identification Of The Target Of Therap Eutic Monoclonal Antibodies Against Mouse Hepatocellular Carcinoma And Preliminary Evaluation Of SLC3A2 As A Potential Diagnostic And Therapeutic Target For Human Hepatocellular Carcinoma

Primary liver cancer(PLC)is the sixth most common malignant tumor in the world,and mortality ranks second among all tumor-induced deaths.The global burden of primary liver cancer is uneven,with China alone accounting for more than 50%in the world.Hepatocellular carcinomas(HCC)is a complex disease,and the main risk factors include chronic hepatitis B virus(HBV)or hepatitis C virus(HCV),alcoholism,obesity,and type 2 diabetes,et al.HCC is also highly heterogeneous,with a poor prognosis and a high probability of recurrence even after surgical resection.The early symptoms of HCC are not obvious,and the diagnosis is difficult,resulting in the majority of patients with advanced HCC.For patients with advanced HCC,conventional treatments such as chemotherapy and radiotherapy are often susceptible to drug resistance,making treatment very limited.At present,the most widely used targeted drug for HCC,sorafenib,is not very effective either,and only prolongs the survival time of patients with advanced HCC for several months.Therefore,it is urgent to develop new and effective HCC treatment drugs,such as small molecule targeted drugs,antibody drugs,microRNAs,etc.,or to explore treatment strategies combined with targeted therapy,immunotherapy and other treatment methods,aiming to improve the clinical efficacy of treating HCC.In the previous study,we used mouse Hepal-6 cells as immunogens and obtained 17 strains monoclonal antibodies specifically against mouse hepatoma cells.Based on these findings,we analyzed the reactivity of these antibodies to hepa1-6 cells by flow cytometry,and found that 3F11 antibody had the highest affinity with hepa1-6 cells.Therefore,3F11 antibody was selected for follow-up research.First,we evaluated the therapeutic effect of the antibody on mouse Nepa1-6 tumor-bearing model,and evaluated the toxicity of the antibody on mice.3F11 showed good antitumor activity and saftety profiles.In order to further explore the target of 3F11 antibody on Hepa1-6 cells,we established antibody target screening platform by using immunoprecipitation and mass spectrum method,and then verified its potential target by in vitro gene transfection and knockout confirmation.Finally,we identified the Solute Carrier Family 3 member 2(SLC3A2)was the target of therapeutic monoclonal antibody 3F11.We used Hepal-6 cDNA and genome as the templates to obtain the SLC3A2 gene,which were sequenced and analyzed to show that the SLC3A2 ORF of Hepal-6 cells were mutated at amino acids 47 and 129.The 129th amino acid mutation of SLC3A2 was verified to be critical for its binding to 3F11 antibody by reverting mutation.SLC3A2 is a target for therapeutic monoclonal antibodies against mouse HCC.Based on the consideration of human homology,we next evaluated the potential of SLC3A2 as a target for human HCC.We firstly examined the expression levels of SLC3A2 in human hepatoma cell lines and primary hepatocytes,and found that the expression of SLC3A2 in human hepatoma cell lines was much higher than that in primary hepatocytes.We next examined the expression levels of SLC3A2 in HCC clinical specimens,SLC3 A2 were expressed at signiciantly higher levels in HCC specimen than in paracancerous specimen.The knockdown of SLC3 A2 in human hepatoma cell lines by using CRISPR-Cas9 gene editing technology significantly inhibited the tumor cell growth and proliferation,indicating that SLC3A2 is important for the growth and proliferation of hepatoma cell lines.In addition,after preliminary studies,we identified a possible ligand of SLC3A2,Galectin-3,which can induce T cell apoptosis and inhibit T cell growth,and we hypothesize that SLC3 A2 may be the ligand of Galectin-3 on T cells.The above results preliminarily indicate that SLC3 A2 has the potential to become a diagnostic and therapeutic target for human HCC.Further research is needed on studing the carcinogenic molecular mechanism and immunoregulatory mechanism of SLC3A2 in the development of hepatocarcinoma,as well as the potential of SLC3A2 as a new target for HCC treatment.This study also facilitates the subsequent study of the molecular mechanism of HCC-targeting antibody,and lays a foundation for the study of potential therapeutic targets for HCC.