Affinity Maturation And Antitumor Activity Of HN3 Monoclonal Antibody

The world wide incidence and mortality of liver cancer ranks the top ten of all cancer types.Currently,only Sorafenib and Lenvanix are recommended for first-line treatment of liver cancer.Sorafenib and Lenvanix are small molecule multi-target tyrosine kinase inhibitors,but their efficacy is limited.Therefore,the development of immunotherapy drugs for liver cancer is highly expected.GPC3 is a member of the acetyl heparan sulfate protein family,which is involved in the regulation of signaling pathways such as Wnt,Hedgehog,bone morphogenetic proteins and FGF,thereby controlling cell proliferation and apoptosis at different developmental stages.GPC3 gene expression is developmental stage and tissue specific,with specific expression in several tissues during embryonic stages and loss of expression in adult tissues.However,GPC3 is specifically expressed in adult tumor tissues such as hepatocellular carcinoma,colorectal carcinoma,and neuroblastoma,while it is low or even absent in normal liver tissues.Upregulated expression of GPC3 in hepatocellular carcinoma promotes proliferation and migration of hepatocellular carcinoma cells and is highly associated with poor patient prognosis.Previous studies by our group have found that the neutralizing antibody HN3 inhibits the proliferation of hepatocellular carcinoma cells by blocking the GPC3-mediated signaling pathway,and that immunotoxins and antibody-drug conjugates（ADCs）targeting GPC3can specifically kill tumor cells.Therefore,GPC3 is an ideal target for immunotherapy of hepatocellular carcinoma.HN3 is a human antibody targeting GPC3,which has been shown to bind to the conformational epitope formed by the N-terminal and C-terminal ends of the GPC3 core protein,blocking the binding of growth factors and triggering intracellular signaling,inactivating YAP and inhibiting the proliferation of hepatoma cells.In vitro,HN3 directly inhibited the growth of GPC3-positive hepatocellular carcinoma cell lines and also inhibited the growth of GPC3-positive xenograft tumors in mice.The HN3-based immunotoxins were more active in killing hepatocellular carcinoma cells,with IC50generally below 100 ng/m L.However,the affinity of the antibody is closely related to its activity,and both antibody neutralizing activity and immunotoxin activity can be improved when the affinity of the antibody is increased.Therefore,in this study,a strategy to optimize the amino acid sequence composition of the CDR region of the antibody was used to improve the affinity of HN3.The main part of this study was to design primers to sequentially mutate the three CDR regions of HN3,and to obtain several mutants with significantly higher affinity than HN3 through the construction and screening of mutant libraries.HN3 was used as a control to verify the binding activity of HN3 mutant naked antibody to GPC3 protein,the binding activity to GPC3-positive cell lines,and the activity to inhibit tumor cell proliferation in vitro.Subsequently,a recombinant immunotoxin based on the HN3mutant was prepared and tested for its affinity,in vitro tumor cell killing activity and in vivo tumor growth inhibitory activity.The following results were obtained:1.192 affinities were obtained by mutating three CDR regions of HN3 and constructing and screening a mutant library The mutant sequences were compared and analyzed,and five representative sequences were selected for subsequent studies.These mutant sequences were compared and analysed,and five representative sequences were selected for follow-up studies:GN4 and GN5 mutated in the CDR3 region,and HL03,HL49 and JW31 mutated in the CDR1,CDR2 and CDR3 regions.2.HN3 and HN3 mutant antibodies were successfully expressed and purified,and the binding of HN3 mutant naked antibodies to GPC3 protein and GPC3-positive cell lines was tested by ELISA and flow cytometry.Five HN3 mutant naked antibodies were able to bind GPC3 protein effectively,with JW31-hFc having an EC₅₀ of 0.042 nmol/L,which was 4.1-fold higher than that of HN3-hFc,and that of HL49-hFc was 0.054nmol/L,which was 3.2-fold higher than that of HN3-hFc.All five HN3 mutant naked antibody proteins were able to bind GPC3-positive cell lines,and HL49-hFc was significantly stronger than HN3-hFc on all GPC3-positive cell lines.3.The inhibition of proliferation of GPC3-positive tumor cells in vitro by HN3mutant naked anti-protein revealed that HL03-hFc was stronger than HN3-hFc.The IC₅₀values of HL03-hFc on Huh7,HepG2 and Hep3B were 6-fold,3.1-fold and 1.9-fold higher than those of HN3-hFc.4.The recombinant immunotoxin was expressed and purified,and the affinity of the HN3 mutant recombinant immunotoxin to the GPC3 protein was tested by ELISA.All five recombinant immunotoxins were able to bind the GPC3 protein effectively,with HL49-PE24 showing the strongest binding activity with an EC₅₀ of 1.729 nmol/L,which was 1.2-fold higher than that of HN3-PE24.Cell killing assays with recombinant immunotoxins showed that GN4-PE24,GN5-PE24 and HL49-PE24 had stronger killing activity than HN3-PE24.The most active immunotoxin was HL49-PE24,with IC₅₀s of5.600 nmol/L,5.842 nmol/L and 1.149 nmol/L on Hep3B,HepG2 and Huh7.The killing activity of GN4-PE24 and GN5-PE24 in different cell lines was 1.5-2.5-fold higher than that of HN3.5.In a tumour suppression assay in G1 tumour-bearing mice,intratumoural and tail vein injections of 5 mg/kg of immunotoxin showed stronger tumour suppressive activity of HL49-PE24 than HN3-PE24,with complete tumour elimination in three out of five HL49-PE24-treated mice.Tail vein injection of the same dose of immunotoxin showed that the anti-tumour response of HL49-PE24 was still stronger than that of HN3-PE24,but weaker than that of intratumoural injection.In conclusion,by mutating the CDR region of HN3,five representative HN3mutants（GN4,GN5,HL03,HL49,JW31）were obtained and naked antibodies and recombinant immunotoxins were prepared in this project.Among them,JW31-hFc and HL49-hFc showed stronger binding activity to GPC3 protein than HN3-hFc,and HL03-hFc inhibited the proliferation of GPC3-positive tumor cells more than HN3-hFc.Among the five HN3 mutant immunotoxins,HL49-PE24,GN4-PE24 and GN5-PE24showed stronger killing activity to GPC3-positive tumor cells than HN3-hFc.In a tumour suppression assay in G1-bearing mice,intra-tumoural injection of 5 mg/kg HL49-PE24resulted in transient tumour elimination in three of the five treated mice and no tumour recurrence was observed in one of the five treated mice at follow-up.These affinity-matured mutants improved the ability to recognize and bind tumor antigens,and in vitro and in vivo tumor suppression assays showed that the anti-tumor activity of naked antibodies and immunotoxins based on these mutants was significantly increased against hepatocellular carcinoma cell lines,which has potential for application in tumor immunotherapy.The results of this study provide a basis for further development of immunotherapeutic techniques or drugs based on HN3 therapeutic antibodies,offering new ideas and experimental approaches for the treatment of hepatocellular carcinoma.