Antibody Gene Therapy For Hepatocellular Carcinoma Using Adenoviral Vectors Targeting Interleukin-17A

Under the current conditions of diagnosis and treatment of liver cancer,only patients(30%)who are diagnosed with early hepatocellular carcinoma(HCC)can undergo curative treatment,while the remaining patients with advanced HCC and relapsed patients can only receive adjuvant therapy.The treatment methods applicable to patients with advanced liver cancer are limited and the therapeutic effect is poor.The median overall survival(MS)of patients receiving adjuvant therapy is less than 6 months,so there is an urgent need for new and more effective treatments methods.Viral vector-based gene therapy has been applied to liver diseases,and has shown good application prospects.Viral vectors have been studied thoroughly,especially adenovirus type 5(Ad5)vector.Ad5 vector has always been the most used type of vector in clinical trials.Because of its hepatotropism,Ad5 vector also has unique advantages in the treatment of liver diseases.In the development of gene therapy,right target is one of the key factors that determine the therapeutic effect.Many studies have shown that interleukin-17A(IL-17A)may play an important role in hepatocellular carcinoma.IL-17A can promote angiogenesis in local tumor tissues and promote the invasion and migration of hepatocytes.The high expression of interleukin-17A in tumor tissues of patients with hepatocellular carcinoma is closely related to tumor progression,poor treatment effects and poor prognosis.Therefore,preventing the synthesis of IL-17A,promoting its degradation or blocking its activity in HCC cancer tissues will inhibit the progression of HCC.Based on the above hypothesis and the mature application of Ad5 vectors in gene therapy,this project aims to develop a gene therapy method targeting interleukin17A with Ad5 vectors and explore its feasibility and therapeutic effects in treatment of hepatocellular carcinoma.Methods:The sequence of recombinant antibody fragments were cloned into Ad5vector.The recombinant antibody fragments needs to be secretory.They should have the ability to bind to IL-17A,and have good stability.After local administration,adenoviral vectors should infect tumor cells in local tissues,and then the recombinant antibody fragments could be secreted.The IL-17A in tumor tissues could be neutralied,and then the tumor growth could be inhibited.The research methods are described:1)The variable regions of heavy chains(VH)and variable regions of light chains(VL)of the IL-17A neutralizing antibody were used for the construction of recombinant antibody fragments.2)Gene synthesis,homologous recombination,double plasmid co-transfection method,Cs CI gradient ultracentrifugation method,and hexon immunoassay were used for plasmid construction and Ad5 vector packaging,concentration,purification and viral titer determination.3)Enzyme linked immunosorbent assay(ELISA)was used to evaluate the function,stability of recombinant antibody fragments.4)Stable HCC cell lines expressing IL-17A were generated by lentiviral vector transduction.Single clones were identified by limited dilution and expansion.Expression of exogenous IL-17A protein in stable cell lines was tested by Quantitative Real-Time-PCR(q RT-PCR)and Western blotting(WB).5)Based on stable cell lines,flow cytometry,transwell,wound healing assay were used to evaluate the function of adenoviral vector in vitro.6)In xenograft models of stable HCC cell lines,immunohistochemistry and small animal imaging methods were used to explore interleukin 17A expression and antitumor effect of adenoviral vectors.The experimental results show:(1)Antibody fragment design,adenovirus vector construction and packaging.1)Four recombinant antibody fragments were designed and the DNA sequences were synthesized.The structures of which are sc Fv,sc Fv-sc Fv,sc Fv-CH3 and sc Fv-Fc.2).The recombinant antibody gene sequences were cloned into a modified p MT shuttle plasmid separately and than then four plasmids pse2784,pse2887,pse2888,and pse2889 were obtained.Those plasmids were identified by restriction enzyme digestion to be 1716 bp,1413 bp,1818 bp,and 1020 bp,which were consistent with the theoretical lengths.The gene sequencing results showed that the constructed sequence was 100%consistency with the designed sequence,which indicatied that the plasmids with recombinant antibody fragment was successfully constructed.3)Four adenoviral vectors were packaged,and their titers were all above10¹⁰ Ifu/ml(Ad IL17-s,3.16×10¹⁰ Ifu/ml;Ad IL17-s C,3.93×10¹⁰ Ifu/ml;Ad IL17-ss,4.11×10¹⁰ Ifu/ml;Ad IL17-s F,9.48×10¹⁰ Ifu/ml).(2)Recombinant antibody fragment function and stability test.1)Four recombinant antibody fragments could be secreted out of the cells and bind to interleukin 17A.The recombinant antibody fragment with sc Fv-Fc structure showed higher stability and the titer of antibody of Ad IL17-s F was higher than 1:1000.2)After 8 times repeated freeze-thaw cycles,the antibody fragments of Ad IL17-s F,Ad IL17-ss,and Ad IL17-s F still had high binding capacity to IL-17A(Ad IL17-s F vs.Ad IL17-s,^****P>0.0001;Ad IL17-s C vs.Ad IL17-s,^####P>0.0001;Ad IL17-ss vs.Ad IL17-s,^????P>0.0001).After 8 days of incubation with serum at 37°C,the antibody fragments of Ad IL17-s F showed higher stability than that of the other three groups(Ad IL17-s F vs.Ad IL17-s,^****P>0.0001;Ad IL17-s F vs.Ad IL17-s C,^####P>0.0001;Ad IL17-s F vs.Ad IL17-ss,^?P>0.05).Conclusion:Antibody fragment sc Fv-Fc of Ad IL17-s F had good stability.(3)The effect of Ad IL17-s F on the function of HCC cell lines in vitro.1)Stable HCC cell lines that secrete IL-17A continuously were constructed.After puromycin screening and monoclonal selection,compared with the control group,the IL-17A were expressed in monoclonal stable cell lines at both m RNA level and protein level.It is indicated that the stable cell lines were successfully constructed.2)Compared with the control group,the stable cell lines showed higher invasive ability(Hep3B:Control vs.IL17⁺,^****P<0.0001;Huh-7:Control vs.IL17⁺,^***P<0.001)and migration ability(Hep3B:Control vs.IL17⁺,^***P<0.001;Huh-7:Control vs.IL17⁺,^***P<0.001).Ad IL17-s F can reverse the enhanced invasion ability of stable stable cell lines(Hep3B-IL17⁺:Control vs.Ad IL17-s F,^**P<0.01;Huh-7-IL17⁺:Control vs.Ad IL17-s F,^**P<0.01)and the migration ability(Hep3B-IL17⁺:Control vs.Ad IL17-s F,^***P<0.001;Huh-7-IL17⁺:Control vs.Ad IL17-s F,^***P<0.001).(4)Evaluation of Ad IL17-s F expression,inhibition of angiogenesis and anti-tumor status in vivo.1)In vivo imaging results show that the Ad IL17-s F could continue to exist for more than 32 days after intratumoral injection(Ad-LUC vs.Control,^****P<0.0001).2)After adenoviral vector local injection in the tumor,the recombinant antibody fragments could be expressed in the tumor tissue(Ad IL17-s F vs.Control,^****P<0.0001).3)Compared with the control group,the angiogenesis in the tumor tissue was inhibited after Ad IL17-s F treatment(^*P<0.05).4)The tumor growth was inhibited compared with the control group(Huh-7-IL17⁺:Control vs.Ad IL17-s F,*P<0.05;Hep3B-IL17⁺:Control vs.Ad IL17-s F,^*P<0.05)and the apoptosis in tumor tissues increased(Ad IL17-s F vs.Control,^*P<0.05).In summary,this is the first time,a new Ad5 vector(Ad IL17-s F)was constructedtargeting interleukin 17A for treatment of hepatocellular carcinoma.The recombinant antibody fragment sc Fv-Fc could be expressed after Ad IL17-s F transduction.The antibody fragments had IL-17A binding ability,and it has good stability even after freeze-thaw cycles and co-incubation with 37?serum for 8 days.Ad IL17-s F could inhibit the invasion and migration of stable HCC cell lines in vitro.After treatment with Ad IL17-s F in vivo,antibody fragments could be expressed in tumor and angiogenesis could be inhibited.The Ad IL17-s F could suppressed tumor growth.This study demonstrated the feasibility of using Ad5 adenoviral vectors carrying a gene encoding IL-17A neutralizing antibody fragments to treat HCC,which confers a new approach for the treatment of solid tumors by neutralizing cytokines from the HCC microenvironment.