Identification Of Single-chain Antibody,Preparation Of Polyclonal Antibody Against GPC3,and Their Application In Detection Of Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of most common cancers in China.The morbidity in Chinese population is about 28.7/10million,and thenumber of the patients is more than half of total patients globally.Most of the patients is diagnosed at the middle and late stage.According to the survey results of the national death causes published by the Ministry of health of China,The mortality of liver cancer isthe second place in various malignant tumors in our country,and the incidence rate is fourth place in china.At present,the main methods of treatment for liver cancer are surgical resection of lesions and chemotherapy,but the recurrence rate is very high,targeting drug sorafenib can only prolong the life of patients and can not completely cure liver cancer.Because the cure rate of early stage liver cancer is very high,and the five year survival rate is about 80%.Therefore,the early diagnosis of liver cancer is particularly important in the treatment of liver cancer.In addition,the development of ADC therapeutic drugs also brings the good news to liver cancer patients.The characteristics of antibody therapy are good specificity and small side effects,and some antibody targeted liver cancer drugs have completed preclinical trials.Objective:Our study is first to clone and express GPC3N-terminal domian(GPC3-ND)in E.coli,prepare polyclonal antibody that target to GPC3 and diagnosis the HCC by anti-GPC3-ND antibody and preliminaryidentifysingle-chain antibodies(scFvs)against the antigen using phage-displayed antibody library.Methods:The DNA fragment encoding GPC3 N-terminal domian(GPC3-ND)was cloned into pET-30a(+)to form expression plasmid using DNA recombinant technology.The his-tagged recombinant protein was expressed by E.coliRosetta with IPTG induction,and was purified by Ni-NTA column.The scFv antibodies against the recombinant GPC3 N-terminal domain were screened using a phage-displayed library previously established in our laboratory.The recombinant GPC3 N-terminal domainwas used to immune Zealand male rabbit for anti-GPC3 polyclonal antibody preparation,wedetectedaffinityof anti-GPC3 antibody withGPC3 by ELISA and WB,and we detected bindingof anti-GPC3 antibody with GPC3 on HCCcell surface by immunofluorescence.Then,we use anti-GPC3 polyclonal antibody to detect expression of GPC3 on liver cancer tissue,that lay the foundation of cheap primary liver cancer diagnosis.The scFv antibodies against the recombinant GPC3-ND were screened using a phage-displayed library previously established in our laboratory.Results:After deleting 26-31 position and 6 proline of GPC3N-terminal domain gene,the recombinant GPC3 N-terminal domain was expressed from E.coliRosetta through pET-30a(+)expression system and purified on a Ni-NTA column.After new Zealand rabbits were immunized with recombinant GPC3 N-terminaldomain,we obtain antibodies specifically bind to the recombinant GPC3 N-terminal domainn.Itshowed that the polyclonal antibody can specifically bind to GPC3 positive primary hepatocellular carcinoma cells and not bind to normal liver cells L02through westernblotting and immunofluorescence technique.Anti-GPC3-ND antibody was used to detect the tissues of primary liver cancerpatients,and the results of IHC were positive,while the IHC tissue of breast cancer patients showed negative results.After screening forth rounds of phage antibody library,we get 1 phage antibody with a complete structure of light chain and heavy chain,which could be read through TG1(Amber codon mutation).Conclusion:We have successfully completed optimizal expression of GPC3and purification,successfully prepared antibodieswith GPC3 specific binding capacity,and this polyclonal antibody successfully detected the expression of GPC3on primary hepatocellular carcinoma tissue.The scFv against GPC3N-terminal domain was preliminarily identified.