Construction And Radioimmunoimaging Of Single-chain Antibody From A Human Hepatocellular Carcinoma Phage Display Library Anainst Glypican-3

Liver cancer is the fifth most prevalent cancer in the world.The incidence and associated morbidity are rising.Hepatocellular carcinoma?HCC?is the major pathological type of liver cancer,accounting for 90%of all the liver cancer,resulting in at least 500000 people deaths every year.The overall 5-year survival rate for patients with liver cancer is about 15%.Liver cancer is not sensitive to most of the chemotherapy drugs.Curative therapeutic approaches are available only for cases in which the diagnosis is done at an asymptomatic early stage,which constitutes only 37%of patients.In vivo imaging and surveillance of individuals with high risk are most utilized strategies for early detection of hepatic nodules.GPC3 is a member of the glypican family that attaches to the cell surface by a glycosylphosphatidylinositol?GPI?anchor.GPC3 is over-expressed in HCC but not expressed or expressed at low levels in normal adult tissue.GPC3 mRNA level is higher than level of AFP in HCC,which is more prominent in small HCC.GPC3 is involved in the occurrence and proliferation of liver cancer.Several studies have confirmed that GPC3is a potential liver cancer therapeutic target.GPC3 as a target for HCC therapy has been investigated with GPC3 vaccine and anti-GPC3 antibodies.These antibodies are in preclinical experimental or clinical trials.However most of the anti-GPC3 antibodies are full-length antibodies originated from mice or human.These monoclonal antibodies have a high molecular weight,low Penetration and targeting,which is not the best antibody to radioimmunoimaging or therapy.Antibody fragments generated from genetic engineer is the tendency of the radioactive immunoimaging or immunotherapy.scFv with small molecular weight,good penetration is applicated in radioimmunoimaging and radioimmunotherapy.We attempted to prepare the human single-chain variable fragment?scFv?antibody by phage display technology with a target of HCC cells expressing GPC3.Panning this libray and labeled it with radionuclide and made radioimmunoimaging in bearing HCC nude mice.ObjectiveConstructing anti-GPC3 human single-chain variable fragment?scFv?antibody from phage display technology and labling anti-GPC3 scFv with radionuclide to made radioimmunoimaging in bearing HCC nude mice.It will provide an effective approach to treat HCC.Methods and ResultsPart?.Construcion of HCC phage antibody libary1.Generation of scFvPeripheral blood from HCC patients separated for B cells to extracted total RNA.Then amplification V_H,V_L by PCR.SOE-PCE connect V_H and V_L.This is the VH-linker-VL libary.Result:The total RNA has two bands 28s and 18s in 1%agar gel electrophoresis.The V_H fragment is about 370bp,V_L fragment is about 360bp and scFv is about 750bp.2.Recombination of scFv Gene Repertoire into pCANTAB-5E by dual-enzyme digestion.Purify scFv transform competent e.coli TG1 by chemical method.Reuslts:The titer of phage antibodies library is 10¹¹cfu/ml.Random digestion reaction showed that the positive insert ratio was 87.5%.Double enzyme digestion PCR of recombinant positive plasmid indicate a clear band in 750bp.I t is confrim the successful recombination of scFv.Part?.Panning of phage antibody library1.Screening phage antibody library with HCC.We use L02 to have a Negative selection first.Then we use HepG2 to enrichment with 3 round.Determination the titer of antibody before and after enrichment.The results show that the phage recovery gradually improve.2.Panning with GPC3.Enrichment of phage antibody libray with GPC3in 3 rounds.The result indicates a high phage recovery every round.3.Production of soluble recombinant antibody.Strong positive recombinant phage antibody infects E.coli HB2151.We get soluble antibody after induction with IPTG.4.Detection soluble antibody with SDS-PAGE and Western blot.And detection immunocompetence and specificity of scFv with ELISA.The coomassie blue staining shows bond on 30KD,which indicates the solubility of scFv.one-way analysis of variance of ELISA shows 0.47±0.11 in HepG2group,0.18±0.05 in SKOV3 group and 0.16±0.04 in PBS group.The result has a statistically significant difference?F=103.934,P=0.000?.LSD Duncan shows a significant difference between HepG2 group and SKOV3?p=0.000?.And a significant difference between HepG2 group and PBS group?p=0.000?.Immunocytochemistry indicate a stain in HepG2 group,however the scFv cannot stain the SKOV3 group and PBS group.The results of immunocytochemistry confirm the specificity of scFv.Part?¹³¹I-scFv Radioimmunoimaging1.¹³¹I was labled with anti-GPC3 scFv by chloramines T method.Detecting the labeling efficiency,specific activity and radioactive chemical purity.And detecting the radioactive chemical purity and stability in blood and room temperature.Then we injected the radionuclide into the nude mice bearing hepatoma and have a SPECT in 4 time points.The results:The labeling efficiency is 80.2±4.0%.The radioactive chemical purity is92.75±3.2%.The specific activity is 3.2±0.2MBq/?g.he radioactive chemical purity in blood and room temperature is above 90%at 4 time points.And there was no statistical significance?P>0.05?.The SPECT display a hot spot in tumor location which was clear visible.ConclusionWe successfully construct human anti-GPC3 HCC human scFv antibodies by phage display technology.We successfully labled 131I with scFv.The labeling efficiency,specific activity and radioactive chemical purity conform the requirements.The antibody is stable in blood and room temperature.The radionuclide is specific and have a good target.