Construction And Identification Of Glypican-3 CAR Lentivirus Expression Vector

Backgroud and Objective The adoptive immunotherapy of T cells modified by chimeric antigen receptor(chimeric antigen receptor, CAR) is a new strategy which is developing rapidly in recent years.CAR gives T lymphocyte lasting vitality,more fertile and targeted killing activity.The unique mechanism and inviting application prospect open up a new stage in tumor biological therapy. It is reported that phosphatidylinositol glycan(glypican- 3, GPC3) is one of tumor associated antigens in hepatocellular carcinoma. Some studies have indicated that GPC3 is highly expressed in HCC,but not in cirrhosis of the liver, hepatitis or adult normal liver tissues. In addition, it is closely related to the development and prognosis of tumor and is the ideal molecular target to targeted therapy cancer. The study constructs lentiviral vector LV-GPC3CAR(the third generation) containing chimeric antigen receptor targeting an anti-GPC3 single-chain antibody. Packing the lentivirus vector, infection of human T cells, which lays the condition for chimeric antigen receptor modified T lymphocyte therapy of tumors.Methods 1. The construction of lentiviral vector LV-GPC3CAR(the third generation) containing chimeric antigen receptor targeting an anti-GPC3 single-chain antibody. 2. After verifying right by double enzyme digestion and sequencing, the lentivirus was obtained by using plp1、plp2、plp/VSVG and LV-GPC3 CAR to transfect 293 T cells. Lentivirus infect 293 T cells. Western-blot test the expression of purpose gene. 3. Human peripheral blood T lymphocytes were isolated and purificated by the CD3 Immunomagnetic Microbeads.The lentivirus was preliminary applicated in human T cells. The infection efficiency of lentivirus was confirmed by FACS examination. Western-blot test the expression of purpose gene.Results 1. Lentiviral vector LV-GPC3 CAR was constructed correctly by double enzyme digestion and the nucleotide sequencing. 2. Packaging lentivirus vector used Lip3000 kit and hole by hole dilution method detecting the titer of lentivirus was 2.7x10^8 TU/ml. Lentivirus infected 293 T cells. After 72 hours of incubation, it is showing the typical morphology of aggregate growth and expression of GFP. Western-blot demonstated 293 T cells expression of GPC3CAR(about 62 k Da) correctly after infection. Immunofluorescence technique detected that LV-GPC3 CAR expressed on the cell membrane. 3. Use IL-2(50U/ml)、anti-CD3(1μg/10ml)and anti-CD28(1μg/10ml) to cultivate T cells. Expression of LV-GPC3 CAR value in human T cells increased with the increase of the MOI, but the cell survival rate is opposite by flow cytometry. Thus, we select the applicable MOI is 20MOI(infection rate was 55.77%,the cell survival rate was 68.80%). Western-blot demonstated T cells expression of GPC3 CAR correctly after infection.Conclusions LV-GPC3 CAR is constructed successfully. GPC3 CAR can exactly express on cell surface. Our study lay the foundation for future research and application in LV-GPC3 CAR modified T lymphocyte therapy of HCC.