The Construction Of Recombinant Lentiviral Vector Expressing HPSE Regulated By HRE Promoter And Its Application In CAR-T Immunotherapy For Solid Tumors

Background:CAR-T immunotherapy refers to the chimeric antigen receptor(CAR)modified T cells,which recognize,bind to tumor antigens,and engage specific tumor killing effects.Recently,CAR-T cells have shown less striking therapeutic effects in solid tumors than in lymphoid malignancies.It has been found that in vitro-cultured CAR-T cells lack expression of the enzyme heparanase(HPSE),which degrades heparan sulfate proteoglycans(HSPG),the main components of extracellular matrix(ECM).This may account for the reduced ability of cultured CAR-T cells to penetrate stroma-rich solid tumors compared with lymphoid tissues.Objectives:A characteristic feature of solid tumour microenvironment is hypoxia.So the aim of this study is to construct a recombinant lentiviral vector with HREs/CMV promoter,which can regulate the expression of HPSE under hypoxia.T cells co-transduced with HRE6 and HER2/CAR have the capacity to express less HPSE in normal and more HPSE in solid tumor hypoxia environment.This strategy will promote T cell infiltration to tumor and antitumor activity while reducing damage to normal tissue.The use of this strategy may enhance the antitumor activity of CAR-T cells in individuals with stroma-rich solid tumors.Methods:First,three recombinant pCDH-HREs/CMV-HPSE recombinant lentiviral plasmids were constructed by molecular biology technique.The plasmids were transfected into 293T cells.Basing on the gray analysis and statistical analysis of Western blot result,we studied whether hypoxia could induce the expression of HPSE and selected a lentiviral plasmid with the best effect for packaging into lentiviral particles.The 3rd generation CAR specific for HER2 was constructed by molecular biology technique,cloned into lentiviral vectors and packaged into lentivirus particles.Second,flow cytometry was used to determine the virus titer and infection efficiency of T cells.The HER2 expression on the surface of tumor cells were evaluated by flow cytometry.Then,Western blot was used to detect the expression of HPSE in HRE6 and HER2/CAR co-transduced T cells under CoCl2 mimetic hypoxia condition.Further more,the specific killing effect of HRE6-HER2/CAR-T cells on HER2+tumor cells were detected.The release of IFN-y were determined by ELISA.The cytotoxicity of HRE6-HER2/CAR-T cells through the ECM to HER2+tumor cells was assessed by transwell cell invasion and cytotoxicity assay.Results:Three pCDH-HREs/CMV-HPSE recombinant lentiviral plasmids were constructed successfully.Hypoxia could induce the expression of HPSE in 293T cells.The effect of pCDH-HRE6/CMV-HPSE recombinant lentiviral plasmid was the best.The 3rd generation CAR specific for HER2 lentiviral vector pCDH-HER2/CAR was constructed successfully.The lentiviral virus titer of HRE6 and HER2/CAR were 3.5×107 TU/mL and 4.5×107 TU/mL.The efficiency of HRE6-T cells and HER2/CAR-T cells were 45.3%and 49.8%.HER2 is highly expressed in human breast cancer cell SKBR3 and low expressed in human breast cancer cell line MDA-MB-231.The infection efficiency of HRE6-HER2/CAR-T cells was 13.4%.Hypoxia could induce the expression of HPSE in HRE6-HER2/CAR-T effectively.HER2/CAR-T and HRE6-HER2/CAR-T could kill HER2+tumor cells SKBR3 specifically in vitro and release a large amount of IFN-y.In the presence of mimetic solid tumour hypoxia microenvironment and ECM,HRE6-HER2/CAR-T could infiltrate through the ECM and kill HER2+tumor cells SKBR3 specifically.Conclusions:In mimetic solid tumour microenvironment,HRE6-HER2/CAR-T could infiltrate through the ECM and kill HER2+tumor cells SKBR3 specifically.This study provided a new possibility for CAR-T immunotherapy in solid tumors,and accumulated a preliminary experimental data which lays the foundation for further study in vivo.