The Construction Of T Cells Expressing Bifunctional Antibody Anti-HER2-anti-CD3 And Its Antitumour Research

Cancer is a serious diseases threatening human life.According to the statistics reported by International Cancer Research Center,more than 14 million cancers were diagnosed and approximately 8.2 million died due to cancer annually in the word.The incidence of cancer has been increasing in recent decades.Human epidermal growth factor 2(HER2)is overexpressed in malignant cancers such as breast cancer,gastric cancer,lung cancer,etc.It plays an important role in the development and progression of some cancers.So HER2 has become an important biomarker and target of therapy in recent years.Genetically engineered T cells,such as chimeric antigen receptor T cells(CAR-T),is a promising precision cell therapy technology.The bispecific antibody,called as bispecific engager(BiTE),which interacts with both cancer antigen and T cell surface receptor to activate T cell,is a precision protein treatment.Both technologies have been achieving great successes in clinic.However,CAR-T cells do not redirect the vast reservoir of resident T cells to tumors,limiting its potential benefits.BiTE can redirect resident T cells to tumors through bridging them,but the short half-life,no bio-distribution,no self-amplify necessitates continuous infusion.In our study,T cells were modified by recombinant lentivirus expressing anti-HER2-anti-CD3 BiTE,designated as HER2-ENG T cell.So the advantages of BiTE and engineered T cell were integrated into a single cell therapy technology.This HER2-ENG T cell can amplify itself and secrete BiTE to lysis HER2 positive tumor.The process and results of the study are demonstrated as follows:1.Construct recombinant virus via homologous recombinant cloning:The PCR product containing anti-HER2-anti-CD3 BiTE gene and a 15 bp extensions homologous to vector at both ends was fused with the linearized pCDH-MCS-EF1-GFP vector in the presence of recombinant enzyme.The resultant plasmid of LV-anti-HER2-anti-CD3 BiTE was sequencing verified and transfected to 293T cells with helper plasmid to generate recombinant lentivirus.The concentration of purified recombinant lentivirus was titrated to 2×108 pfU/mL.2.Construction of HER2-ENG T cell expressing and secreting anti-HER2-anti-CD3.The activated T cell,which original from PBMC stimulated with anti-CD3 and anti-CD28,was transduced with LV-anti-HER2-anti-CD3,which harboring a GFP expressing cassette and the GFP fluorescence can be used to identify and sort T cells with the recombinant virus.Flow-cytometry assay revealed that 28±1.2%(n=3)of T cells was GFP positive after transduced with recombinant virus,and the BiTE mRNA were detected in those T cells by RT-PCR,T cells transduced with GFP lentivirus carry vector were used as a control to study the activity of HER2-ENG T cells.3.HER2-ENG T cells and target cancer cells were co-cultured to evaluate the ability of the HER2-ENG T cells in the lysis of HER2-positive cancer cells in vitro.The co-culture experiments demonstrated that HER2-positive cancer cells stimulated HER2-ENG T to produce high level of IL-2 and IFN-? and caused cytotoxicity specifically in the presence of the HER2-positive cells,such as MCF-7,HER-negative cell MDA-MB-231 did not stimulate HER2-ENG T to kill cancer cells.Moreover,the cultured medium of HER2-ENG T cells facilitated PBMC to kill MCF-7,and the medium of HER2-positive cancer cells stimulated HER2-ENG T was better in facilitating PBMC to kill MCF-7.The results suggested that the HER2-ENG T secreted anti-HER2-anti-CD3 can activated PBMC in the presence of HER2-positive cancer cells.4.The in Vivo antitumor activity of the HER2-ENG T cells.MCF-7 xenograft model was constructed in nude mice.Following subcutaneous injection of 4 × 106 MCF-7 to 4-week-old female nude mice in the right armpit,the formation of micro-tumor was observed after 1 week.Then,the mice were injected with 1 × 107 HER2-ENG T cells(n=5)in the tail vein,and the tumor volume was measured after 4 weeks of cancer inoculation.The tumor size of experimental group which treated with HER2-ENG T cells was smaller than PBS control group and NT(non-transduced T)control group.The results indicated that the significant antitumor activity of the HER2-ENG T cell can be applied to fight against cancer.