Treatment Of Prostate Cancer With CAR-Engineered T Cells Targeted PSCA:A Preclinical Study

Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer-related male death in western world,and is becoming a common cancer increasing in China.The efficiency of early detection of prostate cancer has increased dramatically with a serum test for prostate-specific antigen(PSA).But there is no clear evidence that screening for prostate cancer decreases the risk of death from prostate cancer.Although locally confined disease is treatable,recurrent and metastasized prostate cancer is essentially incurable,including androgen ablation therapy,radiotherapy,chemotherapy with little effect.Recently immunotherapy is an appealing approach to cancer treatment.We hope to improve the treatment effect of prostate cancer with cell immunotherapy.The successful treatment of refractory hematological malignancies with adoptive transfer of chimeric antigen receptor(CAR)-engineered T cells may pave the road to future treatment of solid tumors,using similar approaches.Carefully choosing the target antigen is of outmost importance.As prostate cancer expresses many antigens with limited or no expression in other tissues,these tissue-restricted antigens constitute potential targets for CAR T cell therapy.Early work showed the PSMA and PSCA as the target antigens for CAR T cell therapy with little effect.Our research designed a novel third generation CAR-engineered T cells targeted PSCA,choosing minicircle DNA vector which was a kind of nonviral plasmid as transfection plasmid,by means of nuclefection to make new CAR T cells.Investigate the functions and expression of the new CAR T cells at the same time.To check the PSCA-CAR T cells killing function,the directly method was in vitro killing assay.Target cell lines RT4 and PC-3M were co-cultured with PSCA-CAR T cells in various effector to target cell ratios in the second part experiment.The cytokines secrected levels of IL-2 and IFN-γwere tested meanwhile.The results of in vitro assays were the basis of in vivo,but the difference between in vitro and in vivo might affect the function of PSCA-CAR T cells.So we build humanized mouse models with the NOD/SCID mouse to simulate the microenvironment of tumor patients,and made prostate cancer grafted mouse models with the treatment of PSCA-CAR T cells in the third part experiment.We observed the tumor volumes,tested the expression levels of PSCA-CAR T cells in vivo,and detected the cytokines secrected levels of IL-2 and IFN-γto evaluate the antitumor effect in vivo.Part 1 The experiment on established the third generation chimeric antigen receptor PSCA scFv-CD28-CD137-CD3ζand transfected CAR T cells.Objective:To establish the third generation chimeric antigen receptor PSCA scFv-CD28-CD137-CD3ζ(PSCA-CAR),and tranfer the purpose gene to minicircle DNA vector,then transfected T cells and tested the functions of CAR T cells.Methods:Establish the third generation chimeric antigen receptor PSCA-CAR through whole gene synthesis and put the purpose gene in the pUC57 vecter.The purpose gene PSCA-CAR was transfered to the parental minicircle plasmid by gene engineering technology,then extracted the minicircle DNA from the parental minicircle plasmid.The T cells were transfected by the mincircle DNA vector with the method of nuclefection,to get the PSCA-CAR T cells.The transfection efficiency,the influence of nuclefection to T cell viability,the affect of minnicircle DNA and nuclefection to the expression of T cells molecular pHenotypic were all observed.The expression rates of PSCA-CAR in the T cells membrane were checked by flow cytometry.The PSCA-CAR protein in the T cells was detected by western blot.Results:1 Successfully transferred the PSCA-CAR to the parental minicircle plasmid by gene engineering technology,and extracted the minicircle DNA from the parental minicircle plasmid.There was no gene mutation in the PSCA-CAR with the test of gene sequencing.2 The T cells were successfully transfected by mincircle DNA with the method of nuclefection,and got the PSCA-CAR T cells,transfection efficiency was about 58%,T cell viability was approximately 60.2%.3 The PSCA-CAR T cells proliferation were influenced by the nuclefection temporarily,but the proliferation activity could recover when the cells were cultured with cytokines.4 There were no affects of minnicircle DNA and nuclefection to the expression of the PSCA-CAR T cells molecular pHenotypic.5 The expression of PSCA-CAR in the T cells membrane by flow cytometry was up to 88.96%.6 The PSCA-CAR protein was successfully detected in the PSCA-CAR T cells by western blot.Part 2 The experiment on antitumor effect of PSCA-CAR T cells in vitro.Objective:To observe the antitumor effect of PSCA-CAR T cells in vitro.Methods:Target cell lines RT4(PSCA+)and PC-3M(PSCA-)were co-cultured with PSCA-CAR T cells in various effector to target cell ratios.The killing rates were tested by CCK-8 kit.Quantitative detection of IL-2 and IFN-γlevels in culture medium were measured using ELISA kit.Results:1 There were high killing rates when RT4(PSCA+)were co-cultured with PSCA-CAR T cells.There were no obvious differences of the killing rates between PSCA-CAR T cells and normal T cells when co-cultured with PC-3M(PSCA-).2 The RT4(PSCA+)could markedly stimulate the PSCA-CAR T cells to secret a large amount of IL-2 and IFN-γ,however,the PC-3M(PSCA-)just a little.There were significant differences between RT4(PSCA+)and PC-3M(PSCA-).Part 3 The experiment on antitumor effect of PSCA-CAR T cells in vivo.Objective:To investigate the antitumor effect of PSCA-CAR T cells to the prostate cancer graft mouse in vivo.Methods:To build humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripHeral blood mononuclear cells.Then the expression of human CD3~+T cells and human CD19~+B cells in mouse peripHeral blood were checked by flow cytometry and the human specific ALU gene in the mouse bone marrow cells were decected using PCR technology.Make prostate cancer grafted mouse models when they were humanized,and divided into three groups randomly,with twice treatment of normal saline,normal T cells,PSCA-CAR T cells respectively.We observed the tumor volumes,tested the expression levels of PSCA-CAR T cells in vivo,and detected the cytokines levels of IL-2 and IFN-γ.Tumor tissues were fixed to take pathological examination and marked the PSCA and human CD3 by immunohistochemical.Results:1 The expression levels of human CD3~+T cells and human CD19~+B cells in mouse peripHeral blood tested by flow cytometry were the same as the normal adult.The human specific ALU gene in the mouse bone marrow cells were decected using PCR technology successfully.2 Compared with NS group and normal T cells group,the tumor volumes significant decreased in the PSCA-CAR T cells group.3 The expression of PSCA-CAR T cells were still checked in mouse peripHeral blood after treatment four weeks in the PSCA-CAR T cells group.The IL-2 and IFN-γlevels in the serum measured by ELISA in this group were higher than the other groups.There were significant statistical differences.4 The immunohistochemical results implied PSCA strongly expressed in the prosate tumor tissues in each groups.There was no expression of human CD3 in the tumor tissues in the NS group,and a little expression in the normal T cells group,notably expression in the PSCA-CAR T cells group.Conclusion:1The minicircle DNA vecter carried the PSCA-CAR could effectively transfect human T cells by means of nuclefection to make PSCA-CAR T cells,and a high level expression of PSCA-CAR in the T cells.2 PSCA-CAR T cells could be actived depending on the antigen.PSCA could stimulate PSCA-CAR T cells to secret a large amount of IL-2 and IFN-γin vitro,with strongly resisted tumor cells.3 Humanized mouse models with the NOD/SCID mouse by the means of tail vein injection of human peripHeral blood mononuclear cells were built successfully.Prostate cancer grafted mouse models could be made using the humanized NOD/SCID mouse.PSCA-CAR T cells could effectively migrate to the tumor tissues and played antitumor effect in vivo,which obviously decreased tumor volumes.