Study On The In Vitro And In Vivo Antitumor Activities Of CAR-T Cells Targeting Lung Cancer Biomarker Mesothelin

The death toll of lung cancer is always ranking top among malignant tumors because of its highermorbidity.Over 80 percent of patients have lost the chance for radical surgery because of its hidden symptom.At the same time,lung cancer is a kind of solid tumor with low immunogenicity.Reduced expression of HLA-1 during the evolution of cancer cells leads to not being recognized by dendritic cells and tumor immune escape.Therefore,finding effective treatment is becoming a difficult problem to be solved.In recent years,especially in the study of hematological malignancies,many new breakthroughs and discovers appeared in tumor treatment by using adoptive cellular immunotherapy which induced by the genetic modification of T cells with chimeric antigen receptor.However,the study of chimeric antigen receptor-engineered T cell therapy in the field of solid tumor lags behind due to the limited antigens.Compared with other immunotherapies of tumor,the tumor recognition ability,T cell tumor specific cytotoxicity and the activation of T cell were higher in CAR-T therapy.With the development of recombinant DNA technology,as well as signaling pathway continue to be studied in depth,the signal domain of CAR has been developed from the single in the first generation to multiple structures containing costimulatory molecules such as CD28,CD137(4-1BB),CD134(OX40)and ICOS in the second and third generation.Recently,curative effects on clinical application of antitumor such as lung cancer are achieved by using CAR-T technology.The effective control to malignant tumors,such as leukemia,lymphoma and melanoma shows its therapeutic advantage.However,like other immunotherapies,there exist some problems and inadequacies in CAR-T technology.Off-target effect and insertion mutation are potential risks that should be taken into account.Therefore,more specific antigen,more safe and effective transduction technology should be found for CAR-T to defeat cancer.Tumor relative antigen mesothelin,a 71-KDa precursor protein that is processed to a mature 40-KDa glycosyl-phosphatidylinositol-anchored protein is high expressed in various tumors such as mesothelioma,NSCLC,oesophageal adenocarcinoma and metastatic triple-negative breast cancer,while with a low level in normal tissues.Studies show that mesothelin promoting an aggressive phenotype is strengthened by the clinical observations that patients with mesothelin-positive tumours have poor prognoses.Furthermore,limited expression of mesothelin in ‘normal' pleura,peritoneum,andpericardium provides a safety margin for CAR T-cell therapy.Mesothelin,as a target for immunotherapy,not only play a role in CAR T-cell therapy,but also in others.For example,immunotoxin SS1 P,monoclonal antibody MORAb-009 and antibody-drug conjugate MDX-1382 targets mesothelin to kill cancer.It has been proved that mesothelin is overexpressed in almost lung cancer range from 30% to 70%.Therefore,mesothelin is an excellent target for the tumor immunotherapy in the application of lung cancer clinic trial.Based on the research status of immunotherapy for lung cancer,because of its unique advantage,we detect the tumor cell killing mediated by chimeric antigen receptor-engineered T Cells targeting mesothelin.We utilized the second generation CARs to carry the modified anti-mesothelin antibody and costimulatory molecule 4-1BB.Subsequently,lentivirus was constructed to overexpress MSLN CAR on the surface of T cells.We tested the expression of MSLN CAR by flow cytometry assay.The assay of killing tumor cells by CAR-T was examined to evaluate the tumor antigen recognize ability.To further confirm,a cell line CHO-K1-Mesothelin was built to let mesothelin expressed on the surface of CHO cells.Both Hela cells and CHO-K1-Mesothelin cells were significantly killed by CAR-T.Finally,this effect was proved in mouse PDX model,a common lung cancer model.The change of tumor volume acted as an indicator to evaluate the tumor killing ability in vivo.It was found that the tumor volume in the CAR-T group was significantly smaller than control T cell group within 10 days of cell reinfusion.These results,both in vitro and in vivo,indicate that mesothelin is an efficient target for tumor immunotherapy.In conclusion,through this experiment,we defined that the CAR-T who can target the tumor specific antigen mesothelin has the ability to kill tumor cells overexpressing mesothelin.At the same time,this phenomenon was also observed in mouse PDX model reflected in reduced lung cancer volume.These preclinical studies,both in vitro and in vivo,will provide a theoretical basis for CAR-T therapy to cure mesothelin-overexpressing lung cancer.