The Study Of Chimeric Antigen Receptor T Cells Targeting Mesothelin For The Treatment Of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma is one of gastrointestinal malignants characterized by its indiscernible symptoms at early stages,exceedingly poor prognosis,and dismal overall survival.Over the past three decades,scarce improvement has been made to lower the mortality of patients with pancreatic cancer,and the 5-year survial of them has been maintaining <5%.Hitherto,main clinical treatment strategies still confine to surgical ablation,chemotherapy and radiotherapy,of which,surgical resection is the only curable approach.Unfortunately,more than 80% pancreatic cancer patients have surgically unresectable disease,and chemotherapy and radiotherapy are principal therapies for them.As a consequence,it’s urgent to seek a novel approach for extending survival of these patients.Last several years,chimeric antigen receptor T(CAR T)cell technology has emerged as a hopeful immunotherapeutic candidate in cancer treatment,which has already shown its remarkable clinical utility in the treatment of hematologic malignancies,especially for CD19-CAR T.In contrast,however,the efficacy of CAR T therapy in solid tumors is not as ideal as treating blood malignancies,due to higher hetergenecity of tumor cells,complicated immunesuppressive microenvironment in tumor tissues,and poor infilteration of CAR T cells into tumors.In this study,we constructed a second generation CAR T targeting Region III of mesothelin(MSLN)using piggy Bac transposon-based plasmid transporation technique,and tested the efficacy in treating MSLN+ pancreatic cancer cells both in vitro and in vivo.(1)Meso CAR gene was cloned into carrier plasmid containing piggy Bac transposon for constructing p NB338B-MESO plasmid,which was then transferred into primary T cells.The recombinant MSLN(r MSLN)and α-CD28 antibody cocktail was used to activate and enrich meso CAR T cells after electroporation.The existence of meso CAR was verified by Western blot and RT-PCR.And results of FACS demonstrated that the average positive ratio of meso CAR T reached to >60%;(2)The activation markers of the meso CAR T cells were evaluated,and the results showed that the levels of CD25,CD69,CD137 and CD107α were all up-regulated compared to control T cells activated by α-CD3 antibody.Besides,meso CAR T cells grew significantly faster than the control T cells according to CCK8 results.After stimulation by r MSLN,higher levels of cytokines were detected in the medium of meso CAR T cells;(3)We screened a PANC1 expressing high MSLN and a ASPC1 expressing low MSLN,and RTCA was used to test cytotoxicity of meso CAR T cells to these two PDAC cell lines in vitro.Results demonstrated an MSLN-dependent cytotoxic effect of meso CAR T cells,Furthermore,results of tumor models in vitro suggested that meso CAR T cells possessed stronger ability in suppressing ASPC1 cells,which was selected to construct xenograft models;(4)Luciferase-expressing ASPC1 cells were subcutaneously inoculated into immuno-compromised NSG mice to establish PDAC xenograft mouse models.After treatment,meso CAR T triggers a constant attenuation of tumor burden,and eliminate visible tumors effectively by day 21.IFN-γ level in peripheral blood of meso CAR T group is higher than control T or vehicle group.And meso CAR T cells differtiate into more memory T cells after homing to the spleen in vivo.Moreover,H&E staining showes no difference between these groups,which implies no adverse effect of control T and meso CAR T cells on any of these major organs and indicates the safety of meso CAR T.Here,we constructed meso CAR T cells targeting Region III of MSLN.The meso CAR T cells exhibit excellent ability in proliferation,killing and cytokines secretion in vitro.Likewise,meso CAR T cells inhibit tumor growth in vivo as well,and cause no significant harm to normal organs.Our study provides data of mesoCAR T treating pancreatic cancer for clinical translation from bench to bedside.