Functional Investigation Of Genetic Engineered T Cells Specifically Targeting Triple Negative Breast Cancer

Triple-negative breast cancer（TNBC）is defined as tumors that lack three specific receptors,accounting for 12%¹7%of cases in breast cancers.Additional targets are urgently and strongly sought after in therapy for TNBC.Recent successes of CD19-chiemeric antigen-receptor（CAR）T-based immunotherapy in patients with refractory hematologic malignancies raise the possibility of implementing similar strategies for TNBC.Mesothelin is overexpressed in the majority of TNBC cells.PD-1plays an important role in T cell exhaustion.We hypothesized that TNBC expressing Mesothelin could be a target through Meso-CAR T immunotherapy,coupled with PD-1 blockade to further boost the CAR T’s anti-tumor effects.Due to get Meso-CAR T cell,we constructed Meso-CAR expressing lentivectors to infect human peripheral blood-derived T cells.Several human breast cancer cell lines were analyzed for expression of Mesothelin by Flow cytometry,demonstating that BT549 strongly expressed Mesothelin compared with others.T cell function assays in vitro such as intrecellular cytokine staining,cytotoxic killing,ELISA and CD107a staining were performed to evaluate the engineered T cell.Meso-CAR T cell produced elevated levels of cytokines（IL-2 and IFN-γ）upon coculturing with BT549^Meso+（IL-2:0.55%by ICS,75pg/mL by ELISA;IFN-γ:2.15%by ICS,350pg/mL by ELISA）and Hela^Meso+cells（IL-2:2.1%by ICS,350pg/mL by ELISA;IFN-γ:3.15%by ICS,1100pg/mL by ELISA）,and enhanced target cytotoxicity（E:T=2:1,74%）,demonstrating the Meso-CAR T cells’antigen specificity and potency.Meanwhile,we also determined the feasibility of CRISPR mediatedPD-1 knockout in primary T cells.The plentiCRISPR constructs were built and the lentiviruses were transduced into primary CD4⁺T cells individually.The expression level of PD-1 is usually associated with T cell’s activation status,and CD25 is co-expressed with PD-1 during T cell activation.The efficacy of the gene deletion strategy by Cas9 was confirmed by decreased PD-1-high expressing populations with different efficiencies（PD-1-2:19%,PD-1-6:29%）.These results demonstrate the feasibility to knockout PD1 by CRISPR/Cas9 in primary T cellsAdoptive T cell therapy with CARs has been a focus of intense interests in immunotherapy.Further studies will help develop better strategies more applicable to solid tumors.Our research focused on Meso-CAR T and PD-1 blockade as a part of the overall effort to determine the feasibility of this strategy for treating TNBC patients.