EF1-MSLN CAR vector and pPACKH1 packaging vectors (System Biosciences, California, USA) were transfected on HEK293T cells

EF1-MSLN CAR vector and pPACKH1 packaging vectors (System Biosciences, California, USA) were transfected on HEK293T cells. cell eradication. Our results demonstrated the anti-tumor efficacy of MSLN CAR T cell therapy against pancreatic cancer, suggesting its therapeutic potential. as well as in an pancreatic orthotopic mouse model, to assess the efficacy of CAR T cell treatment strategies. Especially orthotopic pancreatic cancer models demonstrated therapeutic significance of the biologically effective trafficking and therapeutic efficacy. We suggest MSLN CAR T cells can be the promising potential adoptive immunotherapy for the patients with MSLN expressing PDAC . Materials and methods Cell lines and cell culture MIA PaCa-2, AsPC-1, HeLa, OVCAR-3, and HEK293T cell lines were purchased from the American Type Culture Collection (American Type Culture Collection, Manassas, United States). Mesothelin-overexpressing NCR1 MIA PaCa-2 (MIA PaCa-2/MSLN) cell line was obtained from the Mogam Biotechnology Institute (Yongin, Korea). Firefly luciferase (Fluc) or MSLN Fluc vectors were designed, cloned, and packaged into lentivirus. Lentiviral transduction was performed in MIA PaCa-2 and AsPC-1 cells. For the cell culture method, please refer to Supplementary Material. Animals Six-week old female NOG WHI-P97 mice (NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic) were purchased from the Central Institute for Experimental Animals (CIEA) (Kawasaki, Japan). The mice were housed and maintained in specific pathogen-free conditions in facilities according to the institutional guidelines for animal care. All animals were cared for and treated humanely according to the guidelines for the WHI-P97 welfare and use of animals in cancer research, and experimental procedures were approved by the Animal Care and Use Committee of Woojung Bio (IACUC2019-4-31). Expression of MSLN scFv The MSLN-specific IgG was obtained from the Mogam Biotechnology Institute. For full details on MSLN-specific IgG, please refer to the patent [21]. The scFv-encoding genes were amplified using PCR and fused into the pET-22b(+) WHI-P97 vector via an in-fusion reaction. The pET-22b(+) vector carrying the scFv-encoding gene was transformed into BL21 strain. Expressed scFv was purified from the culture broth using TALON metal affinity resin (Takara, Otsu, Japan). Analysis WHI-P97 the binding activity of scFv to MSLN For the analysis of the binding activity of scFv to the recombinant MSLN, the sandwich ELISA was performed using purified scFv coated plates and anti-MSLN biotinylated-antibody (R&D Systems, Minneapolis, MN, USA). For the analysis of scFv binding activity to MSLN on the cells, MSLN-bearing cells were treated with 6x His-tagged anti-MSLN scFv. After subsequent incubation with PE-anti-6x His Tag (BioLegend, San Diego, USA), flow cytometry analysis was performed. For more detail information, please refer to Supplementary Materials. CAR expressing lentivirus production MSLN-CAR constructs included the signal peptide for human CD8, scFv, and the cytoplasmic domain of human CD3 zeta, as well as CD8 or CD28 hinge domains and CD28 or 4-1BB costimulatory domains. All MSLN CAR constructs were incorporated into either the HIV-based lentiviral vector pCDH-MSCV-MCS-EF1-copGFP or pLVX-EF1-IRES-Puro using the In-Fusion HD cloning kit (Clontech, Mountainview, CA, USA). EF1-MSLN CAR vector and pPACKH1 packaging vectors (System Biosciences, California, USA) were transfected on HEK293T cells. Produced lentivirus was quantified by serial dilution and transduction to HEK293T cells. For more detail information, please refer to Supplementary Materials. CAR T cell production Human peripheral blood mononuclear cells (PBMCs) were obtained WHI-P97 from healthy donors under the approval of Public Institutional Bioethics Committee designated by the MOHW (Approval No. P01-201805-31-001). Enriched CD4 or CD8 positive T cells were activated with Transact (Miltenyi Biotec, Bergisch Gladbach, Germany) and transduced with MSLN.