Fab32652p

CAR Mesothelin surface expression on human T-cells was assessed using primary antibody goat anti-human IgG F (ab’)2 Biotin (BioRad, Hercules, CA) along with secondary conjugate Streptavidin APC (BioRad). The following antibodies were also used in FACS buffer in this study: anti-PD1-PE (Biolegends 329906), anti-CCR7-Alexa-fluor 647 (BD 560921), anti-CD8-FITC (BC A07756), anti-CD8-BV421 (BD 562428), anti-HLA-A2-FITC (BD 551,285), anti-CD86-PerCp-Cy5.5 (BD 561129), anti-CLDN6-Dylight 650 (IMAB027), anti-idiotype-IMAB027- Alexa-fluor 647, anti-TIM3-APC (Biolegends 345011) and 7AAD (BC A07704). Mesothelin expression was detected using the PE conjugated anti mesothelin antibody (R&D system FAB32652P). Acquisition and analysis of all samples were performed on a BD FACS CantoI/II (BD Biosciences, San Jose, California) and FlowJo software (v7.6.1).

Human pancreatic tumor cell lines HPAC and Capan-2 (American Type Culture Collection) were cultured in RPMI 1640 medium (Invitrogen) with 10% heat-inactivated fetal calf serum (Bodinco BV) at 37°C in a fully humidified atmosphere containing 5% CO₂. Short tandem repeat (STR) profiling (BaseClear) affirmed identity. Cell lines were quarantined until screening for microbial contamination and mycoplasma had been performed; these tests were negative. In all experiments mice were inoculated subcutaneously with 5 × 10⁶ HPAC or Capan-2 cells. For analysis of receptor expression, cells were incubated with anti-human mesothelin phycoerythrin (catalog number: FAB32652P, R&D systems). Human lung adenocarcinoma epithelial cell line H441 (American Type Culture Collection) served as negative control. Membrane receptor expression was analyzed using flow cytometry (FACSCalibur, BD Biosciences) with Winlist software 6.0 (Verity Software House).