(a) Profiling of a panel of pancreatic ductal adenocarcinoma cells by flow cytometry demonstrates that CDCP1 is highly expressed on PDAC cells. Remarkably, CDCP1 was expressed at much lower levels on non-tumorogenic cells derived from the same tissue origin. (b) A schematic representation of the antibody drug conjugate cell-killing assay. Cells were treated with a primary IgG that targets CDCP1 and a secondary anti-human IgG conjugated to the cytotoxic drug monomethyl auristatin F (MMAF). Cellular viability was quantified by CellTiter-glo after 72 hr incubation with antibody treatment. (c) Dose- dependent antibody drug conjugate-mediated cell killing was only observed in the HPAC tumorigenic cells and not in the non-tumorogenic HPNE cells (n = 3, error bars represent s.d.). (d) Sub-nanomolar treatment with a CDCP1 IgG was sufficient to selectively kill greater than 50% of HPAC cells, but only when in combination with a stoichiometric excess of the secondary antibody drug conjugate (n = 3, error bars represent s.d.). (e) Schematic of the experimental setup for the flow cytometry-based T-cell activation assay used to profile BiTE activity. Cells were incubated with HPAC or HPNE target cells in the presence or absence of antibody treatment. After overnight incubation, T-cell activation was quantified via the expression of an NFAT-dependent GFP reporter gene. (f) Jurkat cells were significantly activated when treated with 1 nM BiTE in the presence of HPAC target cells as compared to HPNE control cells. Importantly, treatment with the CDCP1 BiTE alone or with Fab lacking the CD3 targeting component resulted in no significant T-cell activation. (g) A schematic representation of the in vivo PET imaging study. 89Zr-labeled CDCP1 Fabs were used for PET imaging of PDAC xenograft bearing mice. (h) Representative microPET images of four immunocompromised nu/nu mice bearing cancer xenografts targeted with a 89Zr-labeled CDCP1 Fab. Images over time show the tumor specific expression of CDCP1, as well as the persistent binding of the Fab to the tumor over 24 hr (Left). Importantly, when the same Fab was heat denatured prior to injection or when a negative control xenograft was used, there was no observable uptake of the 89Zr-Fab (Middle and Right). Remarkably, no uptake was observed in the mouse treated with a sub-toxic dose of MEKi prior to imaging, demonstrating the coupling of CDCP1 expression MAPK pathway signaling in vivo. (i) Quantification of tumor specific bio-distribution of the CDCP1 89Zr-Fab in tumor-bearing nu/nu mice (n = 5 per treatment arm) confirms the trends observed by microPET imaging. Tumor localization of 89Zr-Fab was antigen dependent and ablated by specific inhibition of MAPK signaling.