Fig 2: Systematic characterization of the functional importance of the KRAS surfaceome.(a) Experimental strategy for the CRISPRi surfaceome screen. MCF10A Empty Vector or MCF10A KRASG12V cells were transduced with a lentiviral surfaceome sgRNA library containing five guides each for 1605 transmembrane targets. After being cultured for an extended growth period, the abundance of each sgRNA barcode was quantified by deep sequencing. (b) Volcano plots of the CRISPRi experiment showing effects on growth rate (gene phenotype,X-axis) versus –log10(p-value)s (Y-axis). Proteins with a p-value less than 0.05 and a gene phenotype >1 (blue) or <-1 (red) were considered significant. (c) Shown is a representation of CRISPRi phenotypes for RAS pathway control genes (Left), functionally categorized KRASG12V synthetic lethal genes including integrin signaling genes (Middle), and Wnt Signaling genes (Right). KRASG12V synthetic lethal genes were defined as having a p-value<0.025 and a phenotype >0.8 for KRASG12V cells and <0.8 for Empty Vector cells. (d) A Venn diagram comparing KRASG12V synthetic lethal genes and differentially expressed genes. (e) Competitive growth assays performed with KRASG12V cells transduced with the indicated CDCP1 and negative control sgRNAs. Cells were grown in monoculture with growth factors to ensure they maintained sgRNA expression (Left), and in co-culture with cells lacking sgRNA expression without growth factor to determine relative growth rates (Right). (n = 3, error bars represent s.d.).

Fig 3: Systematic approaches to demonstrate the utility of the recombinant antibodies to target and detect RAS signaling in pancreatic ductal adenocarcinoma (PDAC).(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.

Fig 4: CDCP1 complexes are similar between breast and pancreatic cancer cell lines and are stable in a variety of detergents. a MDA-MB-231 or T47D cells were transduced with the indicated adenoviral vectors, CDCP1-containing complexes were isolated with anti-FLAG agarose, and the presence of components of adherens and desmosomal junctions was assessed by immunoblot analysis. b This study was carried out similarly to the study in Fig. 6a, except that the pancreatic cancer cell lines AsPC1 or PANC1 were used. c AsPC1 cells were transduced with control (GFP) or ΔCDCP1 adenoviruses, the cells were extracted with the indicated detergents, and ΔCDCP1-associated proteins were examined by immunoblot (left panel) or total protein levels were assessed by Coomassie staining of the same samples resolved by SDS-PAGE (right panel)

Fig 5: Characterization of E-cadherin/CDCP1-containing protein complexes. a MDA-MB-231 or T47D cell lines stably expressing an E-cadherin-glutathione S-transferase (E-cad-GST) fusion protein were transduced with an adenovirus encoding ΔCDCP1-FLAG or a control adenovirus encoding GFP. Cell extracts were subjected to sequential affinity purification using anti-FLAG agarose followed by glutathione-agarose. b T47D cell extracts were immunoprecipitated with a CDCP1 antibody and the immunoprecipitates were analyzed by immunoblot with the indicated antibodies. c The indicated stable cell lines were transduced with adenoviruses encoding GFP (control) or ΔCDCP1-FLAG and cell extracts were subjected to affinity purification with either anti-FLAG agarose or glutathione agarose. Affinity-purified proteins were analyzed by immunoblot. d HEK 293 cells were transiently transfected with the indicated tagged CDCP1 constructs and treated with the specified DDAs or vehicle. CDCP1 dimerization was monitored by TALON pulldown of His6-tagged proteins followed by purification with anti-FLAG agarose and immunoblot analysis of the sequentially affinity-purified material. e HEK 293 cells were transiently transfected with the indicated plasmid constructs and cell extracts were affinity purified sequentially using TALON resin and anti-FLAG agarose. The affinity-purified material was analyzed by immunoblot