Afuco™ Anti-Human CDCP1 ADCC Recombinant Antibody (RG7287), ADCC Enhanced (CAT#: AFC-140CL)

Figure 1 CDCP1-Src co-transformation is prevented by RG7287 treatment.

5 × 10⁴ NIH3T3 cells were either infected singly with retroviruses (10⁵ virus particles) containing CDCP1 and Src or with both together. After 48 h the cells were expanded to a 10 cm plate and kept in 4% FCS containing medium and either left untreated or incubated with 5 μg/ml of RG7287 antibody (A) and with equivalent amounts of the Fab from RG7287 (B).After 3 weeks,cells were stained with crystal violet and the number of foci was counted.The foci that formed in three separate experiments were counted.The focus formation efficiency in each experiment was calculated by normalizing to the untreated Src and CDCP1 co-infected plates as 100%.The error bars indicate standard deviation.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 2 CDCP1 overexpressing MCF7 cells reach a predefined tumor volume faster than wild type MCF7 cells.

Subcutaneous tumors were generated by injecting 1×10⁷ cells into the flank of SCID beige mice, systemic estrogen was supplemented. The Kaplan–Meier curves compare the times to reach the pre-defined tumor volume of 1000 mm³ between animals with established CDCP1 overexpressing and parental MCF7 tumors (n = 9-10).The time to reach 1000 mm³, is significantly shorter in CDCP1-overexpressing MCF7 tumors (p = 0.0065) compared to wild type MCF7 tumors (33 days versus 42 days). RG7287 treatment of CDCP1-overexpressing MCF7 tumors reversed this feature back to that of the wild type (41 days; p = 0.034). Time to event was analyzed using the pair-wise log-rank test.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 3 Ligation with RG7287 antibody translocates CDCP1 to detergent insoluble fraction.

H322M cells were incubated with either 10 μg/mlof control IgG or (A) RG7287 antibody or (B) the Fab fragment of RG7287 for the indicated times. After lysis in 1% Triton X-100 buffer, the insoluble fraction was prepared and analyzed by Western blotting.The insoluble fraction was isolated and analyzed by Western blotting with antibodies against phospho-and total CDCP1,phospho- and total Src, the lipid raft marker protein flotillin.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 4 CDCP1 translocation to a detergent insoluble fraction is Src dependent.

Prior to a 15 min incubation with 10 μg/ml RG7287 antibody,H322M cells were pre-treated for 1 h with vehicle or the following compounds: Sino5 (4 μM),PP2,or PP3 (20 μM).The insoluble fraction was prepared and analyzed by Western blotting.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 5 RG7287 treatment downregulates CDCP1 in many different tumor cell lines.

(A) H322M, MDA-MB231,Kpl-4, HCT116, HT29,and SW620 cells were incubated with isotype control or RG7287 (10 μg/ml) for 5 h.The cells were lysed with RIPA buffer and analyzed by Western blotting. (B) H322M cells were treated for 1 h with either epoxomicin (1 μM) or chloroquine (1 μM), then 10 μg/ml of RG7287 or the isotype control was added and further incubated for 6 h before lysis in RIPA buffer and analysis by immunoblotting.(C)H322M cells were incubated with 20 μg human RG7287 or isotype control antibodies for the indicated times at 37 °C.Using Accutase the cells were removed from the plate. For each time point 1×106 cells were incubated with phycoerythrin (PE) labeled anti-CDCP1 antibody for 30 min on ice. The median fluorescence intensity (MFI) for the non-internalized control sample that was incubated only with the PE labeled antibody on ice was set to 100%.Histograms of each treatment time point are shown in addition.(D) 2×105 H322M cells were seeded on FCS coated glass coverslips. 10 μg/ml of either human isotype control or RG7287 antibody was added for 5 h.The cells were fixed with 4% PFA,permeabilized and stained for CDCP1 and LAMP1.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 6 Src activation is required for RG7287 induced CDCP1 downregulation.

Src activation is required for RG7287 induced CDCP1 downregulation. Src inhibitors Sino5 (4 μM) and PP2 and the control compound PP3 (20 μM) were added to H322M cells for 1 h prior to the addition of RG7287 antibody (10 μg/ml)for either 1 h or 5 h. The cells were lysed in RIPA buffer and analyzed by immunoblotting.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.

Figure 7 RG7287 inhibits tumor growth in subcutaneous xenograft models.

Established subcutaneous H322M (innoculated with matrigel),Kpl4,and MDA-MB231 (both innoculated without matrigel) tumors in SCID beige mice were treated once weekly (for 6, 3 and 7 weeks, respectively) with vehicle or 10 mg/kg of RG7287 antibody (25 mg/kg in case of Kpl4)and tumor volumes were measured with calipers.(A) The average tumor volume of vehicle and RG7287 treated H322M, Kpl4,and MDA-MB231 xenografts models (n = 10) is plotted over time with standard deviation.(B) Tumors were explanted, fixed in 3.8% buffered formaldehyde solution, and embedded in Paraplast.CDCP1 was detected by immunohistochemistry using the mouse monoclonal antibody MAB2666.(C)Explanted tumors from H322M were lysed in Triton X-100 lysis buffer. The tumor lysates were analyzed for CDCP1 levels and the loading control tubulin by Western blotting.

Kollmorgen,G.,Niederfellner,G.,Lifke,A.,Spohn,G.J.,Rieder,N.,Vega Harring, S.,& Bossenmaier,B.(2013).Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy. Molecular oncology, 7(6),1142-1151.