Inhibition of EGF-induced phosphorylation of ERK1/2 and EGFR by Adnectins was determined using appropriate antibodies26 using an Odyssey? Infrared Imaging Program (LI-COR?). and inhibited down-stream cell signaling and proliferation of individual cancers cell lines (A431, H292, BxPC3 and RH41) with IC50 beliefs which range from 0.1 to 113 nM. Although Adnectins destined to EGFR at a niche site specific from those of anti-EGFR antibodies cetuximab, nimotuzumab and panitumumab, just like the antibodies, the anti-EGFR Adnectins obstructed the binding of RIPK1-IN-3 EGF to EGFR. PEGylated EI-Tandem inhibited the development of both IGF-IR and EGFR powered individual tumor xenografts, induced degradation of EGFR and decreased EGFR phosphorylation in tumors. These total results demonstrate effective engineering of bispecific Adnectins with high potency and preferred specificity. The bispecificity may improve natural activity in comparison to monospecific biologics as tumor development is powered by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy. and expressed as Adnectin proteins. A subset of target binders that blocked EGFR or IGF-IR signaling and had suitable RIPK1-IN-3 biophysical properties were identified (Table 1). These initial clones were optimized for target binding affinity and cellular potency with additional mRNA selection at increasingly lower target concentrations and selection for lower dissociation rate constants. EGFR Adnectins were tested by In-Cell western screening assays for the blockade of phosphorylation of EGFR and ERK, a downstream signaling molecule of EGFR activation. IC50 values obtained during the selection procedures ranged from 9 RIPK1-IN-3 to 250 nM, providing the opportunity for choosing molecules from a wide range of potency values for the construction of bispecific Adnectins. Analogous studies were performed on optimized IGF-IR binders. Optimized EGFR-binding clones (E#1, E#2, E#3 and E#4) inhibited EGFR phosphorylation on Y1068 and downstream phosphorylation of ERK on Y204 of p42/p44 in vitro with IC50 values ranging from 7 to 40 nM, potencies that were more than 100-fold higher than the parental lead clone (Table 1). Table 1 Properties of monospecific and bispecific adnectins HMS174(DE3) or BL212(DE3) (EMD Biosciences) and cells were inoculated MAPK10 in LB medium containing 50 g/mL kanamycin. Expression cultures were grown in Studier ONE auto-induction medium (Novagen/EMD Biosciences?). The clarified lysate was purified using a Ni-NTA Superflow matrix column (Qiagen?) with standard washing and elution methods.23 To allow for PEGylation, the protein was modified near the C-terminus by a serine to cysteine mutation. PEGylation of the protein was accomplished by incubating maleimide-derivatized PEG reagent with the protein solution.24 Progress and confirmation of the PEGylation reaction was confirmed by SDS-PAGE and SEC. Separation of reaction products from precursors was accomplished by ion exchange chromatography. Lipo-polysaccharides were depleted during this process and further removed with SartoBind Q charged membranes (Sartorius AG). Endotoxin levels were determined using the EndoSafe PTS LAL assay (Charles River Laboratories?). The average yield of this purified, PEGylated EI-Tandem from three different preparations was 230 mg per liter of em E. coli /em . Competitive ligand-binding assays. To measure inhibition of EGF binding, A431 cells were plated at 15,000 cells/well in 96-well plates and incubated for 48 h. Cells were washed and incubated with Adnectins diluted in starvation media (DMEM + 0.1% BSA) for 30 min at 37C. Europium (Eu)-labeled EGF (PerkinElmer?) was added at 10 nM and plates were incubated for 3 h at 4C in the dark. After washing, Enhancement solution (PerkinElmer?) was added to the plates and incubated for 1 h at 37C, and the plates were read on the Flexstation II (Molecular Devices?). IC50 values were calculated with Softmax plus software. To measure inhibition of IGF-I binding, MCF-7 cells were plated at 50,000 cells/well in 24-well plates in RPMI 1640 with 10% FBS. After 24 h, cells were washed with RPMI 1640 containing 0.1% BSA and pre-incubated for 30 min on ice with 200 L binding buffer and IGF-IR Adnectin. [125I]-IGF-I, (40 pM 60,000 cpm) from Perkin Elmer, was added and incubated for 3 h on ice. Cells were then washed with PBS containing 0.1% BSA and lysed with 500 L of 0.1% SDS + 0.5 N NaOH. Radioactivity of the lysates was measured using a Wallac 1470 Gamma Counter (Perkin Elmer?) and the data analyzed using SigmaPlot? (Systat?). In-cell western assay. A431 cells were seeded into poly-D-lysine coated 96-well microtiter plates (Becton Dickinson?). Inhibition of EGF-induced phosphorylation of EGFR and ERK1/2 by Adnectins was RIPK1-IN-3 determined using appropriate antibodies26 using an Odyssey? Infrared Imaging System (LI-COR?). Each clone was assayed in duplicate or triplicate and values were normalized to -actin or total ERK1/2 levels. IC50 values were calculated from linear regression analysis of percent inhibition of maximum signal minus background. Size exclusion chromatography and LC-MS. SEC was performed on the purified proteins using a Superdex 200, 5/150 column with UV detection at 214 nm and 280 nm and with fluorescence detection (excitation = 280 nm, emission RIPK1-IN-3 = 350 nm). Adnectins were further analyzed by LC-MS liquid chromatography.