Egfr fc

The peptide mimetic, P28, was synthesized using Liberty Automated Microwave Peptide Synthesizer from CEM Corporation. Purification (>95%) was performed through High Performance Liquid Chromatography using a reverse phase column. For disulfide bond formation, the peptide sample, P28, was dissolved in 0.01M ammonium bicarbonate buffer (pH 8) at a concentration of 0.1 mg/mL and the solution was left to stir in open atmosphere. The progress of the reaction was monitored by analytical HPLC (peak shifted after disulfide bond formation). After the reaction was complete, the peptide solution was purified and checked for mass (loss of 2 protons) using mass spectrometry.
The binding kinetics of growth factor and peptides were measured on a Biacore 3000 instrument (GE, New Jersey) using HBS-EP as running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.15% surfactant P20). EGFR-Fc (R&D) was immobilized to CM5 sensor chip surface using standard amine-coupling chemistry by injecting the following reagents (45 µL) at a flow rate of 5 μL/min: 0.05M N-hydroxysuccinimide, 0.2M N,N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide mixture, EGFR-Fc (10 μg/mL in 10 mM NaAc, pH 5.0), and 1M ethanolamine-HCl (pH 8.5).

The human EGFR-Fc (EGFR-Fc, R&D Systems, cat# 344-ER) or human PD-L1-Fc (PD-L1-Fc, Peprotech, cat# 310–35) chimeras were immobilized (2.5 µg/ml in PBS) on Maxisorp 96-well plates (NUNC Brand Products, cat# 44240) overnight at 4°C. After washing and blocking, conditioned media or purified protein solution (1 µg/ml) was added and incubated for 1 hour at room temperature. The wells were washed and HRP-conjugated anti-poly Histidine (Sigma-Aldrich, cat# A7058), HRP-conjugated anti-FLAG (M2 clone, Sigma-Aldrich, cat# A8592), mouse anti-Myc (clone 9E10, Millipore, cat# 05–419) or HRP-conjugated goat anti-human IgG (GAH) (Sigma-Aldrich, cat# A0170) were added (1 µg/ml). After washing, in the case of mouse anti-Myc, GAM-HRP (1:2,000 dilution) (Jackson ImmunoResearch, cat# 115-085-166) was added for 1 hour at room temperature. Finally, after washing, the plate was developed using 100 μl 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma-Aldrich, cat# T0440) and stopped by 100 μl of 1 N H₂SO₄. Absorbance was read at 450–620 nm using Multiskan FC photometer (Thermo Scientific).

Extracellular domains of EGFR fused with Fc domains of human antibodies (EGFR-Fc, R&D Systems, Inc, USA) were coated overnight at 4 °C onto Nunc MaxiSorp plates (Thermo Fisher Scientific) at a concentration of 2 μg mL^–1. Wells were washed three times with phosphate buffered saline (PBS) and blocked with 2% skimmed milk in PBS for 1 hour. Wells were emptied and incubated with C1C2-nanobodies diluted in 2% skimmed milk in PBS for 1 hour. Plates were washed three times with PBS containing 0.05% v/v Tween20 (PBS-T) and incubated with mouse anti-Myc antibodies (9E10, 1 : 2000) for 1 hour. After washing with PBS-T, plates were incubated with rabbit-anti-mouse HRP (DAKO, P0260, 1 : 1000) for 1 hour. All protein and antibody incubations were performed in triplicates in 2% skimmed milk in PBS at room temperature while shaking. Plates were washed with PBS-T and stained with 100 μL TMB substrate solution (Thermo Fisher Scientific). Reactions were quenched by addition of 50 μL 1 M H₂SO₄, and absorbance at 450 nm was measured using a SpectraMax M2e microplate reader (Molecular Devices, UK). Absorbance values were normalized to control wells to which no C1C2 nanobodies were added. Data were plotted in Graphpad Prism 6 software (Graphpad Software, Inc, USA) and non-linear regression was performed using the ‘one site-specific binding’ option to calculate dissociation constants (K_d).