Figure 1.
Binding specificity of Y-142 to EGFR ligands and to different species of sHB-EGF.
(A) The binding activity of Y-142 to EGFR ligands by ELISA. The various concentrations of Y-142 were incubated in an EGFR ligand-immobilized plate. The binding was then detected with HRP-labeled anti-mouse IgG antibody. Data points represent the mean ± standard deviation (SD) of values acquired in duplicate. (B) The binding activity of Y-142 to human, mouse, and rat HB-EGF by ELISA. The binding activity to different species HB-EGF was measured by ELISA using an electroluminescence-based technology. The various concentrations of Y-142 were incubated in a sHB-EGF-immobilized plate. The binding was detected with sulfo-tagged anti-mouse IgG antibody. Data points represent the mean ± SD of values acquired in duplicate. (C) Amino acid alignment of the EGF-like domain of EGFR ligands. A dot indicates an amino acid different than HB-EGF. A dash represents a gap. Arrowheads labeled with a number indicate the Y-142 binding epitopes identified in Fig. 6. (D) Amino acid alignment of the EGF-like domain of human, mouse, and rat HB-EGF. A dot indicates an amino acid identical to human HB-EGF. Arrowheads labeled with a number indicate the Y-142 binding epitopes identified in Fig. 6.
Figure 2.
Measuring the KD of the Y-142/HB-EGF complex.
Dual-curve KinExA equilibrium titration of sHB-EGF binding to Y-142. KD-controlled data (bottom fitted curve) were acquired by equilibrating sHB-EGF at a concentration range of 4.04 fM–207 pM with 1.03 pM Y-142 binding sites. Antibody-controlled data (top fitted curve) were acquired by equilibrating sHB-EGF at a concentration range of 4.67 fM–239 pM with 35.6 pM Y-142 binding sites. All data points were acquired in duplicate. Both curves were simultaneously fit to a standard positive cooperativity equilibrium model, yielding an effective KD = 1.50 pM (0.31) where the number in parentheses is the 95% confidence interval of the fit, and a Hill coefficient n = 1.68.
Figure 3.
Neutralizing activities of Y-142 against sHB-EGF and ARG signaling.
(A) Inhibitory activity of Y-142 to sHB-EGF binding to EGFR. EGFR-hFc was incubated in an anti-human IgG Fc antibody-coated plate. Y-142 was then incubated at a concentration of 6.7 nM in the presence of 0.63 nM biotinylated sHB-EGF for 1 hour at 37°C. sHB-EGF bound to EGFR-hFc was detected by HRP-labeled streptavidin. sHB-EGF binding to EGFR-hFc in the presence of Y-142 was calculated as a percentage of the “control” sHB-EGF binding to EGFR which occurred without Y-142. Data points represent the mean + SD of values acquired in triplicate. (B) Neutralizing activity of Y-142 against EGFR phosphorylation. SK-OV-3 cells were treated with 10 nM sHB-EGF and 67 nM Y-142. Cell lysates were incubated in an anti-EGFR antibody-coated plate, followed by an incubation with HRP-labeled anti-phosphorytosine antibody. EGFR phosphorylation in the presence of Y-142 was calculated as a percentage of the “control” EGFR phosphorylation which occurred without Y-142. Data points represent the mean + SD of values acquired in triplicate. (C) Neutralizing activity of Y-142 against ERBB4 phosphorylation. Cell lysates of T47D cells as prepared in Fig. 3A were incubated on an anti-ERBB4 antibody-coated plate. The phosphorylation of ERBB4 was detected by a sulfo-tagged anti-phosphotyrosine antibody. ERBB4 phosphorylation in the presence of Y-142 was calculated as a percentage of the “control” ERBB4 phosphorylation which occurred without Y-142. Data points represent the mean + SD of values acquired in duplicate. (D) and (E) Neutralizing activity of Y-142 against (D) ERK1/2 phosphorylation and (E) AKT phosphorylation. In (D) and (E) SK-OV-3 cells treated with 10 nM sHB-EGF and 200 nM Y-142 were stained with an anti-phosphorylated ERK1/2 antibody or an anti-phosphorylated AKT antibody, respectively, followed by an Alexa488-labeled anti-rabbit IgG antibody. Phosphorylated ERK1/2 and phosphorylated AKT were both detected with an ImageXpress Micro instrument and calculated as a percentage of the “control” phosphorylation levels which occurred without Y-142. Data points represent the mean + SD of values acquired in duplicate. (F) Neutralizing activity of Y-142 to ARG. SK-OV-3 cells were treated with 10 nM ARG plus various concentrations of Y-142 (2 nM, 6.7 nM, 20 nM, and 67 nM). Anti-ARG monoclonal antibody (67 nM) was used as a positive control. Cell lysates were incubated in an anti-EGFR antibody-coated plate followed by an incubation with an HRP-labeled anti-phosphorytosine antibody. EGFR phosphorylation was calculated as a percentage of the “control” EGFR phosphorylation which occurred without Y-142. Data points represent the mean + SD of values acquired in triplicate.
Figure 4.
Inhibitory activity of Y-142 against sHB-EGF functions.
(A) and (B) Neutralizing activities of Y-142 against (A) sHB-EGF-induced SK-OV-3 cell proliferation and (B) HUVEC proliferation. SK-OV-3 cells or HUVEC were cultured for 3 days in the presence of sHB-EGF and the indicated concentrations of Y142, cetuximab, or CRM197. Cell proliferation was detected with CellTiter-Glo and calculated as a percentage of the “control” cell proliferation without sHB-EGF. Data points represent the mean ± SD of values acquired in triplicate. (C) Inhibition of HUVEC tube formation by Y-142. HUVEC were cultured on a monolayer of NHDF in the presence of 50 nM sHB-EGF and the indicated concentrations of Y142, cetuximab, CRM197, or bevacizumab for 4 days. HUVEC were then stained with FITC-labeled anti-CD31 antibody. Tube formation (CD31-positive area) was calculated as a percentage of the “control” amount of tube formation in the presence of sHB-EGF. Data points represent the mean ± SD of values acquired in triplicate. (D) Inhibition of VEGF production by Y-142. HUVEC were prepared as in Figure 4C and treated with 50 nM sHB-EGF and the indicated concentrations of Y142, cetuximab, or CRM197 for 4 days. VEGF concentration in the supernatant of co-culture was measured in an electrochemiluminescence-based method. VEGF production was calculated as a percentage of the “control” amount of VEGF produced in the presence of sHB-EGF. Data points represent the mean ± SD of values acquired in triplicate.
Figure 5.
Recognition of a conformational epitope by Y-142.
The binding of Y-142 to a linear or conformational epitope was tested using Western blot. sHB-EGF (A) or ARG (B) was prepared in reducing or non-reducing conditions with or without dithiothreitol, respectively. The sHB-EGF was probed with an anti-HB-EGF polyclonal antibody and with Y-142. The ARG was probed with anti-ARG polyclonal antibody and with Y-142.
Figure 6.
Recognition of F115, G119, Y123, G137, G140, and R142 by Y-142.
Epitope mapping of Y-142 was performed using alanine scanning. Each mutant proHB-EGF expression plasmid was transfected into SW480 cells. The binding activity of Y-142 to the cells was measured in a cell ELISA. The expression level of mutant proHB-EGF was normalized with the binding of anti-HB-EGF polyclonal antibody by each mutant. The binding of Y-142 to mutant proHB-EGF was calculated as a percentage of the “control” binding of Y-142 to wild-type proHB-EGF. Data points represent the mean + SD of values acquired in triplicate.