A potent anti-HB-EGF monoclonal antibody inhibits cancer cell proliferation and multiple angiogenic activities of HB-EGF

Abstract

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers.

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 K_D of the Y-142/HB-EGF complex.

Dual-curve KinExA equilibrium titration of sHB-EGF binding to Y-142. K_D-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 K_D = 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.