Combining anti-ERBB3 antibodies specific for domain I and domain III enhances the anti-tumor activity over the individual monoclonal antibodies

Abstract

Background: Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies.

Methods: We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy.

Results: Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab.

Conclusion: These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.

Figure 1. Ability of single-chain Fv molecules to inhibit cell viability correlates with epitope.

A) Intact (dI-IV) and sub-domains (dI, dI-II, dIII, dIII-IV) of ERBB3 extracellular domain were expressed and subjected to immunoprecipitations with immobilized scFv under conditions of antibody excess. S  =  supernatant (unbound fraction), P  =  pellet (bound fraction).

Figure 2. Binding activity of A5 and F4 anti-ERBB3 IgGs.

A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.

Figure 3. Anti-ERBB3 mAbs inhibit ligand-mediated ErbB3 signaling.

A) Serum-starved BT-474, NCI-N87 and ACHN cells were treated with appropriate antibodies or PBS for 30 minutes followed by HRGβ stimulation for 10 minutes. B) NCI-N87 cells were treated with either the A5/F4 oligoclonal or PBS and stimulated with HRGβ, as appropriate, over a 60 minute time course. Cell lysates were analyzed by western blot and probed with anti-pAKT(S473) as a marker for ligand-induced signaling. Membranes were probed with anti-beta actin mAb that served as loading control.

Figure 4. Effects of anti-ErbB3 mAbs on basal ErbB3 signaling.

BT-474, NCI-N87 and ACHN cells growing in complete media were serum starved for 12–16 hours and treated with 1 µM A5, 1 µM F4, 1.6 nM trastuzumab (T), 100 nM cetuximab (C), PBS or combinations of A5/F4 (AF), A5/F4/trastuzumab (AFT), or A5/F4/Cetuximab (AFC) for a prescribed time. Cell lysates were analyzed by western blot with pERBB3(Y1289) and pAKT(S473) mAbs as markers of ERBB3 signaling, as well as anti-beta actin mAb as a loading control.

Figure 5. Combining A5 and F4 IgGs improves growth inhibition of ERBB3-positive cell lines and synergizes with ERBB-targeted agents.

Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.

Figure 6. The A5/F4 oligoclonal exhibits in vivo efficacy.

NCR nu/nu mice harboring 120–150 mm³ NCI-N87 subcutaneous tumor xenografts were randomly assigned into treatment groups (n≥6/group) and received i.p injections as described in Methods. Means +/−SEM of percentage tumor volume growth is shown for each measurement. Statistical analysis was performed with Wilcoxon Ranked Sum test.