A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties

Abstract

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.

Keywords: Wnt signaling; anti-Frizzled synthetic antibodies; pancreatic ductal adenocarcinoma; phage display.

Figure 1.

Specificities of naïve anti-FZD Fabs. Specificities of Fabs selected for binding to the CRD of FZD7 or FZD4 was assessed by ELISA with each immobilized FZD CRD, and the signals are shown in a purple gradient.

Figure 2.

Inhibition of WNT3A signaling by anti-FZD Abs. HEK293T cells were transfected with a TCF reporter gene and stimulated with WNT3A conditioned media in the presence of indicated Fabs or IgG OMP-18R5 (x-axis). Luciferase activities, normalized to the activity in the presence of a negative control Fab, are shown (y-axis), and inhibition of WNT3A signaling is evidenced by reduced signals.

Figure 3.

Sequences and specificities of optimized anti-FZD Fabs. The sequences of the complementarity-determining regions diversified in the Fab library are shown for Fab F2 and its derivatives. Residues are numbered according to IMGT standards,³⁹ and dashes indicate identity with the F2 sequence. Fab binding was assessed by ELISA with each immobilized FZD CRD, and the signals are shown in a purple gradient.

Figure 4.

Comparison of anti-FZD Ab epitopes. (A) ELISA for detection of solution-phase FZD5 (left and center) or FZD4 (right) binding (y-axis) to immobilized WNT3A, WNT5A or Norrin (x-axis) in the presence of Fab F2 (white bars), Fab F2.A (black bars) or IgG OMP-18R5 (grey bars). Binding was normalized to FZD binding signal in the absence of solution-phase competitor (maximal binding). (B) Sensorgrams for BLI responses (y-axis) monitored in real-time (x-axis). Each sensorgram shows binding of analytes to a tip with immobilized IgG F2.A (top panels) or OMP-18R5 (bottom panels). The vertical line (360 seconds) indicates a switch from solution containing the indicated FZD CRD analyte to a solution containing the indicated analyte IgG. The absence of an increase in BLI response upon switching analytes indicates that the analyte IgG cannot bind to FZD CRD captured by the immobilized IgG. (C) Superposition of the crystal structures of mouse FZD8 (green ribbon) bound to xenopus WNT8 (purple surface, PDB accession 4F0A) and human FZD4 (not shown) bound to human Norrin (cyan surface, PDB accession 5BQC). FZD residues that contact WNT8 but not Norrin are shown as red spheres, and we speculate that the epitopes for Abs F2.A and OMP-18R5 overlap with this region.

Figure 5.

Effects of anti-FZD Abs on PDAC cell signaling and growth. (A) Effects of anti-FZD Abs on expression of the Wnt target genes AXIN2 and NKD1. Gene expression in HPAF-II cells was assessed by RT-qPCR after six days of treatment with 200 nM IgG F2.A or a negative control IgG. Unpaired t-tests, bars represent mean ± s.d. normalized to β-actin expression, n = 2 independent experiments each performed in triplicate. (B) Effects of IgG F2.A on cell cycle phase in HPAF-II and YAPC cells, assessed by DNA content. Cells were treated for six days with 200 nM IgG F2.A or phosphate-buffered saline (untreated) control and percentages of cell populations in G₀/G₁, S and G₂/M phase were determined. Bars represent mean ± s.d., n = 2 independent experiments, with a two-tailed unpaired t-test on proportion of cells in G₀/G₁, compared to control. (C-F) Cell viability assays with the RNF43-mutant PDAC cell lines (C) HPAF-II, (D) AsPC-1, Capan-2 and PaTu8988s, (E) the RNF43 wild-type PDAC cell lines PANC-1 and BxPC-3, and (F) patient-derived RNF43-mutant (GP2A) and RNF43-wildtype (GP3A) PDAC cell lines. Cells were treated with the indicated IgG for 6 days, and viability was determined by Beckman cell counts (GP2A) or Alamar blue assays (all others). Points represent means ± s.d., n = 3 independent experiments. ***P < 0.001, **P < 0.01, and *P < 0.05, two-tailed unpaired t-tests using Holm-Sidak method for multiple comparisons without assuming consistent s.d.

Figure 6.

Effects of IgGs on angiogenesis. (A) Time-course of mean endothelial vessel length for HUVECs treated with phosphate-buffered saline (PBS), siCtrl, siFZD4, siERG, 30 µM Suramin or 1 µM LGK974. Bars represent mean ± s.e.m., n = 4 independent experiments. **P < 0.01, two-tailed, unpaired t-test. (B) Time-course of mean endothelial vessel length for HUVECs treated with PBS or 300 nM IgG F2.A, F2 or OMP-18R5. Bars represent mean ± s.e.m., n = 4 independent experiments. ***P < 0.001, two-tailed, unpaired t-test.