WNT signaling is frequently dysregulated in cancers. However, pan-WNT inhibitors regularly induce adverse effects in patients, mostly notably in bone, creating a need for more specific WNT-pathway targeting strategies. Frizzled-7 (FZD7), a cell-surface receptor for WNT proteins, is a strong target candidate due to its high expression pattern in many tumor types (including but not limited to breast, ovarian, liver, gastric, and skin cancers) and low to modest expression in few normal adult tissues. We validated FZD7 protein expression in primary patient melanomas and breast and ovarian tumor samples, and developed an antibody-drug conjugate (ADC) that targets human FZD7, hereafter referred to as “FZD7 ADC.” Our ADC consists of a chimeric human-mouse IgG1 antibody conjugated to four molecules of antimitotic drug, monomethyl auristatin E (MMAE), by cleavable valine-citrulline linkers. By flow cytometry, we confirmed that the antibody component binds FZD7 and does not cross-react with the other nine human FZD receptors, FZD(1-6,8-10). We have identified MA-148 and PA-1 as human ovary-derived cancer cell lines responsive to our ADC. We also generated a negative control line, MA-148 FZD7-KO, by CRISPR/Cas9 knockout. In a cell viability assay, we demonstrated FZD7 ADC efficacy in inducing direct, FZD7-dependent cytotoxicity. A single dose of ADC killed MA-148 and PA-1 cells in vitro, with an IC50 of ~0.76 ug/mL (~5 nM) in both lines. MA-148 FZD7-KO cells exhibited an IC50 of ~9 ug/mL (~60 nM). Here, we established a therapeutic window in which our ADC specifically kills FZD7-positive cells.
We are currently evaluating FZD7 ADC tumor-killing efficacy in vivo. Because our ADC only binds human FZD7, and not mouse Fzd7, we are utilizing a xenograft model in female nude mice. In a preliminary three-armed study, we established subcutaneous human MA-148-Luciferase (Luc) tumors in mice and treated with a PBS control, 1 mg/kg FZD7 ADC (~0.15 nmole), or 3 mg/kg FZD7 ADC (~0.5 nmole), n = 3-5 per group. We performed an identical study with mice bearing MA-148 FZD7-KO-Luc tumors in parallel. Treatments were delivered twice per week by tail vein injection. Tumor size was measured weekly by an IVIS Spectrum after intraperitoneal luciferin injection. After eight doses over 27 days, MA-148-Luc tumors treated with 3 mg/kg FZD7 ADC completely or partially regressed compared to the control tumors (p = 0.0101 by one-way ANOVA and Tukey's multiple comparisons test). The 1 mg/kg dose did not produce a therapeutic effect in the MA-148-Luc. Importantly, the negative-control MA-148 FZD7-KO-Luc tumors treated with 3- or 1 mg/kg of ADC were not statistically different from the control tumors. Here, we have established a therapeutic FZD7 ADC dose for tumor regression and demonstrated its specificity to FZD7-positive tumors in vivo. Our data show that the FZD7 ADC is an effective strategy to combat cancers expressing FZD7.
Citation Format: Myan Do, Christina C. Wu, Stephen Adams, Dennis Carson, Sunil Advani, Karl Willert. Targeting FZD7-positive cancers using a novel antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1848.