WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/β-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.

Though WNT signaling is frequently dysregulated in solid tumors, drugging this pathway has been challenging due to off-tumor effects. Current clinical pan-WNT inhibitors are non-specific and lead to adverse effects, highlighting the urgent need for more specific WNT-pathway targeting strategies. We identified elevated expression of the WNT receptor Frizzled class receptor 7 (FZD7) in multiple solid cancers in The Cancer Genome Atlas, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival. Moreover, we observed increased FZD7 protein expression in ovarian tumors compared to normal ovarian tissue, indicating that FZD7 may be a tumorspecific antigen. We therefore developed a novel antibody-drug conjugate, septuximab vedotin (F7-ADC), which is composed of a chimeric human-mouse antibody to human FZD7 conjugated to the microtubule-inhibiting drug monomethyl auristatin E (MMAE). F7-ADC selectively binds human FZD7, potently kills ovarian cancer cells in vitro, and induces regression of ovarian tumor xenografts in murine models. To evaluate F7-ADC toxicity in vivo, we generated mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the humantargeting F7-ADC. F7-ADC treatment of these mice did not induce acute toxicities, indicating a potentially favorable safety profile in patients. Overall, our data suggest that the antibody-drug conjugate approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic.

Switchable chimeric antigen receptors (sCAR) provide an important new strategy to precisely regulate CAR-mediated anti-tumor activity. The sCAR system combines a CAR that recognizes peptide neoepitope (PNE) with an injectable “switch” molecule that consists of an anti-tumor Fab linked to PNE. Configurating PNE at different location or chain of the Fab enables us to identify best switches optimal for tumor targeting. Previous studies demonstrate that this sCAR system expressed in T cells provides tight control over anti-tumor activity. Here, we translated this approach to engineer natural killer (NK) cells with the sCAR to provide a universal, targeted cell-therapy approach in a patient-nonspecific manner. First, we engineered human induced pluripotent stem cells (iPSCs) with the sCAR combining the PNE-specific CAR scFv with our previously described NK cell optimized CAR4 signaling motifs consisting of the NKG2D transmembrane domain, 2B4 co-stimulatory domain and the CD3ζ chain. We selected clones that maintained highest level of pluripotency and most stable expression of the sCAR4 on the surface. Next, we generated mature sCAR4-expressing iPSC-derived NK cells that expressed common surface receptors similar to that of donor peripheral blood mononuclear cell-derived NK cells. We then used three panels of switches with specificity to CD19 (consisting of 9 different configurations), to CD33, CD123 and CLL1 (1 configuration each), and to FRIZZLED7 (FZD7; 6 configurations) to target CD19+ B cell lymphoma, acute myeloid leukemia (AML), and ovarian cancer, respectively. All three targets demonstrated switch-specific dose response in killing tumor cell lines. Different configurations conferred variable efficiencies in switch-mediating tumor cell killings and the optimal switch concentrations were found to be different from what was observed previously with sCAR-expressing T cells. In a comparison with the “conventional” (non-switchable) CARs (cCARs), iPSC-NK cells expressing either sCAR4 and treated with an anti-CD19 switch or iPSC-NK cells with an anti-CD19-cCAR4 demonstrated similar level of cytotoxicity against B cell leukemia cells. Finally, we have built a mouse model and are currently testing the iPSC-NK-sCAR system in vivo. Together, this iPSC-NK-sCAR strategy enables close control over CAR-mediated activity with flexibility to target multiple tumor types and a potential to become a novel off-the-shelf therapy. Citation Format: Xiao-Hua Li, Benjamin Goldenson, Jaya Lakshmi Thangaraj, Matthew Gynn, Diana Gumber, Myan Do, Karl Willert, Dan S. Kaufman. Targeting hematological malignancies and solid tumors with switchable chimeric antigen receptor-engineered iPSC-derived natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 559.

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here we describe an engineered protein, called F7L6, comprised of antibody-derived single chain variable fragments that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/beta-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.

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.