Deregulation of the b-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and b-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate b-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate b-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, geneexpression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited b-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of b-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis. Cancer Res; 76(3); 713-23. Ó2015 AACR.

The Wnt/beta-catenin pathway, which signals through the Frizzled (FZD) receptor family and several co-receptors, has long been implicated in cancer. We have developed OMP-54F28, a recombinant fusion protein consisting of the ligand-binding domain of FZD8 and a human IgG1 Fc fragment. OMP-54F28 acts as a decoy receptor in sequestering Wnts and preventing them from binding to FZD receptors and thereby inhibiting Wnt signaling. The Wnt pathway is important for stem cell self renewal, differentiation, tumorigenicity, and epithelial-mesenchymal transition (EMT). Using minimally passaged human patient-derived xenograft tumors, we demonstrate that OMP-54F28 is efficacious as a single agent and in combination with standard of care in four hepatocellular carcinoma (HCC) and two ovarian cancer models. In the HCC models, OMP-54F28 shows tumor growth inhibition (TGI) as a single agent (average of 46%, p<0.05 vs. control) and displays additive TGI in combination with Sorafenib (average of 78%, p<0.05; 48% Sorafenib alone). Also, in the ovarian cancer models, treatment with OMP-54F28 results in TGI as a single agent (average of 32% vs. control) and shows additive TGI in combination with Paclitaxel (average of 78%, p<0.05; 48% with Paclitaxel alone). We also performed in vivo serial transplantation assays and found that OMP-54F28 as a single agent and in combination with standard of care reduces tumor-initiating cell frequency in both HCC and ovarian cancer xenografts. The anti-tumor effect was associated with a decrease in cell proliferation, induction of cell differentiation, and modulation of target Wnt pathway genes. Our data demonstrate the potential therapeutic benefit of targeting Wnt signaling in HCC and ovarian cancer. OMP-54F28 is being tested in Phase 1b clinical studies in these indications. Citation Format: Pete Yeung, Lucia Beviglia, Belinda Cancilla, Cristina Dee-Hoskins, James W. Evans, Marcus M. Fischer, Wan-Ching Yen, Austin Gurney, John Lewicki, Timothy Hoey, Ann M. Kapoun. Wnt pathway antagonist OMP-54F28 (FZD8-Fc) inhibits tumor growth and reduces tumor-initiating cell frequency in patient-derived hepatocellular carcinoma and ovarian cancer xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1907. doi:10.1158/1538-7445.AM2014-1907

The immune checkpoint co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig ITIM domain) is expressed on regulatory T cells (Tregs) and on activated CD4+ T, CD8+ T, and NK cells. We have reported that by blocking TIGIT activity with an IgG2a anti-TIGIT antibody (313R12), CD8+ and CD4+ Tcells and NK cells were activated, resulting in dose-dependent tumor growth inhibition (TGI) in multiple syngeneic mouse models. To explore the pharmacodynamics (PD) and mechanism of action of tumor growth inhibition (TGI) by anti-TIGIT antibodies, we examined the kinetics of immune cell frequency and activation in tumor by flow cytometry, qPCR and immunohistochemistry (IHC). We performed in vivo time course studies in the CT26.WT colon carcinoma model using weekly dosing at 0.1, 0.5 and 12.5 mg/kg anti-TGIT. Mice were sacrificed at 24 hours, 7 days and 14 days after the first dose for biomarker analysis. After 24 hours of treatment, Tregs in the tumor decreased and this reduction of Tregs was sustained at 7 and 14 days. Markers of immune cell activation and exhaustion such as CD69, PD1 and intracellular cytokines were modulated during the course of the study, suggesting a more cytotoxic intratumoral environment after 313R12 treatment. In addition, CD226, a binding partner of TIGIT, was significantly upregulated in T cells, Tregs and NK cells throughout the study, reflecting a feedback loop activated by inhibiting TIGIT activity. The anti-TIGIT antibody used in these studies, 313R12, is effector function competent and is able to induce cell-mediated cytotoxic effector functions upon binding. In order to determine whether effector function is necessary for anti-TIGIT antibody activity, we compared 313R12 with an effector function-deficient molecule, 313R13, in CT26.WT tumors. After 7 days, only 313R12 showed significant TGI compared to control-treated animals, suggesting that effector function is required for efficacy. While the effector function-deficient molecule 313R13 was able to similarly induce some changes in PD biomarkers, including immune cell activation, it required a higher dose than 313R12 to do so. To develop biomarkers for anti-TIGIT, we used microarray analyses to identify anti-TIGIT gene signatures in tumors and blood from multiple syngeneic models. In addition, we developed multiplexed IHC panels (e.g., TIGIT+CD8, TIGIT+FOXP3) to quantify expression of TIGIT and TIGIT ligand-positive immune cells in the tumor and surrounding stroma, and we profiled a panel of 80 human tumors with these panels. In summary, we examined the effects of anti-TIGIT antibodies on preclinical mouse models. Biomarker analysis demonstrated loss of Tregs and activation of T cells and NK cells, as well as effector function, as part of the mechanism of action of the molecule. We have also identified biomarkers that can be used for PD and potential predictive analysis in clinical trial samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Erwan le Scolan, Rose Harris, Reyhaneh Lahmy, Alayne Brunner, Min Wang, Gilbert O'Young, Earth Light Lowe, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Austin Gurney, John Lewicki, Ann Kapoun. Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5627.

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. The Wnt/beta-catenin pathway, which signals through the Frizzled (FZD) receptor family and several co-receptors, has long been implicated in cancer. We have developed ipafricept (FZD8-Fc, OMP-54F28), a recombinant fusion protein consisting of the ligand-binding domain of FZD8 and a human IgG1 Fc fragment. This fusion protein blocks Wnt signaling induced by multiple Wnt family members by binding and sequestering WNT. Using minimally passaged ovarian patient-derived xenograft tumors (PDX), we demonstrate that ipafricept is efficacious in combination with chemotherapy in four of eight ovarian cancer PDX tumors examined. Utilizing an in vivo serial transplantation assay, we quantified a reduction of the tumor initiating cell frequency by ipafricept in combination with paclitaxel. Additionally, we have discovered that pre-treatment with ipafricept several days prior to paclitaxel therapy enhances the activity of both agents when compared to delivering the drugs simultaneously. The anti-tumor effect observed is directly associated with a modulation of Wnt pathway gene sets. In responsive tumors, we discovered that a large number of WNT target genes were significantly down-regulated by ipafricept (e.g, AXIN2, LRP5/6, and FZD8). Conversely, in non-responsive tumors, these genes were either unchanged or up-regulated by the combination therapy. Histologic analysis revealed that total beta-catenin protein levels were reduced by ipafricept alone and in combination with paclitaxel in responsive tumors but were unchanged in non-responsive tumors. We are using these tumors to develop biomarkers that can be used clinically. Our data demonstrates the potential therapeutic benefit of targeting Wnt signaling in ovarian cancer. A Phase 1b clinical trial is currently examining ipafricept in combination with paclitaxel and carboplatin in patients with recurrent platinum-sensitive ovarian cancer. Citation Format: Marcus M. Fischer, Wan-Ching Yen, Chun Zheng, Randall Henner, Fiore Cattaruzza, Tracy Tang, Pete Yeung, Tanuka Biswas, John Lewicki, Austin Gurney, Ann M. Kapoun, Timothy Hoey. Wnt pathway antagonist ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth and reduces tumor-initiating cell frequency in ovarian patient-derived xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2015-4233

Ovarian cancer in a major cause cancer related death in women and this disease is often characterized by resistance to chemotherapy and tumor recurrence. Cancer stem cells (CSCs) have been shown to be relatively insensitive to chemotherapeutic agents. Several lines of evidence indicate that aberrant Notch signaling plays a major role in ovarian cancer progression and resistance to standard therapies. DLL4 is a key ligand that activates the Notch pathway and has been shown to regulate both CSC function and tumor angiogenesis. DLL4 expression in tumor and endothelial cells has also been associated with resistance to bevacizumab treatment. In these studies, we investigated the role of anti-DLL4 in a panel of patient-derived ovarian cancer xenograft models. We utilized anti-human DLL4 (OMP-21M18) and anti-murine DLL4 to block Notch signaling in both the tumor and stromal/vascular cells in the xenografts. We found that anti-DLL4 treatment was broadly efficacious in these ovarian cancer models, significantly inhibiting tumor growth both as a single agent and in combination with paclitaxel. We carried out serial transplantation studies to investigate the effect on cancer stem cells and found that anti-DLL4 in combination with paclitaxel profoundly reduced the tumor initiating cell frequency. In contrast, treatment with paclitaxel alone had the opposite effect and increased ovarian CSC frequency. These data suggest that anti-DLL4 treatment sensitizes chemoresistant ovarian tumorigenic cells to therapy and provide compelling evidence that anti-DLL4 (demcizumab) is a promising strategy for the clinical treatment of ovarian cancer. Citation Format: Wan-Ching Yen, Marcus M. Fischer, Jalpa Shah, Jie Wei, Jennifer Cain, Pete Yeung, Lucia Beviglia, Belinda Cancilla, Ann Kapoun, John Lewicki, Austin Gurney, Timothy Hoey. Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3725. doi:10.1158/1538-7445.AM2013-3725