Marcus Fischer

Targeting Notch Signaling with a Notch2/Notch3 Antagonist (Tarextumab) Inhibits Tumor Growth and Decreases Tumor-Initiating Cell Frequency

Purpose: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. Experimental Design: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. Results: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. Conclusions: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types. Clin Cancer Res; 21(9); 2084–95. ©2015 AACR.

Anti-DLL4 Has Broad Spectrum Activity in Pancreatic Cancer Dependent on Targeting DLL4-Notch Signaling in Both Tumor and Vasculature Cells

Purpose: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy. Experimental Design: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays. Results: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition. Conclusions: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling. Clin Cancer Res; 18(19); 5374–86. ©2012 AACR.

Therapeutic targeting of tumor-derived R-spondin attenuates β-catenin signaling and tumorigenesis in multiple cancer types

Deregulation of the β-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and β-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 β-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate β-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, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited β-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 β-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.

Abstract 1907: 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

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 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

Abstract 3357: Targeting cancer stem cells by an anti-DLL4 antibody inhibits epithelial-to-mesenchymal transition, delays tumor recurrence and overcomes drug resistance in breast and pancreatic cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnAccumulating evidence suggests that tumor growth, recurrence and metastasis are driven by a subset of highly tumorigenic cells referred to as cancer stem cells (CSCs) or tumor initiating cells. Several investigators have demonstrated that CSCs are relatively resistant to chemotherapy and that tumor recurrence and the development of drug resistance after chemotherapy are mediated by residual cancer stem cells. Furthermore, it has been demonstrated that the process of epithelial-to-mesenchymal transition (EMT) contributes to drug resistance and results in cells with CSC-like characteristics. We previously demonstrated that targeting Notch signaling pathway by a novel anti-DLL4 antibody inhibited tumor growth and decreased cancer stem cell frequency. Using patient-derived breast cancer and pancreatic cancer xenograft tumors we found that residual tumors after conventional chemotherapy (paclitaxel for breast cancer and gemcitabine for pancreatic cancer) were enriched with cancer stem cells. Gene expression analysis of cell populations enriched for CSCs revealed that several mesenchymal-associated genes were up-regulated, whereas epithelial gene was down-regulated. As shown by in vivo limiting dilution analyses, treatment with anti-DLL4 antibody decreased cancer stem cell frequency. Anti-DLL4 reversed chemotherapy-induced mesenchymal gene expression and delayed tumor recurrence. The residual tumor cells treated with conventional therapy eventually developed acquired drug resistance following repeated treatment. Compared to the parental xenograft, the resistant tumors had increased cancer stem cell frequency and tumorigenic ability. Treatment with anti-DLL4 antibody inhibited growth of these resistant tumors and re-sensitized them to the chemotherapeutic agent. This effect was in part due to the ability of the antibody to decrease cancer stem cell frequency in resistant tumors. The combination of antibody and chemotherapeutic agent further decreased cancer stem cell frequency compared to the parental tumor. Gene expression analysis demonstrated that resistant tumors had increased expression of many genes associated with EMT, multidrug resistance, DNA repair, and the Notch and Wnt pathways. Importantly, treatment with anti-DLL4 suppressed many gene expression changes in chemo-resistant tumors and resulted in a gene expression profile more like the original (drug sensitive) tumor. Immunohistochemical analysis indicated that anti-DLL4 mediated anti-tumor effect was associated with inhibition of EMT and induction of differentiation markers. Our findings provide a rationale to target cancer stem cells through interference with Notch pathway as a therapeutic approach in patients who are refractory to chemotherapeutic agents.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3357. doi:1538-7445.AM2012-3357

Targeting Cancer Stem Cells and Vasculature by a Novel Anti-Delta-Like 4 Ligand (DLL4) Antibody for Treatment of Triple Negative Breast Cancer.

Triple negative (ER-, PR- Her2-) breast cancer accounts for 10% of all types of breast cancer and is known to particularly aggressive and refractory to current therapies. Chemotherapeutic agents, such as taxanes, are currently the only treatment option for this type of breast cancer, however, these treatments often result in high rates of local and systemic relapse. It has been suggested that cancer stem cells drive tumor growth and progression and are preferentially resistant to many current therapies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway which is known to mediate stem cell self-renewal and vascular development. We hypothesized that targeting cancer stem cells and the tumor vasculature by interfering with the DLL4/Notch pathway will improve treatment outcome. We developed anti-DLL4 antibodies that recognize either the human or murine protein and have potent binding and antagonist activities. We have used these antibodies to investigate the role of DLL4 in triple negative breast cancer and to probe the mechanism in tumor and stromal cells in xenograft models derived from primary breast tumors. These studies showed that anti-DLL4 was efficacious as a single agent and in combination with paclitaxel (Taxol) against triple negative breast tumors including a tumor that developed paclitaxel resistance. Gene expression analysis showed that anti-DLL4 affected vascular-related genes in the stroma and Notch target genes in the tumor and stroma. Furthermore, inclusion of anti-DLL4 delayed breast tumor recurrence following termination of paclitaxel treatment. A tumorigenicity study indicated that treatment with anti-DLL4 decreased cancer stem cell frequency as measured by in vivo limiting dilution assay, whereas paclitaxel alone was ineffective. The combination of anti-DLL4 and paclitaxel further decreased cancer stem cell frequency compared to single agents. Our findings provide a rationale for targeting cancer stem cells and tumor vasculature through inhibition of the DLL4/Notch signaling for breast cancer treatment. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5071.

WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death

WNT antagonism displays marked synergy with taxane chemotherapy and reverses taxane-induced enrichment of cancer stem cells. The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/β-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.

Abstract 2576: Enhanced antitumor efficacy by sequential application of Wnt pathway antagonists in combination with taxanes

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 previously demonstrated that inhibition of Wnt/beta-catenin signaling by vantictumab (anti-Fzd7, OMP-18R5) or ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth, decreases tumorigenicity and induces differentiation in solid tumors. The anti-tumor effect of our Wnt antagonists is most evident in combination with chemotherapeutic agents. We sought to determine if the anti-tumor effect of Wnt pathway inhibitors varied with different chemotherapeutic agents. We compared the growth inhibitory effect of vantictumab and ipafricept with either taxanes (paclitaxel and nab-paclitaxel) or with DNA synthesis inhibitors (gemcitabine and carboplatin) in patient-derived tumor xenografts. We observed enhanced anti-tumor activity when combining vantictumab or ipafricept with nab-paclitaxel or paclitaxel compared to the combination with gemcitabine or carboplatin in pancreatic ductal carcinoma and serous ovarian cancer xenograft models. Histologic analysis in a pancreatic ductal carcinoma indicated that nab-paclitaxel increased mitotic cells and beta-catenin levels. Importantly, the addition of vantictumab to nab-paclitaxel reversed the nab-paclitaxel-induced increase in mitotic cells and beta-catenin expression. A potential mechanism to account for these results involves the observation that Wnt/beta-catenin signaling is under cell cycle control and peaks at the G2/M phase. Taxanes inhibit microtubule function and block the cell cycle at G2/M. In contrast, other chemotherapeutic agents, such as platinum compounds and nucleoside analogs, inhibit DNA synthesis and block cell proliferation at S phase. Our findings suggest that combination of Wnt blockade with chemotherapeutic agents, such as taxanes, that induce G2/M arrest may resulted in enhanced anti-tumor activity. The optimal synergy of anti-Wnt plus taxane combination occurs when the antibody was applied prior to taxane. Further analyses in serous ovarian tumors reveal that pre-treatment with ipafricept resulted in dysregulated beta-cetenin localization within giant multi-nucleated cells and up-regulation of genes associated with negative regulators of G1 progression. Our work provides evidence for the enhanced anti-tumor effect of Wnt pathway inhibitors in combination with taxanes and highlights the importance of preclinical examination to identify the most efficacious combination therapy regimens and the timing of antibody action for Wnt antagonists in combination with taxanes for optimal treatment efficacy. Citation Format: Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Fiore Cattaruzza, Tracy Tang, Pete Yeung, John Lewicki, Austin Gurney, Timothy Hoey. Enhanced antitumor efficacy by sequential application of Wnt pathway antagonists in combination with taxanes. [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 2576. doi:10.1158/1538-7445.AM2015-2576

Abstract 4233: Wnt pathway antagonist ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth and reduces tumor-initiating cell frequency in ovarian patient-derived xenograft models

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

Abstract 3129: Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts

Background: The WNT/ β-catenin signaling pathway has been shown to play a key role in both normal development and tumorigenesis (Polakis, 2007; MacDonald et al., 2009). We have developed a monoclonal antibody, vantictumab, that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, including pancreas, breast, colon and lung. Furthermore, our studies showed that vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012). Material and methods: We set out to identify a predictive biomarker for the response to vantictumab in pancreatic cancer patients by analyzing mRNA-seq gene expression data from 14 patient-derived xenograft (PDX) models. These 14 minimally passaged pancreatic xenograft tumors were tested in vivo and their responses to vantictumab, in combination with the current SOC gemcitabine and nab-paclitaxel were established. Samples from these experiments were collected for Pharmacodynamic (PD) biomarker analysis. We utilized a two-sample Welch9s t-test to identify genes that can distinguish between responders and non-responders and the K-nearest neighbor (KNN, Altman 1992) algorithm for classification. A leave-one-out cross-validation was used to measure area under the ROC curve (Fawcett et al., 2006, AUC), accuracy (ACC), positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the model. Results: PD biomarker analysis confirmed inhibition of genes in Wnt and stem cell pathways by vantictumab in combination with gemcitabine as well as gemcitabine plus nab-paclitaxel. The selected 3-gene signature comprising TGFB3, IGF2 and SMO achieved the best performance (AUC = 0.875, ACC = 0.93, PPV = 0.91, NPV = 1, sensitivity = 1, specificity = 0.75) in the 14 PDX pancreatic tumor models. In addition, a strong correlation between the gene signature biomarker and the ratio of tumor inhibition (RTI) in the pancreatic xenograft experiments was observed. The identified 3-gene biomarker was used to predict the response to vantictumab in combination with gemcitabine and nab-paclitaxel in three additional pancreatic PDX tumor models. The efficacy in the three models was successfully predicted by the biomarker. Conclusions: The 3-gene biomarker is being evaluated in a Phase 1b study of vantictumab in combination with gemcitabine and nab-paclitaxel in previously untreated stage IV pancreatic cancer (NCT02005315). Citation Format: CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Claire Guo, Alayne Brunner, Min Wang, Belinda Cancilla, Austin Gurney, Rainer Brachmann, John Lewicki, Tim Hoey, Ann M. Kapoun. Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3129.