Lucia Beviglia

Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy

Background Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA+CD44+ phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. Methods and Findings Xenogeneic tumors initiated with CoCSC were allowed to reach ∼400 mm3, at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. Conclusions CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

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.

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 2830: Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) by mining gene expression data of human breast cancer xenografts

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The Wnt/β-catenin signaling pathway has been shown to play key roles 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) at a conserved epitope within the extracellular domain. This antibody inhibits the growth of several tumor types, including breast, pancreas, colon and lung. Furthermore, studies also showed that vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care chemotherapeutic agents (Gurney et al., 2012). Predictive biomarkers are central to maximizing clinical benefit by targeting breast cancer patients most likely to respond to vantictumab. We analyzed microarray gene expression data from 8 minimally passaged breast cancer xenograft models (mostly triple-negative) with established in vivo responses to vantictumab combined with paclitaxel (4 responders, 4 non-responders). We utilized support vector machine—recursive feature elimination (SVM-RFE, Guyon et al., 2002) to identify genes that can distinguish between responder and non-responders and SVM for classification. Leave-one-out cross-validation was used to measure positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the models. The selected 6-gene signature achieved the best performance with PPV=NPV=sensitivity=specificity=100% in the 8 breast cancer models. In addition, we observed a strong correlation between the gene signature biomarker and the ratio of tumor volume (RTV) observed in the breast xenograft experiments. The identified 6-gene biomarker was used to predict the response to vantictumab in combination with paclitaxel in 6 additional, HER2-negative breast cancer xenograft models. The efficacy in all 6 models was predicted successfully by the biomarker. Prevalence data for the biomarker will be presented for both HER2-negative and triple-negative breast cancers. The 6-gene biomarker is currently being evaluated in a Phase 1b study of vantictumab in combination with paclitaxel in patients with locally recurrent or metastatic HER2-negative breast cancer. Citation Format: Chun Zhang, Pete Yeung, Lucia Beviglia, Belinda Cancilla, Tracy Tang, Wan-Ching Yen, Austin Gurney, John Lewicki, Timothy Hoey, Ann M. Kapoun. Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) by mining gene expression data of human breast cancer xenografts. [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 2830. doi:10.1158/1538-7445.AM2014-2830

Self-illuminating nanoprobe for in vivo imaging of cancers over-expressing the folate receptor

New in vivo imaging reagents with increased sensitivity and penetration depth are needed to advance our understanding of metastases and accelerate the development of therapeutics. The folate receptor (FR) is a promising imaging target that is up-regulated in many human carcinomas, including cancers of the ovary, breast, pancreas, endometrium, lungs, kidneys, colon, brain, and myeloid cells. Zymera has developed a self-illuminating Bioluminescence Resonance Energy Transfer Quantum Dot (BRET-Qdot) nanoprobe conjugated with folate (BQ-Folate) for in vivo imaging of cancers overexpressing FR. BQ-Folate is a novel nanoprobe formed by co-conjugating Renilla reniformis luciferase enzyme and folate to near-infrared (NIR) emitting quantum dots. The luciferase substrate, coelenterazine, activates the BQ-Folate nanoprobe generating luminescence emission in the near-infrared (NIR) region (655 nm) for increased sensitivity and penetration depth. Because BQ-Folate requires no external light source for light emission, it has significant advantages for challenging in vivo preclinical optical imaging applications, such as the detection of early stage metastases. Zymera and OncoMed Pharmaceuticals have demonstrated that in vivo imaging with the BQ-Folate nanoprobe detected the primary tumor and early stage metastases in an orthotopic NOD/SCID mouse model of human pancreatic cancer.

Abstract 2834: Anti-DLL4 treatment inhibits melanoma tumor growth, recurrence, metastases, and reduces frequency of cancer stem cells in a clinically relevant tumor model in NOD/SCID mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Metastatic burden at the time of diagnosis is often the cause of death in patients suffering from malignant melanoma. More predictive in vivo animal tumor models are needed to elucidate both the efficacy and mechanism of activity for newly developed anti-cancer therapeutic agents. We have developed an orthotopic melanoma model in NOD/SCID mice with intradermal implant of human melanoma cells that are derived directly from human cancer patient specimens. These tumors retain much of the original human cancer heterogeneity, phenotype, and the ability to form metastases in the lungs, lymph nodes, liver, intestines, and brain _ recapitulating the metastatic invasiveness seen in the clinic. We have developed antibodies against the human and the murine Delta-like Notch ligand DLL4, which target both tumor cells and vasculature, and reduce the frequency of cancer stem cells (CSCs) in human tumors implanted into NOD/SCID mice (Hoey et al., Cell Stem Cell 5:1680177, 2009). In our studies with a human melanoma model, we evaluated the anti-tumor and anti-metastatic activities of anti-DLL4 and found a significant inhibition of both primary tumor and metastases in the lungs, liver, intestine and brain. Moreover, in combination with the standard-of-care Paclitaxel, anti-DLL4 treatment caused a greater reduction of tumor growth than either agent alone and an increase in apoptosis of tumor cells compared to control antibody or paclitaxel alone These data were consistent with the results from the limiting dilution assay experiments that demonstrated a significant reduction of tumor initiating cells by the anti-DLL4 alone (1:15) and a further decrease in combination with paclitaxel (1:33) compared to the control antibody (1:5), but no effect by paclitaxel (1:5). In other studies aimed to examine tumor recurrence after excision of the primary tumors, treatment with anti-DLL4, alone and with paclitaxel, prevented recurrence of the primary tumors. In contrast, paclitaxel alone increased the frequency of tumor relapse when compared to the control and other treatments. In addition, anti-DLL4 alone inhibited metastases to lungs, lymph nodes, liver, brain and intestines and this effect was further enhanced by the combination with paclitaxel in the liver and intestines. The results from these studies demonstrate for the first time a significant inhibition of tumor recurrence and metastases in human melanoma by anti-DLL4 and establish a link between reduction of CSC frequency and the concurrent inhibition of metastases. Therefore our findings reveal great potential of our anti-DLL4 antibody as an effective anti-cancer therapy for melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2834. doi:10.1158/1538-7445.AM2011-2834

Abstract 3728: Anti-Notch1 antibody (OMP-52M51) impedes tumor growth and cancer stem cell frequency (CSC) in a chemo-refractory breast cancer xenograft model with an activating Notch1 mutation and screening for activated Notch1 across multiple solid tumor types.

The Notch pathway plays a central role in embryonic development, the regulation of stem and progenitor cells, and is implicated in many human cancers. Notch1 is also known to be activated by oncogenic mutations in tumors including T cell leukemia, chronic lymphocytic leukemia and non-small cell lung cancer. Here we report the discovery of similar mutations in breast cancer. We have developed a primary human xenograft model from a patient bearing an activating Notch1 mutation and have shown that blocking Notch signaling in this model with a novel anti-Notch1 antibody, OMP-52M51, impeded tumor growth and reduced cancer stem cell (CSC) frequency. OMP-52M51 is a humanized antibody that binds with high affinity and selectivity to human Notch1 and antagonizes Notch signaling. Additionally, we sought to determine the frequency of Notch pathway activation across a large panel of human tumors (n>600) by an immunohistochemistry assay that detects the activated form of Notch1 using a polyclonal antibody that specifically recognizes the Notch1 intracellular domain (ICD). Using this test and a rigorous H-score cut-off of 30, we found elevated Notch1 in 7-29% of the following cancers: chemo-resistant breast, gastric, cholangiocarcinoma, esophageal, hepatacellular carcinoma (HCC), and small cell lung cancer (SCLC). The class of patients that showed the highest frequency of elevated Notch1.ICD was chemo-resistant breast cancer (∼30%). This frequency was significantly higher than in unselected breast cancer patients, suggesting that Notch1 signaling plays a significant role in breast cancer chemoresistance. Interestingly, the human xenograft model which showed sensitivity to OMP-52M51 was derived from a patient that failed to respond to pre-operative chemotherapy and developed metastatic disease following surgery. Thus, chemo-resistant breast cancer as well as the other tumors with high Notch1.ICD may represent important classes of patients who could benefit from anti-Notch1 therapy in the clinic. Citation Format: Belinda Cancilla, Jennifer Cain, Min Wang, Lucia Beviglia, Jalpa Shah, Austin Gurney, John Lewicki, Laura Esserman, Tim Hoey, Ann M. Kapoun. Anti-Notch1 antibody (OMP-52M51) impedes tumor growth and cancer stem cell frequency (CSC) in a chemo-refractory breast cancer xenograft model with an activating Notch1 mutation and screening for activated Notch1 across multiple solid tumor types. [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 3728. doi:10.1158/1538-7445.AM2013-3728

Abstract 3725: Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts.

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

Abstract 4330: In vivo evaluation of anti-tumor activity by an anti-VEGF and anti-DLL4 bispecific antibody in a humanized model of skin graft.

Both Notch/Delta-like ligand 4 (DLL4) and vascular endothelial growth factor (VEGF) pathways play a critical role in angiogenesis and tumor growth. Due to differential regulatory effects of VEGF and DLL4 on the vasculature, blockage of DLL4 or VEGF inhibits tumor growth by distinct mechanisms: anti-DLL4 treatment induces an abnormal increase of poorly perfused blood vessels, which results in a nonproductive angiogenesis unable to support tumor growth, whereas the anti-VEGF therapy significantly decreases vasculature reducing the blood supply to tumors. In our study, we have developed a bispecific monoclonal antibody that targets both human DLL4 (hDLL4) and human VEGF (hVEGF). In vitro, this antibody demonstrated nanomolar affinity to hVEGF and hDLL4, and reduced HUVEC proliferation induced by VEGF (EC50 0.86 ug/ml). To test the activity of this bispecific antibody in vivo, we developed a human skin graft model in NOD/SCID mice, and implanted human tumor specimen-derived colon cancer cells intradermally into these skin transplants. The tumor model was selected based on its sensitivity to both the anti-human DLL4 antibody, OMP-21M18, and to the human VEGF inhibitor bevacizumab. The skin graft model provides a suitable human microenvironment for evaluating anti-tumor efficacy and anti-angiogenesis of the bispecific antibody directed against the human component of DLL4 and VEGF and allows a comparison to OMP-21M18 and also to bevacizumab. Each of these treatments was administered to mice intraperitoneally at a dose of 25 mg/Kg weekly. The bispecific antibody caused a significant inhibition of tumor growth (87% TGI) compared to control antibody (p=0.00001), and this effect was superior to either OMP-21M18 (45% TGI) or bevacizumab (70%TGI). The inhibition of tumor growth by the bispecific antibody was consistently associated with increased blood vessels, up-regulated VEGFA and VEGFR2, and enhanced hypoxia and these effects were more pronounced compared to OMP-21M18. As expected, in this model bevacizumab caused a significant decrease of blood vessels, down-regulated VEGFR2, and increased hypoxia. In separate experiments with mice bearing subcutaneous human colon tumors, the bispecific antibody delayed tumor recurrence following termination of chemotherapy and impacted tumorigenicity by decreasing the frequency of tumor initiating cells. These results suggest that our bispecific anti-DLL4 and anti-VEGF antibody is a potential candidate in the treatment of tumors driven by both VEGF and Notch/DLL4 signaling pathways. Citation Format: Lucia Beviglia, Pete Yeung, Wang-Ching Yen, Belinda Cancilla, Sato Aaron, Chris Bond, Janak Raval, Fumiko Axelrod, Cecile Chartier, Shirley Ma, Austin Gurney, John Lewicki, Ann M. Kapoun, Timothy Hoey. In vivo evaluation of anti-tumor activity by an anti-VEGF and anti-DLL4 bispecific antibody in a humanized model of skin graft. [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 4330. doi:10.1158/1538-7445.AM2013-4330

Abstract 3356: Development of a novel Wnt pathway antagonist antibody, OMP-18R5, that reduces tumor initiating cell frequency in breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Wnt/beta-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several co-receptors, is known to play a critical role in several major cancer types. Mutations in the signalling pathway occur in most cases of human colon cancer, and activation of Wnt signalling through various mechanisms has been reported in multiple major tumor types. We have developed a novel Wnt pathway antagonist antibody, OMP-18R5 which was initially identified by binding to Frizzled7, and subsequently found to also bind several other human FZDs through a conserved epitope within the extracellular domain. OMP-18R5 blocks Wnt binding and canonical signalling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, OMP-18R5 inhibited the growth of a broad range of tumor types and has been found to be particularly active in breast cancer. OMP-18R5 treatment reduced tumor growth and the frequency of tumor initiating cells in combination with pacilitaxel. Furthermore, we found that OMP-18R5 treatment can restore chemosensitivity in drug resistant breast tumors. Molecular analyses indicated OMP-18R5 inhibits the expression of EMT markers in breast tumors, thus providing additional evidence linking EMT with resistance to chemotherapy and cancer stem cells. In addition to combination activity with paclitaxel, OMP-18R5 treatment also resulted in increased anti-tumor activity in combination with trastuzumab in a Her2+ breast cancer model. These data suggest that OMP-18R5, which has recently entered Phase 1 clinical testing, may be useful in the treatment of various types of breast cancer. Citation 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 3356. doi:1538-7445.AM2012-3356