William M. Winston

GP369, an FGFR2-IIIb specific antibody, exhibits potent antitumor activity against human cancers driven by activated FGFR2 signaling

Dysregulated fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We surveyed a broad panel of human cancer cell lines for the dysregulation of FGFR2 signaling and discovered that breast and gastric cancer cell lines harboring FGFR2 amplification predominantly express the IIIb isoform of the receptor. Therefore, we used an FGFR2-IIIb-specific antibody, GP369, to investigate the importance of FGFR2 signaling in vitro and in vivo. GP369 specifically and potently suppressed ligand-induced phosphorylation of FGFR2-IIIb and downstream signaling, as well as FGFR2-driven proliferation in vitro. The administration of GP369 in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support the hypothesis that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies.

Neuregulin 1 Expression Is a Predictive Biomarker for Response to AV-203, an ERBB3 Inhibitory Antibody, in Human Tumor Models

Purpose: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. Experimental Design: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. Results: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. Conclusions: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203. Clin Cancer Res; 21(5); 1106–14. ©2014 AACR.

MAP3K11/GDF15 axis is a critical driver of cancer cachexia

Cancer associated cachexia affects the majority of cancer patients during the course of the disease and thought to be directly responsible for about a quarter of all cancer deaths. Current evidence suggests that a pro‐inflammatory state may be associated with this syndrome although the molecular mechanisms responsible for the development of cachexia are poorly understood. The purpose of this work was the identification of key drivers of cancer cachexia that could provide a potential point of intervention for the treatment and/or prevention of this syndrome.

23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity

Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit. Mol Cancer Ther; 14(8); 1858–67. ©2015 AACR.

Abstract 2509: AV-203, a humanized ERBB3 inhibitory antibody inhibits ligand-dependent and ligand-independent ERBB3 signaling in vitro and in vivo

ERBB3 is a member of the EGFR receptor tyrosine kinase (RTK) family. Agents targeting the family of EGFR RTKs have become widely used for the treatment of lung, colon, breast, gastric, and head and neck cancers. Among EGFR family members, ERBB3 is of special interest because of its ability to activate the survival pathway driven by PI3K, its essential role in HER2 mediated cancers, and its involvement in tumor progression and drug resistance. The ERBB3 receptor is expressed on cells from cancers of the head and neck, lung, breast, ovaries, prostate, colon, pancreas, and gastrointestinal tract. Its expression is often linked to poor prognosis. In addition, it has been implicated in the development of resistance to current anti-cancer agents including receptor-targeted tyrosine kinase inhibitors (TKI). ERBB3 lacks detectable tyrosine kinase activity and its activation requires the heterodimerization of ERBB3 with RTK partners, i.e., HER2, EGFR or MET. ERBB3 recruitment in heterodimer complexes takes place when RTKs are overexpressed or amplified or stimulated by ligands, i.e. Neuregulin-1 (NRG1) or epidermal growth factor (EGF). Because of the lack of ERBB3 kinase activity, antibody therapies directed against the extracellular domain of ERBB3 seem to be the most effective method to disrupt the function of ERBB3. Herein, characterization of AV-203, a humanized ERBB3 inhibitory antibody, is presented. AV-203 is a humanized immunoglobulin G1/kappa antibody that targets the ERBB3 RTK. AV-203 binds to human ERBB3 with high affinity (KD = 76 pM at 37°C) characterized by fast association and slow dissociation rates. AV-203 also binds to cynomolgus monkey ERBB3 but not to mouse ERBB3, allowing toxicological assessment of the antibody in this species. AV-203 is a potent inhibitor of ERBB3 activation and its downstream signaling molecule AKT in response to both ligands, NRG1 and EGF. In ligand-independent settings, AV-203 inhibits the steady state activation of ERBB3/AKT which depends on the presence of an overexpressed RTK such as HER2. AV-203 can prevent the ERBB3/HER2 heterodimer formation and completely inhibit the proliferation in response to NRG1 ligand in human breast cancer cell line, MCF7. AV-203 down regulates ERBB3 receptor in vitro and in vivo. Finally, AV-203 inhibits tumor growth in a broad spectrum of xenograft models in which ERRB3 is activated by its ligand NRG1 or by HER2 overexpression such as the pancreatic cancer BxPC3 or the HER2 amplified breast cancer MDA-MB-453 xenograft models, respectively. In conclusion, AV-203 demonstrated high affinity binding to ERBB3, potent inhibition of NRG1 binding and of ERBB3 activation in ligand-dependent and ligand-independent manners both in vitro and in vivo. First in human trial of AV-203 in cancer patients is planned for 2012. 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 2509. doi:1538-7445.AM2012-2509

Abstract 1153: Effective treatment of cancer associated cachexia by AV-380, a GDF15 inhibitory antibody

Background: One of the most lethal and debilitating effects of cancer is the development of cancer-related anorexia-cachexia syndrome (CACS). It affects the majority of advanced cancer patients and is thought to be responsible for up to ∼30% of all cancer deaths. CACS is a complex metabolic syndrome associated with malnutrition and severe involuntary weight loss due to the loss of muscle and fat tissue, as well as the clinical manifestation of anemia, inflammation and suppression of immune functions. The precise molecular mechanisms responsible for cancer cachexia are poorly understood, thus limiting the development of effective therapeutics. Current evidence suggests that a pro-inflammatory state may be responsible for many of the symptoms associated with CACS. Growth Differentiation Factor 15 (GDF15) is a pro-inflammatory cytokine whose circulating levels are significantly increased in cachectic cancer patients and several animal models of cancer cachexia. Here we demonstrate that the inhibition of GDF15 function results in the compete reversion of the phenotypic and metabolic changes associated with CASC. Methods: Mice bearing HT-1080 human fibrosarcoma xenografts have increased plasma GDF15 levels and develop cachexia were used in this study. Tumor bearing animals were treated either with AV-380, a GDF15 neutralizing antibody or IgG1 control. The effect on body weight, muscle/fat mass and organ sizes were assessed. Metabolic changes induced by the treatment were measured by a comprehensive laboratory animal monitoring system (CLAMS). Results: Treatment with AV-380 restored body weight, muscle and fat mass as well as normal organ sizes. Analyses of the CLAMS data demonstrated that mice receiving AV-380 reversed the phenotypic and metabolic changes associated with the cachexia induced by this tumor model. Moreover, AV-380 treated mice showed an increase in locomotor activity and energy expenditure, achieving levels similar to non tumor bearing control animals. Conclusions: Inhibition of GDF15 function completely reverted body weight loss, restored normal body composition and triggered a catabolic to anabolic metabolic switch in this cancer cachexia model. The data highlights the therapeutic potential of the GDF15 inhibitory antibody AV-380 for the treatment for CACS. Citation Format: Lorena Lerner, Nianjun Tao, Brian Krieger, Richard Nicoletti, Bin Feng, Nesreen Ismail, William Winston, Yanyu Zhang, Jinwei Jiang, Solly Weiler, Jeno Gyuris. Effective treatment of cancer associated cachexia by AV-380, a GDF15 inhibitory antibody. [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 1153. doi:10.1158/1538-7445.AM2015-1153

Abstract A5: Tivozanib, a selective VEGFR TKI, potently blocks angiogenesis and growth in tumors that express a high level of VEGF-C and are refractory to VEGF-A blockade.

Background: Scientific understanding of the role of VEGF-A in tumor angiogenesis has led to the development of antiangiogenic therapies, such as bevacizumab, that selectively target VEGF-A. However, clinical trials across multiple cancer types have resulted in limited positive outcomes. VEGF-C is thought to be a potent lymphangiogenic growth factor and plays a role in tumor angiogenesis through VEGFR3; it has also been shown to bind to VEGFR2, which is important in tumor angiogenesis. Nevertheless, a direct role of VEGF-C in driving tumor angiogenesis has not been established. To explore the potential of VEGF-C as a driver of tumor angiogenesis and its implication in developing antiangiogenic therapies, we assessed the activity of tivozanib, a potent and selective TKI for VEGFR1, 2 and 3, and a VEGF-A targeted antibody in animal tumor models that exhibit distinct VEGF-C and VEGF-A expression. Method: A total of 107 independently derived murine breast tumors were expanded in vivo to establish population-based primary tumor models. The tumors were characterized for their angiogenesis phenotypes, including expression of angiogenic factors, microvasculature (demonstrated by quantitative CD31 IHC analysis), and myeloid infiltration, a previously identified mediator of tivozanib resistance. To examine the role of VEGF-C vs. VEGF-A in tumor angiogenesis and in affecting response to anti-VEGF therapies, a panel of representative tumors were tested with the cross species anti-VEGF-A antibody B20–4.1 and tivozanib. Tivozanib was also compared with B20–4.1 in blocking VEGF-C stimulated VEGFR2 or VEGFR3 activation in endothelial cells, and in VEGF-C stimulated in vivo angiogenesis in matrigel assay. Results: Three distinct subtypes of tumors were identified: those that expressed high level of VEGF-A and low VEGF-C, or high VEGF-C and low VEGF-A, or both high VEGF-A and high VEGF-C. These tumors exhibited characteristic VEGF driven microvasculature. When treated with systemic administration of B20–4.1 or tivozanib, the tumors expressing high VEGF-A showed comparable response to both agents, while the tumors expressing high VEGF-C but low VEGF-A only responded to tivozanib. In tumors that express both high VEGF-A and high VEGF-C, B20–4.1 treatment resulted in only modest response, while tivozanib completely blocked tumor progression accompanied by vast central necrosis. Pharmacodynamic analysis in representative tumors revealed tumor death associated with progressive hypoxia in tivozanib-treated tumors, consistent with antiangiogenic mechanism of action. Consistent with these findings, tivozanib, but not B20–4.1, potently blocked VEGF-C stimulated VEGFR2 and VEGFR3 activation in endothelial cells, and effectively blocked VEGF-C induced in vivo angiogenesis in a matrigel assay. Conclusion: These data suggest that VEGF-C can play a direct role in tumor angiogenesis, and that tumor expressing high level of VEGF-C are sensitive to VEGFR TKI tivozanib but are refractory to VEGF-A targeted antibodies. These findings provides further scientific evidence that pan-VEGFR TKIs may have broader activity than agents that selectively target VEGF-A. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A5.

Abstract 644: Anti-tumor activities of antibodies targeting the RON receptor and a biomarker of response

RON is a receptor tyrosine kinase of the MET family. Stimulation by its ligand, Macrophage Stimulating Protein (MSP), activates a signaling cascade leading to cell growth, migration, invasion and resistance to apoptosis. In animal models, RON overexpression in breast and lung results in tumor growth and metastasis. RON receptor activation in animal models also play a role in tumor-host interactions such as osteolytic bone destruction and tumor associated macrophage infiltration. RON overexpression has been demonstrated in several solid tumors including pancreatic, breast, ovarian and colon. RON overexpression is correlated with disease progression and shorter survival in ovarian and colon cancer. Several isoforms of RON have been reported, including a potentially oncogenic form, RON Δ160 in CRC. Given the strong evidence for the involvement of RON in numerous aspects of tumor biology, investigating an anti-RON antibody as cancer therapy is warranted. We have identified and characterized a panel of antagonistic murine anti-human RON antibodies. Humanization of two antibodies resulted in Superhumanized™ anti-RON antibodies that are capable of inhibiting MSP dependent RON downstream signaling, cell migration and invasion in vitro. The anti-RON antibodies have subnanomolar binding affinity to wildtype RON and RON Δ160 receptors. The lead antibody is capable of internalizing and degrading the receptor in vitro and in vivo. The antibodies are capable of inhibiting growth of engineered murine models that are driven by wildtype or RON Δ160 receptor, as well as traditional human cancer xenografts. Given the complex role of RON in tumor biology, identification of response biomarkers is crucial for identifying the patient populations most likely to benefit from treatment. A multi-gene biomarker potentially predictive of tumor response to RON antibody, the RON pathway index, was tested and validated using a panel of human cancer cell line xenografts. Current results demonstrate a statistically significant correlation between the degree of tumor growth inhibition by anti-RON antibody treatment and RON pathway index value. Thus, we have identified a biomarker of tumor response to anti-RON antibody that can potentially help us identify tumor types or tumor subtypes of interest in the clinic. 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 644. doi:10.1158/1538-7445.AM2011-644

Abstract 642: Novel EGFR inhibitory antibodies directed against EGFR mutants with potentially reduced toxicity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Non-small cell lung cancers (NSCLC) driven by ligand-independent activating mutations in epidermal growth factor receptor (EGFR) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs). About half of acquired resistance to EGFR-TKI therapy results from a secondary point mutation in the EGFR tyrosine kinase domain at amino acid position 790 (T790M). T790M mutants also display reduced sensitivity to Cetuximab treatment. We have investigated the molecular mechanism responsible for the reduced Cetuximab sensitivity and found that T790M mutant receptors primarily exist and signal as monomers. We have exploited this characteristic of the T790M mutant to isolate novel EGFR inhibitory antibodies with activities against all EGFR variants. In addition, as opposed to other EGFR inhibitory antibodies, these novel antibodies have reduced ligand binding inhibitory activity and minimal inhibitory effect on EGF induced human primary keratinocyte proliferation, suggesting a potentially reduced skin toxicity profile. The broad activity profile, combined with potentially reduced skin toxicity, suggests that these antibodies will have great potential for combinability with other therapeutic agents. 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 642. doi:10.1158/1538-7445.AM2011-642

Abstract 4586: Essential role of fibroblast growth factor receptor 2 (FGFR2) in tumorigenesis of human cancers with activated FGFR2 signaling demonstrated by functional blocking antibodies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Aberrant activation of fibroblast growth factor receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification have been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We identified a broad panel of human cancer cell lines with dysregulated FGFR2 signaling and examined the sensitivity of these human cell lines to monoclonal antibodies specifically targeting FGFR2. These FGFR2 antibodies potently suppressed ligand-induced phosphorylation of FGFR2 and downstream signaling, as well as cell proliferation in vitro. The administration of FGFR2 monoclonal antibodies in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies. 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 4586. doi:10.1158/1538-7445.AM2011-4586