Nick Loizos

Platelet-derived growth factor receptor alpha (PDGFRα) targeting and relevant biomarkers in ovarian carcinoma

OBJECTIVE Platelet-derived growth factor receptor alpha (PDGFRα) is believed to be associated with cell survival. We examined (i) whether PDGFRα blockade enhances the antitumor activity of taxanes in ovarian carcinoma and (ii) potential biomarkers of response to anti-PDGFRα therapy. METHODS PDGFRα expression in 176 ovarian carcinomas was evaluated with tissue microarray and correlated to survival outcome. Human-specific monoclonal antibody to PDGFRα (IMC-3G3) was used for in vitro and in vivo experiments with or without docetaxel. Gene microarrays and reverse-phase protein arrays with pathway analyses were performed to identify potential predictive biomarkers. RESULTS When compared to low or no PDGFRα expression, increased PDGFRα expression was associated with significantly poorer overall survival of patients with ovarian cancer (P=0.014). Although treatment with IMC-3G3 alone did not affect cell viability or increase apoptosis, concurrent use of IMC-3G3 with docetaxel significantly enhanced sensitization to docetaxel and apoptosis. In an orthotopic mouse model, IMC-3G3 monotherapy had no significant antitumor effects in SKOV3-ip1 (low PDGFRα expression), but showed significant antitumor effects in HeyA8-MDR (high PDGFRα expression). Concurrent use of IMC-3G3 with docetaxel, compared with use of docetaxel alone, significantly reduced tumor weight in all tested cell lines. In protein ontology, the EGFR and AKT pathways were downregulated by IMC-3G3 therapy. MAPK and CCNB1 were downregulated only in the HeyA8-MDR model. CONCLUSION These data identify IMC-3G3 as an attractive therapeutic strategy and identify potential predictive markers for further development.

A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC50 = 0.06 nM) and strongly blocks its interaction with SCF (IC50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α

Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors. Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor. Results: In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin. Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847–57. ©2017 AACR.

Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. Summary: This study validates the FGF9 lung adenocarcinoma mouse model as a tool to screen and evaluate potential therapeutics that are designed to inhibit FGF9 or its target receptor, FGFR3.

Abstract C159: A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

Stem cell factor receptor also known as c-Kit is a receptor tyrosine kinase that mediates cell growth, survival, and differentiation signals in response to its ligand stem cell factor (SCF). Aberrant c-Kit expression and/or activation through mutations or autocrine/paracrine signaling mechanisms occur in various malignancies and promote tumor development. Specific therapeutic targeting of c-Kit in cancer is warranted given its cancer role. In this study, we characterize the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds human c-Kit receptor with high affinity (EC50= 0.06nM) and strongly blocks its interaction with SCF (IC50= 0.41nM) in solid phase assays. Flow cytometry shows CK6 binding to the cell surface of small cell lung carcinoma (SCLC), melanoma, leukemia, and other human c-Kit expressing tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways in these tumor cell lines. Reduced levels of phosphorylated c-Kit, mitogen-activated protein kinase (MAPK) and protein kinase B/Akt were observed. Given these findings, we evaluated the antitumor growth efficacy of CK6 in several human xenograft tumor models in vivo. CK6 monotherapy treatment significantly suppressed tumor growth of NCI-H526 SCLC (T/C%= 50) and Malme-3M Melanoma (T/C%= 58) xenograft models. The combination of CK6 with standard of care (SOC) chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, led to enhanced tumor growth inhibition (SCLC T/C%= 12; melanoma T/C%= 38) compared to CK6 monotherapy or SOC alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human cancers harboring c-Kit receptor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C159. Citation Format: Maria Lebron, Laura Brennan, Chris Damoci, Marie Prewett, Marina Starodubtseva, Michael Amatulli, Yiwei Zhang, Douglas Burtrum, Paul Balderes, Kris Persaud, David Surguladze, Nick Loizos, Keren Paz, Helen Kotanides. A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C159.

Phase 1 study of narnatumab, an anti-RON receptor monoclonal antibody, in patients with advanced solid tumors

SummaryPurpose Macrophage-stimulating 1-receptor (RON) is expressed on macrophages, epithelial cells, and a variety of tumors. Narnatumab (IMC-RON8; LY3012219) is a neutralizing monoclonal antibody that blocks RON binding to its ligand, macrophage-stimulating protein (MSP). This study assessed safety, maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and efficacy of narnatumab in patients with advanced solid tumors. Methods Narnatumab was administered intravenously weekly at 5, 10, 15, or 20 mg/kg or every 2 weeks at 15, 20, 30, or 40 mg/kg in 4-week cycles. Results Thirty-nine patients were treated, and 1 dose-limiting toxicity (DLT) (grade 3 hyponatremia, 5 mg/kg) was reported. The most common narnatumab-related adverse events (AEs) were fatigue (20.5%) and decreased appetite, diarrhea, nausea, and vomiting (10.3% each). Except for 2 treatment-related grade 3 AEs (hyponatremia, hypokalemia), all treatment-related AEs were grade 1 or 2. Narnatumab had a short half-life (<7 days). After Cycle 2, no patients had concentrations above 140 μg/mL (concentration that demonstrated antitumor activity in animal models), except for 1 patient receiving 30 mg/kg biweekly. Eleven patients had a best response of stable disease, ranging from 6 weeks to 11 months. Despite only 1 DLT, due to suboptimal drug exposure, the dose was not escalated beyond 40 mg/kg biweekly. This decision was based on published data reporting that mRNA splice variants of RON are highly prevalent in tumors, accumulate in cytoplasm, and are not accessible by large-molecule monoclonal antibodies. Conclusions Narnatumab was well tolerated and showed limited antitumor activity with this dosing regimen.

Abstract C129: Antitumor efficacy and non-clinical safety of the high affinity anti-FGFR4 antibody H4: Implications for targeting the FGF19-FGFR4 axis in oncology

While FGFR4 and its ligand FGF19 represent a promising target for cancers in which FGF19 is overexpressed or gene amplified, inhibition of this pathway carries a potential toxicity risk due to the role of FGFR4 in bile acid homeostasis. Treatment with an anti-FGF19 antibody caused significant toxicity in cynomolgus monkeys that was attributed to perturbations in the enterohepatic circulation of bile acids. Given that FGF19 signals through other FGFRs besides FGFR4, we hypothesized that the toxicity profile of an anti-FGFR4 antibody might be different than that of an anti-FGF19 antibody. To test this we set out to identify an anti-FGFR4 antibody that inhibits FGF19 binding and displays anti-tumor activity in models overexpressing FGF19, and then determine its toxicity profile. A phage screening effort yielded several anti-FGFR4 antibodies including H4, a high affinity fully human IgG1. H4 binds to the extracellular domain of FGFR4 (Kd = 12 pM) but does not cross react with FGFR1, FGFR2, or FGFR3. Furthermore, H4 efficiently blocks FGF19 binding to FGFR4 (IC50 = 589 pM) and inhibits FGF19-mediated downstream signaling in Hep3B2 cells. In human liver cancer cells that overexpress FGF19, H4 inhibits proliferation (IC50 250-1,100 nM). Anti-tumor activity was evaluated using cell line-derived xenograft tumors established from the FGF19 overexpressing and gene amplified cell lines HuH-7 and Hep3B2. H4 inhibited tumor growth in these models (T/C of 50% and 8%, respectively), and the antitumor effect was accompanied by alterations in several biomarkers associated with FGFR4 pathway inhibition including increased expression of CYP7A, the gene encoding the rate-limiting enzyme of bile acid synthesis. Finally, to assess the potential toxicity of H4 treatment, single dose intravenous toxicology studies were conducted in rats (20, 60, and 200 mg/kg) and cynomolgus monkeys (5, 20, and 100 mg/kg). Although H4 binds with similar affinity to monkey and rat FGFR4, the toxicology profiles were distinct. H4 was well tolerated in rats with no significant findings up to 200 mg/kg. Effects in monkeys included sporadic malformed feces, reduced food intake, elevated serum ALT (up to 44 fold over baseline) and AST activities, and elevated fecal bile acid concentrations at all doses. Hyperplasia of the gall bladder epithelium occurred at 100 mg/kg. There were no microscopic findings in the liver. Qualitatively this toxicity profile is similar to that of the anti-FGF19 antibody, although the maximum tolerated dose (MTD) of H4 in the monkey after a single dose was considered to be >100 mg/kg. These data are relevant for drug development in Oncology in light of the efforts aimed at developing anti-FGFR4 antibodies and small molecule inhibitors specific for FGFR4. Citation Format: Timothy R. Mack, Colleen Burns Burns, Xuemei Guo, William John Feaver, Marie Prewett, Jennifer Gruber, Amelie Forest, Armando R. Irizarry, Ruslan Novosiadly, Nick Loizos, Andrew Dropsey. Antitumor efficacy and non-clinical safety of the high affinity anti-FGFR4 antibody H4: Implications for targeting the FGF19-FGFR4 axis in oncology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C129.