James R. Tonra

Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth.

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.

IMC-A12, a human IgG1 monoclonal antibody to the insulin-like growth factor I receptor.

Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.

Synergistic Antitumor Effects of Combined Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor-2 Targeted Therapy

Purpose: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. Experimental Design: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. Results: Monotherapy ED50 values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1α potentially acting as molecular links between EGFR and VEGFR2 inhibition. Conclusions: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Anti-Vascular Endothelial Growth Factor Receptor-1 Antagonist Antibody as a Therapeutic Agent for Cancer

Purpose: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. Experimental Design: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. Results: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD = 54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 μg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. Conclusions: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

Targeting the platelet-derived growth factor receptor α with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: Implications as a potential therapeutic target

Platelet-derived growth factor receptor α (PDGFRα) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRα, which did not cross-react with the β form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRα. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRα. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P = 0.0004) and SKLMS-1 (P < 0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRα.

Tumor Growth Inhibition with Cetuximab and Chemotherapy in Non–Small Cell Lung Cancer Xenografts Expressing Wild-type and Mutated Epidermal Growth Factor Receptor

Purpose: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non–small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. Experimental Design: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. Results: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. Conclusions: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.

Functional significance of VEGFR-2 on ovarian cancer cells

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780‐par and HeyA8 ovarian cancer cell lines expressed VEGFR‐1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR‐2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR‐2 expression, whereas only 15% showed moderate to high VEGFR‐1 expression. By immunofluorescence, little or no VEGFR‐2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR‐2 specific antibody (1121B), murine VEGFR‐2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR‐1 antibody had no effect. Treatment with 1121B effectively blocked VEGF‐induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR‐2 is present on most ovarian cancer cells. The observed anti‐tumor activity of VEGF‐targeted therapies may be mediated by both anti‐angiogenic and direct anti‐tumor effects.

Tumors Established with Cell Lines Selected for Oxaliplatin Resistance Respond to Oxaliplatin if Combined with Cetuximab

Purpose: To establish whether cetuximab, a chimeric IgG1 antibody targeting epidermal growth factor receptor, has the potential to restore responsiveness to oxaliplatin in preclinical cancer models, as has been shown with irinotecan in irinotecan refractory metastatic colorectal cancer patients. Experimental Design: The effects of cetuximab and oxaliplatin, alone or in combination, were tested in vitro and in vivo using human colorectal cancer cell lines selected for oxaliplatin resistance, as well as parental control cell lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu athymic mice, as well as activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair genes, density of apurinic/apyrimidinic DNA damage, and accumulation of platinum-DNA adducts in vitro. Results: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft models was greater than that of monotherapies and independent of the responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced expression of excision repair cross-complementation group 1 and XPF, which are key components of the nucleotide excision repair pathway involved in the excision of platinum-DNA adducts. In addition, cetuximab reduced expression of XRCC1, a component of the base excision repair pathway responsible for the repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair protein levels were downstream to effects on mitogen-activated protein kinase and AKT pathway activation. In line with effects on DNA repair protein expression, cetuximab increased the accumulation of platinum and apurinic/apyrimidinic sites on DNA during oxaliplatin treatment. Conclusions: Cetuximab has the potential to salvage the benefits of oxaliplatin in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair capacity.

Tumor necrosis factor-alpha and interleukin-1 antagonists alleviate inflammatory skin changes associated with epidermal growth factor receptor antibody therapy in mice.

Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.

Abstract 2590: KD019: Blood brain barrier penetrant HER2/neu, Src, and EGFR inhibitor

KD019 is an orally bioavailable small molecule inhibitor of molecular drivers of cancer growth and progression, including HER2/neu, Src family nonreceptor tyrosine kinases (Src) and EGFR. This profile of activity underlies the robust activity of KD019 in animal models of cancer. Although alternative agents targeting these tyrosine kinase receptors are available, only KD019 targets all of these pathways without the need for a combination strategy. A more important shortcoming of alternative agents is a lack of blood brain barrier (BBB) penetration (Brain/Plasma concentration ratio In the present work, KD019 is demonstrated to achieve concentrations in the brain equivalent to that in blood utilizing quantitative whole body autoradiography (QWBA). Lister Hooded partially pigmented rats were administered a single dose of 14C labeled KD019 and tissue radioactivity was evaluated utilizing phosphor-storage imaging plates and a Fuji FLA-5100 fluorescent image analyzing system (Quotient Bioresearch, Rushden, UK). Brain/blood radioactivity ratio was approximately 1 at 6-24 hours after dosing indicating significant brain penetration of KD019. BBB penetrance was further supported by a study performed in CD1 mice utilizing LC/MS/MS to determine tissue KD019 concentrations following a single oral dose of 100 mg free base/kg unlabeled drug (Pharmaron, Beijing, China). Brain/plasma KD019 ratios were 2.3-4.4 from 1-24 hours after dosing with brain concentrations of 2137-8253 ng/g tissue, compared to 0.03-0.07 for the EGFR/HER2 inhibitor lapatinib with brain concentrations of 0-286 ng/g tissue. To demonstrate that the penetration of KD019 into the brain parenchyma would translate into anticancer effects, KD019 efficacy was tested in a GL261 orthotopic syngeneic model of glioma (Molecular Imaging, Michigan, USA). GL261-luc2 luciferase expressing cells were implanted intracranially (2 mm right lateral and 1 mm anterior from Bregma, 2-3 mm down from burr hole) into C57BL/6 albino mice. KD019 dosed orally once daily at 75 mg base/kg on Days 8-12 and 15-19 after intracranial implantation significantly reduced the intracranial tumor growth as evaluated by bioluminescence, and extended median survival time by 20%. In conclusion, KD019 is a BBB penetrant TKI with an important and novel profile of kinase activity that, based on the data presented, will be advanced further in nonclinical and clinical experiments aiming at benefiting patients with intracranial tumors, a significant unmet clinical need. KD019 is currently being tested in combination with trastuzumab in patients with HER2-positive breast cancer and brain metastases. Citation Format: James R. Tonra, Masha Poyurovsky, Kevin G. Liu, Jeegar Patel, Nishta Rao, Robert Tilton, John L. Ryan, Mark S. Berger, Larry Witte, Ji-In Kim, Samuel D. Waksal. KD019: Blood brain barrier penetrant HER2/neu, Src, and EGFR inhibitor. [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 2590. doi:10.1158/1538-7445.AM2015-2590