Brian Belmontes

AMG 479, a fully human anti–insulin-like growth factor receptor type I monoclonal antibody, inhibits the growth and survival of pancreatic carcinoma cells

Pancreatic carcinoma is a leading cause of cancer deaths, and recent clinical trials of a number of oncology therapeutics have not substantially improved clinical outcomes. We have evaluated the therapeutic potential of AMG 479, a fully human monoclonal antibody against insulin-like growth factor (IGF) type I receptor (IGF-IR), in two IGF-IR–expressing pancreatic carcinoma cell lines, BxPC-3 and MiaPaCa2, which also differentially express insulin receptor (INSR). AMG 479 bound to IGF-IR (KD 0.33 nmol/L) and blocked IGF-I and IGF-II binding (IC50 < 0.6 nmol/L) without cross-reacting to INSR. AMG 479 completely inhibited ligand-induced (IGF-I, IGF-II, and insulin) activation of IGF-IR homodimers and IGF-IR/INSR hybrids (but not INSR homodimers) leading to reduced cellular viability in serum-deprived cultures. AMG 479 inhibited >80% of basal IGF-IR activity in BxPC-3 and MiaPaCa2 xenografts and prevented IGF-IR and IGF-IR/INSR hybrid activation following challenge with supraphysiologic concentrations of IGF-I. As a single agent, AMG 479 inhibited (∼80%) the growth of pancreatic carcinoma xenografts, and long-term treatment was associated with reduced IGF-IR signaling activity and expression. Efficacy seemed to be the result of two distinct biological effects: proapoptotic in BxPC-3 and antimitogenic in MiaPaCa2. The combination of AMG 479 with gemcitabine resulted in additive inhibitory activity both in vitro and in vivo. These results indicate that AMG 479 is a clinical candidate, both as a single agent and in combination with gemcitabine, for the treatment of patients with pancreatic carcinoma.[Mol Cancer Ther 2009;8(5):1095–105]

Selective and Potent Raf Inhibitors Paradoxically Stimulate Normal Cell Proliferation and Tumor Growth

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., V600EB-Raf A375, IC50 on cells = 2 nmol/L; ED50 on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor–induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients. Mol Cancer Ther; 9(8); 2399–410. ©2010 AACR.

Broad Antitumor Activity in Breast Cancer Xenografts by Motesanib, a Highly Selective, Oral Inhibitor of Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors

Purpose: Angiogenesis plays a critical role in breast cancer development and progression. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates endothelial cell proliferation and survival. We investigated the effects of motesanib, a novel, oral inhibitor of VEGF receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit receptor, on the growth of xenografts representing various human breast cancer subtypes. Experimental Design: Athymic nude mice were implanted with MCF-7 (luminal) or MDA-MB-231 (mesenchymal) tumor fragments or Cal-51 (mixed/progenitor) tumor cells. Once tumors were established, animals were randomized to receive increasing doses of motesanib alone or motesanib plus cytotoxic chemotherapy (docetaxel, doxorubicin, or tamoxifen). Results: Across all three xenograft models, motesanib treatment resulted in significant dose-dependent reductions in tumor growth, compared with vehicle-treated controls, and in marked reductions in viable tumor fraction and blood vessel density. No significant effect on body weight was observed with compound treatment compared with control-treated animals. Motesanib did not affect the proliferation of tumor cells in vitro. There was a significantly greater reduction in xenograft tumor growth when motesanib was combined with docetaxel (MDA-MB-231 tumors) or with the estrogen receptor modulator tamoxifen (MCF-7 tumors), compared with either treatment alone, but not when combined with doxorubicin (Cal-51 tumors). Conclusions: Treatment with motesanib alone or in combination with chemotherapy inhibits tumor growth in vivo in various models of human breast cancer. These data suggest that motesanib may have broad utility in the treatment of human breast cancer.

Ganitumab (AMG 479) Inhibits IGF-II–Dependent Ovarian Cancer Growth and Potentiates Platinum-Based Chemotherapy

Purpose: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. Experimental Design: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I–, IGF-II–, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. Results: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho–IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I–, IGF-II–, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. Conclusions: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho–IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947–58. ©2014 AACR.

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Δ746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 μg/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation. [Mol Cancer Ther 2009;8(6):1536–46]

Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte–Associated Protein Blockade

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models. Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses. Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy. Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190–202. ©2017 AACR.

Antibody-mediated neutralization of autocrine Gas6 inhibits the growth of pancreatic ductal adenocarcinoma tumors in vivo

Gas6 and its receptors Axl, Mer and Tyro‐3 (TAM) are highly expressed in human malignancy suggesting that signaling through this axis may be tumor‐promoting. In pancreatic ductal adenocarcinoma (PDAC), Gas6 and the TAM receptor Axl are frequently co‐expressed and their co‐expression correlates with poor survival. A strategy was devised to generate fully human neutralizing antibodies against Gas6 using XenoMouse® technology. Hybridoma supernatants were selected based on their ability to inhibit Gas6 binding to the receptor Axl and block Gas6‐induced Axl phosphorylation in human cells. Two purified antibodies isolated from the screened hybridomas, GMAB1 and GMAB2, displayed optimal cellular potency which was comparable to that of the soluble extracellular domain of the receptor Axl (Axl‐Fc). In vivo characterization of GMAB1 was conducted using a pharmacodynamic assay that measured inhibition of Gas6‐induced Akt activation in the mouse spleen. Treatment of mice with a single dose (100–1000 µg) of GMAB1 led to greater than 90% inhibition of Gas6‐induced phosphorylated Akt (pAkt) for up to 72 hr. Based on the target coverage observed in the PD assay, the efficacy of GMAB1 was tested against human pancreatic adenocarcinoma xenografts. At doses of 50 µg and 150 µg, twice weekly, GMAB1 was able to inhibit 55% and 76% of tumor growth, respectively (p < 0.001 for both treatments vs. control Ig). When combined with gemcitabine, GMAB1 significantly inhibited tumor growth compared to either agent alone (p < 0.001). Together, the data suggest that Gas6 neutralization may be important as a potential strategy for the treatment of PDAC.

Abstract 21: Selective and potent inhibitors of the mutant B-Raf pathway paradoxically stimulate the MAPK pathway in wild-type B-Raf cells

B-Raf is a member of the Raf family of serine/threonine kinases, which also include A-Raf and C-Raf. While all 3 Raf kinases stimulate the MAPK signaling cascade, B-Raf is the most catalytically active. B-Raf hyperactivity through mutation or over-expression is reported in many solid and hematologic malignancies. An especially high frequency (60%) of activating B-Raf mutations is found in melanoma, with the kinase domain mutant (V600E) accounting for greater than 95% of these. The identification and development of B-Raf inhibitors that selectively block signaling of the V600E B-Raf dependent MAPK pathway may have therapeutic value in these and other malignancies driven by activation of B-Raf. We report here on the characterization of novel, potent and selective small molecule Raf kinase inhibitors demonstrating potent cellular activity. Compound 1 (Smith, DeMorin et al. 2009) exhibits excellent potency in cells harboring V600E B-Raf (P-ERK IC 50 ranging from 1-14nM) however, cells that contain wild type B-Raf are significantly less sensitive to inhibition of MAPK signaling by compound 1 (P-ERK IC 50 ranging from 260nM to > 1μM). This shift in potency between V600E B-Raf versus wt B-Raf cell lines is also observed with cellular viability. In contrast to cells harboring B-Raf mutation, exposure to selected Raf inhibitors resulted in a dose-dependent, and sustained activation of MAPK signaling, increased Raf basal kinase activity and heterodimer stabilization in all wild type B-Raf cell lines examined . In the mutant KRAS MIA PaCa-2 and A549, Raf inhibition by selected compounds led to entry into the cell cycle, 80% increased proliferation, 40% increased colony formation and significantly stimulated tumor growth in vivo (Beltran et al, AACR 2010). Inhibition with structurally distinct Raf inhibitors or isoform specific siRNA knock-down of Raf demonstrated that these effects were mediated directly through Raf. Either A-Raf or C-Raf, mediated the Raf-inhibitor-induced MAPK pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in both malignant and normal cells such as HUVEC cells. Indeed mouse treatment with any of the efficacious inhibitors resulted in hyperplasia observed in the epithelial layer of mouse esophagus and stomach. These data suggest that mutant versus wild type B-Raf MAPK pathways are distinctly regulated in cells. An implication of these results is that certain Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients. Smith, A. L., F. F. DeMorin, et al. (2009). “Selective Inhibitors of the Mutant B-Raf Pathway: Discovery of a Potent and Orally Bioavailable Aminoisoquinoline.” Journal of Medicinal Chemistry 52(20): 6189-6192. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 21.

Abstract 2027: Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models

Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC Evasion of apoptosis represents an essential hallmark in the progression of many cancers. The Bcl-2 family of proteins plays a central role in regulating the apoptotic process. Targeting pro-survival Bcl-2 family members like Mcl-1 with small molecule inhibitors represents a viable therapeutic approach for the treatment of cancer. This study evaluated the in vitro and in vivo activity of AMG 176, a potent and selective Mcl-1 inhibitor currently in Phase I clinical development. AMG 176 binds with high affinity and selectivity to the BH3-binding groove of Mcl-1. In a cell based split-luciferase complementation assay, AMG 176 disrupted the interaction between Mcl-1 and Bak, leading to downstream activation of the intrinsic apoptosis pathway as measured by increased caspase activity and subsequent effects on viability. Oral administration of AMG 176 to mice bearing OPM-2 multiple myeloma xenografts resulted in a dose-dependent increase in activated Bak with a clear PK/PD relationship. Dosing regimens (20-60 mg/kg PO, QD) evaluating discontinuous and continuous administration of AMG 176 in established OPM2 xenografts demonstrated robust tumor growth inhibition with complete tumor regression at an elevated dose. Efficacy in this model was achieved at doses in agreement with those eliciting induction of apoptotic markers. Treatment of tumor cell lines with Compound A, a close structural analog of AMG 176, revealed a dose- and time-dependent increase in Mcl-1 protein levels that was reversible upon compound washout. Subsequent experiments performed with cycloheximide suggested that elevations in Mcl-1 protein levels were due to an increase in Mcl-1 protein half-life, likely driven by the compounds ability to disrupt proteasome-mediated degradation. Compound A was also used to characterize the kinetics of activating apoptosis. These studies revealed a rapid induction of apoptosis and loss of viability in Mcl-1 dependent multiple myeloma and AML cell lines. In cell lines highly dependent on Mcl-1, treatment with Compound A for as little as two hours was sufficient to achieve complete cell killing. Cell line profiling studies (>200 lines) revealed robust effects on cell viability in a subset of solid tumor cell lines and cell lines of hematological origin, including multiple myeloma, acute myeloid leukemia and non-Hodgkin lymphoma. Subsequent association analysis with molecular profiling endpoints identified an inverse correlation between BCLxL expression and sensitivity to Mcl-1 inhibition. Combination screens with Compound A revealed multiple highly synergistic combinations including compounds targeting the MAPK pathway, standard of care chemotherapeutics and agents targeting additional pro-survival members of the BCL-2 family. In conclusion, AMG 176 is a potent and selective Mcl-1 inhibitor, with significant in vitro and in vivo activity in Mcl-1 dependent cancer models. Citation Format: Sean R. Caenepeel, Brian Belmontes, Jan Sun, Angela Coxon, Gordon Moody, Paul E. Hughes. Preclinical evaluation of AMG 176, a novel, potent and selective Mcl-1 inhibitor with robust anti-tumor activity in Mcl-1 dependent cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2017-2027

Abstract C172: Efficacy of AMG 479, alone and in combination with cisplatin, in ovarian carcinoma xenograft models

AMG 479, a fully human monoclonal antibody raised to the type I insulin‐like growth factor receptor (IGF1R), is currently being studied in the treatment of patients with ovarian carcinoma. We have previously shown that AMG 479 inhibits the growth of pancreatic and sarcoma tumor models by inhibiting IGF1R signaling through ligand blockade and receptor internalization and degradation. In the present study, we tested the ability of AMG 479 to inhibit the growth of four human ovarian carcinoma xenografts (OV‐90, SK‐OV3, TOV‐21G, and OV‐CAR3) in nude mice. Recent evidence has shown that multiple players in the IGF‐axis are differentially regulated in human ovarian serous carcinomas, suggesting that the IGF1R pathway may play an important role in the growth of these tumors. The OV‐90 model showed exquisite sensitivity to single‐agent AMG 479, reaching significant (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C172.