Selam Ogbagabriel

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]

Tumor penetration and epidermal growth factor receptor saturation by panitumumab correlate with antitumor activity in a preclinical model of human cancer

BackgroundSuccessful treatment of solid tumors relies on the ability of drugs to penetrate into the tumor tissue.MethodsWe examined the correlation of panitumumab (an anti-epidermal growth factor [EGFR] antibody) tumor penetration and EGFR saturation, a potential obstacle in large molecule drug delivery, using pharmacokinetics, pharmacodynamics, and tumor growth rate in an A431 epidermoid carcinoma xenograft model of human cancer. To determine receptor saturation, receptor occupancy, and levels of proliferation markers, immunohistochemical and flow cytometric methods were used. Pharmacokinetic data and modeling were used to calculate growth characteristics of panitumumab-treated tumors.ResultsTreatment with panitumumab in vivo inhibited pEGFR, Ki67 and pMAPK levels vs control. Tumor penetration and receptor saturation were dose- and time-dependent, reaching 100% and 78%, respectively. Significant tumor inhibition and eradication (p < 0.05) were observed; plasma concentration associated with tumor eradication was estimated to be 0.2 μg/ml. The tumor inhibition model was able to describe the mean tumor growth and death rates.ConclusionsThese data demonstrate that the antitumor activity of panitumumab correlates with its ability to penetrate into tumor tissue, occupy and inhibit activation of EGFR, and inhibit markers of proliferation and MAPK signaling.

Abstract A182: U3–1287 (AMG 888), a fully human anti-HER3 mAb, demonstrates in vitro and in vivo efficacy in the FaDu model of human squamous cell carcinoma of the head and neck (SCCHN).

Background: Anti-EGFR mAbs have demonstrated clinical utility in SCCHN; however, most patients are resistant or acquire resistance to therapy. In preclinical models, activation of HER3 is a resistance mechanism to current HER inhibitors. Recent data (Wilson et al 2011) demonstrated that SCCHN cancer cell lines and primary tumors express both HER3 and heregulin, the ligand for HER3. This autocrine loop is one potential mechanism for the lack of sensitivity to anti-EGFR mAbs observed in SCCNH patients. Herein, we report in vitro and in vivo activity of U3–1287 (AMG 888) as a single agent and in combination with panitumumab, an anti-EGFR mAb, in the FaDu preclinical model of human SCCHN. Methods: FaDu (SCCHN) cells were treated with U3–1287 (AMG 888), panitumumab, the combination or control mAbs for 4 hours. To determine in vitro efficacy on cell proliferation, FaDu cells were treated with 1 μg/mL U3–1287 (AMG 888), 1 μg/ml panitumumab, the combination or control mAbs in serum containing medium. After 72 hours, the growth of treated cells was measured by ATPLite assay. Levels of pHER3, pEGFR, pERK1/2 and pAKT were measured by Western blot analyzes to determine the inhibition of HER3 oncogenic signaling. To determine in vivo efficacy, mice bearing ∼200 mm3 FaDu xenografts were treated 2×/week with U3–1287 (AMG 888), panitumumab, the combination or control mAbs. Results: Treatment with either U3–1287 (AMG 888) or panitumumab resulted in inhibition of pHER3 and pAKT or pEGFR and pERK1/2, respectively. Treatment with the combination resulted in inhibition of pHER3, pEGFR, pAKT and pERK1/2. Although treatment with panitumumab or U3–1287 (AMG 888) alone significantly inhibited in vitro cell proliferation versus control mAb treated cells (57% (p Conclusions: U3–1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling in FaDu SCCHN cells resulting in in vitro and in vivo growth inhibition. SCCHN xenografts were sensitive to U3–1287 (AMG 888) treatment as single agent or in combination with panitumumab. Combination treatment with U3–1287 (AMG 888) and panitumumab resulted durable regressions which was not observed with either single agent. These data together with the HER3/heregulin autocrine loop detected in patient samples (Wilson et al 2011) provides evidence for the potential clinical investigation of U3–1287 (AMG 888) in SCCHN. 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 A182.

Abstract LB-306: U3-1287 (AMG 888), a fully human anti-HER3 mAb, inhibits HER3 activation and induces HER3 internalization and degradation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Understanding the mechanism of action (MOA) of novel therapeutic agents is instrumental for their recommended use in the clinic. U3-1287 (AMG 888) is a novel, fully human mAb that targets HER3, a member of the EGFR family involved in tumor growth and resistance to anti-HER inhibitors. To understand its MOA, we evaluated HER3 internalization, degradation, and downstream signaling in tumor cell lines in response to U3-1287 (AMG 888) treatment. Methods: Confocal microscopy was used to locate antibody/receptor complexes. MDA-MB-453 and MDA-MB-175 breast cancer cells were serum starved overnight and treated on ice with 10 ug/mL of quantum dot 585 (Invitrogen) conjugated U3-1287 (AMG 888) and 2 ug/mL of quantum dot 655 (Invitrogen) conjugated HER2 Ab5 (Labvision). Images were taken at time 0 and after 1 hour incubation at 37°C. In addition, cell surface reduction of HER3 was measured by flow cytometry. T47D breast cancer cells were incubated at 37°C for 0, 1, 4 and 24 hours with either U3-1287 (AMG 888), c225, trastuzumab or c2C4 (10 µg/ml). The cells were re-stained with the same primary antibodies (10 µg/ml) at 4°C, and analyzed. To evaluate the consequence of longer term U3-1287 (AMG 888) treatment, BxPC3 pancreatic cells were treated from 0-96 hours in vitro and A549 NSCLC xenografts were treated for 2 weeks in vivo. Total HER3, pHER3 and pAKT were measured by Western blot and HER3 mRNA was measured by RT-PCR. Results: U3-1287 (AMG 888) treatment of MDA-MB-453 and MDA-MB-175 cells resulted in the internalization of HER3 but not HER2. Treatment of T47D cells with trastuzumab, c2C4 or c225, U3-1287 (AMG 888) resulted in a 8, 4, 0 and 73% reduction of cell surface HER2, HER2, HER1 and HER3, respectively at the 1 hr time point. A ∼75% reduction of HER3 was maintained during the 24 hr treatment with U3-1287 (AMG 888). Although a 60% reduction in total HER3 was not observed until 4 hours post treatment of BxPC3 cells with U3-1287 (AMG 888), pHER3 and pAKT were reduced >90% within 15 minutes. Neither U3-1287 (AMG 888) nor heregulin (the ligand for HER3) treatment resulted in a significant change in HER3 mRNA levels over the 96 hr treatment period. Analyses of A549 tumor xenograft tissue demonstrated a dose dependent loss of total HER3 and pHER3 in response to U3-1287 (AMG 888) administration versus IgG control treatment. Conclusions: We demonstrate that U3-1287 (AMG 888)-induced inhibition of HER3 signaling and HER3 degradation are separate events, whereby loss of HER3 receptor in response to U3-1287 (AMG 888) treatment is preceded by U3-1287 (AMG 888)-mediated inhibition of HER3 activation. HER3 transcription is not affected by U3-1287 (AMG 888) or heregulin in BxPC3 cells. These findings suggest that U3-1287 (AMG 888) mediates both inhibition of HER3 downstream signaling and HER3 degradation and that these mechanisms may contribute to the activity of U3-1287 (AMG 888) in the clinic. 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 LB-306.

Abstract B167: Identifying first in human (FIH) doses and schedule of U3‐1287 (AMG 888), a fully human anti‐HER3 mAb, based on preclinical pharmacokinetic (PK), pharmacodynamic (PD) and efficacy data

Background: HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. Although HER3 lacks intrinsic kinase activity, it serves as a scaffold for PI3K/AKT signaling for the HER family via heterodimeric interactions. The goal of this study was to use preclinical modeling to predict a minimal effective dose regimen for objective response using preclinical PK and BxPC3 xenograft mice anti‐tumor efficacy and PD data. Materials and Methods: U3‐1287 (AMG 888) concentration‐time data obtained from mice (0.025 to 2.5 mg/mouse), rats (1 to 100 mg/kg) and monkeys (1 to 200 mg/kg) were combined and analyzed using a target‐mediated drug disposition model. Animal PK parameters were scaled based on body weight to predict human PK characteristics. An Emax model was used to relate drug concentration and inhibition of pHER (measure by ELISA) in BxPC3 xenograft tumors. A PK/PD/efficacy model (based on Simeoni et al 2004) was used to analyze tumor growth data from mice bearing BxPC3 pancreatic xenografts (∼200 mm3) treated twice per week at 25, 100, 200, or 500 g for a 1 month. The model was validated with tumor growth data following additional doses of 400 g biweekly and 200 g biweekly, weekly and twice a week. The relationship between drug concentration, the inhibition of pHER3 in animals and interspecies PK scaling was used to select the minimal effective dose for the first in human study. Results: U3‐1287 (AMG 888) treatment of BxPC3 xenografts resulted in a statistically significant inhibition of tumor growth and pHER3 levels in a dose and schedule dependent manner (p Conclusion: The anti‐tumor efficacy in the BxPC3 pancreatic xenograft model was correlated with an increased serum concentration of U3‐1287 (AMG 888) and a decrease in pHER3 levels, allowing for the development of a PK/PD/Efficacy relationship. This relationship combined with interspecies PK scaling was used to determine a dose and schedule for U3‐1287 (AMG 888) to investigate in a FIH study. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B167.

Abstract B171: U3‐1287 (AMG 888), a fully human anti‐HER3 mAb, demonstrates in vitro and in vivo efficacy in NSCLC models

Background: EGFR TKIs have demonstrated activity in NSCLC, however most patients are resistant or acquire resistance to therapy. HER3 is a member of the EGFR family of receptors and is considered a target for NSCLC treatment. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism (de novo and acquired) to current HER inhibitors. Herein, we report the activity of U3‐1287 (AMG 888) in preclinical models of human NSCLC. We also evaluate human NSCLC tumor specimens for detectable pHER3, pAKT and HRG, the ligand for HER3. Methods: To determine the inhibition of HER3 oncogenic signaling, A549 and Calu‐3 NSCLC cells were treated with 100 nM of U3‐1287 (AMG 888), panitumumab, lapatinib or control for 1 hour. To determine in vitro efficacy on anchorage independent growth, A549 cells were treated with 10 g/mL U3‐1287 (AMG 888), other HER family Abs or control mAb in serum containing medium. Tumor cell colonies formed in the absence of exogenous ligand for 10 days and were stained with MTT and quantified using a Scanalyzer HTS camera imaging system. To determine in vivo efficacy, mice bearing ∼200 mm3 A549 or Calu‐3 NSCLC xenografts were treated 2x/week with anti‐HER family inhibitors or control. A549 xenograft tumors were analyzed for the inhibition of pHER3 byWestern blotting. The anti‐tumor effect of U3‐1287 (AMG 888) with EGFR inhibitors was tested in the Calu‐3 NSCLC xenograft model. Levels of pHER3, pAKT and HRG in 20 NSCLC patient specimens were determined by Western blotting. Results: Treatment with U3‐1287 (AMG 888) resulted in inhibition of pHER3 in both NSCLC cell lines. pAKT was inhibited in the A549 cell line with U3‐1287 (AMG 888) as a single agent and in A549 and Calu‐3 cells to a greater extent when combined with another HER family inhibitor. U3‐1287 (AMG 888) inhibited colony growth by 50% (p Conclusions: U3‐1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling and cell growth in NSCLC cell lines in vitro and in vivo. NSCLC xenografts are sensitive to U3‐1287 (AMG 888) treatment as single agent or in combination with the anti‐EGFR agents. The preclinical data together with the detectable levels of activated HER3 in patient samples provide evidence for the potential clinical application of U3‐1287 (AMG 888) in NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B171.

Abstract B161: U3‐1287 (AMG 888), a fully human anti‐HER3 mAb, demonstrates preclinical efficacy in HER2+ and HER2− breast cancer models

Background: HER3 is required for proliferation in HER2 amplified (HER2 + ) breast cancer cell lines, but may also be important in the HER2 negative (HER2 − ) setting. Though HER3 lacks kinase activity, it is a scaffold for PI3K signaling for the HER family and other receptor kinases via heterodimeric interactions. In preclinical models, activation of HER3 is a resistance mechanism to current HER family inhibitors. We report the efficacy of U3‐1287 (AMG 888) in HER2 + and HER2 − preclinical breast cancer models. We also evaluate human breast tumor samples for detectable levels of pHER3 and pAKT. Methods: Levels of pHER3, pERK and pAKT were determined by western blotting. To determine the inhibition of HER3 oncogenic signaling, HER2 + (SKBR3, MDA‐MB‐453 and HCC1569) and HER2 − (MDA‐MB‐175 VII) breast cancer cells were treated with 10 µg/ml of U3‐1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (250 to 1500 nM) or controls prior to heregulin (HRG) stimulation. To determine the activity on cell proliferation in the presence of 0.4% FBS, breast cancer cell lines were incubated with 10 µg/ml of U3‐1287 (AMG 888), cetuximab, c2C4, trastuzumab, lapatinib (50 to 1500 nM) or control for 1 hour prior to HRG stimulation. After 4 days, the growth of treated cells was measured with alamarBlue™. To determine the activity on anchorage‐independent growth, breast cancer cells were treated with 2 to 5 g/ml U3‐1287 (AMG 888), anti‐HER antibodies, 500 nM lapatinib, or control. Tumor cell colonies formed in the absence or presence of HRG for 6 to 10 days and were stained with MTT for 4 to 6 hours and quantified. Results: Treatment of breast cancer cell lines with U3‐1287 (AMG 888) resulted in an inhibition of pHER3 and pAKT. In cell proliferation assays, U3‐ 1287 (AMG 888) reduced heregulin‐stimulated SkBR‐3 proliferation up to 40% (p + and 10%, 35% of the HER2 − human tumor samples had detectable pHER3 and pAKT, respectively. Conclusions: U3‐1287 (AMG 888) inhibits proximal and distal HER3 oncogenic signaling in breast cell lines in vitro. Breast cancer cells are sensitive to U3‐1287 (AMG 888) treatment as single agent and in combination with anti‐HER agents. The preclinical data together with detectable levels of pHER3 in patient samples provide evidence for the potential clinical application of U3‐1287 (AMG 888) in HER2 + and HER2 − breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B161.

Abstract A150: Molecular mechanisms mediating the pharmacodynamic interactions between oxaliplatin (Ox) and epidermal growth factor receptor (EGFR) inhibitors in KRAS mutant colorectal cancer (CRC) cells.

Background: Platinum agents are a standard of care for treatment many cancers. Clinical data has shown that patients (pts) with CRC with mutant (MT) KRAS do not respond to EGFR inhibitors (Amado et al 2009). Furthermore, results from phase 3 clinical trials with erlotinib, cetuximab, or panitumumab have shown that pts whose tumors express MT KRAS have shorter progression-free survival and overall survival when an EGFR inhibitor is added to a platinum-containing regimen vs chemotherapy alone (Eberhard et al 2005, Bokemeyer et al 2011, Douillard et al 2010). However, KRAS status is not predictive of outcomes in pts receiving Ox- or irinotecan-containing regimens without an EGFR inhibitor (Richman et al 2009). This negative interaction has not been observed in pts whose tumors express either wild type (WT) KRAS receiving an EGFR inhibitor and chemotherapy or whose tumors express MT KRAS receiving an EGFR inhibitor with irinotecan (Peeters et al 2010, Van Cutsem et al 2009). Our goal was to gain a molecular mechanistic understanding of the negative interaction between EGFR inhibitors and Ox in KRAS MT CRC cells. Methods: To investigate the negative interaction between Ox and EGFR inhibitors, isogenic MT and WT KRAS-expressing HCT116 CRC cells were treated with Ox, SN-38 (the active metabolite of irinotecan), panitumumab, or gefitinib as single agents or in combination for 72 hrs. Viability was measured using an ATPlite assay. To determine the cellular distribution of EGFR, cells were serum-starved, labeled with a fluorescent anti-EGFR mAb, and visualized by a confocal microscopy. To analyze the effects of single agent or combination treatment on downstream phosphorylation of the PI3K or MAPK pathways, cells were treated for 24 hrs and phospho-proteins were detected by Western blotting. To investigate whether negative interaction between Ox and EGFR inhibitors could be reversed, cells were treated with Ox and gefitinib in combination with inhibitors to MEK, PI3K or Src for 72 hrs. Results: Treatment with gefitinib, but not panitumumab, reversed the anti-proliferative effects of Ox in the MT KRAS-expressing CRC cells vs Ox alone (p Conclusion: We developed a preclinical model to further understand the negative interaction between MT KRAS-expressing cancer cells, platinum agents, and EGFR inhibitors. Feedback though AKT may contribute to the increased resistance to Ox in KRAS MT cells. Inhibitors of MEK, PI3K, or Src reversed the negative interaction between the EGFR inhibitors and Ox in a mutant-KRAS genetic background. 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 A150.