Thore Hettmann

Monoclonal Antibody Blockade of the Human Eag1 Potassium Channel Function Exerts Antitumor Activity

The potassium channel ether à go-go has been directly linked to cellular proliferation and transformation, although its physiologic role(s) are as of yet unknown. The specific blockade of human Eag1 (hEag1) may not only allow the dissection of the role of the channel in distinct physiologic processes, but because of the implication of hEag1 in tumor biology, it may also offer an opportunity for the treatment of cancer. However, members of the potassium channel superfamily are structurally very similar to one another, and it has been notoriously difficult to obtain specific blockers for any given channel. Here, we describe and validate the first rational design of a monoclonal antibody that selectively inhibits a potassium current in intact cells. Specifically blocking hEag1 function using this antibody inhibits tumor cell growth both in vitro and in vivo. Our data provide a proof of concept that enables the generation of functional antagonistic monoclonal antibodies against ion channels with therapeutic potential. The particular antibody described here, as well as the technique developed to make additional functional antibodies to Eag1, makes it possible to evaluate the potential of the channel as a target for cancer therapy.

Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

Background During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective–retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. Methods HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. Results The subgroup of patients with high HRG mRNA levels (“HRG-high”) demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies. Conclusions The prospective–retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined. ClinicalTrials.gov Number NCT02134015.

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 2519: U3-1565, a fully human anti-HB-EGF monoclonal antibody, inhibits oncogenic signaling and tumor cell growth in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: U3-1565 is a fully human monoclonal antibody directed against human heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family of ligands that bind to and activate the EGFR and HER4. HB-EGF-mediated activation of its target receptors results in oncogenic signaling. Methods: To determine inhibition of basal EGFR phosphorylation, human tumor cell lines were treated with U3-1565 or IgG2 and pEGFR levels were analyzed by ELISA. Inhibition of in vivo angiogenesis was analyzed by implanting HUVEC-based spheroids under the skin of SCID mice, which were treated with U3-1565, bevacizumab or PBS and analyzed for human neovasculature. To determine in vivo efficacy, SCID mice bearing EFO27-CL58 ovarian cancer xenografts were treated weekly with U3-1565, Cisplatin or with a combination of U3-1565 and Cisplatin. To perform xenograft analysis tumor-bearing mice were treated with U3-1565, cetuximab and erlotinib and primary xenograft tissue was collected, lyzed and pEGFR and pERK levels were analyzed by Western blotting. To determine expression of HB-EGF and pEGFR in human tumor tissue, Immunohistochemistry (IHC) stainings of human carcinoma samples were performed. Results: Inhibition of basal pEGFR levels by U3-1565 in comparison to control IgG2 treatment was demonstrated in various cancer cell lines. U3-1565 showed dose-dependent inhibition of HB-EGF-stimulated pHER4 and pERK activation in vitro (data not shown). Using a HUVEC-based spheroid in vivo system, inhibition of human endothelial cell sprouting by U3-1565 was shown. In a HB-EGF overexpressing ovarian cancer xenograft model U3-1565 demonstrated in vivo tumor cell growth inhibition as a single agent. Combination treatment of U3-1565 with Cisplatin resulted in tumor regression during the treatment phase and prevented re-growth of xenograft tumors after treatment stop. Analysis of EFO27-CL58 tumor xenograft tissue demonstrated strong reduction of pEGFR and partially reduced pERK levels after U3-1565 in vivo treatments. IHC staining of fresh human tumor samples revealed an overlapping expression pattern of HB-EGF and pEGFR. Conclusions: Our results demonstrate that U3-1565 inhibits basal activation of EGFR activation in vitro and reduces neo-vasculature formation (angiogenesis) in vivo. In a tumor xenograft model U3-1565 demonstrated tumor cell growth arrest as a single agent or in combination with Cisplatin or erlotinib. Analysis of xenograft material after in vivo exposure to U3-1565 strongly suggest that U3-1565s anti-tumor activity is based on inhibition of EGFR activation and prevention of oncogenic MAPK signaling. These data together with the evidence that human HB-EGF and activated EGFR are coexpressed in human tumor samples provide preclinical rational to develop U3-1565 as a clinical mAb candidate to treat cancer in human patients. 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 2519. doi:1538-7445.AM2012-2519