Iring Heisler

Anetumab Ravtansine - a Novel Mesothelin-Targeting Antibody-Drug Conjugate Cures Tumors with Heterogeneous Target Expression Favored by Bystander Effect

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody–drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC50 of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors. Mol Cancer Ther; 13(6); 1537–48. ©2014 AACR.

BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I

The activation of the transcription factor hypoxia‐inducible factor‐1 (HIF‐1) plays an essential role in tumor development, tumor progression, and resistance to chemo‐ and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase‐driven HIF‐1 reporter cell line under hypoxia. The high‐throughput screening led to the identification of a class of aminoalkyl‐substituted compounds that inhibited hypoxia‐induced HIF‐1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87‐2243 was found to inhibit HIF‐1α and HIF‐2α protein accumulation under hypoxic conditions in non‐small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF‐1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87‐2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase‐2. Antitumor activity of BAY 87‐2243, suppression of HIF‐1α protein levels, and reduction of HIF‐1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87‐2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87‐2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87‐2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia‐induced HIF‐1 activity in tumors might be an interesting therapeutic approach to overcome chemo‐ and radiotherapy‐resistance of hypoxic tumors.

Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Significance This paper reports the first structure of WT-human glucose transporter 1 (hGLUT1), to our knowledge, cocrystallized with inhibitors. The structures provide a template to develop therapeutic inhibitors applicable to cancers, because cancer cells become dependent on greatly increased glucose consumption. This dependence results in up-regulation of glucose transporter expression, especially hGLUT1. The bound inhibitors include the natural compound cytochalasin B and two of a series of previously undescribed organic compounds that bind in the submicromolar range. Our results emphasize that modulation of glucose import by hGLUTs should focus on making good interaction points for compounds and that the actual chemical backbone of the inhibitor is of less importance. Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1–4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

Identification of novel GLUT inhibitors

The compound class of 1H-pyrazolo[3,4-d]pyrimidines was identified using HTS as very potent inhibitors of facilitated glucose transporter 1 (GLUT1). Extensive structure-activity relationship studies (SAR) of each ring system of the molecular framework was established revealing essential structural motives (i.e., ortho-methoxy substituted benzene, piperazine and pyrimidine). The selectivity against GLUT2 was excellent and initial in vitro and in vivo pharmacokinetic (PK) studies are encouraging.

Abstract 1442: Effects of selective and broad glucose transporter (GLUT) inhibition on glucose distribution in tumor bearing mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose uptake in malignant cells has been associated with upregulated expression of glucose transporters, mainly overexpression of GLUT1 and/or GLUT3. There is limited knowledge about how selective (e.g. GLUT1) versus broad (multi-) GLUT inhibition affects glucose homeostasis in tumor bearing mice. Using potent small molecule inhibitors, we compared [14C]-2-Deoxy-D-Glucose (2-DG) distribution after selective GLUT1 versus GLUT1, 3 and 4 [multi-GLUT] inhibition and versus control in human NSCLC NCI-H460 tumor bearing mice. A single dose of a GLUT1 selective and a multi-GLUT inhibitor were administered to NCI-H460 tumor bearing NMRI nu/nu mice. At the respective Cmax concentrations, a bolus of 2-DG was rapidly injected intra-peritoneally and the distribution of metabolically stable 2-DG was obtained using whole-body autoradiography after 15 min and 120 min. With the multi-GLUT inhibitor only a very short inhibition of 2-DG uptake was observed in the NCI-H460 tumors while a long-lasting inhibition was detected in heart, brain and brown fat tissue. In contrast, a long-lasting inhibition of 2-DG uptake was observed in NCI-H460 tumors for the selective GLUT1 inhibitor. 2-DG concentrations were reduced in the brain following administration of the selective GLUT1 inhibitor at 15 min and returned to normal levels at 120 min while the tumor 2-DG concentration stayed low. The 2-DG findings go in parallel with the histopathological findings present in the brain and heart after treatment with the multi-GLUT inhibitor. However, similar histopathological findings have not been observed in the brain and heart after treatment with the selective GLUT-1 inhibitor. Therefore, selective GLUT1 inhibition is associated with a sustained low 2-DG concentration in the NCI-H460 tumors while only minor changes in glucose homeostasis were observed in other organ systems. Citation Format: Melanie Heroult, Wolfram Steinke, Anna-Lena Frisk, Sandra Borkowski, Kirstin Meyer, Heike Petrul, Iring Heisler, Maria Quanz, Roland Neuhaus, Bernd Buchmann, Thomas Mueller, Marcus Bauser, Andrea Haegebarth, Michael Brands, Karl Ziegelbauer. Effects of selective and broad glucose transporter (GLUT) inhibition on glucose distribution in tumor bearing mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2014-1442