Esther Zwick-Wallasch

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.

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

Abstract The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.

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

Abstract 3148: Immunohistochemical analysis of HB-EGF expression in breast, colon, gastric, hepatocellular, lung, ovarian, pancreatic and prostate cancer

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family of proteins. It is synthesized as a transmembrane protein, which can be cleaved at the cell surface to release the soluble ectodomain sHB-EGF. HB-EGF has been implicated in several physiological and pathological processes, including tumorigenesis and metastasis. Anti-HB-EGF therapeutic monoclonal antibodies are currently under investigation and hold promise for treating multiple types of cancer. In order to investigate the relationship between HB-EGF expression in cancer and response to therapy, an immunohistochemical assay to HB-EGF was developed and validated. Antigen retrieval and antibody titration studies were performed for six antibodies in order to select an antibody with proper performance characteristics. The antibodies were tested in characterized xenografts, characterized cell lines, placenta (HB-EGF-positive) and/or ovarian cancer. Of the antibodies tested, the AF-259-NA goat polyclonal antibody demonstrated the appropriate staining pattern in xenografts, cell lines and placenta. The optimized immunohistochemistry assay was used to stain cancer samples, and the frequency of moderate (2+) or strong (3+) staining in cancer cells was as follows: gastric = 50%, hepatocellular = 50%, lung = 40%; ovarian = 38%, colon = 37%, breast-ER+ = 20%, bladder = 10%; breast-HER2+ = 10%, pancreatic = 10%, breast-triple negative = 0%, prostate = 0%. When compared by average H-score, the cancer ranked in the following order (H-score in parenthesis): Hepatocellular (69), ovarian (37), gastric (23), breast-HER2+ (14), breast-ER+ (12), pancreatic (10), lung (5), breast-triple negative (2), colon (2), bladder (1), prostate (1). For comparison, H-scores for normal tissues were as follows: Normal bladder epithelium (74), stomach (24), colon (8), prostate (3), ovary (1), liver (1), breast (0), lung (0) and epidermis (0). In summary, immunohistochemistry using the AF-259-NA antibody produces the expected staining pattern in characterized samples and can be applied to human cancer samples to evaluate the relationship between clinical response and HB-EGF expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3148. doi:10.1158/1538-7445.AM2011-3148