Abstract 1832: Anti-nicastrin monoclonal antibodies as therapeutics for invasive breast cancer: Characterization and function

Abstract

Abstract Background: Nicastrin (NCT) is a crucial structural and functional constituent of the gamma-secretase (GS) enzyme which activates a range of substrates (Notch1-4, CD-44, Her-4, E-cadherin, EpCAM etc.), with a causative role in development and progression of malignant disease. NCT is the only GS component with a single-pass transmembrane- and a large extracellular domain (ECD). This makes NCT a potential target for monoclonal antibody (Ab) therapy. NCT has a GS-substrate recognition role, while other relevant functional regions of NCT ECD are still unknown. We have previously determined that NCT is overexpressed in breast cancers (BC) where its abundance confers worse overall survival in the estrogen receptor negative patients and that its genetic silencing inhibit BC cell invasion (Filipovic et al, Br Can Res Tr 2011). Methods: Using genetic immunization with NCT cDNA, we raised and cloned rat hybridomas expressing anti-NCT monoclonal Abs (NMA). Four clones (NMA1-4) were selected and purified, based on their ability to recognise cell surface NCT on BC cells and potency to inhibit BC cell invasion/proliferation in vitro. Non-cell based binding of whole Ab IgGs was measured by ELISA. Cell-based binding was assessed by FACS. BIACore 3000 was used for initial epitope mapping. Antigen for ELISA and BIACore was the NCT-ECD protein.Results: From the four clones, three induce functional cellular effects (NMA2-4). NMA1 clone recognises cellular, cell surface NCT but has no functional effects on BC cells and was used as negative control in all experiments. In MDA-MB-231 cells, Abs NMA2 and NMA4 were equally potent at inhibiting invasion (>60%). NMA4 and NMA2 both arrested MDA-MB-231 cell proliferation up to 50%. In 3D Matrigel, NMA2 and NMA4 conserved growth inhibitory effect and induced re-polarization of BC acini. By ELISA, the highest Kd was determined for NMA4. Cell-based quantitative FACS on non-permeabilized MDA-MB231s allowed determination of Ab binding affinity to the endogenous antigen. Binding affinity could be ranked in the following order: NMA1 = NMA2 = NMA4 > NMA3. BIACore competitive binding determined that NMA2 and NMA4 Abs bind spatially distinct regions on NCT. Cell fractionation and western blotting showed that NMA4 recognizes cell-membrane NCT. NMA2 and NMA4 reduced Notch activation and vimentin levels in MDA-MB231 cells by >50%. Conclusion: Our results suggest that anti-NCT Abs may be potential therapeutics for invasive breast cancer. Data will be presented which compare the relationship between BIAcore binding affinity, cross-competition for NCT binding site and functional inhibition of cell proliferation and invasion, indicating relevance of distinct NCT domains for distinct cellular processes. 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 1832. doi:1538-7445.AM2012-1832