Abstract 908: Development of a cell proliferation assay to be used as a read-out system for determining the in vivo potency of bevacizumab in neutralizing the biological activity of VEGF in cancer patients
Authors: Wentink Madelon Q. , Broxterman Henk J. , Gruijl Tanja D. de , Pili Roberto , Vliet Hans J. van der , Griffioen Arjan W. , Verheul Henk M.W. 
Published: May 2015

Abstract
INTRODUCTION: Blocking of vascular endothelial growth factor (VEGF) by the monoclonal antibody bevacizumab or by VEGF-Trap is a validated clinical approach to block tumor angiogenesis. For these anti-angiogenic therapeutics there is still a major lack of knowledge on biological determinants that are predictive for clinical benefit in cancer patients. We reasoned that a test that would assess the inhibition of the biological activity of VEGF by bevacizumab in the patients’ plasma or serum during therapy would serve as a biomarker for response. We tested such an approach by measuring the inhibition of VEGF-dependent cell proliferation by patients’ serum using the Ba/F3-R2 cell line, which is a murine pre-B lymphocyte cell line stably transfected with a fusion protein, consisting of the extracellular domain of hVEGFR2 and the transmembrane and cytoplasmic domain of the mouse erythropoietin receptor. When grown in medium devoid of mIL-3, this cell line is solely dependent on VEGF for proliferation and survival.
METHODS: Ba/F3-R2 cells (licensed by the Ludwig Institute for Cancer Research, New York, NY) were seeded at a density of 200.000 cells/mL and cultured for 72 hours in the presence of either A)medium containing hVEGF pre-incubated for 1 hour with various concentrations of bevacizumab (Avastin, Roche /Genentech) or B) naïve serum or serum ( 5 or 10%) collected after one cycle of bevacizumab from 7 patients with metastatic colorectal cancer that was pre-incubated with VEGF. Serum VEGF concentrations of these patients were previously determined by ELISA Cell proliferation was subsequently quantified with the cell proliferation agent WST-1 (Roche).
RESULTS: The optimal VEGF concentration to study bevacizumab-mediated modulation of VEGF dose-dependent proliferation of Ba/F3-R2 cells was 1.25 ng/ml. At this concentration, Ba/F3-R2 cell proliferation was inhibited for 50-80% using a clinically effective bevacizumab concentration of 10 μg/mL.
Co-incubation of VEGF with naïve serum samples did not have any inhibitory effect on Ba/F3-R2 cell proliferation, while 30-90% inhibition of Ba/F3-R2 cell proliferation was observed when VEGF was co-incubated with serum of 7 patients who had received one cycle of bevacizumab treatment. Furthermore, a significant negative correlation (R2 = 0.85, p = 0.0030) was found between free -VEGF concentrations in serum after one cycle of treatment with bevacizumab and proliferation inhibition of Ba/F3-R2 cells.
CONCLUSIONS: We have developed an assay that can be used to reproducibly determine the efficacy of bevacizumab in neutralizing the biological activity of VEGF both in vitro and in patient-derived serum samples before and during treatment with bevacizumab. We propose that this assay could serve as a potential biomarker to predict response to bevacizumab and possibly other agents that target VEGF.
Citation Format: Madelon Q. Wentink, Henk J. Broxterman, Tanja D. de Gruijl, Roberto Pili, Hans J. van der Vliet, Arjan W. Griffioen, Henk M.W. Verheul. Development of a cell proliferation assay to be used as a read-out system for determining the in vivo potency of bevacizumab in neutralizing the biological activity of VEGF in cancer patients. [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 908. doi:10.1158/1538-7445.AM2014-908