Abstract A58: Antibodies to amphiregulin, an abundant growth factor in patients' fluids, inhibits tumor growth
Authors: Lindzen Moshit , Carvalho Silvia , Yarden Yosef 
Published: May 2016

Abstract
It is well established that growth factors of the EGF family are involved in tumor progression and, accordingly, antibodies that intercept a cognate receptor, EGFR/ERBB1, or a co-receptor, HER2, have been approved for cancer therapy. So far, no anti-ligand antibody has been clinically approved. Hence, our studies currently address the therapeutic potential of intercepting one or more ligands of the EGF family of growth factors. We began by assaying the repertoire of EGF-like ligands present in body fluids collected from advanced stage ovarian and lung cancer patients. Analysis of ovarian ascites fluids identified AREG as the most frequently secreted factor expressed (86%), with relatively high concentrations of the growth factor (10-1800 pg/ml). A parallel analysis of pleural effusions from lung cancer patients showed similar results. The analysis also identified TGF-alpha, HB-EGF, EGF, NRG1 and BTC, but they were not only detected in fewer patients, their levels rarely exceeded 200 pg/ml. Taken together, these results identifed AREG as a most prevalent EGF-like growth factor of human cancer.
The majority of body fluids we analyzed were derived from patients treated with chemotherapy, therefore, we examined the possibility that high level secretion of AREG by cancer cells might be attributable to prior exposure to chemotherapeutic agents, such as cisplatin. Hence, we introduced a reporter DNA construct containing the promoter region of AREG into ovarian and lung cancer cells. Upon treatment of cells with increasing concentrations of cisplatin, we observed increases in the reporter activity. Moreover, exposure of both cancer cells to the chemotherapeutic agent was followed by increased secretion of AREG, but not EGF, TGF-alpha or HB-EGF, to the medium, further supporting specificity of the cisplatin effect to the AREG promoter.
Because AREG depletion from human ovarian cells retarded their tumorigenic growth as xenografts in mice, we generated a neutralizing monoclonal anti-AREG antibody. As expected the antibody intercepted AREG-induced phosphorylation of EGFR. This prompted us to investigate the impact on tumorigenic growth of human cancer cells. In the first set of experiments we used three different anti-AREG antibodies to treat mice injected with three different human cancer cell lines: MLS (ovarian), BxPC3 (pancreas) and Cal27 (head and neck). With all cell lines we observed different levels of tumor growth inhibition. Because antibody AR30 showed partial inhibition of ovarian tumor growth, our next set of animal studies tested the prediction that chemotherapy-induced up-regulation of AREG secretion supports tumor growth under platinum-based treatment. Accordingly, mice were treated without or with AR30, along with cisplatin. The results showed that as single agents, both cisplatin and the AR30 antibody only mildly inhibited MLS tumors. Nonetheless, the combination of cisplatin and AR30 almost completely inhibited tumor growth.
In summary, we detected relatively high concentrations of AREG in the majority of advanced ovarian and lung cancer patients. Intercepting AREG using anti-AREG antibodies inhibited growth of xenografts and strongly enhanced chemotherapy efficacy. Taken together, these results raise the possibility that AREG and other binders of EGFR might serve as potential targets for cancer therapy.
Citation Format: Moshit Lindzen, Silvia Carvalho, Yosef Yarden. Antibodies to amphiregulin, an abundant growth factor in patients' fluids, inhibits tumor growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A58.