Abstract 2537: Antibody dependent cytotoxicity is enhanced by Ari-4175 through NK cell activation
Authors: MacFarlane Alexander , Bagnyukva Tetyana , Zhang Jiping , Campbell Kerry , Jones Barry , Bachovchin William , Borghaei Hossein 
Published: May 2015

Abstract
We previously reported that the prolyl peptidase inhibitor, Ari-4175, showed dramatic anti-tumor effects in KRAS mutant colorectal cancer xengrafts when given either alone or in combination with cetuximab. In vitro data suggest that the therapeutic effect of 4175 might partially be due to the augmentation of ADCC through elevating expression of CD16 on NK cells. However, our data indicates that 4175 does not have a direct effect on isolated NK cells. Therefore, we believe that the observed effect on NK cells and augmentation of ADCC is as a result of activation of another cell type possibly working through the production of a cytokine. We noted a significant expansion of neutrophils in 4175-treated mice that occurred prior to increased expression of CD69 and CD16 on NK cells. Therefore, we hypothesized that neutrophils might be stimulated by 4175, which results in subsequent activation of NK cells to enhance their ADCC response.
Ari-4175 was administered orally to nude mice at 200 µg q.d. x5 days/week. Peripheral blood or spleens were assayed for immune parameters, ex vivo, at various time points. Expression of surface markers on myeloid and NK cells were monitored by flow cytometry. Natural cytotoxicity and ADCC were assayed using HCT116 cells and cetuximab in a flow cytometry-based assay. Neutrophil depletion experiments were accomplished by administration of an anti Ly-6G antibody (clone 1A8, cat#BE0075-1, BioXCell), specific for neutrophils, (1 mg per mouse, injected on the days 0, 2, and 4). An Isotype control antibody (rat IgG2a; cat#BE0089, BioXCell) was given at the same dose and by the sameschedule.
We were able to effectively eliminate neutrophils and the previously reported cell population (CD45+ CD3- NKp46- CD11c- CD19- CD11b+ Ly6C+ Ly6G+) that appeared to be upregulated following treatment with 4175. Preliminarily, depletion of neutrophils and this cell subset did not have any impact on ADCC augmentation or activation of NK cells. ADCC was significantly greater with 4175 and cetuximab treatment compared to 4175 alone with or without neutrophil depletion. Additionally, results of xenograft studies with panitumomab, an anti EGFR antibody that does not induce ADCC, and 4175 seem to suggest that there is less activity with this combination than with 4175 and cetuximab which is capable of inducing ADCC.Our results indicate that the immunomodulatory effects of Ari-4175 on NK cells appear to be independent of neutrophils. However, in these experiments treatment with 4175 and neutrophil depletion were initiated at the same time. Additional experiments to better elucidate the mechanism of action of this agent are in progress.
Citation Format: Alexander MacFarlane, Tetyana Bagnyukva, Jiping Zhang, Kerry Campbell, Barry Jones, William Bachovchin, Hossein Borghaei. Antibody dependent cytotoxicity is enhanced by Ari-4175 through NK cell activation. [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 2537. doi:10.1158/1538-7445.AM2014-2537