Abstract 3971: Impact of various first- and second-generation tyrosine-kinase inhibitors on frequency and functionality of immune cells.
Authors: Pawlowski Nina , Hoerzer Helen , Singh-Jasuja Harpreet , Hilf Norbert 
Published: August 2014

Abstract
Combining immunotherapeutic approaches with targeted therapies like tyrosine-kinase inhibitors (TKIs) may give additional benefits to patients with VEGF-driven tumors (e.g. RCC). Previously, we have investigated the impact of first-generation TKIs sorafenib and sunitinib demonstrating distinct, in part opposing effects on immune cells (Hipp et al, Blood 2008, 111(12): 5610ff). Meanwhile, second-generation TKIs (pazopanib, axitinib, tivozanib) were approved or are expected to receive approval soon. In contrast to the multi-targeting first-generation TKIs, these TKIs act more selectively, therefore potentially inducing less off-target toxicities. However, so far little is known about the immunomodulatory properties of these new TKIs. Thus, we compared the influence of two first- and three second-generation TKIs on frequency and function of immune cells and on immune responses in mice.
C57BL/6 mice were orally treated with vehicle, 25 mg/kg axitinib, 20 mg/kg tivozanib, 30 mg/kg pazopanib, 30 mg/kg sorafenib or 40 mg/kg sunitinib (daily for one week). Twelve hours after the last application, splenocytes were analyzed for CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Tivozanib and (as published before) sunitinib significantly reduced the frequency of Tregs among CD4+ cells, while no effect was observed for axitinib, pazopanib and sorafenib. Moreover, percentages of CD4+ as well as CD8+ T cells were increased in tivozanib- and sunitinib-treated mice.
The functional status of isolated immune cells from TKI pre-treated mice was assessed in a mixed lymphocyte reaction (MLR). CD4+ T-cell proliferation of cells isolated from sorafenib-treated mice was diminished, while CD8+ T-cell proliferation of cells from sunitinib-treated mice was increased. In vitro activation assays revealed that - irrespective of the used TKI - the treatment led to reduced responsiveness of immune cells in terms of cytokine secretion and expression of surface activation markers.
Finally, C57BL/6 mice were immunized subcutaneously twice (days 1 and 8) with ovalbumin peptide vaccine alone (negative control) or combined with poly-IC (positive control). To analyze the effect of TKIs in vivo, mice were pretreated (day -7 to -3) or concurrently treated (day 1 - 13) with axitinib, tivozanib or pazopanib in addition to vaccinations with peptide plus poly-IC. On day 15, induction of peptide-specific T cells was analyzed by MHC multimer staining as well as by IFN-γ ELISPOT. Pretreatment or concurrent treatment with all three second-generation TKIs did not negatively impact the induction of immune responses - in contrast to concurrent treatment with sorafenib reported before.
In conclusion, these results suggest that immunotherapy and second-generation TKIs can be combined in the treatment of cancer, but the influence of these TKIs on the immune system requires further assessment in clinical trials.
Citation Format: Nina Pawlowski, Helen Hoerzer, Harpreet Singh-Jasuja, Norbert Hilf. Impact of various first- and second-generation tyrosine-kinase inhibitors on frequency and functionality of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2013-3971