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. 2017 May:46:112-123.
doi: 10.1016/j.intimp.2017.02.028. Epub 2017 Mar 7.

Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo

Melek M E Sunay  1 , Jeremy B Foote  2 , James M Leatherman  3 , Justin P Edwards  3 , Todd D Armstrong  3 , Christopher J Nirschl  3 , Jessica Hicks  4 , Leisha A Emens  5
Affiliations

Sorafenib combined with HER-2 targeted vaccination can promote effective T cell immunity in vivo

Melek M E Sunay et al. Int Immunopharmacol. 2017 May.

Abstract

The tumor microenvironment (TME) is established and maintained through complex interactions between tumor cells and host stromal elements. Therefore, therapies that target multiple cellular components of the tumor may be most effective. Sorafenib, a multi-kinase inhibitor, alters signaling pathways in both tumor cells and host stromal cells. Thus, we explored the potential immune-modulating effects of sorafenib in a murine HER-2-(neu) overexpressing breast tumor model alone and in combination with a HER-2 targeted granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting vaccine (3T3neuGM). In vitro, sorafenib inhibited the growth of HER-2 overexpressing NT2.5 tumor cells, inducing apoptosis. Sorafenib also interfered with ERK MAPK, p38 MAPK, and STAT3 signaling, as well as cyclin D expression, but did not affect HER-2 or AKT signaling. In vivo, single agent sorafenib disrupted the tumor-associated vasculature and induced tumor cell apoptosis, effectively inducing the regression of established NT2.5 tumors in immune competent FVB/N mice. Immune depletion studies demonstrated that both CD4+ and CD8+ T cells were required for tumor regression. Sorafenib treatment did not impact the rate of tumor clearance induced by vaccination with 3T3neuGM in tumor-bearing FVB/N mice relative to either sorafenib treatment or vaccination alone. In vivo studies further demonstrated that sorafenib enhanced the accumulation of both CD4+ and CD8+ T cells into the TME of vaccinated mice. Together, these findings suggest that GM-CSF-secreting cellular immunotherapy may be integrated with sorafenib without impairing vaccine-based immune responses.

Keywords: Breast cancer; Immunity; Immunotherapy; Sorafenib; Vaccine.

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Conflict of interest statement

Conflict of interest

Under a licensing agreement between Aduro Biotech and the Johns Hopkins University, the University and Dr. Emens are entitled to milestone payments and royalty on sales of the GM-CSF-secreting breast cancer vaccine. The terms of these arrangements are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

Figures

Fig. 1
Fig. 1
Sorafenib inhibits growth of HER2-overexpressing cells in vitro. (A) NT2.5 cells were treated in vitro with varying concentrations of sorafenib ranging from 0 to 10 μM and analyzed for growth by MTT assay 24, 48 or 72 hour post-treatment. (B) NT2.5 cells were treated with sorafenib for 24 h and stained for Annexin V and 7-AAD and analyzed by flow cytometry. (C) NT2.5 cells were treated with sorafenib for 2 h and then cells were harvested, quantified for protein and analyzed by Western blot for HER2 pathway targets MEK1/2, ERK1/2, p38, STAT3, and AKT. (D) NT2.5 cells were treated with 5 μM and 10 μM sorafenib or (E) MAPK inhibitors for 6 to 7 h and then cells were harvested for protein and analyzed by Western blot for cyclin D1, D2, D3, BCL2 and BCLXL expression. All figures are representative of 2–3 independent experiments.
Fig. 2
Fig. 2
Sorafenib inhibits growth of breast tumor cells in vivo. FVB/N mice (n = 10) were tumor challenged at Day 0 and began sorafenib or vehicle treatment on Day 10 and were followed for tumor growth (A) and overall survival (B). (C) Tumors from FVB/N mice were prepared for histological examination 3 weeks after drug treatment as described in (A). Representative samples of mice treated with vehicle (top) or sorafenib (bottom) are shown with H&E staining or immunohistochemistry to detect endothelial cells (PECAM/CD31) or apoptotic cells (activated caspase 3), at 10× magnification. Staining was quantified using Nikon Elements software.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig. 3
Fig. 3
T cells are required for sorafenib-mediated targeting of NT2.5 cells. The experiment in Fig. 2A was repeated in the setting of T cell depletion. Additional groups were included where CD4+ or CD8+ T cells were depleted individually (Sor-CD4, Sor-CD8) or together (Sor-CD4/CD8) prior to the initiation of sorafenib therapy, and then followed for tumor growth (A). NK cells and macrophages were also depleted (B). In a separate experiment, FVB/N (n = 6) were tumor challenged at Day 0 and began sorafenib or vehicle treatment on Day 10. Treatment was ceased in sorafenib-treated mice that completely rejected tumors. After one week, mice were re-challenged on the contralateral side 28 days post tumor clearance and followed for tumor growth at the original site and the re-challenge site. Tumor outgrowth, (C) and survival (D) are shown.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig. 4
Fig. 4
Sorafenib can be effectively combined with vaccine in FVB/N mice. FVB/N mice (n = 10) were tumor challenged on Day 0, vaccinated on Day 7 and then given daily (×5) sorafenib or vehicle treatment beginning on Day 7. Animals were followed for tumor growth (A) and tumor-free survival (B). FVB/N mice (n = 10) were tumor challenged, and on Day 7 were vaccinated and daily sorafenib or vehicle treatment was initiated. Two weeks post-vaccination, splenic CD8+ effector T cells were isolated and used for IFNγ ELISPOT with (C) NT2.5B7.1 or 3T3neuB7.1 or (D) p50 or NP peptide-pulsed T2dq cells. Data is representative of 3 independent experiments of 10 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig. 5
Fig. 5
Sorafenib treatment increases numbers of IFNγ producing CD4+ T and total CD8+ T cells in the TME. FVB/N mice (n = 10–25) were tumor challenged at Day 0, began sorafenib or vehicle treatment with or without 3T3GM or 3T3GMneu vaccination on Day 10 and at Day 22 (A) numbers of total CD4+ T cells, (B) IFNγ producing CD4+ T cells, (C) CD8+ T cells, and (D) IFNγ producing CD8+ T cells were evaluated within the tumor infiltrate by flow cytometry and intracellular cytokine staining. Data is cumulative from 3 experiments of 3–5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig. 6
Fig. 6
Sorafenib therapy does not affect HER-2 specific CD8+ T cell priming, migration or function. (A) FVB/N mice (n = 13/group) were tumor challenged (Day 0), then vaccinated and treated with either daily doses of sorafenib or vehicle beginning on 7 days after tumor implantation. HER-2 specific CD8+ T cells were adoptively transferred into tumor bearing FVB/N mice 24 h after vaccination and mice were euthanized 5 days later to assess HER-2 specific CD8+ T cell expansion, migration and IFNγ production. (B) Tumor weights in each treatment group were assessed. (C) Numbers of adoptively transferred Thy1.2+ CD8+ T cells in the spleen and (D) tumor were quantified. (E) IFNγ production from Thy1.2+ CD8+ T cells isolated from tumor draining lymph nodes was evaluated after a 6 hour co-culture with either p50 or NP pulsed T2Dq antigen presenting cells. Samples were analyzed by flow cytometry, with the number of positive cells normalized to cell number (spleen) or tissue weight (tumor). Data is cumulative from 3 experiments of 3–5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig. 7
Fig. 7
Sorafenib monotherapy activates and increases IL-12 production by tumor associated macrophage. At 7 days post-NT.25 tumor implantation, tumor-bearing FVB-N mice were treated with daily doses of either vehicle or 30 mg/kg sorafenib. Treatment was continued for 12 days. On Day 12 post-therapy tumors were harvested and TIL was evaluated for (A) Tumor associated macrophage (TAM) identified as CD11b+ Ly6C- Ly6G- F480+ myeloid cells. (B) Numbers of TAMs and their (C) MHCII expression were evaluated in vehicle and sorafenib treated mice. CD11b+ F480+ MHCII+ myeloid cells were isolated from vehicle and sorafenib treated, tumor bearing FVB/N mice at 12 days post treatment. (D) Expression of IL-12 and IL-10 mRNA was evaluated by QPCR. (E–F) CD4+ T cells isolated from (E) spleens and (F) TIL were evaluated for the TH1 polarizing transcription factor T-bet and the TH2 polarizing transcription factor Gata-3. Data is representative of 2–3 independent experiments of 5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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