Combined vaccine+axitinib therapy yields superior antitumor efficacy in a murine melanoma model

Abstract

Axitinib, a tyrosine kinase inhibitor of vascular endothelial growth factor receptors, has demonstrated modest efficacy when applied as a single agent in the setting of advanced-stage melanoma. On the basis of the reported ability of axitinib to 'normalize' the tumor vasculature, we hypothesize that combination therapy using axitinib plus specific peptide-based vaccination would promote superior activation and recruitment of protective T cells into the melanoma microenvironment, leading to enhanced treatment benefit. Using a subcutaneous M05 (B16.OVA) melanoma model, we observed that a treatment regimen consisting of a 7-day course of axitinib (0.5 mg/day provided orally) combined with a subcutaneous vaccine [ovalbumin (OVA) peptide-pulsed syngenic dendritic cells adenovirally engineered to produce IL-12p70] yielded superior protection against melanoma growth and extended overall survival when compared with animals receiving either single modality therapy. Treatment benefits were associated with: (a) a reduction in suppressor cell (myeloid-derived suppressor cells and Treg) populations in the tumor, (b) activation of tumor vascular endothelial cells, and (c) activation and recruitment of type-1, vaccine-induced CD8 T cells into tumors. These results support the therapeutic superiority of combined vaccine+axitinib immunotherapy and the translation of such approaches into the clinic for the treatment of patients with advanced-stage melanoma.

Conflict of interest statement

Conflicts of interest: None declared

Figures

Figure 1. Combined vaccine + axitinib therapy elicits superior anti-tumor efficacy in an established murine melanoma model

C57BL/6 mice were injected s.c. with MO5 (B16.OVA) tumor cells. After 7 days, mice bearing established tumors were randomized into groups of 5 animals each with comparable mean tumor sizes. Animals were then left untreated, or they received a s.c. vaccine (syngenic DC.IL12 pulsed with the OVA257–264 and OVA323–339 peptides on days 7 and 14), axitinib alone (0.5 mg/day provided via oral gavage on days 7–13) or the combination of vaccine + axitinib, as outlined in Materials and Methods. Tumor growth was monitored every 2–3 days and is reported as mean +/− SD for one representative experiment of 3 performed in panel A. In B, aggregate survival data from all 3 experiments is provided in a Kaplan-Meier plot. *p < 0.05 versus all other groups.

Figure 2. Specific Type-1 CD8+ T cell infiltration is enhanced following combined vaccine + axitinib therapy

Melanomas and TDLN were harvested on the indicated days from mice treated as outlined in Fig. 1. After enzymatic digestion/mechanical disruption, single-cell suspensions were analyzed for absolute numbers of bulk and OVA-specific (i.e. those binding the PE-conjugated H-2Kb/SIINFEKL tetramer) CD8+ tumor-infiltrating lymphocytes (TIL) by flow cytometry. An example of the flow profile data/gating obtained for the day 14 VAC + AX cohort is provided in panel A. In B, absolute cell numbers (mean ± SD) are reported. These were determined by multiplying the percentage of either gated CD8+ or tetramer-positive CD8+ T cells (based on flow cytometry analysis) by the total number of viable cells obtained from each tissue. *p < 0.05 compared to all other cohorts. In C, MACS-isolated CD8+ T cells from TIL and TDLN were longitudinally analyzed for their ability to produce IFN-γ in response to syngenic DC pulsed with OVA254–262 (SIINFEKL) peptide for 48 hours at a 10:1 E:T ratio. Cell-free supernatants were harvested and IFN-γ levels (pg/ml) were assessed using cytokine-specific ELISA, with mean +/− SD values reported from triplicate determinations. *p < 0.05 versus all groups. T cell reactivity against DC not pulsed with OVA peptide was < 50 pg/ml for all groups.

Figure 3. Combination vaccine + axitinib therapy reduces and/or prevents accumulation of MDSC and Treg suppressor cells in the melanoma microenvironment to a greater extent than the component single modality therapies

Isolated melanomas harvested and processed as described in Fig. 2 were analyzed by flow cytometry for the presence of regulatory cells expressing either a Treg (CD4+Foxp3+) or an MDSC (CD11b+Gr1+) phenotype as described in Materials and Methods. Panel A provides an example of data obtained from day 14 untreated tumors, demonstrating the gating region for Treg and MDSC quantitation. In B, absolute numbers of cells bearing Treg and MDSC (mean +/−SD) phenotypes were determined by multiplying the (flow-based) percentage of double-positive cells by the overall number of viable cells obtained from tumor tissue. *p < 0.05 compared to all other groups.

Figure 4. Combined vaccine + axitinib therapy is more effective than single modality treatment in promoting a “Type-1” melanoma microenvironment

A, Total RNA was purified from single cell suspensions of MO5 tumors harvested on the indicated days as described in Fig. 2. RT-PCR was then performed using primer pairs specific to the Type-1 associated markers Tbet, IFN-γ, CXCR3, and CXCL10. PCR products were resolved through electrophoresis, imaged, and band intensities were normalized to the intensity of β-actin-specific amplification from paired samples. Data is presented as the fold change in transcript levels (mean ± SD) compared to baseline values from the same cohort on the day of treatment initiation (i.e. day 7 post-tumor challenge). *p < 0.05 versus all groups. In B, RT-PCR analysis was performed using Tbet- and Foxp3-specific primer pairs as outlined in panel A and Materials and Methods, with the mean ratio of Tbet/Foxp3 transcript levels reported (+/− SD) on the indicated days after treatment initiation. In C, day 20 melanomas were harvested from all groups and tissue sections analyzed by immunofluorescence microscopy for expression of CD31 and VCAM-1 as outlined in Materials and Methods. Cell nuclei were imaged by staining with DAPI. Mean (+/− SD) absolute numbers of CD31+VCAM-1+ cells per high power field (HPF) over over 10 HPFs/slide are reported. *p < 0.05 versus all other groups.