Survivin-targeted immunotherapy drives robust polyfunctional T cell generation and differentiation in advanced ovarian cancer patients

Abstract

DepoVax™ is an innovative and strongly immunogenic vaccine platform. Survivin is highly expressed in many tumor types and has reported prognostic value. To generate tumor-specific immune response, a novel cancer vaccine was formulated in DepoVax platform (DPX-Survivac) using survivin HLA class I peptides. Safety and immune potency of DPX-Survivac was tested in combination with immune-modulator metronomic cyclophosphamide in ovarian cancer patients. All the patients receiving the therapy produced antigen-specific immune responses; higher dose vaccine and cyclophosphamide treatment generating significantly higher magnitude responses. Strong T cell responses were associated with differentiation of naïve T cells into central/effector memory (CM/EM) and late differentiated (LD) polyfunctional antigen-specific CD4+ and CD8+ T cells. This approach enabled rapid de novo activation/expansion of vaccine antigen-specific CD8+ T cells and provided a strong rationale for further testing to determine clinical benefits associated with this immune activation. These data represent vaccine-induced T cell activation in a clinical setting to a self-tumor antigen previously described only in animal models.

Trial registration: ClinicalTrials.gov NCT01416038.

Keywords: DepoVax; T cells; cancer; immunotherapy; survivin.

Figures

Figure 1.

Cyclophosphamide and the dose of vaccine affect the strength of immune response as seen by IFNγ ELISPOT and tetramer staining. PBMCs from cohorts A (A), B (B) and C (C) were stimulated overnight with survivin peptides in an IFNγ ELISPOT assay. Data presented represent the number of spot forming units (SFU) per million PBMC from individual patients over time. Statistically significant differences were established by general linear model: C vs. A, p = 0.015; C vs. B, p = 0.013. (D) Patient PBMCs were stained with corresponding HLA-matched tetramer reagent ex vivo (left panels) or stimulated with indicated peptides for 10 d and were stained with corresponding tetramer reagent (right panels) to detect CD8+ T cells with peptide-specific T cell receptor repertoires. HIV tetramer served as a negative control and CMV-specific tetramer was used on a known CMV-positive donor PBMC as internal positive assay control (data not shown). Data represented as percentage of live gated CD3+CD8+ cells that were positive for tetramer staining and the baseline value (Study Day 0) is subtracted from each post-vaccination time points for each subject. The data shows the results at different time points for each patient.

Figure 2.

Survivin antigen-specific CD8+ T cells are detected in the blood of DPX-Survivac vaccine recipients. PBMCs were tested for the presence of peptide-specific CD8+ T cells using MHC-tetramer reagents designed using HLA-A1, -A2 and -A3 survivin peptides used in DPX-Survivac. Assay was performed on non-stimulated PBMC (ex vivo) and after 10 d of stimulation in vitro in the presence of HLA-matched survivin peptide(s) and low concentrations of IL-2 (10 U/mL) and IL-15 (10 ng/mL). Live lymphocyte gate was used to further identify CD3+CD8+ T cells that were positive for tetramer staining. Individual patient data from all patients in cohort C are shown. The HLA type of the tetramer used is shown under the patient identification number at the left of the figure.

Figure 3.

DPX-Survivac vaccination induces polyfunctional T cells and the strength of immune response correlates with Progression Free Survival. (A) PBMCs were tested for the presence of multiple cytokine secreting polyfunctional T cells by intracellular staining. After 6 h stimulation with survivin peptides, cells were stained for surface phenotypic markers and intracellular cytokines (IFNγ, TNF-α, IL-2 and others). Flow cytometry analysis was used to detect multiple cytokine production by effector memory (EM; CD27−CD45RA−), central memory (CM; CD27+CD45RA−) and late differentiated (LD; CD27−CD45RA+) CD4+ or CD8+ T cells at each time point. Each pie chart shows the relative levels of polyfunctional T cells in cohorts B and C at baseline and post-vaccination time points (mean values of all subjects). Arcs indicate the frequency of IFNγ+ T cells that can also concurrently secrete one or more additional cytokines (TNF-α/IL-2). (B) PBMCs were tested for the presence of polyfunctional T cells of different phenotypes by intracellular cytokine staining. Mean frequency of each phenotype of CD4+/CD8+ T cells capable of multi-cytokine secretion were measured in cohort C to understand the kinetics of changing functional T cell phenotypes following DPX-Survivac treatment. Data shown represent values after the background staining is subtracted (background on all samples <0.02%).