Phase I/II study of GM-CSF DNA as an adjuvant for a multipeptide cancer vaccine in patients with advanced melanoma

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by a > or =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.

Figure 1. Immunization with GM-CSF DNA followed by tyrosinase and gp100 peptides induced peptide-specific CD8+ T cells assessed by tetramer binding and IFNγ production

PBMCs were collected pre-vaccination, and at several time points during or after vaccination (C = week 7, D = week 11, and E = week 17) and analyzed by tetramer and ICS IFNγ assays. (a) Two patients with positive tetramer assays – patient # 4, Tyrosinase time points D and E; patient # 20 gp100 time point D, and (b) the corresponding ICS IFNγ assays are shown. Only patient # 20 – time points C and D – are positive. Pre-vac, pre-vaccination.

Figure 2. GP100 tetramer-reactive CD8+ cells in the responder population have an effector memory phenotype

PBMCs were analyzed by tetramer assay after in vitro culture using gp100_209-217 ITDQVPFSV peptide. (a) Dot plots from patient # 20 at time point D are shown. (b) Contour plots from patient # 20 at time point D show CD3+CD8+ T cells analyzed for tetramer reactivity. Upper plots gated CD3+CD8+tetramer+ T cells; lower row plots gated on CD3+CD8+tetramer-T cells.

Figure 3. Phenotypic characterization of cells secreting IFNγ in ICS assays

ICS assays were performed with CD45RO, CD62L, CD127, CD107a, and granzyme B. (a) Representative dot plots from patient # 20 at time point D are shown. (b) Contour plots from patient # 20 at time point D show the gated CD3+CD8+IFN-γ+ T cells. Upper plots gated on CD3+CD8+IFNγ+ T cells; lower row plots gated on CD3+CD8+IFNγ- T cells.

Figure 4. Polyfunctional antigen specific CD8+ T cells are induced following immunization

(a) Representative dot-plots from sample patient #12 at time point C. Single function gates were set based on negative control (unstimulated sample, bottom row) and were placed consistently across samples. (b) Responses in patient #12 are shown at all 5 time points. Every possible combination of responses is shown on the x axis. Responses are grouped and color coded according to the number of functions. Bars indicate the percentage of the total response contributed by CD8+ T cells with a given functional response. (c) Each pie represents a time point in patient #12 and each slice of the pie represents the fraction of the total response that consists of CD8+ T cells positive for a given number functions.