Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant

Abstract

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.

Figures

Figure 1

Typical local erythematous skin reaction in the area of imiquimod application (representative patient).

Figure 2

NY-ESO-1 antibody responses. Extrapolated reciprocal titers (y axis) for each patient at each vaccination time point (x axis) as measured by ELISA using NY-ESO-1 recombinant protein (blue circles) and NY-ESO-1 peptide pool (red squares). For responders, the mean maximum titer was 1:1,400 using NY-ESO-1 protein, 1:1,500 using NY-ESO-1 peptides. Time points indicated by vaccination cycle (C) and day (D), or by follow-up visit (FU1, FU2).

Figure 3

Intracellular cytokine staining. Following a 1 wk IVS with pooled NY-ESO-1 overlapping peptides, cells were re-stimulated with antigen and stained for intracellular IFNγ. (a) Gating for flow cytometry. Cell aggregates were excluded by gating for singlets (FSC-height vs. FSC-area), and CD4+ T cells were selected by sequentially gating for lymphocytes, CD3+ cells, and CD8− CD4+ cells. This gating strategy removes most autofluorescent dead or dying cells and minimizes non-specific background. (b) Quantification of IFNγ-secreting NY-ESO-1-specific CD4+ T cells. Representative pre- (upper) and post- (lower) vaccine samples for Patient 6 are shown. Plots on the right show T cells re-stimulated with pooled NY-ESO-1 overlapping peptides. Plots on the left show parallel pre-sensitized T cell controls that did not receive a second stimulation. For all plots, CD4 staining is shown on the y axis and IFNγ staining is shown on the x axis. (c) Summary of results for all patients. Filled bars show the percentage of CD4+ T cells secreting IFNγ following re-stimulation with the NY-ESO-1 overlapping peptide pool. Open bars represent the values for parallel controls that did not receive a second stimulation. The threshold for detection for this assay, defined as 0.1% of CD4+ T cells, is indicated by a horizontal line in each graph. Time points are indicated by vaccination cycle (C1, C2, C3 or C4) and day (D01 or D10), or as follow-up visits (FU1, 1-month follow-up; FU2, 2-month follow-up).

Figure 4

Epitope mapping of cellular and humoral reactivities to NY-ESO-1 (a) Mapped CD4+ T cell responses in patients with reactivity to pooled NY-ESO-1 peptides. The percentage of IFNγ secreting CD4+ T cells responding to a particular AA sequence is shown by patient. (b) Mapping of B cell responses for subjects reactive with pooled NY-ESO-1 peptides. The reciprocal antibody titer for antibody to individual NY-ESO-1 peptide sequences is shown by patient.

Figure 5

(a) Representative H&E stained sections of control skin (left), imiquimod treated skin (center) and vaccine site (right), 40x magnification. (b) Representative immunohistochemistry sections for each of the tested markers (at 200x magnification, untreated skin (left), vaccine/imiquimod site (middle)) and graphs (right) showing cell counts for all individual patients (* p<0.05, ** p<0.01 compared to untreated control skin).