Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients

Abstract

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1-expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1-specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28-). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1-specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1-specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1-specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1-specific CD8+ T cells after peptide-based vaccine strategies.

FIGURE 1. Expansion of ex vivo detectable NY-ESO-1-specific CD8+ T cells following immunizations with the analogue peptide NY-ESO-1 157-165V, CpG and Montanide

CD8+ T cells were isolated from PBMCs of patients obtained at different time points during vaccination. Numbers of NY-ESO-1-specific CD8+ T cells were assessed by ex vivo flow cytometry using PE-conjugated HLA-A2/NY-ESO-1 157-165 tetramers. (A) Dot plots from total CD8+ T cells of MP 6, 7 and 8 before and after vaccination. Numbers indicate percentages of ex vivo detectable NY-ESO-1 157-165-specific CD8+ T cells. As negative control, we evaluated the number of HIV-pol specific CD8+ T cells using HLA-A2/HIV-pol 476-484 tetramers. HIV-pol-specific CD8+ T cells were undetectable in all three patients. (B) Kinetics of HLA-A2/NY-ESO-1 tetramer⁺ (tet+) CD8+ T cells following immunizations. All three patients immunized with peptide NY-ESO-1 157-165V, CpG and Montanide (arm 3) exhibited an increase in the frequency of NY-ESO-1-specific CD8+ T cells detectable already after one month of treatment. (C) As control, kinetics of HLAA2/NY-ESO-1 tetramer⁺ (tet+) CD8+ T cells is also shown in patients who were not immunized with the analogue peptide NY-ESO-1 157-165V (arms 1 and 2). No significant increase in NY-ESO-1-specific CD8+ T cells could be detected following immunizations. Vertical lines, time points of vaccinations.

FIGURE 2. Maturational status of spontaneous and vaccine-elicited NY-ESO-1-specific CD8+ T cells

CD8+ T cells from the three patients in arm 3 were isolated from PBMCs pre- and post-immunizations and used in ex vivo multiparemeter flow cytometry assays combining HLA-A2/NY-ESO-1 157-165 tetramers and antibodies against multiple cell surface markers as reported in MATERIAL AND METHODS. Numbers in each quadrant represent percentages of tetramers⁺ cells for each phenotypically distinct CD8+ T cell subset defined by the expression of the following surface markers: (A) CD45RA/CCR7 (A), (B) CD27/CD28; and (C) CD127. (D) Dot plots showing ex vivo expression of granzyme B, granzyme A, perforin and PD-1 on HLA-A2/NY-ESO-1 157-165 tetramer⁺ CD8+ T cells before and after vaccination for MP 7.

FIGURE 3. Ex vivo assessment of NY-ESO-1-specific CD8+ T cells following immunizations

CD8+ T cells were isolated pre- (solid bars) and post-vaccination (white bars) from PBMCs of MP 1, 2 and 3 in arm 1 (A), MP 4 and 5 in arm 2 (B) and MP 6, 7 and 8 in arm 3 (C), and incubated in the presence of T2 cells pulsed either with peptide NY-ESO-1 157-165 or peptide NY-ESO-1 157-165V in ex vivo IFN-γ ELISPOT assays. (D) Ex vivo recognition of peptide-pulsed T2 cells and HLA-A2⁺ NY-ESO-1⁺ melanoma cells by NY-ESO-1-specific CD8+ T cells from two melanoma patients in arm 3, pre- and post-vaccination. Briefly, HLA-A2/NY-ESO-1 tetramer⁺ (tet+) CD8+ T cells were sorted by flow cytometry from PBMCs of MP 6 and 7 (arm 3) as described in MATERIAL AND METHODS. One thousand CD8+ T cells were incubated in a 24h IFN-γ ELISPOT assay in the presence of T2 cells pulsed with either peptide NY-ESO-1 157-165 (10μg/ml) or peptide NY-ESO-1 157-165V (10μg/ml). The CD8+ T cells were also incubated in the presence of the HLA-0201⁺, NY-ESO-1⁺, melanoma cell line UPCI-MEL 285.1 cells +/− anti-HLA-A,-B,-C antibodies (W6/32). The HLA-0201⁺, NY-ESO-1⁻ , melanoma cell line, UPCI-MEL 136.1 was used as control. IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars). P values < 0.05 were considered significant and are indicated with asterisks.

FIGURE 4. Functional studies of NY-ESO-1-specific CD8+ T cell clones derived from patients immunized with peptide NY-ESO-1 157-165V, CpG and Montanide

(A) Recognition of peptide-pulsed-T2 cells and the HLA-A2⁺, NY-ESO-1⁺, melanoma cell line UPCI-MEL 285 by CD8+ T cell clones 80/1, 93/13 and 95/3 isolated from PBMCs of MP 6, 7 and 8, respectively. One thousand CD8+ T cell clones were incubated in a 24-h IFN-γ ELISPOT assay in the presence of T2 cells pulsed either with the wild-type peptide NY-ESO-1 157-165 or the analogue peptide NY-ESO-1 157-165V (10 μg/ml), or UPCI-MEL 285 cells ± anti-HLA-DR antibodies (L243) or ± anti-HLA-A, -B, -C antibodies (W6/32). The HLA-A2⁺, NY-ESO-1⁻, melanoma cell line UPCI-MEL 136 was used as a negative control. (B) Lysis of the HLA-A2⁺ NY-ESO-1⁺ melanoma cell line UPCI-MEL 285 by clones 80/1, 93/13 and 95/3 at indicated effector-to-target cell ratios +/− L243 and W6/32 antibodies. ⁵¹Cr-release was measured after 4 h of incubation. (C) Reactivity of CD8+ T cell clones 80/1, 93/13 and 95/3 to titrated doses of peptides NY-ESO-1 157-165 and NY-ESO-1 157-165V assessed in a 24hr IFN-γ ELISPOT assay. Each column represents the mean spot number of triplicates ± SD (bars). P values < 0.05 were considered significant and are indicated by asterisks.