Vaccination with agonist peptide PSA: 154-163 (155L) derived from prostate specific antigen induced CD8 T-cell response to the native peptide PSA: 154-163 but failed to induce the reactivity against tumor targets expressing PSA: a phase 2 study in patients with recurrent prostate cancer

Abstract

We conducted a clinical trial of peptide prostate specific antigen (PSA): 154-163 (155L) vaccination in human leukocyte antigen (HLA)-A2 patients with detectable and rising serum PSA after radical prostatectomy for prostate cancer (Clinicaltrials.gov identifier NCT00109811). The trial was a single dose-level, phase 2 pilot trial of 1 mg of PSA: 154-163 (155L) emulsified with adjuvant (Montanide ISA-51). The primary endpoint was the determination of immunogenicity of the vaccine; secondary outcomes were determination of toxicity and effect on serum PSA. The vaccine was given subcutaneously 7 times on weeks 0, 2, 4, 6, 10, 14, and 18. Peptide-specific CD8 T-cell responses in the peripheral blood mononuclear cells (PBMC) of patients were measured by interferon (IFN)-gamma enzyme-linked immunosorbent spot assay. CD8 T-cell cultures were also established by in vitro stimulation with the peptide presented by autologous dendritic cells. Five patients were enrolled and completed all vaccinations. No IFN-gamma response to PSA: 154-163 (155L) was detected in unfractioned PBMC in any patient either before or after vaccination. Three of 5 patients demonstrated strong IFN-gamma responses to PSA: 154-163 (155L) and native PSA: 154-163 peptides in CD8 T-cell cultures derived from postvaccination PBMC. However, peptide-specific T cells failed to recognize HLA-A2 positive targets expressing endogenous PSA. There were no significant changes in serum PSA level in any subject. No serious adverse events were observed. PSA: 154-163 (155L) is not an effective immunogen when given with Montanide ISA-51. The PSA: 154-163 peptide is poorly processed from endogenous PSA and therefore represents a cryptic epitope of PSA in HLA-A2 antigen-presenting cells.

Figures

FIGURE 1. Change in serum PSA over time

Serum PSA levels were measured on the day of vaccination according to study protocol. Each line corresponds to individual patient. Arrows indicate time points of peptide vaccination.

FIGURE 2. Peptide-specific CD8 T-cell response in prostate cancer patients before and after vaccination with peptide PSA154–165(155L) (peptide PSA3A) in Montanide ISA-51

Cell lines were developed from CD8 T cell-enriched baseline (pre-vac) and post-vaccine (post-vac) PBMC by stimulation with PSA3A peptide presented by autologous DC. The cultures were tested for the reactivity with the specific peptide or PSA-expressing cell line after one or two cycles of in vitro stimulation by IFN-γ ELISPOT assay. T cells at 50,000 cells/well were co-cultured with irradiated HEK293-A2 cells in the presence (black bars) or absence (gray bars) of specific peptide for 48 hr or HEK293-A2-PSA cells (dark gray bars). Results for three responding patients are shown. The assays were developed as described in Materials and Methods. The data are mean ±SD of triplicate determinations. * p<0.0001 (2-sided t-test).

FIGURE 3. Comparison of the response to PSA3A and native PSA3 peptide

CD8 T cell cultures from three responding patients were generated as described in Figure 2. A. CD8 T cells were co-cultured with irradiated HEK293-A2 cells in the absence of peptide (gray bars) or presence of PSA3A (black bars) or PSA3 (white bars) peptide (25 µg/ml). B. CD8 T cells from patient Pr159 were co-cultured with HEK293-A2 cells in the presence of increasing concentrations of peptide PSA3A (black circles) or PSA3 (white circles). Frequency of IFN-γ-producing cells was assessed by ELISPOT assay. The data represent mean ±SD of triplicate determinations.

FIGURE 4. MHC restriction of PSA3A peptide-specific CD8 T cell response

CD8 T cell line from patient Pr150 was tested in IFN-γ ELISPOT assay against HLA-A2 expressing OVCAR3 tumor cells pulsed with PSA3A peptide in the presence of anti-HLA class I, anti-HLA-DR or isotype control antibodies. The data are mean ±SD of triplicate determinations.

FIGURE 5. Failure of LNCaP cells recognition by peptide-specific CD8 T cell cultures

The PSA154–165(155L) peptide-specific CD8 T cell cultures were developed as described in Materials and Methods (two rounds of stimulation for Pr159 or multiple stimulations with peptide and cloning for Pr142 clone 1). T cells were co-cultured with irradiated HEK293-A2 cells in the presence or absence of the specific peptide or LNCaP cells for 48 hr. Representative experiments are shown. * Too numerous to count.

FIGURE 6. Characterization of OVCAR3-PSA target cell line

A. HLA-A2 expression. OVCAR3-EV and OVCAR3-PSA cell lines were incubated for 48 hr with or without rhIFN-γ (5ng/ml), harvested and stained with anti-HLA-A2-FITC monoclonal antibody or FITC-labeled isotype control antibody. B. Presentation of the endogenously-processed viral peptide. CD8 T cell line was developed from Pr159 PBMC by stimulation with influenza M1matrix protein 58–65 peptide as described in the legend to Figure 2 and tested for the reactivity with influenza A virus-infected OVCAR3-EV (depicted) or OVCAR-PSA (not shown) cells in the IFN-γ ELISPOT assay. Data are mean ±SD of triplicate determinations. * p<0.0001 (2-sided t-test).

FIGURE 7. Lack of recognition of endogenously-expressed PSA by PSA3A peptide-specific CD8 T cells

A. PSA3A peptide-specific CD8 T cell lines derived by two cycles of in vitro stimulation with PSA3A peptide were tested for the reactivity with OVCAR3-EV in the presence or absence of specific peptide, or OVCAR3-PSA cells in IFN-γ ELISPOT assay. Results for CD8 T cell lines from three responding patients are shown. Data are mean ±SD of triplicate determinations. * p<0.0001 (2-sided t-test). B. The response of individual clones developed from PSA3A peptide-specific CD8 to PSA3A peptide and PSA-expressing HLA-A2-positive tumor cell line. Peptide-specific CD8 T-cell clones were developed from advanced PSA3A peptide-specific CD8 cultures of all responding patients (Pr142, Pr143, Pr150, Pr159) and tested in IFN-γ ELISPOT assay. Representative clones from each patient are shown.