Vaccination with synthetic analog peptides derived from WT1 oncoprotein induces T-cell responses in patients with complete remission from acute myeloid leukemia

Abstract

A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 microg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-gamma release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-gamma-secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia.

Trial registration: ClinicalTrials.gov NCT00398138.

Figures

Figure 1

Survival curves for vaccinated patients. (A) Disease-free survival. (B) Overall survival.

Figure 2

WT1 transcript levels in vaccinated patients. (A) Relapsed patients: increases in WT1 transcripts were large at the time of or just before relapse. The absolute changes occur over orders of magnitude and are shown using a logarithmic scale. (B) Remission patients: variations in transcript levels were comparatively small, either trending toward decrease or stable at very low levels. Minor variations are best appreciated using a linear scale.

Figure 3

CD4+ proliferation. (A) CD4+ T cells from pre (i), postvaccine 3 (ii), and postvaccine 6 (iii) vaccinations from patient 5 (A0201+) were incubated with indicated peptides at 20 μg/mL or 50 μg/mL for 5 days, and 1 μCi [3H]-thymidine was added to the cultures for 20 hours. The cell proliferation was determined by [3H]-thymidine incorporation. Data are mean ± SD from quadruplicate cultures. After 3 vaccinations, cell proliferation increased 54-fold to 331, 37-fold to 427, 4.2-fold to 122A1, and 2.6-fold to 122A (P = .032) at a concentration of 50 μg/mL peptides tested. There was no significant dose dependency of the peptides, and similar responses were also seen after 6 vaccinations. (B) Time course of CD4+ response: CD4+ T-cell responses of 3 patients who completed 12 vaccinations were calculated by the fold increase of the CD4+ T-cell proliferation against 331, 427, and 122A1 peptides over irrelevant peptide B2A2 long at a concentration of 50 μg/mL. Responses at T9 and T12 were not tested for patient 2 (A0201+) because of clinical relapse before those time points. CD4+ T-cell responses were elicited and maintained throughout vaccination, although the magnitude to each peptide with respect to vaccination times varied among patients.

Figure 4

IFN-γ secretion. CD3+ T cells from patient 5 were stimulated twice with WT1-A (native), WT1-A1 (analog) (A), 122A (native), or 122A1 (analog) (B) peptides. IFN-γ–secreting T cells were measured by ELISPOT assay after challenge with the indicated peptides. Controls were: no peptide (only CD14+ APCs) or with irrelevant Ewing sarcoma–derived peptide (EW) (A) or JAK-2 derived peptide (JAK-2 DR) (B). Data are mean ± SD from quadruplicate cultures from prevaccine (i), postvaccine 3 (ii), and postvaccine 6 (iii). Results indicate that a WT1-A–specific response can be generated not only by the HLA-A0201 restricted peptide but also by the HLA-DR peptide (WT1 122A1) that contains the embedded short sequence, demonstrating the processing and presentation of the WT1-A epitope.

Figure 5

Tetramers. CD3+ T cells from the same culture described in Figure 3 were stained with WT1-A/HLA-A0201 tetramer with mAbs to CD8 and other T-cell markers. Percentage of tetramer-positive CD8+ T cells (number shown in upper left corner of each histogram) were gated on CD3+ events after passing through the small lymphocyte gate. Cells from prevaccine, postvaccine 3, and postvaccine 6 are shown as T0, T3, and T6, respectively. The data are representative staining from triplicate cultures. After vaccination, a robust increase in percentage of WT1-specific CD8+ T cells was noted in cultures with WT1-A, WT1-A1, and 122A1 peptides (top axis). Peptide 122A stimulation induced a weak but significant response.

Figure 6

Cytotoxicity assay. CD3+ T cells from patient 5 were stimulated with WT1-A or WT1A1 peptides twice as described in Figure 5. Target cells used included the ALL derived 697 cell line (A0201+; WT1+) and the B-cell lymphoma cell line SKLY-16 (A0201+; WT1−). The cytotoxicity of the T cells was measured using a standard 51Cr release assay. The SKLY-16 cells pulsed with WT1-A (SKLY-WT1A) or an irrelevant Ewing sarcoma–derived HLA-A0201 binding peptide (SKLY-EW) were used as positive and negative controls for the specificity of killing. Effector/target (E:T) ratios are indicated on the x-axis. Data demonstrate T cell–specific killing against WT1 plus HLA-matched targets.