Synthetic tumor-specific breakpoint peptide vaccine in patients with chronic myeloid leukemia and minimal residual disease: a phase 2 trial

Abstract

Background: Imatinib is the current standard frontline therapy for chronic myelogenous leukemia (CML). In the majority of patients, imatinib induces a complete cytogenetic response (CCyR); however, complete molecular responses are infrequent. The Bcr-Abl fusion creates a unique sequence of amino acids that could constitute a target for immunomodulation.

Methods: A mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences was used to vaccinate patients with CML in CCyR who were receiving imatinib therapy and who had stable Bcr-Abl transcript levels.

Results: Ten patients were enrolled, all with b2a2 transcripts (including 2 patients who had coexpression of b2a2 and b3a2). Patients had received imatinib for a median of 62 months. Three of 10 patients achieved 1-log reduction in Bcr-Abl transcript levels, including the 2 patients who had received previous interferon therapy, and 3 other patients achieved a major molecular response. The vaccine was tolerated well, and there were no grade > or =3 adverse events. Vaccination did not affect the leukocyte profiles in peripheral blood except for regulatory T cells, which were down-regulated briefly during the late stage of vaccination in patients who achieved approximately 1-log reduction in Bcr-Abl transcript levels.

Conclusions: The current data suggested that vaccination-related transient disruption of immune tolerance may contribute to the reduction in Bcr-Abl transcripts. Clinically, this Bcr-Abl peptide vaccine may transiently improve the molecular response in a subset of patients with CML.

FIGURE 1

Vaccination was associated with increased glucocorticoid-induced tumor necrosis factor receptor (GITR) expression by regulatory T cells (T_R) concurrent with a decrease in the percentage of T_R. Peripheral blood mononuclear cells (PBMCs) were collected from imatinib-treated patients with chronic myeloid leukemia (CML) before they were vaccinated with human leukocyte antigen (HLA)-matched CML breakpoint heteroclitic peptides and at 3 months, 6 months, 9 months, and 12 months after vaccination. Patients who acquired a minimum 1-log reduction in BCR-ABL transcripts by Month 15 of follow-up were classified as responders (circles; n = 3 patients) and nonresponders (triangles; n = 7 patients). Staining and flow cytometric analysis for T_R markers was performed in batches according to the manufacturer’s protocol (eBioscience; San Diego, Calif) with surface marker staining before permeabilization and staining with the forkhead box P3 (FoxP3) antibody (eBioscience). Responders had a significantly greater percentage of T_R cells that expressed GITR at 6 months after vaccination (left plot) with a concurrent diminishing percentage of CD4-positive/CD25(high)FoxP3-positive cells in PBMCs (right plot) (asterisk: P < .05; Mann-Whitney U test). Plots show the mean ± standard error of the mean. The table below the plots represents individual responses by responders and nonresponders at each point of assessment.

FIGURE 2

Vaccination had a minimal effect on leukocyte distribution. Leukocyte subsets were analyzed fresh by flow cytometry before vaccination with human leukocyte antigen (HLA)-matched chronic myeloid leukemia (CML) breakpoint heteroclitic pep-tides and at 3 months, 6 months, 9 months, and 12 months after vaccination. Patients who acquired a minimum of 1-log reduction in BCR-ABL transcripts by Month 15 of follow-up were classified as responders (circles; n = 3 patients) and nonresponders (triangles; n = 7 patients). A cross-sectional analysis indicated that CD3-positive/CD4-positive lymphocytes were significantly lower in nonresponders than in responders at 9 months (P = .033). Longitudinal analysis revealed a significant drop in the myeloid dendritic cell (mDC) count among responders (P = .009). No other significant differences or changes were observed. WBC indicates white blood cells; CD3, T cells; CD19, B cells; NK, natural killer cells; NKT, natural killer T cells; +, positive; CD8, suppressor/cytotoxic T cells; CD4, helper T cells; DC, dendritic cells; pDC, plasmacytoid dendritic cells.

FIGURE 3

Proliferation of peptide-stimulated CD4-positive T cells increased significantly 3 months after vaccination. CD4-positive T cells were isolated from peripheral blood mononuclear cells by positive selection using an AutoMACS cell separator (Miltenyi Biotec, Auburn, Calif). Then, 10⁵ CD4-positive T cells were incubated with a mixture of vaccine peptides in quadruplicate wells for 4 days, and 1 µCi of ³H-thymidine was added to each well and incubated for an additional 24 hours. The amount of ³H-thymidine incorporation was measured on Day 4 using a scintillation counter. The proliferation of CD4-positive T cells, measured as the count per million (cpm), increased significantly at 3 months after vaccination (asterisk: P = .05; Wilcoxon signed-rank test for paired samples), and the trend was maintained at 12 months. Nevertheless, no statistically significant difference was observed between responders and nonresponders at baseline, at 3 months after vaccination, or at 12 months after vaccination.