A phase I/IIa clinical trial of immunotherapy for elderly patients with acute myeloid leukaemia using dendritic cells co‐pulsed with WT1 peptide and zoledronate (2011)

Immunotherapy, which is less toxic and is tumoricidal through mechanisms different from those of chemotherapy, has the potential capacity to confer clinical benefits on elderly patients with acute myeloid leukaemia (AML), in whom therapy-related mortality and resistance to chemotherapy are considered to be major reasons for dismal prognosis. We conducted a phase I/IIa clinical trial of immunotherapy for elderly patients with AML, using dendritic cells (DCs) pulsed with HLA-A*2402-restricted modified WT1235–243 peptide (CYTWNQMNL) and zoledronate. Wilms' tumour 1 (WT1) is a transcription factor highly expressed in AML (Oka et al, 2008). HLA-A*2402-restricted modified WT1235–243 peptide, in which an anchor residue of the natural WT1235–243 peptide (CMTWNQMNL) was substituted to enhance the affinity to the HLA-A*2402 molecule, has a more potent capacity to activate WT1-specific cytotoxic T lymphocytes (CTLs) in vitro than the natural peptide (Tsuboi et al, 2002). The peptide vaccine using the modified peptide has shown antitumor activity in patients, indicating that modified peptide-induced CTLs can kill tumour cells that present the natural peptide (Oka et al, 2004). Zoledronate is an aminobisphosphonate that induces the accumulation of isopentenyl pyrophosphate, a ligand of Vγ9Vδ2 T-cell receptor, in a variety of cells (Gober et al, 2003). We and others have shown that addition of zoledronate to tumour antigen-loaded DCs trigger activation of Vγ9Vδ2 T cells, which produce interferon (IFN)-γ and stimulate the interacting DCs, leading to enhanced activation of tumour antigen-specific CD8+ T cells (Fiore et al, 2007; Takahara et al, 2008). As Vγ9Vδ2 T cells are located in secondary lymphoid organs (Dieli et al, 2003), DCs loaded with tumour antigen and zoledronate may strongly activate antigen-specific CD8+ T cells in draining lymph nodes, owing to the helper effect of Vγ9Vδ2 T cells. The study protocol was approved by the Ethics Committee, Graduate School and Faculty of Medicine, Kyoto University. Written informed consent was obtained from each patient before enrollment in accordance with the Declaration of Helsinki. Three HLA-A*2402-positive patients with ages of 69–77 were enrolled (Table I). WT1 mRNA expressions in the patients' leukemic cells were confirmed by real-time quantitative polymerase chain reaction. Following recovery of normal hematopoeisis after chemotherapies, monocytes were collected by apheresis and elutriation, and cultured with 800 IU/ml granulocyte-macrophage colony-stimulating factor and 500 IU/ml interleukin-4 to generate immature DCs. DCs were matured with 10 ng/ml tumour necrosis factor-α plus 1 μg/ml prostaglandin E2. The majority of DCs were co-pulsed with 10 μmol/1 modified WT1 peptide and 0·1 μmol/1.zoledronate (WT1/ZOL-DC), while the rest of the DCs were pulsed with 2 μg/ml keyhole limpet hemocyanin (KLH) (KLH-DCs). DCs were frozen, and thawed at the time of DC administration. We administered WT1/ZOL-DCs intradermally (1 × 107 cells) and intravenously (1 × 107 cells) in an attempt to stimulate WT1-specific T cells in draining lymph nodes as well as in bone marrow, which is the main tumour site in leukaemia and has been reported to preferentially contain leukaemia-specific T cells (Melenhorst et al, 2009). In addition, 5 × 106 KLH-DCs were intradermally administered to monitor immunogenicity of DCs. The administration of DCs was repeated every 2 weeks for a total of five administrations. The vaccinations were well tolerated and safe. Whereas Patient #1 dropped out of the study after the third vaccination owing to rapid growth of leukemic cells with no inductions of immune responses to either KLH or the modified WT1 peptide, Patients #2 and #3, who completed the five vaccinations, exhibited inductions of immune responses to both KLH and the modified WT1 peptide as detected by skin delayed type hypersensitivity tests and/or IFN-γ ELISPOT assays (data not shown). WT1-specific immune responses were also assessed by HLA tetramer staining, using an HLA-A*2402/modified WT1 peptide tetramer (Fig. 1). In either of the two patients, tetramer-positive T cells were not detected in peripheral blood mononuclear cells (PBMCs) without in vitro expansion at any time points. In both patients, however, tetramer-positive CD8+ T cells became detectable 2 weeks after the 4th vaccination in PBMCs stimulated with the modified peptide for a week in vitro. In addition, albeit at much lower frequencies, tetramer-positive T cells were also detected in natural peptide-stimulated PBMCs. Interestingly, whereas tetramer-positive T cells rapidly declined thereafter in modified peptide-stimulated PBMC, tetramer-positive T cells persisted until 3 months after the 5th vaccination in natural peptide-stimulated PBMCs. Stimulation with the natural peptide also expanded tetramer-positive T cells from bone marrow (Fig. 1C). We assessed numbers of γδ T cells in peripheral blood and bone marrow and serum IFN-γ levels to determine whether the vaccinations induced activation of γδ T cells in vivo. However, we could not detect it using these assays (data not shown). In association with the inductions of an immune response to WT1, either transient decrease in leukemic cells (Patient #2) or disease stabilization (Patient #3) was observed (Table 1), suggesting that the vaccinations may have impacted on the clinical courses of these patients. (A) HLA tetramer staining of PBMCs from Patient #2. At indicated time points, PBMCs were stimulated with DCs pulsed with the WT1 modified peptide [WT1 (modified)] or with the natural peptide [WT1 (natural)] for a week, then stained with PE-conjugated HIV/HLA-A*2402 tetramer as a negative control or PE-conjugated WT1 modified peptide/HLA-A*2402 tetramer and FITC-conjugated anti-CD8 mAb, and analysed by flow cytometry. Dead cells were excluded by propidium iodide staining. Numbers indicate percentages of tetramer-positive cells within the CD8+ population. (B) HLA tetramer staining of PBMCs from Patient #3. At indicated time points, PBMCs were stimulated and analysed as in (A). (C) HLA tetramer staining of bone marrow mononuclear cells in Patient #2. Three months after the 5th vaccination, the cells were stimulated in vitro with DCs pulsed with the WT1 natural peptide for a week, and then stained and analysed as in (A). These data indicate that vaccination with the modified WT1 peptide expands modified peptide-reactive T cells, a small fraction of which cross-reacts with the natural peptide. Notably, whereas modified peptide-specific T cells rapidly declined after the termination of vaccination, natural peptide-reactive T cells survived longer in vivo. Although the reason is not clear, it might be due to persistent stimulation with the endogenous cognate antigen derived from residual leukemic cells, as observed in CD8+ T cell memory maintained by persistent antigenic stimulation in chronic viral infection (Shin et al, 2007). Thus, although vaccination with a modified peptide may expand many 'useless' T cells that do not recognize the natural peptide, a small fraction of T cells that cross-react to the natural peptide may preferentially survive and can be exploited as effector cells upon boosting. A recent report on immunotherapy using WT1 mRNA-electroporated DCs indicates the promise of DC-based immunotherapy for AML after reducing a tumour burden by chemotherapy (Van Tendeloo et al, 2010). Further strengthening of vaccination, such as a combination of other treatments to block immunosuppressive pathways, may lead to an improved clinical outcome. We thank Maki Utsumi for her excellent technical assistance and physicians who referred patients. This study was supported by MEDINET, Co., Ltd. Coordination Support and Training Program for Translational Research from Ministry of Education, Culture, Sports, Science, and Technology of Japan. The Third Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor, and Welfare, Japan. MEDINET, Co., Ltd.