Effectiveness of WT1 Peptide Vaccination Combined with Imatinib in a Patient with CML in Chronic Phase.

Abstract 4253

Cytotoxic T lymphocytes (CTLs) are presumed to kill the relevant antigen-expressing leukemia cells including leukemic stem cells, which display intrinsic resistance against tyrosine kinase inhibitors such as imatinib in CML patients. In order to clarify the safety and effectiveness of WT1 peptide vaccination for the patients with CML, we started WT1 peptide vaccination in combination with imatinib therapy for a CML patient who could not acquire a major molecular response by the administration of imatinib. In addition, we tried to evaluate the kinetics of WT1-specific CTLs in peripheral blood (PB) during WT1 peptide vaccination. A 51 years-old male with CML in chronic phase had been treated with 400 mg imatinib for 4 years. BCR-ABL transcripts decreased transiently to less than 700 copies in 1 μg RNA (3-log reduction: 300 copies in 1 μg RNA extracted from PB cells; median in our laboratory) but gradually increased to more than 1,000 copies thereafter. Since the patient was HLA-A*2402⁺ and an informed consent was obtained, HLA-A*2402-restricted 9mer WT1 peptides (CYTWNQMNL; a.a. 235-243), which had been identified to possess an anti-tumor immunogenicity, were administered subcutaneously at the dose of 1 mg/day every 2 weeks in combination with 400 mg imatinib for first 5 months and thereafter every 4 weeks for 12 months. No adverse effects due to WT1 peptide vaccination was observed except for skin induration and redness at the sites of WT1 peptide injection. Although BCR-ABL transcripts increased up to more than 2,000 copies after transient decrease to less than 1,000 copies by the administration of WT1 peptides every 2 weeks, the transcripts have decreased to less than 500 copies by the administration of WT1 peptides every 4 weeks. The appearance of WT1-specific CTLs in PB was confirmed by evaluating the frequency of MHC/WT1 tetramer⁺CD8⁺ T cells by using MLPC (mixed lymphocyte peptide culture). MLPC was performed by culturing 1-3 ×10⁵ PB mononuclear cells (MNCs) in 200 l of 5% autologous serum-containing medium with 10 μg WT1 peptides in 96 well plates. Frequency of MHC/WT1 tetramer⁺ cells in PB-CD8⁺ cells was calculated by the following formula. Number of wells containing a lump of tetramer⁺CD8⁺ cells / [(Number of PB-MNCs seeded in a well of MLPC) x (total number of wells for MLPC) x (ratio of PB-CD8⁺ cells in PB-MNCs)]. Although WT1-specific CTLs were not detected in PB before WT1 peptide vaccination, the CTLs appeared after the second vaccination and maintained at the level of nearly 15/10⁶ CD8⁺ cells thereafter. The MHC/WT1 tetramer⁺ cells showed cytotoxicity against only leukemia cells expressing WT1 and HLA-A*2402. The cytotoxicity was blocked clearly by adding unlabeled cold target cells in cold inhibition test and blocked also by antibodies against MHC class I but not by antibodies against MHC class II. In vitro study demonstrated that the stimulation with WT1 peptides made WT1-specific CTLs, which were generated by MLPC, less reactive to MHC/WT1 tetramers and the unreactivity lasted at least for 2 weeks. The present study showed that WT1 peptide vaccination for an imatinib-pretreated CML patient is feasible and effective, which is due to the generation of WT1-specific CTLs with cytotoxicity against WT1-expressing leukemia cells. In addition, since the CTLs are suggested to get tolerant to WT1 peptides presented by leukemia cells by the exposure with WT1 peptides, the interval of peptide administration has to be considered for the clinical efficacy of peptide vaccination. The present findings including in vivo and in vitro studies revealed that the administration of the peptides every 4 weeks is superior to every 2 weeks.