An Immunogenic Peptide Derived from NM23-H2 Is Expressed on Bcr/abl⁺ Cells.

Objective: Most tumors express antigens which, when presented by MHC molecules, can be recognized by cytotoxic T-lymphocytes. These tumor-associated-antigens (TAA) are considered to be key determinants in the graft-versus-tumor effect after allogeneic hematopoietic cell transplantation (HCT) and are therefore potential candidates for tumor vaccination. Unfortunately only small numbers of TAA have been isolated to date. In this project we looked for immunogenic peptides presented by bcr/abl⁺ cells of an HLA-A32 CML patient.

Methods: Leukemia-specific mixed lymphocyte leukemia cell cultures (MLLC) were generated by co-culturing irradiated bcr/abl+ cells from the patient with peripheral blood mononuclear cells (PBMC) from the HLA-identical, related stem cell donor. The cultures were re-stimulated once per week with irradiated leukemic cells and IL-2. A cDNA library was constructed from bcr/abl⁺ leukemic cells in pcDNA3.1 and divided into pools of 100 cDNAs. 293T cells were then co-transfected with each pool together with the appropriate HLA-cDNA and tested 48 h later in an IFN-gamma Elispot assay using CD8⁺ sorted MLLC cells. Positive cDNA pools were divided into sub-pools of 10 colonies and re-screened. Positive sub-pools were finally separated into single clones and screened again. The entire open reading frame of NM23-H2 was amplified by PCR, cloned into an expression vector and transfected together with HLA-A32 cDNA into 293T cells to confirm reactivity in the Elispot assay. The immunogenicity of synthetic peptides was determined by direct loading of HLA-A32 expressing APCs. Peptides (10cg) were also tested in the ELISPOT assays with CD8⁺ sorted PBMC of the patients at different time points after HCT.

Results: An MLLC responder cell population recognized bcr/abl⁺ cells (CD14⁺, CD34⁺ subpopulations and EBV) without recognising bcr/abl⁻ cells (CD4⁺ and CD8⁺ T cells or PHA blasts). The reaction was completely blocked by two monoclonal antibodies against HLA class I antigens. Screening of the cDNA library identified 3 HLA-A32 restricted positive single clones, all of which were 600 bp long. Sequencing revealed each to be identical to NM23-H2 (An NDP kinase and transcriptional activator of c-myc) without any mutations or translocations. Although no anchors for HLA-A32 have yet been defined, two viral peptides known to be restricted by HLA-A32 have tryptophan at position 9. Three Nm23 peptides which share this feature (NM23-H2_70–78, NM23-H2_125–133, NM23-H2_141–149) were identified, synthesized and tested in the ELISPOT assay. Of these, only the Nm23-H2_70–78 (SGPVVAMVW) generated a positive reaction. Specific recognition of SGPVVAMVW was half-maximal at a concentration of 1 nM. Subsequently, we looked for reactivity against the peptide in vivo, stimulating CD8+ sorted PBMC at different time points after HCT. While no reaction was detectable either before or up to 3 months after HCT, positive reactions were observed thereafter.

Conclusion: Peptide Nm23_70–78 derived from NM23-H2, is immunogenic and a potential candidate for specific immunotherapy of CML patients.