[Purpose & background] CML66 is a newly identified cancer-testis antigen by SEREX method in post-transplant CML patient who had a second remission by DLI for relapse. Thus CML66 is initially considered to be implicated in graft-versus-leukemia (GvL) effect against CML, while its’ physiological function remains unknown. The identification by SEREX means its’ immunogenicity to produce antibody, however the T-cell response specific for CML66, particularly its’ ability to generate cytotoxic T-lymphocyte (CTL) against leukemia still remains to be verified. Thus we explored a CTL-epitope of CML66 to induce epitope-specific CTL which can kill human leukemia cells, because of the exploration of its’ clinical applicability as an anticancer vaccine for the immunotherapy.

[Methods] At first, we synthesized a variety of CML66-derived 9 aminoacid peptides (9 mer) that had computedly-predicted high binding affinity to HLA-A*2402 molecule. CD8+ T lymphocytes from an HLA-A*2402+healthy donor were co-cultured with autologous monocyte-derived mature dendritic cells (mDCs). CD8+T lymphocytes were repeatedly stimulated with peptide-loaded mDCs. Thereafter, the target epitope-specificity of growing cells was examined by a standard 51Cr-release assay. Additionally, the blocking tests by using anti-HLA class I and anti-class II monoclonal antibody (mo.ab.) were conducted to confirm its’ HLA-A*2402-restricted fashion. Next, CML66 mRNA expression level of target cells including myeloid leukemia cell line cells and primary leukemia cells was examined by real-time semi-quantitative PCR (RQ-PCR). The relative expression level of CML66 mRNA was determined by comparative Ct method.

[Result] We identified two CML66-derived 9 mer epitopes with high binding affinity to HLA-A*2402 measured by using HLA-A*2402 gene transfected T2 (T2-A24) cell. One of 2 epitopes, the epitope of CML66; aa70–78: WIQDSVYYI generated the epitope-specific CTL, in vitro, and those CTL exerted anti-leukemia activity against human myeloid leukemia cell line cells in an HLA-A*2402-restricted fashion, but not any cytotoxicity against normal cells. Furthermore, the HLA-A*2402 restriction was confirmed by blocking test by HLA-class I and II mo.ab. Next CML66 mRNA expression level was revealed high in myeloid leukemia cell line cells but low in normal cells, which were compared to that of K562 cell line cell. In primay leukemia cells, acute myelogenous leukemia(AML) cells and acute lymphoblastic leukemia(ALL) cells showed the high expression level of CML66 mRNA. Regarding to the FAB classification of AML, the expression level of CML66 mRNA tended to be higher in subsets ranging from M1 to M4, particularly M2 cells. Even by small number, it was of interest that the expression level of CML66 mRNA in primary chronic myelogenous leukemia (CML) cells was high in cells from blastic phase, but low in cells from chronic phase. This finding may suggest the correlation between CML66 and growth activity of tumor cells.

[Conclusion] We identified the novel HLA-A*2402 restricted CTL-epitope derived from CML66; aa70–78: WIQDSVYYI, which may be a promising and secure target for immunotherapy against acute leukemias and aggressive CML.

Author notes

Disclosure: No relevant conflicts of interest to declare.