Peptides from Nme2 Are Preferentially Expressed By Chronic Phase CML Cells, Recognized By T Cells and Restricted By the Most Frequent HLA-Antigens

Nme2 (non metastasis protein 2) is a nucleoside diphosphate kinase that plays multiple roles in signalling and metabolism. We previously identified Nme2 as a tumor antigen in chronic myeloid leukaemia (CML) and found Nme2 protein over-expression to be a universal and specific feature of Bcr/Abl⁺ cells in chronic-phase of the disease. Nme2 up-regulation in Bcr/Abl⁺ cells is post-transcriptional and is reversed by TKI treatment. Furthermore, the IL-3 independence induced by Bcr/Abl transfection of Ba/F3 cells is also accompanied by Nme2 over-expression and is reversed by specific miRNA-mediated down-regulation of Nme2, suggesting that Nme2 contributes to Bcr/Abl driven leukemogenesis. Here, we have investigated the potential of Nme2 as a target for CML specific immunotherapy by assessing it's antigenicity in common HLA backgrounds.

Peripheral blood mononuclear cells (PBMCs) were obtained from 40 patients with CML and 24 healthy donors who carried the common HLA-A02 (n=21/7); A03 (n=12/8) and A24 (n=8/9) antigens. Together, these represent the most common HLA types in the Caucasian population. Samples were obtained before (n = 13) and after (n = 36) hematopoietic stem cell transplantation (HCT) and Nme2 specific cytotoxic T lymphocytes (CTL) were generated using artificial antigen presenting cells (aAPC) engineered to express both Nme2 and the appropriate HLA-A antigen. PBMC were primed with aAPC and then re-stimulated weekly. Following CD8⁺ T-cell selection on day 14, IFNγ production in response to Nme2 expressing stimulator cells was evaluated in Elispot assays. In addition Nme2 reactivity was investigated in peripheral blood cells of CML patients by Elispot assays at various time points up to 10 years after HCT.

The frequency of Nme2 specific T cells was significantly higher in CML patients pre HCT (39.3 specific T cells/10⁵ CD8⁺ cells, p=0.004) than in healthy donors (2.17 specific T cells/10⁵ CD8⁺ cells). A further rise in specific T cell response was detected in CML patients post HCT (142 specific T cells/10⁵ CD8⁺ cells, p=0.0006), with a significant increase being evident by day +50 after HCT. The subsequent activity varies, with a weakly pronounced second peak occurring around 3 years post-transplant. However, Nme2 specific T cells persisted over the entire observation period with a significant Nme2 specific T cell response still being detectable nearly 10 years post transplant. The highest Nme2 specific T cell activity was found in HLA-A03 positive patients (187 T cells/10⁵ CD8⁺ cells) and the lowest in the HLA-A02 background (102 T cells/10⁵ CD8⁺ cells, p=0.00001). Restriction by HLA-A24 was associated with an intermediate activity (141 Nme2 specific T cells/10⁵ CD8⁺ cells). We identified 8 patients in which an increase of Bcr-Abl transcript level from 0% Bcr-Abl/Abl to a detectable amount (0.28%, range 0.001 to 5.017% Bcr-Abl/Abl) was observed. In all cases, a transient increase in Nme2 reactive T cells (2.7 fold, p=0.007) was associated with re-entry into molecular remission. Furthermore, Nme2 specific T cells were lower before relapse (88 T cells/10⁵ CD8⁺ cells) than in patients with long lasting molecular remission (186 T cells/10⁵ CD8⁺ cells, p=0.026).Finally, we successfully generated Nme2 specific CTLs from healthy donors (4/5) as well as from patients with CML who had received HCT (10/10). CTLs showed a mean expansion rate of 23 (healthy donors) to 30 fold (CML patients) and HLA class I restricted Nme2 specific recognition. Partially HLA matched, primary CML cells were targeted by these Nme2 specific CTLs (6/6), regardless of the presence of the T315I Bcr/Abl mutation that bestows resistance to tyrosine kinase inhibitors (TKI).

In summary, we show here that Nme2 is a strong inducer of immune response in transplanted CML patients carrying common HLA class I alleles, implying that the immune response to Nme2 may be involved in the GvL effect post HCT. Our results suggest that Nme2 specific CTLs may be of therapeutic relevance in eradicating the residual leukemic cells after therapy with TKI or HCT.