Generation of Specific Human CD8+ T Cell Responses to the Myeloproliferative Disorder Associated V617F Mutated JAK2 Kinase by Use of Analog Peptide Vaccine Candidates.

The recent identification of the JAK2 V617F mutation in myeloproliferative diseases including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis has the potential to serve as a new therapeutic target for these disorders. We hypothesized that the mutated JAK2 kinase sequence could generate new immunogenic peptide-epitopes that could be recognized by the human immune system. Therefore, such sequences could be potential vaccine candidates. The myeloproliferative diseases, especially PV and ET, are ideal diseases for a vaccine approach as they are indolent disorders and also not suitable for use of possibly toxic chemotherapies. Using computer based predictive algorithms, potentially immunogenic nonamer peptides spanning the V617F region of JAK2 were identified. Since JAK2 is a normally expressed self-protein, the goal is to break immune tolerance to the mutated JAK2 sequence without allowing significant recognition of the native sequence. Therefore, synthetic analogs of the V617F mutated peptides with stronger predictive binding to MHC were designed by introducing single amino-acid substitutions into the HLA-A0201 and A0301 binding positions. Corresponding non-mutated native sequences showed little predicted binding. The binding of the mutated and synthetic analog peptides was confirmed in vitro using an MHC class I stabilization assay on TAP deficient cells. Native sequences bound poorly as expected. JAK2 peptides derived from V617F sequences as well as their analogs were used to elicit MHC-restricted, peptide-specific CTL responses in an in vitro model using T-cells from either HLA-A0201 or A0301 healthy donors after stimulation with autologous monocyte-derived dendritic cells. The analog peptides were shown to be immunogenic in vitro, and activated CD8+ T cells that recognized the synthetic analog peptide pulsed targets as determined by an IFN-gamma ELISPOT assay. In addition, and most importantly, these T cells cross-reacted with cellular targets that were pulsed with the mutated V617F peptide associated with the myeloproliferative diseases, but not with the native JAK2 peptide. These analog peptides thus satisfied our initial goals for a vaccine candidate. Further studies with these peptides and others eliciting CD4+ responses will determine their potential for successful use as vaccine candidates