HLA Ligandome Analysis in Primary Polycythemia Vera (PV) Samples Shows No Tumor-Association of Naturally Presented JAK2 Ligands but Reveals a Vast Array of Novel PV-Associated HLA Ligands

Polycythemia vera (PV) is a myeloproliferative disorder mainly driven by the JAK2V617F mutant kinase. Except allogeneic stem cell transplantation, the current standard therapies, including novel therapies aiming for specific inhibition of JAK2 that are presently under investigation, do not offer a chance for cure. Of note, clear clinical benefits of unspecific immunotherapy with pegylated IFN-α suggest that PV might also be targeted by specific peptide based vaccination. Here we aimed to identify tumor associated HLA ligands of PV with special focus on peptides derived from JAK2. Therefore we analyzed HLA class I and II ligands of primary PV samples using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry.

We analyzed the HLA class I ligandomes of 7 PV patients with the JAK2V617F mutation as well as of 4 patients with secondary (JAK2V617F negative) polycythemia. As controls, we profiled the ligandomes of 40 healthy volunteers (30 blood and 10 bone marrow samples). We identified more than 7,000 unique HLA class I ligands representing 4,435 proteins on primary PV samples. Moreover, 3 different HLA ligands derived from JAK2 on PV (KYLINLETL, HLA-A*24; KIGDFGLTK, HLA-A*03; GQGTFTKIFK, HLA-A*11) were identified. None of these ligands was found to be PV-exclusive, with JAK2 being represented on 3/4 (75%) of PV samples (JAK2V617F positive) and 18/30 (60%) healthy controls. No JAK2-derived peptides were identified on JAK2V617F negative patients, and no peptides covering the JAK2V617F mutation were identified. Since our previous work strongly suggests that tumor exclusive representation of an antigen is a prerequisite for the immunogenicity of its corresponding T cell epitopes, JAK2-derived wild-type peptides are unlikely to constitute optimal vaccines in PV. On the other hand, comparative profiling of the HLA class I ligandomes identified 935 antigens to be exclusively presented in JAK2V617F positive PV with 12 ligandome-derived tumor-associated antigens (LiTAAs) representing 26 different HLA ligands with exclusive representation in ≥30% of PV patients. Overlap analysis of PV patients with and without JAK2V617F mutation revealed 92% of the exclusive antigens to be represented on both groups, while only 8% and 0% showed exclusive representation in JAK2V617F positive and negative PV patients, respectively.

As CD4⁺ T cells play important indirect and direct roles in anti-tumor immunity, we further applied our approach to HLA class II ligandomes of 8 PV patients (6 with, 2 without JAK2V617F mutation) and 18 HV (13 blood and 5 bone marrow samples) Using this approach, we identified more than 7,700 different naturally presented HLA class II ligands (1,700 source proteins). Notably, no JAK2-derived HLA class II peptides were identified in PV patients and HV ligandomes. Applying the same antigen-ranking strategy as described for HLA class I, we identified 5 additional HLA class II LiTAAs (exclusive in >30% of PV ligandomes) represented by 19 corresponding LiTAPs (ligandome-derived tumor-associated peptides). Overlap analysis of PV-exclusive source proteins revealed 2 proteins (POLA1, polymerase (DNA directed), alpha 1 and RHOQ, ras homolog family member Q) to be exclusively represented both in HLA class I and II ligandomes of PV patients.

The novel HLA class I and II ligandome-derived tumor associated antigens identified in this study may constitute prime candidates for peptide-based immunotherapy of PV.