Mapping The HLA Ligandome Of Chronic Lymphocytic Leukemia – Towards Peptide Based Immunotherapy

Accelerated clonal evolution and inhibition of immune effector functions are fundamental drawbacks of chemotherapeutic treatment of chronic lymphocytic leukemia (CLL) which contribute to increased clinical aggressiveness of relapsed disease. Anticancer immune responses such as graft versus leukemia effects and remissions after donor lymphocyte infusions, on the other hand, have been correlated to long-term CLL-free survival. Clonotype analysis in these cases suggested clonally expanded CD8⁺ T cells recognizing tumor associated antigens (TAAs) presented by HLA as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine-based immunotherapy. We here report on our approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative and semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for the identification of ligandome derived TAAs (LiTAAs) and informed selection of peptide vaccine candidates. HLA class I ligands were isolated from MACS-sorted CLL cells as well as from normal B cells or PBMC of healthy volunteers using a standard immunoaffinity purification protocol. Liquid chromatography coupled mass spectrometry (LC-MS/MS) peptide analysis was performed on a LTQ Orbitrap hybrid mass spectrometer followed by database assisted processing of fragment spectra. Semi-quantitative data analysis provided information regarding the abundance of HLA ligands in the respective ligandomes. In addition, HLA surface expression on CLL cells and autologous normal B cells was quantitatively determined using a flow cytometric assay. Selected peptides were characterized functionally in IFN-γ ELISPOT assays using PBMC of healthy volunteers and CLL patients. No significant difference in HLA class I surface expression between CLL cells and autologous normal B cells was observed. So far, we were able to map the HLA class I peptidomes of 25 CLL patients and 35 healthy controls. In total, we were able to identify more than 25,000 different HLA ligands representing >8,500 different source proteins. More than 15,000 different ligands were derived from CLL cells representing a total of 6,500 source proteins. A twofold data mining approach was used to identify both, broadly presented LiTAAs suited for off-the-shelf vaccine development, and LiTAAs showing patterns of patient-specific overrepresentation allowing for actively personalized target identification. The former strategy enabled us to pinpoint the most frequently and abundantly represented targets from the bulk of over 2,000 source proteins, which were exclusively represented in the ligandomes of CLL cells. Several published CLL-associated antigens/epitopes were found to be presented (e.g. Pim-1 Oncogene, SET nuclear oncogene, Mucin-1), which served to validate our methodological approach as proof of principle. Beyond that we identified a vast array of novel proteins that are broadly and exclusively represented in the HLA peptidome of CLL cells. Based on these findings we selected HLA-A*02, A*03 and B*07 restricted ligands derived from top ranking LiTAAs (e.g.TP53I11, PARP3, CDCA7L) for immunological characterization. Using patient PBMC, we observed frequent, reproducible and specific immune recognition of the selected peptides by CD8⁺ T cells in recall ELISPOT assays. The observed reactivity to CLL-associated self-peptides indicates their potential as therapeutic vaccines while underlining the validity of our target identification and selection strategy. Currently we are expanding our analyses to cover a comprehensive spectrum of HLA types with the goal to develop a clinically applicable CLL-specific multi-peptide vaccine.