Mapping the Impact of Proteasome Inhibitor Therapy on the Immunopeptidome of Multiple Myeloma: Mass Spectrometry Identifies Robust Targets for T Cell Immunotherapy

Recent studies underscore that multiple myeloma is an immunogenic disease and suggest that it can be effectively treated by T cell based immunotherapy via immunomodulation. This strategy might be synergistically complemented by therapeutic vaccination, which may help induce and guide specific anti-cancer T cell responses. We have recently conducted a study which directly characterized the antigenic landscape of myeloma by mass spectrometric analysis of naturally presented HLA ligands and identified a panel of T cell epitopes characterized by exquisite myeloma-association (Walz, Stickel et. al., Blood 2015). As standard of care in myeloma includes proteasome inhibitor therapy and the proteasome plays a central role in the generation of MHC-presented peptides, it is of great importance to thoroughly characterize and take into account the effects of this treatment on the antigenic landscape of myeloma cells and implement only robustly presented targets for peptide vaccine design. This is even more important

Here we present a mass spectrometry-based study, which longitudinally and semi-quantitatively maps the effects of treatment with the 2^nd generation proteasome inhibitor carfilzomib in an in vitro model of multiple myeloma. We observed considerable plasticity of the HLA class I ligandome of MM.1S cells after treatment with carfilzomib with 17.9±1.1.% (mean of 3 biological replicates ± SD) of HLA ligands showing significant modulation (fold change ≥ 4, P ≤ 0.01) at t_24h compared to mock-treated controls (down-modulated: 11.5±1.1%, up-modulated: 6.3±0.6%). We were able to longitudinally tracke the abundance of 28 previously defined myeloma antigens, confirming robust (16/28, 57.1%) or even increased presentation (8/28, 28.6%) under treatment for the majority of these peptides. However, - importantly - we observed highly distortive effects of carfilzomib treatment on the HLA allotype distribution of target cells, which manifested as a marked reduction of HLA ligands restricted by HLA-A*23:01 and A*24:02 (-62.5±1.8% and -57.0±0.6%, respectively, at t=24h after treatment). These findings indicate strong allotype-specific effects of carfilzomib on the antigenic landscape of myeloma cells, which we interpret to be a direct reflection of the mechanism of action of this drug. As a significant proportion of the U.S. population are carriers of the affected alleles (A*23:01: 8.2%; A*24:02: 22.6%), these findings could have broad implications for the design or implementation of antigen-specific therapies in patients under proteasome inhibitor treatment. Furthermore, these findings might indicate the possibility of altered cancer immunosurveillance as a consequence of proteasome inhibitor therapy.