HLA Ligandome Analysis in Multiple Myeloma Reveals Novel Naturally Presented Myeloma-Associated Antigens Showing Stable Expression Under Proteasome Inhibitor Therapy
Authors: Walz Simon D. , Kowalewski Daniel J. , Schuster Heiko , Weisel Katja , Salih Helmut R. , Kanz Lothar , Rammensee Hans-Georg , Stevanovic Stefan , Stickel Juliane S. 
Published: October 2019

Abstract
Results of allogeneic transplantation and reports that multiple myeloma (MM) cells can act as antigen presenting cells, but also the favorable immune effector-to-target cell ratio in the minimal residual disease (MRD) setting suggest that MM, which previously was considered to be weakly immunogenic, may be targeted effectively by T cell based immunotherapy. Following conventional therapy, peptide-based immunotherapy might offer a low side-effect opportunity to extend time to disease progression and possibly eradicate MRD. Therefore we aim to develop a multi-peptide vaccine for the immunotherapy of MM patients based on naturally processed and presented HLA ligands.
In a first step we analyzed HLA surface expression on MM cells, which constitutes a prerequisite for effective T cell recognition. In MM patients (n=20), HLA class I expression was found to be heterogeneous with a mean of 415,000 ± 55,000 molecules on malignant cells. This expression was significantly (p<0.005, unpaired t-test) higher than that on autologous healthy B cells (200,000 ± 20,000), T cells (165,000 ± 15,000) and hematopoietic progenitor cells (205,000 ± 30,000). In addition, HLA class I expression on primary MM cells was also found to be significantly higher than that on plasma cells of healthy volunteers (HV, n=15) (290,000 ± 25,000, p<0.05). Mean HLA-DR expression on myeloma cells (25,000 ± 7,000) was significantly lower compared to HLA class I expression (p<0.0001) without significant difference compared to expression levels on autologous bone marrow cells as well as normal plasma cells of HVs (40,000 ± 5,000).
Using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry, we identified more than 15,000 different HLA class I ligands from 8 primary MM samples, 2 plasma cell leukemia samples and 5 myeloma cell lines (MCLs). Comparative mapping of MM HLA ligandomes and the ligandomes of 40 HVs (30 blood and 10 bone marrow samples) revealed 15 ligandome-derived tumor-associated antigens (LiTAAs) represented by 36 corresponding HLA peptides (LiTAPs), showing exclusive representation in >30% of myeloma samples. Among them we identified 3 novel HLA peptides derived from Multiple Myeloma SET domain(MMSET), which is an established driver of myeloma cell proliferation, stimulating the expression of c-myc.
We further studied the effect of the second-generation proteasome inhibitor carfilzomib on HLA surface expression and HLA ligandome composition. Notably, a slight albeit not significant increase in mean HLA class I expression was observed after 24h (t24) and 48h (t48) of carfilzomib treatment in MCL (n=5, fold change t24=1.4, pt24=0.37, fold change t48=1.6, pt48=0.25) as well as in primary myeloma cells in vitro (n=7, fold change t24=1.2, pt24=0.21, fold change t48=1.1, pt48=0.46). No significant loss or down regulation of HLA class I expression on MM cells was observed in ex vivo analysis of primary samples obtained from 2 myeloma patients before therapy and after 4 weeks of carfilzomib treatment.
In order to analyze the impact of proteasome inhibition on the composition of the HLA class I ligandome we incubated MCLs (U266, MM.1S) with carfilzomib and performed quantitative mass spectrometry at baseline as well as at t24 and t48. On U266, 4.6% (4.0%) and 4.4% (8.6%) of total identified peptides (n=2444) were significantly up- or down regulated after 24h/48h of treatment with carfilzomib, respectively, whereas on MM.1S (n=1693) 1.6% (12.4%) and 10.8% (18.0%) were significantly regulated after 24h/48h. Focusing on the LiTAPs presented on these MCLs, 76.5% (88.2%) on U266 and 83.3% (66.7%) on MM.1S remained stably expressed after incubation with carfilzomib. Further analyses evaluating overall changes in peptide motifs and the occurrence of novel, cryptic peptides after carfilzomib treatment are ongoing.
Taken together our data demonstrate that myeloma cells are valid targets for T cell based immunotherapy and provide a panel of novel, naturally presented myeloma-associated antigens, which exhibit stable presentation under proteasome inhibitor therapy.

Disclosures
No relevant conflicts of interest to declare.