IMiDs and Celmods Enhance Antigen Presentation Via MHC Class 1 - a Potential Novel Immunostimulatory Effect
Authors: Martin Shannon , Chrisochoidou Yakinthi , Margarido Andreia S. , Sialana Fernando , Morales Salomon , Leiro Marc , Li Yigen , Bird Sarah , Mitsopoulos Konstantinos , Arwert Esther , Pawlyn Charlotte 
Published: December 2024

Introduction
Immunomodulatory drugs (IMiDs) such as Lenalidomide (Len) have a direct impact on myeloma (MM) cells by functioning as a molecular glue, binding to a CRL4CRBN E3 ubiquitin ligase (cereblon) and leading to degradation of neo-substrates including ikaros (IKZF1) and aiolos (IKZF3). This results in downregulation of IRF4/MYC and MM cell death. IMiDs have also been shown to have direct action on the immune system, including T cells via upregulation of IL-2. We hypothesized that the enhanced degradation of neo-substrates in the tumour cell could be associated with a third mechanism of action, through altered antigen presentation of the neo-substrate degradation products as peptides via MHC class I (MHC-I), leading to upregulation of CD8+ T cell immunosurveillance. Additionally, we hypothesized that the novel cereblon binding agents (CELMoDs), such as Mezigdomide (Mezi), would lead to enhanced altered antigen presentation and a greater induced T cell response compared to IMiDs.
Methods
H929 cells were treated with either 10uM Len, 0.01uM Mezi or DMSO. IKZF1/IKZF3 degradation was measured via Western blot, after 2 and 24hrs. Global HLA levels were measure via flow cytometry after 24 hours of drug exposure with a pan HLA-ABC antibody. Subsequently, 1x10^8 H929 cells per condition were incubated with 10uM Len, 0.01uM Mezi or DMSO for 2 hours and 24hours. Cells were harvested and lysed in 1% CHAPS buffer. MHC-I peptide complexes were then isolated via incubation with bead/antibody complex, and the peptides then eluted and dried. MHC-peptides were labelled with tandem mass tags (TMT) to facilitate relative quantification via mass spectrometry (TMT-MS2). Predicted binding was analysed using NetMHC website. Normalised peptide abundance values were compared to their relative DMSO control using Log2 fold change (FC).
Results
Degradation of IKZF1 and IKZF3 was confirmed at 2 and 24hrs. There was greater degradation with Mezi compared to Len and at the longer time point. Global MHC-I expression did not change following incubation with Len and Mezi, as seen by flow. This indicates that any upregulated peptides subsequently identified by MS were not due to upregulation of MHC-I itself.
After isolation of MHC-I bound peptides and MS identification, a total of 7809 peptides (between 8-15 amino acids in length) from 3901 protein sources were identified across all samples. Most of the peptides were 9-mers (53.7%), consistent with the literature for MHC-I isolated peptides. 82% of peptides had high predicted binding for all the MHC-I alleles for H929 (HLA-A03:01, HLA-A24:02, HLA-B07:02, HLA-B18:01, HLA-C07:02).
All peptides with high predicted binding and consistently upregulated by a log2FC of at least 0.3 in all three replicates were considered further. Both Len and Mezi led to enhanced antigen presentation compared to DMSO, with more unique peptides presented at 24hrs vs 2hrs and with Mezi vs Len (Len 2hrs 2 peptides, Len 24hrs 24 peptides, Mezi 2hrs 10 peptides, Mezi 24hrs 40 peptides). 3 peptides (derived from proteins IKZF3, TMEM147 and ITGB7) were seen to have an increased expression across more than one treatment condition, and3 additional peptide sequences (derived from proteins APOL6 and ITGB7) corresponded to the same protein source across different treatment conditions.
All peptides derived from the IKZF3 and IKZF1 proteins were explored in more detail. Across the dataset 4 distinct peptides from IKZF1 (x1) and IKZF3 (x3) were identified. Their pulldown was enriched after incubation with Len (IKZF1: mean Log2 FC 2hrs -0.04, 24 hrs 0.44, IKZF3 mean Log2 FC 2hrs -0.12, 0.07, 0.34, 24 hrs -0.09, 0.16, 0.40) and Mezi (IKZF1: mean Log 2 FC 2hrs 0.3, 24hrs 0.43, IKZF3 mean Log2 FC 2hr 0.17, 0.49, 0.53, 24 hrs 0.18, 0.43, 0.63) compared to DMSO control, most marked for the 24hour time point and with Mezi. These, and the additional peptides of interest from protein sources TMEM147, ITGB7 and APOL6 will be analysed in immunogenicity assay to confirm stimulation of CD8+ T cells.
Discussion
Our data suggest that myeloma cells can present peptides from the known neo-substrates IKZF1 and IKZF3, along with additional peptides, after IMiD/CELMoD therapy via MHC-I. This occurred to a greater extent with the CELMoD Mezi compared to the IMiD Len. Experiments to assess the immunogenicity of the presented peptides are ongoing. These data highlight a potential novel mechanism by which IMiDs and CELMoDs may stimulate the immune system.