MM-210 Targeting Proteasome-Dependent Antigen Presentation to Enhance T Cell–Mediated Tumor Killing

Abstract

Cancer immunotherapy has emerged as a leading strategy in the war on cancer; however, only a minority of patients exhibit maximal benefits and durable responses. Proteasomes are central components of the proteolytic processing machinery required to generate antigenic peptides presented by MHC class I molecules to cytotoxic T lymphocytes (CTLs). Since cancer cells downregulate the generation and presentation of antigenic peptides to escape anti-tumor immunity, we hypothesized that proteasomal generation of antigenic peptides could be pharmacologically enhanced to increase tumor antigen presentation and enhance CTL-mediated tumor lysis. We performed a cell-based, high-throughput screen (HTS) using ~3,400 FDA-approved drugs and bioactive molecules to identify agents that increased proteasomal peptide-hydrolyzing activity. We then determined whether "hits" identified in the HTS also increased the presentation of a specific MHC class I antigen, SIINFEKL, which is derived from chicken ovalbumin. We engineered a T cell that specifically expresses a T-cell receptor (TCR) that recognizes SIINFEKL. Tumor cells were pre-treated with hits identified in the HTS, most notably the histone deacetylase (HDAC6) inhibitors Tubastatin A and ACY-1215, and then incubated with the engineered T cells to determine the effect on T cell-mediated tumor lysis. We found that Tubastatin A and ACY-1215 potently increased proteasome activity in the cell-based assay. We employed the lymphoma cell line EG.7-ova that constitutively expresses the chicken ovalbumin gene. Proteasomal degradation of ovalbumin generates a peptide, SIINFEKL, that is presented at the tumor surface complexed with the MHC class I molecule. Treatment of EG. 7-ova cells with HDAC6 inhibitors enhanced the presentation of the SIINFEKL:MHC class I complex nearly 3-fold. CTLs were then engineered to express a TCR that specifically recognizes SIINFEKL. When EG.7-ova cells were pre-treated with HDAC6 inhibitors and then co-cultured with SIINFEKL-restricted CTLs, tumor lysis was also increased up to 3-fold. Western blotting showed that treatment of MM cells with HDAC6 inhibitors increased immunoproteasome levels and catalytic activity. Taken together, our results endorse a paradigm-shifting approach to exploit pharmacologics that enhance the proteasomal generation of tumor antigens as an anti-myeloma strategy. Pharmacologic modulation of proteasome activity potently stimulates the presentation of tumor antigens and represents an actionable approach to trigger anti-myeloma immunity.