Background Clinical data demonstrates increased antigen presentation diversity is a key factor in determining response rates to checkpoint inhibitors.1 In addition to tumour mutational burden/microsatellite instability, increased HLA heterozygosity and HLA evolutionary diversity are non-overlapping factors recently identified to further diversify the immunopeptidome and improve clinical response to checkpoint therapies.2 3 Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides loaded into classical and nonclassical class I MHC molecules.4 5 Ablation of mouse ERAAP modifies the immunopeptidome, resulting in improved immunogenicity, generation of CD8 T cell responses and tumor growth inhibition.6 7 Recently identified selective small molecules potently inhibit ERAP1 across key species and haplotypes.8 We report the further profiling of lead candidate ERAP1 inhibitors in human primary T cell in vitro assays and in vivo tumor models in mice.
Methods Human cancer cell lines treated with ERAP1 inhibitors in vitro or in vivo in xenograft mouse models were assessed by immunopeptidomics9 to profile peptide repertoire changes. Novel or upregulated peptides were also tested in human immunogenicity assays. FACS analysis of T cells stimulated with Tyrosinase mRNA transfected human dendritic cells ± ERAP1 inhibition was to assess T cell repertoire changes. ERAP1 inhibitor and anti PD-1 mAb combination was assessed in syngeneic mouse tumor models to investigate tumour growth inhibition and PD end-points (e.g. IHC).
Results Analysis of human cervical, lung, colorectal and melanoma cell lines carrying distinct HLA haplotypes demonstrates a consistent and profound effect of ERAP1 inhibition on the immunopeptidome. Novel and upregulated cancer associated antigens identified in association with multiple different HLA-A and B alleles stimulate IFNγ production in primary naïve human T cell immunogenicity assays. The impact of ERAP1 inhibition on the T cell repertoire to the melanoma antigen tyrosinase is ongoing. The combination of ERAP1 inhibitor and anti PD-1 mAb led to significant tumor growth inhibition in the CT26 syngeneic mouse tumor model that correlated with increased infiltration of T cells to the tumor. Further PD end-points to be analysed include immune gene array and TCR Vbeta repertoire.
Conclusions Grey Wolf ERAP1 inhibitors significantly modify the immunopeptidome both in vitro and in vivo across a broad range of HLA and tumor types. Combination of these inhibitors with anti PD-1 leads to significant T cell infiltration and tumor growth inhibition. Thus, ERAP1 mediated modulation of the immunopeptidome has the potential to drive anti tumor T cell responses and be a transformative immunotherapy.
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