Effects of modulation of ERAP1 activity in antigen processing and presentation

Abstract

ERAP1 plays a key role in the antigen processing and presentation pathway as a peptide editor, allowing for the binding of neoantigens to MHC class I (MHC I). ERAP1 is polymorphic, showing different peptide trimming function across its variants. However, the effect of this variation in the trimming function of ERAP1 is still not fully understood. The purpose of the study is to characterise the effect of the different ERAP1 variants and their inhibition on the peptide repertoire presented by tumour cells, providing valuable insights into how this process works. This characterisation will also enhance the pharmacological modulation of ERAP1, which has already been proven to promote T cell and NK-mediated anti-tumour cytotoxic response.
The (mouse) ERAAP gene was knocked out (KO) by CRISPR-Cas9 in the CT26 and 4T1 cell lines. After ERAAP KO, MHC I kinetics were assessed by investigating the dissociation of peptide-MHC complexes at the cell surface. The assessment of MHC I assembly showed that in the absence of ERAAP, peptide/MHC complexes formed were more unstable, representing the presentation of peptides with lower binding affinity for MHC I. Proteins of human ERAP1 allotypes were produced and assessed for their trimming activity levels of two substrates displaying a wide range of trimming activities across allotypes. In addition, the same ERAP1 allotypes were then re-expressed in the ERAAP KO cell lines through retroviral transduction revealing a strong recovery in MHC I expression levels suggesting the formation of new peptide-MHC complexes. Lastly, immunopeptidomic analysis comparing the CT26 WT and ERAAP KO was performed showing a distinct subset of 529 peptides presented exclusively in the KO cell line. Peptides from immunopeptidomic analysis were then administered ex-vivo displaying strong immunogenicity for a handful of candidates.
In conclusion, modulation of ERAP1 activity significantly alters the presented peptide repertoire, uncovering potentially immunogenic neoantigens.

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Identifiers

ORCID for Shami Alvarez Mourid: orcid.org/0000-0003-3311-3370

ORCID for Edd James: orcid.org/0000-0001-8638-7928

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