In this short presentation, ESR4 describes the process followed to obtain monoclonal antibodies from hybridoma cell lines, together with a brief overview of the next steps in the course of characterizing the immunopeptidome of the A375 human melanoma cell line after genetic and pharmacological inhibition of ERAP1, one of the two main aminopeptidases found to act in the endoplasmic reticulum.

The human immune system utilises small peptides bound to major histocompatibility complexes I (MHC I) - found on the cell surface –to distinguish healthy cells from those that are either infected or cancerous. The MHC-bound peptides are recognised by T- cells in a process called antigen presentation. The set of peptides presented to the immune system, known as the immunopeptidome, is generated intracellularly by proteolytic cleavage by the proteasome and other proteases, followed often by trimming excess N-terminal amino acids by endoplasmic reticulum aminopeptidases (ERAPs). ERAPs have been correlated with tumour escape or immune system hyperstimulation. Recent results have revealed that shifts in the immunopeptidome due to genetic variation or pharmacological modulation of ERAPs can enhance or down-regulate adaptive immune responses, which makes them promising targets for cancer immunotherapy and treatments of autoimmune diseases. In light of these results, the proteomic characterization of the immunopeptidome after a pharmacological or genetic alteration of ERAP activity is of very high importance. However, for the MS-based characterisation of the immunopeptidome, MHC I molecules with their bound peptides need to be purified by immunoaffinity chromatography, which requires monoclonal antibodies specific to MHC I molecules.

M1 aminopeptidases consisting of endoplasmatic reticulum aminopeptidases 1 and 2 (ERAP 1 and ERAP2) and insulin-regulated aminopeptidase (IRAP) are a subfamily of aminopeptidases that play a critical role in the process of antigenic peptides generation and the overall control of adaptative immune system response. The regulation of such enzymes could be a new way to approach autoimmune disease treatment and improve immunotherapy against cancer. Therefore, targeting ERAPs and IRAP has raised significant interest during the last decade because of their potential applications.1

These aminopeptidases are a group of metalloproteases that contain a zinc atom in the active site which is involved in the cleavage of the N-terminal amide bond of peptides. Different methods have been used for the design of inhibitors that could present selectivity using several chelating groups, such as carboxylic acids or phosphorus-containing groups. Phosphinic peptide analogues are pseudopeptides that contain a phosphinic group in lieu of the scissile peptide bond of a natural peptide that chelates with the zinc atom in the active site. This phosphinic group in the carboxylic position, together with the carbon in the place of the amine group mimic the structure of a natural peptide’s transition state during the amide bond cleavage, which results in efficiently potent as well as selective inhibitors.2

Phosphinic peptide inhibitors have already shown good potency and selectivity against other metalloproteases such as Aminopeptidase A or MMP-12.3,4 Regarding ERAPs/IRAP, several potent phosphinic inhibitors have already been reported, however no selective inhibitor has been developed so far for these enzymes.5

In the present work, phosphinic peptide libraries have been designed and synthesized, using combinatorial and solid-phase techniques. These libraries have set the basis for the detailed study of structure-activity relationships for ERAPs/IRAP, aiming to the identification of patterns that will lead to the discovery of selective inhibitors of target enzymes.

ERAP1 trimming of antigenic peptides has an important role in anticancer immune responses, by generating optimal peptides for MHC-I loading and presentation to T cells. Previous studies showed that colon carcinomas tend to overexpress ERAP1 and that ERAP1 is able to destroy by over-trimming strong cancer-related antigens, such as GSW11. Moreover, ERAP1 knockdown by RNAi induces a strong anticancer immune reaction against CT26 colon carcinoma cells in vivo and stimulates an immunological memory. We established an ERAP1 knock-out MC38 colon carcinoma cell line and will employ a syngeneic mouse model to analyse differences in tumour progression and anticancer immune responses between wild-type MC38 cells and ERAP1-KO MC38 cells. A CRISPR-Cas9 approach was used to knock out the ERAP1 gene in MC38. Single-cell clones were isolated by limiting dilution and confirmed for ERAP1 knock-out by PCR, Sanger Sequencing and Western Blot. Flow cytometry was used to analyse the difference in MHC-I expression between WT and ERAP1-KO MC38 cells. We obtained one ERAP1-KO MC38 clone where the absence of ERAP1 protein was confirmed by Western Blot and Sanger sequencing. Even if stimulated with Interferon γ ERAP1 knock-out cells didn’t show any expression of ERAP1 protein. Flow cytometry analysis showed an average 20% reduction in surface expression of MHC class I proteins H-2Kb and H-2Db on ERAP1 Knock-out cells compared to MC38 WT cells. Future work will investigate the role of ERAP1 on tumour growth rate and immune cells phenotype of WT vs KO MC38 injected in the sub cute of C57BL/6 mice.

ERAP1, is an enzyme that plays a key role in the antigen processing and presentation pathway as an editor of the peptide repertoire, allowing for the binding of neoantigens to MHC I.  The trimming mechanism of ERAP1 is still not fully understood, if we characterise the effect of ERAP1 allotypes and their inhibition on the peptide repertoire we will gain valuable insights to elucidate this process. It has been seen that the pharmacological modulation of this enzyme has the potential to promote T cell and NK-mediated anti-tumour cytotoxic response (Cifaldi et al. 2011).

By assessing the effect of ERAP1 inhibition on the peptide repertoire presented by tumour cells, and the effect of ERAP1 allotypes and their inhibition on the peptide repertoire we will be able to gain a better understanding of the trimming mechanism of ERAP1 and its implications in cancer.   

We have been able to successfully knockout the ERAP1 gene in CT26 cells and optimised a methodology for knockout detection and validation consisting in Sanger sequencing, Western Blot, MHC I expression and TOPO cloning. We did further analysis to assess the MHC I kinetics and stability of the knockout cell line by investigating the dissociation of pMHC complexes at the cell surface using Brefeldin A decay assay and an acid-stripping test. We now plan to insert the different human allotypes back into the CT26 ERAAP-KO cell line using retroviral transduction, determine the effect of ERAP1 inhibitors such as leucinethiol on the trimming activity of relevant ERAP1 alleles and manipulate ERAP1 function to modulate the presentation of peptide epitopes in the CT26 cell line as well as other cell lines. 

Endoplasmic reticulum aminopeptidases (ERAPs) are multifunctional metalloproteases involved in antigen processing and presentation. By trimming the N-terminus of peptide precursors, ERAPs succeed to generate mature antigenic epitopes ready for loading upon major histocompatibility complex class I (MHC-I) molecules and ultimately eliciting T-cytotoxic cellular responses. The connection of ERAP1/2 genetic variants with putative therapeutic applications in inflammatory and proliferative disorders has often been at the centre of our efforts by developing small-molecule probes that can fine-tune immune dysregulation. Targeted and controlled regulation of ERAP1 constitutes a challenging task because of the high structural similarity and broad substrate specificity inside the M1 enzyme family. As a result, the identification and multiparametric optimization of potent and novel chemical scaffolds for ERAP1 selective modulation is imperative. Here we present different strategies to access new ERAP1 inhibitors. First, carboxylic acids and bioisosteres are an important class of bioactive compounds that can be optimized efficiently to target metalloproteases. Structure-activity relationships (SAR) in combination with computer modelling and silico docking studies could fill the gaps in our existing chemical series. Second, Kinetic target-guided synthesis (KTGS) and fragment-based drug discovery (FBDD) will be used to generate hits and leads for ERAP1 in a systematic and refined manner.