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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 naive 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. References Rizvi N, Hellmann MD, Snyder A, et al. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science. 2015;348(6230):124–128. Chowell D, Morris LGT, Grigg CM, et al. Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy. Science 2018;359 (6375):582–587. Chowell D, Krishna C, Pierini F, et al. Evolutionary divergence of HLA class I genotype impacts efficacy of cancer immunotherapy. Nature Medicine 2019;25(11):1715–1720. Shastri N, Nagarajan N, Lind KC, et al. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum. Curr Opin Immunol 2014; 26:123–127. Mpakali A, Maben Z, Stern LJ, et al. Molecular pathways for antigenic peptide generation by ER aminopeptidase 1. Mol Immunol 2018; 13:50–57. James E, Bailey I, Sugiyarto G, et al. Induction of protective antitumor immunity through attenuation of ERAAP function. J Immunol 2013;190(11):5839–5846. Manguso RT, Pope HW, Zimmer MD, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 2017;547(7664):413–418. Leonard, H Remtulla A, Poynton F, et al. AACR Annual Meeting 2020. Purcell AW, Ramarathinam SH, Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 2019;14(6):1687–1707.

Clinical data demonstrates increased antigen presentation diversity is an important factor in determining response rates to checkpoint inhibitors. In addition to tumor mutational burden, increased HLA heterozygosity and HLA evolutionary diversity are non-overlapping factors which further diversify the immunopeptidome and improve clinical response to checkpoint therapies. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides loaded into classical and nonclassical MHC Class I molecules. Human genome-wide association studies have identified single nucleotide polymorphisms within ERAP1 that are associated with immune-related diseases, such as ankylosing spondylitis, providing human genetic validation for ERAP1's role in human disease and antigen presentation. Further, ablation of mouse ERAAP modifies the immunopeptidome, resulting in improved immunogenicity, generation of CD8+ T cell responses and tumor growth inhibition. Grey Wolf Therapeutics have developed highly potent and selective ERAP1 inhibitors. These inhibitors demonstrate significant modulation of the cancer-related antigen repertoire across diverse ERAP1 and HLA genotypes and cancer-type backgrounds, both in vitro and in vivo. These changes in the antigen repertoire drive changes in T cell activation and response, leading to increased T cell infiltration into CT26 syngeneic tumors and T cell receptor (TCR) diversification when combined with anti-PD-1. We have identified immune related markers that are modulated following ERAP1 inhibition in syngeneic tumor models which have the potential to be used as biomarkers. Importantly, ERAP1 inhibitor induced immunopeptidome and T cell changes lead to significant tumor growth inhibition in syngeneic mouse models when combined with anti-PD-1. In parallel, we have demonstrated the ability of ERAP1 inhibitor induced novel cancer associated antigens to stimulate human CD8+ T cell responses. Extensive assessment of the potential of ERAP1 inhibitors to enhance tumor immune responses in combination with additional therapies (e.g. chemotherapy and radiotherapy), across different tumor microenvironments, is ongoing. These data provide the foundation from which we plan to explore the potential of our first-in-class ERAP1 inhibitor development candidate in the clinic. Citation Format: Andrew Leishman, Fergus Poynton, Nicola Ternette, Elisa Lori, Camila de Almeida, Henry Leonard, Emma Reeves, Edd James, Kristopher Clark, Carmen Tong, Jason Shiers, Martin Quibell, Peter Ian Joyce. First in class inhibitors of ERAP1 have the potential to be a transformative immunotherapy in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1715.

Immune checkpoint therapy has changed the cancer treatment paradigm, however the majority of patients respond poorly to T cell checkpoint blockade alone. Emerging evidence suggests factors that increase the diversity of the immunopeptidome such as high tumor mutation burden and HLA-I evolutionary divergence (HED), improve clinical response to checkpoint blockade providing an impetus to develop strategies that increase the presentation of cancer associated antigens to the immune system. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase that trims peptides loaded into classical and nonclassical MHC molecules. Human genome-wide association studies have identified single nucleotide polymorphisms within ERAP1 that are associated with immune-related diseases, such as ankylosing spondylitis, providing human genetic validation for ERAP1's role in human disease and antigen presentation. Furthermore, in mice, genetic ablation, reduction or pharmacological inhibition of ERAP1 changes the immune repertoire, creating novel antigens with improved immunogenicity that lead to the generation of productive CD8 T cell responses and tumor growth inhibition in various syngeneic models. We have generated and profiled highly potent, species cross reactive and selective ERAP1 inhibitors through a suite of in vitro and in vivo assays to develop these as novel immunotherapy agents. In vitro CETSA measurements show potent cellular target engagement that correlates with in vitro SPR and enzyme potency. Using the model antigen SIINFEKL and Class I MHC expression as key pharmacodynamic measurements, we demonstrate Class I modulation in vitro and in vivo in line with published data from ERAP1 knockout mice. These assays are being used to elucidate the kinetics and temporal dynamics of the antigen change to optimise dosing schedules for in vivo studies. Implementation of our immunopeptidomics pipeline has demonstrated treatment with ERAP1 inhibitors both generate novel antigens in vitro and in vivo and lead to profound and consistent increased overall immunopeptidome length in mouse and human cells. To establish the functional consequence of these changes, we developed a human primary T cell immunogenicity platform. The immunogenic potential of these novel cancer associated antigens has been confirmed by the ability to stimulate IFNγ production in naïve T cells and suggests responses to these antigens could reinvigorate anti-tumor responses. Extensive assessment of mouse and human CD8 T cells responses is ongoing, in order to characterise and select Grey Wolf Therapeutics' first lead ERAP1 inhibitor for use as monotherapy or in combination with other immunotherapies such as checkpoint blockade. Citation Format: Jamie Ware, Patrick McIntyre, Kristopher Clark, Carmen Tong, Jason Shiers, Elisa Lori, Camila de Almeida, Emma Reeves, Henry Leonard, Alihussein Remtulla, Michael Ford, Nicola Ternette, Fergus Poynton, Edd James, Lesley Young, Martin Quibell, Peter I. Joyce. Potent oral ERAP1 inhibitors modify the immunopeptidome in vivo and are novel immunotherapy agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5551.

Optimal peptide loading on the MHC I molecule is essential for antigen presentation to CD8+ cytotoxic T lymphocytes. Endoplasmic Reticulum Aminopeptidase associated with Antigen Processing (ERAAP) is responsible for N-terminal trimming of peptides to optimal length for stable loading and presentation through MHC I. In humans, ERAAP is also known to play a role in pro-inflammatory cytokine receptor cleavage as well as regulating blood pressure and angiogenesis. Recently, a single nucleotide polymorphism (SNP) linkage analysis study has identified ERAAP as being associated with increased risk of the autoimmune inflammatory disorder, ankylosing spondylitis (AS). Here, we show that SNP mutation of ERAAP alters its ability to trim peptides. Transfection of ERAAP-/- cells with ERAAP molecules incorporating each of the SNPs identified in the linkage analysis show a hierarchy of reduced function. More pronounced functional phenotypes are revealed with additional mutations suggesting a cumulative effect. Although the mechanism for the effect of SNP mutation on ERAAP function is unclear, it appears that they cause a dramatic effect on peptide trimming.

Depletion of regulatory T cells in the CT26 murine tumour model stimulates a robust protective T cell response which is also protective to challenge with other tumours of different histological origins. We have characterised a cross-protective antigen, GSW11 (GGPESFYCASW) presented by H-2Dd. This antigen was found to be encoded within the env (gp90) gene of an endogenous retrovirus emv-1 (MuLV), previously shown to encode other CT26 antigens. Further characterisation of GSW11-specific T cell responses has shown them to be suppressed by Tregs and are absent in CT26 challenged mice. In comparison, AH1-specific T cells (AH1 is a previously characterised CT26 antigen) are not suppressed. Here we show that the GSW11-specific T cell responses are present in CT26 challenged Treg replete mice for a limited period in the tumour of challenged mice. Characterisation of the AH1 and GSW11-specific T cells infiltrating the CT26 tumour reveal differences in expression of several molecules associated with cell survival and activation. These expression profiles provide an intriguing explanation for the differential suppression of tumour-specific T cells in vivo.

Regulatory T cells (Tregs) play a pivotal role in the suppression of tumour antigen responses. It is well established that balb/c mice depleted of Tregs, using the anti-CD25 antibody pc61, are capable of rejecting the colon carcinoma line CT26. Mice challenged with CT26 in these circumstances have also been shown to reject a further challenge with CT26 or a second tumour line. The antigens responsible for this rejection are only partially characterised, with MHC class 1 antigens known to be restricted to all three H2-D molecules. Our laboratory has identified a Dd restricted antigen, GSW11, which is largely responsible for the cross protective response. Investigation into the processing of GSW11 revealed a sensitivity to over-processing by Eraap. In an attempt to increase the immunogenicity we generated Eraap knockdown CT26 using a plasmid knockdown system. These CT26 derivatives with reduced Eraap mRNA and protein levels were found to stimulate a peptide specific hybridoma more than wild type CT26. In addition, balb/c mice challenged with Eraap knockdown CT26 gained an 80% protection verses 0% mice injected with wild type CT26. Further experimentation is being undertaken to elucidate the exact mechanisms behind this protection.

There is accumulating evidence that CD4+ CD25+ regulatory T cells (Treg) play an important role in anti-tumour immunity by preventing effective T cell responses to tumour antigens. Tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. The depletion of Tregs prior to challenge with the murine colorectal tumour, CT26, stimulates a robust, protective T cell response which is also protective to challenge with other tumours of different histological origins, such as B cell lymphomas and a renal cell carcinoma. This cross protection has not been seen with other tumour cell lines. We have identified a CT26-derived cross-protective antigen, GSW11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. This protein has previously been shown to encode CT26-specific CD8 and CD4 antigens, implicating it as a 'hot-spot' for CT26 tumour antigens. Interestingly, we have identified a truncated version of gp70 which may be responsible for generation of GSW11. Expression studies have revealed increased gp70 expression in CT26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (GSW11) generated.