Vaccine Can Induce CD4-Mediated Responses to Homocitrullinated Peptides via Multiple HLA-Types and Confer Anti-Tumor Immunity

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Katherine Cook, Wei Xue, Suha Atabani, Peter Symonds, Abdullah Al Omari, Ian Daniels, Sabaria Shah, Ruhul Hasan Choudhury, Daisy Weston, Rachael Metheringham, Victoria Brentville and Lindy Durrant

ABSTRACT: Homocitrullination is the post translation modification (PTM) of the amino acid lysine to homocitrulline also referred to as carbamylation. This PTM has mainly been studied in relation to autoimmune diseases including rheumatoid arthritis. Homocitrullination of lysines alters their charge which can lead to generation of neoepitopes that are differentially presented by MHC-II and induce modification-specific immune responses. Homocitrullination is often considered a process which triggers autoimmune disease by bypassing self-tolerance however, we suggest that homocitrullination may also have an alternative role in immune responses including protection against cancer. Here we demonstrate that immune responses to homocitrullinated peptides from three different proteins can be induced via multiple HLA-types. Immunization of Balb/c or HLA-transgenic DR4 and DR1 mice can induce modification-specific CD4 mediated IFNγ responses. Healthy human donors show a clear repertoire for the homocitrullinated Vimentin peptide (Vim116-135Hcit), with modification-specific and oligoclonal responses. Importantly, in vivo homocitrulline specific Vim116-135Hcit, Cyk8 371-388Hcit and Aldo 140-157Hcit responses are able to confer an anti-tumor effect in the murine B16 melanoma model. The Vim116- 135Hcit anti-tumor response was dependent upon tumor expression of MHC-II suggesting the direct recognition of PTMs on tumor is an important anti-tumor mechanism. Cancer patients also have a CD4 repertoire for Vim116-135Hcit. Together these results suggest that homocitrulline-specific immune responses can be generated in healthy mice and detected in human donors through a variety of HLA-restrictions. Immunization can induce responses to Vim116-135Hcit, Aldolase 140-157Hcit and Cyk8 371-388Hcit which provide anti-tumor therapy across several HLA-types. Our results advance our understanding of homocitrulline-specific immune responses, with implications for a number of fields beyond autoimmunity, including tumor immune surveillance.

PAD-2- mediated citrullination of nucleophosmin provides an effective target for tumor immunotherapy

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Ruhul H Choudhury, Peter Symonds, Samantha J Paston, Ian Daniels, Katherine W Cook, Mohamed Gijon, Rachael L Metheringham, Victoria A Brentville, Lindy G Durrant

Background The enzymatic conversion of arginine to citrulline is involved in gene and protein regulation and in alerting the immune system to stressed cells, including tumor cells. Nucleophosmin (NPM) is a nuclear protein that plays key roles in cellular metabolism including ribosome biogenesis, mRNA processing and chromatin remodeling and is regulated by citrullination. In this study, we explored if the same citrullinated arginines within NPM are involved in gene regulation and immune activation.
Methods HLA-DP4 and HLA-DR4 transgenic mice were immunized with 22 citrullinated NPM overlapping peptides and immune responses to the peptides were assessed by ex vivo ELISpot assays. Antitumor immunity of NPM targeted vaccination was assessed by challenging transgenic mice with B16F1 HHDII/iDP4, B16F1 HHDII/ PAD2KOcDP4, B16F1 HHDII and Lewis lung carcinoma cells/cDP4 cells subcutaneously. Peripheral blood mononuclear cells isolated from healthy donors were stimulated with NPM266-285cit peptides with/without CD45RO+memory cell depletion to assess if the responses in human were naïve or memory.
Results In contrast to NPM regulation, which is mediated by peptidylarginine deiminase (PAD4) citrullination of arginine at position 197, only citrullinated NPM266-285 peptide induced a citrulline-specific CD4 T cell response in transgenic mice models expressing human HLA-DP4 or HLA-DR4. Vaccinations with the NPM266-285cit peptide stimulated antitumor responses that resulted in dramatic tumor therapy, greatly improved survival, and protected against rechallenge without further vaccination. The antitumor response was lost if MHCII expression on the tumor cells was knocked out demonstrating direct presentation of the NPM266-285cit epitope in tumors. This antitumor response was lost in B16 tumors lacking PAD2 enzyme indicating NPM266cit is citrullinated by PAD2 in this model. Assessment of the T cell repertoire in healthy individuals and patients with lung cancer also showed CD4 T cells that respond to NPM266-285cit.The proliferative CD4 responses displayed a Th1 profile as they were accompanied with increased IFNγ and granzyme B expression. Depletion of CD45RO+ memory cells prior to stimulation suggested that responses originated from a naïve population in healthy donors.
Conclusion This study indicates PAD2 can citrullinate the nuclear antigen NPM at position 277 which can be targeted by CD4 T cells for antitumor therapy. This is distinct from PAD4 citrullination of arginine 197 within NPM which results in its transport from the nucleoli to the nucleoplasm.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy

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Katherine W Cook, Wei Xue, Peter Symonds, Ian Daniels, Mohamed Gijon, David Boocock, Clare Coveney, Amanda K Miles, Sabaria Shah, Suha Atabani, Ruhul H Choudhury, Poonam Vaghela, Daisy Weston, Rachael L Metheringham, Victoria A Brentville and Lindy G Durrant.
Background  Homocitrullination is the post-translational modification of lysine that is recognized by T cells.
Methods  This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry.
Results  Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected  B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment.
Conclusion  We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

A novel bivalent DNA vaccine encoding both spike protein receptor-binding domain and nucleocapsid protein of SARS-CoV-2 to elicit T cell and neutralising antibody responses that cross react with variants

A novel bivalent DNA vaccine encoding both spike protein receptor-binding domain and nucleocapsid protein of SARS-CoV-2 to elicit T cell and neutralising antibody responses that cross react with variants

Brentville VA, Vankemmelbeke M, Metheringham RL, Symonds P, Cook KW, Urbanowicz R, Tsoleridis T, Coleman C, Chang K-C, Skinner A, Dubinina E, Daniels I, Shah S, Dixon JE, Pockley AG, Adams SE, Paston SJ, Daly JM, Ball J and Durrant LG.

ABSTRACT: The efficacy of vaccines targeting SARS-CoV-2 is becoming apparent now that the mRNA and adenovirus vector vaccines that have been approved for emergency use are showing promise. However, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. To improve longevity and future protection against variants, we have designed a DNA vaccine encoding both the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and its nucleocapsid (N) protein, the latter of which is highly conserved amongst beta coronaviruses. The vaccine elicits strong pro-inflammatory CD4 Th1 and CD8 T-cell responses to both proteins, with these responses being significantly enhanced by fusing the nucleocapsid sequence to a modified Fc domain. We have shown that the vaccine also stimulates high titre antibody responses to RBD which efficiently neutralise in both a pseudotype and live virus neutralisation assay and show cross reactivity with S proteins from the emerging variants Alpha (B.1.1.7) and Beta (B.1.351). This DNA platform can be easily adapted to target variant RBD and N proteins and we show that a vaccine variant encoding the B.1.351 RBD sequence stimulates cross-reactive humoral and T-cell immunity. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for targeting emerging SARS-CoV-2 variants.

Cancer Vaccines, Adjuvants, and Delivery Systems

Cancer Vaccines, Adjuvants, and Delivery Systems

Samantha J. Paston, Victoria A. Brentville, Peter Symonds and Lindy G. Durrant

ABSTRACT: Vaccination was first pioneered in the 18th century by Edward Jenner and eventually led to the development of the smallpox vaccine and subsequently the eradication of smallpox. The impact of vaccination to prevent infectious diseases has been outstanding with many infections being prevented and a significant decrease in mortality worldwide. Cancer vaccines aim to clear active disease instead of aiming to prevent disease, the only exception being the recently approved vaccine that prevents cancers caused by the Human Papillomavirus. The development of therapeutic cancer vaccines has been disappointing with many early cancer vaccines that showed promise in preclinical models often failing to translate into efficacy in the clinic. In this review we provide an overview of the current vaccine platforms, adjuvants and delivery systems that are currently being investigated or have been approved. With the advent of immune checkpoint inhibitors, we also review the potential of these to be used with cancer vaccines to improve efficacy and help to overcome the immune suppressive tumor microenvironment.

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor response

Victoria A Brentville, Rachael L Metheringham, Ian Daniels, Suha Atabani, Peter Symonds, Katherine W Cook, Mireille Vankemmelbeke, Ruhul Choudhury, Poonam Vaghela, Mohamed Gijon, Ghislaine Meiners, Willem-Jan Krebber, Cornelis J M Melief, Lindy G Durrant

ABSTRACT: Background  Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes.

Post-translational modifications such as citrullination are excellent targets for cancer therapy

Post-translational modifications such as citrullination are excellent targets for cancer therapy

V.A. Brentville, M. Vankemmelbeke, R.L. Metheringham, L.G. Durrant

ABSTRACT: Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4+ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress.  Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumour microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4+ T cell responses that would recognize these siPTM’s produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy

Victoria A Brentville, Peter Symonds, Katherine  W Cook, Ian Daniels, Tracy Pitt, Mohamed Gijon, Poonam Vaghela, Wei Xue, Sabaria Shah, Rachael E Metheringham, and Lindy Durrant

ABSTRACT: Post-translational modifications are induced in stressed cells which cause them to be recognised by the system. One such modification is citrullination where the positive charged arginine is modified to a neutral citrulline. We demonstrate most healthy donors show an oligoclonal CD4 response in vitro to at least one citrullinated vimentin or enolase peptide. Unlike rheumatoid arthritis patients, these T cell responses were not restricted by HLA-DRB1 shared epitope (SE) alleles, suggesting they could be presented by other MHC class II alleles. As HLA-DP is less polymorphic than HLA-DR, we investigated whether the common allele, HLA-DP4 could present citrullinated epitopes. The modification of arginine to citrulline enhanced binding of the peptides to HLA-DP4 and enhanced high-frequency CD4 responses in HLA-DP4 transgenic mouse models. Our previous studies have shown that tumours present citrullinated peptides restricted through HLA-DR4 which are good target for anti-tumour immunity. In this study, we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival, p < 0.0001) against established B16 tumours d and against the LLC/2 lung cancer model (p = 0.034) both expressing HLA-DP4. Since most tumours do not constitutively express MHC class II molecules, models were engineered that expressed MHC class II under the control of an IFNγ inducible promoter. Immunisation with citrullinated peptides resulted in 90% survival (p < 0.001) against established B16 HHD tumour expressing IFNγ inducible DP4. These studies show that citrullinated peptides can be presented by a range of MHC class II molecules, including for the first time HLA-DP4, and are strong targets for anti-tumour immunity.

CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, Paris, 25-28 September 2019

Post-translationally modified antigens are good targets for cancer immunotherapy but some patients have antigen specific T-regs that may need to be neutralized

Suha Atabani, Victoria Brentville, Ian Daniels, Ruhul Choudhury, Katherine Cook, Poulam Patel and Lindy Durrant

Improving selection criteria for post translationally modified CD4 epitopes using computer algorithms.

K Cook, P Symonds, A Skinner, S Shah, R Metheringham, S Paston, V Brentville and L Durrant

Carbamylation of lysine residues mediated by MDSCs in the tumour environment make excellent targets for CD4 T cell mediated cancer immunotherapy

K Cook, W Xue, I Daniels, P Symonds, M Gijon, D Boocock, C Coveney, A Miles, P Vaghela, R Choudhury, S Shah, S Atabani, R Metheringham, V Brentville and L Durrant

Targeting citrullinated vimentin and enolase with cytotoxic CD4 T cells, relies upon MHC-II expression by tumors, reduces myeloid suppressor cells and directly kills tumor cells

V Brentville, R Metheringham, I Daniels, S Atabani, P Symonds, K Cook, R Choudhury, P Vaghela, M Gijon, G Meiners, W-J Krebber, CJM Melief and L Durrant

Citrullinated glucose-regulated protein 78 is a candidate target for cancer immunotherapy

V Brentville, J Chua, S Atabani, P Symonds, K Cook, R Choudhury, I Daniels, S Shah and L Durrant

An ultraspecificmonoclonal antibody recognises a novel marker on stem memory T cells and induce cell proliferation and differentiation in vitro and in vivo

J Chua, E Cid, M Vankemmelbeke, R McIntosh, R Metheringham, I Daniels, V Brentville and L Durrant