ABSTRACT:  Presentation at the 20th International Congress of the Society for Melanoma Research - Philadelphia, November 6th - 9th, 2023

Heather Shaw, Poulam Patel, Miranda Payne, Satish Kumar, Sarah Danson, Dennis Hadjiyiannakis, Clare Barlow, Martin Highley, Amna Sheri, Amanda Fitzpatrick, Ioannis Karydis, Maria Marples, Robert Miller, Fayaz Master and Lindy Durrant

A DNA plasmid melanoma cancer vaccine, SCIB1, combined with nivolumab + ipilimumab in patients with advanced unresectable melanoma: Efficacy and safety results from the open-label Phase 2 SCOPE trial

Heather Shaw, Poulam Patel, Miranda Payne, Satish Kumar, Sarah Danson, Martin Highley, Clare Barlow, Robert Miller, Fayaz Master and Lindy Durrant

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Sabaria Shah, Katherine W Cook, Peter Symonds, Juliane Weißer, Anne Skinner, Abdullah Al Omari, Samantha J Paston, Ian Pike, Lindy G Durrant, Victoria A Brentville

ABSTRACT:
Background: Post-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered immune responses. One such modification is carbamylation (homocitrullination) that results in the formation of homocitrulline (Hcit) residues in a non-enzymatic reaction of cyanate with the lysine residues in the polypeptide chain. Homocitrullination occurs in the tumor microenvironment and CD4-mediated immune responses to Hcit epitopes can target stressed tumor cells and provide a potent antitumor response in mouse models.
Methods: Homocitrullinated peptides were identified and assessed in vitro for HLA-A2 binding and in vivo in human leukocyte antigen (HLA) transgenic mouse models for immunogenicity. CD8 responses were assessed in vitro for cytotoxicity and in vivo tumor therapy. Human tumor samples were analyzed by targeted mass spectrometry for presence of homocitrullinated peptides.
Results: Homocitrullinated peptides from aldolase and cytokeratin were identified, that stimulated CD8-mediated responses in vivo. Modified peptides showed enhanced binding to HLA-A2 compared with the native sequences and immunization of HLA-A2 transgenic mice generated high avidity modification specific CD8 responses that killed peptide expressing target cells. Importantly, in vivo the homocitrullinated aldolase specific response was associated with efficient CD8 dependent antitumor therapy of the aggressive murine B16 tumor model indicating that this epitope is naturally presented in the tumor. In addition, the homocitrullinated aldolase epitope was also detected in human tumor samples.
Conclusion: This is the first evidence that homocitrullinated peptides can be processed and presented via MHC-I and targeted for tumor therapy. Thus, Hcit-specific CD8 T-cell responses have potential in the development of future anticancer therapy.

Modi-2, a vaccine targeting homocitrullinated self-epitopes, stimulates potent CD4-mediated anti-tumour responses as a therapy for solid cancers

Abdullah Al-Omari, Katherine Cook, Peter Symonds, Anne Skinner, Yaling Zhu, Vince Coble, Nazim Uddin, Priscilla Ranglani, Adrian Parry, Sally Adams, Geoffrey Lynn, Lindy Durrant and Victoria Brentville

Vaccination stimulating post-translational modification specific Th1 responses repolarises the tumour environment to reduce suppressive LAP expressing T cells

Suha Atabani, Katherine Cook, Peter Symonds, Ian Daniels, Ruhul Choudhury, Alissa Wright, Anne Skinner, Victoria Brentville and Lindy Durrant

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Sabaria Shah, Abdullah Al-Omari, Katherine W Cook, Samantha J Paston, Lindy G Durrant and Victoria A Brentville

ABSTRACT:
Complex cellular interactions between the immune system and cancer can impact tumour development, growth, and progression. T cells play a key role in these interactions; however, the challenge for T cells is to recognize tumour antigens whilst minimizing cross-reactivity with antigens associated with healthy tissue. Some tumour cells, including those associated with viral infections, have clear, tumour-specific antigens that can be targeted by T cells. A high mutational burden can lead to increased numbers of mutational neoantigens that allow very specific immune responses to be generated but also allow escape variants to develop. Other cancer indications and those with low mutational burden are less easily distinguished from normal tissue. Recent studies have suggested that cancer-associated alterations in tumour cell biology including changes in post-translational modification (PTM) patterns may also lead to novel antigens that can be directly recognized by T cells. The PTM-derived antigens provide tumour-specific T-cell responses that both escape central tolerance and avoid the necessity for individualized therapies. PTM-specific CD4 T-cell responses have shown tumour therapy in murine models and highlight the importance of CD4 T cells as well as CD8 T cells in reversing the immunosuppressive tumour microenvironment. Understanding which cancer-specific antigens can be recognized by T cells and the way that immune tolerance and the tumour microenvironment shape immune responses to cancer is vital for the future development of cancer therapies.

Modi-1, anti citrullinated neoepitope vaccine alone and combined with checkpoint inhibitors in patients with head and neck, breast, renal and ovarian cancers: ModiFY Phase 1/2 basket clinical trial: report after completion of monotherapy dose finding

Christian Ottensmeier, David J Pinato, Rebecca Herbertson, Anne Armstrong, Stefan Symeonides, Poulam Patel, Sarah J Danson, James Korolewicz, David Cameron, Robert Miller, Fayaz Master, Samantha Paston, Lindy Durrant

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

ABSTRACT:
Although the efficacy of vaccines targeting SARS-CoV-2 is apparent now that the approved mRNA and adenovirus vector vaccines are in widespread use, the longevity of the protective immune response and its efficacy against emerging variants remains to be determined. We have therefore designed a DNA vaccine encoding both the SARS-CoV-2 spike receptor binding domain (‘RBD’) and nucleocapsid proteins, 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 in mice and rats, with 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 in mice that efficiently neutralise in pseudotype and live virus neutralisation assays and show cross reactivity with spike proteins from the variants B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). The vaccine also showed good protection in a viral challenge model in ACE2 receptor transgenic mice. This DNA platform can be easily adapted to target variant proteins and we show that a vaccine variant encoding the Beta variant sequence stimulates cross-reactive humoral and T cell responses. These data support the translation of this DNA vaccine platform into the clinic, thereby offering a particular advantage for rapidly targeting emerging SARS-CoV- 2 variants.

Modi-1, anti citrullinated neoepitope vaccine alone and combined with checkpoint inhibitors in patients with Head and Neck, Breast, Renal and Ovarian Carcinoma: Protocol for the ModiFY Phase I/II Basket Clinical Trial

Lindy Durrant, Fayaz Master, Samantha Paston, Robert Miller, David J Pinato, Rebecca Herbertson, Anne Armstrong, Stefan Symeonides, Christian Ottensmeier

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Ruhul H. Choudhury, Ian Daniels, Poonam Vaghela, Suha Atabani, Thomas Kirk, Peter Symonds, Katherine W. Cook, Abdullah Al-Omari, Daisy Weston, Sabaria Shah, David Hutchinson, Samantha J. Paston, Rachael L. Metheringham, Victoria A. Brentville, Lindy G. Durrant

ABSTRACT:
Citrullination and homocitrullination are stress induced post-translational modifications (siPTMs) which can be recognized by T cells. Peripheral blood mononuclear cells isolated from healthy donors and rheumatoid arthritis (RA) patients were stimulated with nine siPTM-peptides. CD45RA/CD45RO depletion was employed to determine if peptide-specific responses are naïve or memory. Human leucocyte antigen (HLA)-DP4 and HLA-DR4 transgenic mice were immunized with siPTM-peptides and immune responses were determined with ex vivo ELISpot assays. The majority (24 out of 25) of healthy donors showed CD4 T cell-specific proliferation to at least 1 siPTM-peptide, 19 to 2 siPTM-peptides, 14 to 3 siPTM-peptides, 9 to 4 siPTM-peptides, 6 to 5 siPTM-peptides and 4 to 6 siPTM-peptides. More donors responded to Vim28-49cit (68%) and Bip189-208cit (75%) compared with Vim415-433cit (33%). In RA patients, the presentation of citrullinated epitopes is associated with HLA-SE alleles; however, we witnessed responses in healthy donors who did not express the SE allele. The majority of responding T cells were effector memory cells with a Th1/cytotoxic phenotype. Responses to Vim28-49cit and Eno241-260cit originated in the memory pool, while the response to Vim415-433cit was naïve. In the HLA-DP4 and HLA-DR4 transgenic models, Vim28cit generated a memory response. Peptide-specific T cells were capable of Epstein–Barr virus transformed lymphoblastoid cell line recognition suggesting a link with stress due to infection. These results suggest siPTM-peptides are presented under conditions of cellular stress and inflammation and drive cytotoxic CD4 T cell responses that aid in the removal of stressed cells. The presentation of such siPTM-peptides is not restricted to HLA-SE in both humans and animal models.

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Victoria Anne Brentville, Peter Symonds, JiaXin Chua, Anne Skinner, Ian Daniels, Katherine Wendy Cook, Sasa Koncarevic, Roxana Martinez-Pinna, Sabaria Shah, Ruhul Hasan Choudhury, Poonam Vaghela, Daisy Weston, Abdullah Al-Omari, James Davis and Lindy G. Durrant

ABSTRACT:
Introduction: Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.
Methods: A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.
Results: We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide.
Conclusion: We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.