Autophagy, citrullination and cancer

A cell needs to maintain a balance between biosynthesis and degradation of cellular components to maintain homeostasis. There are 2 pathways, the proteasome, which degrades short-lived proteins, and the autophagy/lysosomal pathway, which degrades long-lived proteins and organelles. Both of these pathways are also involved in antigen presentation or the effective delivery of peptides to MHC molecules for presentation to T cells. Autophagy (macroautophagy) is a key player in providing substantial sources of citrullinated peptides for loading onto MHC-II molecules to stimulate CD4(+) T cell responses. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. We therefore investigated if citrullinated peptides could stimulate CD4(+) T cell responses that would recognize these modifications produced during autophagy within tumor cells. Focusing on the intermediate filament protein VIM (vimentin), we generated citrullinated VIM peptides for immunization experiments in mice. Immunization with these peptides induced CD4(+) T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 d after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. These results show how CD4(+) cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides produced during autophagy may offer especially attractive vaccine targets for cancer 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

Post-translational modifications are induced in stressed cells which cause them to be recognised by the immune 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-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 induced 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 targets for anti-tumour immunity. In this study we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival,

Targeting gp100 and TRP-2 with a DNA vaccine: incorporating T cell epitopes with a human IgG1 antibody induces potent T cell responses that are associated with favourable clinical outcome in a phase I/II trial

A DNA vaccine, SCIB1, incorporating two CD8 and two CD4 epitopes from TRP-2/gp100 was evaluated in patients with metastatic melanoma. Each patient received SCIB1 via intramuscular injection with electroporation. The trial was designed to find the safest dose of SCIB1 which induced immune/clinical responses in patients with or without tumour. Fifteen patients with tumor received SCIB1 doses of 0.4-8 mg whilst 20 fully-resected patients received 2-8 mg doses. Twelve patients elected to continue immunization every 3 months for up to 39 months. SCIB1 induced dose-dependent T cell responses in 88% of patients with no serious adverse effects or dose limiting toxicities. The intensity of the T cell responses was significantly higher in patients receiving 4 mg doses without tumor when compared to those with tumor (p< 0.01). In contrast, patients with tumor showed a significantly higher response to the 8 mg dose than the 4 mg dose (p< 0.03) but there was no significant difference in the patients without tumor. One of 15 patients with measurable disease showed an objective tumor response and 7/15 showed stable disease. 5/20 fully-resected patients have experienced disease recurrence but all remained alive at the cut-off date with a median observation time of 37 months. A positive clinical outcome was associated with MHC-I and MHC-II expression on tumors prior to therapy (p=0.027). We conclude that SCIB1 is well tolerated and stimulates potent T cell responses in melanoma patients. It deserves further evaluation as a single agent adjuvant therapy or in combination with checkpoint inhibitors in advanced disease

Citrullinated α-enolase is an effective target for anti-cancer immunity

Targeting post-translationally modified epitopes may provide a new strategy for generating tumor specific immune responses. Citrullination is the post-translational modification of arginine to citrulline catalyzed by peptidylarginine deaminase (PAD) enzymes. Presentation of citrullinated peptides on MHC-II has been associated with autophagy. Tumors upregulate autophagy and present citrullinated peptides in response to stresses including nutrient deprivation, oxygen deprivation, redox stress and DNA damage, making them good targets for immune attack. The ubiquitous glycolytic enzyme α-enolase (ENO1) is often citrullinated and degraded during autophagy. Immunization of mice with two citrullinated ENO1 peptides (ENO1 241-260cit253 or 11-25cit15) induced strong Th1 responses that recognize the post-translationally modified, but not the wild type unmodified epitope. ENO1 11-25cit15 induced tumor therapy of melanoma cells in C57Bl/6 (B16F1 50% survival p = 0.0026) and ENO1 241-260cit253 in HLA-DR4 transgenic mice (B16-DR4 50% survival p = 0.0048). In addition, ENO1 241-260cit253 induced therapy of pancreatic (Pan02-DR4 50% survival p = 0.0076) and lung (LLC/2-DR4 40% survival p = 0.0142) tumors in HLA-DR4 transgenic mice. The unmodified epitope induced no anti-tumor response. Minimal regression of class II negative B16 or LLC/2 tumor was seen, confirming direct recognition of MHC-II was required. Most tumors only express MHC-II in the presence of IFNγ; an IFNγ inducible model showed strong responses, with rejection of tumors in up to 90% of animals (p = 0.0001). In humans, a repertoire to ENO1 241-260cit253 was observed in healthy donors. This response was CD4 mediated and seen in people with a variety of HLA types suggesting a broad application for this vaccine in human cancer therapy

Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T-cell-mediated antitumor immunity

Stressful conditions in the harsh tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. However, autophagy also causes post-translational modification of proteins that are recognized by the immune system. In particular, modified self-antigens can trigger CD4(+) T-cell responses that might be exploited to boost antitumor immune defenses. In this study, we investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. Focusing on the intermediate filament protein vimentin, which is frequently citrullinated in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, we generated citrullinated vimentin peptides for immunization experiments in mice. Immunization with these peptides induced IFN?- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8(+) T cells. These results show how CD4 cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides may offer especially attractive vaccine targets for cancer therapy

A clinical trial of a DNA vaccine (SCIB1) that targets dendritic cells in vivo in fully resected melanoma patients; a vaccine to prevent disease recurrence?

Background: SCIB1 is a DNA vaccine encoding a human IgG1 antibody with CDRs that contain four epitopes from two melanoma antigens (three from gp100 and one from TRP2). The vaccine elicits potent anti-tumour responses by stimulating high frequency, high avidity T-cells via both direct and cross-presentation of antibody. A clinical study in stage III/IV melanoma patients, all with tumour present at study entry, showed that 2-8mg doses could induce T-cell responses in 7/9 patients with no associated toxicity. Encouragingly overall survival was 31 months. This study addresses the question as to whether SCIB1 can be used as an adjuvant therapy in fully resected melanoma patients to prevent further disease. Methods: Sixteen patients with fully resected stage III (n=9) or stage IV (n=7) melanoma were immunised with 4mg of SCIB1 by intramuscular electroporation at 3 weekly intervals and subsequently at 3 and 6 months. Patients could continue treatment for 5 years. Results: All 16 patients showed vaccine-epitope-specific T-cell responses (i.e. proliferation ex vivo and/or γIFN Elispot responses in-vitro). Twelve patients responded to all four epitopes, two patients to three epitopes, one to two epitopes and one to a single epitope. Five patients remain in the continuation phase - all show strong T-cell memory responses following boosting. At present, median survival time is 37 months from trial entry and 41.5 months from diagnosis of metastases. Overall survival is 100% for both groups. Five patients relapsed at 1, 4, 14, 17 and 18 months but have shown no further recurrences at follow-up. Conclusion: These results show that a DNA vaccine encoding epitopes from melanoma antigens can induce measurable T-cell responses and, furthermore, it may confer protection from recurrence of melanoma with little associated toxicity. SCIB1 deserves further evaluation as an adjuvant therapy

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

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 Bip and used HLA transgenic mice to assess immunogenicity by ELISpot assay. Vaccine efficacy was assessed in tumour therapy studies using HLA matched B16 melanoma expressing constitutive or IFNγ inducible MHC-II as represented by most human tumours. To determine the mechanism behind the therapy, immune cell infiltrates were analysed using flow cytometry and therapy studies in the presence of MPO inhibitor and T cell depletion performed. We assessed the T cell repertoire to homocitrullinated peptides in cancer patients and healthy donors using flow cytometry. Results: Homocitrulline peptide vaccination stimulated strong CD4 T cell responses and induced significant anti-tumour therapy in an established tumour model. The anti-tumour response was dependent upon CD4 T-cells and the effect was driven mainly via direct tumour recognition, as responses were only observed if the tumours were induced to express MHC-II. In vitro proliferation assays show that healthy donors and cancer patients have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by myeloperoxidase (MPO) inhibition reduced tumour therapy by the vaccine induced T cells (P=0.0018). Analysis of the tumour 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 homocitrulline 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 tumour and likely reversed the immunosuppressive environment. Conclusion: We propose that MPO, potentially produced by MDSCs, catalyses the build-up of cyanate in the TME which diffuses into tumour 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 tumours containing high levels of MPO expressing MDSCs

A novel HAGE/WT1-ImmunoBody® vaccine combination enhances anti-tumour responses when compared to either vaccine alone

Many cancers, including myeloid leukaemia express the cancer testis antigen (CTA) DDX43 (HAGE) and/or the oncogene Wilms’ tumour (WT1). Here we demonstrate that HAGE/WT1-ImmunoBody® vaccines derived T-cells can kill ex-vivo human CML cell lines expressing these antigens and significantly delay B16/HHDII+/DR1+/HAGE+/WT1+ tumour growth in the HHDII/DR1 mice and prolonged mouse survival in the prophylactic setting in comparison to non-immunised control mice. We show that immunisation of HHDII/DR1 mice with HAGE- and WT1-ImmunoBody® DNA vaccines in a prime-boost regime in two different flanks induce significant IFN-γ release by splenocytes from treated mice, and a significant level of cytotoxicity against tumour targets expressing HAGE/WT1 in vitro. More importantly, the combined HAGE/WT1 ImmunoBody® vaccine significantly delayed tumour growth in the B16/HHDII+/DR1+/HAGE+/WT1+ tumour model and prolonged mouse survival in the prophylactic setting in comparison to non-immunised control mice. Overall, this work demonstrates that combining both HAGE- and WT1-ImmunoBody® into a single vaccine is better than either vaccine alone. This combination vaccine could be given to patients whose cancer expresses HAGE and WT1 in parallel with existing therapies in order to decrease the chance of disease progression and relapse

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

IntroductionPost 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.MethodsA 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.ResultsWe 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&lt;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.ConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation