Immune responses to citrullinated and homocitrullinated peptides in healthy donors are not restricted to the HLA SE shared allele and can be selected into the memory pool.

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.

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

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.

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.

The Clinical and Prognostic Significance of Protein Arginine Deiminases 2 and 4 in Colorectal Cancer.

INTRODUCTION: Protein arginine deiminases (PADIs) are a family of enzymes that catalyse the post-translational modification of proteins. Association between PADI expression and clinicopathology, protein expression, and outcome was determined. METHODS: PADI2 and PADI4 expression was assessed immunohistochemically in a cohort of colorectal cancer (CRC) patients. RESULTS: CRC tissues expressed variable levels of PADI2 which was mainly localised in the cytoplasm and correlated with patient survival (p = 0.005); high expression increased survival time from 43.5 to 67.6 months. Expression of cytoplasmic PADI2 correlated with the expression of nuclear ß catenin, PADI4, and alpha-enolase. In contrast, expression of nuclear PADI2 correlated with a decrease in survival (p = 0.010), with high expression decreasing survival from 76.4 to 42.9 months. CRC tissues expressed variable levels of PADI4 in both the nucleus and cytoplasm. Expression of cytoplasmic PADI4 correlated with survival (p = 0.001) with high expression increasing survival time from 48.1 to 71.8 months. Expression of cytoplasmic PADI4 correlated with expression of nuclear ß catenin, alpha-enolase (p ≤ 0.0001, p = 0.002), and the apoptotic related protein, Bcl-2. Expression of nuclear PADI4 also correlated with survival (p = 0.011), with high expression of nuclear PADI4 increasing survival time from 55.4 to 74 months. Expression of nuclear PADI4 correlated with p53, alpha-enolase, and Bcl-2. Multivariate analysis showed that TNM stage, cytoplasmic PADI2, and PADI4 remained independent prognostic factors in CRC. Both PADI2 and PADI4 are good prognostic factors in CRC. CONCLUSION: High expression of cytoplasmic PADI2, PADI4, and nuclear PADI4 were associated with an increase in overall survival.

Citrullinated Epitopes Identified on Tumour MHC Class II by Peptide Elution Stimulate Both Regulatory and Th1 Responses and Require Careful Selection for Optimal Anti-Tumour Responses.

Background: Somatic mutations or post-translational modifications of proteins result in changes that enable immune recognition. One such post-translational modification is citrullination, the conversion of arginine residues to citrulline. Citrullinated peptides are presented on MHC class II (MHCII) via autophagy which is upregulated by cellular stresses such as tumourigenesis. Methods: Peptides were eluted from B16 melanoma expressing HLA-DP4 and analysed by mass spectrometry to profile the presented citrullinated repertoire. Initially, seven of the identified citrullinated peptides were used in combination to vaccinate HLA-DP4 transgenic mice. Immune responses were characterised from the combination and individual vaccines by ex vivo cytokine ELISpot assay and assessed for tumour therapy. Results: The combination vaccine induced only weak anti-tumour therapy in the B16cDP4 melanoma model. Immune phenotyping revealed a dominant IFNγ response to citrullinated matrix metalloproteinase-21 peptide (citMMP21) and an IL-10 response to cytochrome p450 peptide (citCp450). Exclusion of the IL-10 inducing citCp450 peptide from the combined vaccine failed to recover a strong anti-tumour response. Single peptide immunisation confirmed the IFNγ response from citMMP21 and the IL-10 response from citCp450 but also showed that citrullinated Glutamate receptor ionotropic (citGRI) peptide stimulated a low avidity IFNγ response. Interestingly, both citMMP21 and citGRI peptides individually, stimulated strong anti-tumour responses that were significantly better than the combined vaccine. In line with the citGRI T cell avidity, it required high dose immunisation to induce an anti-tumour response. This suggests that as the peptides within the combined vaccine had similar binding affinities to MHC-II the combination vaccine may have resulted in lower presentation of each epitope and weak anti-tumour immunity. Conclusion: We demonstrate that tumours present citrullinated peptides that can stimulate Th1 and regulatory responses and that competition likely exists between similar affinity peptides. Characterisation of responses from epitopes identified by peptide elution are necessary to optimise selection for tumour therapy.

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.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor 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 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.

Helicase antigen (HAGE)-derived vaccines induce immunity to HAGE and ImmunoBody®-HAGE DNA vaccine delays the growth and metastasis of HAGE-expressing tumors in vivo.

The management of patients with triple-negative breast cancer (TNBC) continues to pose a significant clinical challenge. Less than 30% of women with metastatic TNBC survive 5 years, despite adjuvant chemotherapy and the initial higher rates of clinical response that can be achieved with neoadjuvant chemotherapy. ImmunoBody is a plasmid DNA designed to encode a human antibody molecule with complementarity-determining regions engineered to express cytotoxic and helper T-cell epitopes derived from the cancer antigen of interest. The helicase antigen (HAGE) is a cancer testis antigen, which is expressed in TNBC. Herein, we have identified a 30-amino-acid-long HAGE-derived sequence containing human leukocyte antigen (HLA)-A2- and HLA-DR1-restricted epitopes and demonstrated that the use of this sequence as a peptide (with CpG/incomplete Freund's adjuvant) or incorporated into an ImmunoBody vaccine can generate specific interferon-γ-secreting splenocytes in HHDII+ DR1+ mice. T-cell responses elicited by the ImmunoBody-HAGE vaccine were superior to peptide immunization. Moreover, splenocytes from ImmunoBody-HAGE-vaccinated mice stimulated in vitro could recognize HAGE+ tumor cells and the human TNBC cell line MDA-MB-231. More importantly, the growth of implanted HHDII+ DR1+ HAGE+ Luc+ B16 cells.

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

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. METHODS: We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry. RESULTS: The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients. CONCLUSIONS: Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

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 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 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, p < 0.0001) against established B16 tumours 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.

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.

SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors.

PURPOSE: We have previously shown that supraoptimal signaling of high avidity T cells leads to high expression of PD-1 and inhibition of proliferation. This study was designed to see if this effect could be mitigated by combining a vaccine that stimulates high avidity T cells with PD-1 blockade. EXPERIMENTAL DESIGN: We investigated the anti-tumor effect of a huIgG1 antibody DNA vaccine (SCIB1) and PD-1 blockade. RESULTS: Vaccination of HLA-DR4 transgenic mice with SCIB1 induced high frequency and avidity T cell responses that resulted in survival (40%) of mice with established B16F1-DR4 tumors. SCIB1 vaccination was associated with increased infiltration of CD4 and CD8 T cells within the tumor but was also associated with upregulation of PD-L1 within the tumor environment. PD-1 blockade also resulted in increased CD8 T cell infiltration and an anti-tumor response with 50% of mice showing long term survival. In line with our hypothesis that PD-1/PD-L1 signaling results in inhibition of proliferation of high avidity T cells at the tumor site, the combination of PD-1 blockade with vaccination, enhanced the number and proliferation of the CD8 tumor infiltrate. This resulted in a potent anti-tumor response with 80% survival of the mice. CONCLUSIONS: There is a benefit in combining PD-1 blockade with vaccines that induce high avidity T cell responses and in particular with SCIB1.

SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade.

Checkpoint blockade has demonstrated promising antitumor responses in approximately 10-40% of patients. However, the majority of patients do not make a productive immune response to their tumors and do not respond to checkpoint blockade. These patients may benefit from an effective vaccine that stimulates high-avidity T cell responses in combination with checkpoint blockade. We have previously shown that incorporating TRP-2 and gp100 epitopes into the CDR regions of a human IgG1 DNA (ImmunoBody®: IB) results in significant tumor regression both in animal models and patients. This vaccination strategy is superior to others as it targets antigen to antigen-presenting cells and stimulates high-avidity T cell responses. To broaden the application of this vaccination strategy, 16 NY-ESO-1 epitopes, covering over 80% of HLA phenotypes, were incorporated into the IB (SCIB2). They produced higher frequency and avidity T cell responses than peptide vaccination. These T cells were of sufficient avidity to kill NY-ESO-1-expressing tumor cells, and in vivo controlled the growth of established B16-NY-ESO-1 tumors, resulting in long-term survival (35%). When SCIB2 was given in combination with Treg depletion, CTLA-4 blockade or PD-1 blockade, long-term survival from established tumors was significantly enhanced to 56, 67 and 100%, respectively. Translating these responses into the clinic by using a combination of SCIB2 vaccination and checkpoint blockade can only further improve clinical responses.

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.

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 IFNγ- 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.

Novel prostate acid phosphatase-based peptide vaccination strategy induces antigen-specific T-cell responses and limits tumour growth in mice.

Treatment options for patients with advanced prostate cancer remain limited and rarely curative. Prostatic acid phosphatase (PAP) is a prostate-specific protein overexpressed in 95% of prostate tumours. An FDA-approved vaccine for the treatment of advanced prostate disease, PROVENGE® (sipuleucel-T), has been shown to prolong survival, however the precise sequence of the PAP protein responsible for the outcome is unknown. As the PAP antigen is one of the very few prostate-specific antigens for which there is a rodent equivalent with high homology, preclinical studies using PAP have the potential to be directly relevant to clinical setting. Here, we show three PAP epitopes naturally processed and presented in the context of HHDII/DR1 (114-128, 299-313, and 230-244). The PAP-114-128 epitope elicits CD4(+) and CD8(+) T-cell-specific responses in C57BL/6 mice. Furthermore, when immunised in a DNA vector format (ImmunoBody®), PAP-114-128 prevents and reduces the growth of transgenic adenocarcinoma of mouse prostate-C1 prostate cancer cell-derived tumours in both prophylactic and therapeutic settings. This anti-tumour effect is associated with infiltration of CD8(+) tumour-infiltrating lymphocytes and the generation of high avidity T cells secreting elevated levels of IFN-γ. PAP-114-128 therefore appears to be a highly relevant peptide on which to base vaccines for the treatment of prostate cancer.

High avidity cytotoxic T lymphocytes can be selected into the memory pool but they are exquisitely sensitive to functional impairment.

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 µg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.

Vaccines as early therapeutic interventions for cancer therapy: neutralising the immunosuppressive tumour environment and increasing T cell avidity may lead to improved responses.

IMPORTANCE OF THE FIELD: Considerable progress has been made in identifying the antigens recognised by the immune system. This has led to the success of monoclonal antibody therapy and the recent approval of prophylactic vaccines that give excellent protection against cervical cancer. Provenge will shortly be the first therapeutic vaccine to be approved. AREAS COVERED IN THIS REVIEW: Our aim is to discuss the recent success with prophylactic cancer vaccines for prevention of cancer and the progress with therapeutic vaccines design to eradicate established tumours. Therapeutic vaccines need to stimulate high-avidity T cell responses that can recognise and kill tumours. How this can be achieved in cancer patients is discussed. The immunosuppressive tumour environment also needs to be modified to allow extravasation and efficacy of the vaccine induced T cells. WHAT THE READER WILL GAIN: An insight into the limitations of present cancer vaccine approaches and how they can be manipulated to give more effective anti-tumour responses. TAKE HOME MESSAGE: A combination of more effective vaccines that stimulate high-avidity T cells, in combination with drugs or monoclonal antibodies that neutralize immunosuppressive factors within the tumour environment are needed to improve the efficacy of immunotherapy of cancer.

DNA vaccination with T-cell epitopes encoded within Ab molecules induces high-avidity anti-tumor CD8+ T cells.

Stimulation of high-avidity CTL responses is essential for effective anti-tumor and anti-viral vaccines. In this study we have demonstrated that a DNA vaccine incorporating CTL epitopes within an Ab molecule results in high-avidity T-cell responses to both foreign and self epitopes. The avidity and frequency was superior to peptide, peptide-pulsed DC vaccines or a DNA vaccine incorporating the epitope within the native Ag. The DNA Ab vaccine was superior to an identical protein vaccine that can only cross-present, indicating a role for direct presentation by the DNA vaccine. However, the avidity of CTL responses was significantly reduced in Fc receptor gamma knockout mice or if the Fc region was removed suggesting that cross presentation of Ag via Fc receptor was also important in the induction of high-avidity CTL. These results suggest that generation of high-avidity CTL responses by the DNA vaccine is related to its ability to both directly present and cross-present the epitope. High-avidity responses were capable of efficient anti-tumor activity in vitro and in vivo. This study demonstrates a vaccine strategy to generate high-avidity CTL responses that can be used in anti-tumor and anti-viral vaccine settings.