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,

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

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

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

DNA vaccines

Patients are beginning to benefit from antibody based, cellular and vaccine approaches that are effective against genetically diverse and therapy-resistance cancers. BCG immunotherapy is now being used as a first line treatment for human bladder cancer and the introduction of prophylactic vaccination against Hepatitis B and HPV cancers is starting to show positive results. Following recent FDA approval for a vaccination against prostate cancer, and optimistic results in clinical trials for a vaccine targeting cancer antigens in lung cancer, cancer immunotherapy is now significantly impacting patient clinical management. Tumor Immunology and Immunotherapy provides an up-to-date and comprehensive account of cancer immunity and immunotherapy. It discusses our adaptive and innate immunity to cancer, the mechanisms underpinning our immune response, current approaches to cancer immunotherapy, and how tumour and host responses can circumvent effective anti-cancer immunity. The book examines recent results, publications and current areas of interest including 'immune editing' and the specific issues that are affecting the research and development of vaccines, providing insight into how these problems may be overcome, as viewed by world leaders in the field. Tumor Immunology and Immunotherapy will appeal to clinicians working in oncology and cancer immunotherapy, and research scientists including PhD and masters students, post-doctoral researchers and senior investigators

High avidity CTL responses can be modulated by the doses of TCR stimulus.

<p>A, LPS blasts were stained for expression of CD11c, MHC class I, CD80, CD86 (filled histograms) compared to control (open histograms). B, High avidity CTL from DNA immunization were stimulated <i>in vitro</i> with supraoptimal (100 µg/ml) and optimal (10 ng/ml) dose peptide pulsed LPS blasts. After 6 days <i>in vitro</i> cultures were assessed for peptide specific responses against peptide (1 µg/ml) pulsed RMAS cells and tumor cell recognition. C, i) CTL cultures were analyzed for avidity of epitope specific responses by measuring responses to increasing peptide concentration on RMAS cells in IFNγ elispot assay, ii) normalization of responses. D, supraoptimal (diamonds) and optimal (squares) dose peptide stimulated CTL were assayed for cytotoxicity against B16F1 (closed symbols) or MeWo (open symbols) by chromium release assay at 50∶1, 25∶1 and 12.5∶1 effector:target ratios. Data is representative of at least three independent experiments. E, CTL cultures stimulated with supraoptimal and optimal dose peptide pulsed LPS blasts were analyzed for sensitivity to CD3 stimulation in IFNγ elisa assay (normalized data is shown). Data is representative of at least three independent experiments.</p

High avidity responses induced by DNA immunization are efficiently maintained into memory.

<p>A, schematic of immunization regime. B, Mice immunized with DNA were boosted at days 42 or 64 and frequency of immune responses analysed in IFNγ elispot assay at days 20, 48 and 70. C, Analysis of avidity of responses by peptide titration in IFNγ elispot assay, D, normalization of responses to increasing peptide concentration. E, Analysis of memory phenotype of antigen specific CTL by combination staining for CD62L and CD127 markers. Data is representative of at least three independent experiments.</p

Low avidity responses show limited anti-tumor efficacy.

<p>A, Recognition of B16F1 and MeWo cells by immunized splenocytes in IFNγ elispot assay. B, schematic of immunization regime. C, Tumor volume at day 16 post tumor challenge. D, Survival analysis of mice immunized with DNA and boosted with DNA or peptide followed by challenge with B16F1 tumor.</p

Supraoptimal dose antigen promotes T cell impairment and death.

<p>Splenocytes from DNA immunization were stimulated <i>ex vivo</i> with supraoptimal (100 µg/ml) and optimal (10 ng/ml) dose peptide pulsed LPS blasts. A, CFSE labeled splenocytes were assayed for proliferation of antigen specific CTL after 6 days <i>in vitro</i> culture. CFSE intensity is shown gated on pentamer positive CD8 cells. CTL were stained for B, the uptake of 7-AAD (plots gated on Pentamer+ CD8+ cells) * p = 0.0093, C, the expression of PD-1 (plots gated on Pentamer+ cells), D) average data and E, expression of PD-1 and LAG-3 (plots gated on Pentamer+ CD8+ cells), F) average data. Data is representative of at least three independent experiments.</p