Anti-tumour immune responses to modified self-epitopes

Abstract

The present invention relates to modified citrullinated enolase peptides that can be used as targets for cancer immunotherapy. These peptides can be used as vaccines or as targets for monoclonal antibody (mAb) therapy. Such vaccines cur mAbs may be used in the treatment of cancer.

Claims

1. A method for the treatment of cancer in a subject in need thereof comprising administering to the subject a nucleic acid encoding a citrullinated peptide, wherein the citrullinated peptide comprises, consists essentially of, or consists of: i) an amino acid sequence selected from the group consisting of: TABLE-US-00010 (SEQ ID NO: 1) VIGMDVAASEFFcitSGKYDLD, (SEQ ID NO; 2) VIGMDVAASEFYcitSGKYDLD, (SEQ ID NO; 3) EVDLFTSKGLFcitAAVPSGAS, (SEQ ID NO: 4) EVDLYTAKGLFcitAAVPSGAS, (SEQ ID NO: 5) KGVPLYcitHIADLAGNSEVIL, (SEQ ID NO; 6) KGVPLYcitHIADLAGNPEVIL, (SEQ ID NO; 7) VGDDLTVTNPKcitIAKAVNEK, (SEQ ID NO; 8) VGDDLTVTNPKcitIAKAASEK, (SEQ ID NO: 9) IFDScitGNPTVEVDLF, or (SEQ ID NO: 10) IFDScitGNPTVEVDLY, wherein “cit” represents citrulline, or ii) the amino acid sequence of i), with the exception of 1, 2 or 3 amino acid substitutions, and/or 1, 2 or 3 amino acid insertions, and/or 1, 2 or 3 amino acid deletions in a non-citrulline position.

2. The method of claim 1, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, ovarian cancer, endometrial carcinoma, pancreatic cancer, leukemia, melanoma, head and neck cancer or lung cancer.

3. The method of claim 1, wherein the subject is a human or non-human animal.

4. The method of claim 1, wherein the nucleic acid is administered as a pharmaceutical composition comprising the nucleic acid in combination with a pharmaceutically acceptable carrier.

5. The method of claim 3, wherein the subject is a human.

6. The method of claim 5, wherein the subject expresses HLA-DP4.

7. The method of claim 2, wherein the cancer is breast cancer.

8. The method of claim 7, wherein the cancer is estrogen receptor negative breast cancer.

9. The method of claim 2, wherein the cancer is ovarian cancer.

10. The method of claim 2, wherein the cancer is pancreatic cancer.

11. The method of claim 10, wherein pancreatic cancer is pancreatic ductal adenocarcinoma.

12. The method of claim 1, wherein the citrullinated peptide comprises the amino acid sequence VIGMDVAASEFFcitSGKYDLD (SEQ ID NO: 1), wherein “cit” represents citrulline.

13. The method of claim 1, wherein the citrullinated peptide consists essentially of the amino acid sequence VIGMDVAASEFFcitSGKYDLD (SEQ ID NO: 1), wherein “cit” represents citrulline.

14. The method of claim 1, wherein the citrullinated peptide consists of the amino acid sequence VIGMDVAASEFFcitSGKYDLD (SEQ ID NO: 1), wherein “cit” represents citrulline.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Sequence alignments of Human Enolase.

(2) Alignment of Human Enolase Subunits ENOA (ENO1, α), ENOB (ENO3, β), ENOG (ENO2, γ) and ENO4 depicting homology. Light grey homologous regions, dark grey not homologous with ENO4.

(3) FIGS. 2A-2C: Screening IFNγ responses to peptide pools

(4) Transgenic mouse strains with human DR4 (FIG. 2A) or DR1/HHD (FIG. 2B) and parental C57BL/6 (FIG. 2C) mice were used to screen IFNγ responses to peptide. Mice were immunised with pools of 4-6 non-overlapping Human citrullinated Enolase peptides. 14 days after immunisation, mice were sacrificed and splenocytes were harvests. E vivo responses to stimulation with Human and mouse equivalent peptides were assessed by IFNγ Elispot. Media only responses were used as a negative control. For each pool n=3. Statistical significance of peptide responses compared to media responses for each pool was determined by ANOVA with Dunnett's post-hoc test *p<0.5, **p<0.01, ***p<0.001.

(5) FIGS. 3A-3C: Human Enolase 241-260 citrullinated peptide induces strong IFN responses A single immunisation of human 241cit peptide was given to transgenic DR4 mice. Ex vivo Elispot was used to determine the IFNγ (FIG. 3A) and IL-10 (FIG. 3B) responses generated to the human and mouse equivalent citrullinated (cit) peptides and the wild type (wt) sequences. IFNγ responses to citrullinated peptides in the presence of MHC class II blocking antibody (L243) were also assessed by Elispot (FIG. 3C). For all assays media only responses were used as a negative control. For each experiment n=3. p values are shown.

(6) FIGS. 4A-4C: Multiple citrullinated Enolase peptides induce low level IFNγ responses

(7) Mice were given a single immunisation of citrullinated human Enolase peptides corresponding to positions 21-40 (FIG. 4A), 126-145 (FIG. 4B) and 316-335 (FIG. 4C). E vivo Elispot was used to determine IFNγ responses in DR4 and DR1/HHD mice. Responses are shown to human and mouse citrullinated peptides and their wild type equivalents when available. Media only responses were used as a negative control. For each peptide n=3. Statistical significance of responses compared to media control were determined by ANOVA with Dunnett's post-hoc test *p<0.5, **p<0.01, ***p<0.001.

(8) FIG. 5: Enolase 241cit peptide induces responses in DP4 mice.

(9) Transgenic DP4 mice were immunised with three doses of Enolase 241cit peptide over three weeks. Splenocytes were collected 21 days after the initial dose was administered. E vivo IFNγ Elispot were used to determine the response to Enolase 241 peptides.

(10) FIGS. 6A-6F: Human Enolase 241cit peptide provides an in vivo survival advantage in anti-tumour studies

(11) Immunoblot (FIG. 6A) of lysates from B16F1 (Lane 1), ID8 (Lane 2), TrampC1 (Lane 3), Pan02 (Lane 4), LLC/2 (Lane 5), RTLCL (Lane 6), HeLa (Lane 7) cell lines against ladder probed for α Enolase (ENO1) and β actin. The bands correspond to the expected size for ENO-1 (47 kDa) and β-actin (42 kDa).

(12) DR4 mice were challenged with B16DR4 tumour. Survival (FIG. 6B) and tumour size at day 17 post tumour implant (FIG. 6C) are shown for unimmunised control animals and animals immunised with Enolase 241cit peptide four days after tumour implant. n=10, results shown are from two independent experiments.

(13) DR4 mice were challenged with B16 tumour expressing IFNγ inducible DR4. Survival (FIG. 6D) and tumour size at day 17 post tumour implant (FIG. 6E) are shown for unimmunised control animals and animals immunised with Enolase 241cit peptide four days after tumour implant. n=10. Surviving mice from the immunised group were rechallenged with the same tumour cell line at day 42 post initial tumour implant (indicated by arrow). Survival data for rechallenged mice and a previous unchallenged control group are shown (FIG. 6F). Significant p values are shown. For tumour volume medians and p values are shown as determined by Mann Whitney U test.

(14) FIGS. 7A and 7B: Enolase 241cit peptide provides a survival advantage in an LLC2 tumour model

(15) DR4 mice were challenged with the Lewis lung carcinoma cell line LLC2. Four days after tumour challenge mice were immunised with human Enolase 241cit peptide. Survival data for mice challenged with wild type LLC2 (FIG. 7A) or LLC2 transfected with constitutive chimeric DR4 (FIG. 7B) are shown. Statistical differences between immunised and unimmunised control mice were determined by Mantel-Cox test, p values are shown, n=10.

(16) FIGS. 8A-8D: ENO1 DNA vaccination induces a similar response to Enolase241cit peptide

(17) DR4 mice were given two immunisations of ENO1 DNA bullets using a gene gun. After 14 days after the second immunisation mice were sacrificed and splenocytes were harvested. Er vivo Elispots were performed to determine IFNγ (FIG. 8A) and IL-10 (FIG. 8B) responses to stimulation with Enolase 241 peptides. Mice were challenged with B16DR4 tumour cell line which constitutively expressed DR4 and after 4 days were immunised with ENO1 DNA, survival data (FIG. 8C) and tumour volume at day 11 (FIG. 8D) are shown.

(18) FIGS. 9A-9D: Adjuvant effects the response induced to Enolase 241cit peptide.

(19) DR4 mice were given a single immunisation of human Enolase 241cit in the presence of adjuvant CpG/MPLA or IFA. IFNγ (FIG. 9A) and IL-10 (FIG. 9B) responses were determined by ex vivo Elispot. For these studies n=3. IFNγ responses in mice given a single or three immunisation of 5 μg or 25 μg of Enolase 241cit peptide in the presence of GM-CSF were determined (FIG. 9C). For these studies n=3. DR4 mice were also challenge with B16DR4 tumour and survival (FIG. 9D) of unimmunised control animals or animals immunised with 3 doses of 5 μg of Enolase 241cit peptide with GM-CSF beginning four days after tumour implant were determined. n=10.

(20) FIG. 10: Responses develop rapidly suggesting a pre-existing Enolase 241cit response.

(21) DR4 mice were immunised with a single dose of Enolase 241cit peptide in CpG/MPLA 2, 6 or 14 days before mice were sacrificed and ex vivo Elispots were used to determine the IFN responses. n=3, p values represent significant difference compared to peptide responses at day 2.

(22) FIGS. 11A-11C: Enolase 241cit peptide induces responses in Human PBMCs.

(23) PBMCs were isolated from 6 healthy donors and cultured with media, human Enolase 241cit or Enolase 241 wt peptide. Thymidine assays were performed to determine proliferation after 4, 7 and 11 days (FIG. 11A). HLA typing was performed on each donor. Supernants from donor 4 on day 11 were collected and analysed for cytokine levels using luminex (FIG. 11B). Data shown represents response above media control background for each cytokine. PBMCs from donor 4 were labelled with CFSE prior to stimulation with wild type and citrullinated peptides. The CD4 and CD8 populations within the CFSE labelled cell population was assessed for the peptide stimulated samples by flow cytometry at day 7 and day 10 (FIG. 11C).

(24) FIG. 12: Alignment of Human Enolase α (ENOA) subunit with equivalent sequences from other species (Mouse, Rat, Cow, Pig, Horse, Chicken, Cat, Dog, Rabbit and Sheep) depicting homology.

(25) FIG. 13: Alignment of) Human Enolase β (ENOB) subunit with equivalent sequences from other species (Mouse, Rat, Cow, Pig, Horse, Chicken, Cat, Dog, Rabbit and Sheep) depicting homology.

(26) FIG. 14: Alignment of Human Enolase γ (ENOG) subunit with equivalent sequences from other species (Mouse, Rat, Cow, Pig, Horse, Chicken, Cat, Dog, Rabbit and Sheep) depicting homology.

(27) FIGS. 15A-15C: Human Enolase 241-260 citrullinated peptide induces CD4 responses.

(28) DR4 transgenic mice were immunized with human 241cit peptide. E vivo Elispot was used to determine the IFNγ responses generated to the human and mouse equivalent citrullinated (cit) peptides and the wild type (wt) sequences. IFNγ responses to citrullinated peptides in the presence of MHC class II blocking antibody (L243), CD4 blocking antibody or CD8 blocking antibody (FIG. 15A) or in CD4 depleted or enriched cell fractions (FIG. 15B) were assessed. IFNγ responses to shorter peptide sequences were also tested (FIG. 15C). For all assays media only responses were used as a negative control. For each experiment n=3. p values are shown.

(29) FIGS. 16A-16F: Characterisation of responses in HHDII/DP4 mice.

(30) Transgenic DP4 mice were immunised with three doses of human (FIGS. 16A-16E) or mouse (FIG. 16F) Enolase 241cit peptide over three weeks. Splenocytes were collected 21 days after the initial dose was administered. E vivo IFNγ Elispot were used to determine the response to Enolase 241 peptides. Responses to the human peptide were tested for avidity by peptide titration (FIG. 16A), IL-10 secretion (FIG. 16B) and Granzyme B secretion (FIG. 16C). IFNγ responses in the presence of CD4 blocking antibody (FIG. 16D) and to shorter peptide sequences were also tested (FIG. 16E). For all assays media only responses were used as a negative control. For each experiment n=3. p values are shown.

(31) FIG. 17: Human Enolase 241cit peptide provides an in vivo survival advantage in B16 melanoma anti-tumour studies in HHDII/DP4 transgenic mice.

(32) DP4 transgenic mice were challenged with B16DP4 tumour. Survival is shown for unimmunised control animals and animals immunised with Enolase 241cit peptide four days after tumour implant. n=10. Statistical differences between immunised and unimmunised control mice were determined by Mantel-Cox test, p values are shown, n=10.

(33) FIG. 18: Enolase 241cit peptide provides a survival advantage Pan02 (pancreatic) tumour model.

(34) DR4 transgenic mice were challenged with the Pan02 pancreatic carcinoma line expressing constitutive DR4. Four days after tumour challenge mice were immunised with human Enolase 241cit peptide and survival monitored. Statistical differences between immunised and unimmunised control mice were determined by Mantel-Cox test, p values are shown, n=10.

(35) FIGS. 19A-19D: Response can be induced to Enolase 241cit peptide in combination with a variety of adjuvants.

(36) DR4 (FIGS. 19A & 19B) or HHDII/DP4 (FIGS. 19C & 19D) transgenic mice were immunised with human Enolase 241cit in the presence of adjuvant CpG/MPLA (6 μg each), IFA, Poly I:C (10 μg) or Imiquimod (25 μg). IFNγ (FIGS. 19A & 19C) and IL-10 (FIGS. 19B & 19D) responses were determined by ex vivo Elispot. For these studies n=3.

(37) FIGS. 20A-20C: Enolase 241cit specific responses reactivated from normal donors produce predominantly Th1 cytokines and are CD4 mediated.

(38) PBMCs from donors 1 and 4 were cultured with human enolase 241cit or wt peptide and IFNg release measured in IFNγ elispot at day 13 (FIG. 20A). IFN responses were analysed by intracellular cytokine staining in combination with CD4 and CD8 markers (FIG. 20B). Supernatant from PBMC cultures of donors 1, 4 and 7 were analysed for cytokine levels at days 2, 5 and 12 by luminex assay (FIG. 20C).

(39) FIGS. 21A-21B: Eno 21cit peptide induces CD4 responses in mice.

(40) C57Bl/6 mice were immunized with human enolase 21cit peptide. Er vivo Elispot was used to determine the IFNγ responses generated to the human citrullinated (cit) peptide and the wild type (wt) sequences (FIG. 21A). IFNγ responses to citrullinated peptides in the presence of CD4 blocking antibody or CD8 blocking antibody (FIG. 21B) were assessed. For all assays media only responses were used as a negative control. For each experiment n=3. p values are shown.

(41) FIGS. 22A-22C: Eno 11cit peptide induces CD4 responses in mice.

(42) C57Bl/6 mice were immunized with human enolase 11cit peptide. Er vivo Elispot was used to determine the IFNγ responses generated to the human and mouse citrullinated (cit) peptides and the human wild type (wt) sequences (FIG. 22A). IL-10 responses to the human cit peptide were assessed (FIG. 22B). IFNγ responses to human citrullinated peptide in the presence of CD4 blocking antibody or CD8 blocking antibody (FIG. 22C) were assessed. For all assays media only responses were used as a negative control. For each experiment n=3. p values are shown.

METHODS

(43) 2.1. Commercial mAbs

(44) Anti-HLA-DR antibody (clone L243) was purified from HB-55 hybridoma cells (ATCC, USA) culture supernatant by sepharose protein G affinity chromatography. The antibody Rabbit monoclonal [EPR10864 (B)] to ENO1 was used. Anti-mouse CD4 (clone GK1.5) and anti-mouse CD8 (clone 2.43) were purchased from BioXcell, USA.

(45) 2.2. Cell Lines

(46) The murine melanoma B16F1 and murine Lewis lung carcinoma LLC/2 cell lines were obtained from the ATCC. The murine Pan02 cell line was obtained from the National Cancer Institute tumour repository. The B16F1 cell line is cultured in RPMI medium 1640 (GIBCO/BRL) and LLC/2 and Pan02 in DMEM. Both are supplemented with 10% FCS, L-glutamine (2 mM) and sodium bicarbonate buffered unless otherwise stated.

(47) 2.3. Immunogens

(48) 2.3.1. Peptides

(49) Peptides >90% purity were synthesized by Genscript (New Jersey, USA). Stored lyophilized in 0.2 mg aliquots at −80° C. On day of use they were reconstituted to the appropriate concentration in PBS.

(50) 2.4. Plasmids

(51) The mammalian expression vector pCMVSPORT6 encoding murine alpha Enolase (ENO-1) full length cDNA (IMAGE ID 5376359) was obtained from Source Bioscience.

(52) To construct the plasmid pVITRO2 Human HLA-DP4, the nucleotide sequence encoding the full length human HLA-DPA*0103 alpha chain flanked by FspI/EcoRI and the HLA-DPB*0401 beta chain flanked by BamHI/SalI restriction sites were synthesized. Following sequence confirmation, the HLA-DPA*0103 chain was cloned into the FspI/EcoRI mcs2 of the vector pVITRO2-hygro-mcs (Invivogen). The HLA-DPB*0401 chain was subsequently inserted into the BamHI/SalI mcs1 of the mammalian expression vector alongside the alpha HLA-DPA*0103 chain present within mcs2.

(53) To generate the HHDII plasmid, cDNA was synthesized from total RNA isolated from EL4-HHD cells. This was used as a template to amplify HHD using the forward and reverse primers and sub cloned into pCR2.1. The HHD chain, comprising of a human HLA-A2 leader sequence, the human β2-microglobulin (β2M) molecule covalently linked via a glycine serine linker to the α 1 and 2 domains of human HLA-0201 MHC class molecule and the α3, transmembrane and cytoplasmic domains of the murine H-2db class 1 molecule, was then inserted into the EcoRV/HindIII sites of the mammalian expression vector pCDNA3.1 obtained from Invitrogen.

(54) To generate the plasmid pVitro 2 Chimeric HLA-DR401 cDNA was generated from mRNA isolated from the splenocytes of transgenic HLA-DR4 mice. This was used as a template to amplify the chimeric alpha and beta chains separately using forward and reverse primers that incorporated a FspI/EcoRI and BamHI/SalI sites respectively. On sequence confirmation full length chimeric alpha chain comprising of murine H2-Ea with human HLA-DRA alpha 1 domain was ligated into the FspI/EcoRI mcs2 of the vector pVITRO2-hygro-mcs (Invivogen). The beta chain comprising of murine H2-Eb with human DRB1*0401 Beta 1 domain was then inserted into the BamHI/SalI mcs1 of the vector alongside the chimeric alpha chain.

(55) To construct the IFNγ inducible plasmid pDCGAS chimeric HLA-DR401, the chimeric alpha and beta chains, were cloned into the pDCOrig vector described elsewhere (Metheringham et al., 2009) in replacement of the heavy and light chain. The IFNγ inducible promoter consisting of a TATA box and the GAS (IFNγ activated sequence) direct repeat enhancer element was amplified by PCR utilizing the vector pGAS-Luc (Agilent) as a template. The CMV promoter within each cassette was excised and replaced with the IFNγ inducible promoter driving expression of the HLA-DR401 chains within the pDCOrig vector backbone.

(56) Endotoxin free plasmid DNA was generated using the endofree Qiagen maxiprep kit (Qiagen, Crawley)

(57) 2.5. Transfection

(58) LLC2 cells were transfected using the Lipofectamine transfection reagent (Invitrogen) with 4 μg of the plasmid pVitro 2 Chimeric HLA-DR401 that encodes both full length chimeric alpha and beta chains according to the manufacturer's instructions. The B16F1 cell line previously knocked out for murine MHC class II by Zinc finger Technology (Sigma Aldrich) was transfected with either the pDC GAS chimeric HLA-DR401 or the pVitro 2 chimeric HLA-DR401 plasmids where chimeric HLA-DR401 is under expression of the IFNγ inducible promoter or the constitutive promoter that drive high level expression respectively.

(59) Transfected cells were selected by growth in the presence of Hygromycin B (300 μg/ml) or zeocin (300 μg/ml). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the anti-human HLA-DR PE-Cy7 conjugated antibody (clone L243) from eBioscience. Cells transfected with the IFNγ inducible plasmid where incubated overnight in the absence or presence of murine IFNγ (30 ng/ml, Gibco life technologies) prior to staining with the antibody.

(60) The B16F1 cell line previously knocked out for murine MHC class I and II by Zinc finger Technology (Sigma Aldrich) was transfected using the Lipofectamine transfection reagent (Invitrogen) with 4 μg of each of the plasmids pCDNA3 HHDII and pVITRO2 Human HLA-DP4 plasmids. Transfected cells were selected by growth in the presence of G418 (500 μg/ml) and Hygromycin B (300 μg/ml). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the anti-human beta 2 microglobulin FITC and anti-human HLA-DR/DP/DQ (clone WR18) PE antibodies from Serotec and Abcam respectively.

(61) 2.6 Western Blotting

(62) Cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Sigma) and proteins separated on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) followed by transfer onto PVDF membrane. The membrane was blocked for 1 hour with 3% BSA then probed with antibodies to human/mouse ENO-1 (clone EPR10863(B), Abcam) 1 in 1000 and β actin (clone AC-15, Sigma) 1 in 15000. Proteins were visualised using the fluorescent secondary antibody IRDye 800RD against rabbit (for ENO-1) and IRDye 680RD secondary anti mouse (for β actin). Membranes were imaged using a Licor Odyssey scanner.

(63) 2.7. Immunisations

(64) 2.7.1. Immunisation Protocol

(65) C57BL/6 mice (Charles River, UK), HLA-DR4 mice (Taconic, USA), HHDII/DR1 mice (Pasteur institute, France) and the HHD/HLA-DP4 transgenic strain of mouse as described in patent WO2013/017545 A1 (EMMA repository, France) were used, aged between 8 and 12 weeks, and cared for by the staff at Nottingham Trent University. All work was carried out under a Home Office project licence. Peptides were dissolved in PBS to 1 mg/ml and then emulsified (a series of dilutions) with different adjuvants: CpG and MPLA 6 μg/mouse of each (Invivogen, UK), Incomplete Freund's 50 μl/mouse (Sigma, UK), poly I:C 10 μg/mouse (Invivogen, UK), Imiquimod 25 μg/mouse (Invivogen, UK) and GMCSF 10 μg/mouse (Peprotech, UK). Peptides (25 μg/mouse) were injected subcutaneously at the base of the tail. DNA (1 μg/mouse) was coated onto 1.0 μm gold particles (BioRad, Hemel Hempstead, UK) using the manufacturer's instructions and administered intradermally by genegun (BioRad). Homspera (10 nM/mouse) (PeptideSynthetics, UK) was injected intradermally with genegun immunisation. Mice were immunized at either day 0 for peptide immunisation or days, 0 and 7 for genegun immunisation, unless otherwise stated. Spleens were removed for analysis at day 14 for peptide and day 20 for peptide or genegun immunisation unless stated otherwise.

(66) For tumour challenge experiments mice were challenged with 2.5×10.sup.4 B16 DR4 cells or 1×10.sup.6 LLC2/LLC2 DR4 cells, 2.5×10.sup.5 Pan02 DR4 cells in matrigel or 4×10.sup.5 B16 HHDII DP4 cells subcutaneously on the right flank 3 days prior to primary immunisation and then were immunised as above. Tumour growth was monitored at 3-4 days intervals and mice were humanely euthanized once tumour reached ≥10 mm in diameter.

(67) 2.8. Analysis of Immune Response

(68) 2.8.1. Ex Vivo Elispot Assay

(69) Elispot assays were performed using murine IFNγ, IL-17 and IL-10 capture and detection reagents according to the manufacturer's instructions (Mabtech, Sweden). In brief, anti-IFNγ, IL-17 and IL-10 antibodies were coated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at a variety of concentrations) and 5×10.sup.5 per well splenocytes were added to the wells of the plate in triplicate. Tumour target cells were added where relevant at 5×10.sup.4/well in triplicate and plates incubated for 40 hrs at 37° C. After incubation, captured IFN and IL-10 were detected by biotinylated anti-IFNγ and IL-10 antibodies and developed with a streptavidin alkaline phosphatase and chromogenic substrate. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd).

(70) 2.8.2 Luminex Multiplexed Assay

(71) A three-step indirect procedure was used for the multiplexed Luminex assay (Invitrogen) for IgG antibodies to IL-10, IL-17, IFNy, TNFα, IL-2 & IL-4. Standard, control, and unknown sera were diluted 1:2 in 50% assay diluent buffer (Invitrogen) & 50% serum free RPMI. Serial standard dilutions were included in each assay. Each dilution of standard, control, and unknown sera was mixed with a set of coupled Luminex microspheres in 96-well filtration plates (Millipore Multiscreen; Millipore Corporation, Bedford, Mass.) and incubated for 2 hours at room temperature with shaking. Microspheres were collected by vacuum filtration and washed with PBST. Biotinylated detector antibody was added to each well for 1 hour at room temperature with shaking. Microspheres were collected by vacuum filtration and washed with PBST. Streptavidin conjugated R-phycoerythrin—was added to each well. Following a 30 minute incubation and a wash step, microspheres were resuspended in PBST, and read in a Biorad BioPlex Luminex analyzer equipped with an XY platform. Data acquisition and analysis performed with Luminex software (BioPlex Systems)

(72) 2.8.3 Proliferation Assay (Thymidine)

(73) PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque (Sigma) gradient centrifugation. PBMC (1.5×10.sup.6 cells/well) were stimulated with single peptides (final concentration 10 μg/ml) in RPMI containing 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPES and Penicillin-streptomycin (1%) in a final volume of 2 ml. Stimulation with purified protein derivative, PPD (final concentration 10 μg/ml) served as a positive control for the proliferative capacity of PBMC. As a negative control PBMC were incubated with medium alone. The PBMC were cultured at 37° C. in an atmosphere of 5% CO.sub.2 for 4, 7 and 11 days. To assess proliferation at these times points 100 μl in triplicate from each culture was aliquoted into a round bottom well of a 96 well plate and 3H-thymidine added (0.0185 MBq/well) and incubated at 37° C. for a further 8 hours. The cultures were harvested onto unifilter plates and incorporation of .sup.3H-thymidine was determined by β-scintillation counting. The results were assessed by calculating the stimulation index (SI) as the ratio of the mean of counts per minute (cpm) of epitope-stimulated to the mean of unstimulated cultures. The proliferative assay was considered positive when SI >2.5.

(74) 2.8.4 Proliferation Assay (CFSE)

(75) PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque (Sigma) gradient centrifugation. PBMC (1.5×10.sup.6 cells/well) were stimulated with single peptides (final concentration 10 μg/ml) in RPMI containing 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPES and Penicillin-streptomycin (1%) in a final volume of 2 ml. As a negative control PBMC were incubated with medium alone. The PBMC were cultured at 37° C. in an atmosphere of 5% CO.sub.2 for 7 and 10 days. To assess proliferation at these times points cells were sampled and stained with surface marker CD4 and CD8 antibodies labelled with PE-Cy5 and efluor 450 respectively. After staining cells were fixed and analysed on a Milteny MACSQuant flow cytometer.

(76) 2.8.5 PBMC Culture and IFNγ Elispot

(77) PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque (Sigma) gradient centrifugation. PBMC (1.5×10.sup.6 cells/well) were stimulated with single peptides (final concentration 10 μg/ml) in RPMI containing 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPES, Penicillin-streptomycin (1%), 10 ng/ml recombinant human IL-15 and 5 ng/ml recombinant human IL-7 in a final volume of 2 ml. Recombinant human IL-2 was added on day 3 at 20 IU/ml. On day 13 cells were washed and added to human IFN elispot assay. Elispot assays were performed using human IFNγ capture and detection reagents according to the manufacturer's instructions (Mabtech, Sweden). In brief, anti-IFNγ antibody was coated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at 10 μg/ml) and 1×10.sup.5 per well PBMCs were added to the wells of the plate in quadruplicate and plates incubated for 20 hrs at 37° C. After incubation, captured IFNγ was detected by biotinylated anti-IFN antibody and developed with a streptavidin alkaline phosphatase and chromogenic substrate. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd).

(78) 2.8.6 Intracellular Cytokine Analysis

(79) PBMC cultures were set up as detailed above. On day 14 PBMCs were washed and cultured with synthetic peptide (10 μg/ml) in the presence of brefeldin A for 20 hrs at 37° C. Cells were stained with cell surface markers CD4 and CD8 using PE-Cy5 and efluor450 labelled antibodies respectively. Cells were subsequently fixed and permeabilised and stained with IFNγ PE-Cy7 labelled antibody. After staining cells were fixed and analysed on Miltenyi MACSQuant flow cytometer.

(80) 2.9 Immunohistochemical Analysis

(81) Normal and tumour tissue binding was by immunohistochemistry (IHC) as described previously (Durrant et al., 2006). Immunohistochemical staining was performed on 4 μm sections using Novolink polymer detection system (Leica Biosystems, RE7150-K). Briefly, slides were deparaffinised with xylene and rehydrated through three changes of alcohol, the antigen-retrieval was performed in citrate buffer (pH 6.0) for 20 min using a microwave oven. Endogenous peroxidase activity was blocked by Peroxidase Block for 5 min. Slides were washed with TBS (pH 7.6), followed by the application of Protein Block for 5 min. Following another TBS wash, primary antibody, optimally diluted in Leica antibody diluent (RE7133), was applied and incubated for 60 min. The anti-ENO1 rabbit monoclonal EPR10864 (B) was used at 1/200. Slides were washed with TBS followed by incubation with Post-Primary Block for 30 min followed by a TBS wash. Novolink polymer was applied for 30 min. DAB working solution was made up of 1:20 DAB chromogen in DAB substrate buffer was prepared and applied for 5 min. Slides were counterstained with Novolink haematoxylin for 6 min, and dehydrated.

(82) The TMA slides were initially assessed by light microscope assessment of staining quality and specificity. Slides were then scanned into high-resolution digital images (0.45 μm/pixel) using a NanoZoomer slide scanner (Hamamtsu Photonics, Welwyn Garden City, UK) and accessed using a web-based interface NDP viewer (Nanozoomer Digital Pathology). They were scored at 920 magnification using a minimum of 2400 high-resolution screen (91920 1080). Cases were scored without knowledge of the ENO1 status and patient outcome and were scored by two people (MG and MM). Assessment of staining was based on a semi-quantitative approach using a modified histochemical score (H-score) taking the intensity of staining and the percentage of stained cells into account. For the intensity, a score index of 0, 1, 2, and 3 corresponding to negative, weak, moderate, and strong staining intensity was used, and the percentage of positive cells at each intensity was estimated subjectively. Statistical analysis was performed using SPSS 13.0 (SPSS Inc, Chicago). Stratification cut-points for the survival analysis were determined using X-Tile software (Camp et al., 2004) and P values of <0.05 were considered significant.

(83) Patient Cohorts

(84) The study populations include cohorts of consecutive series of 462 archived colorectal cancer (Simpson et al., 2010) specimens (1994-2000; median follow up 42 months; censored December 2003; patients with lymph node positive disease routinely received adjuvant chemotherapy with 5-flurouracil/folinic acid), 350 ovarian cancer (Duncan et al., 2007) samples (1982-1997; median follow up 192 months: censored November 2005:patients with stage II to IV disease received standard adjuvant chemotherapy which in later years was platinum based), 142 gastric cancer (Abdel-Fatah et al., 2013) samples (2001-2006; median follow up 66 months; censored January 2009; no chemotherapy) 68 pancreatic and 120 billary/ampullary cancer (Storr et al., 2012) samples (1993-2010:median 45 months; censored 2012; 25-46% of patients received adjuvant chemotherapy with 5-flurouracil/folinic acid and gemcitabine) 220 non-small cell lung cancers (January 1996-July 2006: median follow up 36 months censored May 2013; none of the patients received chemotherapy prior to surgery but 11 patients received radiotherapy and 9 patients received at least 1 cycle of adjuvant chemotherapy post-surgery) obtained from patients undergoing elective surgical resection of a histologically proven cancer at Nottingham or Derby University Hospitals. No cases were excluded unless the relevant clinico-pathological material/data were unavailable. This retrospective study was based on a consecutive series of 902 patients with primary invasive breast carcinomas who were diagnosed from 1987 to 1998 and entered onto the Nottingham Tenovus Primary Breast Carcinoma series. This is a well characterised series of patients under the age of 71 years (median 55 years) with long term follow up. All patients were treated in a uniform way in a single institution and have been investigated for a wide range of protein expression.

(85) All patients received standard surgical treatment of either mastectomy or wide local excision with radiotherapy. Before 1988, patients did not receive systemic adjuvant therapy. From 1988 onwards, patients were selected for systemic adjuvant treatment on the basis of NPI score and hormone receptor status. Patents with a NPI<3.4 received no adjuvant therapy; those with an adjuvant score higher than 3.4 received tamoxifen if they were estrogen receptor positive (±goserelin if premenopausal) or classical cyclophosphamide, methotrexate and fluorouracil if they were ER negative and fit enough to tolerate chemotherapy. Survival data was maintained prospectively. Breast cancer specific survival (BCSS) was defined as the time (in years) from the date of the primary surgical treatment to the time of death from breast cancer. Survival was censored if the patient was still alive, lost to follow up (n=73) or died from other causes.

Example 1. Sequence Alignment and Homology of Enolases

(86) In mammals there are four isoforms of the enolase enzyme, ENO1 (A); ENO2 (B), ENO3 (G) and ENO4 which are encoded by four distinct genes. They are highly conserved and have a high degree of amino acid homology (FIG. 1).

Example 2. CD4 Responses to Citrullinated Enolase

(87) The human alpha-Enolase peptide sequence was broken down into overlapping 20-mers. Any 20-mer containing an arginine was selected and the arginine residues were replaced with citrulline (cit). The selected 20mer peptides are summarised in Table 1.

(88) TABLE-US-00003 TABLE 1 Enolase peptides utilised. Enolase peptide (aa co- Peptide sequences ordinates) Human peptide Mouse homologue  1-20 MSILKIHA-CIT-EIFDSRGNPTV (SEQ MSILRIHA-CIT-EIFDSRGNPTV ID NO: 18) (SEQ ID NO: 19)  6-25 IHA-CIT-EIFDS-CIT-GNPTVEVDLF IHA-CIT-EIFDS-CIT- (SEQ ID NO: 20) GNPTVEVDLY (SEQ ID NO: 21) 21-40 EVDLFTSKGLF-CIT-AAVPSGAS EVDLYTAKGLF-CIT-AAVPSGAS (SEQ ID NO: 22) (SEQ ID NO: 23) 26-45 TSKGLF-CIT-AAVPSGASTGIYE (SEQ TAKGLF-CIT-AAVPSGASTGIYE ID NO: 24) (SEQ ID NO: 25) 36-55 PSGASTGIYEALEL-CIT-DNDKT (SEQ — ID NO: 26) 46-65 ALEL-CIT-DNDKT-CIT- ALEL-CIT-DNDKT-CIT- YMGKGVSKA (SEQ ID NO: 27) FMGKGVSQA (SEQ ID NO: 28) 56-75 -CIT-YMGKGVSKAVEHINKTIAP -CIT-FMGKGVSQAVEHINKTIAP (SEQ ID NO: 29) (SEQ ID NO: 30) 121-140 AGAVEKGVPLY-CIT-HIADLAGN — (SEQ ID NO: 31) 126-145 KGVPLY-CIT-HIADLAGNSEVIL (SEQ KGVPLY-CIT-HIADLAGNPEVIL ID NO: 32) (SEQ ID NO: 33) 171-190 LPVGAANF-CIT-EAM-CIT-IGAEVYH LPVGASSF-CIT-EAM-CIT- (SEQ ID NO: 34) IGAEVYH (SEQ ID NO: 35) 176-195 ANF-CIT-EAM-CIT-IGAEVYHNLKNV SSF-CIT-EAM-CIT- (SEQ ID NO: 36) IGAEVYHNLKNV (SEQ ID NO: 37) 241-260 VIGMDVAASEFF-CIT-SGKYDLD VIGMDVAASEFY-CIT-SGKYDLD (SEQ ID NO: 38) (SEQ ID NO: 39) 246-265 VAASEFF-CIT-SGKYDLDFKSPD VAASEFY-CIT- (SEQ ID NO: 40) SGKYDLDFKSPD(SEQ ID NO: 41) 256-275 KYDLDFKSPDDPS-CIT-YISPDQ (SEQ KYDLDFKSPDDPS-CIT-YITPDQ ID NO: 42) (SEQ ID NO: 43) 261-280 FKSPDDPS-CIT-YISPDQLADLY (SEQ FKSPDDPS-CIT-YITPDQLADLY ID NO: 44) (SEQ ID NO: 45) 316-335 VGDDLTVTNPK-CIT-IAKAVNEK VGDDLTVTNPK-CIT-IAKAASEK (SEQ ID NO: 46) (SEQ ID NO: 47) 321-340 TVTNPK-CIT-IAKAVNEKSCNCL TVTNPK-CIT-IAKAASEKSCNCL (SEQ ID NO: 48) (SEQ ID NO: 49) 326-345 K-CIT-IAKAVNEKSCNCLLLKVN K-CIT-IAKAASEKSCNCLLLKVN (SEQ ID NO: 50) (SEQ ID NO: 51) 361-380 QANGWGVMVSH-CIT-SGETEDTF QSNGWGVMVSH-CIT-SGETEDTF (SEQ ID NO: 52) (SEQ ID NO: 53) 366-385 GVMVSH-CIT-SGETEDTFIADLV — (SEQ ID NO: 54) 391-410 GQIKTGAPC-CIT-SE-CIT-LAKYNQL — (SEQ ID NO: 55) 396-415 GAPC-CIT-SE-CIT-LAKYNQLL-CIT- GAPC-CIT-SE-CIT-LAKYNQIL- IEE (SEQ ID NO: 56) CIT-IEE (SEQ ID NO: 57) 401-420 SE-CIT-LAKYNQLL-CIT-IEEELGSK SE-CIT-LAKYNQIL-CIT- (SEQ ID NO: 58) IEEELGSK (SEQ ID NO: 59) 406-425 KYNQLL-CIT-IEEELGSKAKFAG KYNQIL-CIT-IEEELGSKAKFAG (SEQ ID NO: 60) (SEQ ID NO: 61) 416-434 ELGSKAKFAG-CIT-NF-CIT-NPLAK ELGSKAKFAG-CIT-SF-CIT- (SEQ ID NO: 62) NPLAK (SEQ ID NO: 63)  indicates mouse and human sequences are homologous aa that alter in the mouse sequence are highlighted in bold

(89) Screening of Enolase Peptide Responses

(90) Screening was performed to identify potential citrullinated Enolase epitopes in mice. Mice were immunised with pools of 4-6 human citrullinated peptides. To reduce the effect of possible cross reactivity the peptides within each pool were chosen so that they did not contain any overlapping amino acid sequences. Each pool was administered as a single immunisation containing 20 μg of each peptide and CpG/MPLA as an adjuvant. After 14 days the mice were culled and the immune responses to each peptide within the immunising pool were assessed by ex vivo Elispot (FIG. 2). In addition, the splenocytes were screened against the murine equivalent sequences. We have previously shown that citrullinated peptides can induce responses in the transgenic DR4 mouse strain. Given that different mouse strains have different MHC repertoires a number of strains were used for screening. Peptide responses were assessed in C57BL/6 mice as well as transgenic strains expressing human DR4 or HHD/DR1 in a C57BL/6 background (see materials and methods).

(91) Significant IFNγ responses were detected to a number of different peptides. In the DR4 mice the pool containing the Enolase 241-260 citrullinated peptide induced a significant response to human 241cit (p<0.05) and mouse 241cit (p<0.0001). No other peptides showed significant IFNγ responses in DR4 mice. In the HHD/DR1 mice, the pool with Enolase peptide 126-145 induced a significant response to human 126cit (p<0.05) but not mouse 126cit. The pool with Enolase peptide 316-335 induced a significant response to human 316cit (p<0.05) but not mouse 316cit. The pool containing the peptide Enolase 1-20 did not induced a significant response to human peptide but did induce a response to mouse 1cit (p<0.05). In the C57BL/6 mice, the pool containing the peptide Enolase 21-40 induced a significant response to human 21cit (p<0.05) but not mouse 21cit. The pool with Enolase peptide 126-145 induced a significant response to mouse 126cit (p<0.05) but not human 126cit. The pool with Enolase peptide 261-280 induced a significant response to human 261cit (p<0.05) but not mouse 261cit. This suggests that peptides 21-40, 126-145, 241-260 and 316-335 justified further investigation.

(92) From the initial screen Enolase 241cit immunisation in DR4 mice induced the strongest immune response. Therefore, this peptide was investigated further. DR4 mice were given a single immunisation with 25 μg of the human 241cit peptide and CpG/MPLA. E vivo elispot on splenocytes showed a significant IFNγ response to citrullinated peptides compared to media controls for both the mouse (p=0.0008) and human (p=0.0124) sequence (FIG. 3A). The mouse sequence is actually the same sequence as aa241-260 from ENO2 and ENO3 so is still a self-antigen. Interestingly, neither the human or mouse wild type (wt) sequence, where the arginine residue at position 253 has not been replaced with a citrulline, produced an immune response. This confirmed that Enolase 241cit induced a citrulline specific IFNγ response in DR4 mice.

(93) To determine the type of cytokine response generated by Enolase 241cit peptide ex vivo IL-10 was also assayed. No significant increase in IL-10 production was observed in ELISpot assays in response to peptide stimulation (FIG. 3B).

(94) Previously citrullinated peptide specific responses have been shown to be CD4 mediated. To determine whether the response to 241cit is CD4 dependent an Elispot assay was performed with a human MHC class II blocking antibody (clone L243) (FIG. 3C). IFN response were significantly reduced by L243 in response to both human Enolase 241cit (p=0.0171) and mouse 241cit (p=0.0023). To further confirm that 241cit specific responses were CD4 mediated Elispot assay was performed including a murine CD4 or CD8 blocking antibody (clone GK1.5 or clone 2.43 respectively) or using CD4 enriched or depleted cell fractions. Responses to human enolase 241cit were significantly reduced in the presence of the CD4 blocking antibody but not the CD8 blocking antibody (FIG. 15A). Responses were also present in the CD4 enriched fraction but not in the CD4 depleted fraction (FIG. 15B). Since Enolase 241-260 is a long peptide we sought to determine the optimal 15mer sequence that the responses recognize. Two 15mer peptides spanning the 241-260 sequence were tested for responses with aa241-255 stimulating specific responses but no response to aa246-260 (FIG. 15C).

(95) To confirm lower frequency responses seen in initial screens, DR4, C57Bl/6 and HHD/DR1 mice were given a single immunisation of the human citrullinated Enolase peptides corresponding to the sequences at positions 21-40, 126-145 and 316-335 (FIG. 4). Enolase 21cit induced a low level but significant response in DR4 mice to the mouse (p<0.01) but not to the human sequence. In C57Bl/6 mice strong IFNγ responses were observed that respond to the citrullinated but not the wt peptide (FIG. 21A). These appear to be CD4 mediated as they are efficiently blocked by CD4 blocking antibody but not a CD8 blocking antibody (FIG. 21B). Enolase 126cit induced a low level significant response to the human peptide in DR4 mice (p<0.05) but not in DR1/HHD mice. Enolase 316cit induced a moderate response to the human but not the mouse sequence in both DR4 (p<0.01) and DR1/HHD (p<0.01) mice.

(96) Enolase sequences were also subject to in silico analysis for peptide sequences with high binding affinity to human and murine MHC class II using the online IEDB prediction program. This suggested the aa11-25 sequence to be strong for murine MHC class II (I-Ab) therefore the citrullinated aa11-25 peptide was tested for responses in C57Bl/6 mice. Mice showed IFN responses to this citrullinated peptide that cross reacted with the equivalent sequence from the murine sequence with minimal reactivity to the wt peptide (FIG. 22A). No IL-10 responses were seen to the citrullinated enol 1 peptide (FIG. 22B). The enolase 11cit specific IFN response was mediated by CD4 cells as blocking these with a CD4 blocking antibody abrogated the response whereas use of a CD8 blocking antibody had no effect on the response (FIG. 22C).

(97) To determine whether HLA-DP4 might also be able to present the Enolase 241cit peptide transgenic DP4 mice were utilised. DP4 mice were immunised with three doses of either human or mouse Enolase 241cit peptide. IFNγ responses were determined by Elispot (FIG. 5). Mice immunized with human Enolase 241cit peptides showed responses to both Human Enolase 241cit (p<0.0001) and mouse 241cit (p<0.0001) showed increased IFNγ responses when compared to the wild type peptides (FIG. 5A). These responses show an avidity of between 1 and 0.1 ug/ml peptide (FIG. 16A). Cells from mice immunized with the human 241-260cit peptide show granzyme B release but no IL-10 in response to the cit peptide but not the wt peptide (FIGS. 16B and C). Responses specific for the human 241-260 cit peptide in the DP4 mice are also blocked by a CD4 blocking antibody but not CD8 blocking antibody and show cross reactivity to a shorter peptide spanning aa241-255 (FIGS. 16D and E). Immunisation of DP4 transgenic mice with the murine Enolase 241-260 peptide also induces responses specific to the citrullinated peptide but not the wildtype (FIG. 16F).

Example 3: Cit Enolase Peptide Presented on Tumour Cells can be Targeted for Tumour Therapy

(98) We had already established by Western blotting that the melanoma B16F1 and Lewis lung Carcinoma cell lines constitutively express Alpha Enolase (FIG. 6A). Next, the anti-tumour effect of Enolase 241cit peptide immunisation was assessed in vivo. The effect of Enolase immunisation on the growth of the mouse B16 melanoma cell line transfected with constitutive human DR4 (B16DR4) was assessed. Mice were challenged with B16DR4, 3 days prior to immunisation with Enolase 241cit peptide. Enolase 241cit peptide immunised mice showed a significant survival advantaged over control mice (FIG. 6B). Unimmunised mice showed 15% survival after 45 days whereas Enolase 241cit immunised mice showed 50% survival (p=0.0001). The tumour volume (FIG. 6C) was also significantly lower in the Enolase 241cit immunised mice (median 0 mm.sup.3) compared to the control group (median 49 mm.sup.3) at day 17 post tumour implant (p=0.0043).

(99) Since responses to the 241-260cit epitope have also been demonstrated in DP4 mice, DP4 transgenic mice were challenged with the mouse B16 melanoma line expressing constitutive human DP4 (B16DP4) and subsequently immunized with Enolase 241cit peptide. Enolase 241cit peptide immunized mice showed a significant survival advantage (p=0.0058) over unimmunized mice with survival rates after 60 days of 70% and 10% respectively (FIG. 17).

(100) To determine whether survival is effected by the constitutive expression of MHC class II in this tumour cell line, the anti-tumour effect was assessed in B16 cells where the HLA-DR4 expression is IFNγ inducible (iDR4). Mice were challenged with B16iDR4 4 days prior to immunisation with Enolase 241cit peptide (FIG. 6D). Survival was significantly increased in the Enolase 241cit immunised group (90%) compared to the unimmunised control animals (survival 0%) at day 42 (p<0.0001). The tumour volume at day 17 post tumour implant (FIG. 6E) was also significantly lower in the Enolase 241cit (median 0 mm.sup.3) compared to unimmunised control mice (median 65 mm.sup.3, p=0.0048). Given the high survival percentage in the immunised group these mice were rechallenged with B16iDR4 at day 42 to see if memory had been established. New untreated mice were also challenged with tumour as a control group. Enolase immunised survivors showed a significant survival advantage on rechallenge compared to previously untreated control mice (FIG. 6F). 39 days after the rechallenge survival in the Enolase 241cit immunised group was 67% while all of the control group were dead by day 29 (p=0.0112).

(101) To determine whether this anti-tumour effect is specific to the B16DR4 model, mice were next challenged with the Lewis lung carcinoma cell line LLC2 (FIG. 7A). Preliminary studies suggested that a higher implant cell number was required to obtain consistent growth of the LLC2 cells compared to B16 cells (data not shown). For this reason, in our hands the LLC2 model is more aggressive than the B16 model. Mice were challenged with parental LLC2 or LLC2 transfected to constitutively express DR4 (LLCDR4) four days before immunisation with Enolase 241cit peptide. Survival data shows that Enolase 241cit immunisation provided a survival advantage against the LLCDR4 tumour (p=0.0142) with 40% of mice surviving today 58 compared to the control where all mice died by day 48. However, in mice challenged with the parental LLC2 tumour while Enolase 241cit immunised mice showed a small but significant increase in survival time (p=0.0005). These results suggest that human DR4 expression on tumour cells is important for tumour rejection in this model.

(102) Enolase is also expressed by the pancreatic tumour line Pan02 (FIG. 6A). This line was engineered to constitutively express HLA-DR4 and DR4 transgenic mice were challenged with tumour followed 4 days later by immunization with Enolase 241cit peptide. Enolase 241cit peptide immunized mice show significantly enhanced survival (p=0.0076) in the Pan02 DR4 model compared to unimmunized control DR4 mice. 50% of mice show survival at day 60 compared to none of the unimmunized mice (FIG. 18).

Example 4. DNA Immunisation Results in Responses to Citrullinated Enolase

(103) As APCs can constitutively citrullinate epitopes it was possible that a DNA construct encoding Enolase may be citrullinated and stimulate a response. HLA-DR4 transgenic mice were therefore immunised with a DNA construct encoding mouse enolase. Stimulated T cells from these mice were screened in vitro for IFNγ, responses to both citrullinated and uncitrullinated enolase 241 peptide. FIG. 8A shows that mice only responded to the citrullinated mouse peptide (mean: IFNγ 180/million splenocytes; p=0.0001). No I1-10 response was observed (FIG. 8B).

(104) Next, the anti-tumour effect of Enolase DNA immunisation was assessed in vivo. Mice were challenged with B16DR4 four days prior to immunisation with Enolase DNA. Enolase DNA immunised mice showed a significant survival advantaged over control mice (FIG. 8C). Unimmunised showed 15% survival after 45 days whereas Enolase DNA immunised mice showed 60% survival (p=0.0001). The tumour volume (FIG. 8D) was also significantly lower in the Enolase DNA immunised mice (median 20 mm.sup.3) compared to the control group (median 150 mm.sup.3) at day 17 post tumour implant (p=0.0088).

Example 5. Determination of Whether CD4 Responses to Enolase Peptides Vary when Combined with Different Adjuvants and at Different Doses

(105) Enolase 241cit peptide induces a strong IFNγ response when administered as a single 25 ug dose with the adjuvant CpG/MPLA. The effect of adjuvant and dose regime on the response generated was investigated. Mice were immunised with a single dose of Enolase 241cit peptide with either CpG/MPLA or incomplete Freund's adjuvant (IFA) as the adjuvant. IFNγ responses to Enolase 241cit peptides were detected by Elispot when CpG/MPLA (p=0.0028) was the adjuvant but no IFNγ response was seen when IFA was used as an adjuvant (FIG. 9A). IL-10 responses were detected by Elispot when Enolase 241cit peptide was administered with IFA (p<0.0001) but not CpG/MPLA (FIG. 9B). In addition to these adjuvants responses in combination with other TLR agonists Poly I:C (TLR3) and imiquimod (TLR7) were assessed in both DR4 and DP4 transgenic mice. The combination with poly I:C induces IFNγ responses in both mouse strains but no IL-10 (FIG. 19). Enolase 241cit peptide combined with imiquimod induces IFNγ responses in DP4 transgenic mice but not in DR4 transgenic mice (FIG. 19). This suggests that the type of cytokine response generated by immunisation with Enolase 241cit peptide can be strongly affected by the adjuvant selected.

(106) Next, dose responses to immunisation with GM-CSF were assessed. Mice were given a single or three immunisations with either 25 μg or 5 μg of Enolase 241cit peptide. IFN responses were assessed by Elispot (FIG. 9C). Detectable responses could be observed after 1 or 3 doses with 25 μg of peptide. Next mice were challenged with B16DR4 and then given three doses of 5 μg of Enolase in GM-CSF over three weeks, to determine whether this is sufficient to induce an anti-tumour response (FIG. 9D). Enolase 241cit immunised mice had a significant survival advantage over control mice (p=0.0045) with 70% of animals surviving at day 45 compared to 0% in the control group by day 28.

Example 6. Enolase 241cit Memory Responses

(107) The ability of different adjuvants to polarise the responses to immunisation with Enolase 241cit peptide may suggest plasticity of the T-cell population involved. This may indicate a pre-existing or memory response. Therefore, next the speed with which an Enolase cit response developed was determined. Mice were immunised with a single dose of Enolase 241cit peptide in CpG/MPLA 2, 6 or 14 days before being sacrificed. Ex vivo Elispots were used to determine the IFNγ responses (FIG. 10). Immunisation with the mouse version of the peptide induced an IFNγ response which could be detected 2 days later. There was no significant difference between the responses seen after 2, 6 or 14 days. Immunisation with the human Enolase 241cit peptide led to an IFNγ response which was detectable after 6 days. Responses were significantly increased after 6 (p=0.0009) and 14 (p=0.0092) days when compared to responses after 2 days. These results suggest that there may be a pre-existing response to Enolase 241cit peptide which is specific to the endogenous murine peptide.

Example 7. Responses in Healthy Human Donors

(108) Mouse response to Enolase 241cit peptide can also be detected as early as 2 days after immunisation. This raised the question of whether humans have a pre-existing response to Enolase 241cit peptides which can be detected. To investigate this PBMCs were isolated from 6 healthy donors and cultured in the presence of Human Enolase peptides. Thymidine proliferation assays were performed on the cells after 4, 7 and 11 days and the proliferation index for each was calculated (FIG. 11A). 5/6 of the donors showed proliferation to Enolase 241cit peptide on at least one of the samples days. For example, Donor 1 showed a proliferative response to Enolase 241cit at day 11 (mean 20.4) and day 7 (mean 28.6) but not at day 4 (mean 0.8). Responses to Enolase 241 wt were consistently low at day 11 (mean 0.9), 7 (mean 1.2) and 4 (mean 0.3). In contrast Donor 2 showed only a low level response at day 11 (mean 2.7) and Donor 6 was a non-responder. For each donor HLA types were determined and are shown on the figure.

(109) Donor 4 gave a high proliferation index at day 4 (mean 12.5) and day 7 (mean 28) and day 11 (4.4). This donor was chosen for further analysis. Supernatants were taken from cells at each time point and cytokine levels were analysed by Luminex. The response above the background level of the media only control was calculated for each cytokine (FIG. 11B). IFNγ and IL-10 gave citrullinated peptide specific responses which increased over time. Some increase in IL-17, Granzyme B and TNFα levels were seen in wild type stimulated cells however these responses were higher in the citrullinated peptide stimulated samples.

(110) Next, PBMCs from donor 4 were labelled with Carboxyfluorescein succinimidyl ester (CFSE) prior to ex vivo culture in the presence of peptides. On day 7 and 10 cells were removed and stained with anti-CD8 and anti-CD4 fluorochome conjugated antibodies and analysed by flow cytometry (FIG. 11C). Of the proliferating CFSE.sup.low population between 73-96% of cells were CD4+ and 0-2% were CD8+. Enolase 241cit peptide showed increased proportions of CFSE.sup.lowCD4.sup.+ cells compared to Enolase 241 wt peptide. At day 10, 15% of the Enolase 241cit lymphocytes are CFSE.sup.lowCD4.sup.+ whereas 1% of the Enolase 241 wt peptides are CFSE.sup.lowCD4.sup.+. IFNγ responses have also been shown by IFNγ elispot assay in which PBMCs cultured for 13 days in Enolase 241 cit or wt peptides were restimulated with citrullinated or wt Enolase 241 peptide and cytokine release measured. FIG. 20A shows results of the IFNγ elispot assay on donors 1 and 4. Cells from both donors show responses to the citrullinated peptide but not the wt peptide. Further analysis of these responses by intracellular cytokine staining reveals IFNγ responses to be CD4 mediated. FIG. 20B shows intracellular cytokine staining on PBMCs from donor 4 cultured for 13 days in Enolase 241cit peptide and restimulated with either citrullinated or wt peptide. IFNγ positive CD4 cells are observed upon stimulation with the citrullinated peptide (0.44%) but not the wt peptide.

(111) Luminex data from cultures on 3 donors shows IFNγ responses to the citrullinated Enolase 241 peptide with minimal response to the wt peptide and low level IL-10, TNFα or IL-17 responses (FIG. 20C).

(112) These results suggest that healthy humans are able to develop a CD4 proliferative response to Enolase 241cit peptide which is citrulline specific and capable of producing Th1 cytokines.

Example 8. Immunohistochemistry

(113) Citrullination is carried out by PAD enzymes and in particular the PAD2 and PAD4 enzymes. These require high levels of calcium and are usually activated in dead or dying cells or cells undergoing autophagy. Healthy cells should not express citrullinated proteins but tumours due to either hypoxia or nutritional stress will activate autophagy and citrullinated enolase. Colorectal, gastric, lung, breast and ovarian tumours were therefore stained for expression of enolase.

(114) Colorectal Tumours:

(115) 232 colorectal tumours were stained with an ENO-1 specific monoclonal antibody (Table 2). 28% of tumours failed to stain, 56% showed weak staining (Hscore 1-100), 13% moderate staining (Hscore 101-200) and 3% showed strong staining (Hscore 201-300) were most cells stained intensely.

(116) TABLE-US-00004 TABLE 2 Immunohistochemical staining of Colorectal tumour array for Eno-1 Negative Low Moderate High Total H- 0 1-100 101-200 201-300 SCORE cores 655    129    30    8    232  28%  56% 13% 3%

(117) Gastric Tumours:

(118) 70 gastrctumours were stained with an ENO-1 specific monoclonal antibody (Table 3). 16% of tumours failed to stain, 62% showed weak staining (Hscore1-100), 19% moderate staining (Hscore101-200) and 3% showed strong staining (Hscore 201-300) were most cells stained intensely.

(119) TABLE-US-00005 TABLE 3 Immunohistochemical staining of gastric tumour array for Eno-1 Negative Low Moderate High Total H 0  1-100 101-200 201-300 SCORE cores 11    44    13    2    70 16% 62% 19% 3%

(120) Non-Small Cell Lung Tumours:

(121) 223 non-small cell lung tumours were stained with an ENO-1 specific monoclonal antibody (Table 4). 20% of tumours failed to stain, 59% showed weak staining (Hscore 1-100), 17% moderate staining (Hscore 101-200) and 4% showed strong staining Hscore 201-300 were most cells stained intensely.

(122) TABLE-US-00006 TABLE 4 Immunohistochemical staining of non-small cell lung tumours tumour array for Eno-1 Negative Low Moderate High Total H- 0 1-100 101-200 201-300 SCORE cores 45 132 37 9 223 % 20 59 17 4

(123) Ovarian Tumour:

(124) 223 ovarian tumours were stained with an ENO-1 specific monoclonal antibody (Table 5). 42% of tumours failed to stain, 51% showed weak staining (Hscore1-100), 2% moderate staining (Hscore 101-200) and 5% showed strong staining (Hscore 201-300) were most cells stained intensely.

(125) TABLE-US-00007 TABLE 5 Immunohistochemical staining of Ovarian tumour array for Eno-1 Negative Low Moderate High Total H- 0 1-100 101-200 201-300 SCORE cores 93 115 5 10 223 % 42 51 2 5

(126) Breast Tumours:

(127) 858 breast tumours were stained with an ENO-1 specific monoclonal antibody (Table 6). 28% of tumours failed to stain, 19% showed weak staining (Hscore 1-100), 36% moderate staining (Hscore 101-200) and 17% showed strong staining (Hscore 201-300) were most cells stained intensely.

(128) TABLE-US-00008 TABLE 6 Immunohistochemical staining of Breast tumour array for Eno-1 Negative Low Moderate High Total H- 0 1-100 101-200 201-300 SCORE cores 239    165    310    144    858 %  28%  19%  36%  17%

(129) Oestrogen Receptor Negative Breast Tumours:

(130) 249 oestrogen receptor negative breast tumours were stained with an ENO-1 specific monoclonal antibody (Table 7). 8% of tumours failed to stain, 14% showed weak staining (Hscore 1-100), 55% moderate staining (Hscore 101-200) and 23% showed strong staining (Hscore 201-300) were most cells stained intensely.

(131) TABLE-US-00009 TABLE 7 Immunohistochemical staining of Oestrogen receptor negative breast tumour array for Eno-1 Negative Low Moderate High Total H- 0  1-100 101-200 201-300 SCORE cores 19    36    136    58    249 %  8% 14%  55% 23%

Example 9 Homology of Enolase Between Different Species

(132) Enolases are highly conserved between, mouse, dog sheep, cows, horse, pig and humans (FIGS. 12-14). As the vaccine induces T cell responses in humans and mice and anti-tumour responses in mice, it can be assumed similar responses will be seen in other species.

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