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EPITOPES

20220288178 · 2022-09-15

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to epitopes containing homocitrulline (Hcit) that can be used as targets for cancer immunotherapy. The homocitrullinated T cell epitope has (i) a predicted binding score to MHC class II or class I of <30 using the online IEDB prediction program (http://www.iedb.org/) and (ii) at least 5 consecutive amino acids that form a spiral conformational structure. These modified peptides can be used as vaccines or as targets for T cell receptor (TCR) and adoptive T cell transfer therapies.

    Claims

    1. A homocitrullinated T cell epitope having (i) a predicted binding score to WIC class II or WIC class I of <30 using the online IEDB prediction program (http://www.iedb.org/) and (ii) at least 5 consecutive amino acids that form a spiral conformational structure.

    2. The epitope of claim 1, which is from a cytoplasmic protein.

    3. The epitope of claim 1, which comprises, consists essentially of, or consists of i) one or more of the following amino acid sequences, wherein one or more of the lysine (K) residues is replaced with homocitrulline: TABLE-US-00019 NYIDKVRFLEQQNKILLAEL (Vimentin 116-135), LARLDLERKVESLQEEIAFLK (Vimentin 215-235), QIDVDVSKPDLTAALRDVRQQ (Vimentin 255-275), EAEEWYKSKFADLSEAAN (Vimentin 286-303 Hcit), LPLVDTHSKRTLLKTVETRDGQV (Vimentin 431-454), FKNTRTNEKVELQELNDRFA (Vimentin 96-115) TNEKVELQELNDRFANYIDKVR (Vimentin 101-122) KVRFLEQQNKLLAE (Vimentin 120-134) DVRQQYESVAAKNLQEAE (Vimentin 271-288) EAEEWYKSKFADLSEAANRN (Vimentin 286-305) FSLADAINTEFKNTRTNEKVELQ (Vimentin 86-108) KMALDIEIATYRKLLEGEE (Vimentin 390-408) IGGVILFHETLYQKADDGRP (Aldolase 74-93), KDGADFAKWRCVLKIGEH (Aldolase 140-157) LSDHHIYLEGTLLKPNMVT (Aldolase 217-235) HACTQKFSHEEIAMATVTA (Aldolase 238-256) KCPLLKPWALTFSYGRALQ (Aldolase 289-307) DLKRCQYVTEKVLAAVYKA (Aldolase 198-216) AAQEEYVKRALANSLACQGK (Aldolase 323-342) KVLAAVYKALSDHHIYLEG (Aldolase 208-226) YVTEKVLAAVYKALSD (Aldolase 204-219 Hcit) VLAAVYKAL (Aldolase 209-217 Hcit) KFASFIDKVRFLEQQNKMLE (Cytokeratin 8 101-120) LEQQNKMLETKWSLLQQQKT (Cytokeratin 8 112-131) KMLETKWSL (Cytokeratin 8 117-125) EINKRTEMENEFVLIKKDVDE (Cytokeratin 8 182-202) LREYQELMNVKLALDIEI (Cytokeratin 8 371-388) KLALDIEIATYRKLLEGEE (Cytokeratin 8 381-399) ETKWSLLQQQKTARSNMDNMF (Cytokeratin 8 120-140) EQIKSLNNKFASFIDKVRFL (Cytokeratin 8 93-112) ENEFVLIKKDVDEAYMNKV (Cytokeratin 8 190-208) GKHGDDLRRTKTEISEM (Cytokeratin 8 294-310) RQLREYQELMNVKLALEI (Cytokeratin 8 369-388) EIEGLKGQRASLEAAIADA (Cytokeratin 8 320-338) EQRGELAIKDANAKLSELEA (Cytokeratin 8 339-358) NDPSVQQDIKFLPFKVVEKKT (BiP 104-124) EISAMVLTKMKETAEA (BiP 144-159) GEDFDQRVMEHFIKLYKKKTG (BiP 255-275) QKLRREVEKAKRALSSQHQAR (BiP 286-306) EDFSETLTRAKFEELNMDLFR (BiP 316-336) EELNMDLFRSTMKPVQKVL (BiP 328-346) RIPKIQQLVKEFFNGKEPSRG (BiP 367-387) TVTIKVYEGERPLTKDNHLLG (BiP 460-480) RNELESYAYSLKNQIGDK (BiP 562-579) KKELEEIVQPHSKLYGSAG (BiP 620-639) PLRPQNYLFGCELKADK (NPM 11-27) EGSPIKVTLATLKMSVQPTVSL (NPM 68-89) EEEDVKLLS1SGKRSAPGGGS (NPM 129-149) SKGQESFKKQEKTPKTPKG (NPM 222-240) GGSLPKVEAKFINYVKNCFR (NPM 258-277) AKFINYVKNCFRMTDQEAIQDL (NPM 266-287) MSILKIHAREIFDSRG (Alpha enolase 1-16) NDKTRYMGKGVSKAVEHI (Alpha enolase 52-69) TENKSKFGANAILGVSLAVCKA (Alpha enolase 100-121) GSHAGNKLAMQEFMILPVGAA (Alpha enolase 156-176) REAMRIGAEVYHNLKNVIK (Alpha enolase 179-197) NVTKEKYGKDATNVGDEGG (Alpha enolase 194-212) DVAASEFFRSGKYDLDFKSP (Alpha enolase 245-264) PDQLADLYKSFIKDYPVVS (Alpha enolase 273-291) WGAWQKFTASAGIQVVG (Alpha enolase 301-317) NKSCNCLLLKVNQIGSVTE (Alpha enolase 333-352) RSERLAKYNQLLRIEEELGS (Alpha enolase 400-419) GSKAKFAGRNFRNPLAK (Alpha enolase 418-434) EPSQMLKHAVVNLINYQD (Beta-Catenin 127-144) EKLLWTTSRVLKVLSVCSSNK (Beta-Catenin 334-354) TLHNLLLHQEGAKMAVRL (Beta-Catenin 258-275) AKMAVRLAGGLQKMVALLNK (Beta-Catenin 269-288) KTNVKFLAITTDCLQILAYG (Beta-Catenin 288-307) TYEKLLWTTSRVLKVLSV (Beta-Catenin 332-349) TSRVLKVLSVCSSNKPAIV (Beta-Catenin 340-358) YGLPWVKLLHPPSHWPL (Beta-Catenin 489-506) HWPLIKATVGLIRNLALCPA (Beta-Catenin 503-522) IENIQRVAAGVLCELAQDK (Beta-Catenin 607-625) GVATYAAAVLFRMSEDKP (Beta-Catenin 650-667) IDLKDKYKNIGAKLVQDVAN (HSP60 84-103) TVLARSIAKEGFEKISKGAN (HSP60 117-136) GEALSTLVLNRLKVGLQVVA (HSP60 280-299) TTSEYEKEKLNERLAKLS (HSP60 381-398) GIIDPTVKVRTALLDAAGVA (HSP60 517-536), or ii) one or more of the amino acid sequences 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-lysine position.

    4. The epitope of claim 3, which comprises, consists essentially of, or consists of i) one or more of the following amino acid sequences: TABLE-US-00020 NYID-Hcit-VRFLEQQN-Hcit-ILLAEL Vimentin 116-135 Hcit), LARLDLER-Hcit-VESLQEEIAFL-Hcit Vimentin 215-235 Hcit), QIDVDVS-Hcit-PDLTAALRDVRQQ Vimentin 255-275 Hcit), EAEEWY-Hcit-S-Hcit-FADLSEAAN Vimentin 286-303 Hcit), LPLVDTHS-Hcit-RTLL-Hcit-TVETRDGQV Vimentin 431-454 Hcit), F-Hcit-NTRTNE-Hcit-VELQELNDRFA Vimentin 96-115 Hcit) TNE-Hcit-VELQELNDRFANYID-Hcit-VR Vimentin 101-122 Hcit) KVRFLEQQN-Hcit-LLAE Vimentin 120-134 Hcit) DVRQQYESVAA-Hcit-NLQEAE Vimentin 271-288 Hcit) EAEEWY-Hcit-S-Hcit-FADLSEAANRN Vimentin 286-305 Hcit) FSLADAINTEF-Hcit-NTRTNE-Hcit-VELQ Vimentin 86-108 Hcit) Hcit-MALDIEIATYR-Hcit-LLEGEE Vimentin 390-408 Hcit) IGGVILFHETLYQ-Hcit-ADDGRP Aldolase 74-93 Hcit), Hcit-DGADFA-Hcit-WRCVL-Hcit-IGEH Aldolase 140-157 Hcit) LSDHHIYLEGTLL-Hcit-PNMVT Aldolase 217-235 Hcit) HACTQ-Hcit-FSHEEIAMATVTA Aldolase 238-256 Hcit) Hcit-CPLL-Hcit-PWALTFSYGRALQ Aldolase 289-307 Hcit) DL-Hcit-RCQYVTE-Hcit-VLAAVY-Hcit-A Aldolase 198-216 Hcit) AAQEEYV-Hcit-RALANSLACQG-Hcit Aldolase 323-342 Hcit) Hcit-VEAAVY-Hcit-ALSDHHIYLEG Aldolase 208-226 Hcit) YVTE-Hcit-VLAAVY-Hcit-ALSD Aldolase 204-219 Hcit) VLAAVY-Hcit-AL (Aldolase 209-217) KFASFID-Hcit-VRFLEQQN- Hcit-MLE (Cytokeratin 8 101-120 Hcit) LEQQN-Hdt-MLET-Hcit- WSLLQQQ-Hcit-T (Cytokeratin 8 112-131 Hcit) Hcit-MLET-Hcit-WSL (Cytokeratin 8 117-125) EIN-Hcit-RTEMENEFVLI- Hcit-Hcit-DVDE (Cytokeratin 8 182-202 licit) LREYQELMNV-Hdt-LALDIEI (Cytokeratin 8 371-388 Hcit) Hcit-LALDBEIATYR-Hdt- LLEGEE (Cytokeratin 8 381-399 Hcit) ET-Hdt-WSLLQQQ-Hcit- TARSNMDNMF (Cytokeratin 8 120-140 Hcit) EQI-Hcit-SLNN-Hcit- FASFID-Hcit-VRFL (Cytokeratin 8 93-112 Hcit) ENEFVLI-Hcit-Hcit- DVDEAYMN-Hcit-V (Cytokeratin 8 190-208 Hcit) G-Hcit-HGDDLRRT-Hcit- TEISEM (Cytokeratin 8 294-310 Hcit) RQLREYQELMNV-Hcit-LALEI (Cytokeratin 8 369-388 Hcit) EIEGL-Hcit-GQRASLEAAiADA (Cytokeratin 8 320-338 Hcit) EQRGELAI-Hcit-DANA- Htit-LSELEA (Cytokeratin 8 339-358 Hcit) NDPSVQQDI-Hcit-FLPF-Hcit- VVE-Hcit-Hcit-T (BiP 104-124 Hcit) EISAMVLT-Hcit-M-Hcit-ETAEA (BiP 144-159 Hcit) GEDFDQRVMEHFI-Hcit-LY-Hcit- Hcit-Hcit-TG (BiP 255-275 Hcit) QKLRREVEKAKRALSSQHQAR (BiP 286-306 Hcit) EDFSETLTRA-Hcit-FEELNMDLFR (BiP 316-336 Hcit) EELNMDLFRSTM-Hcit-PVQ- Hcit-VL (BiP 328-346 Hcit) RIP-Hcit-IQQLV-Hcit- EFFNG-Hcit-EPSRG (BiP 367-387 Hcit) TVTI-Hcit-VYEGERPLT- Hcit-DNHLLG (BiP 460-480 Hcit) RNELESYAYSL-Hcit- NQIGD-Hcit- (BiP 562-579 Hcit) Hcit-Hcit-ELEEIVQPIIS- Hcit-LYGSAG (BiP 620-639 Hcit) PLRPQNYLFGCEL-Hcit-AD-Hcit- (NPM 11-27 Hcit) EGSPIKVTLATLKMSVQPTVSL (NPM 68-89 Hcit) EEEDV-Hcit-LLSISG-Hcit- RSAPGGGS (NPM 129-149 Hcit) S-Hcit-GQESF-Hcit-Hcit-QE- Hcit-TP-Hcit-TP-Hcit-G (NPM 222-240 Hcit) GGSLP-Hcit-VEA-Hcit- FINYV-Hcit-NCFR (NPM 258-277 Hcit) A-Hcit-FINYV-Hcit- NCFRMTDQEAIQDL (NPM 266-287 Hcit) MSIL-Hcit-IHAREIFDSRG (Alpha enolase 1-16 Hcit) ND-Hcit-TRYMG-Hcit-GVS-Hcit-AVEHI (Alpha enolase 52-69 Hcit) TEN-Hcit-S-Hcit-FGANAILGVSLAVC-Hcit-A (Alpha enolase 100-121 Hcit) GSHAGN-Hcit-LAMQEFMILPVGAA (Alpha enolase 156-176 Hcit) REAMRIGAEVYHNL-Hcit-NVI-Hcit- (Alpha enolase 179-197 Hcit) NVI-Hcit-E-Hcit-YG-Hcit-DATNVGDEGG (Alpha enolase 194-212 Hcit) DVAASEFFRSG-Hcit-YDLDF-Hcit-SP (Alpha enolase 245-264 Hcit) PDQLADLY-Hcit-SFI-Hcit-DYPWS (Alpha enolase 273-291 Hcit) WGAWQ-Hdt-FTASAGIQVVG (Alpha enolase 301-317 Hcit) N-Hcit-SCNCLLL-Hcit-YNQIGSVTE (Alpha enolase 333-352 Hcit) RSERLA-Hcit-YNQLLRIEEELGS (Alpha enolase 400-419 Hcit) GS-Hcit-A-Hcit-FAGRNFRNPLA-Hcit- (Alpha enolase 418-434 Hcit) EPSQML-Hcit-HAVVNLINYQD (Beta-Catenin 127-144 Hcit) E-Hcit-LLWTTSRVL-Hcit-VLSVCSSN-Hcit (Beta-Catenin 334-354 Hcit) TLHNLLLHQEGA-Hdt-MAVRL (Beta-Catenin 258-275 Hcit) A-Hcit-MAVRLAGGLQ-Hcit-MVALLN-Hcit (Beta-Catenin 269-288 Hcit) -Hcit-TNV-Hcit-FLAJTTDCLQILAYG (Beta-Catenin 288-307 Hcit) TYE-Hcit-LLWTTSRVL-Hcit-VLSV (Beta-Catenin 332-349 Hcit) TSRVX-Hcit-VTSVCSSN-Heit-PAIV (Beta-Catenin 340-358 Hcit) YGLPVVV-Hcit-LLHPPSHWPL (Beta-Catenin 489-506 Hcit) HWPLI-Heit-ATVGLIRM-ALCPA (Beta-Catenin 503-522 Hcit) IENIQRVAAGVLCELAQD-Hcit- (Beta-Catenin 607-625 Hcit) GvatyAAAVLFRMSED-Hcit-P (Beta-Catenin 650-667 Hcit) IDLKDKYKNIGAKLVQDVAN (HSP60 84-103 Hcit) TVLARSIA-Hcit-EGFE-Hcit-IS-Hcit-GAN (HSP60 117-136 Hcit) GEALSTLVLNRL-Hcit-VGLQVVA (HSP60 280-299 Hcit) TTSEYE-Hcit-E-Hcit-LNERLA-Hcit-LS (HSP60 381-398 Hcit) GIIDPTV-Hcit-VRTALLDAAGVA (HSP60 517-536 Hcit) wherein “Hcit” represents homocitrulline, or ii) one or more of the amino acid sequences 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-homocitrulline position.

    5. A complex of the antigen of claim 1 and an MHC molecule, optionally wherein the MHC molecule is MHC class II, optionally selected from HLA-DR4, DR1 and DP4, or MHC class I, optionally HLA-A2.

    6. A binding moiety that binds the polypeptide of claim 1.

    7. The binding moiety of claim 6, which binds the polypeptide when it is in complex with MHC.

    8. The binding moiety of claim 7, wherein the binding moiety is a T cell receptor (TCR) or an antibody.

    9. The binding moiety of claim 8, wherein the TCR is on the surface of a cell.

    10. The binding moiety of claim 8, wherein the TCR comprises an alpha chain variable domain and a beta chain variable domain with the following CDRs: TABLE-US-00021 TCR Beta chain Abha chain 9 CDR1: ENHRY CDR1: TISGTDY CDR2: SYGVKD CDR2: GLTSN CDR3: AISERRDQETQY CDR3: ILRDVYDYKLS 0 CDR1: DFQATT CDR1: VTNFRS CDR2: SNEGSKA CDR2: LTSSGIE CDR3: SAPIHTDTQY CDR3: AVHDAGNMLT 1 CDR1: SGHDY CDR1: SSVSVY CDR2: FNNNVP CDR2: YLSGSTLV CDR3: ASRGGLASNEQF CDR3: AVSEGGGSYIPT 2 CDR1: LNHDA CDR1: DSASNY CDR2: SQIVND CDR2: IRSNVGE CDR3: ASSLGTFYEQY CDR3: AASGNTNAGKST 3 CDR1: YFSETQ CDR1: TISGTDY CDR2: SGHRS CDR2: GLTSN CDR3: CDR3: ASSLGVMVVSTDTQY ILRDRVSNFGNEKLT 4 CDR1: SGHAT CDR1: VSGNPY CDR2: FQNNGV CDR2: YITGDNLV CDR3: ASSPTQGASYEQY CDR3: AVRDAGYSTLT 6 CDR1: ENHRY CDR1: DSASNY CDR2: SYGVKD CDR2: IRSNVGE CDR3: AISERRDQETQY CDR3: AASIDRDDKII 7 CDR1: MDHEN CDR1: VSGLRG CDR2: SYDVKM CDR2: LYSAGEE CDR3: ATTQGSYNEQF CDR3: AVQAGSYIPT 9 CDR1: DFQATT CDR1 SSVPPY CDR2: SNEGSKA CDR2: YTSAATLV CDR3: SARTSGTNTQY CDR3: AVSGRNDYKLS 0 CDR1: MNHEY CDR1: TISGTDY CDR2: SVGEGT CDR2: GLTSN CDR3: ASSRSWTASGYT CDR3: ILRDGSGNEKLT 1 CDR1: SGHNS CDR1: DSASNY CDR2: FNNNVP CDR2 IRSNVGE CDR3: ASSVAQLAGKGEQF CDR3 AASIDRDDKII 2 CDR1: SNHLY CDR1 DSAIYN CDR2: FYNNEI CDR2: IQSSQRE CDR3: ASRRVMGYGYT CDR3 ALNSGGSNYKLT 3 CDR1: DFQATT CDR1 TISGTDY CDR2: SNEGSKA CDR2 GLTSN CDR3: SAGRAGTSGTYEQY CDR3 ILRSNFGNEKLT 4 CDR1: LNHDA CDR1 NYSPAY CDR2: SQIVND CDR2 IRENEKE CDR3: ASSGGQFNQPQH CDR3 ALGOTGANNLF 5 CDR1: KGHSH CDR1 TISGNEY CDR2: LQKENI CDR2 GLKNN CDR3: ASSPEALANTGELF CDR3 IVRVGYNNNDMR 6 CDR1: KGHDR CDR1 TISGTDY CDR2: SFDVKD CDR2 GLTSN CDR3: ATSDPSGPPYEQY CDR3 ILRAQGGSEKLV 7 CDR1: SNHLY CDR1 IQSSORE CDR2: FYNNEI CDR2 DSAIYN CDR3: ASRAGTGIGGYT CDR3 AVYSGGSNYKLT CDR1: MDHEN CDR1 TSESDYY CDR2: SYDVKM CDR2: QEAYKQQN CDR3: ASSLLGSSPLH CDR3 AYRSYNOGGKLI CDR1: LGHDT CDR1 TISGTDY CDR2: YNNKEL CDR2 GLTSN CDR3: ASSQEPSTHNEQF CDR3: ILKNYGGSQGNLI CDR1: MNHNY CDR1 YSGSPE CDR2: SVGAGI CDR2 HISR CDR3: ASSPGQPYGYT CDR3: ALSGPSYGQNFV CDR1: MNHNS CDR1 ATGYPS CDR2: SASEGT CDR2 ATKADDK CDR3: ASEGLASYNEQF CDR3: ALTGGGYQKVT CDR1: MNHEY CDR1 TSINN CDR2: SMNVEV CDR2 IRSNERE CDR3: ASSFREGEKLF CDR3: ATAMNTGFQKLV CDR1: LGHNA CDR1 NSMFDY CDR2: YNFKEQ CDR2 ISISSIKDK CDR3: CDR3: ASSREGLAGLNEQF AASGWGDGGATNKLI

    11. (canceled)

    12. A method of treating or preventing cancer in a subject in need thereof comprising administering said subject the polypeptide of claim 1.

    13. A pharmaceutical composition comprising a polypeptide of claim 1 together with a pharmaceutically acceptable carrier.

    14. A method of identifying a binding moiety that binds a complex as claimed in claim 5, the method comprising contacting a candidate binding moiety with the complex and determining whether the candidate binding moiety binds the complex.

    15. A pharmaceutical composition comprising the complex of claim 5 together with a pharmaceutically acceptable carrier.

    Description

    EXAMPLES

    [0092] The present invention will now be described further with reference to the following examples and the accompanying drawings.

    [0093] FIG. 1: Chemical reaction conversion of lysine to Hcit (Jaisson, Pietrement, and Gillery 2011) Increased urea or breakdown of thiocyanate leads to accumulation of cyanate and increased carbamylation of lysine forming Hcit residues.

    [0094] FIG. 2: Sequence logo representation of the binding motifs for HLA-DR, HLA-A and HLA-B molecules a: Sequence logo representation of the binding motifs for 6 HLA-DR molecules using NNAlign. On the positive y-axis, the amino acids enriched at each peptide position and on the negative y-axis the corresponding depleted amino acids. MHC class II DR molecules have a binding motif with interactions at P1, P4, P6 and P9. The height of a column in the sequence logo indicates the importance of a certain position in defining the motif, and the height of each letter in the column the amino acid preference at that position (Andreatta et al. 2011). Sequence logos are calculated using the WebLogo program (Crooks et al. 2004).b: Sequence logo representation of the binding motifs for 5 HLA-A molecules using NetMHCpan. On the positive y-axis, the amino acids enriched at each peptide position and on the negative y-axis the corresponding depleted amino acids. MHC class I A molecules have a binding motif with interactions at P2 and P9. The height of a column in the sequence logo indicates the importance of a certain position in defining the motif, and the height of each letter in the column the amino acid preference at that position (Andreatta et al. 2011). c: Sequence logo representation of the binding motifs for 4 HLA-B molecules using NetMHCpan. On the positive y-axis, the amino acids enriched at each peptide position and on the negative y-axis the corresponding depleted amino acids. MHC class I B molecules have a binding motif with interactions at P2 and P9. The height of a column in the sequence logo indicates the importance of a certain position in defining the motif, and the height of each letter in the column the amino acid preference at that position (Andreatta et al. 2011).

    [0095] FIG. 3: Sequences of human aldolase, vimentin, cytokeratin 8, BiP, NPM, alpha-Enolase, pcatenin and HSP60

    [0096] a: Amino acid sequence of human Aldolase

    [0097] Aldolase (also known as Fructose-bisphosphate ALDOA) is a glycolytic enzyme. Vertebrates encode three forms of this enzyme; ALDOA A encoded by ALDOA is expressed predominantly in muscle, ALDOA B (ALDOB) in liver and ALDOA C (ALDOC) in the brain. The sequences alignment was performed for ALDOA, ALDOB and ALDOC.

    [0098] b: Amino acid sequence of human ALDOA A

    [0099] c: Amino acid sequence of human vimentin

    [0100] d: Amino acid sequence of human Cytokeratin 8

    [0101] e: Amino acid sequence of human BiP

    [0102] f: Amino acid sequence of human NPM

    [0103] g: Amino acid sequence of human alpha-enolase

    [0104] h: Amino acid sequence of human β-catenin

    [0105] i: Amino acid sequence of human HSP60

    [0106] FIG. 4: Screening of IFNγ ELISpot responses generated by peptides containing homocitrulline residues

    [0107] Transgenic HLA-DR4, HLA-HHDII/DR1 or HLA-HHDII/DP4 mice were screened for responses to peptides containing homocitrulline residues. All mice received 3 doses of peptides with CpG/MPLA as an adjuvant. IFNγ responses were then assessed by ex vivo ELISpot. Responses to vimentin peptides containing Hcit were seen in HLA-DR4 (a) and HLA-HHDII/DR1 (c) mice. Responses to ALDOA peptides containing Hcit were seen in HLA-HHDII/DP4 (b) and HLA-HHDII/DR1 (d) mice. Statistical analysis was performed, p values are represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    [0108] FIG. 5: IFNγ responses to vimentin are strain-specific

    [0109] Ex vivo IFNγ ELISpot responses to vimentin 116-134 Hcit peptide were also assessed in HLA-HHDII/DP4 and C57131/6 mice (a). Responses to vimentin 116-134wt peptide were assessed in HLA-HHDII/DR1 and HLA-DR4 mice (b). In addition, IL-10 (c) and IL-17 (d) responses were in response to vimentin 116-135 Hcit peptides were determined. For all studies mice were immunised with three doses of peptide with CpG/MPLA as an adjuvant and responses were assessed at day 21. None of the responses were statistically significant.

    [0110] FIG. 6: IFNγ responses to ALDOA are strain-specific

    [0111] Ex vivo IFNγ ELISpot responses to ALDOA 74-93 Hcit and ALDOA 140-157 Hcit were assessed in HLA-DR4 mice (a). Ex vivo ELISpot was also used to assess IL-10 (b) and IL-17 (c) responses to ALDOA peptides in HLA-HHDII/DP4 mice. For all studies mice were immunised with three doses of peptide with CpG/MPLA as an adjuvant and responses were assessed at day 21. Significant p values are shown for peptide compared to media only control stimulation.

    [0112] FIG. 7: Characterisation of Hcit-specific IFNγ responses in mice

    [0113] IFNγ ELISpot responses in splenocytes from mice immunised with vimentin 116-135 Hcit peptide were assessed in transgenic HLA-DR4 (a) and HLA-HHDII/DR1 (b) mice. Responses in mice immunised with ALDOA 74-93 Hcit and ALDOA 140-157 Hcit peptides were assessed in transgenic HLA-HHDII/DP4 (c) and HLA-HHDII/DR1 (d) mice. The wt peptides were included as controls. Mice were given three immunisations of peptides with CpG/MPLA and spleens were harvested on day 21. Splenocytes were restimulated with peptides alone or with peptides in combination with anti-CD4 or anti-CD8 blocking antibodies. Significant p values are shown for Hcit peptides compared to wt peptides and to peptide plus blocking antibodies.

    [0114] FIG. 8: Sequence alignment T cell epitopes containing homocitrulline for different species Alignment of Hcit T cell epitopes from human vimentin (a), ALDOA (b), enolase (c), Bip (d), β-catenin (e), Cyk8 (f) and NPM (g) and HSP60 (h) subunit with equivalent sequences from other species (Mouse, Rat, Cow (Bovine), Pig, Horse, Cat, Dog, Rabbit and Sheep) depicting homology.

    [0115] FIG. 9: In vitro carbamylation of vimentin and ALDOA

    [0116] B16F1 cells or recombinant vimentin or recombinant ALDOA A protein were carbamylated by incubation in the presence of potassium cyanate (KCNO). Carbamylation was then assessed using carbamylation ELISA (a). P values are shown compared untreated to treated samples. Error bars show standard deviation. Carbamylated recombinant proteins were assessed by mass spectrometry on a SCIEX 6600 TripleTof mass spectrometer via a Duospray (TurboV) source with a 50 um electrode and lysine residues from ALDOA (b) or vimentin (c) were assessed for the carbamylation modification.

    [0117] FIG. 10: Screening of IFN y ELISpot responses to cytokeratin 8 (Cyk8) peptides

    [0118] Transgenic HLA-HHDII/DP4 mice were screened for responses to pools of Hcit Cyk8 peptides containing homocitrulline residues. All mice received 3 doses of peptides with CpG/MPLA as an adjuvant. IFN γ responses were then assessed by ex vivo ELISpot (a). Responses to Cyk8 101Hcit, 112Hcit, 371 Hcit and 381Hcit were assessed for wild type cross reactivity after 3 immunisations (b). Statistical analysis was preformed, p values are represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

    [0119] FIG. 11: Characterisation of Cytokeratin 8 (Cyk8) Hcit peptide responses in HHDII/DP4 mice

    [0120] HHDII/DP4 mice were immunised with individual Hcit peptides. Ex vivo ELISpot were then performed on day 21. Splenocytes were restimulated with media, hcit peptide, wild type (wt) peptides, or Hcit peptides in the presence of CD4/CD8 blocking antibodies (a-b, d-e). Immunisation with the shorter Cy8 117Hcit peptide also induced an immune response (f).

    [0121] FIG. 12: Characterisation of Cytokeratin 8 (Cyk8) Hcit peptide responses in HHDII/DR1 mice

    [0122] HHDII/DR1 mice were immunised with pooled peptides ex vivo ELISpot were then performed on day 21 to screen for responses (a). Mice were then immunised with individual Hcit peptides (b-d) or wt peptides (e). In ELISpots splenocytes were restimulated with media, hcit peptide, wild type (wt) peptides, or Hcit peptides in the presence of CD4/CD8 blocking antibodies.

    [0123] FIG. 13: Screening of IFNγ ELISpot responses generated by peptides containing homocitrulline residues in HLA-HHDII/DP4 mice

    [0124] Transgenic HLA-HHDII/DP4 mice were screened for responses to peptides containing homocitrulline residues that were selected based spiral shape and predicted HLA-DP4 binding. All mice received 3 doses of peptides with CpG/MPLA as an adjuvant. IFNγ responses were then assessed by ex vivo ELISpot. Responses to Bip (a), Enolase (b), NPM (c), Vimentin (d) and aldolase (e and 0 peptides containing Hcit were seen. Responses to Hcit aldolase peptides (e and 0 were assessed for responses in the presence of CD4 or CD8 blocking antibodies and for cross reactivity to wildtype (wt) peptides.

    [0125] FIG. 14: Responses to Homoctirullinated peptides can be cultured

    [0126] Splenocytes from transgenic HLA-HHDII/DP4 mice immunised with Hcit peptides were then grown ex vivo in the presence of Hcit peptides. IFNγ responses were then reassessed by ELISpot. Responses to Bip (a), Enolase (b), NPM (c) and Vimentin (d) peptides containing Hcit were seen. Responses were also assessed in the presence of CD4 or CD8 blocking antibodies and for cross reactivity to wildtype (wt) peptides.

    [0127] FIG. 15: Screening of IFNγ ELISpot responses generated by peptides containing homocitrulline residues in HLA-HHDII/DR1 mice

    [0128] Transgenic HLA-HHDII/DR1 mice were screened for responses to aldolase peptides containing homocitrulline residues that demonstrated responses in HHDII/DP4 mice. All mice received 3 doses of peptides with CpG/MPLA as an adjuvant. IFNγ responses were then assessed by ex vivo ELISpot. Responses to Aldolase 204 Hcit (A) and the shorter Aldolase 209hcit (B) peptide were assessed.

    [0129] FIG. 16: Humans have a repertoire for Hcit peptides

    [0130] PBMCs were isolated from 14 healthy donors and 11 cancer patients and assessed for responses to Hcit peptides by CFSE proliferation assays. PBMCs were CD25-depleted and then CFSE-labelled and stimulated with 1M/ml of peptides. Proliferation was assessed on day 10. Example plots are shown for healthy donor BD0051 (A) and lung cancer patient LG10 (B). Summary graphs show that the majority of peptide-induced proliferation is found in the CD4+ cells for both healthy donors (C) and patients (D). CD4 proliferation in each donor was assessed in comparison to the media control for healthy donors (E) and cancer patients (F).

    [0131] FIG. 17: Analysis of cytokine expression performed on donors

    [0132] Example plots from patient LG10 showing staining for IFNγ, GraB and CD134 on the proliferating CFSE.sup.low CD4+ population after stimulation with peptides for 10 days (A). Summary of flow cytometry data showing the percentage of proliferating CD4+ cells which express GraB, CD134 and IFNγ among the healthy donor (B) and patient (C) responders who showed proliferation following peptide stimulation.

    [0133] FIG. 18: Humans have a repertoire for Cyk8 Hcit peptides

    [0134] PBMCs were isolated from 7 healthy donors and assessed for responses to Hcit peptides by CFSE proliferation assays. PBMCs were CD25-depleted and then CFSE-labelled and stimulated with 10 μg/ml of peptides. Proliferation was assessed on day 10.

    [0135] FIG. 19: Increase survival in mice immunised with Hcit peptides

    [0136] In vivo tumour survival studies were carried out by implanting mice with HLA matched transgenic B16F1 cells s.c. on the right flank on day 1. On day 4, 11 and 18 mice were immunised with peptide with CpG/MPLA. Tumour growth was assessed after immunisation with vimentin 116-135 Hcit in HLA-HHDII/DR1 (a) or HLA-DR4 mice (b). Tumour growth after immunisation with ALDOA peptides were assessed in HLA-HHDII/DP4 (c) and HLA-HHDII/DR1 (d) mice. Tumour growth was also assessed after immunisation with vimentin 116-135 Hcit in HLA-DR4 mice implanted with B16F1 with an IFNγ inducible DR4 cell line (e). Statistical analysis was performed comparing survival in immunised mice to survival in control mice, significant p values are shown.

    [0137] FIG. 20: Increase survival in mice immunised with Cyk8 Hcit peptides

    [0138] In vivo tumour survival studies were carried out by implanting mice with HLA matched transgenic B16F1 cells s.c. on the right flank on day 1. On day 4, 11 and 18 mice were immunised with peptide with CpG/MPLA. Tumour growth was assessed after immunisation with Cyk8 Hcit peptides in HLA-HLA-HHDII/DR1 mice.

    [0139] FIG. 21: Increased survival in mice immunised with Hcit peptides in IFNγ inducible HLA tumour models

    [0140] In vivo tumour survival studies were carried out by implanting HLA-DP4 transgenic mice with HLA matched transgenic B16F1 cells s.c. on the right flank on day 1 where HLA is under the control of IFNγ inducible promoter. On day 4, 11 and 18 mice were immunised with peptide with CpG/MPLA. Tumour growth was assessed after immunisation with Cyk8 371Hcit peptide (a) or Bip 562Hcit peptide (b), Vimentin86Hcit, NPM 266Hcit or Enolase 156Hcit in HLA-HHDII/DP4.

    [0141] FIG. 22: MPO is produced by MDSCs in the anti-tumour environment

    [0142] Flow cytometry was used to assess MPO expression on B16F1 cells (a) or CD45+ tumour-infiltrating cells (b). Spleen, blood and tumour cells were stained to determine expression of MPO on CD45+ cells (c). The proportion of MPO+ cells which express G-MDSC (Ly6G+Ly6C.sup.low) or M-MDSC (Ly6G-Ly6C.sup.high) markers was assessed in the spleen and tumour (d). Tumour infiltrating MPO+ cells from the Ly6G-Ly6C.sup.high population were assessed for CD115 and F4/80 expression (d).

    [0143] FIG. 23: The role of cell populations in the anti-tumour effect seen in response to Hcit ALDOA peptides

    [0144] Anti-Ly6G (a) or Anti-Ly6C (b) antibodies were injected in to mice during anti-tumour studies to determine the role of these cell populations in the anti-tumour effect seen in response to Hcit ALDOA peptides. P values shown compare survival in the groups given the combination of peptide and antibody to groups given peptide alone or antibody alone. The percentage of MPO+ cells in TILs from control mice or mice given anti-Ly6C Ab and ALDOA immunisations are shown (c).

    [0145] FIG. 24: In vitro carbamylation: In vitro grown bone marrow derived MDSCs (BM-MDSCs) produce MPO and leading to carbamylation of B16 cells

    [0146] BM-MDSCs were derived from mice. Staining was performed to identify MPO+ cells in both the M-MDSCs and G-MDSC populations both unstimulated and after LPS stimulation (a). Co-culture of B16 cells with MDSCs was performed in the presence of potassium thiocyanate and H.sub.20.sub.2. The level of Carbamyation was then assessed by staining with an anti-carbamylysine antibody (b).

    [0147] FIG. 25: Tumour survival is dependent on expression of MHCII

    [0148] In vivo tumour survival studies were also carried out using B16F1 cell lines which lack appropriate MHCII expression. Transgenic B16F1 cells were implanted s.c. on the right flank on day 1. On day 4, 11 and 18 mice were immunised with peptide with CpG/MPLA. For vimentin 116-135 Hcit, anti-tumour studies were performed using B16F1 lacking DR4 (a) and with B16F1 HHDII lacking DR1 (b) in the appropriate mouse strains. For ALDOA 74-93Hcit and ALDOA 140-157 Hcit survival was determined after implant with B16F1 HHDII lacking DP4 expression (c). Statistical analysis was performed comparing survival in immunised mice to survival in control mice, significant p values are shown.

    [0149] FIG. 26: Tumour survival is dependent on CD4 responses

    [0150] In vivo tumour survival studies were also carried out using B16F1 HHDII/iDP4 cell lines in the presence of CD4 or CD8 depletion antibodies. Transgenic B16F1 HHDII/DP4 cells were implanted s.c. on the right flank on day 1. On day 4, 8 and 11 mice were immunised with peptide with CpG/MPLA and given i.p. injections of depletion antibodies. Statistical analysis was performed comparing survival in immunised mice to survival in immunised mice also given antibodies, significant p values are shown.

    [0151] FIG. 27: Tree map is another illustrative approach to show diversity to ALDOA 74-93hcit.

    [0152] Tree map of the CD4 sorted CFSE high (A) and the CFSE low (B) TRA chain in response to ALDOA 74-93hcit. Tree map of the CD4 sorted CFSE high (C) and the CFSE low (D) TRB chain in response to ALDOA 74-93hcit. Each rounded rectangle represents a unique entry: V-J-uCDR3, where the size of the spot denotes the relative frequency.

    [0153] FIG. 28: TCR9 a) Sequence 3 hTRBV10-3-CDR3(AISERRDQETQY), b) Sequence 4 hTRAV26-2-CDR3 (ILRDVYDYKLS). CDRs are shown in bold.

    [0154] FIG. 29: TCR10 a) Sequence 15 hTRBV20-1-CDR3 (SAPIHTDTQY) b) Sequence 16 hTRAV36/DV7-CDR3 (AVHDAGNMLT). CDRs are shown in bold.

    [0155] FIG. 30: TCR11 a) Sequence 17 hTRBV12-4-CDR3 (ASRGGLASNEQF), b) Sequence 18 hTRAV8-6-CDR3 (AVSEGGGSYIPT). CDRs are shown in bold.

    [0156] FIG. 31: TCR12 a) Sequence 19 hTRBV19-CDR3 (ASSLGTFYEQY) b) Sequence 20 hTRAV13-1-CDR3 (AASGNTNAGKST). CDRs are shown in bold.

    [0157] FIG. 32: TCR13 a) Sequence 21 hTRBV5-1-CDR3 (ASSLGVMVVSTDTQY) b) Sequence 22 hTRAV26-2-CDR3 (ILRDRVSNFGNEKLT). CDRs are shown in bold.

    [0158] FIG. 33: TCR14 a) Sequence 23 hTRBV11-2-CDR3 (ASSPTQGASYEQY), b)Sequence 24 hTRAV3-CDR3 (AVRDAGYSTLT). CDRs are shown in bold.

    [0159] FIG. 34: TCR16 Sequence 3 hTRBV10-3-CDR3 (AISERRDQETQY, FIG. 17a) a) Sequence 25 hTRAV13-1-CDR3 (AASIDRDDKII). CDRs are shown in bold.

    [0160] FIG. 35: TCR17 a) Sequence 26 hTRBV28-CDR3 (ATTQGSYNEQF) b) Sequence 27 hTRAV20-CDR3 (AVQAGSYIPT). CDRs are shown in bold.

    [0161] FIG. 36: Tree map is another illustrative approach to show diversity to ALDOA 140-157hcit.

    [0162] Tree map of the CD4 sorted CFSE high (A) and the CFSE low (B) TRA chain in response to ALDOA 74-93hcit. Tree map of the CD4 sorted CFSE high (C) and the CFSE low (D) TRB chain in response to ALDOA 140-157hcit. Each rounded rectangle represents a unique entry: V-J-uCDR3, where the size of the spot denotes the relative frequency.

    [0163] FIG. 37: TCR19 a) Sequence 28 hTRBV20-1-CDR3 (SARTSGTNTQY), b) Sequence 29 hTRAV8-4-CDR3 (AVSGRNDYKLS). CDRs are shown in bold.

    [0164] FIG. 38: TCR20 a) Sequence 30 hTRBV6-3-CDR3 (ASSRSWTASGYT), b) Sequence 31 hTRAV26-2-CDR3 (ILRDGSGNEKLT)). CDRs are shown in bold.

    [0165] FIG. 39: TCR21 a) Sequence 32 hTRBV12-3-CDR3 (ASSVAQLAGKGEQF), Sequence 25 hTRAV13-1-CDR3 (AASIDRDDKII (FIG. 23a). CDRs are shown in bold.

    [0166] FIG. 40: TCR22 a) Sequence 33 hTRBV2-CDR3 (ASRRVMGYGYT), b) Sequence 34 hTRAV21-CDR3 (ALNSGGSNYKLT). CDRs are shown in bold.

    [0167] FIG. 41: TCR23 a) Sequence 35 hTRBV20-1-CDR3 (SAGRAGTSGTYEQY), b) Sequence 36 hTRAV26-2-CDR3 (ILRSNFGNEKLT). CDRs are shown in bold.

    [0168] FIG. 42: TCR24 a) Sequence 37 hTRBV19-CDR3 (ASSGGQFNQPQH), b) Sequence 38 hTRAV6-CDR3 (ALGQTGANNLF). CDRs are shown in bold.

    [0169] FIG. 43: TCR25 a) Sequence 39 hTRBV18-CDR3 (ASSPEALANTGELF), b) Sequence 40 hTRAV26-1-CDR3 (IVRVGYNNNDMR). CDRs are shown in bold.

    [0170] FIG. 44: TCR26 a) Sequence 41 hTRBV24-1-CDR3 (ATSDPSGPPYEQY), b) Sequence 42 hTRAV26-2-CDR3 (ILRAQGGSEKLV). CDRs are shown in bold.

    [0171] FIG. 45: TCR27 a) Sequence 43 hTRBV2-CDR3 (ASRAGTGIGGYT)), b) Sequence 44 hTRAV21-CDR3 (AVYSGGSNYKLT). CDRs are shown in bold.

    [0172] FIG. 46: Tree map is another illustrative approach to show diversity vimentin 116-135hcit.

    [0173] Tree map of the CD4 sorted CFSE high (A) and the CFSE low (B) TRA chain in response to vimentin 116-135hcit. Tree map of the CD4 sorted CFSE high (C) and the CFSE low (D) TRB chain in response to vimentin 116-135hcit. Each rounded rectangle represents a unique entry: V-J-uCDR3, where the size of the spot denotes the relative frequency.

    [0174] FIG. 47: TCR1 a) Sequence 1 hTRBV28-CDR3(ASSLLGSSPLH), b) Sequence 2 hTRAV38-2/DV8-CDR3 (AYRSYNQGGKLI). CDRs are shown in bold.

    [0175] FIG. 48: TCR4 a) Sequence 5 hTRBV3-1-CDR3 (ASSQEPSTHNEQF), b) Sequence 6 hTRAV26-2-CDR3 (ILKNYGGSQGNLI). CDRs are shown in bold.

    [0176] FIG. 49: TCR5 a) Sequence 7 hTRBV6-6-CDR3 (ASSPGQPYGYT), b) Sequence 8 hTRAV16-CDR3 (ALSGPSYGQNFV). CDRs are shown in bold.

    [0177] FIG. 50: TCR6 a) Sequence 9 hTRBV6-1-CDR3 (ASEGLASYNEQF), b) Sequence 10 hTRAV9-2-CDR3 (ALTGGGYQKVT). CDRs are shown in bold.

    [0178] FIG. 51: TCR7 a) Sequence 11 hTRBV27-CDR3 (ASSFREGEKLF), b) Sequence 12 hTRAV17-CDR3 (ATAMNTGFQKLV). CDRs are shown in bold.

    [0179] FIG. 52: TCR8 a) Sequence 13 hTRBV4-2-CDR3 (ASSREGLAGLNEQF), b) Sequence 14 hTRAV29/DV5-CDR3 (AASGWGDGGATNKLI). CDRs are shown in bold.

    [0180] FIG. 53: HLA-DP4 binding a) binding of HepB 181-193 but not negative control peptides to HLA-DP4, b) competition of biotinylated HepB peptide binding with unlabelled HepB peptide, c) competition of homocitrulline containing and wild type aldolase peptides with biotinylated HepB peptide for binding to HLA-DP4, d) competition of homocitrulline containing cytokeratin 8 peptides with biotinylated HepB peptide for binding to HLA-DP4, e) competition of homocitrulline containing and wild type vimentin peptide with biotinylated HepB peptide for binding to HLA-DP4.

    METHODS

    [0181] Cell Lines and Culture

    [0182] The murine melanoma B16F1 cell line (ATCC-CRL-6323) was obtained from the American Tissue Culture Collection (ATCC). B16F1 was cultured in RPMI medium 1640 (GIBCO/BRL) supplemented with 10% fetal calf serum (FCS), L-glutamine (2mM) and sodium bicarbonate buffered to pH7. The cell line utilised were mycoplasma free, authenticated by suppliers (STR profiling), and are used within ten passages.

    [0183] Plasmids and Transfections

    [0184] Cell lines were transfected using the Lipofectamine Transfection Reagent (Invitrogen) utilising the protocol previously described (Brentville et al. 2016). B16F1 cells were knocked out for murine MHC-I and/or MHC-II using ZFN technology (Sigma) and transfected with constitutive HLA-DR4, HLA-DR1 or HLA-DP4 using the the pVitro 2 chimeric plasmid. Cells were also transfected with the HHDII plasmid comprising of a human HLA-A2 leader sequence, the human β2-microglobulin (β2M) molecule covalently linked via a glycine serine linker to the α1 and 2 domains of human HLA-0201 MHC class 1 molecule and the α3, transmembrane and cytoplasmic domains of the murine H-2Db class 1 molecule, where relevant as previously described (Xue et al. 2016). B16F1 cells were also transfected with the IFNγ-inducible HLA-DR4 or HLA-DP4 using the pDC GAS chimeric HLA-DR401 or HLA-DP4 plasmids where chimeric HLA-DR401 or HLA-DP4 are under expression of the IFNγ-inducible promoter. Plasmid details and transfection protocol have previously been described in full (Brentville et al. 2016; Brentville et al. 2019). Transfected cells were grown in the presence of zeocin (300 μg/ml), hygromycin B (300 μg/ml) or G418 (500 μg/ml).

    [0185] In Vitro Carbamylation and Detection

    [0186] Carbamylation of proteins was performed following the protocol previously described (Shi et al. 2011). Briefly, foetal calf serum, recombinant human ALDOA (Sigma) or vimentin (Abcam) proteins or B16F1 cell lysate prepared by 4 cycles of freeze/thaw were in vitro carbamylated by incubating in the presences of 1M KCNO at 37° C. for 10 hrs. For B16F1 cell lines, cells were allowed to adhere to flasks and media was then supplemented with KCNO to a final concentration of 1M overnight. After treatment cells were removed from flask using a cell scraper and lysed by freeze/thaw. All samples were then extensively dialysed in dH.sub.2O. OXIselect carbamylation ELISA (Cell Biolabs) was used to detect carbamylation following the manufacturer's instructions.

    [0187] Mass Spectrometry

    [0188] Samples were prepared by trypsin digest at a ratio of 1:50 trypsin to protein overnight at 37° C. Samples were then dried under vacuum and resuspended in 0.1% formic acid/5% acetonitrile in LCMS grade water before MS analysis. For MS Analysis, samples were injected via autosampler (Eksigent Ekspert nanoLC 425 LC system utilising a 1-10 μl/min pump module running at 5 μl/min) with a 2 min wash trap/elute configuration onto a YMC Triad C18 column (300 um i.d., 3 μm particle size, 15 cm) in a column oven at 35° C. Samples were gradient eluted over an 87 min runtime into a SCIEX 6600 TripleTof mass spectrometer via a Duospray (TurboV) source with a 50 μm electrode. The 6600 was set up in IDA mode (Independent Data Acquisition/Data Dependent Acquisition) for 30 ions per cycle fragmentation. Total cycle time 1.8 s, TOFMS scan 250 ms accumulation; 50 ms for each product ion scan.

    [0189] Data was analysed using PEAKS Studio 8.0 (Bioinformatic Solutions Inc. Waterloo, Canada) searching the SwissProt human (Uniprot manually annotated/curated) database, 25 ppm parent mass error tolerance, 0.1 Da fragment mass error tolerance searching for modifications for citrullination (R), deamidation (NQR), oxidation (M). Sites were identified as a confident modification site with a minimum ion intensity of 5%.

    [0190] Immunogens

    [0191] Peptides of >90% purity were synthesized by Genscript (New Jersey, USA) and stored lyophilised in 0.2 mg to 0.4 mg aliquots at −80° C. On the day of use they were reconstituted to the appropriate concentration with phosphate buffered saline (PBS).

    [0192] Immunisation Protocol

    [0193] C57BL/6 mice (Charles River, UK), HLA-DR4 mice (Model #4149, Taconic, USA), HLA-A2/DR1 (HHDII/DR1, Pasteur Institute), HLA-A2.1+/+ HLA-DP4+/+ hCD4+/+ (HLA-HHDII/DP4) transgenic mice (EM:02221, European Mouse Mutant Archive) described in patent WO2013/017545 Al (EMMA repository, France) were used, aged between 8 and 12 weeks. Peptides were dissolved in PBS to 1 mg/ml and then emulsified (a series of dilutions) with CpG ODN 1826 and MPLA 6 μg/mouse of each (Invivogen, UK). Peptides (25 μg/mouse) were injected subcutaneously at the base of the tail. Mice were immunised at days 1, 7 and 14 for peptide immunisation. Spleens were removed for analysis at day 21 unless stated otherwise.

    [0194] For tumour challenge experiments, mice were challenged with B16F1 cells s.c. on the right flank 3 days before primary immunisation (unless stated otherwise) and subsequently immunised as above. Tumour implants were carried out at dose of 2.5×10.sup.4 cells/mouse for B16F1, 2.5×10.sup.4 cells/mouse for B16F1 DR4, 4×10.sup.5 cells/mouse for B16F1 HHDII/DP4, 5×10.sup.5 cells/mouse for B16 HHDII/DR1, 1×10.sup.5 cells/mouse for B16F1 HHDII/inducibleDP4, 5×10.sup.4 cells/mouse for B16F1 inducible DR4. For studies involving depletion antibodies, 250 μg anti-mouse Ly6C antibody (clone Monts, BioXcell) was administered i.p. on days 8, 10, 12, 15, 17 and 19. 400 μg anti-mouse Ly6G antibody (clone 1A8, Biolegend) was administered i.p. on day 8 followed by 250 μg on days 11, 15 and 18. Anti-human CD4 antibody (clone OKT4, BioXcell) or anti-mouse CD8 antibody (clone 2.43, BioXcell) were administered at 500 μg dose on day 4 followed by 300 μg dose on days 8 and 11. Tumour growth was monitored twice weekly and mice were humanely euthanised once tumour reached mm in diameter.

    [0195] Analysis of Immune Response—Ex Vivo ELISpot Assay

    [0196] 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 specific antibodies were coated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at various concentrations) and 5×10.sup.5 per well splenocytes were added to wells of the plate in quadruplicate 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 IFNγ, IL-10 and IL-17 were detected by biotinylated anti-IFNγ, IL-10 and IL-17 antibodies and developed with a streptavidin alkaline phosphatase and chromogenic substrate. Lipopolysaccharide (LPS; 5 μg/ml) was used as a positive control. For MHC blocking studies, anti-CD4 blocking antibody (GK1.5) or anti-CD4 blocking antibody (RPA-T4) for HHDII/DP4 mice which express human CD4 and anti-CD8 blocking antibody (2.43) from Bioxcell were used at 20 μg/ml. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd).

    [0197] Isolation and Analysis of Animal Tissue

    [0198] Spleens were disaggregated and treated with red cell lysis buffer for 2 mins. Tumours were harvested and mechanically disaggregated. Cells were then stained with 1:50 dilution of anti-CD45 (efluor 450, clone 30-F11), anti-CD11b (PE-Cy7, clone M1/70), anti-Ly6C (APC, Clone HK1.4) and anti-Ly6G (FITC, Clone RB6-8C5) (Thermofisher). Cells were washed, fixed and permeabilized using intracellular fixation/permeablization buffers (ThermoFisher) according to the manufactures instructions. Intracellular staining for cytokines was performed using a 1:10 dilution of anti-MPO (PE, clone HM105, Hycult Biotech). Stained samples were analysed immediately on a MACSQuant 10 flow cytometer equipped with MACSQuant software version 2.8.168.16380.

    [0199] In Vitro Production of MDSCs and Co-Culture with B16

    [0200] Bone marrow derived MDSCs were produced by isolating bone marrow from mice and then culturing in the presence of GM-CSF (1 ng/ml) and IL-18 (50 ng/ml) for 6 days. For co-culture experiments B16 cells were seeded in a monolayer and then incubated with MDSCs at a ratio of 1:10. Potassium thiocyanate (200 uM) and H2O2 (10 uM) were added when stated. Cells were then incubated overnight before staining. CarbLy staining was perfromed using Rabbit Anti-Carbamylation (Homocitrulline) Polyclonal Antibody (#CAY22428-1ea#) at 1/50 for 1 hours followed by donkey anti-rabbit-A647 conjugated secondary (#ab150063) used 1/1000 for 1 hour.

    [0201] Peripheral Blood Mononuclear Cell (PBMC) Isolation

    [0202] Demographics of healthy donors and patients are given in Table 5a and b. Peripheral blood samples were drawn into lithium heparin tubes (Becton Dickinson) and processed immediately following venepuncture. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque. Proliferation and cultured ELISpot assay of PBMCs were performed immediately after isolation. For CD25 depletion PBMCs were processed as above and enriched using anti-CD25 microbeads and MACS Cell Separation Columns (Miltenyi).

    [0203] Proliferation Assay-Carboxyfluorescein Succinimidyl Ester (CFSE)

    [0204] Briefly, a 50 μM stock solution in warm PBS was prepared from a master solution of 5 mM in dimethyl sulfoxide (DMSO). CFSE was rapidly added to PBMCs (5×10.sup.6 cells/ml loading buffer (PBS with 5% v/v heat inactivated FCS)) to achieve a final concentration of 5 μM. PBMCs were incubated at room temperature in the dark for 5mins after which non-cellular incorporated CFSE was removed by washing twice with excess ('10 v/v volumes) of loading buffer (300 g×10 mins). Cells were made up in complete media to 1.5×10.sup.6/mL and plated and stimulated with vehicle (negative control), PHA (positive control, final concentration 10 μg/ml) or peptide (10 μg/ml) as described above.

    [0205] On day 10, 500 μl of cells were removed from culture, washed in PBS and stained with 1:50 dilution of anti-CD4 (PE-Cy5, clone RPA-T4, ThermoFisher), anti-CD8 efluor 450, clone RPA-T8, ThermoFisher) and anti-CD134 (PE-Cy7, Clone REA621, Miltenyi). Cells were washed, fixed and permeabilised using intracellular fixation/permeablisation buffers (ThermoFisher) according to the manufactures instructions. Intracellular staining for cytokines was performed using a 1:50 dilution of anti-IFNγ (clone 4S.B3, ThermoFisher) or anti-Granzyme B (PE, Clone GB11, Thermofisher). Stained samples were analysed immediately on a MACSQuant 10 flow cytometer equipped with MACSQuant software version 2.8.168.16380.

    [0206] FACS Cell Sorting

    [0207] On day 10, the contents of the culture wells were mixed gently, pooled (according to peptide stimulation) and washed in PBS (300 g×10 mins). Pellets were gently re-suspended in 500 μL of PBS containing 10 μl of anti CD4 eFluo450 (clone RPA-T4, ThermoFisher, cat no 48-0049-42) and 10 μL of anti-CD8 APC (clone RPA-T8, ThermoFisher, cat 17-0088-41). Cells were stained at 4° C. for 30 mins before being washed (5 min×300 g) in 1.0 ml of PBS and resuspended in 300 μl of FACS sorting buffer (PBS supplemented with 1 mM EDTA, 25 mM HEPES and 1%v/v HI FCS). 10 μl of sample was removed from each stained sample and 90 μl of FACS sorting buffer added. 10,000 events were collected on a MACSQuant Analyser 10 flow cytometer to determine proliferation. The remaining cells were used for bulk FACS sorting.

    [0208] Cells were sorted using sterile conditions in a MoFlo XDP High Speed Cell Sorter machine. All samples were sorted into 1.0 ml of RNA protect (5 parts Protect, Qiagen:1 part FACS sorting buffer, Sigma) separating the CD4+ve/CFSEhigh and CD4+ve/CFSElow populations. Sorted cells (bulk) were stored at −80° C.

    [0209] Determination of the α and β chain pairing of TCRs recognising peptides containing homocitrulline. Sorted cells (bulk) from CD4.sup.+ve/CFSE.sup.high and CD4.sup.+ve/CFSE.sup.low populations in RNA protect were shipped to iRepertoire Inc (Huntsville, Ala., USA) for NGS sequencing of the TCRA and TCRB chain to confirm expansion of TCR's in the CD4.sup.+ve/CFSE.sup.low cells, proliferating to the peptide in contrast to the non proliferating CD4.sup.+ve/CFSE.sup.high population. In brief RNA was purified from sorted cells, RT-PCR was performed, cDNA was then subjected to Amplicon rescued multiplex PCR (ARM-PCR) using human TCR α and β 250 PER primers (iRepertoire Inc., Huntsville, Ala., USA). Information about the primers can be found in the United States Patent and Trademark Office (U.S. Pat. Nos. 7,999,092 and 9,012,148B2). After assessment of PCR/DNA samples, 10 sample libraries were pooled and sequenced using the Illumina MiSeq platform (Illumina, San Diego, Calif., USA). The raw data was analysed using IRweb software (iRepertoire). V, D, and J gene usage and and CDR3 sequences were identified and assigned and tree maps generated using iRweb tools. Tree maps show each unique CDR3 as a coloured rectangle, the size of each rectangle corresponds to each CDR3 abundance within the repertoire and the positioning is determined by the V region usage.

    [0210] To elucidate the cognate pairing and sequencing of TCRα and TCRβ chains IRepertoire used their iPair™ technology, the CD4.sup.+ve/CFSE.sup.low populations of cells (bulk sorted, that were simultaneously bulk sequenced) were seeded at 1 cell/well into a iCapture 96 well plate. RT-PCR is performed and the PCRα and β chains where amplified from the single cells using Amplicon rescued multiplex PCR (arm-PCR). Data was analysed utilising the iPair™ Software program for frequency of specific chain pairing and the sequences ranked on comparison to bulk data.

    [0211] DP4 Preparation for Binding Assay

    [0212] 2× T175 gave 2.5×10.sup.7 B16 HHDII/DP4 (B16F1 B2M H2Ab1 dKO A35 HHDII/DP4 H7/2E9/F9 p14) cells. This was enough for 5 preps using the following protocol with Mem-PER™ Plus Membrane Protein Extraction Kit (thermo-scientific cat #89842).

    [0213] 5×10.sup.6 cells were resuspended in the growth media by scraping the cells off the surface of the plate with a cell scraper and then centrifuged at 300 g for 5 mins. The cell pellet was washed with 3 ml of Cell Wash Solution and centrifuged at 300 g for 5 mins. The supernatant was removed and discarded. The cells were resuspended in 1.5 ml of Cell Wash Solution and transfer to a 2 ml centrifuge tube and centrifuged at 300 g for 5 mins. The supernatant was discarded. 0.75 ml of permeabilization buffer was added to the cell pellet and vortexed briefly to obtain a homogeneous cell suspension and incubated for 10 mins at 4° C. with constant mixing. The permeabilized cells were centrifuge for 15 mins at 16,000 g before carefully removing the supernatant containing cytosolic proteins and transfering to a new tube. 0.5 ml of solubilization buffer was added to the pellet and resuspend prior to incubating at 4° C. for 30 minutes with constant mixing prior to centrifugation at 16,000 g for 15 mins at 4° C. The supernatant containing solubilized membrane and membrane-associated proteins was transfered to a new tube. Aliquots were frozen at −80° C. for future use. 12.5 μl Hait™ Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100×) thermo-scientific (Catalog number 78445) was added prior to freezing.

    [0214] DP4 Binding ELISA.

    [0215] High binding plates were coated with streptavidin (Sigma S4762 at 1 mg/ml) 1/500 in PBS and incubated overnight at 4° C. The plates were blocked with 1% BSA in PBS for 4 hrs at room temp and washed 3× PBS/0.5% Tween. 450 μl of cell prep lysate was incubated with 50 μg biotinylated peptide and incubated for 4 hrs at 37° C.: Lysate/peptide mix was added to the plates at 100 μl/well and incubated for 4 hrs at room temperature. Plates were washed 3× with PBS/0.5% Tween. 100 μl per well of Leinco anti-Human HLA-DP4 clone B7/21 #H260 1 mg/ml at 1/500 dilution in 1% BSA/PBS was added to the lysate and incubated for 1 hr at room temperature before washing 3× PBS/0.5% Tween. Goat anti-mouse IgG3-HRP at 0.5 mg/ml 1/500 100 μl per well (Invitrogen #M32607) diluted in 1% BSA/PBS) was added for 1 hr. The plates were washed and 150 μl of TMB substrate was added to each well. The reaction was stopped with 50 μl of 2N H.sub.2SO.sub.4 and the plates read at 450 nm.

    [0216] DP4 Competition Assay.

    [0217] High binding plates were coated with streptavidin (Sigma S4762 at 1 mg/ml) 1/500 in PBS and incubated overnight at 4° C. The plates were blocked with 1% BSA in PBS for 4 hrs at room temp and washed 3×PBS/0.5% Tweenx. 450 μl of cell prep lysate (B16F1 B16 HHDII/DP4 H7/2E9/F9) was mixed with 50 μg test peptide (see below) or 10 μg of unlabelled Hep B peptide for 30 mins. 10 μg biotinylated Hep B peptide was added & incubated for 4 hrs at 37° C.:

    TABLE-US-00009 Hep B 181-192 GFFLLTRILTIPQ Fibrinogen 78-91 NQDFTN-cit-INKLKNS cit (Hu) Collagen II 1236-1249 LQYM-cit-ADQAAGGLR.  cit (Hu) Aldolase A 74-93 IGGVILFHETLYQ-hcit-ADDGRP Aldolase A 74-93 WT IGGVILFHETLYQKADDGRP Aldolase A 140-157 hcit-DGADFA-hcit- WRCVL-hcit-IGEH Aldolase A 140-157 WT KDGADFAKWRCVLKIGEH Aldolase A 217-235 LSDHHVYLEGTLL- hcit-PNMVT Aldolase A 238-256 HACTQ-hcit- FSH(N)EEIAMATVTA Aldolase A 289-307 hcit-CPLL-hcit- PWALTFSYGRALQ Cytokeratin 8 101-120 KFASFID-Hcit- VRFLEQQN-Hcit-MLE Cytokeratin 8 112-131 LEQQN-hcit-MLET- hcit-WSLLQQQ-hcit-T Cytokeratin 8 182-202 EIN-hcit-RTEMENEFVLI- hcit-hcit-DVDE Cytokeratin 8 371-388 LREYQELMNV-hcit-LALDIEI Cytokeratin 8 381-399 hcit-LALDIEIATYR- hcit-LLEGEE Vimentin 116-135 NYID-hcit-VRFLEQQN- hcit-ILLAEL Vimentin 116-135 WT NYID-hcit-VRFLEQQN- hcit-ILLAEL

    [0218] Lysate/peptide mix was added to the plates at 100 μ/well and incubated for 4 hrs at room temperature. Plates were washed 3× with PBS/0.5% Tween. 100 μl per well of Leinco anti-Human HLA-DP4 clone B7/21 #H260 1 mg/ml at 1/500 dilution in 1% BSA/PBS was added and incubated for 1 hr at room temperature prior to washing 3× PBS/0.5% Tween. Goat anti-mouse IgG3-HRP at 0.5mg/ml 1/500 100 μl per well (Invitrogen #M32607) diluted in 1% BSA/PBS) was added for 1 hr at room temperature prior to washing and adding 150 μl of TMB substrate to each well. The reaction was stopped with 50 μl of 2N H.sub.2SO.sub.4 and plates read plate at 450 nm.

    [0219] Statistical Analysis

    [0220] Statistical analysis was performed using GraphPad Prism software version 7. Comparative analysis of the ELISpot results was performed by applying paired or unpaired ANOVA or Student t test as appropriate with values of p calculated accordingly. Comparison of tumour survival was assessed by log-rank test. p<0.05 values were considered statistically significant and p<0.01 values were considered highly significant.

    Example 1

    CD4 Responses to Homocitrullinated Vimentin 116-135

    [0221] In silico bioinformatic analysis of vimentin (Table 2) was performed to identify peptide sequences with high binding affinity to human MHC class II using the online IEDB prediction program (http://www.iedb.org/). The top binding affinity peptides whose core binding region contained a lysine and demonstrated homology between human and mouse were selected. The lysine residues were replaced with homocitrulline (Hcit). The selected peptides are summarised in Table 1, PepFold (spiral) analysis was done retrospectively.

    [0222] Screening of Vimentin Peptide Responses

    [0223] Screening was performed to identify potential homocitrullinated vimentin epitopes in mice. Mice were immunised with pools of homocitrullinated peptides. To reduce the effect of possible cross reactivity, the peptides within the pool were chosen so that they did not contain any overlapping amino acid sequences. Each pool was administered as three immunisations containing 20 μg of each peptide and CpG/MPLA as an adjuvant on day 1, 8 and 15. On day 21 the mice were culled and the immune responses to each peptide within the immunising pool were assessed by ex vivo ELISpot. Given that different mouse strains have different MHC repertoires a number of strains were used for screening. Peptide responses were assessed in transgenic strains expressing human DR4 or HHDII/DR1 in a C57BL/6 background (see methods).

    [0224] Significant IFNγ responses were detected to peptide vimentin 116-135 Hcit. In both HLA-DR4 (FIG. 4a) and HHDII/DR1 (FIG. 4c) mice, the pool containing the Hcit vimentin 116-135 peptide induced a significant response to vimentin 116-135 Hcit. No other peptides showed significant IFNγ responses in HLA-DR4 or HHDII/DR1 mice. Our predictions therefore only identified 1/5 peptides which could induce a T cell response. In addition, responses to vimentin 116-135 Hcit were tested in HHDII/DP4 and C57Bl/6 mice but no IFNγ responses were observed (FIG. 5a). This result suggested the homocitrullinated vimentin peptide 116-135 justified further investigation. In addition, immunisation of HHDII/DR1 or HLA-DR4 mice with the unmodified peptide (vimentin 116wt) failed to induce any responses (FIG. 5b). Thus a key characteristic of immunogenic peptides is they express homocitrulline.

    TABLE-US-00010 TABLE 2 Vimentin peptides. DP4 DP4 DR4 DR4 DR1 DR1 T predic- predic- predic- predic- predic- predic- cell coordin- se- tion ted tion ted tion ted re- Protein ates quence score cores score cores scores cores Spiral sponse Vimentin 116-135 NYID-hcit-  2.12-17.18 IDKVRFLEQ  5.06-38.50 VRFLEQQNK 11.76-14.32 FLEQQNKIL custom-character yes VRFLEQQN-  2.12 YIDKVRFLE  5.06 FLEQQNKIL 14.32-58.24 VRFLEQQNK hcit- 30.02 KVRFLEQQN 38.50 YIDKVRFLE 58.24 RFLEQQNKI ILLAEL 30.02 RFLEQQNKI Vimentin 215-235 LARLDLER- 21.67-49.11 KVESLQEEI 24.51-30.76 LERKVESLQ 76 ERKVESLQE custom-character   no hcit- 44.08-60.82 LERKVESLQ 26.21 ARLDLERKV 79.7-81.37 ARLDLERKV VESLQE 57.44-60.82 ARLDLERKV 26.21-30.76 LDLERKVES 79.70-81.37 DLERKVESL EIAFL- hcit Vimentin 255-275 QIDVDVS- 82.48-88.15 VSKPDLTAA 17.21-30.56 SKPDLTAAL 58.03 SKPDLTAAL custom-character no hcit- 17.21 IDVDVSKPD 58.21-62.81 KPDLTAALR PDLTAALR 23.61 VDVSKPDLT 58.03-61.06 VDVSKPDLT DVRQQ 30.56 VSKPDLTAA Vimentin 286-303 EAEEWY-  2.06-2.22 KSKFADLSE  4.76-9.29 KFADLSEAA 18.99 KFADLSEAA custom-character no hcit-  2.06-5.69 WYKSKFADL  9.29-11.26 YKSKFADLS 18.99-41.08 YKSKFADLS S-hcit-  3.31-5.69 KFADLSEAA FADLSE AAN Vimentin 431-454 LPLVDTHS- 35.09-40.42 THSKRTLLI  9.5 IKTVETRDG 24.07-34.8 KRTLLIKTV custom-character no hcit- 41.46-60.30 TLLIKTVET  9.5 LIKTVETRD 31.48-41.38 THSKRTLLI RTLLI- 41.46-49.99 HSKRTLLIK 10.96-12.02 TLLIKTVET 24.07-44.99 TLLIKTVET hcit- 59.59-60.30 LLIKTVETR  9.5-12.02 LLIKTVETR TVETRD 22.22-26.93 THSKRTLLI GQV  

    [0225] To further characterise the responses induced by the carbamylated peptides mice were immunised with Hcit peptides and splenocytes assessed for cross reactivity to the wt peptides and responses to the Hcit peptides in the presence of CD4 and CD8 blocking antibodies. Vimentin 116-135 Hcit IFNγ response in DR4 (FIG. 7a) and HHDII/DR1 (FIG. 7b) transgenic mice showed no cross reactivity to the unmodified wt peptide. Thus a key characteristic of immunogenic peptides is they express homocitrulline. The vimentin 116-135 Hcit response was significantly higher than the wt response in both DR4 (p<0.0001) and HHDII/DR1 (p<0.0001) mice. Cross reactivity was seen to a shorter Hcit peptide sequence spanning amino acid 120-134 in both DR4 (p=0.0001) and HHDII/DR1 (p=0.0005) mice. In both strains the vimentin 116-135 Hcit responses were significantly decreased with the addition of CD4 blocking antibody (p=0.0054 and p<0.0001, respectively) but not in the presence of the CD8 blocking antibody. These abrogated responses reveal that the responses are mediated by CD4 cells. Thus a key characteristic of the response is it is mediated by CD4 T cells. Splenocytes from mice showed no production of IL-10 and IL-17 in response to immunisation with vimentin 116-135 Hcit peptide (FIG. 5c & d).

    [0226] Thus the Minimal Epitope in Both DR4 and DR1 is Vimentin 120-134 with Homocitrulline at Positions 120 and 129.

    [0227] Next, we tested if vimentin can be carbamylated at our key residues. The recombinant protein was treated in vitro with potassium cyanate and carbamylation was assessed by ELISA. Carbamylation was significantly increased after treatment with potassium cyanate demonstrating that vimentin contains lysines that can be subject to homocitrullination (FIG. 9a). Next, carbamylated recombinant protein sample was analysed by mass spectrometry to determine the presence of homocitrulline molecules. Mass spectrometry demonstrates that vimentin (FIG. 9c) protein can be homocitrullinated at a number of sites. Together these results show that vimentin could undergo carbamylation at the correct sites.

    Example 2

    CD4 Responses to Homocitrullinated ALDOA

    [0228] In silico bioinformatic analysis of ALDOA (Table 3) was performed to identify peptide sequences with high binding affinity to human MHC class II using the online IEDB prediction program (http://www.iedb.org/). The top binding affinity peptides whose core binding region contained a lysine and demonstrated homology between human and mouse were selected. The lysine residues were replaced with Hcit. The selected peptides are summarised in Table 3. PepFold (spiral) analysis was done retrospectively.

    TABLE-US-00011 TABLE 3 ALDOA peptides utilised. DP4 DP4 DR4 DR4 DR1 DR1 predic- predic- predic- predic- predic- predic- coordin- se- tion ted tion ted tion ted T cell Protein ates quence score cores score cores scores cores Spiral response ALDOAA 74-93 IGGVIL  2.5- FHETLYQKA 30.22 FHETLYQKA 33.91-60.75 FHETLYQKA custom-character yes FHETLY  8.04 LFHETLYQK 30.22 LFHETLYQK 46.61-60.75 LFHETLYQK Q-hcit-  3.09- ADDGRP  8.04 ALDOAA 140-157 hcit- 22.10- FAKWRCVLK 49.80- FAKWRCVLK 59.86-65.39 AKWRCVLKI custom-character yes DGADFA- 28.10 AKWRCVLKI 57.56 59.86-65.39 FAKWRCVLK hcit- 23 35- DFAKWRCVL WRCVL- 28.10 hcit- 22.10- IGEH 28.10 ALDOAA 217-235 LSDHH  4.92- YLEGTLLKP  4.87- IYLEGTLLK 13.40 GTLLKPNMV custom-character no VYLEG  6.66 IYLEGTLLK  7.88 YLEGTLLKP TLL-  6.66  7.88 hcit- PNMVT ALDOAA 238-256 HACTQ- 13.18- TQKFSHEEI  4.11 KFSHEEIAM 46.73 TQKFSHEEI custom-character yes hcit- 17.73 KFSHEEIAM FSH(N) 13.18 EEIAMA 17.73 TVTA ALDOAA 289-307 hcit- 14.38- KPWALTFSY 11.15- LLKPWALTF 24.99-28.52 CPLLKPWAL custom-character no CPLL- 19.06 LLKPWALTF 20.21 hcit- 19.06 PWALT FSYGR ALQ

    [0229] Screening of ALDOA peptide responses

    [0230] Screening was performed to identify potential homocitrullinated ALDOA epitopes in transgenic HHDII/DP4 and HHDII/DR1 mice. Mice were immunised with pools of human homocitrullinated 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 three immunisations containing 20 μg of each peptide and CpG/MPLA as an adjuvant. After 21 days the mice were culled and the immune responses to each peptide within the immunising pool were assessed by ex vivo ELISpot in HHDII/DP4 (FIG. 4b) and HHDII/DR1 mice (FIG. 4d). Peptides spanning amino acids 74-93 (Ald 74Hcit), 140-157 (Ald 140Hcit), and 238-256 (Ald 238Hcit) all showed stimulation of IFNγ responses in HHDII/DP4 transgenic mice whereas only Ald 140Hcit stimulated responses in HHDII/DR1 transgenic mice. Our predictions therefore identified 3/5 peptides which could induce a T cell response. No responses were seen to ALDOA 74-93Hcit or 140-157 in DR4 mice (FIG. 6a). Of the responding peptides ALDOA 74-93 Hcit and 140-157 Hcit peptides are homologous in human and mice therefore these two peptides were selected for further investigation. Minimal IL-10 (FIG. 6b) or IL-17 (FIG. 6c) responses were seen to Aldolase peptides in HHDII/DP4 mice.

    [0231] The responses to ALDOA 74-93 Hcit and 140-157 Hcit immunisation were characterised for cross reactivity to the wt peptide sequence. In HHDII/DP4 mice, limited responses were seen to either wt peptide with Ald 74 Hcit and Ald 140 Hcit responses significantly higher than the 74-93wt (p<0.0001) or 140-157wt (p<0.0001) (FIG. 7c). Thus a key characteristic of immunogenic peptides is they express homocitrulline. As with the vimentin responses those to the ALDOA peptide were significantly reduced in the presence of CD4 blocking antibodies for both ALDOA 74-93Hcit (p<0.0001) and ALDOA 140-157Hcit (p<0.0001). Thus a key characteristic of the response is it is mediated by CD4 T cells. In HHDII/DR1 mice immunised with ALDOA 140-157 Hcit IFNγ responses to Ald 140 Hcit were significantly higher than the responses to ALDOA 140-157wt (FIG. 7d; p=0.0047). Thus a key characteristic of immunogenic peptides is they express homocitrulline.

    [0232] In mammals there are three isoforms of the ALDOA enzyme, ALDOA (A); ALDOB (B) and ALDOC (C) which are encoded by three distinct genes. They are highly conserved and have a high degree of amino acid homology (FIG. 3a). ALDOA 74-93 is highly conserved in all three isotypes with only 5 amino acid difference between isotype A and B and only 4 between A and C. ALDOA 140-157 is highly conserved in all three isotypes with only 7 amino acid difference between isotype A and B and only 2 between A and C. Vimentin and ALDOA are also highly conserved between, mouse, rat, chicken, dog, sheep, cow, horse, pig and humans (FIG. 8). 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.

    [0233] Next, we tested if ALDOA can be carbamylated at our key residues. The recombinant protein was treated in vitro with potassium cyanate and carbamylation was assessed by ELISA. Carbamylation was significantly increased after treatment with potassium cyanate demonstrating that ALDOA contains lysines that can be subject to homocitrullination (FIG. 9a). Next, carbamylated recombinant protein sample was analysed by mass spectrometry to determine the presence of homocitrulline molecules. Mass spectrometry demonstrates that ALDOA (FIG. 9b) protein can be homocitrullinated at a number of sites. Together these results show that ALDOA could undergo carbamylation at the correct sites.

    Example 3

    Predictions for Identifying Homocitrulline Containing T Cell Targets

    [0234] As In silico bioinformatic analysis of vimentin and ALDOA (Table 2 and 3) using IEDB prediction program (http://www.iedb.org/) identified 10 peptides but only four of these were immunogenic. We sort to combine IEDB with another prediction tool. Computer modelling was done using PEP-FOLD3, a novel computational framework hosted by the University of Paris (http://mobyl.rpbs.univ-paris-diderot.fr/portal.py#forms::PEP-FOLD3). The software allows both de novo free or biased prediction for linear peptides between 5 and 50 amino acids, and the generation of native-like conformations of peptides interacting with a protein when the interaction site is known in advance. Structures are calculated as a result of over 100 different simulations per peptide.

    [0235] Interestingly we have shown that peptides found to give a high frequency T cell response share a common structure with a spiral like structure of at least 5 amino acids within he peptide; lack of a spiral is associated with negative responses in T cell assays. However, strong predicted MHC binding does not automatically mean a repertoire exists to the peptide, rather informs the selection of more effective peptides for further study based on high binding affinity, the presence of homocitrulline residues in the core region, the peptide having apredominately spiral conformational structure upon 3D modelling, and the existence of homology between the mouse and human sequences.

    [0236] Using this combination of predictions, 5 CyK8 peptides containing homocitrulline were synthesised (Table 4). Screening was performed to identify potential homocitrullinated Cyk8 epitopes in transgenic HHDII/DP4. Mice were immunised with pools of human homocitrullinated 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 three immunisations containing 20 μg of each peptide and CpG/MPLA as an adjuvant. After 21 days the mice were culled and the immune responses to each peptide within the immunising pool were assessed by ex vivo ELISpot in HHDII/DP4 (FIG. 10a). Peptides spanning amino acids 101-120 (Cyk8 108,117Hcit), 112-131 (CyK8 117,122,130 Hcit),182-201 (CyK8 185,197,198 Hcit), 371-388 (CyK8 381hcit) and 381-399 (CyK8 381,393Hcit) all showed stimulation of IFNγ responses in HHDII/DP4 transgenic mice. The responses to CyK8 101-120 Hcit, 112-131 Hcit, 182-201 Hcit, 371-388 Hcit and 381-399 Hcit immunisation were characterised for cross reactivity to the wt peptide sequence. In HHDII/DP4 mice, limited cross reactive responses were seen any wt peptide with CyK8 112 Hcit, CyK371 and CyK8 381 Hcit responses significantly higher than the 112-131wt (p<0.0001), 371-388 (p<0.0001) or 381-399wt (p<0.01) (FIG. 10b). Thus a key characteristic of immunogenic peptides is they express homocitrulline. All of these peptides (100%) were immunogenic. Thus a high predicted HLA-DP4 binding affinity (<30), the presence of homocitrulline residues in the core region, the peptide being amphipathic in nature with a predominantely spiral conformational structure upon 3D modelling, and the existence of homology between the mouse and human sequences defines immunogenicity. When this model was applied retrospectively to include the vimentin and ALDOA peptides 8/9 (88%) were correctly predicted.

    [0237] Responses to Cyk8 101 Hcit, 112Hcit, 371 Hcit and 381Hcit were assessed in the presence of CD4 or CD8 blocking antibodies. Responses to Cyk8 101Hcit, 371Hcit and 381Hcit were inhibited upon inclusion of the CD4 blocking antibody but not with CD8 blocking antibody (FIG. 11a,d,e). Response to Cyk8 112Hcit was inhibited in the presence of CD8 blocking antibody but not with CD4 blocking antibody. (FIG. 11b). The Cyk8 112Hcit peptide sequence was analysed for predicted sequences to bind to HLA-A2 as the HHDII/DP4 mice possess HLA-A2 MHC class I allele. The shorter Cyk8 117-125Hcit peptide was tested for stimulation of immune responses in HHDII/DP4 mice and showed the generation of responses to the Cyk8 117Hcit peptide. These responses were blocked in the presence of CD8 blocking antibody but not in the presence of CD4 blocking antibody suggesting a CD8 mediated response to this peptide (Figure lit). Responses to homocitrullinated Cyk8 peptides show both CD4 and CD8 mediated responses in HHDII/DP4 mice.

    [0238] The five selected homocitrullinated Cyk8 peptides were also screened in HHDII/DR1 transgenic mice. Significant responses were detected to Cyk8 371Hcit and 112Hcit peptides (p<0.0001) which showed minimal cross reactivity to the wt peptide sequences (p<0.002) (FIG. 12a). Response to Cyk8 112Hcit peptide in the HHDII/DR1 mice was lost in the presence of CD8 blocking antibody but less effect of the CD4 blocking antibody was seen (FIG. 12b). This response also demonstrated cross reactivity to the shorter Cyk8 117Hcit peptide (FIG. 12b). Immunisation with the shorter Cyk8 117-125Hcit peptide showed the generation of responses to the Cyk8 117Hcit peptide that cross reacted with the Cyk8 112Hcit peptide, this Cyk8 117Hcit responses was also lost in the presence of CD8 blocking antibody (FIG. 12c). Response to the Cyk8 371 Hcit peptide in HHDII/DR1 mice was lost in the presence of the CD4 blocking antibody but not with the CD8 blocking antibody (FIG. 12c). Responses to the Cyk8 112Hcit and Cyk8 371 Hcit could be maintained in vitro and exhibited similar response characteristics compared to ex vivo analysis further confirming the CD8 and CD4 mediated responses respectively (FIG. 12d). Responses to homocitrullinated Cyk8 peptides show both CD4 and CD8 mediated responses.

    [0239] The ability of the wt peptides to stimulate a response was tested with the Cyk8 112Hcit peptide in the HHDII/DR1 mice. Cyk8 112wt peptide was administered as three immunisations containing 25 μg of peptide and CpG/MPLA as an adjuvant. After 21 days the mice were culled and the immune responses to peptide were assessed by ex vivo ELISpot. HHDII/DR1 mice showed no specific response to the immunising Cyk8 112wt peptide or to the Cyk8 112Hcit and Cyk8 117Hcit peptides (FIG. 12e).

    [0240] Additional peptides within vimentin and ALDOA and peptides within BiP, nucleophosmin, enolase, β-catenin and HSP60 have also been identified using this motif (Table 5).

    [0241] A number of peptides were chosen to test and HHDII/DP4 mice were immunised with pools of human homocitrullinated 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 three immunisations containing 20 μg of each peptide and CpG/MPLA as an adjuvant. After 21 days the mice were culled and the immune responses to each peptide within the immunising pool were assessed by ex vivo ELISpot (FIG. 13). Peptides from BiP spanning amino acids 316-336, 328-346 and 562-579, from nucleophosmin (Npm) spanning amino acids 11-27, 258-277 and 266-287, from enolase spanning amino acids 156-176, 245-264 and 400-419, from vimentin spanning amino acids 86-108 and 390-408 and from aldolase spanning amino acids 204-219 and 209-217 showed stimulation of IFNγ responses in HHDII/DP4 transgenic mice. The responses were characterised for cross reactivity to the wt peptide sequence. In HHDII/DP4 mice, limited cross reactive responses were seen to any wt peptide with BiP 328Hcit, 562Hcit, Npm 258Hcit, Npm 266Hcit and aldolase 204Hcit responses significantly higher than the corresponding wt peptides (p<0.05) (FIG. 13a-e). Thus the prediction model applies also to a wider peptide panel and a key characteristic of the immunogenic peptides is they express homocitrulline.

    [0242] To further characterise the responses induced by the carbamylated (homocitrulline containing) peptides mice were immunised with Hcit peptides and splenocytes assessed after 7 days culture for responses to the Hcit peptide in the presence of CD4 or CD8 blocking antibodies. Responses to aldolase 204Hcit showed loss of response in the presence of both CD4 and CD8 blocking antibodies although this was partial loss of response. Thus suggesting this sequence may contain both CD4 and CD8 responses (FIG. 13e and f). The response to the shorter aldolase 209Hcit peptide was lost in the presence of the CD8 blocking antibody suggesting a CD8 mediated response to this peptide (FIG. 13f). Responses to BiP 316Hcit, 328Hcit and 562Hcit all showed loss of response in the presence of CD4 blocking antibody but not with CD8 blocking antibody (FIG. 14a). Responses to enolase 156Hcit, 245Hcit and 400Hcit were also lost in the presence of CD4 blocking antibody but not in the presence of CD8 blocking antibody (FIG. 14b). The same loss of response in the presence of CD4 blocking antibody was seen for the vimentin 86Hcit and 390Hcit responses (FIG. 14d). The Npm responses to 11 Hcit and 258Hcit also show blocking of the response in the presence of the CD4 blocking antibody but not with the CD8 blocking antibody (FIG. 14c), however the Npm 266Hcit response is lost in the presence of the CD8 blocking antibody but not with the CD4 blocking antibody (FIG. 14c).

    [0243] Since responses to homocitrullinated aldolase 204-219 in HHDII/DP4 mice suggested the presence of a HHDII (HLA-A2) restricted CD8 response this peptide was tested for responses in HHDII/DR1 mice alongside the shorter 209-217 Hcit peptide that was shown to elicit a CD8 mediated response in HHDII/DP4 mice. Both peptides stimulated strong responses to the homocitrulline peptides with lower reactivity to the wt sequences in HHDII/DR1 mice (FIG. 15).

    [0244] Thus a key characteristic of most responses is they are mediated by CD4 T cells. In addition, Hcit specific responses can also be mediated by CD8 T cells.

    TABLE-US-00012 TABLE 4 Homocitrulline binding predictions for CyK8. DP4 DP4 DR4 DR4 DR1 DR1 predic- predic- predic- predic- predic- predic- Prot- coordi- se- tion ted tion ted tion ted T cell ein nates quence score cores score cores scores cores Spiral response Cyk 8 101-120 KFASFID-  0.97-17.18 IDKVRFLEQ  5.06 FLEGGNKML 12.27 FLEQQNKML custom-character Yes Hcit-  0.97-1.68 SFIDKVRFL 25.16-37.08 IDKVRFLEG 12.27-66.46 VRFLEQGNK VRFLEQQN-  6.08 FiDKVRFLE 25.16-26.26 FASFIDKVR 63.29 FASFIDKVR Hcit-  5.06-37.08 VRFLEQQNK 65.13 KVRFLEQQN MLE Cyk8 112-131 LEQQN-  3.16-4.72 KMLETKWSL 12.19-24.67 MLETKWSLL 31.04-44.89 KWSLLQQQK custom-character Yes hcit-  3.16-4.72 MLETKWSLL 12.19-15.38 LETKWSLLQ 44.89-46.47 MLETKWSLL MLET- 12.96-15.38 KMLETKWSL 31.04-44.89 LETKWSLLQ hcit- 24.67 QNNMLETKW WSLLQQQ- hcit-T Cyk8 182-202 EIN- 27.44-41.36 MENEFVLIK 69.5-75.9 MENEFVLIK custom-character Yes hcit- 36.01-41.15 FVLiKKDVD RTEMEN 33.01 INKRTEMEN EFVLI- hcit- hcit- DVDE Cyk 8 371-388 LREYQ 24.36-26.69 LMNVKLALD  4.42-5.8 YQELMNVKL 6.74-20.92 YQELMNVKL custom-character Yes ELMNV- 24.36-26.69 ELMNVKLAL  5.76 LMNVKLALD 6.74-20.92 ELMNVKLAL hcit- LALDIEI Cyk 8 381-399 hcit- 18.10-24.62 IEIATYRKL 12.39-27.03 LDIEIATYR 39.89- IEIATYRKL custom-character Yes LALDIE 24.62 IATYRKLLE 23.47-27.68 IATYRKLLE 27.66 IATYR- 27.68 YRKLLEGEE hcit- LLEGEE

    TABLE-US-00013 TABLE 5  Homocitrulline binding predictions for Vimentin, ALDOA, cytokeratin 8, Bip, NPM, enolase, βcatenin and HSP60. DP4 DR1 pre- DP4 DR4 DR4 pre- DR1 dic- pre- Predic- pre- dic- pre- coordi- tion dicted tion dicted tion dicted Protein nates sequence score cores score cores scores cores Spiral ALDOAA 198-216 DL-  8.92- CQYVTEKVL  1.65- YVTEKVLAA 10.18- YVTEKVLAA custom-character Hcit- 11.09 YVTEKVLAA  1.78 13.7 RCQYVTE-  8.92- Hcit- 11.09 VLAAVY- Hcit- A ALDOAA 323-342 AAQEEYV- 45.05- KRALANSLA  7.44- VKRALANSL  3.72- VKRALANSL custom-character Hcit-RALA 55.91 YVKRALANS  7.59 KRALANSLA 12.6 KRALANSLA NSLACQG- 52.36- EEYVKRALA  7.44- YVKRALANS  3.72- Hcit 56.49  7.59 8.47 45.05-  7.44 52.36 ALDOAA 208-226 Hcit- 29.94- VLAAVYKAL  5.93- YKALSDHHI  5.79- YKALSDHHI custom-character VLAAVY- 32.49 YKALSDHHI  7.72 VYKALSDHH  6.42 VLAAVYKAL Hcit- 39.24-  5.93-  6.39- ALSDHHIY 42.43  7.72  6.42 ALDOAA 204-219 YVTEKVLAA 16.41- VLAAVYKAL  3.95 YVTEKVLAA 15.29- VLAAVYKAL custom-character VYKALSD 19.85 10.46 KVLAAVYKA 20.07 YVTEKVLAA 25.20 LAAVYKALS  1.34- LAAVYKALS 14.77 20.07 Vimentin 86-108 FSLADAIN 25.28- AINTEFKNT  4.34- FKNTRTNEK 73.49 EFKNTRTNE custom-character TEF-Hcit- 29.44 FKNTRTNEK  4.52 INTEFKNTR 73.49- NTEFKNTRT NTRTNE- 25.28- EFKNTRTNE 17.84- 79.9 FKNTRTNEK Hcit- 27.76 TRTNEKVEL 19.26 73.49 VELQ 25.28- 27.76 25.32- 27.76 Vimentin 390-408 Hcit- 18.19- IEIATYRKL 23.74- IATYRKLLE 40.8- IEIATYRKL custom-character MALDIEIA 24.62 IATYRKLLE 27.68 YRKLLEGEE 57.66 TYR-Hcit- 24.62 27.68 LLEGEE Cyto- 120-140 ET- 55.49 ETKWSLLQQ  0.5-2.17 LQQQKTARS  8.47-13.91 KWSLLQQQK custom-character keratin Hcit- 55.49-75.39 WSLLQQQKT  9.25-63.26 LQQQKTARS 8 WSLLQQQ- 63.26-87.43 LQQQK1ARS  8.47-13.91 WSLLQQQKT Hcit- TARSNMDNMF Cyto- 93-112 EQI- 19.63-25.41 KFASFIDKV  1.98-5.39 IKSLNNKFA 11.25-20.28 iKSLNNKFA custom-character keratin Hcit- 19.63-27.86 SLNNKFASF 17.73-20.24 FASFIDKVR 11.25-49.25 LNNKFASFI 8 SLNN- 17.73-20.24 LNNKFASFI Hcit- FASFID- Hcit- VRFL Cyto- 190-208 ENEFVLI- 67.48-75.49 FVLIKKDVD 11.45 IKKDVDEAY 78.64-86.64 KKDVDEAYM custom-character keratin Hcit- 67.48-67.96 EFVLIKKDV 11.45 FVUKKDVD 78.64-84.99 FVLIKKDVD 8 Hcit- DVDEAYMN- Hcit- V Cyto- 294-310 G- 72.15-81.41 LRRTKTEiS  3.19-3.29 I.RRTKTEIS 76.17-85.76 LRRTKTEiS custom-character keratin Hcit- 76.17-85.34 RRTKTEISE 8 HGDDLRRT- Hcit- TEISEM Cyto- 369-388 RQLREYQEL 24.36-26.78 ELMNVKLAL  4.42-5.82 YQELMNVKL 6.6-20.92 YQELMNVKL custom-character keratin MNV-Hcit- 24.36-26.78 LMNVKLALD 6.74-20.92 ELMNVKLAL 8 LALDIEI Cyto- 320-338 EiEGL- 59.6-65.73 LKGQRASI.E 13.22 LKGQRASl.E 8.36-13.25 LKGQRASLE custom-character keratin Hcit- 8.36-8.42 IEGLKGQRA 8 GQRASLE AAIADA Cyto- 339-358 EQRGELAS- 75.97-77.15 DANAKLSEL 13.67 IKDANAKLS 27.63-49.46 IKDANAKLS custom-character keratin Hcit- 95.14-96.26 LAIKDANAK 13.67-13.76 AIKDANAKL 27.63-49.46 LAIKDANAK 8 DANA- 95.14-95.26 AIKOANAKL Hcit- LSELEA BiP 104-124 NDPSVQQDI-  3.13-5.48 IKFLPFKVV  8.87-8.95 FLPFKVVEK 26.97-54.82 DIKFLPFKV custom-character Hcit-  3.13-3.36 QDIKFLPFK  8.88-29.27 DIKFLPFKV 26.97 IKFLPFKVV FLPF-  3.62-7.91 QQDIKFLPF 22.3-30.85 VQQDIKFLP 81.79 SVQQDIKFL Hcit- 22.3-29.27 IKFLPFKVV VVE- Hcit- Hcit- T BiP 144-159 EiSAMVLT- 42.22-42.30 MVLTKMKET  8.68-8.69 ISAMVLTKM 36.03 ISAMVLTKM custom-character Hcit- 42.22-42.30 SAMVLSKMK 36.03 LTKMKETAE M- Hcit- ETAEA BiP 255-275 GEDFDQR  7.94-12.95 RVMEHFIKL 17.21 FIKLYKKKT 32.16-38.72 FIKLYKKKT custom-character VMEHFI- 12.95 QRVMEHFIK 17.21-40.78 VMEHFIKLY 38.72-65.12 QRVMEHFIK Hcit- LY- Hcit- Hcit- Hcit- TG BiP 286-306 Q- 70.38-84.43 KRALSSQHQ 15.95-19.49 LRREVEKAK 34.03-65.3 AKRALSSQH custom-character Hcit- 78.03-85.41 EKAKRALSS 19.45-23.67 VEKAKRALS 34.03-65.3 VEKAKRALS LRREVE- 83.26-85.26 LRREVEKAK 23.67 AKRALSSQH 34.03-39.52 KRALSSQHQ Hcit- 23.67 KRALSSQHQ 43.46-45.85 KLRREVEKA A- 43.46-56.99 REVEXAKRA Hdt- RALSSQ HQAR BiP 316-336 EDFSET  0.13-1.31 LTRAKFEEL 11.45-15.14 FSETLTRAK 53.82-68.1 FEELNMDLF custom-character LTRA- 21.98-31.82 RAKFEELNM 54.2-65.21 SETLTRAKF Hcit- 21.98-31.82 LTRAKFEEL 57.7-77.4 LTRAKFEEL FEELNM DLFR BiP 328-346 EELNMDL 19.99-23.39 LFRSTMKPV  6.65-13.67 MDLFRSTMK 28.99-39.69 LFRSTMKPV custom-character FRSTM- 21.61-23.39 FRSTMKPVQ  6.65-8.29 FRSTMKPVQ 35.68-36.69 MDLFRSTMK Hcit- 28.99 FRSTMKPVQ PVQ- Hcit- VL BiP 367-387 RIP-  4.15-27.73 QLVKEFFNG  9.73 FFNGKEPSR 47.06-62.09 FFNGKEPSR custom-character Hdt-  4.15-20.84 LVKEFFNGK  9.73-24.14 LVKEFFNGK 65.99-73.97 IQQLVKEFF IQQLV- 15.33-24.14 IQQLVKEFF 71.21-73.97 LVKEFFNGK Htit- EFFNG- Hcit- EPSRG BiP 460-480 TVTI- 17.85-20.08 KVYEGERPL 10.02-27.01 YEGERPLTK 44.67-63.59 KVYEGERPL custom-character Hcit- 17.85-51.73 YEGERPLTK 44.67-65.03 YEGERPLTK WEGERPLT- Hcit- DNHLLG BiP 562-579 RNELESY 21.01 LESYAYSLK 12.81-16 YAYSLKNQI 32.42-43.8 YAYSLKNQI custom-character AYSL- 21.01 YAYSLKNQI 14.01 LESYAYSLK 32.42-43.8 AYSLKNQIG Hcit- NQIGD- Hcit BiP 620-639 Hcit- 32.06-47.49 IVQPIISKL  6.28-7.76 VQPIISKLY 25.51-36.44 IVQPIISKL custom-character Hcit- 39.29-47.49 VQPIISKLY  7.72-19.66 IVQPIISKL 25.51-26.88 VQPIISKLY ELEEIV QPIIS- Hcit- LYGSAG Nucleo- 11-27 PLRPQNYLFG 26.13-33.98 YLFGCELKA 10.18-10.36 YLFGCELKA 41.72-42.71 YLFGCELKA custom-character phosmin CEL-Hcit- 26.54 LFGCELKAD 10.18-10.2 LRPQNYLFG AD- Hcit- Nucleo- 68-89 EGSPI- 32.93 KMSVQPTVS  5.42-6.05 KVTLATLKM 17.14-18.61 KMSVQPTVS custom-character phosmin Hcit- 33.41-45.82 KVTLATLKM  5.15-12.57 LKMSVQPTV 19.12-33.26 KVTLATLKM VTLATL- 32.93 LKMSVQPTV  5.5-7.38 IKVTLATLK 18.61-20.48 LATLKMSVQ Hcit- 33.41-40.80 TLATLKMSV  7.38-12.57 LATLKMSVQ 17.14-20.48 TLKMSVQPT MSVQPTVSL Nucleo- 129-149 EEEDV- 65.37-77.59 LLSISGKRS  4.11 VKILSISGK 5.03-17.94 LLSISGKRS custom-character phosmin Hcit- 66.93-77.59 ISGKRSAPG  4.11-9.89 LLSISGKRS LLSISG- 65.37-75.07 KLLSISGKR  9.89 ISGKRSAPG Hcit- RSAPGGGS Nucleo- 222-240 S- 73.23-91.45 FKKQEKTPK 13.76 FKKQEKTPK 76.73-78.7 FKKQEKTPK custom-character phosmin Hcit- 73.23-85.41 ESFKKQEKT 76.73-78.7 KKQEKTPKT GQESF- 82.11-85.41 KKQEKTPKT Hcit- Hcit- QE- Hcit- TP- Hcit- TP- Hcit- G Nucleo- 258-277 GGSLP- 24.31-30.51 KVEAKFINY 14.7-25.2 FINYVKNCF 51.23-60.28 VEAKFINYV custom-character phosmin Hcit- 24.31-30.51 VEAKFINYV 25.2-34.52 EAKFINYVK 51.23-59.06 LPKVEAKFI VEA- 28.92-30.51 FINYVKNCF 31.9-43.83 LPKVEAKFI Hcit- 25.2-43.83 VEAKFINYV FINYV- Hcit- NCFR Nucleo- 266-287 A-  2.93-3.06 KFINYVKNC 11.37 FINYVKNCF 40.86-50.73 FINYVKNCF custom-character phosmin Hcit-  2.93-14.95 YVKNCFRMT 15.24-21.39 YVKNCFRMT 40.86-55.78 YVKNCFRMT FINYV- 11.37-20.77 INYVKNCFR Hcit- NCFRMTD QEAIQDL Alpha 1-16 MSIL- 26.7-29.02 ILKIHAREI 14.35-25.75 ILKIHAREI 17.82 MSILKIHAR custom-character Enolase Hcit- 26.7-29.02 LKIHAREIF 14.35 MSILKIHAR 17.82-35.56 ILKIHAREI IHAREI FDSRG Alpha 52-69 ND- 59.77-60.48 KTRYMGKGV 10.02-10.25 MGKGVSKAV 21.68-25.27 YMGKGVSKA custom-character Enolase Hcit- 59.77-68.62 YMGKGVSKA 10.25 RFMGKGVSK 21.68-23.87 KTRYMGKGV TRYMG- 10.02-10.25 YMGKGVSKA Hcit- GVS- Hcit- AVEHI Alpha 100-121 TEN- 13.21-26.83 KFGANAILG 11.17 KFGANAILG 12.06-24.07 KSKFGANAI custom-character Enolase Hcit- S- Hcit- FGANAIL GVSLAVC- Hcit- A Alpha 156-176 GSHAGN-  1.2-22.16 KLAMQEFMI 12.67-32.56 KLAMQEFMI 34.28-46.65 KLAMQEFMI custom-character Enolase Hcit- 22.16 HAGNKLAMQ LAMQEFMI LPVGAA Alpha 179-197 REAMRIGA 25.95-28.39 VYHNLKNVI  3.51-30.06 VYHNLKNVI 52.39 VYHNLKNVI custom-character Enolase EVYHNL-  3.51 YHNLKNVIK 12.91 YHNLKNVIK Hcit- NVI- Hcit- Alpha 194-212 NVI- 83.85-94.96 YGKDATNVG  3.14-3.29 YGKDATNVG 72.08-72.12 KYGKDATNV custom-character Enolase Hcit- 83.85-85.66 VIKEKYGKD 72.08-72.12 YGKDATNVG E- 92.59-94.96 KYGKDATNV Hcit- YG- Hcit- DATNV GDEGG Alpha 245-264 DVAASEFF 10.04-19.07 FRSGKYDLD 22.59-24.17 FRSGKYDLD 54.29-81.42 FRSGKYDLD custom-character Enolase RSG-Hcit- 10.04-19.07 FFRSGKYDL 38.34 SEFFRSGKY 54.29-81.42 FFRSGKYDL YDLDF- 81.42 SEFFRSGKY Hcit- 69.53-81.42 ASEFFRSGK SP Alpha 273-291 PDQLADLY- 19.25-24.65 ADLYKSFIK  2.4-22.77 YKSFIKDYP 40.44 KSFIKDYPV custom-character Enolase Hcit- 19.25-22.11 DLYKSFIKD  2.4 FIKDYPVVS 63.72-66.68 LADLYKSFI SFI- 20.41-24.65 LYKSFIKDY 22.03-27.67 LYKSFIKDY 60.86-66.68 ADLYKSFIK Hcit- 27.67 LADLYKSFI 40.44 FIKDYPVVS DYPVVS Alpha 301-317 WGAWQ- 26.6-30 KFTASA6IQ  5.6-9.22 WQKFTASAG  6.34 WQKFTASAG custom-character Enolase Hcit-  6.34 AWQKFTASA FTASAGIQWG Alpha 333-352 NE- 39.34-62.75 CLLLKVNQI  9.38-19.95 LLKVNQIGS 14.35-15.37 CLLLKVNQI custom-character Enolase Hcit- 39.34-42.6 LLKVNQIGS 12.48-19.95 CLLLKVNQI SCNCLLL- 39.34-62.75 SCNCLLLKV Hcit- VNQIGSVTE Alpha 400-419 RSERLA- 11.8-12.36 RLAKYNQLl 23.15-31.22 LAKYNQLLR 45.27-52.87 RLAKYNQLL custom-character Enolase Hcit- 11.8-18.57 AKYNQLLRI 58.12-59.75 AKYNQLLRI YNQLLRI EEELGS Alpha 418-434 GS- 16.18-19.61 KAKFAGRNF 19.53-33.35 KFAGRNFRN 76.13 KFAGRNFRN custom-character Enolase Hcit- 16.18 KFAGRNFRN A- Hcit- FAGRNF RNPLA- Hcit- Beta 127-144 EPSQML- 33.28-41.88 LKHAWNLI  3.3-3.45 LKHAWNLI 16.66-22.24 LKHAWNLI custom-character catenin Hcit-HA 33.28-41.88 MLKHAVVNL 16.66-19.78 AWNLINYQ VVNLINYQD Beta 334-354 E-  0.74-12.86 WTTSRVLKV  1.86-4.47 VLKVLSVCS  9.62 KLLWTTSRV custom-character catenin Hdt- 10.61-61.82 ITSRVLKVL  1.88-4.47 LKVLSVCSS  9.62-17.94 WTTSRVLKV LLVvTTSRVL-  4.77-7.6 WTTSRVLKV 12.82-17.94 SRVIKVLSV Hdt- 12.82-15.58 VLKVLSVCS VLSVCSSN- Hcit Beta 258-275 TLHNLLI 55.35-73.17 LLHQEGAKM  5.06 LLHQEGAKM 12.43 LHQEGAKMA custom-character catenin HQEGA-Hcit-  5.06 LHQEGAKMA 12.43-13.1 LLHQEGAKM MAVRL Beta 269-288 A- 18.5-35.63 LQKMVALLN  4.73-7.08 LQKMVALLN 26.97 GLQKMVALL custom-character catenin Hcit- 18.5-32.91 LAGGLQKMV  4.73-31.97 VRLAGGLQK 19.62-27.23 VRLAGGLQK MAVRLAGGLQ- 18.5-35.63 GLQKMVALL 30.74-31.35 LAGGLQKMV 23.3-26.97 LAGGLQKMV Hcit- 32.91 VRLAGGLQK 26.97 LQKMVALLN MVALLN- Hcit Beta 288-307 -Hcit-  8.87-10.9 NVKFLAITT custom-character catenin TNV- Hcit- FLAITTDC LQILAYG Beta 332-349 TYE-  0.73-1.12 WTTSRVLKV  3.19-7.23 WTTSRVLKV  9.62-11.15 KLLWTTSRV custom-character catenin Hcit-  3.24 YEKLLWTTS  9.62 WHSRVLKV LLWTTSRVL- Hcit- VLSV Beta 340-358 TSRVL- 59.83-63.44 LKVLSVCSS  1.6-1.88 VLSVCSSNK 12.82-34.92 LKVLSVCSS custom-character catenin Hcit- 48.36-66.69 KVLSVCSSN  1.6-1.88 LKVLSVCSS 12.82-34.92 VLKVLSVCS VLSVCSSN- 59.83-69.02 RVLKVLSVC 12.82-15.54 SRVLKVLSV Hcit- PAIV Beta 478-497 catenin Beta 489-506 YGLPVW- 43.17-61.12 PVVVKLLMP  4.66-4.96 VVKLLHPPS 16.56-20.53 VVKLLHPPS custom-character catenin Hcit- 43.17-49.37 LPVVVKLLH 16.56-20.53 VKLLHPPSH LLHPPSH 61.92 VVVKLLHPP WPL Beta 503-522 HWPLI- 12.35-30.05 IKATVGLIR 10.06-13.34 WPLIKATVG 22.88-24.83 LIKATVGLI custom-character catenin Hcit- 12.35-30.05 LIKATVGLI 22.88 HWPLIKATV ATVGLIRNL ALCPA Beta 607-625 LENIQRVAA 39.59 VLCELAQDK custom-character catenin GVLCELAQD- Hcit- Beta 650-667 GVATYAAA  9.35-12.15 VLFRMSEDK custom-character catenin VLFRMSED- Hcit- P HSP60 117-136 TVLARSIA-  8.56-21.55 IAKEGFEKI 21.85-52.27 IAKEGFEKI 45.06-65.33 VLARSIAKE custom-character Hcit- 21.85-42.65 LARSIAKEG 45.06-71.85 LARSIAKEG EGFE- 31.74 EGFEKISKG 71.85-83.97 SIAKEGFEK Hcit- 71.85-83.97 RSIAKEGFE IS- Hcit- GAN HSP60 280-299 GEALSTLV 10.52-13.78 STLVLNRLK  7-7.76 LVLNRLKVG 19.9-22.24 LNRLKVGLQ custom-character LNRL- 19.17-27.64 RLKVGLQVV 11.03-13.12 LNRLKVGLQ 22.24 TLVLNRLKV Hcit- 19.17-27.64 LVLNRLKVG VGLaVVA 23.62-27.64 TLVLNRLKV HSP60 381-398 TTSEYE- 18.89-23.14 KLNERLAKL 27.33-36.43 YEKEKLNER 51.23-59.99 KLNERLAKL custom-character Hcit- 29.24 TTSEYEKEK 27.33 KLNERLAKL 74.42-77.04 YEKEKLNER E- 18.89-29.24 YEKEKLNER 51.23-77.04 KEKLNERLA Hcit- I LNERLA- Hcit- LS HSP60 517-536 GIIDPTV- 18.25-30.13 KVVRTALLD 14.63-17.84 TKVVRTALL 15.82-21.8 PTKVVRTAL custom-character Hcit- 18.25-30.13 TKVVRTALL 17.84 IDPTKVVRT 20.49-21.8 TKVVRTALL VRTALLD 21.19 IDPTKVVRT AAGVA HSP60 84-103 IDL- 50.61-63.16 YKNIGAKLV  4.84-11.64 YKNIGAKLV  0.42-2.37 KYKNIGAKL custom-character Hcit- 50.61-63.16 KNIGAKLVQ  0.42-2.37 YKNIGAKLV D- Hcit- Y-Hcit- NIGA- Hcit- LVQDVAN

    Example 4

    Responses in Healthy Human Donors and Cancer Patients

    [0245] Determination as to the existence of a repertoire of T cells for carbamylated epitopes in humans was investigated using PBMCs from normal, healthy donors. PBMCs were isolated and CD25-depleted. Cells were labelled with CFSE and proliferation was monitored after stimulation with homocitrulline peptides. Example plots are shown for one healthy donor and one lung cancer patient (FIGS. 106a and b). Most healthy donors tested showed a CD4+ T cell proliferative response that was above twice the background for at least one of the peptides tested (FIGS. 16c and e). Across the healthy donors, ALDOA 74-93Hcit (p=0.0079), ALDOA 140-157 Hcit (p=0.0122) and vimentin 116-135Hcit (p<0.0001) induced significant CD4 proliferation. In conclusion, healthy donors show a repertoire of CD4 T cells that can respond to the carbamylated peptides.

    [0246] In addition to healthy donors (Table 6a) we examined the repertoire of responses in three ovarian, one breast and seven lung cancer patients (Table 6b). Seven of eleven patients tested showed proliferative CD4 responses to one or more of the carbamylated peptides (FIGS. 16d and f). Across the patients, ALDOA 74-93 Hcit (p=0.0353) induced significant proliferative responses when compared to the media only control. Vimentin 116-135 Hcit responses were just short of significance (p=0.0605) This suggests that cancer patients also have repertoires of CD4 T cells that are capable of responding to the carbamylated peptides and would support the targeting of carbamylated vimentin and ALDOA for cancer therapy. The proliferative responses were predominantly CD4 mediated as shown in FIG. 16d.

    [0247] Analysis of cytokine expression was also performed on majority of donors (example plots shown FIG. 17a). For each peptide, the donors that showed proliferative responses above twice the background were assessed for expression of CD134, IFNγ and GraB. The percentage of proliferating CD4 cells expressing each marker was determined. This staining showed variable expression of these markers, however the detection of IFNγ, granzyme B and CD134 suggest that these cells are a cytotoxic Th1 phenotype (FIGS. 17b and c).

    [0248] Determination as to the existence of a repertoire of T cells for carbamylated Cyk8 epitopes in humans was investigated using PBMCs from seven normal, healthy donors. PBMCs were isolated and CD25-depleted. Cells were labelled with CFSE and proliferation was monitored after stimulation with Cyk8 Hcit peptides. Most healthy donors tested showed a CD4+ T cell proliferative response that was above twice the background for at least one of the peptides tested (FIG. 18). In conclusion, healthy donors show a repertoire of CD4 T cells that can respond to the carbamylated Cyk8 peptides.

    TABLE-US-00014 TABLE 6 Details of healthy donors and cancer patients A. Healthy Donors ID Sex HLA type BD0015 F HLA-A: *03, *24, HLA-B: *07, *15, HLA-C: *03, *07, HLA-DR: *04, *15, HLA-DQ: *03, *06, HLA-DP: *04 BD0022 F HLA-A: *01, *02, HLA-B: *35, *50, HLA-C: *06, *12, HLA-DR: *04, *07, HLA-DQ: *02, *03, HLA-DP: *02, *04 BD0041 F HLA-A: *01, *24, HLA-B: *07*40, HLA-C: *03*07, HLA-DR: *04*11, HLA-DQ: *03, HLA-DP: *02*04 BD0050 F HLA-A: *24, *26, HLA-B: *35*45, HLA-C: *04*06, HLA-DR: *07*11 , *52b, *53a, HLA-DQ: *02*03, HLA-DP: *02*04 BD0010 F HLA-A: *02, *11, HLA-B: *40*44, HLA-C: *03*16, HLA-DR: *13, *16, *52c, *51 b, HLA-DQ: *05*06, HLA-DP: *04*10 BD0095 M Not available BD0017 F Not available BD0001 M HLA-A: *02, *32, HLA-B: *8, *44, HLA-C: N/A, HLA-DR: *03, *07, HLA-DQ: *02, HLA-DP: N/A BD0051 F HLA-A: *11, *68, HLA-B: *07*15, HLA-C: *05*07, HLA-DR: *13, *15, *52b, *51 a, HLA-DQ: *06, HLA-DP: *04*19 BD0044 F Not available BD0014 F Not available BD0025 F HLA-A: *02, *29, HLA-B: *07, *57, HLA-C: *06, *07, HLA-DR: *01 , *07, *53a, HLA-DQ: *03, *05, HLA-DP: *03, *13 BD0038 F HLA-A: *26, *33, HLA-B: *40*58, HLA-C: *03, HLA-DR: *09*11, HLA-DQ: *03, HLA-DP: *04*05 BD0016 M HLA-A: *01, *02, HLA-B: *08, *44, HLA-C: *05, *07, HLA-DR: *03, *15, *51a, *52a, HLA-DQ: *02, *06, HLA-DP: *01, *04 BD0007 F HLA-A: *01, *32, HLA-B: *08, *15, HLA-C: *07, HLA-DR: *03, *13, *51a, *51c, HLA-DQ: *02, *06, HLA-DP: *04, *13 B. Cancer Patients ID LG6 LG8 LG9 LG10 LG12 Age 72 71 79 67 71 Sex Female Male Male Male Female Smoking status Ex-smoker Ex-smoker Ex-smoker Smoker Ex-smoker HLA type N/A N/A N/A N/A N/A Indication/ Adenocarcinoma/ Adeno- Adeno Adeno- SCLC/ Treatment currently carcinoma/ carcinoma/ carcinoma/ Chemotherapy none, previous Tyrosine Chemotherapy Checkpoint chemotherapy and Kinase Inhibitor checkpoint inhibitor inhibitors and Steroid ID LG19 OV19 OV21 OV22 BR7 Age 65 60-70 72 76 51 Sex Male Female Female Female Female Smoking status Ex-smoker N/A N/A N/A N/A HLA type HLA A: A*01 *03 HLA B: B*07, *08 HLA C: C*07 HLA DR: *04*15, *51a, *53a HLA DQ: *03 *06 HLA-DP: *02, *04 N/A HLA A: A*0201 HLA B: B*14, *15 HLAC: C*03, *08 HLA DR: *03, *52a, *52b HLA DQ: *02 HLA-DP: *01,*04 HLA A: A*01 *03 HLA B: B*18, *44 HLA C:C*05, *12 HLA DR: *04, *15, *53a, *51a HLA DQ: *03, *06 HLA-DP: *04, *05 HLA A: A*11 ,*25 HLA B: B*18, *38 HLA C: CM2 HLA DR: *11, *15,*52b, *51 a HLA DQ: *03, *06 HLA-DP: *04 Indication/ Treatment Patient with metastatic adenocarcinoma of the lung (liver and lymph node involvement) who has PDL1 staining of 95% of the tumour cells, EGFR mutation and ALK rearrangement negative. The patient was bled prior to the 6.sup.th cycle of first line pembrolizumab. Last CT scan 2 months prior showed evidence of disease response Stage 3C low grade serous ovarian adenocarcinoma. BRCA negative. Patient with stage 3C high grade papillary serous ovarian carcinoma, BRCA-ve who finished 6 cycles of carboplatin and caelyx on the 14.sup.th of March 2019 and had partial response to treatment on CT scan Patient with stage 4 primary peritoneal adenocarcinoma, who was treatment naïve Patient with triple negative metastatic breast cancer. Finished Pembrolizumab treatment Apr. 2018. Previous capecitabine and carboplatin. Bled prior to starting Eribulin

    Example 5

    To Determine the α and β Chain Pairing of TCRs Recognising Peptides Containing Homocitrulline

    [0249] The CD4 T cells that proliferated (CFSE Low) in response to ALDOA 74-93 Hcit were analysed for their TCR expression in comparison to the non proliferating cells (CFSE High). Examination of TCR clonality of the responding CD4 T cells revealed a bias of TCR Vβ and Vα sequences among CD4+ proliferating cells from donor BD00016 ALDOA 74 Hcit (FIG. 27a-d). These responses appear oligoclonal with a couple of dominant TCRVβ and TCRVα chains compared to the non-proliferating CD4s from the same cultures thus suggesting a focussed TCR repertoire.

    [0250] To identify the correct pairing of the TCRVβ and TCRVα chains, single proliferating cells were sorted into 96 individual wells and TCRα and TCRβ chains were sequenced using iPair™ technology. 76/92 wells contained both TCRVβ and TCRVα chains, 89 wells contained sequences identified from bulk analysis. Out of these 38/89 wells contained the TRB chain and 11/89 wells the TRA chain only. 40/89 wells contained both TRA and TRB chains (Table 7). Remaining wells with paired chains contain either a TRA or TRB or both sequence/s of high bulk rank >30 with a frequency of less than <10%.

    TABLE-US-00015 TABLE 7 iPair™ sequencing and analysis of the TCRα and β chains ALDOA 74 Hcit FRE- QUEN- CY (40 CDR1 CDR2 CDR3 Read Bulk Unique/ SEQ WELLS) WELLS Peptide Peptide Peptide V Gene D Gene JGene C Gene Count Rank shared ID TCR 8 B11/D02/ ENHRY SYGVKD AISERRDQETQY hTR8V10-3 * hTR8J2-5 hTRBC 9659 1 S 3 9 G10/B801/ TISGTDY GLTSN ILRDVYDYKLS hTRAV26-2 * hTRAJ20 hTRBC 4355 1 S 4 DE12/DE 09/DF06/ DH01 10 C04/E02/ DFQATT SNEGSKA SAPIHTDTQY hTR8V20-1 * hTR8J2-3 hTBDC 1503 5 S 15 10 F02/H98/ VTNFRS LTSSGIE AVHPAGNMLT hTRAV36/ * hTRAJ39 hTRBC 12749 3 S 16 H11/DB10/ DV7 DB12/DE02/ DE03/DF08 3 G08/DF04/ SGHDY FNNNVP ASRGGLASNEQF hTR8V12-4 hTR8D2 hTRBJ2-1 hTRBC 404 4 S 17 11 DG06 SSVSVY YLSGSTLV AVSEGGGSYIPT hTRAV8-6 * hTRAJ6 hTRAC 15860 2 S 18 5 C02/DA04/ LNHDA SQIVND ASSLGTFYEQY hTR8V19 hTR8D1 hTR8J2-7 hTRBC 900 3 S 19 12 DC07/ DSASNY IRSNVGE AASGNTNAGKST hTRAV13-1 * hTRAJ27 hTRAC 5387 7 S 20 20 DE01/ DH10 7 C03/E05/ SGHRS YFSETQ ASSLGVMVVS hTRBVS-1 * hTRBJ2-3 hTRBC 1244 12 U 21 13 E08/F03/ TDTQV F08/ TISGTDV GLTSN ILRDRVSNF hTRAV26-2 * hTRAJ4B hTRAC 11109 6 S 22 DF10/ GNEKLT DG07 1 A06 SGHAT FQNNGV ASSPTQGASYEQY hTRBV11-2 hTRBD1 hTRBJ2-7 hTRBC 4927 2 S 23 14 VSGNPY YITGDNLV AVRDAGYSTLT hTRAV3 * hTRAJ11 hTRAC 3557 10 S 24 1 DC02 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2-S hTRBC 10415 1 S 3 15 DSASNY IRSNVGE AASGNTNAGKST hTRAV13-1 * hTRAJ27 hTRAC 52 7 S 20 1 G01 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2-5 hTRBC 1437 1 S 3 16 DSASNY IRSNVGE AASIDRDDKII hTRAV13-1 * hTRAJ30 hTRAC 9890 58 U 25 1 E06 MDHEN SYDVKM ATTQGSYNEQF hTR3V28 hTRBD1 hTRBJ2-1 hTRBC 5226 7 S 26 17 VSGLRG LYSAGEE AVQAGSYIPT hTRAV20 * hTRAJG hTRAC 8047 8 S 27

    [0251] The full sequences of these TCRs are shown in FIGS. 28-35.

    [0252] The CD4 T cells that proliferated in response to ALDOA 140-157 Hcit were analysed for their TCR expression in comparison to the non proliferating cells. Examination of TCR clonality of the responding CD4 T cells revealed a bias of TCR Vβ and Vα sequences among CD4+ proliferating cells from donor BD00016 ALDOA 140-157 Hcit (FIG. 36a-d). These responses appear oligoclonal with a couple of dominant TCRVβ and TCRVα chains compared to the non-proliferating CD4s from the same cultures thus suggesting a focussed TCR repertoire.

    [0253] To identify the correct pairing of the TCRVβ and TCRVα chains, single proliferating cells were sorted into 96 individual wells and TCRα and TCRβ chains were sequenced using iPair™ technology. 60/75 wells contained both TCRVβ and TCRVα chains, 66 wells contained sequences identified from bulk analysis. Out of these 20/66 wells contained the TRB chain and 12/66 wells the TRA chain only. 34/66 wells contained both TRA and TRB chains (Table 8). Remaining wells with paired chains contain either a TRA or TRB or both sequence/s of high bulk rank >30 with a frequency of less than <10%.

    TABLE-US-00016 TABLE 8 iPair™ sequencing and analysis of the TCRα and β chains ALDOA 140 Hcit FRE- QUEN- CY (34 CDR1 CDR2 Read Bulk Unique/ SEQ WELLS) WELLS Peptide Peptide CDR3 Peptide V Gene D Gene J Gene C Gene Count Rank shared ID TCR 3 G07/DA04/ EMHRY SYGVKD AISERRDQETQV hTRBV10-3 * hTRBJ2-5 hTRBC 3159 1 S 8 18 DE06 TISGTDY GLTSN ILRDVYDVKLS hTRAV26-2 * hTRAJ20 hTRAC 12015 6 S 4 9 B01/B06/ DFQATT SNEGSKA SARTSGTNTQY Htrbv20-3 hTRBD2 hTRBJ2-3 hTRBC 3305 2 S 28 19 C02/E01/ SSVPPY YTSAATLV AVSGRNDYKLS hTRAV8-4 * hTRAJ20 hTRAC 12349 1 29 E04/E11/ H08/DE09/ DG11 6 B03/C01/ MNHEY SVGEGT ASSRSWTASGVT hTRBV6-3 hTRBD1 hTRBJ1-2 hTRBC 2389 3 S 30 20 C03/DB06/ TISGTDY GLTSN ILRDGSGNEKLT hTRAV26-2 * hTRAJ48 HTRAC 13952 2 S 31 DC05/DC09 2 B08/E12 SGHNS FNNNVP ASSVAQLAGKGE hTRBV12-3 hTRBD2 hTRBJ2-1 hTRBC 3352 5 S 32 21 DSASNY IRSNVGE QFAASIDRDDKI hTRAV13-1 * hTRAJ30 hTRAC 8867 3 S 25 I 3 A1G/D02/ SNHLY FYNNEI ASRRVMGYGVT hTRBV2 * hTRBJ1-2 hTRBC 5484 4 S 33 22 F09 DSAIVN IQSSQRE ALNSGGSNYKL hTRAV21 * hTRAJ53 hTRAC 8520 11 S 34 T 1 F02 DFQATT SNEGSKA SAGRAGTSGTYE hTRBV20-3 hTRBD2 hTRBJ2-7 HTRBC 1569 11 U 35 23 TISGTDY GLTSN QYILRSNP hTRAV26-2 * hTRAJ48 hTRAC 12730 4 S 36 GNEKLT 1 E03 LNHDA SQIVND ASSGGQFNQPQH hTRBV10 hTRBD1 hTRBJ1-5 HTRBC 1303 9 S 37 24 NYSPAY IRENEKE ALGQTGAN hTRAV6 * hTRAJ36 hTRAC 10507 8 U 38 NLF 2 G11/DC02 KGHSH IQKENI ASSPEALANTGELF hTRBV18 * hTRBJ2-2 hTRBC 2473 8 S 39 25 TISGNEY GLKNN IVRVGYNN hTRAV26-1 * hTRAJ43 hTRAC 4309 7 S 40 NDMR 2 DU/DA12 KGHDR SFDVKD ATSDPSGPPYEQY hTRBV24-1 hTRBD2 hTRBJ2-7 hTRBC 480 21 S 41 26 TISGTDY GLTSN ILRAQGGS hTRAV26-2 * hTRAJ57 hTRAC 13807 10 S 42 EKLV 1 B07 SNHLV FYNNEI ASRAGTGIGGVT hTRBV2 hTRBD1 hTRBJ1-2 hTRBC 2294 10 S 43 27 DSAIYN IQSSQRE AVYSGGSNY hTRAV21 * hTRAJ53 hTRAC 12152 17 S 44 RLT

    [0254] The full sequences of these TCRs are shown in FIGS. 37-45.

    [0255] The CD4 T cells that proliferated in response to vimentin 116-135 Hcit were analysed for their TCR expression in comparison to the non proliferating cells. Examination of TCR clonality of the responding CD4 T cells revealed a bias of TCR Vβ and Vα sequences among CD4+ proliferating cells from donor BD00016 ALDOA 140-157 Hcit (FIG. 46a-d). These responses appear oligoclonal with a couple of dominant TCRVβ and TCRVα chains compared to the non-proliferating CD4s from the same cultures thus suggesting a focussed TCR repertoire.

    [0256] To identify the correct pairing of the TCRVβ and TCRVα chains, single proliferating cells were sorted into 96 individual wells and TCRα and TCRβ chains were sequenced using iPair™ technology. 70/80 wells contained both TCRVβ and TCRVα chains, 73 wells contained sequences identified from bulk analysis. Out of these 34/73 wells contained the TRB chain and 9/73 wells the TRA chain only. 30/73 wells contained both TRA and TRB chains (Table 9). Remaining wells with paired chains contain either a TRA or TRB or both sequence/s of high bulk rank >30 with a frequency of less than <10%.

    TABLE-US-00017 TABLE 9 iPair™ sequencing and analysis of the TCRα and β chains for vimentin 116 Hcit FRE- QUEN- CY (30 CDR1 CDR2 CDR3 Read Bulk Unique/ SEQ wells) WELSS Peptide Peptide Peptide V Gene D Gene J Gene C Gene Count Rank shared ID TCR 6 A01/A06/ MDHEN SYDYKM ASSLLGSSPLH hTRBV28 hTRBD2 hTRBJ1-6 hTRBC 236 7 S 1 1 B11/C05/ TSESDY QEAYKQ AYRSYNQGGKLI hTRAV38-2/ * HTRAJ23 hTRAC 17129 1 S 2 E02/H07 Y N DV8 1 D12 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2-5 hTRBC 545 1 S 3 2 TISGTDY GLTSN ILRDVYDYKLS hTRAV26-2 * HTRAJ20 hTRAC 12336 2 S 4 1 G02 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2-5 hTRBC 58 1 S 3 3 TISGTDY QEAYKQQ AYRSYNQGGKLI hTRAV38-2/ * HTRAJ23 hTRAC 3641 1 S 2 N DV8 2 D11/F01 LGHDI YNNKEL ASSQEPSIHNEQF hTRBV3-1 * hTRBJ2-1 hJRbC 822 8 S 5 4 TISGTDY GLTSN ILKNYGGSQGNLI hTRAV26-2 * hTRAJ42 hTRAC 10799 3 S 6 1 D09 MNHNY SVGAGI ASSPGQPYGYT hTRBV6-6 hTRBD1 hTRBJ1-2 hTRBC 972 4 U 7 5 YSGSPE HISR ALSGPSYGQNFV hTRAV16 HTRAJ26 hTRAC 6779 6 S 8 1 C03 MNHNS SASEGT ASEGLASYNEQF hTRBV6-1 hTRBD2 hTRBJ2-1 hTRBC 6119 6 S 9 6 ATGYPS ATKADDK ALTGGGYQKVT hTRAV9-2 * HTRAJ13 hTRAC 3386 169 U 10 2 A05/H06 MNREY SMNVEV ASSFREGEKLF hTRBV27 hTRBD2 hTRBJ1-4 hTRBC 516 61 U 11 7 TSINN IRSNERE ATAMNTGFQKLV hTRAV17 * hTRAJ8 hTRAC 12085 8 S 12 3 B05/ LGHNA YNFKEQ ASSREGLAGLNEQF hTRBV4-2 hTRBD1 hTRBJ2-1 hTRBC 10614 26 S 13 8 E01/F10 NSMFDY LSISSIK AASGWGDGGA hTRAV29/ * HTRAJ32 hTRAC 2057 122 S 14 DK TNKLI DV5

    [0257] The full sequences of these TCRs are shown in FIGS. 47-52.

    Example 6

    Immunisation with Homocitrulline Peptides Provides Efficient Therapy of the Aggressive B16 Melanoma

    [0258] To determine if the epitopes that stimulate T cell responses are presented on MHC-II within the tumour environment, our homocitrullinated peptides were checked for tumour therapy in B16 tumour models. Mice were implanted with tumour cells on day 1 and then immunised with peptides and CpG/MPLA on days 4, 11 and 18.

    [0259] HHDII/DR1 (FIG. 19a) or DR4 (FIG. 19b) transgenic mice were challenged with B16F1 HHDII/DR1 or B16F1 DR4 tumours respectively prior to immunisation with vimentin 116-135Hcit peptide and tumour growth and survival were monitored. HHDII/DR1 mice show significantly enhanced survival of 50% compared to control mice (p<0.0001) and wildtype peptide treated mice (p=0.0051). Wild type peptide with CpG/MPLA did not show a significant anti-tumour response. DR4 mice show a similar response with overall survival of 70% in vimentin 116-135Hcit peptide immunised mice which was significantly increased when compared to control mice (p=0.0042).

    [0260] HHDII/DP4 mice were challenged with B16 HHDII/DP4 tumour cells and then immunised with ALDOA homocitrulline peptides (FIG. 19c). Compared to control mice, the mice treated with either ALDOA 74-93Hcit (90% p<0.0001) or ALDOA 140-157Hcit (60% p=0.0179) showed a significant increase in survival. The combination of both ALDOA homocitrulline peptides also showed significant survival (90% p<0.0001). In HHDII/DR1 mice, immunisation with ALDOA 140Hcit peptide led to a significant increase in survival (p=0.0027) when compared to the control mice (FIG. 19d). ALDOA 74-93Hcit was not tested as it did not induce an immune response in this mouse strain.

    [0261] HHDII/DR1 mice were implanted with B16HHDII/DR1 tumour cells on day 1 and then immunised with Cyk8 371Hcit, 112hcit or both peptides and CpG/MPLA on days 4, 11 and 18. Significant prevention of tumour growth was seen by both the individual peptide vaccinations (p<0.05) and the combination (p<0.01) (FIG. 20).

    [0262] This data suggests that ALDOA 74-93Hcit and 140-157Hcit peptides, the vimentin 116-135 Hcit peptide and Cyk8 371-388Hcit and 112-131Hcit are naturally presented and can be targeted for tumour therapy by CD4 or CD8 T cell responses. However, most melanoma tumour cells do not express MHC class II unless stimulated with IFNγ. To mimic the naturally occurring tumour, we engineered B16F1 cells to express HLA-DR4 under control of mouse IFNγ-inducible promoter. The HLA-DR4 expression level can be upregulated in the presence of mouse IFNγ (Brentville et al. 2016; Cook et al. 2018). HLA-DR4 mice were then implanted with this IFNγ-inducible B16 DR4 tumour followed by immunisation with vimentin 116-135Hcit peptide (FIG. 19e). Mice immunised with the vimentin 116-135Hcit peptide showed a significant enhancement of survival (40%) over unimmunised control mice (p=0.0102). This suggests that a vimentin 116-135 Hcit specific response is able to produce enough IFNγ in vivo to upregulate HLA-DR4 expression in the B16 tumour model and promote an anti-tumour effect.

    [0263] In another similar model, HHDII/DP4 mice were implanted with B16F1 cells expressing HLA-DP4 under control of mouse IFNγ-inducible promoter (B16HHDII/iDP4) on day 1 and then immunised with Cyk8 371Hcit peptide, BiP 526Hcit, Enolase 156Hcit, NPM 266Hcit or Vimentin 86Hcit peptides and CpG/MPLA on days 4, 11 and 18. Significant prevention of tumour growth was seen (p=0.0273, p=0.0027, p=0.0154, p=0.0102 and p=0.0008 respectively) (FIG. 21).

    [0264] This data suggests that homocitrullinated Cyk8 371, BiP 526, Enolase 156, NPM 266 and Vimentin 86 peptides are naturally presented by tumours and can be targeted by CD4 T cell responses.

    Example 7

    Tumour Cells Do Not Express MPO but Neutrophils and MDSCs are a Source of MPO Within the Tumour Environment

    [0265] Having demonstrated that peptides containing homocitrulline can induce tumour therapy we next looked to determine the source of carbamylation in the tumour environment. Our in vitro data suggests that cells cultured in the presence of cyanate can undergo intracellular carbamylation. In some inflammatory conditions cyanate levels are increased as a result of the actions of MPO. Therefore, we assessed MPO expression on tumours. Staining of in vitro cultured tumour lines revealed that cells did not express MPO (FIG. 22a). MPO is known to be expressed by some immune cells, therefore tumours grown in vivo were also assessed for MPO expression. Mice were implanted with B16 tumours which were allowed to grow to 10 mm diameter and then extracted, disaggregated and analysed by flow cytometry. MPO expression was absent from the CD45−ve fraction which includes tumour cells but was present on populations of cells expressing CD11b within the CD45+ve fraction (FIG. 22b). Analysis of markers Ly6C and Ly6G reveals a population of CD11b+MPO+ cells that express either Ly6G and lower levels of Ly6C or no Ly6G and higher levels of Ly6C (FIG. 22b). These populations have been characterised in the literature as granulocytic (G-MDSCs) and monocytic (M-MDSCs), respectively (Rose, Misharin, and Perlman 2012; Bronte et al. 2016). The Ly6G+ population could also contain neutrophils as both G-MDSCs and neutrophils express this marker pattern.

    [0266] To assess if the population of cells within tumour expressing MPO was different from those in the spleen, MPO producing cells were assessed in these tissues. MPO levels were elevate in the tumour when compared to the spleen (FIG. 22c). Staining of splenocytes and tumour infiltrating lymphocytes (TILs) revealed differences in which cell types are producing MPO (FIG. 22d). In the spleen, MPO is predominantly produced by Ly6G+Ly6C.sup.low cells (median 65% of MPO+ cells) with only minimally contribution of Ly6G-Ly6C.sup.hi cells (median 12% of MPO+ cells). In contrast, in the tumour, MPO is produced predominantly by Ly6G+Ly6C.sup.low cells (median 22%) and more Ly6G-Ly6C.sup.hi cells (median 36%) with a smaller contribution of the Ly6G.sup.−Ly6C.sup.low cells. This suggests that in the tumour both the G-MDSC-like and M-MDSC-like populations could contribute to carbamylation. To verify if the M-MDSCs were monocyte or macrophage derived they were stained for the macrophage markers CD115 and F4/80 (FIG. 22e). The Ly6G-Ly6C.sup.hi cells were negative for the macrophage markers suggesting that they indeed myeloid derived.

    [0267] Next, we aimed to determine if these cell populations play a role in carbamylation in the tumour microenvironment and are therefore necessary for the anti-tumour effect. HHDII/DP4 mice were implanted with tumour and then immunised with ALDOA homocitrulline peptides. Mice where then either untreated or treated with Ly6G or Ly6C depletion antibodies to remove either the neutrophils/G-MDSCs or monocytes/M-MDSCs, respectively. Peptide vaccination was associated with 100% survival whereas peptide and Ly6G+ antibody was associated with a slight but significant reduction in the anti-tumour effect (80% survival, p=0.04) (FIG. 23a). However, the combination of vaccination and Ly6G antibody was still significantly better than antibody alone (0% survival, p=0.0017). This suggests that the Ly6G+ population has a small role in the anti-tumour effect.

    [0268] Next, antibodies were used to deplete the Ly6C+ population (FIG. 23b). Ly6C depletion alone increased survival when compared to control (20% survival, p=0.0012). For this study vaccination alone gave 80% survival (p<0.0001). However, the combination of peptide vaccination and Ly6C antibody significantly reduced the survival seen with vaccine alone (survival 40%, p=0.0480). Survival in the group given vaccine and antibody was comparable with survival seen with the Ly6C antibody treatment alone. Staining of tumours after Ly6C antibody treatment shows a decrease in the level of MPO expressing CD45+ cells (FIG. 23c). Together these results indicate a role for Ly6C+ cells in tumours as the source of MPO which in turn leads to carbamylation of proteins in tumours. The results may also suggest that the Ly6C+ population plays a role in promoting tumour growth given that depletion of these cells increases survival.

    [0269] To provide further evidence to support the ability of MDSCs to mediate carbamylation MDSCs were generated in vitro from bone marrow derived cells in the presence of GMCSF and 1L18. Levels of were measured on G-MDSCs and M-MDSCs by flow cytometry staining. In the presence of the media alone or the LPS stimulation both G-MDSCs and M-MDSCs show evidence of MPO production (FIG. 24a). These results indicate a role for MDSCs as a source of MPO which is necessary for carbamylation.

    [0270] In vitro cultured MDSCs were co incubated with B16 tumour cells to determine if carbamylation can be induced within the tumour cells. B16 tumour cells are known to lack expression of MPO but upon co incubation with MPO expressing MDSCs and the additional of the MPO substrates KSCN and H.sub.2O.sub.2 they show increased levels of carbamylation (FIG. 24b). These results indicate that the MPO produced by MDSCs can act to carbamylate proteins within tumour cells

    Example 8

    Tumour Therapy is Mainly Mediated by the Direct Action of CD4 Cells Upon Tumour Cells Presenting Peptide on MHC-II

    [0271] MDSCs appear to be important for carbamylation in the tumour environment, therefore it is possible that vaccine induce CD4 cells do not directly interact with tumour cells. We next aimed to determine whether tumours cells need to present MHC-II in order for immunisation with carbamylated peptides to have an effect on survival. HLA-DR4 transgenic mice were implanted with tumour cells that were not able to express HLA-DR4 (FIG. 25a). In this model, mice immunised with vimentin 116-135 Hcit peptide showed a survival rate of 20%, which is lower than seen against B16F1 DR4 cells, but was still a significant increase over the control (p=0.0010). This suggests that the vimentin 116-135 Hcit response has an indirect effect upon the tumours in the HLA-DR4 model possibly through the recognition of tumour infiltrating APCs and secretion of proinflammatory cytokines. However, in HHDII/DR1 mice implanted with B16F1 HHDII cells that cannot express DR1, immunisation with vimentin 116-135Hcit peptide provided no survival advantage over the control mice (FIG. 25b). Therefore, direct recognition of tumour cells through MHC-II seems to have a major role in the anti-tumour effect, as tumour therapy is dramatically enhanced in models where the appropriate MHC class II molecule is expressed on the tumour. This is also true in the ALDOA immunised mice. The anti-tumour effect of the carbamylated ALDOA peptides was completely lost in a model where tumour cells could not express HLA-DP4 (FIG. 25c). Therefore, these studies provide evidence for the presentation of the carbamylated peptides by tumour cells on MHC class II molecules and the direct recognition of these by the infiltrating CD4 T cells.

    [0272] To provide further evidence for the role of CD4 T cell responses in tumour therapy HHDII/DP4 mice were implanted with B16HHDII/iDP4 tumour cells on day 1 and then immunised with peptides and CpG/MPLA on days 4, 8 and 11. Concurrent with peptide immunisation mice were also treated with CD4 or CD8 depleting antibody and tumour growth monitored. Mice immunised with the aldolase Hcit peptides showed 70% tumour free survival (p<0.0001) compared to 10% survival in control unimmunised mice (FIG. 26). Immunisation with the aldolase wt peptides showed no difference in tumour survival compared to control mice indicating that the tumour therapy response is Hcit specific. Depletion of CD8 T cells had no effect upon the tumour therapy mediated by the aldolase Hcit peptides. However, depletion of CD4 T cells caused a significant loss of tumour therapy (p=0.0124) providing evidence for an essential role of the CD4 T cell response in the tumour therapy mediated by the aldolase Hcit peptides. Therefore, this study provides further evidence for the presentation of the carbamylated peptides by tumour and the direct role of CD4 T cells in tumour therapy.

    [0273] To show whether cyanate/isocyanic acid can cross cell membranes B16F1 melanoma cell line was cultured in vitro in the presence or absence of potassium cyanate which is in dynamic equilibrium with isocyanic acid. As a positive control lysates were also produced from in vitro cultured B16F1 cells and then treated with or without KCNO. Carbamylation was significantly increased in both the whole cell and cell lysates after incubation with KCNO (FIG. 9a). This shows that proteins from whole tumour cells can undergo carbamylation, implying that cyanate/isocyanic acid can cross the cell membrane to induce intracellular carbamylation.

    [0274] B16DP4 tumours were also lysed and analysed by mass spectroscopy for carbamylation of HSP60. K191, K202, K205, K218, K222, K359, K481 and K58 were all carbamylated.

    Example 9

    Homocitrulline Peptides Bind to HLA-DP4

    [0275] A known hepB HLA-DP4 binding peptide and 3 peptides which do not bind to HLA-DP4 were biotinylated and incubated with the HLA-DP4 preparation (FIG. 53a). Biotinylated Hep B bound strongly (OD 0.850), in contrast the negative peptides showed no significant binding over background.

    [0276] To ensure that the biotinylation of Hep B had not interfered with its binding, HLA-DP4 was incubated with equal amounts of biotinylated and unlabelled Hep B peptide. Unlabelled peptide competed equally reducing binding by 47% (FIG. 53b).

    [0277] The biotinylated HepB peptide was then incubated with a 5 fold excess of the unlabelled homocitrulline peptides. All peptides inhibited the binding of biotinylated Hep B to HLA-DP4 but to varying levels (FIG. 53c-e). Aldolase A 74-93Hcit, Aldolase A 140-157 Hcit, Aldolase A 217-235 Hcit, Aldolase A 238-256 Hcit, Cyk8 101-120 Hcit, Cyk8 112-131 Hcit, Cyk8 182-202 Hcit, Cyk8 371-388 Hcit and Cyk8 281-399 Hcit all showed >than 60% inhibition (Table 10). These results suggest that TCRs that recognise HLA-DP4 complexed with any of these peptides would be useful for tumour therapy. All of the Hcit peptides showed strong inhibition of biotinylated Hep B to HLA-DP4 than the wild type peptides with the exception of vimetin 116 which also failed to induce a T cell response in HLA-DP4 transgenic mice.

    TABLE-US-00018 TABLE 10 Competitive binding of homocitrulline containing peptides with Hep B viral peptide to HLA-DP4 Competition % inhibition Biotin Hep-B Biotin Hep-B + HepB 47 Biotin Hep-B + Aldolase A 74-93 Hcit 69 Biotin Hep-B + Aldolase A 74-93 WT 27 Biotin Hep-B + Aldolase A 140- 157 Hcit 76 Biotin Hep-B + Aldolase A 140- 157 WT 29 Biotin Hep-B + Aldolase A 217- 235 Hcit 84 Biotin Hep-B + Aldolase A 238- 256 Hcit 73 Biotin Hep-B + Aldolase A 289- 307 Hcit 45 Biotin Hep-B + Cyk8 101-120 Hcit 71 Biotin Hep-B + Cyk8 112-131 Hcit 64 Biotin Hep-B + Cyk8 182-202 Hcit 61 Biotin Hep-B + Cyk8 371-388 Hcit 72 Biotin Hep-B + Cyk8 281-399 Hcit 60 Biotin Hep-B + Vim 116-135 Hcit 44 Biotin Hep-B + Vim 116-135 WT 48

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