ANTI-TUMOUR RESPONSE TO MODIFIED SELF-EPITOPES
20230192790 · 2023-06-22
Assignee
Inventors
- Linda Gillian Durrant (Nottingham, GB)
- Victoria Anne Brentville (Nottingham, GB)
- Rachael Louise Metheringham (Nottingham, GB)
Cpc classification
G01N2333/978
PHYSICS
G01N2800/52
PHYSICS
C07K14/4748
CHEMISTRY; METALLURGY
G01N2333/47
PHYSICS
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
Anti-tumour immune responses to modified self-epitopes. The present invention relates to the use of tumour-associated epitopes in medicine and in particular in the treatment of cancer. The epitopes stimulate an immune reaction against the tumour and have a modification selected from deimination of arginine to citrulline, nitration of tyrosine, oxidation of tryptophan and deamination of glutamine or asparagine. The invention also relates to nucleic acids comprising sequences that encode such epitopes for use in the treatment of cancer.
Claims
1.-18. (canceled)
19. A tumour-associated T cell epitope which stimulates an immune reaction against the tumour for use in medicine, the epitope having a modification of deimination of arginine to citrulline.
20. A tumour-associated T cell epitope which stimulates an immune reaction against the tumour, optionally and a nucleic acid comprising a sequence that encodes such an epitope, for use in a method of treating cancer, wherein the epitope has a modification of deimination of arginine to citrulline.
21. The epitope for use of claim 20, wherein the nucleic acid is targeted to a cell that expresses a PAD enzyme.
22. The epitope for use of claim 21, wherein the cell that expresses a PAD enzyme is an antigen presenting cell (APC), and the nucleic acid is translated and post-translationally citrullinated within the APC to stimulate T cells that are specific to the citrullinated epitope.
23. The epitope for use of claim 22, wherein the nucleic acid is targeted to an APC by intradermal injection.
24. The epitope for use of claim 21, wherein the nucleic acid does not encode a leader sequence.
25. The epitope for use of claim 19, wherein the epitope is derived from NY-ESO-1, MMP7, cytokeratins, ING4, MUC1, CEA.CAMS, CD59, bcl2, β-catenin, CXCL8, CXCL 10, CXCL 12, α enolase, myelin basic protein, histone, nucleophosmin, B23, co-activator complex, anti-thrombin, aggregan, elongation factor 1α, adenylcyclase associated protein (CAP1), glucose regulated protein, ALDH2, cartilage intermediate layer protein (CLIP), aldolase, phosphoglycerate kinase 1 (PGK1), calreticulin, HSP60, far upstream element-binding proteins 1 and 2 (FUSE-BPs), asporin, cathepsin D, heparin binding protein, β-actin, F-actin, capping protein α-1 subunit (CapZα-1), albumin, ghistamine receptor, protein disulphide-isomerase ER60 precursor, mitochondrial aldehyde dehydrogenase (ALDH2), BiP (GRP78), HSP90 or GSK3β.
26. The epitope for use of claim 19, wherein the epitope is derived from vimentin.
27. The epitope for use of claim 26, wherein the epitope comprises at least one of the following sequences: TABLE-US-00017 YVTTSTRTYSLGSALR, VRLRSSVPG, RLRSSVPGV, and FSSLNLRET, wherein one or more of the R residues is substituted for citrulline.
28. The epitope for use of claim 27, wherein the epitope comprises at least one of the following sequences: TABLE-US-00018 RSYVTTSTRTYSLGSALRPSTS (vim28-49), SAVRLRSSVPGVR (vim65-77) LPNFSSLNLRETNLDSLPL (vim415-433).
29. The epitope for use of claim 26, wherein the epitope comprises at least one of the following sequences: TABLE-US-00019 RSSVPGVRL, SAVRLRSSV, ATRSSAVRL, YATRSSAVRLRSSVPGVRL (vim 61-79), RSSVPGVRL, GVRLLQDSV, RLRSSVPGVRLLQDSVDFS (vim 69-87), QLKGQGKSR, KSRLGDLYE, EQLKGQGKSRLGDLYEEEM (vim 125-154), ELRRQVDQL, EMBELRRQV, DLYEEEMRELRRQVDQLTN (vim 148-166), QLTNDKARV, VEVERDNLA, LTNDKARVE, VDQLTNDKARVEVERDNLA (vim 161-179), EVERDNLAE, NDKARVEVERDNLAEDIMR (vim 166-184), IMRLREKLQ, DNLAEDIMRLREKLQEEML (vim 176-194), QREEAENTL, KLQEEMLQR, EKLQEEMLQREEAENTLQS (vim 187-205), and/or FRQDVDNAS, ENTLQSFRO, EAENTLQSFRQDVDNASLA (vim 198-216), wherein one or more of the R residues is substituted for citrulline.
30. The epitope for use of claim 26, wherein the nucleic acid encodes full length vimentin.
31. The epitope for use of claim 19, wherein the epitope comprises a sequence selected from: TABLE-US-00020 IQKLYGKRS, preferably SQDDIKGIQKLYGKRS (MMP7-247), NILTIRLTAA, preferably PGVLLKEFTVSGNILTIRLTAADHR (NYESO-1-119), ILTIRLTAA, preferably PGVLLKEFTVSGNILTIRLTAADHR (NYESO-1-119), EIRELQSQ, preferably EEEIRELQSQISDTSVVLS (cytokeratin 8 229-247), AKQDMARQLREYQEL, preferably AKQDMARQLREYQELMNVKL (cytokeratin 8: 363-382), AKQDMARQ, preferably LQRAKQDMARQLREYQELM (cytokeratin 8: 360-378), ISSSSFSRV, preferably PGSRISSSSFSRVGSS (cytokeratin 8: 29-44), PRAFSSRS, preferably STSGPRAFSSRSYTSGPG (cytokeratin 8: 13-30), EAALQRAKQ, preferably ELEAALQRAKQDMARQL (cytokeratin 8: 355-371), LEVDPNIQAVRTQE, preferably LEVDPNIQAVRTQEKEQI (cytokeratin 8: 78-95), QKKLKLVRT, preferably AQKKLKLVRTSPEYGMP (ING4 158-174), LKLVRTSPE, preferably AQKKLKLVRTSPEYGMP (ING4 158-174), and KKLKLVRTS, preferably AQKKLKLVRTSPEYGMP (ING4 158-174), YMSSARSLS, MSSARSLSS, TEYMSSARS, preferably KLATEYMSSARSLSSEEK (ING4 44-58) FDLFENRKK, preferably RAPFDLFENRKKKNN (HSP90-346-360) YLNFIRGVV or FIRGVVDSE, preferably IPEYLNFIRGVVDSE (HSP90 378-392) LRYYTSASG, LLRYYTSAS or LSELLRYYT, preferably RKKLSELLRYYTSASGDEMVSL (HSP90-456-477) RRRLSELLRYHTSQS (HSP90 beta 456-460) VGVFKNGRV or FKNGRVEII, preferably YSCVGVFKNGRVEII (BiP39-53) YFNDAQRQA, preferably VPAYFNDAQRQATKDA (BiP 172-186) FEIDVNGIL or VTFEIDVNG, preferably EVTFEIDVNGILRVT (BiP 497-511) ITNDQNRLT, preferably KITITNDQNRL TPEE (BiP 522-536) LQIVARLKN or VARLKNNNR, preferably NCALQIVARLKNNNR (CXCL 12-54-68) VEIIATMKK or RVEIIATMK, preferably CPRVEIIATMKKKGE (CXCL 10 57-71) wherein one or more of the R residues is substituted for citrulline.
32. An epitope consisting of the sequence YVTTSTRTYSLGSALR, optionally consisting of the sequence TABLE-US-00021 RSY-VTTSTRTYSLGSALRPSTS (vim28-49), and/or a nucleic acid encoding such an epitope, for use in a method of treating cancer.
33. The epitope for use of claim 20, wherein the cancer is melanoma, breast, endometrial, colorectal or ovarian cancer.
34. The epitope for use of claim 19, wherein the use is human or veterinary use.
35. A peptide consisting of the amino acid sequence YVTTSTcitTYSLGSALcit, or comprising, or consisting of the amino acid sequence TABLE-US-00022 citSYVTTSTcitTYSLGSALcitPSTS (vim28-49), wherein cit represents citrulline.
Description
EXAMPLES
[0079] The present invention will now be described further with reference to the following examples and the accompanying drawings.
[0080]
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
[0089] HHDII/DRI transgenic mice were injected on day 0 with 2.5×10.sup.4 B16 HHDII NYESO-1 cells. Mice were immunised at day 3, 10 and 17 with antibody DNA hulgGI DNA containing the helper epitope NYESO-1 119-143 in the CDRLI site via gene gun. (i) Responses specific for the helper epitope were analysed ex vivo at day 20 by IFNγ Elispot assay against the wild type, citrullinated NYESO-1 119-143 helper peptides and an irrelevant control. Responses are measured as spots/million splenocytes. (ii) Survival of animals immunised with NYESO-1 antibody DNA alone or with homspera adjuvant or with homspera alone.
[0090]
[0091] HLA-DR4 transgenic mice were immunised with a DNA construct encoding murine vimentin via gene gun or Vim 28-49 and 415-433 citrullinated peptides in CpG/MPLA s.c. once a week for 3 consecutive weeks. On day 19, splenocytes were analysed in vitro against vimentin 28-49 and 415-433 wild type and citrullinated peptides at 5 μM concentration by IFN gamma elispot assay.
[0092]
[0093]
[0094]
[0095]
[0096] HLA-DR4 transgenic mice were immunised with 25 μg of vimentin 415-433 human citrullinated peptide at day 0. On day 14, splenocytes were analysed in vitro against human and murine vimentin citrullinated 415 peptides at 5 μM concentration by (i) IFNγ (ii) IL-17 elispot assays. Responses are measured as average spots/million splenocytes.
[0097]
[0098]
[0099] HLA-DR4 (
[0100]
[0101] HLA-DR4 transgenic mice were immunised with 25 μg murine vimentin 415-433 citrullinated peptide at day 0. T regulatory cell depletion was carried out using an anti-CD25 monoclonal antibody (PC61) three days prior to the immunisation. On day 14, splenocytes were analysed by IFNγ elispot assay against titrating concentrations of citrullinated murine vimentin 415 peptide.
[0102]
[0103] Human citrullinated vimentin 415-433 and 28-49 peptides (25 μg) in Alum, IFA, GMCSF, MPLA and CpG or TMX201 adjuvant was administered s.c. Fourteen days after immunisation splenocytes were analysed for specific responses to the helper epitope by (i) IFNγ, (ii) IL-17 and (iii) IL-IO elispot assays against wild type and citrullinated peptides and an irrelevant control. Responses are measured as spots/million splenocytes.
[0104]
[0105] HLA-DR4 transgenic mice were immunised with 25 μg of human citrullinated vimentin 415 peptide at day 0, 7 and 14. Half of the mice also received anti-CTLA-4 mab at days 7 and 14. On day 21, splenocytes were analysed in vitro against (i) citrullinated vimentin 415 at 5 μM concentration by IFNγ elispot assay. (ii) Avidity of epitope specific responses were measured to increasing peptide concentration in IFNγ elispot assay. Responses are measured as spots/million splenocytes.
[0106]
[0107]
[0108]
[0109]
[0110] (i) HLA-DR4 transgenic mice were immunised on days 0, 7 and 14 with citrullinated human vimentin 415-433 peptide in CPG and MPLA. On day 19, splenocytes were stimulated in vitro with human citrullinated Vimentin 415-433 peptide pulsed blasts. Six days post stimulation CTL lines were assessed by chromium release assay for ability to lyse DR4 splenocytes pulsed with citrullinated human Vimentin 415-433 peptide, T2 DR4 tumour cells pulsed with citrullinated human vimentin 415-433 peptide and T2 DR4 cells alone. Responses are measured as % cytotoxicity. P values indicated on graph are for the target to effector ratio 10:1. P values for 20:1 and 40:1 are all highly significant P<0.0001.
[0111] (ii) HHD/DR1 transgenic mice were immunised on days 0, 7 and 14 with citrullinated human vimentin 28-49 peptide in CPG and MPLA. On day 19, splenocytes were stimulated in vitro with human citrullinated vimentin 28-49 peptide pulsed blasts. Six days post stimulation CTL lines were assessed by chromium release assay for ability to lyse T2 DR1 tumour cells pulsed with citrullinated human vimentin 28-49 peptide, T2 DR1 cells alone and T2 cells alone. Responses are measured as % cytotoxicity. P values indicated on the graph are significant and for the target to effector ratio 12.5:1.
[0112] P values for 25:1 (T2DR1 P=0.0017, T2 DR1+vim28 cit P<0.0001) and 50:1 (T2DR1
[0113] P=0.0008, T2 DR1+vim28 cit P=0.0005) are all highly significant.
[0114]
[0115] HLA-DR4 transgenic mice were injected on day 0 with 2.5×10.sup.4 B16F1-DR4 cells. (
[0116]
[0117] HLA-DR4 transgenic mice were injected on day 0 with 2.5×10.sup.4 B16F1-DR4 cells. Mice were immunised sc with (
[0118]
[0119] HLA-DR4 transgenic mice were injected on day 0 with 2.5×10.sup.4 B16F1 cells (
[0120]
[0121]
[0122]
[0123]
[0124] Human 415 vimentin peptide (25 μg) in MPLA and CPG adjuvant was administered s.c. Fourteen days after immunisation splenocytes were analysed for specific responses to the helper epitopes by (i) IFNγ, (ii) IL-17 and (iii) IL-10 elispot assays against helper peptide and an irrelevant control in HLA-DR4 mice. Responses are measured as spots/million splenocytes.
[0125]
[0126] Citrullinated ING4 peptide 158-174 peptide (25 μg) in MPLA and CPG adjuvant was administered s.c on days 0, 7 and 14. Seven days after the last immunisation splenocytes were analysed for specific responses to the helper epitopes by IFNγ elispot assays against helper peptide in the presence or absence of an HLA-DR blocking monoclonal antibody in HLA-A2/DR1 mice. Responses are measured as spots/million splenocytes.
[0127]
[0128]
[0129]
[0130] Methods
[0131] 2.1. Commercial mAbs
[0132] The primary rabbit anti-human Vimentin (clone EPR3776), rabbit anti-human PAD2 (clone pab0197), rabbit anti-human citrulline (clone ab6464) were all purchased from Abeam. The primary rabbit anti-human PAD-4 (clone pab 0199) was obtained from Covalab and the anti-human HLA-DR PE-Cy7 conjugated antibody (clone L243) from eBioscience. Anti-CD25 antibody (clone PC61), anti-IFNγ antibody (clone XMGI.2), anti-IL-17 (clone 17F3) antibody and anti-CD4 (clone GKI.5) antibody were purchased from BioXcell. Anti-CTLA4 antibody was purified from HB304 hybridoma cells culture supernant (ATCC, USA) by sephorose protein G affinity chromatography. Anti-HLA-DR antibody (clone L243) was purified from HB-55 hybridoma cells (ATCC, USA) culture supernatant by sepharose protein G affinity chromatography. Rabbit mAb anti HMGB1 (clone D3E5) was purchased from Cell Signaling Technology.
[0133] 2.2. Cell Lines
[0134] The T cell/B cell hybrid cell line T2 [80] stably transfected with functional class II DR4 (DRB1*0401; T2 DR4) or DR1 (DRB1*0101;T2DR1) have been described [82, 83] and was kindly provided by Dr. Janice Blum and Professor Lawrence Stern. The murine melanoma B16F1 and B16F10 cell lines were obtained from the ATCC. All cell lines were cultured in RPMI medium 1640 (GIBCO/BRL) supplemented with 10% FCS, L-glutamine (2 mM) and sodium bicarbonate buffered unless otherwise stated. HB304 hybridoma cells were cultured in Hybridoma SFM (Invitrogen, UK).
[0135] To generate tumour targets presenting citrullinated epitopes for in vitro assays cells were treated with 0.1M citric acid (pH3.0) containing 1% BSA at 4° C. for 2 mins. Cells were subsequently washed with media and cultured in absence of serum for 20 hrs at 37° C. Autophagy and PAD inhibitors, 3-methyladenine (Sigma) and CI-amidine (Calbiochem), were added for the 20 hr culture in serum free media at final concentrations of 10 mM and 50 μg/ml respectively.
[0136] 2.3. Immunogens
[0137] 2.3.1. Peptides
[0138] Peptides >90% purity were synthesized by Peptide Synthetics (Fareham, UK). Stored lyophilized in 0.2 mg aliquots at −80° C. On day of use they were reconstituted to the appropriate concentration in 10% dimethyl formamide.
[0139] 2.4. Plasmids
[0140] Generation of antibody DNA constructs have been described in detail elsewhere [84]. In brief to generate the antibody DNA constructs, epitopes were incorporated into complementary determining regions of the heavy and light variable regions of the antibody chains using standard molecular biological techniques. The HLA-DR4 restricted helper CD4 epitopes from the epitopes tyrosinase 448-462 (DYSYLQDSDPDSFQD) (SEQ ID NO: 89), the murine and human gp100 44-59 epitope (WNRQLYPEWTEVQGSN (SEQ ID NO: 90)/WNRQLYPEWTEAQRLD) (SEQ ID NO: 91) and the I-Ab restricted epitope from HepB nucleoprotein 128-140 (TPPAYRPPNAPIL) (SEQ ID NO: 92) were inserted in replacement of CDRL1 of the kappa chain. Similarly, the HLA-DR1 restricted helper CD4 epitopes from the epitopes MMP7 247-262 (SQDDIKGQKLYGKRS) (SEQ ID NO: 93), SSX2 33-48 (KEEWEKMKASEKIFY) (SEQ ID NO: 94), NYESO-1 87-111 (LLEFYLAMPFATPMEAELARRSLAQ) (SEQ ID NO: 95) and 119-143 (PGVLLKEFTVSGNILTIRLTAADHR) (SEQ ID NO: 4) were incorporated into CDRL1 while the modified epitope from epitope triosephosphate isomerise 23-37 (GELIGILNAAKVPAD) (SEQ ID NO: 96) was inserted in replacement of CDRL3 of the kappa chain. The HLA-DR4 restricted CD4 vimentin 415-433 (LPNFSSLNLRETNLDSLPL) (SEQ ID NO: 97) and HLA—DR1 28-49 (RSYVTTSTRTYSLGSALRPSTS) (SEQ ID NO: 98) restricted epitopes were both incorporated into CDRH3. The DR1 CD4 NYESO-1 87-111(LLEFYLAMPFATPMEAELARRSLAQ) (SEQ ID NO: 95) was also encoded within the extended sequence 83-111 cloned into the CDRH3 site of the antibody DNA double expression vector. The human IgG1 and murine IgG2a ImmunoBody vectors containing all three vimentin epitopes were also generated. The HLA-DR1 28-49(RSYVTTSTRTYSLGSALRPSTS) (SEQ ID NO: 98), 65-77 (SAVRLRSSVPGVR) (SEQ ID NO: 59) and the HLA-DR4 restricted human CD4 vimentin 415-433 (LPNFSSLNLRETNLDSLPL) (SEQ ID NO: 97) epitopes were incorporated into the CDRH1, CDRH2 and CDRH3 sites respectively.
[0141] The plasmid pVax1Murine Vimentin full length was generated by amplification of the full length sequence using as a template cDNA from mRNA that had been isolated from the B16F1 cell line. Forward and reverse primers utilised were designed to incorporate a HindIII and BamHI site respectively. On amplification and confirmation of wild type sequence full length murine vimentin was incorporated into the HindIII/BamHI sites of the multiple cloning site within the mammalian expression vector pVaxI (Invitrogen).
[0142] To generate the plasmid pVitro 2 Chimeric HLA-DR401 cDNA was generated from mRNA isolated from the splenocytes of transgenic HLA-DR4 mice. This was used as a template to amplify the chimeric alpha and beta chains separately using forward and reverse primers that incorporated a fspI/EcoRI and BamHI/SalI sites respectively. On sequence confirmation full length chimeric alpha chain comprising of murine H2-Ea with human HLA-DRA alpha 1 domain was ligated into the fspI/EcoRI mcs2 of the vector pVITRO2-hygro-mcs (Invivogen). The beta chain comprising of murine H2-Eb with human DRB1*0401 Beta 1 domain was then inserted into the BamHI/SalI mcs1 of the vector alongside the chimeric alpha chain.
[0143] To generate the HHD plasmid cDNA was synthesized from total RNA isolated from EL4-HHD cells. This was used as a template to amplify HHD using the forward and reverse primers and subcloned into pCR2.1. The HHD chain, comprising of a human HLA-A2 leader, the human B2 microglobulin molecule covalently linked via a glycine serine linker to the α 1 and 2 domains of human HLA-0201 MHC class1 molecule and the α3, transmembrane and cytoplasmic domains of the murine H-2db class 1 molecule, was then inserted into the EcoRV/HindIII sites of the mammalian expression vector pCDNA3.1 obtained from invitrogen.
[0144] To construct the mammalian double expression plasmid that encodes murine Tap2 and NYESO-1 full length chains, NYESO-1 was amplified from the IMAGE clones 40146393 obtained from geneservice with forward and reverse primers that incorporated a BamH1/XhoI site respectively. On sequence confirmation full length NYESO-1 was ligated into the BamHI/XhoI multiple cloning site of the antibody DNA double expression vector in replacement of the light chain. Murine Tap2 was amplified from the image clone 6530488 after removal of a HindIII site from encoding sequence and incorporation of this site before the start codon, and cloned into the expression vector pOrigHIB using HindIII/EcoRV. Murine Tap2 was then transferred in replacement of the heavy chain using HindIII/AvrII into the double expression vector alongside full length NYESO-1.
[0145] In order to knockdown expression of murine B2 microglobulin and murine MHC class II in the cell line B16F10 RNA interference was utilized. Complimentary oligos that target sequence 266 of murineB2 microglobulin and 159 of murine MHC class II were annealed and inserted separately into pCDNA6.2 GW miR (Invitrogen). The pre-miRNA expression cassette containing miRNA 266 was excised using BamHI/XhoI and ligated into the XhoI/BglII site of pCDNA6.2 GW miR 159 in order to chain the two miRNA's and express them in one primary transcript within the same vector.
[0146] Endotoxin free plasmid DNA was generated using the endofree Qiagen maxiprep kit (Qiagen, Crawley).
[0147] 2.5. Transfection
[0148] B16F10 cells were transfected successively using Lipofectamine transfection reagent with expression vectors encoding full length NY-ESO-land Murine Tap2, HHDII and a siRNA to knockdown expression of murine MHC class II and murine (32 microglobulin. Transfected cells were selected by growth in the presence of Zeocin (300 μg/ml), G418 (500 μg/ml) and Blasticidin (4 μg/ml) respectively. Lines were cloned by limiting dilution and expression was confirmed by flow cytometry.
[0149] B16F1 cells were transfected using the Lipofectamine transfection reagent (Invitrogen) with 4 μg of the plasmid pVitro 2 Chimeric HLA-DR401 that encodes both full length chimeric alpha and beta chains according to the manufacturer's instructions. Transfected cells were selected by growth in the presence of Hygromycin B (200 μg/ml). Lines were cloned by limiting dilution and expression was confirmed by flow cytometry using the anti-human HLA-DR PE-Cy7 conjugated antibody (clone L243) from eBioscience.
[0150] 2.6 HLADR0401 Binding Studies
[0151] In brief, peptides of interest were mixed with a predetermined concentration biotinylated HA.sub.306-318 reference peptide at increasing concentrations and added to plate bound HLA DR0401. Amounts of biotinylated reference peptide binding to HLA DR0401 was quantified using streptavidin linked enzyme step followed by detection with chromogenic substrate. Maximal binding is taken as the value achieved by biotinylated HA 306-318 peptide alone. As a positive control unlabelled HA 306-318 peptide was used to compete with the biotinylated version.
[0152] 2.7. Immunisations
[0153] 2.7.1. Immunisation Protocol C57BL/6 mice (Charles River, UK), HLA-DR4 mice (Taconic, USA), HHDII mice (Pasteur institute, France) and HHDII/DRI mice (Pasteur institute, France) were used, aged between 8 and 12 weeks, and cared for by the staff at Nottingham Trent University. All work was carried out under a Home Office project license. Peptides were dissolved in 10% Dimethylformamide to 1 mg/ml and then emulsified (a series of dilutions) with different adjuvants: CpG and MPLA 6 μg/mouse of each (Invivogen, UK), Incomplete Freund's SOW/mouse (Sigma, UK), and GMCSF 10 μg/mouse (Peprotech, UK). Peptides (25 μg/mouse) were injected subcutaneously at the base of the tail. DNA (1 μg/mouse) was coated onto 1.0 μm gold particles (BioRad, Hemel Hempstead, UK) using the manufacturer's instructions and administered intradermally by genegun (BioRad). Homspera (10 nM/mouse) (PeptideSynthetics, UK) was injected intradermally with genegun immunisation. Mice were immunized at either day 0 for peptide immunisation or days, 0, 7, and 14 for peptide and genegun immunisation. Spleens were removed for analysis at day 14 for peptide and day 20 for peptide or genegun immunisation unless stated otherwise. 400 μg Anti-CD25 antibody (PC61) was administered i.p. in saline 3 days prior to the immunisation. 200 μg Anti-CTLA-4 antibody (UC 10-4F, 10-11) was administered i.p. in saline at day 7 and 14 with either genegun or peptide immunisation.
[0154] For tumour challenge experiments mice were challenged with 2.5×10.sup.4 B16 HHDII NYESO/TAP2 siβ2m 1F10 cells or B16 DR4 2E7 cells subcutaneously on the right flank 3 days prior to primary immunisation and then were immunised as above. Anti-IFNγ antibody (300 μg/dose), anti-IL-17 antibody (200 μg/dose) and anti-HLA-DR antibody (300 μg/dose) were administered i.p. in saline at days 2, 7, 11 and 14 post tumour implant. Anti-CD4 antibody (500 μg/dose) was administered i.p.in saline at days 2 and 8 post tumour implant. Tumour growth was monitored at 3-4 days intervals and mice were humanely euthanized once tumour reached ≥10 mm in diameter.
[0155] 2.8. Analysis of Immune Response
[0156] 2.8.1. Ex Vivo Elispot Assay
[0157] Elispot assays were performed using murine IFNγ, IL-17 and IL-10 capture and detection reagents according to the manufacturer's instructions (Mabtech, Sweden). In brief, anti-IFNγ, IL-17 and IL-10 antibodies were coated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at a variety of concentrations) and 5×10.sup.5 per well splenocytes were added to the wells of the plate in triplicate. Tumour target cells were added where relevant at 5×10.sup.4/well in triplicate and plates incubated for 40 hrs at 37° C. After incubation, captured IFNγ, IL-2, IL-17 and IL-10 were detected by biotinylated anti-IFNγ, IL-17 and IL-10 antibodies and developed with a strepatavidin alkaline phosphatase and chromogenic substrate. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd). Functional avidity was calculated as the concentration mediating 50% maximal effector function using a graph of effector function versus peptide concentration.
[0158] 2.8.2 Ex Vivo Depletion of CD8 and CD4 Cells from Splenocyte Cultures
[0159] Splenocytes were subject to positive isolation of CD4 or CD8 cells using antibody coated magnetic beads (Miltenyi Biotech) according to manufacturer's instructions. For MEW class II blocking studies 20 μg/ml anti-HLA-DR (clone L243) antibody was added to elispot assays.
[0160] 2.8.3 Granzyme B ELISA
[0161] Supernatant from ex vivo IFNγ elispot assays on splenocytes was removed after 40 hrs and assessed for Granzyme B by elisa assay (R&D systems) according to manufacturer's instructions.
[0162] 2.8.4 Luminex Multiplexed Assay
[0163] A three-step indirect procedure was used for the multiplexed Luminex assay (Invitrogen) for IgG antibodies to IL-10, IL-17, IFNγ, TNFα, IL-2 & IL-4. Standard, control, and unknown sera were diluted 1:2 in 50% assay diluent buffer (Invitrogen) & 50% serum free RPMI. Serial standard dilutions were included in each assay. Each dilution of standard, control, and unknown sera was mixed with a set of coupled Luminex microspheres in 96-well filtration plates (Millipore Multiscreen; Millipore Corporation, Bedford, Mass.) and incubated for 2 hours at room temperature with shaking. Microspheres were collected by vacuum filtration and washed with PBST. Biotinylated detector antibody was added to each well for 1 hour at room temperature with shaking. Microspheres were collected by vacuum filtration and washed with PB ST. Streptavidin conjugated R-phycoerythrin—was added to each well. Following a 30 min incubation and a wash step, microspheres were resuspended in PBST, and read in a Biorad BioPlex Luminex analyzer equipped with an XY platform. Data acquisition and analysis performed with Luminex software (BioPlex Systems).
[0164] 2.8.5 Proliferation Assay
[0165] PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque (Sigma) gradient centrifugation. PBMC (1.5×10.sup.6 cells/well) were stimulated with single peptides (final concentration 10 μg/ml) in RPMI containing 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPES and Penicillin-streptomycin (1%) in a final volume of 2 ml. Stimulation with purified protein derivative, PPD (final concentration 10 μg/ml) served as a positive control for the proliferative capacity of PBMC. As a negative control PBMC were incubated with medium alone. The PBMC were cultured at 37° C. in an atmosphere of 5% CO.sub.2 for 4, 7 and 11 days. To assess proliferation at these times points 100 μl in triplicate from each culture was aliquoted into a round bottom well of a 96 well plate and .sup.3H-thymidine added (0.0185 MBq/well) and incubated at 37° C. for a further 8 hours. The cultures were harvested onto unifilter plates and incorporation of .sup.3H-thymidine was determined by β-scintillation counting. The results were assessed by calculating the stimulation index (SI) as the ratio of the mean of counts per minute (cpm) of epitope-stimulated to the mean of unstimulated cultures. The proliferative assay was considered positive when SI >2.5.
[0166] 2.8.6 .sup.51Cr-Release Assay
[0167] Target cells were labelled for 1 hr with 1.85 MBq sodium (.sup.51Cr) chromate (Amersham, Essex, UK) with or without 10 μg/ml peptide. Post incubation they were washed 3 times in RPMI. Targets 5×10.sup.3/well of a 96-well V-bottomed plates were set up and co incubated with different densities of effector cells in a final volume of 200 μl of RPMI, 10% FCS (Sigma), 20 mM HEPES buffer, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. After 20 hrs at 37° C., 50 μl of supernatants were removed from each well and transferred to a Lumaplate (Packard, Rigaweg, the Netherlands). Plates were read on a Topcount Microplate Scintillation Counter (Packard). Percentage specific lysis was calculated using the following formula: specific lysis=100×[(experimental release-spontaneous release)/(maximum release-spontaneous release)].
[0168] 2.9 Immunohistochemical Analysis
[0169] Tissue array sections were first deparaffinised with xylene, rehydrated through graded alcohol and immersed in methanol containing 0.3% hydrogen peroxide for 20 mins to block endogenous peroxidase activity. In order to retrieve antigenicity, sections were immersed in 500 ml of pH6.0 citrate buffer and heated for 20 mins on the 6.sup.th sense setting of a microwave. Endogenous avidin/biotin binding was blocked using an avidin/biotin blocking kit (Vector Labs). In order to block non-specific binding of the primary antibody, all sections were then treated with 100 μl of 1/5 normal horse serum (NETS) in PBS for 15 mins. Test sections were incubated with 100 μl of primary antibody diluted in PBS for 1 hr at 22° C. or overnight at 4° C. Positive control tissue comprised whole sections of colorectal cancer tissue stained with 02-microglobulin at 1/1000 dilution (in PBS; Dako). The primary antibody was omitted from the negative control, which was left incubating in NHS. After washing with PBS, all sections were incubated with 100 μl of biotinylated goat anti-mouse/rabbit immunoglobulin (Dako) diluted 1:100 in NHS, for 30 mins. Sections were washed again in PBS and incubated with 100 μl of pre-formed streptavidin-biotin/HRP complex (Dako) for 60 mins at room temperature (RT). Subsequently, visualisation of epitope expression was achieved using DAB. Finally, sections were lightly counterstained with haematoxylin (Dako), dehydrated in alcohol, cleared in xylene (GentaMedica, York, UK) and mounted with distyrene, plasticizer and xylene (DPX; BDH).
[0170] Evaluation of staining: In order to allow permanent storage of the slides, they were imaged at ×20 using a NanoZoomer 2.0 slide imaging system (Hamamatsu, Higashi-ku, Japan). Expression of markers on the tissue was analysed using the images in the NanoZoomer Digital Pathology Virtual Slide Viewer (Hamamatsu). Screening of marker expression was performed concurrently by two investigators with previous experience of scoring, blinded to the clinical information. For H score, cores were briefly analysed and representative cores of negative, weak, moderate and strong cores were used as guides for the whole tissue micro-array (TMA). As well as the intensity of the staining, the percentage of positively stained tumour cells was estimated. The two scores were then combined to form the H score, where H=percentage cells stained X intensity (range=0-300). Using the NanoZoomer Slide Viewer, the area of both tumour and stroma were measured and number of positive cells in each area counted. A value of positive cells per mm.sup.2 was then calculated.
[0171] 2.9.1. Colorectal Tumour TMA
[0172] Antisera were Screened for Tumour Binding on a Gastric Cancer TMA.
[0173] Patient study and design: The study population comprised a series of 462 consecutive patients undergoing elective surgical resection of a histologically proven sporadic primary colorectal cancer at the University Hospital, Nottingham, UK (Table 1). These patients were treated between 1st January 1994 and 31st December 2000; this time period allowed meaningful assessment of the prognostic markers studied. All patients treated during this time-frame were considered eligible for inclusion in the study. Tumours were classified as mucinous carcinoma, when more than 50% of tumour volume consisted of mucin.
TABLE-US-00008 TABLE 1 Clinicopathological variables for colorectal TMA patient cohort (n = 462) Frequency of total Variable Categories cohort (%) Gender Male 266 (58) Female 199 (42) Age (years) Median 72 Range 58-93 Status Alive 169 (37) Dead 293 (63) Tumour Grade Well differentiated 29 (6) Moderately differentiated 353 (77) Poorly differentiated 71 (15) Unknown 8 (2) Tumour Site Colon 238 (52) Rectum 181 (39) Unknown 43 (9) TNM Stage 0 (T.sub.is) 3 (1) 1 69 (15) 2 174 (28) 3 155 (33) 4 54 (12) Unknown 7 (2) Extramural Negative 224 (48) Vascular Invasion Positive 128 (28) Unknown 110 (24) Histological Type Adenocarcinoma 392 (85) Mucinous carcinoma 51 (11) Columnar carcinoma 4 (1) Signet ring carcinoma 7 (1) Unknown 8 (2)
[0174] Clinicopathology: Only cases where the relevant pathological material was unavailable were excluded from the study. Follow-up was calculated from time of resection of the original tumour with all surviving cases being censored for data analysis at 31st December 2003, this produced a median follow up of 37 months (range 0-116) for all patients and 75 months (range 36-116) for survivors.
[0175] A prospectively maintained database was used to record relevant clinicopathological data, with data provided from the UK Office for National Statistics; this was available in more than 99% of cases. The information collected was independently validated through case note review of deceased patients. Disease specific survival was used as the primary end point; however, data was also collected on the various other relevant clinical and histopathological parameters these are summarised in Table 2.3. Adjuvant chemotherapy consisting of FOLFOX was reserved for those patients with positive lymph nodes, although, surgical and adjuvant treatment was at the discretion of the supervising physician. Prior ethical review of the study was conducted by the Nottingham Local Research and Ethics Committee, who granted approval for the study.
[0176] Construction of the array blocks incorporated a wide spectrum of electively resected colorectal tumours and was found to be broadly representative of the colorectal cancer population in the UK. 266 (58%) patients were male and 196 (42%) female. The median age at the time of surgery was 72 years, consistent with a median age at diagnosis of colorectal cancer of 70-74 years in the UK [85]. 69 (15%) tumours arrayed were tumour, node metastasis (TNM) stage 1, 174 (38%) stage 2, 155 (34%) stage 3 and 54 (11%) stage 4; there were 3 cases of in-situ disease. These figures are comparable with national figures for distribution of stage 1-4 at diagnosis of 11, 35, 26 and 29% respectively [85]. The majority of tumours (392, 85%) were adenocarcinomas, and were most frequently of a moderate histological grade (353, 77%). 128 (28%) tumours were noted to have histological evidence of extramural vascular invasion, 224 (48%) had no evidence of vascular invasion, and this information was not available in 110 (24%) cases. At the time of censoring for data analysis 228 (49%) patients had died from their disease, 64 (14%) were deceased from all other causes, and 169 (37%) were alive. The median five-year disease-specific survival for the cohort was 58 months, comparable with the national average of approximately 45% five-year survival for colorectal cancer in the UK [86].
[0177] 2.9.2. Ovarian Cancer TMA
[0178] Antisera were screened for tumour binding on an ovarian cancer TMA. The ovarian cancer TMA represents a cohort of 362 patients with primary ovarian cancer treated at Nottingham University Hospitals between 2000 and 2007. Staging of the cancers was performed using the International Federation of Obstetrics and Gynaecology (FIGO) criteria. All patients included in this study were treated according to the current standard chemotherapy regimens with either single agent carboplatin in 65 patients (41.4%) or platinum-based combination chemotherapy in 89 patients (56.7%), with 3 patients refusing chemotherapy. Platinum-resistant cases were defined as patients who progressed on first-line platinum chemotherapy during treatment or who relapsed within 6 months after treatment. All patients underwent surgery; over 44% of cases (n=69) were deemed to be sub optimally debulked (tumour remaining <1 cm) after initial surgery. Patients were followed-up by physical examination, computed tomography, and CA-125 levels. Haematoxylin and eosin-stained sections from the tumours of these patients were reviewed by a gynaepathologist blinded to the clinical data and pathological diagnosis. For each tumour, a review of its type and differentiation was also carried out by SD. Clinical data associated with each case was collected and recorded from the patients' notes or via the hospital's electronic records (NotIS). Such information included: patients' age at diagnosis, FIGO stage, extent of surgical cyto-reduction, and the type, duration and response to chemotherapy. Details of adjuvant treatment, disease-specific survival (DSS) and overall survival (OS) were documented for all patients. Survival was calculated from the operation date until 30th of May 2008 when any remaining survivors were censored. Median follow up was 36 months. Ethical approval to collect the samples and relevant data for the study was granted by the Nottinghamshire Local Research Ethics Committee.
[0179] 2.9.3. Normal Tissue TMA
[0180] The normal tissue TMA contained 59 cores representing 38 normal organs. Each core was categorised according to the origin of the sample; normal tissue from a non-cancer patient, normal tissue from a cancer patient, but the cancer involves an unrelated organ, normal tissue adjacent to the cancer. The tissue type and category is detailed in Table 2.
TABLE-US-00009 TABLE 2 Details of normal tissue TMA and tissue type Tissue type Age Gender Placenta 29 F Esophagus 23 M Rectum 24 F Gallbladder 24 M Skin 83 F Adipose 26 M Heart 27 M Skeletal 26 M Bladder 36 F Ileum 62 M Spleen 30 M Brain 68 M Jejunum 56 M Stomach 66 M Breast 27 F Kidney 56 M Testis 32 M Cerebellum 73 F Liver 30 M Thymus 28 M Cervix 30 F Lung 24 M Smooth 23 M Muscle Colon 28 M Ovary 50 F Tonsil 28 F Diaphram 26 M Pancreas 50 M Uterus 40 F Duodenum 24 M Thyroid 26 M
Example 1. CD4 Responses in Wild Type Mice to Self and Foreign Epitopes
[0181] T cell responses to tumour associated epitopes are often weak or non-existent due to tolerance and T cell deletion within the thymus. Nonetheless we screened a variety of self and foreign CD4 epitopes for their ability to stimulate helper responses. As previous studies have shown that antibody DNA constructs gave the strongest immune responses [84], a variety of CD4 foreign and self-epitopes were incorporated into separate constructs and screened in wild type mice. Table 3 lists the sequences of all the epitopes and their mouse homologs where appropriate.
TABLE-US-00010 TABLE 3 CD4 epitopes SYMPATHEI SEQUENCE COORDINATE CORE SCORE HLA Self Murine WNRQLYPEWTEVQGSN 44-59 LYPEWTEV 26 DR4 gp100 (SEQ ID NO: 90) Q (SEQ ID NO: 99) Vimentin SAVRLRSSVPGVR 65-77 VRLRSSVP 23 DR1 65-77 (SEQ ID NO: 59) G (SEQ ID NO: 57) Vimentin RSYVTTSTRTYSLGSALR 28-49 TYSLGSAL 27/20 DR1 28-49 PSTS (SEQ ID NO: 98) R (SEQ ID DR4 NO: 100) YVTTSTRT Y (SEQ ID NO: 101) Self in core regions Vimentin LPNSSLNLRETNLDSLPL 415-433 FSSLNLRE 26 DR4 415-433 (SEQ ID NO: 61) T (SEQ ID NO: 60) ING4 AQKKLKLVRTSPEYGMP 158-174 QKKLKLV 22 DR1 158-174 (SEQ ID NO: 21) RT (SEQ ID 20 DR4 NO: 20) LKLVRTSP E (SEQ ID NO: 22) KKLKLVRT S (SEQ ID NO: 23) Foreign in core regions Human WNRQLYPEWTEAQRLD 44-59 LYPEWTEA 26 DR4 gp100 (SEQ ID NO: 91) Q (SEQ ID NO: 102) MMP-7 SQDDIKGIQKLYGKRS 247-262 IQKLYGKR 35 DR1 247-262 (SEQ ID NO: 2) S (SEQ ID NO: 1) Tyrosinase DYSYLQDSDPDSFQD 448-462 YLQDSDPD 22 DR4 (SEQ ID NO: 89) S (SEQ ID NO: 103) mTPI GELIGILNAAKVPAD (SEQ 23-37 IGILNAAKV 36 DR1 ID NO: 96) (SEQ ID NO: 104) NYESO-1 LLEFYLAMPFATPMEAELA 87-111 FYLAMPFAT 26/22 DR1 87-111 RRSLAQ (SEQ ID NO: 95) (SEQ ID NO: 105) NYESO-1 PGVLLKEFTVSGNILTIRLT 119-143 ILTIRLTAA 23/22 DR1 119-143 AADHR (SEQ ID NO: 4) (SEQ ID DR4 NO: 5) NILTIRLTA (SEQ ID NO: 106) SSX2 34-48 KEEWEKMKASEKIFY 34-48 WEKMKASEK 26 DR1 (SEQ ID NO: 94) (SEQ ID NO: 107) Hepatitis B TPPAYRPPNAPIL 128-140 YRPPNAPIL I-Ab Nucleoprotein (SEQ ID NO: 92) (SEQ ID (helper) NO: 108) Arginine residues are underlined Non homologous residues are in italics
[0182]
Example 2. DNA Immunisation Result in Responses to Citrullinated Vim 28, Vim 415, MMP7, and NY-ESO-1
[0183] RA patients have been shown to make T cell responses to citrullinated vimentin epitopes. As APCs can constitutively citrullinate epitopes it was possible that the antibody DNA constructs were being citrullinated and this was stimulating the response. HLA-DR4 transgenic mice were therefore immunised with an antibody-DNA construct encoding the self-vim 28 epitope. Stimulated T cells from these mice were screened in vitro for IFNγ, IL-17 and IL-2 responses to both citrullinated and uncitrullinated vim 28 peptide.
[0184] In contrast, when MMP7 247-262 was incorporated into a DNA vaccine (
[0185] When mice were injected with B16 HHDII NYESO-1 tumour cells and then immunised with NYESO-1 119 incorporated into an antibody-DNA vaccine (
[0186] As antigen presenting cells can constitutively citrullinate epitopes it was interesting to see if citrullinated self-epitope specific responses could be induced using full length antigen delivered as a DNA vaccine. HLA-DR4 transgenic mice were therefore immunised with DNA construct encoding the whole murine vimentin antigen. Stimulated T cells from these mice were screened in vitro for IFNγ responses to both citrullinated and uncitrullinated vim 28-49 and 415-433 peptides.
[0187] These results suggest that epitopes from the antibody-DNA constructs are being citrullinated and that the T cells recognising these modified peptides have not been deleted/anergised.
Example 3. Peptide Immunisation Results in Responses to Citrullinated Vim 28, Vim 415 and Vim 65
[0188] To determine if this was restricted to DNA vaccines or whether the citrullinated peptides could also stimulate this repertoire, mice were immunised with wild type and citrullinated vim 28-49 and citrullinated vim 415-433 peptides in combination with CpG and MPLA adjuvants.
[0189]
[0190]
Example 4. Determination of Whether CD4 Responses to Self-Epitopes are a Naïve, Memory or Treg Response
[0191] The mice made a potent IFNγ and IL-17 response to a single immunisation of human vim 415 cit suggesting that it was boosting a memory or a Treg response. To determine if this was a natural Treg response that was being converted to an IFNγ/IL-17 response, mice were depleted of natural Tregs with anti-CD25 mAb and immunised with mouse vim 415 cit. Natural Treg depletion had no influence on the frequency or the avidity of the response suggesting that natural Tregs were not the responding population (
[0192] Anti-CTLA-4 mabs can block the interaction of CTLA-4 with its cognate receptor CD80/86 thus preventing the inhibition of T cells induced by this ligand. Immunisation of mice with vim 415 cit peptide in the presence of an anti-CTLA-4 mab significantly increased the avidity of the T cell response from 10.sup.−6M to 10.sup.−8M (
Example 5. Cancer Patients Response to Self-Peptides
[0193] Nine melanoma patients were screened for their responses to a series of self-peptides (
TABLE-US-00011 TABLE 4 Haplotypes of cancer patients Pt019 A2 A3 B7 B55 DR7 DR16 Pt020 A2 B27 B40 DR3 DR13 Pt021 A3 A25 B44 B35 DR1 DR13 Pt023 A2 A3 B7 B35 DR1 DR15 Pt028 A2 A24 B7 B35 DR1 DR13 Pt029 A2 A25 B15 DR15 Pt032 A1 A11 B51 B18 DR15 Pt033 B7 DR11 DR15 Pt034 A11 B7 B35 DR1 DR4 Pt035 A29 A30 B50 DR1 DR7
Example 6. Expression of PAD Enzymes, Vimentin and Citrulline
[0194] Citrullination is carried out by PAD enzymes and in particular the PAD2 and PAD4 enzymes. These require high levels of calcium and are usually activated in dead or dying cells. It therefore seemed unlikely that healthy tumours cells would express citrullinated proteins. Colorectal and ovarian tumours and normal tissues were therefore stained for vimentin, citrullination and expression of the PAD2 and PAD4 enzymes.
[0195] Normal Tissues
[0196] Expression of vimentin, PAD2, PAD4 and citrulline is shown for normal tissues in Table 5.
[0197] Mesenchymal cells such as connective tissue cells, blood cells and neuronal cells all express vimentin as a cytoskeletal protein. Most of the cells within spleen, thyroid, testes, cervix, ovary, tonsils, uterus, lung, thymus and breast stained strongly for vimentin. Weak staining of less than 50% of cells was seen in placenta, rectum, colon, pancreas and the duodenum skeletal and smooth muscle, gall bladder, oesophagus, kidney, liver, bladder, ileum, jejunum, stomach. No staining was observed in skin, adipose tissue, skeletal muscle, rectum, brain, cerebellum, diaphragm or heart.
[0198] The majority of liver cells stained strongly with anti-PAD2 mAb. The majority of smooth muscle cells and brain cells stained weakly. Over half of the cells within the cerebellum, pancreas and testes stained strongly with PAD2. Less than 50% of the cells within gall bladder, ileum, jejunum, stomach, and colon stained strongly for PAD2. Whereas oesophagus, rectum, skeletal muscle, bladder, breast, kidney, placenta, heart, diaphragm, duodenum thyroid and lung cells stained less than 50% of their cells weakly. Skin, adipose tissue, spleen, tonsils, thymus, ovary and uterus were negative.
TABLE-US-00012 TABLE 5 Expression of Vimentin, PAD2, PAD4 and citrullinated proteins in normal tissues Vimentin PAD-2 PAD-4 Anti-citrulline HMGB1 Intensity Area Intensity Area Intensity Area Intensity Area Intensity Area Placenta + 25-50% + 25-50% + 25-50% − 0% + 75-100% Oesophagus + 1-25% + 25-50% + 25-50% +/− 1-25% + 50-75% Rectum − − + 25-50% − − +/− 1-25% −/+ 25-50% Gallbladder + 1-25% ++ 1-25% + 1-25% +/− 1-25% + 75-100% Skin − − − − − − +/− 75-100% −/+ 1-25% Adipose − − − − − − − 0% − 0% Heart − − + 25-50% + 50-75% +/− 75-100% −/+ 1-25% Skeletal + 1-25% + 1-25% − − +/− 1-25% + 75-100% Muscle Bladder + 1-25% + 1-25% − − +/− 1-25% + 50-75% Ileum + 25-50% ++ 1-25% + 1-25% +/− 1-25% + 50-75% Spleen ++ 75-100% − − − − − − + 75-100% Brain − − + 75-100% + 75-100% +/− 75-100% −/+ 25-50% Jejunum + 1-25% ++ 1-25% − − +/− 25-50% −/+ 25-50% Stomach + 1-25% ++ 25-50% ++ 1-25% − − −/+ 0-25% Breast ++ 50-75% + 1-25% + 25-50% + 50-75% + 25-50% Kidney + 1-25% + 1-25% − − +/− 75-100% −/+ 1-25% Testis ++ 75-100% ++ 50-75% + 50-75% + 75-100% −/+ 25-50% Cerebellum − 0% ++ 50-75% ++ 75-100% ++ 75-100% ++ 75-100% Liver + 1-25% ++ 75-100% ++ 75-100% ++ 75-100% + 75-100% Thymus ++ 50-75% − − − − + 1-25% + 50-75% Cervix + 75-100% + 1-25% − − − − −/+ 0-25% Lung + 50-75% + 25-50% − − + 75-100% + 25-50% Smooth + 1-25% + 75-100% − − + 75-100% −/+ 0-25% Muscle Colon ++ 25-50% ++ 50-75% ++ 25-50% + 25-50% + 50-75% Ovary + 75-100% − − − − − − −/+ 0-25% Tonsil ++ 75-100% − − − − − − + 50-75% Diaphragm − − + 1-25% + 1-25% + 1-25% −/+ 0-25% Pancreas + 1-25% ++ 50-75% − − + 75-100% + 50-75% Uterus ++ 75-100% − − − − + 1-25% −/+ 0-25% Duodenum ++ 25-50% + 25-50% + 25-50% ++ 50-75% + 50-75% Thyroid ++ 75-100% + 25-50% + 1-25% ++ 25-50% −/+ 25-50%
[0199] The majority of liver and cerebellum cells stained strongly with anti-PAD4 mab. The majority of brain and heart cells stained weakly. Less than 50% of the cells within colon and stomach stained strongly for PAD4 whereas less than 50% of the cells within ileum, diaphragm, duodenum, thyroid, testes, breast, gallbladder, oesophagus stained weakly. Skin, skeletal and smooth muscle, bladder, spleen, jejunum, kidney, liver, cervix, lung, ovary, tonsils, pancreas, uterus, rectum, adipose tissue, thymus and uterus were negative.
[0200] Cerebellum and liver stained strongly with anti-citrulline mab. The majority of skin, heart, kidney lung, pancreas and brain cells stained weakly. Greater than 50% of duodenum stained strongly and greater than 50% of breast and testes cells stained weakly. Less than 50% of the cells within the thymus and colon and jejunum stained weakly. Less than 25% of cells within the thymus stained strongly and greater than 25% within oesophagus, rectum, gallbladder, skeletal muscle, bladder, ileum, thymus, tonsil, diaphragm, and uterus stained weakly. Spleen, skin, stomach, ovary, tonsil, adipose tissue, placenta and cervix were negative for citrulline.
[0201] Ovarian Tumours
[0202] Ovarian tumours are of mesenchymal origin and would therefore be expected to express vimentin. PAD4 is expressed weakly by normal ovary. 219 ovarian tumours were stained with a vimentin specific mAb. Only 9/219 (4%) of tumours failed to stain, a further 16/219 (7%) stained weakly, whereas 194/219 (89%) stained strongly. Kaplan Meier survival analysis showed there was no correlation with vimentin expression and survival. There was a weak correlation between expression of vimentin and the stress related protein ULBP1 (p=0.017), PAD4 (p=0.018) and CEA-CAM4 (p=0.033).
[0203] 219 ovarian tumours were stained with a PAD4 specific mAb (
[0204] Although tumours express PAD4 it should only be activated in dying cells. To assess if this is true ovarian tumours were stained with an anti-citrulline peptide specific mAb. Only 7/228 (3%) of tumours failed to stain, a further 34/228 (15%) stained weakly whereas 187/228 (82%) stained strongly. However, not all of the cells within a tumour stained. In 69/228 (30%) less than 25% of cells stained. 83/228 (36%) stained between 25-50% of cells and, as previously, these were mainly of stromal origin. In 53/228 (23%) 50-75% of the cells stained including some epithelial cells and in 16/228 (7%) of tumours greater than 75% of cells stained. Kaplan Meier survival analysis showed there was no correlation with citrulline expression and survival. There was a correlation between intensity of expression of citrulline and CEA-CAMS (p=0.037), BCL2 (p=0.011) and Lewis.sup.y (p=0.053). There was a correlation between percentage of cells expressing citrulline and grade (p=0.034), BCL2 (p=0.035), CD59 (p=0.049) and ULBP1 (p=0.044).
[0205] 360 ovarian tumours were stained for PAD2. 9% could not be evaluated due to the absence of enough tissue core or no evaluable tumour cells (i.e. all stroma) in the core. Of the 329 evaluable ovarian tumours stained with a PAD2 specific mAb, all tumours expressed PAD2. A further 277/329 (84%) stained weakly, 52/329 (16%) stained strongly. Kaplan Meier (
[0206] 360 ovarian tumours were stained for HMGB1. 10% could not be evaluated due to the absence of enough tissue core or no evaluable tumour cells (i.e. all stroma) in the core. Of the 316 evaluable Ovarian tumours stained with a HMGB-1 specific mAb, only 23/360 (7%) tumours failed to stain. A further 42/316 (13%) stained weakly, 52/329 (87%) stained strongly. Kaplan Meier (
[0207] When tumour cell expression of high and low PAD2 was compared with high and low HMGB1 expression (
[0208] Colorectal Tumours
[0209] Colorectal tumours are of epithelial origin and are not expected to express vimentin unless they are undergoing epithelial to mesenchymal transition. Expression of PAD2 and PAD4 was seen in normal colon.
[0210] 282 colorectal tumours were stained with a vimentin specific mAb. Only 25/282 (9%) of tumours failed to stain, a further 4/282 (1%) stained weakly whereas 253/282 (90%) stained strongly. However not all of the cells within a tumour stained. 114/282 (40%) less than 25% of cells stained and these were all stromal cells. 68/282 (24%) between 25-50% of cells stained and again these were mainly of stromal origin. 42/282 (15%) 50-75% of the cells stained including some epithelial cells and in 33/282 (12%) of tumours greater than 75% of cells stained. Kaplan Meier survival analysis showed that there was no correlation with vimentin intensity or percentage of cells stained and survival. There was a correlation between expression of vimentin and TRAIL R2 (p=0.003), IL-17 in tumours (p=0.021), MUC1 p=0.001), PAD2 (p=0.025) and PAD4 (p=<0.0001).
[0211] 296 colorectal tumours were stained with a PAD2 specific mAb (
[0212] 291 colorectal tumours were stained with a PAD4 specific mAb (
TABLE-US-00013 TABLE 6 Means for Survival Time of colorectal cancer patients expressing PAD4. Mean(a) Intensity of 95% Confidence Interval staining Estimate Std. Error Upper for PAD4 Lower Bound Upper Bound Lower Bound Bound .00 44.813 10.483 24.267 65.358 1.00 74.140 4.262 65.786 82.494 2.00 78.125 4.632 69.047 87.203 Overall 74.391 3.104 68.307 80.475 (a)Estimation is limited to the largest survival time if it is censored.
[0213] a Estimation is limited to the largest survival time if it is censored.
[0214] Tumour stage and vascular invasion also correlate with patient survival. In a multivariate model TNM stage (p=<0.0001), vascular invasion (p=<0.0001) and PAD4 expression (p=0.017) where independent predictors of patient survival.
[0215] There was a correlation between expression of PAD4 and BCL2 (p=0.01), β-catenin (p=0.001), number of CD8 T cells (p=0.006), MUC1 (p=0.000), CEA.CAM5 (p=0.000), CD59 (p=0.038) vimentin (p=0.000) and PAD2 (p=0.000).
[0216] Although tumours express PAD4, it should only be activated in dying cells. To assess if this is true colorectal tumours were stained with an anti-citrulline peptide specific mAb. All of the tumours stained 41/316 (13%) stained weakly whereas 275/316 (87%) stained strongly. Kaplan Meier survival analysis showed there was a weak correlation with citrulline expression and survival (p=0.078). There was a correlation between expression of citrulline and radiation therapy (p>0.001).
Example 7. Anti-Tumour Responses to Citrullinated Peptides
[0217] Both mice and humans show responses to citrullinated self-peptides and DNA vaccines. However, if these epitopes are not citrullinated in tumours then T cells will have no anti-tumour activity.
[0218] As human tumours express vimentin, PAD2/4 and citrulline the anti-tumour response of citrullinated vimentin was assessed in a mouse model. Splenocytes from mice immunised with both citrullinated vimentin 415-433 and 28-49 peptides were assessed for ability to respond to B16 tumour cells in vitro.
[0219] Mice immunised with either vim 415 cit or vim 28 cit were assessed for their ability to kill T2 tumour cells transfected with human DR4 in vitro.
[0220] HLA/DR4 transgenic mice were implanted with B16 tumours transfected with DR4. They were immunised with vim 415-433 citrullinated peptide or DNA vaccine encoding vim 415-433 sequence and tumour growth was monitored. Mice immunised with either vim 415-433 cit peptide or vim 415-433 encoded within a DNA vaccine stimulate strong anti-tumour responses (
[0221] Both vim 415-433 and vim 28-49 citrullinated peptides induce high frequency IFNγ responses. Blockade of IFNγ in vivo abrogates both vim 415-433 and vim 28-49 citrullinated peptide specific anti-tumour responses (
[0222] To determine the importance of direct tumour recognition by vim 415-433 and vim 28-49 cit specific responses mice were challenged with the B16 tumour lacking expression of HLA-DR0401 and subsequently immunised with Vim 415-433 or vim 28-49 citrullinated peptides. Mice immunised with vim 28-49 citrullinated peptides show delayed tumour growth and enhanced survival compared to control (
Example 8. Homology of Vimentin Between Different Species
[0223] Vimentin is highly conserved between chicken, mouse, dog sheep, cows, horse, pig and humans (
Example 9. Vimentin Responses Restricted Through Other HLA Haplotypes
[0224] HLA-DR4 mice made a potent IFNγ and IL-17 response to a single immunisation of human vim 415 cit peptide. To determine if this or other citrullinated vimentin epitopes could induce immune responses in other haplotypes a range of 20 mer peptides (table 8) covering the whole span of vimentin and incorporating every arginine replaced with citrulline residues was screened for IFNγ/IL-17 responses in HLA-DR1 and C57/B1 mice (
[0225]
TABLE-US-00014 TABLE 7 Citrullinated vimentin IFNγ/IL-17 responses Amino acid Significant Significant position response response within Core in HLA- in C57B1 Antigen sequence Peptide regions DR1 mice mice Vimentin 1-19 MSTRSVSSSSYRRMFGGPG − − 1 (SEQ ID NO: 109) Vimentin 3-22 RSVSSSSYRRMFGGPGTAS − − 2 (SEQ ID NO: 110) Vimentin 14-32 MFGGPGTASRPSSSRSYVT − − 3 (SEQ ID NO: 111) Vimentin 19-33 GTASRPSSSRSYVTTSTRT − − 4 (SEQ ID NO: 112) Vimentin 26-44 SSRSYVTTSTRTYSLGSAL − − 5 (SEQ ID NO: 113) Vimentin 36-54 RTYSLGSALRPSTSRSLYA − − 6 (SEQ ID NO: 114) Vimentin 41-59 GSALRPSTSRSLYASSPGG − − 7 (SEQ ID NO: 115) Vimentin 55-66 SSPGGVYATRSSAVRLRSS − − 8 (SEQ ID NO: 116) Vimentin 61-79 YATRSSAVRLRSSVPGVRL RSSVPGVRL + − 9 (SEQ ID NO: 65) (SEQ ID NO: 62) SAVRLRSSV (SEQ ID NO: 63) ATRSSAVRL (SEQ ID NO: 64) Vimentin 69-87 RLRSSVPGVRLLQDSVDFS RSSVPGVRL + − 10 (SEQ ID NO: 68) (SEQ ID NO: 62) GVRLLQDSV (SEQ ID NO: 67) Vimentin 91-109 AINTEFKNTRTNEKVELQE − − 11 (SEQ ID NO: 117) Vimentin 103-121 EKVELQELNDRFANYIDKV − − 12 (SEQ ID NO: 118) Vimentin 113-131 RFANYIDKVRFLEQQNKIL − − 13 (SEQ ID NO: 119) Vimentin 125-154 EQLKGQGKSRLGDLYEEEM QLKGQGKSR + − 14 (SEQ ID NO: 71) (SEQ ID NO: 69) KSRLGDLYE (SEQ ID NO: 70) Vimentin 148-166 DLYEEEMRELRRQVDQLTN ELRRQVDQL + − 15 (SEQ ID NO: 74) (SEQ ID NO: 72) EMRELRRQV (SEQ ID NO: 73) Vimentin 161-179 VDQLTNDKARVEVERDNLA QLTNDKARV + + 16 (SEQ ID NO: 78) (SEQ ID NO: 75) VEVERDNLA (SEQ ID NO: 76) LTNDKARVE (SEQ ID NO: 77) Vimentin 166-184 NDKARVEVERDNLAEDIMR EVERDNLAE − + 17 (SEQ ID NO: 80) (SEQ ID NO: 79) Vimentin 176-194 DNLAEDIMRLREKLQEEML IMRLREKLQ − + 18 (SEQ ID NO: 82) (SEQ ID NO: 81) Vimentin 187-205 EKLQEEMLQREEAENTLQS QREEAENTL − + 19 (SEQ ID NO: 85) (SEQ ID NO: 83) KLQEEMLQR (SEQ ID NO: 84) Vimentin 198-216 EAENTLQSFRQDVDNASLA FRQDVDNAS − + 20 (SEQ ID NO: 88) (SEQ ID NO: 86) ENTLQSFRQ (SEQ ID NO: 87) Vimentin 211-229 DNASLARLDLERKVESLQE − − 21 (SEQ ID NO: 120) Vimentin 262-280 KPDLTAALRDVRQQYESVA − − 22 (SEQ ID NO: 121) Vimentin 295-312 FADLSEAANRNNDALRQAK − − 23 (SEQ ID NO: 122) Vimentin 301-319 AANRNNDALRQAKQESTEY − − 24 (SEQ ID NO: 123) Vimentin 311-329 QAKQESTEYRRQVQSLTCE − − 25 (SEQ ID NO: 124) Vimentin 334-352 KGTNESLERQMREMEENFA − − 26 (SEQ ID NO: 125) Vimentin 355-373 AANYQDTIGRLQDEIQNMK − − 27 (SEQ ID NO: 126) Vimentin 370-388 QNMKEEMARHLREYQDLLN − − 28 (SEQ ID NO: 127) Vimentin 392-410 ALDIEIATYRKLLEGEESR − − 29 (SEQ ID NO: 128) Vimentin 401-419 RKLLEGEESRISLPLPNFS − − 30 (SEQ ID NO: 129) Vimentin 415-433 LPNFSSLNLRETNLDSLPL − − 31 (SEQ ID NO: 61) Vimentin 431-449 LPLVDTHSKRTLLIKTVET − − 32 (SEQ ID NO: 130) Vimentin 441-459 TLLIKTVETRDGQVINETS − − 33 (SEQ ID NO: 131) Arginine residues substituted with citrulline are underlined
Example 10. How to Screen for Citrullinated T Cell Epitopes
[0226] Any citrullinated protein that has been described in the literature can be a potential target for T cells. However, it must first have the capacity to be presented on MHC class I and/or class II MHC antigens and it must be recognised by a T cell receptor. Antigen presenting cells constitutively undergo autophagy and it is within these double membrane autophagosomes that sufficient intracellular Ca.sup.2+ can accumulate to active PAD enzymes, citrullinate epitopes which are then presented on MHC antigens. Finally, to be an anti-tumour target, tumour cells must also induce citrullination within autophagosomes and present the same modified epitope on MHC antigens. Therefore, to identify citrullinated epitopes that can still stimulate anti-tumour immunity, it is necessary to screen target proteins for induction of T cell responses and for tumour recognition.
[0227] a) In Vitro T Cell Proliferation of Human Peripheral Blood by Citrullinated Peptides.
[0228] Human peripheral blood can be stimulated in vitro as outlined in Example 5. Citrullinated 20mer peptides spanning the whole protein can be screened for T cell proliferation. Sorting of CD4 and CD8 T cells can identify CD4 and CD8 epitopes and the HLA restriction can be identified by HLA typing the donor.
[0229] b) Stimulate Cells from Conventional or HLA Transgenic Mice In Vitro or In Vivo with 20 Mer Citrullinated Peptides which Span the Whole of a Target Protein
[0230] To determine if citrullinated cytokeratin-8 epitopes could induce immune responses, a range of 20 mer peptides (Table 8) covering the whole span of cytokeratin-8 and incorporating arginines in the predicted core binding region replaced with citrulline residues were screened for IFNγ/IL-17 responses in HLA-DR1 and C57 BI mice (
TABLE-US-00015 TABLE 8 Citrullinated cytokeratin IFNγ/IL-17 responses Significant Significant response in response HLA-DR1 in C57B1 Antigen Coordinates Peptides Core regions mice mice Cytokeratin 229-247 EEEIRELQSQISDTSVVLS EIRELQSQI + − 1 (SEQ ID NO: 7) (SEQ ID NO: 132) Cytokeratin 363-382 AKQDMARQLREYQELMNVKL ARQLREYQE + − 2 (SEQ ID NO: 9) (SEQ ID NO: 133) AKQDMARQL (SEQ ID NO: 134) Cytokeratin 360-378 LQRAKQDMARQLREYQELM AKQDMARQL + − 3 (SEQ ID NO: 11) (SEQ ID NO: 134) ARQLREYQE (SEQ ID NO: 133) Cytokeratin 324-342 LKGQRASLEAAIADAEQRG − − 4 (SEQ ID NO: 135) Cytokeratin 239-257 ISDTSVVLSMDNSRSLDMD − − 5 (SEQ ID NO: 136) Cytokeratin 137-156 DNMFESYINNLRRQLETLGQ − − 6 (SEQ ID NO: 137) Cytokeratin 388-407 IATYRKLLEGEESRLESGMQ − − 7 (SEQ ID NO: 138) Cytokeratin 264-281 KAQYEDIANRSRAEAESM − − 8 (SEQ ID NO: 139) Cytokeratin 460-478 AVVVKKIETRDGKLVSESS − − 9 (SEQ ID NO: 140) Cytokeratin 99-117 NNKFASFIDKVRFLEQQNK − − 10 (SEQ ID NO: 141) Cytokeratin 202-221 EAYMNKVELESRLEGLTDEI − − 11 (SEQ ID NO: 142) Cytokeratin 208-226 VELESRLEGLTDEINFLRQ − − 12 (SEQ ID NO: 143) Cytokeratin 29-44 PGSRISSSSFSRVGSS ISSSSFSRV + − 13 (SEQ ID NO: 13) (SEQ ID NO: 12) Cytokeratin 13-30 STSGPRAFSSRSYTSGPG GPRAFSSRS + − 14 (SEQ ID NO: 15) (SEQ ID NO: 144) Cytokeratin 174-193 DFKNKYEDEINKRTEMENEF − − 15 (SEQ ID NO: 145) Cytokeratin 355-371 ELEAALQRAKQDMARQL EAALQRAKQ + − 16 (SEQ ID NO: 17) (SEQ ID NO: 16) Cytokeratin 78-95 LEVDPNIQAVRTQEKEQI NIQAVRTQE + − 17 (SEQ ID NO: 19) (SEQ ID NO: 146) DPNIQAVRT (SEQ ID NO: 147) Cytokeratin 297-316 KHGDDLRRTKTEISEMNRNI − − 18 (SEQ ID NO: 148) Arginine residues substituted to citrulline are underlined
[0231] c) Screen Known Citrullinated Epitopes for T Cell Responses
[0232] ING4 protein is citrullinated by PAD4 at position 133 in its NLS region. This prevents its association with p53 which is essential for p53 activation. Citrullinated ING4 protein is rapidly degraded suggesting it may be abundantly expressed on MHC class II. ING4 peptide AQKKLKLVRTSPEYGMP (SEQ ID NO: 21) failed to stimulate any immune response in HLA-DR1 transgenic mice but AQKKLKLVcitTSPEYGMP (SEQ ID NO: 162) stimulated an IFNγ/IL-17 response (mean 200 spots/million splenocytes) to the citrullinated peptide but no response to the wild type peptide.
[0233] The response to the citrullinated peptides was blocked with a MHC class II blocking mab (
[0234] d) Screen any Protein for Citrullinated T Cell Epitopes.
[0235] As we have shown in example 2, immunising with DNA encoding a whole antigen results in responses to citrullinated peptides. Here we show that if we immunise with DNA encoding whole antigen and screen against all possible citrullinated 20mer peptides only T cell responses to the citrullinated epitopes presented by the HLA molecules stimulate an immune response. The panel of citrullinated peptides is detailed in Table 7.
[0236] e) Screen Predicted Peptides Selected Based on MHC Binding Score and Arginine Residues within the Core Region.
[0237] We have shown that responses can be induced to known citrullinated epitopes but here we also demonstrate that peptide epitopes can be selected based on predicted MHC binding scores and presence of arginine residues within the core MHC binding region. For this example peptides were selected from BiP, HSP90, CXCL10, CXCL12 and ING4 that had high predicted binding to HLA-DR4 using the SYFPEITHI prediction algorithm (www.syfpeithi.de) and then further restricted through the presence of arginines in the core binding region (determined using IEDB prediction algorithm (www.iedb.org) (Table 9). Peptides containing all arginines changed to citrulline were tested.
TABLE-US-00016 TABLE 9 Predicted HLA-DR4 peptides Response SYMPATHEI in HLA- SEQUENCE COORDINATE CORE SCORE DR4 mice HSP90 346- RAPFDLFENRKKKNN 346-360 FDLFENRK 28 + 360 (SEQ ID NO: 29) K (SEQ ID NO: 28) HSP90 378- IPEYLNFIRGVVDSE 378-392 YLNFIRGV 28 − 392 (SEQ ID NO: 32) V, (SEQ ID NO: 30) FIRGVVDS E (SEQ ID NO: 31) HSP90 456- RKKLSELLRYYTSA 456-477 LRYYTSAS 26/22/20 + 477 SGDEMVSL G, (SEQ ID (SEQ ID NO: 36) NO: 33) LLRYYTSA S, (SEQ ID NO: 34) LSELLRYY T (SEQ ID NO: 35) HSP90beta RRRLSELLRYHTSQS 546-470 26 456-470 (SEQ ID NO: 37) BiP 39-53 YSCVGVFKNGRVEII 39-53 VGVFKNG 26 + (SEQ ID NO: 40) R (SEQ ID NO: 150) FKNGRVEI I (SEQ ID NO: 151) BiP 172-186 VPAYFNDAQRQATKDA 172-186 YFNDAQR 28 + (SEQ ID NO: 42) QA (SEQ ID NO: 41) BiP 522-536 KITITNDQNRLTPEE 522-536 ITNDQNRL 26 - (SEQ ID NO: 46) T (SEQ ID NO: 45) ING4 44-58 KLATEYMSSARSLSSEEK 44-58 YMSSARSL 26/22 + (SEQ ID NO: 27) S, (SEQ ID NO: 24) MSSARSLS S, (SEQ ID NO: 25) TEYMSSAR S (SEQ ID NO: 26) CXCL10 57-71 CPRVEIIATMKKKGE 57-71 VEIIATMK 26 − (SEQ ID NO: 52) K, (SEQ ID NO: 50) RVEIIATM K (SEQ ID NO: 51) CXCL12 54-68 NCALQIVARLKNNNR 54-68 LQIVARLK 26 − (SEQ ID NO: 49) N, (SEQ ID NO: 47) VARLKNN NR (SEQ ID NO: 48)
[0238] HLA-DR4 transgenic mice were immunised on up to three occasions with citrullinated peptides and responses assessed ex vivo by IFNg elispot against relevant citrullinated and unmodified peptides.
[0239] To prove that the T cells recognise tumours, they can be screened for recognition of tumour target cells, acid stripped to encourage MHC recycling and serum starved to induce autophagy (
Example 11
[0240] Previous studies have shown that it is possible to determine the differentiation of naïve CD4 cells to different helper phenotypes depending upon their cytokine milieu present when they are stimulated. In contrast, we have shown for the first time in
REFERENCES
[0241] 1. Gao, F. G., et al., Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell. Cancer Res, 2002. 62(22): p. 6438-41. [0242] 2. Janssen, E. M., et al., CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature, 2003. 421(6925): p. 852-6. [0243] 3. Fearon, E. R., et al., Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell, 1990. 60(3): p. 397-403. [0244] 4. Baxevanis, C. N., et al., Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J Immunol, 2000. 164(7): p. 3902-12. [0245] 5. Cella, M., et al., Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med, 1996. 184(2): p. 747-52. [0246] 6. Ridge, J. P., F. Di Rosa, and P. Matzinger, A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature, 1998. 393(6684): p. 474-8. [0247] 7. Schoenberger, S. P., et al., T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature, 1998. 393(6684): p. 480-3. [0248] 8. Ayyoub, M., et al., An immunodominant SSX-2-derived epitope recognized by CD4+ T cells in association with HLA-DR. J Clin Invest, 2004. 113(8): p. 1225-33. [0249] 9. Halder, T., et al., Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3. Cancer Res, 1997. 57(15): p. 3238-44. [0250] 10. Pardoll, D. M. and S. L. Topalian, The role of CD4+ T cell responses in antitumor immunity. Curr Opin Immunol, 1998. 10(5): p. 588-94. [0251] 11. Topalian, S. L., MHC class II restricted tumor antigens and the role of CD4+ T cells in cancer immunotherapy. Curr Opin Immunol, 1994. 6(5): p. 741-5. [0252] 12. Muranski, P., et al., Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood, 2008. 112(2): p. 362-73. [0253] 13. Paludan, C., et al., Epstein-Barr nuclear antigen 1-specific CD4(+) Th1 cells kill Burkitt's lymphoma cells. J Immunol, 2002. 169(3): p. 1593-603. [0254] 14. Quezada, S. A., et al., Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J Exp Med, 2010. 207(3): p. 637-50. [0255] 15. Xie, Y., et al., Naive tumor-specific CD4(+) T cells differentiated in vivo eradicate established melanoma. Journal of Experimental Medicine, 2010. 207(3): p. 651-667. [0256] 16. Brandmaier, A. G., et al., High-avidity autoreactive CD4+ T cells induce host CTL, overcome T(regs) and mediate tumor destruction. J Immunother, 2009. 32(7): p. 677-88. [0257] 17. Lauwen, M. M., et al., Self-tolerance does not restrict the CD4+ T-helper response against the p53 tumor antigen. Cancer Res, 2008. 68(3): p. 893-900. [0258] 18. Touloukian, C. E., et al., Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice. J Immunol, 2000. 164(7): p. 3535-42. [0259] 19. Mohanan, S., et al., Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis. Biochem Res Int, 2012. 2012: p. 895343. [0260] 20. Nomura, K., Specificity and mode of action of the muscle-type protein-arginine deiminase. Arch Biochem Biophys, 1992. 293(2): p. 362-9. [0261] 21. Stensland, M. E., et al., Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4. Biol Chem, 2009. 390(2): p. 99-107. [0262] 22. Klareskog, L., et al., Immunity to citrullinated proteins in rheumatoid arthritis. Annual Review of Immunology, 2008. 26: p. 651-675. [0263] 23. Migliorini, P., et al., The immune response to citrullinated antigens in autoimmune diseases. Autoimmunity Reviews, 2005. 4(8): p. 561-564. [0264] 24. Nijenhuis, S., et al., Autoantibodies to citrullinated proteins in rheumatoid arthritis: clinical performance and biochemical aspects of an RA-specific marker. Clinica Chimica Acta, 2004. 350(1-2): p. 17-34. [0265] 25. Sebbag, M., et al., Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins. European Journal of Immunology, 2006. 36(8): p. 2250-2263. [0266] 26. Vossenaar, E. R., et al., Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin. Arthritis Res Ther, 2004. 6(2): p. R142-50. [0267] 27. Vossenaar, E. R., et al., Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages. Annals of the Rheumatic Diseases, 2004. 63(4): p. 373-381. [0268] 28. Birnboim, H. C., et al., Cutting edge: MHC class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response. J Immunol, 2003. 171(2): p. 528-532. [0269] 29. Herzog, J., et al., Activated antigen-presenting cells select and present chemically modified peptides recognized by unique CD4 T cells. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(22): p. 7928-7933. [0270] 30. Bronte, V., et al., Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers. Journal of Experimental Medicine, 2005. 201(8): p. 1257-1268. [0271] 31. Arentz-Hansen, H., et al., The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med, 2000. 191(4): p. 603-12. [0272] 32. Pudney, V. A., et al., DNA vaccination with T-cell epitopes encoded within Ab molecules induces high-avidity anti-tumor CD8(+) T cells. European Journal of Immunology, 2010. 40(3): p. 899-910. [0273] 33. DURRANT, L. G. M., Rachael Louise; PUDNEY, Victoria Anne; NUCLEIC ACIDS, 2008. [0274] 34. Feitsma, A. L., et al., Identification of Citrullinated Vimentin Peptides as T Cell Epitopes in HLA-DR4-Positive Patients With Rheumatoid Arthritis. Arthritis and Rheumatism, 2010. 62(1): p. 117-125. [0275] 35. Kryszke, M. H. and P. Vicart, Regulation of the expression of the human vimentin gene: Application to cellular immortalization. Pathologie Biologie, 1998. 46(1): p. 39-45. [0276] 36. Paulin, D., EXPRESSION OF THE GENES-CODING FOR THE HUMAN INTERMEDIATE FILAMENT PROTEINS. Pathologie Biologie, 1989. 37(4): p. 277-282. [0277] 37. Ramaekers, F. C., et al., Use of antibodies to intermediate filaments in the characterization of human tumors. Cold Spring Harb Symp Quant Biol, 1982. 46 Pt 1: p. 331-9. [0278] 38. Thomas, P. A., et al., Association between keratin and vimentin expression, malignant phenotype, and survival in postmenopausal breast cancer patients. Clinical Cancer Research, 1999. 5(10): p. 2698-2703. [0279] 39. Conforti, G., et al., DIFFERENT VIMENTIN EXPRESSION IN 2 CLONES DERIVED FROM A HUMAN COLOCARCINOMA CELL-LINE (LOVO) SHOWING DIFFERENT SENSITIVITY TO DOXORUBICIN. British Journal of Cancer, 1995. 71(3): p. 505-511. [0280] 40. Moran, E., et al., Co-expression of MDR-associated markers, including P-170, MRP and LRP and cytoskeletal proteins, in three resistant variants of the human ovarian carcinoma cell line, OAW42. European Journal of Cancer, 1997. 33(4): p. 652-660. [0281] 41. Gilles, C., et al., Vimentin expression in cervical carcinomas: association with invasive and migratory potential. J Pathol, 1996. 180(2): p. 175-80. [0282] 42. Williams, A. A., et al., CD 9 and vimentin distinguish clear cell from chromophobe renal cell carcinoma. BMC Clin Pathol, 2009. 9: p. 9. [0283] 43. Gustmann, C., et al., CYTOKERATIN EXPRESSION AND VIMENTIN CONTENT IN LARGE CELL ANAPLASTIC LYMPHOMAS AND OTHER NON-HODGKINS-LYMPHOMAS. American Journal of Pathology, 1991. 138(6): p. 1413-1422. [0284] 44. Yamamoto, Y., K. Izumi, and H. Otsuka, AN IMMUNOHISTOCHEMICAL STUDY OF EPITHELIAL MEMBRANE ANTIGEN, CYTOKERATIN, AND VIMENTIN IN PAPILLARY THYROID-CARCINOMA—RECOGNITION OF LETHAL AND FAVORABLE PROGNOSTIC TYPES. Cancer, 1992. 70(9): p. 2326-2333. [0285] 45. Coppola, D., et al., Prognostic significance of p53, bcl-2, vimentin, and S100 protein-positive Langerhans cells in endometrial carcinoma. Human Pathology, 1998. 29(5): p. 455-462. [0286] 46. Fuyuhiro, Y., et al., Clinical Significance of Vimentin-positive Gastric Cancer Cells. Anticancer Research, 2010. 30(12): p. 5239-5243. [0287] 47. Takemura, K., et al., EXPRESSION OF VIMENTIN IN GASTRIC-CANCER-A POSSIBLE INDICATOR FOR PROGNOSIS. Pathobiology, 1994. 62(3): p. 149-154. [0288] 48. Ivaska, J., Vimentin: Central hub in EMT induction? Small Gtpases, 2011. 2(1): p. 51-53. [0289] 49. Vuoriluoto, K., et al., Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene, 2011. 30(12): p. 1436-1448. [0290] 50. Hill, J. A., et al., Cutting edge: The conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule. J Immunol, 2003. 171(2): p. 538-541. [0291] 51. Ireland, J., J. Herzog, and E. R. Unanue, Cutting edge: Unique T cells that recognize citrullinated peptides are a feature of protein immunization. J Immunol, 2006. 177(3): p. 1421-1425. [0292] 52. Asaga, H., et al., Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils. Journal of Leukocyte Biology, 2001. 70(1): p. 46-51. [0293] 53. Nagata, S. and T. Senshu, PEPTIDYLARGININE DEIMINASE IN RAT AND MOUSE HEMATOPOIETIC-CELLS. Experientia, 1990. 46(1): p. 72-74. [0294] 54. Loos, T., et al., Citrullination of CXCL10 and CXCL11 by peptidylarginine deiminase: a naturally occurring posttranslational modification of chemokines and new dimension of immunoregulation. Blood, 2008. 112(7): p. 2648-2656. [0295] 55. Proost, P., et al., Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation. Journal of Experimental Medicine, 2008. 205(9): p. 2085-2097. [0296] 56. Struyf, S., et al., Citrullination of CXCL12 Differentially Reduces CXCR4 and CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1 Activity via CXCR4. J Immunol, 2009. 182(1): p. 666-674. [0297] 57. Ireland, J. M. and E. R. Unanue, Autophagy in antigen presenting cells results in presentation of citrullinated peptides to CD4 T cells. Journal of Experimental Medicine, 2011. 208(13): p. 2625-2632. [0298] 58. Asaga, H., M. Yamada, and T. Senshu, Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun, 1998. 243(3): p. 641-6. [0299] 59. Mor-Vaknin, N., et al., Vimentin is secreted by activated macrophages. Nature Cell Biology, 2003. 5(1): p. 59-63. [0300] 60. Vossenaar, E. R., et al., PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays, 2003. 25(11): p. 1106-18. [0301] 61. Kaufmann, S. H. E., The contribution of immunology to the rational design of novel antibacterial vaccines. Nature Reviews Microbiology, 2007. 5(7): p. 491-504. [0302] 62. Kubilus, J., R. F. Waitkus, and H. P. Baden, PARTIAL-PURIFICATION AND SPECIFICITY OF AN ARGININE-CONVERTING ENZYME FROM BOVINE EPIDERMIS. Biochimica Et Biophysica Acta, 1980. 615(1): p. 246-251. [0303] 63. Senshu, T., et al., Studies on specificity of peptidylarginine deiminase reactions using an immunochemical probe that recognizes an enzymatically deiminated partial sequence of mouse keratin K1. Journal of Dermatological Science, 1999. 21(2): p. 113-126. [0304] 64. Chang, X., et al., Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis. Rheumatology, 2005. 44(1): p. 40-50. [0305] 65. Chang, X. and J. Han, Expression of peptidylarginine deiminase type 4 (PAD4) in various tumors. Mol Carcinog, 2006. 45(3): p. 183-96. [0306] 66. Guo, Q. and W. Fast, Citrullination of inhibitor of growth 4 (ING4) by peptidylarginine deminase 4 (PAD4) disrupts the interaction between ING4 and p53. J Biol Chem, 2011. 286(19): p. 17069-78. [0307] 67. Karlin, S. and S. F. Altschul, Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci USA, 1993. 90(12): p. 5873-7. [0308] 68. Altschul, S. F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10. [0309] 69. Altschul, S. F., et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997. 25(17): p. 3389-402. [0310] 70. Myers, E. W. and W. Miller, Approximate matching of regular expressions. Bull Math Biol, 1989. 51(1): p. 5-37. [0311] 71. Torelli, A. and C. A. Robotti, ADVANCE and ADAM: two algorithms for the analysis of global similarity between homologous informational sequences. Comput Appl Biosci, 1994. 10(1): p. 3-5. [0312] 72. Pearson, W. R. and D. J. Lipman, Improved tools for biological sequence comparison. Proc Natl Acad Sci USA, 1988. 85(8): p. 2444-8. [0313] 73. Remington, R., Remington's pharmaceutical sciences. 16th ed. 1980: Mack Pub. Co. [0314] 74. Stewart, Solid phase peptide synthesis. 2nd ed. 1984, Rockford: Illinois Pierce Chemical Company. [0315] 75. Bodanzsky, B., The practice of peptide synthesis. 1984, New York: Springer Verlag. [0316] 76. Sambrook, J., Fritsch and Maniatis, Molecular cloning: A laboratory manual. 2nd ed. 1989: Cold Spring Harbor Laboratory Press. [0317] 77. Ausubel, J., Short protocols in molecular biology. 1992: John Wiley & Sons [0318] 78. Pluckthun, A., Antibody engineering: advances from the use of Escherichia coli expression systems. Biotechnology (N Y), 1991. 9(6): p. 545-51. [0319] 79. Reff, M. E., High-level production of recombinant immunoglobulins in mammalian cells. Curr Opin Biotechnol, 1993. 4(5): p. 573-6. [0320] 80. Trill, J. J., A. R. Shatzman, and S. Ganguly, Production of monoclonal antibodies in COS and CHO cells. Curr Opin Biotechnol, 1995. 6(5): p. 553-60. [0321] 81. Palena, C., et al., Strategies to target molecules that control the acquisition of a mesenchymal-like phenotype by carcinoma cells. Exp Biol Med (Maywood), 2011. 236(5): p. 537-45. [0322] 82. Denzin, L. K., et al., Assembly and intracellular transport of HLA-DM and correction of the class II antigen processing defect in T2 cells. Immunity, 1994. 1(7): p. 595-606. [0323] 83. Kovats, S., et al., Presentation of abundant endogenous class II DR-restricted antigens by DM-negative B cell lines. Eur J Immunol, 1997. 27(4): p. 1014-21. [0324] 84. Metheringham, R. L., et al., Antibodies designed as effective cancer vaccines. MAbs, 2009. 1(1): p. 71-85. [0325] 85. Quinn M J B P, B.A., Kirby E A, Jones J, Cancer trends in england and wales, 1950-1999, in London: Stationery Office2001. p. 206-207. [0326] 86. NICE, Improving outcomes in colorectal cancers: Manual update, 2004: London: National Institute for Clinical Excellence.