Epitopes
12458688 ยท 2025-11-04
Assignee
Inventors
- Wei Xue (Oxfordshire, GB)
- Katherine Wendy Cook (Oxfordshire, GB)
- Linda Gillian Durrant (Oxfordshire, GB)
- Victoria Anne Brentville (Oxfordshire, GB)
Cpc classification
C12Y401/02013
CHEMISTRY; METALLURGY
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to epitopes containing homocitrulline (Heit) that can be used as targets for cancer immunotherapy. The homocitrullinated T cell epitope has (i) a predicted binding score to MIC 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 method of treating cancer in a subject identified as having a tumor environment comprising myeloperoxidase (MPO)-expressing cells, wherein the MPO-expressing cells produce isocyanic acid that can diffuse into tumor cells and result in the carbamylation of epitopes in tumor cells in the tumor environment, wherein the resulting carbamylated epitopes each comprise at least one lysine amino acid residue that has been converted to homocitrulline and are presented on the surface of the tumor cells via MHC class I or II complexes, the method comprising administering to the subject a composition that comprises peptides comprising at least one of the carbamylated epitopes in an amount effective to induce production and/or secretion of IFN-gamma, granzyme B, and/or CD134 in T cells that bind the MHC class I or II complexes presented on the surface of the tumor cells.
2. The method of claim 1, wherein the subject has a solid tumor or a haematological neoplasia.
3. The method of claim 1, wherein the subject has pancreatic cancer, renal cancer, melanoma, head and neck cancer, breast cancer, lung cancer, Burkitt's lymphoma, chronic lymphocytic leukaemia, melanoma, pancreatic adenocarcinoma, breast cancer, colon cancer, acute lymphoblastic leukaemia or acute myeloid leukaemia.
4. The method of claim 1, wherein the T cells are CD4 T cells and/or CD8 T cells.
5. The method of claim 1, wherein the composition further comprises an adjuvant or pharmaceutically acceptable vaccine component.
6. The method of claim 5, wherein the adjuvant is a cytosine-phosphate-guanosine dinucleotide (CpG) oligonucleotide or monophosphoryl-lipid A (MPLA).
7. The method of claim 1, wherein the composition further comprises a CpG oligonucleotide adjuvant and/or a MPLA adjuvant.
8. The method of claim 1, wherein the MPO-expressing cells are monocytic and granulocytic myeloid derived suppressor cells (MDSC).
Description
EXAMPLES
(1) The present invention will now be described further with reference to the following examples and the accompanying drawings.
(2)
(3)
(4)
(5) 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. b: Amino acid sequence of human ALDOA A c: Amino acid sequence of human vimentin d: Amino acid sequence of human Cytokeratin 8 e: Amino acid sequence of human BiP f: Amino acid sequence of human NPM g: Amino acid sequence of human alpha-enolase h: Amino acid sequence of human -catenin i: Amino acid sequence of human HSP60
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(7) 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. IFN 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.
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(9) Ex vivo IFN ELISpot responses to vimentin 116-134 Hcit peptide were also assessed in HLA-HHDII/DP4 and C57BI/6 mice (a). Responses to vimentin 116-134 wt 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.
(10)
(11) Ex vivo IFN 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.
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(13) IFN 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.
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(15) Alignment of Hcit T cell epitopes from human vimentin (a), ALDOA (b), enolase (c), Bip (d), -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.
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(17) 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.
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(19) 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 responses were then assessed by ex vivo ELISpot (a). Responses to Cyk8 101Hcit, 112Hcit, 371Hcit 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
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(21) 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).
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(24) 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. IFN responses were then assessed by ex vivo ELISpot. Responses to Bip (a), Enolase (b), NPM (c), Vimentin (d) and aldolase (e and f) peptides containing Hcit were seen. Responses to Hcit aldolase peptides (e and f) were assessed for responses in the presence of CD4 or CD8 blocking antibodies and for cross reactivity to wildtype (wt) peptides.
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(26) Splenocytes from transgenic HLA-HHDII/DP4 mice immunised with Hcit peptides were then grown ex vivo in the presence of Hcit peptides. IFN 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.
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(28) 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. IFN responses were then assessed by ex vivo ELISpot. Responses to Aldolase 204 Hcit (A) and the shorter Aldolase 209hcit (B) peptide were assessed.
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(30) 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 10 g/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).
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(32) Example plots from patient LG10 showing staining for IFN, 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 IFN among the healthy donor (B) and patient (C) responders who showed proliferation following peptide stimulation.
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(34) 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 g/ml of peptides. Proliferation was assessed on day 10.
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(36) 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 IFN inducible DR4 cell line (e). Statistical analysis was performed comparing survival in immunised mice to survival in control mice, significant p values are shown.
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(38) 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.
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(40) 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 IFN 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.
(41)
(42) 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).
(43)
(44) 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).
(45)
(46) 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.2O.sub.2. The level of Carbamyation was then assessed by staining with an anti-carbamylysine antibody (b).
(47)
(48) 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.
(49)
(50) 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.
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(78) Methods
(79) Cell Lines and Culture
(80) 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 (2 mM) and sodium bicarbonate buffered to pH7. The cell line utilised were mycoplasma free, authenticated by suppliers (STR profiling), and are used within ten passages.
(81) Plasmids and Transfections
(82) 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 2-microglobulin (2M) molecule covalently linked via a glycine serine linker to the 1 and 2 domains of human HLA-0201 MHC class 1 molecule and the 3, transmembrane and cytoplasmic domains of the murine H-2 Db class 1 molecule, where relevant as previously described (Xue et al. 2016). B16F1 cells were also transfected with the IFN-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 IFN-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 g/ml), hygromycin B (300 g/ml) or G418 (500 g/ml).
(83) In Vitro Carbamylation and Detection
(84) 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.
(85) Mass Spectrometry
(86) 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 l/min pump module running at 5 l/min) with a 2 min wash trap/elute configuration onto a YMC Triart C18 column (300 um i.d., 3 m particle size, 15 cm) in a column oven at 35 C. Samples were gradient eluted over an 87 min runtime into a SCIEX 6600 Triple Tof mass spectrometer via a Duospray (TurboV) source with a 50 m electrode. The 6600 was set up in IDA mode (Independent Data Acquisition/Data Dependent Acquisition) for 30 ions per cycle fragmentation. Total cycle time 1.8s, TOFMS scan 250 ms accumulation; 50 ms for each product ion scan.
(87) 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%.
(88) Immunogens
(89) 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).
(90) Immunisation Protocol
(91) 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 A1 (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 g/mouse of each (Invivogen, UK). Peptides (25 g/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.
(92) 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 2.510.sup.4 cells/mouse for B16F1, 2.510.sup.4 cells/mouse for B16F1 DR4, 410.sup.5 cells/mouse for B16F1 HHDII/DP4, 510.sup.5 cells/mouse for B16 HHDII/DR1, 110.sup.5 cells/mouse for B16F1 HHDII/inducibleDP4, 510.sup.4 cells/mouse for B16F1 inducible DR4. For studies involving depletion antibodies, 250 g anti-mouse Ly6C antibody (clone Monts, BioXcell) was administered i.p. on days 8, 10, 12, 15, 17 and 19. 400 g anti-mouse Ly6G antibody (clone 1A8, Biolegend) was administered i.p. on day 8 followed by 250 g 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 g dose on day 4 followed by 300 g dose on days 8 and 11. Tumour growth was monitored twice weekly and mice were humanely euthanised once tumour reached 15 mm in diameter.
(93) Analysis of Immune Response-Ex Vivo ELISpot Assay
(94) ELISpot assays were performed using murine IFN, IL-17 and IL-10 capture and detection reagents according to the manufacturer's instructions (Mabtech, Sweden). In brief, anti-IFN, IL-17 and IL-10 specific antibodies were coated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at various concentrations) and 510.sup.5 per well splenocytes were added to wells of the plate in quadruplicate Tumour target cells were added where relevant at 510.sup.4/well in triplicate and plates incubated for 40 hrs at 37 C. After incubation, captured IFN, IL-10 and IL-17 were detected by biotinylated anti-IFN, IL-10 and IL-17 antibodies and developed with a streptavidin alkaline phosphatase and chromogenic substrate. Lipopolysaccharide (LPS; 5 g/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 g/ml. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd).
(95) Isolation and Analysis of Animal Tissue
(96) 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.
(97) In Vitro Production of MDSCs and Co-Culture with B16
(98) 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 M) and H.sub.2O.sub.2 (10 M) 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.
(99) Peripheral Blood Mononuclear Cell (PBMC) Isolation
(100) 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).
(101) Proliferation Assay-Carboxyfluorescein Succinimidyl Ester (CFSE)
(102) Briefly, a 50 M stock solution in warm PBS was prepared from a master solution of 5 mM in dimethyl sulfoxide (DMSO). CFSE was rapidly added to PBMCs (510.sup.6 cells/ml loading buffer (PBS with 5% v/v heat inactivated FCS)) to achieve a final concentration of 5 M. PBMCs were incubated at room temperature in the dark for 5 mins after which non-cellular incorporated CFSE was removed by washing twice with excess (10 v/v volumes) of loading buffer (300g 10 mins). Cells were made up in complete media to 1.5106/mL and plated and stimulated with vehicle (negative control), PHA (positive control, final concentration 10 g/ml) or peptide (10 g/ml) as described above.
(103) On day 10, 500 l 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-IFN (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.
(104) FACS Cell Sorting
(105) On day 10, the contents of the culture wells were mixed gently, pooled (according to peptide stimulation) and washed in PBS (300g 10 mins). Pellets were gently re-suspended in 500 L of PBS containing 10 l of anti CD4 eFluo450 (clone RPA-T4, ThermoFisher, cat no 48-0049-42) and 10 L 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 300g) in 1.0 ml of PBS and resuspended in 300 l of FACS sorting buffer (PBS supplemented with 1 mM EDTA, 25 mM HEPES and 1% v/v HI FCS). 10 l of sample was removed from each stained sample and 90 l 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.
(106) 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/CFSE.sup.low populations. Sorted cells (bulk) were stored at 80 C.
(107) Determination of the and chain pairing of TCRs recognising peptides containing homocitrulline. Sorted cells (bulk) from CD4+ve/CFSEhigh and CD4+ve/CFSE.sup.low populations in RNA protect were shipped to iRepertoire Inc (Huntsville, AL, USA) for NGS sequencing of the TCRA and TCRB chain to confirm expansion of TCR's in the CD4+ve/CFSE.sup.low cells, proliferating to the peptide in contrast to the non proliferating CD4+ve/CFSEhigh 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 and 250 PER primers (iRepertoire Inc., Huntsville, AL, USA). Information about the primers can be found in the United States Patent and Trademark Office (U.S. Pat. Nos. 7,999,092 and 9,012,1482). After assessment of PCR/DNA samples, 10 sample libraries were pooled and sequenced using the Illumina MiSeq platform (Illumina, San Diego, CA, 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.
(108) To elucidate the cognate pairing and sequencing of TCRa and TCRB 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 TCR and 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.
(109) DP4 Preparation for Binding Assay
(110) 2T175 gave 2.510.sup.7 B16 HHDII/DP4 (B16F1 2M 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).
(111) 510.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 16,000g 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 16,000g 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 l Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) thermo-scientific (Catalog number 78445) was added prior to freezing.
(112) DP4 Binding ELISA.
(113) 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 l of cell prep lysate was incubated with 50 g biotinylated peptide and incubated for 4 hrs at 37 C.: Lysate/peptide mix was added to the plates at 100 l/well and incubated for 4 hrs at room temperature. Plates were washed 3 with PBS/0.5% Tween. 100 l 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 lgG3-HRP at 0.5 mg/ml 1/500 100 l per well (Invitrogen #M32607) diluted in 1% BSA/PBS) was added for 1 hr. The plates were washed and 150 l of TMB substrate was added to each well. The reaction was stopped with 50 l of 2N H.sub.2SO.sub.4 and the plates read at 450 nm.
(114) DP4 competition assay.
(115) 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 3PBS/0.5% Tweenx. 450 l of cell prep lysate (B16F1 B16 HHDII/DP4 H.sub.7/2E9/F9) was mixed with 50 g test peptide (see below) or 10 g of unlabelled Hep B peptide for 30 mins. 10 g biotinylated Hep B peptide was added & incubated for 4 hrs at 37 C.:
(116) TABLE-US-00009 HepB 181-192 GFFLLTRILTIPQ(SEQIDNO:191) Fibrinogen 78-91 NQDFTN-cit-INKLKNS cit(Hu) (SEQIDNO:1628) CollagenII 1236-1249 LQYM-cit-ADQAAGGLR. cit(Hu) (SEQIDNO:1629) AldolaseA 74-93 IGGVILFHETLYQ-hcit-ADDGRP (SEQIDNO:15) AldolaseA 74-93WT IGGVILFHETLYQKADDGRP (SEQIDNO:15) AldolaseA 140-157 hcit-DGADFA-hcit-WRCVL-hcit-IGEH (SEQIDNO:16) AldolaseA 140-157WT KDGADFAKWRCVLKIGEH (SEQIDNO:16) AldolaseA 217-235 LSDHHVYLEGTLL-hcit-PNMVT (SEQIDNO:194) AldolaseA 238-256 HACTQ-hcit-FSH(N)EEIAMATVTA (SEQIDNO:195) AldolaseA 289-307 hcit-CPLL-hcit-PWALTFSYGRALQ (SEQIDNO:19) Cytokeratin8 101-120 KFASFID-Hcit-VRFLEQQN-Hcit-MLE (SEQIDNO:25) Cytokeratin8 112-131 LEQQN-hcit-MLET-hcit-WSLLQQQ-hcit-T (SEQIDNO:26) Cytokeratin8 182-202 EIN-hcit-RTEMENEFVLI-hcit-hcit-DVDE (SEQIDNO:28) Cytokeratin8 371-388 LREYQELMNV-hcit-LALDIEI (SEQIDNO:29) Cytokeratin8 381-399 hcit-LALDIEIATYR-hcit-LLEGEE (SEQIDNO:30) Vimentin 116-135 NYID-hcit-VRFLEQQN-hcit-ILLAEL (SEQIDNO:3) Vimentin 116-135WT NYID-hcit-VRFLEQQN-hcit-ILLAEL (SEQIDNO:3)
(117) Lysate/peptide mix was added to the plates at 100 l/well and incubated for 4 hrs at room temperature. Plates were washed 3 with PBS/0.5% Tween. 100 l per well of Leinco anti-Human HLA-DP4 clone B7/21 #H.sub.260 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 lgG3-HRP at 0.5 mg/ml 1/500 100 l per well (Invitrogen #M32607) diluted in 1% BSA/PBS) was added for 1 hr at room temperature prior to washing and adding 150 l of TMB substrate to each well. The reaction was stopped with 50 l of 2N H.sub.2SO.sub.4 and plates read plate at 450 nm.
(118) Statistical Analysis
(119) 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
(120) 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 (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.
(121) Screening of Vimentin Peptide Responses
(122) 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 g 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).
(123) Significant IFN responses were detected to peptide vimentin 116-135 Hcit. In both HLA-DR4 (
(124) TABLE-US-00010 TABLE2 Vimentinpeptides. DP4 DP4 DR4 DR4 DR1 DR1 T coor- predic- predic- predic- predic- predic- predic- cell din- se- tion ted tion ted tion ted Spi- re- Protein ates quence score cores score cores scores cores ral sponse Vimentin 116-135 NYID- hcit- 2.12- 17.18 IDKVRFLEQ (SEQID 5.06- 38.50 VRFLEQQNK (SEQID 11.76- 14.32 FLEQQNKIL (SEQID yes VRFLEQQN- 2.12 NO:197) 5.06 NO:201) 14.32- NO:202) hcit- 30.02 YIDKVRFLE 38.50 FLEQQNKIL 58.24 VRFLEQQNK ILLAEL 30.02 (SEQID (SEQID 58.24 (SEQID (SEQID NO:198) NO:202) NO:201) NO:3) KVRFLEQQN YIDKVRFLE RFLEQQNKI (SEQID (SEQID (SEQID NO:199) NO:198) NO:200) RFLEQQNKI (SEQID NO:200) Vimentin 215-235 LARLDLER- hcit- 21.67- 49.11 KVESLQEEI (SEQID 24.51- 30.76 LERKVESLQ (SEQID 76 79.7- ERKVESLQE (SEQID
no VESLQE 44.08- NO:203) 26.21 NO:204) 81.37 NO:207) EIAFL- 60.82 LERKVESLQ 26.21- ARLDLERKV 79.70- ARLDLERKV hcit 57.44- (SEQID 30.76 (SEQID 81.37 (SEQID (SEQID 60.82 NO:204) NO:205) NO:205) NO:4) ARLDLERKV LDLERKVES DLERKVESL (SEQID (SEQID (SEQID NO:205) NO:206) NO:208) Vimentin 255-275 QIDVDVS- hcit- PDLTAALR 82.48- 88.15 VSKPDLTAA (SEQID NO:209) 17.21- 30.56 17.21 SKPDLTAAL (SEQID NO:210) 58.03 58.21- 62.81 SKPDLTAAL (SEQID NO:210)
no DVRQQ 23.61 IDVDVSKPD 58.03- KPDLTAALR (SEQID 30.56 (SEQID 61.06 (SEQID NO:5) NO:211) NO:213) VDVSKPDLT VDVSKPDLT (SEQID (SEQID NO:212) NO:212) VSKPDLTAA (SEQID NO:209) Vimentin 286-303 EAEEWY- hcit- 2.06- 2.22 KSKFADLSE (SEQID 4.76- 9.29 KFADLSEAA (SEQID 18.99 18.99- KFADLSEAA (SEQID
no S-hcit- 2.06- NO:214) 9.29- NO:216) 41.08 NO:216) FADLSEAAN 5.69 WYKSKFADL 11.26 YKSKFADLS YKSKFADLS (SEQID 3.31- (SEQID (SEQID (SEQID NO:6) 5.69 NO:215) NO:217) NO:217) KFADLSEAA (SEQID NO:216) Vimentin 431-454 LPLVDTHS- hcit- 35.09- 40.42 THSKRTLLI (SEQID 9.5 9.5 IKTVETRDG (SEQID 24.07- 34.8 KRTLLIKTV (SEQID
no RTLLI- 41.46- NO:218) 10.96- NO:221) 31.48- NO:224) hcit- 60.30 TLLIKTVET 12.02 LIKTVETRD 41.38 THSKRTLLI TVETRDGQV 41.46- (SEQID 9.5- (SEQID 24.07- (SEQID (SEQID 49.99 NO:219) 12.02 NO:222) 44.99 NO:218) NO:196) 59.59- HSKRTLLIK TLLIKTVET TLLIKTVET 60.30 (SEQID (SEQID (SEQID 22.22- NO:220) NO:219) NO:219) 26.93 LLIKTVETR LLIKTVETR (SEQID (SEQID NO:223) NO:223) THSKRTLLI (SEQID NO:218)
(125) 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 IFN response in DR4 (
(126) Thus the minimal epitope in both DR4 and DR1 is vimentin 120-134 with homocitrulline at positions 120 and 129.
(127) 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 (
Example 2. CD4 Responses to Homocitrullinated ALDOA
(128) 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 (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.
(129) TABLE-US-00011 TABLE3 ALDOApeptidesutilised. DP4 DP4 DR4 DR4 DR1 DR1 T coor- predic- predic- predic- predic- predic- predic- cell din- se- tion ted tion ted tion ted re- Protein ates quence score cores score cores scores cores Spiral sponse ALDOAA 74-93 IGGVIL FHETLY Q-hcit- 2.5- 8.04 3.09- FHETLYQKA (SEQID NO:811) 30.22 30.22 FHETLYQKA (SEQID NO:811) 33.91- 60.75 46.61- FHETLYQKA (SEQID NO:811) yes ADDGRP 8.04 LFHETLYQK LFHETLYQK 60.75 LFHETLYQK (SEQID (SEQID (SEQID (SEQID NO:15) NO:225) NO:225) NO:225) ALDOAA 140-157 hcit- DGADFA- hcit- WRCVL- 22.10- 28.10 2335- 28.10 FAKWRCVLK (SEQID NO:228) AKWRCVLKI 49.80- 57.56 FAKWRCVLK (SEQID NO:228) 59.86- 65.39 59.86- 65.39 AKWRCVLKI (SEQID NO:226) FAKWRCVLK
yes hcit- 22.10- (SEQID (SEQID IGEH 28.10 NO:226) NO:228) (SEQID DFAKWRCVL NO:16) (SEQID NO:227) ALDOAA 217-235 LSDHH VYLEG TLL- 4.92- 6.66 6.66 YLEGTLLKP (SEQID NO:229) 4.87- 7.88 7.88 IYLEGTLLK (SEQID NO:230) 13.40 GTLLKPNMV (SEQID NO:231)
no hcit- IYLEGTLLK YLEGTLLKP PNMVT (SEQID (SEQID (SEQID NO:230) NO:229) NO:194) ALDOAA 238-256 HACTQ- hcit- FSH(N) EEIAMA 13.18- 17.73 13.18 17.73 TQKFSHEEI (SEQID NO:232) KFSHEEIAM 4.11 KFSHEEIAM (SEQID NO:233) 46.73 TQKFSHEEI (SEQID NO:232)
yes TVTA (SEQID (SEQID NO:233) NO:195) ALDOAA 289-307 hcit- CPLL- hcit- 14.38- 19.06 19.06 KPWALTFSY (SEQID NO:234) 11.15- 20.21 LLKPWALTF (SEQID NO:235) 24.99- 28.52 CPLLKPWAL (SEQID NO:236)
no PWALTFS LLKPWALTF YGRALQ (SEQID (SEQID NO:235) NO:19)
Screening of ALDOA Peptide Responses
(130) 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 g 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 (
(131) 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 74-93 wt (p<0.0001) or 140-157 wt (p<0.0001) (
(132) 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 (
(133) 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 (
Example 3. Predictions for Identifying Homocitrulline Containing T Cell Targets
(134) As In silico bioinformatic analysis of vimentin and ALDOA (Table 2 and 3) using IEDB prediction program (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 (mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/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.
(135) 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.
(136) 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 g 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 (
(137) Responses to Cyk8 101Hcit, 112Hcit, 371Hcit 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 (
(138) 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) (
(139) The ability of the wt peptides to stimulate a response was tested with the Cyk8 112Hcit peptide in the HHDII/DR1 mice. Cyk8 112 wt peptide was administered as three immunisations containing 25 g 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 112 wt peptide or to the Cyk8 112Hcit and Cyk8 117Hcit peptides (
(140) Additional peptides within vimentin and ALDOA and peptides within BiP, nucleophosmin, enolase, -catenin and HSP60 have also been identified using this motif (Table 5).
(141) 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 g 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 (
(142) 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 (
(143) 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 HHDIVDP4 mice. Both peptides stimulated strong responses to the homocitrulline peptides with lower reactivity to the wt sequences in HHDIVDR1 mice (
(144) Thus a key characteristic of most responses is they are medlated by CD4 T cells. In addition, Heit specific responses can also be mediated by CDS T cells.
(145) TABLE-US-00012 TABLE4 HomocitrullinebindingpredictionsforCyK8. DP4 DP4 DR4 DR4 DR1 DR1 T predic- predic- predic- predic- predic- predic- cell Prot- coordi- se- tion ted tion ted tion ted re- ein nates quence score cores score cores scores cores Spiral sponse Cyk8 101-120 KFASFID- Hcit- VRFLEQQN- 0.97- 17.18 0.97- IDKVRFLEQ (SEQID NO:197) 5.06 25.16- 37.08 FLEGGNKML (SEQID NO:239) 12.27 12.27- 66.46 FLEQQNKML (SEQID NO:239) Yes Hcit- 1.68 SFIDKVRFL 25.16- IDKVRFLEG 63.29 VRFLEQGNK MLE 6.08 (SEQID 26.26 (SEQID 65.13 (SEQID (SEQID NO:237) 5.06- NO:197) NO:201) NO:25) FiDKVRFLE 37.08 FASFIDKVR FASFIDKVR (SEQID (SEQID (SEQID NO:238) NO:240) NO:240) VRFLEQQNK KVRFLEQQN (SEQID (SEQID NO:201) NO:199) Cyk8 112-131 LEQQN- hcit- 3.16-4.72 3.16-4.72 KMLETKWSL (SEQID 12.19- 24.67 MLETKWSLL (SEQID 31.04- 44.89 KWSLLQQQK (SEQID
Yes MLET- NO:27) 12.19- NO:241) 44.89- NO:244) hcit- MLETKWSLL 15.38 LETKWSLLQ 46.47 MLETKWSLL WSLLQQQ- (SEQID 12.96- (SEQID 31.04- (SEQID hcit-T NO:241) 15.38 NO:242) 44.89 NO:241) (SEQID 24.67 KMLETKWSL LETKWSLLQ NO:26) (SEQID (SEQID NO:27) NO:242) QNNMLETKW (SEQID NO:243) Cyk8 182-202 EIN- hcit- RTEMEN EFVLI- 27.44- 41.36 36.01- 41.15 MENEFVLIK (SEQID NO:245) FVLIKKDVD 69.5- 75.9 MENEFVLIK (SEQID NO:245)
Yes hcit- 33.01 (SEQID hcit- NO:246) DVDE INKRTEMEN (SEQID (SEQID NO:28) NO:247) Cyk8 371-388 LREYQ ELMNV- 24.36- 26.69 LMNVKLALD (SEQID 4.42-5.8 5.76 YQELMNVKL (SEQID 6.74- 20.92 YQELMNVKL (SEQID
Yes hcit- 24.36- NO:248) NO:250) 6.74- NO:250) LALDIEI 26.69 ELMNVKLAL LMNVKLALD 20.92 ELMNVKLAL (SEQID (SEQID (SEQID (SEQID NO:29) NO:249) NO:248) NO:249) Cyk8 381-399 hcit- LALDIE 18.10- 24.62 IEIATYRKL (SEQID 12.39- 27.03 LDIEIATYR (SEQID 39.89- 27.66 IEIATYRKL (SEQID
Yes IATYR- 24.62 NO:251) 23.47- NO:253) NO:251) hcit- IATYRKLLE 27.68 IATYRKLLE LLEGEE (SEQID 27.68 (SEQID (SEQID NO:252) NO:252) NO:30) YRKLLEGEE (SEQID NO:254)
(146) TABLE-US-00013 TABLE5 HomocitrullinebindingpredictionsforVimentin,ALDOA, cytokeratin8,Bip,NPM,enolase,cateninandHSP60. 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-Hcit- RCQYVTE- 8.92- 11.09 CQYVTEKVL (SEQID 1.65- 1.78 YVTEKVLAA (SEQID 10.18- 13.7 YVTEKVLAA (SEQID Hcit- 8.92- NO:255) NO:256) NO:256) VLAAVY- 11.09 YVTEKVLAA Hcit-A (SEQID (SEQID NO:256) NO:20) ALDOAA 323-342 AAQEEYV- Hcit-RALA 45.05- 55.91 KRALANSLA (SEQID 7.44- 7.59 VKRALANSL (SEQID 3.72- 12.6 VKRALANSL (SEQID
NSLACQG- 52.36- NO:257) 7.44- NO:260) 3.72- NO:260) Hcit 56.49 YVKRALANS 7.59 KRALANSLA 8.47 KRALANSLA (SEQID 45.05- (SEQID 7.44 (SEQID (SEQID NO:21) 52.36 NO:258) NO:257) NO:257) EEYVKRALA YVKRALANS (SEQID (SEQID NO:259) NO:258) ALDOAA 208-226 Hcit- VLAAVY- Hcit- 29.94- 32.49 39.24- VLAAVYKAL (SEQID NO:24) 5.93- 7.72 5.93- YKALSDHHI (SEQID NO:261) 5.79- 6.42 6.39- YKALSDHHI (SEQID NO:261)
ALSDHHIY 42.43 YKALSDHHI 7.72 VYKALSDHH 6.42 VLAAVYKAL (SEQID (SEQID (SEQID (SEQID NO:22) NO:261) NO:262) NO:24) ALDOAA 204-219 YVTEKVLAA VYKALSD 16.41- 19.85 VLAAVYKAL (SEQID 3.95 10.46 YVTEKVLAA (SEQID 15.29- 20.07 VLAAVYKAL (SEQID
(SEQID NO:24) 25.20 NO:256) 1.34- NO:24) NO:23) KVLAAVYKA 14.77 YVTEKVLAA (SEQID 20.07 (SEQID NO:263) NO:256) LAAVYKALS LAAVYKALS (SEQID (SEQID NO:264) NO:264) Vimentin 86-108 FSLADAIN TEF-Hcit- NTRTNE- 25.28- 29.44 25.28- AINTEFKNT (SEQID NO:265) 4.34- 4.52 17.84- FKNTRTNEK (SEQID NO:266) 73.49 73.49- 79.9 EFKNTRTNE (SEQID NO:267)
Hcit-VELQ 27.76 FKNTRTNEK 19.26 INTEFKNTR 73.49 NTEFKNTRT (SEQID 25.28- (SEQID (SEQID (SEQID NO:13) 27.76 NO:266) NO:269) NO:270) 25.32- EFKNTRTNE FKNTRTNEK 27.76 (SEQID (SEQID NO:267) NO:266) TRTNEKVEL (SEQID NO:268) Vimentin 390-408 Hcit- MALDIEIA TYR-Hcit- 18.19- 24.62 24.62 IEIATYRKL (SEQID NO:251) 23.74- 27.68 27.68 IATYRKLLE (SEQID NO:252) 40.8- 57.66 IEIATYRKL (SEQID NO:251)
LLEGEE IATYRKLLE YRKLLEGEE (SEQID (SEQID (SEQID NO:14) NO:252) NO:254) Cyto- keratin 120-140 ET- Hcit- 55.49 55.49- ETKWSLLQQ (SEQID 0.5-2.17 LQQQKTARS (SEQID 8.47- 13.91 KWSLLQQQK (SEQID
8 WSLLQQQ- 75.39 NO:271) NO:273) 9.25- NO:244) Hcit- 63.26- WSLLQQQKT 63.26 LQQQKTARS TARSNMDNMF 87.43 (SEQID 8.47- (SEQID (SEQID NO:272) 13.91 NO:273) NO:31) LQQQKTARS WSLLQQQKT (SEQID (SEQID NO:273) NO:272) Cyto- keratin 93-112 EQI-Hcit- SLNN-Hcit- 19.63- 25.41 KFASFIDKV (SEQID 1.98- 5.39 IKSLNNKFA (SEQID 11.25- 20.28 IKSLNNKFA (SEQID
8 FASFID- 19.63- NO:274) 17.73- NO:276) 11.25- NO:276) Hcit-VRFL 27.86 SLNNKFASF 20.24 FASFIDKVR 49.25 LNNKFASFI (SEQID (SEQID 17.73- (SEQID (SEQID NO:32) NO:275) 20.24 NO:240) NO:277) LNNKFASFI (SEQID NO:277) Cyto- keratin 8 190-208 ENEFVLI- Hcit-Hcit- DVDEAYMN- 67.48- 75.49 67.48- FVLIKKDVD (SEQID NO:246) 11.45 11.45 IKKDVDEAY (SEQID NO:279) 78.64- 86.64 78.64- KKDVDEAYM (SEQID NO:280)
Hcit-V 67.96 EFVLIKKDV FVUKKDVD 84.99 FVLIKKDVD (SEQID (SEQID (SEQID (SEQID NO:33) NO:278) NO:246) NO:246) Cyto- keratin 294-310 G-Hcit- HGDDLRRT- 72.15- 81.41 LRRTKTEIS (SEQID 3.19- 3.29 LRRTKTEIS (SEQID 76.17- 85.76 LRRTKTEIS (SEQID
8 Hcit-TEISEM NO:281) NO:281) 76.17- NO:281) (SEQID 85.34 RRTKTEISE NO:34) (SEQID NO:282) Cyto- keratin 369-388 RQLREYQEL MNV-Hcit- 24.36- 26.78 ELMNVKLAL (SEQID 4.42- 5.82 YQELMNVKL (SEQID 6.6- 20.92 YQELMNVKL (SEQID
8 LALDIEI 24.36- NO:249) NO:250) 6.74- NO:250) (SEQID 26.78 LMNVKLALD 20.92 ELMNVKLAL NO:283) (SEQID (SEQID NO:248) NO:249) Cyto- keratin 320-338 EiEGL-Hcit- GQRASLE 59.6- 65.73 LKGQRASLE (SEQID 13.22 LKGQRASLE (SEQID 8.36- 13.25 LKGQRASLE (SEQID
8 AAIADA NO:284) NO:284) 8.36- NO:284) (SEQID 8.42 IEGLKGQRA NO:36) (SEQID NO:285) Cyto- keratin 339-358 EQRGELAS- Hcit-DANA- 75.97- 77.15 DANAKLSEL (SEQID 13.67 13.67- IKDANAKLS (SEQID 27.63- 49.46 IKDANAKLS (SEQID
8 Hcit-LSELEA 95.14- NO:286) 13.76 NO:289) 27.63- NO:289) (SEQID 96.26 LAIKDANAK AIKDANAKL 49.46 LAIKDANAK NO:37) 95.14- (SEQID (SEQID (SEQID 95.26 NO:287) NO:288) NO:287) AIKDANAKL (SEQID NO:288) BiP 104-124 NDPSVQQDI- Hcit-FLPF- Hcit-VVE- 3.13-5.48 3.13-3.36 3.62-7.91 IKFLPFKVV (SEQID NO:290) 8.87-8.95 8.88- 29.27 FLPFKVVEK (SEQID NO:293) 26.97- 54.82 26.97 DIKFLPFKV (SEQID NO:294)
Hcit-Hcit-T QDIKFLPFK 22.3- DIKFLPFKV 81.79 IKFLPFKVV (SEQID (SEQID 30.85 (SEQID (SEQID NO:38) NO:291) 22.3- NO:294) NO:290) QQDIKFLPF 29.27 VQQDIKFLP SVQQDIKFL (SEQID (SEQID (SEQID NO:292) NO:295) NO:296) IKFLPFKVV (SEQID NO:290) BiP 144-159 EiSAMVLT- Hcit-M- 42.22- 42.30 MVLTKMKET (SEQID 8.68-8.69 ISAMVLTKM (SEQID 36.03 36.03 ISAMVLTKM (SEQID
Hcit-ETAEA 42.22- NO:297) NO:299) NO:299) (SEQID 42.30 SAMVLSKMK LTKMKETAE NO:39) (SEQID (SEQID NO:298) NO:300) BiP 255-275 GEDFDQR VMEHFI-Hcit- 7.94- 12.95 RVMEHFIKL (SEQID 17.21 17.21- FIKLYKKKT (SEQID 32.16- 38.72 FIKLYKKKT (SEQID
LY-Hcit- 12.95 NO:301) 40.78 NO:303) 38.72- NO:303) Hcit-Hcit-TG QRVMEHFIK VMEHFIKLY 65.12 QRVMEHFIK (SEQID (SEQID (SEQID (SEQID NO:40) NO:302) NO:304) NO:302) BiP 286-306 Q-Hcit- LRREVE-Hcit- 70.38- 84.43 KRALSSQHQ (SEQID 15.95- 19.49 LRREVEKAK (SEQID 34.03- 65.3 AKRALSSQH (SEQID
A-Hdt-RALSSQ 78.03- NO:305) 19.45- NO:307) 34.03- NO:309) HQAR 85.41 EKAKRALSS 23.67 VEKAKRALS 65.3 VEKAKRALS (SEQID 83.26- (SEQID 23.67 (SEQID 34.03- (SEQID NO:41) 85.26 NO:306) 23.67 NO:308) 39.52 NO:308) LRREVEKAK AKRALSSQH 43.46- KRALSSQHQ (SEQID (SEQID 45.85 (SEQID NO:307) NO:309) 43.46- NO:305) KRALSSQHQ 56.99 KLRREVEKA (SEQID (SEQID NO:305) NO:310) REVEXAKRA (SEQID NO:311) BiP 316-336 EDFSETLTRA- Hcit-FEELNM DLFR 0.13-1.31 LTRAKFEEL (SEQID NO:312) 11.45- 15.14 21.98- FSETLTRAK (SEQID NO:313) 53.82- 68.1 54.2- FEELNMDLF (SEQID NO:315)
(SEQID 31.82 RAKFEELNM 65.21 SETLTRAKF NO:42) 21.98- (SEQID 57.7- (SEQID 31.82 NO:314) 77.4 NO:316) LTRAKFEEL LTRAKFEEL (SEQID (SEQID NO:312) NO:312) BiP 328-346 EELNMDL FRSTM-Hcit- PVQ-Hcit-VL 19.99- 23.39 21.61- LFRSTMKPV (SEQID NO:317) 6.65- 13.67 6.65-8.29 MDLFRSTMK (SEQID NO:319) 28.99- 39.69 35.68- LFRSTMKPV (SEQID NO:317)
(SEQID 23.39 FRSTMKPVQ FRSTMKPVQ 36.69 MDLFRSTMK NO:43) (SEQID (SEQID 28.99 (SEQID NO:318) NO:318) NO:319) FRSTMKPVQ (SEQID NO:318) BiP 367-387 RIP-Hdt- IQQLV-Htit- EFFNG-Hcit- 4.15- 27.73 4.15- QLVKEFFNG (SEQID NO:320) 9.73 9.73- 24.14 FFNGKEPSR (SEQID NO:322) 47.06- 62.09 65.99- FFNGKEPSR (SEQID NO:322)
EPSRG 20.84 LVKEFFNGK 15.33- LVKEFFNGK 73.97 IQQLVKEFF (SEQID (SEQID 24.14 (SEQID 71.21- (SEQID NO:44) NO:321) NO:321) 73.97 NO:323) IQQLVKEFF LVKEFFNGK (SEQID (SEQID NO:323) NO:321) BiP 460-480 TVTI-Hcit- WEGERPLT- Hcit-DNHLLG 17.85- 20.08 17.85- KVYEGERPL (SEQID NO:324) 10.02- 27.01 YEGERPLTK (SEQID NO:325) 44.67- 63.59 44.67- KVYEGERPL (SEQID NO:324)
(SEQID 51.73 YEGERPLTK 65.03 YEGERPLTK NO:45) (SEQID (SEQID NO:325) NO:325) BiP 562-579 RNELESYAYSL- Hcit-NQIGD- 21.01 21.01 LESYAYSLK (SEQID 12.81-16 14.01 YAYSLKNQI (SEQID 32.42- 43.8 YAYSLKNQI (SEQID
Hcit NO:326) NO:327) 32.42- NO:327) (SEQID YAYSLKNQI LESYAYSLK 43.8 AYSLKNQIG NO:46) (SEQID (SEQID (SEQID NO:327) NO:326) NO:328) BiP 620-639 Hcit-Hcit- ELEEIVQPIIS- 32.06- 47.49 IVQPIISKL (SEQID 6.28-7.76 7.72- VQPIISKLY (SEQID 25.51- 36.44 IVQPIISKL (SEQID
Hcit-LYGSAG 39.29- NO:329) 19.66 NO:330) 25.51- NO:329) (SEQID 47.49 VQPIISKLY IVQPIISKL 26.88 VQPIISKLY NO:47) (SEQID (SEQID (SEQID NO:330) NO:329) NO:330) Nucleo- phosmin 11-27 PLRPQNYLFG CEL-Hcit- AD-Hcit- (SEQID 26.13-33.98 26.54 YLFGCELKA (SEQID NO:331) LFGCELKAD 10.18-10.36 10.18-10.2 YLFGCELKA (SEQID NO:331) LRPQNYLFG 41.72-42.71 YLFGCELKA (SEQID NO:331)
NO:48) (SEQID (SEQID NO:332) NO:33) Nucleo- phosmin 68-89 EGSPI-Hcit- VTLATL-Hcit- MSVQPTVSL (SEQID 32.93 33.41- 45.82 32.93 KMSVQPTVS (SEQID NO:339) KVTLATLKM 5.42-6.05 5.15- 12.57 5.5-7.38 KVTLATLKM (SEQID NO:334) LKMSVQPTV 17.14- 18.61 19.12- 33.26 KMSVQPTVS (SEQID NO:339) KVTLATLKM
NO:49) 33.41- (SEQID 7.38- (SEQID (SEQID 40.80 NO:334) 12.57 NO:335) 18.61- NO:334) LKMSVQPTV IKVTLATLK 20.48 LATLKMSVQ (SEQID (SEQID 17.14- (SEQID NO:335) NO:337) 20.48 NO:338) TLATLKMSV LATLKMSVQ TLKMSVQPT (SEQID (SEQID (SEQID NO:336) NO:338) NO:340) Nucleo- phosmin 129-149 EEEDV-Hcit- LLSISG-Hcit- RSAPGGGS 65.37- 77.59 66.93- LLSISGKRS (SEQID NO:341) 4.11 4.11-9.89 9.89 VKILSISGK (SEQID NO:344) 5.03- 17.94 LLSISGKRS (SEQID NO:341)
(SEQID 77.59 ISGKRSAPG LLSISGKRS NO:50) 65.37- (SEQID (SEQID 75.07 NO:342) NO:341) KLLSISGKR ISGKRSAPG (SEQID (SEQID NO:343) NO:342) Nucleo- phosmin 222-240 S-Hcit-GQESF- Hcit-Hcit-QE- Hcit-TP-Hcit- 73.23- 91.45 73.23- FKKQEKTPK (SEQID NO:345) 13.76 FKKQEKTPK (SEQID NO:345) 76.73- 78.7 76.73- FKKQEKTPK (SEQID NO:345)
TP-Hcit-G 85.41 ESFKKQEKT 78.7 KKQEKTPKT (SEQID 82.11- (SEQID (SEQID NO:51) 85.41 NO:346) NO:347) KKQEKTPKT (SEQID NO:347) Nucleo- phosmin 258-277 GGSLP-Hcit- VEA-Hcit- 24.31- 30.51 KVEAKFINY (SEQID 14.7- 25.2 FINYVKNCF (SEQID 51.23- 60.28 VEAKFINYV (SEQID
FINYV-Hcit- 24.31- NO:348) 25.2- NO:350) 51.23- NO:349) NCFR 30.51 VEAKFINYV 34.52 EAKFINYVK 59.06 LPKVEAKFI (SEQID 28.92- (SEQID 31.9- (SEQID (SEQID NO:52) 30.51 NO:349) 43.83 NO:351) NO:352) FINYVKNCF 25.2- LPKVEAKFI (SEQID 43.83 (SEQID NO:350) NO:352) VEAKFINYV (SEQID NO:349) Nucleo- phosmin 266-287 A-Hcit- FINYV-Hcit- NCFRMTDQEAIQ 2.93-3.06 2.93- 14.95 KFINYVKNC (SEQID NO:353) 11.37 15.24- 21.39 FINYVKNCF (SEQID NO:350) 40.86- 50.73 40.86- FINYVKNCF (SEQID NO:350)
DL YVKNCFRMT 11.37- YVKNCFRMT 55.78 YVKNCFRMT (SEQID (SEQID 20.77 (SEQID (SEQID NO:53) NO:354) NO:354) NO:354) INYVKNCFR (SEQID NO:355) Alpha Enolase 1-16 MSIL-Hcit- IHAREIFDSRG 26.7- 29.02 ILKIHAREI (SEQID 14.35- 25.75 ILKIHAREI (SEQID 17.82 17.82- MSILKIHAR (SEQID
(SEQID 26.7- NO:356) 14.35 NO:356) 35.56 NO:358) NO:54) 29.02 LKIHAREIF MSILKIHAR ILKIHAREI (SEQID (SEQID (SEQID NO:357) NO:358) NO:356) Alpha Enolase 52-69 ND-Hcit- TRYMG-Hcit- 59.77- 60.48 KTRYMGKGV (SEQID 10.02- 10.25 MGKGVSKAV (SEQID 21.68- 25.27 YMGKGVSKA (SEQID
GVS-Hcit- 59.77- NO:359) 10.25 NO:361) 21.68- NO:360) AVEHI 68.62 YMGKGVSKA 10.02- RFMGKGVSK 23.87 KTRYMGKGV (SEQID (SEQID 10.25 (SEQID (SEQID NO:55) NO:360) NO:362) NO:359) YMGKGVSKA (SEQID NO:360) Alpha Enolase 100-121 TEN-Hcit-S- Hcit-FGANAIL 13.21- 26.83 KFGANAILG (SEQID 11.17 KFGANAILG (SEQID 12.06- 24.07 KSKFGANAI (SEQID
GVSLAVC- NO:363) NO:363) NO:364) Hcit-A (SEQID NO:56) Alpha Enolase 156-176 GSHAGN-Hcit- LAMQEFMILPVG AA 1.2- 22.16 22.16 KLAMQEFMI (SEQID NO:365) 12.67- 32.56 KLAMQEFMI (SEQID NO:365) 34.28- 46.65 KLAMQEFMI (SEQID NO:365)
(SEQID HAGNKLAMQ NO:57) (SEQID NO:366) Alpha Enolase 179-197 REAMRIGA EVYHNL-Hcit- 25.95- 28.39 VYHNLKNVI (SEQID 3.51- 30.06 VYHNLKNVI (SEQID 52.39 12.91 VYHNLKNVI (SEQID
NVI-Hcit- NO:367) 3.51 NO:367) NO:367) (SEQID YHNLKNVIK YHNLKNVIK NO:58) (SEQID (SEQID NO:368) NO:368) Alpha Enolase 194-212 NVI-Hcit-E- Hcit-YG- Hcit- 83.85- 94.96 83.85- YGKDATNVG (SEQID NO:369) 3.14-3.29 YGKDATNVG (SEQID NO:369) 72.08- 72.12 72.08- KYGKDATNV (SEQID NO:371)
DATNVGDEGG 85.66 VIKEKYGKD 72.12 YGKDATNVG (SEQID 92.59- (SEQID (SEQID NO:59) 94.96 NO:370) NO:369) KYGKDATNV (SEQID NO:371) Alpha Enolase 245-264 DVAASEFF RSG-Hcit- YDLDF- 10.04- 19.07 10.04- FRSGKYDLD (SEQID NO:372) 22.59- 24.17 38.34 FRSGKYDLD (SEQID NO:372) 54.29- 81.42 54.29- FRSGKYDLD (SEQID NO:372)
Hcit-SP 19.07 FFRSGKYDL SEFFRSGKY 81.42 FFRSGKYDL (SEQID (SEQID (SEQID 81.42 (SEQID NO:60) NO:373) NO:374) 69.53- NO:373) 81.42 SEFFRSGKY (SEQID NO:374) ASEFFRSGK (SEQID NO:375) Alpha Enolase 273-291 PDQLADLY- Hcit-SFI- 19.25- 24.65 ADLYKSFIK (SEQID 2.4- 22.77 YKSFIKDYP (SEQID 40.44 63.72- KSFIKDYPV (SEQID
Hcit-DYPVVS 19.25- NO:376) 2.4 NO:379) 66.68 NO:382) (SEQID 22.11 DLYKSFIKD 22.03- FIKDYPVVS 60.86- LADLYKSFI NO:61) 20.41- (SEQID 27.67 (SEQID 66.68 (SEQID 24.65 NO:377) 27.67 NO:380) 40.44 NO:381) LYKSFIKDY LYKSFIKDY ADLYKSFIK (SEQID (SEQID (SEQID NO:378) NO:378) NO:376) LADLYKSFI FIKDYPVVS (SEQID (SEQID NO:381) NO:380) Alpha Enolase 301-317 WGAWQ- Hcit- 26.6-30 KFTASAGIQ (SEQID 5.6-9.22 WQKFTASAG (SEQID 6.34 6.34 WQKFTASAG (SEQID
FTASAGIQWG NO:383) NO:384) NO:384) (SEQID AWQKFTASA NO:62) (SEQID NO:385) Alpha Enolase 333-352 NE-Hcit- SCNCLLL- Hcit- 39.34-62.75 39.34-42.6 39.34-62.75 CLLLKVNQI (SEQID NO:387) 9.38-19.95 12.48-19.95 LLKVNQIGS (SEQID NO:388) 14.35-15.37 CLLLKVNQI (SEQID NO:387)
VNQIGSVTE LLKVNQIGS CLLLKVNQI (SEQID (SEQID (SEQID NO:386) NO:388) NO:387) SCNCLLLKV (SEQID NO:389) Alpha 400-419 RSERLA- 11.8- RLAKYNQLL 23.15- LAKYNQLLR 45.27- RLAKYNQLL
Enolase Hcit-YNQLLRI 12.36 (SEQID 31.22 (SEQID 52.87 (SEQID EEELGS 11.8- NO:390) NO:392) 58.12- NO:390) (SEQID 18.57 AKYNQLLRI 59.75 AKYNQLLRI NO:64) (SEQID (SEQID NO:391) NO:391) Alpha Enolase 418-434 GS-Hcit-A- Hcit-FAGRNF RNPLA-Hcit- 16.18-19.61 16.18 KAKFAGRNF (SEQID NO:393) 19.53-33.35 KFAGRNFRN (SEQID NO:394) 76.13 KFAGRNFRN (SEQID NO:394)
(SEQID KFAGRNFRN NO:65) (SEQID NO:394) Beta catenin 127-144 EPSQML- Hcit-HA 33.28- 41.88 LKHAWNLI (SEQID 3.3-3.45 LKHAWNLI (SEQID 16.66- 22.24 LKHAWNLI (SEQID
VVNLINYQD 33.28- NO:395) NO:395) 16.66- NO:395) (SEQID 41.88 MLKHAVVNL 19.78 AWNLINYQ NO:66) (SEQID (SEQID NO:396) NO:397) Beta catenin 334-354 E-Hdt- LLVvTTSRVL- 0.74- 12.86 WTTSRVLKV (SEQID 1.86- 4.47 VLKVLSVCS (SEQID 9.62 9.62- KLLWTTSRV (SEQID
Hdt- 10.61- NO:398) 1.88- NO:400) 17.94 NO:402) VLSVCSSN- 61.82 ITSRVLKVL 4.47 LKVLSVCSS 12.82- WTTSRVLKV Hcit (SEQID 4.77- (SEQID 17.94 (SEQID (SEQID NO:399) 7.6 NO:401) 12.82- NO:398) NO:67) WTTSRVLKV 15.58 SRVIKVLSV (SEQID (SEQID NO:398) NO:403) VLKVLSVCS (SEQID NO:400) Beta 258-275 TLHNLLI 55.35- LLHQEGAKM 5.06 LLHQEGAKM 12.43 LHQEGAKMA
catenin HQEGA-Hcit- 73.17 (SEQID 5.06 (SEQID 12.43-13.1 (SEQID MAVRL NO:404) NO:404) NO:405) (SEQID LHQEGAKMA LLHQEGAKM NO:68) (SEQID (SEQID NO:405) NO:404) Beta catenin 269-288 A-Hcit- MAVRLAGGLQ- 18.5- 35.63 LQKMVALLN (SEQID 4.73-7.08 4.73- LQKMVALLN (SEQID 26.97 19.62- GLQKMVALL (SEQID
Hcit-MVALLN- 18.5- NO:406) 31.97 NO:406) 27.23 NO:408) Hcit 32.91 LAGGLQKMV 30.74- VRLAGGLQK 23.3- VRLAGGLQK (SEQID 18.5- (SEQID 31.35 (SEQID 26.97 (SEQID NO:69) 35.63 NO:407) NO:409) 26.97 NO:409) 32.91 GLQKMVALL LAGGLQKMV LAGGLQKMV (SEQID (SEQID (SEQID NO:408) NO:410) NO:407) VRLAGGLQK LQKMVALLN (SEQID (SEQID NO:409) NO:406) Beta catenin 288-307 -Hcit-TNV- Hcit- FLAITTDC LQILAYG 8.87-10.9 NVKFLAITT (SEQID NO:410)
(SEQID NO:70) Beta catenin 332-349 TYE-Hcit- LLWTTSRVL- 0.73-1.12 WTTSRVLKV (SEQID 3.19-7.23 3.24 WTTSRVLKV (SEQID 9.62- 11.15 KLLWTTSRV (SEQID
Hcit-VLSV NO:398) NO:398) 9.62 NO:402) (SEQID YEKLLWTTS WHSRVLKV NO:71) (SEQID (SEQID NO:411) NO:398) Beta catenin 340-358 TSRVL-Hcit- VLSVCSSN- Hcit-PAIV 59.83- 63.44 48.36- LKVLSVCSS (SEQID NO:401) 1.6-1.88 1.6-1.88 VLSVCSSNK (SEQID NO:413) 12.82- 34.92 12.82- LKVLSVCSS (SEQID NO:401)
(SEQID 66.69 KVLSVCSSN LKVLSVCSS 34.92 VLKVLSVCS NO:72) 59.83- (SEQID (SEQID 12.82- (SEQID 69.02 NO:812) NO:401) 15.54 NO:400) RVLKVLSVC SRVLKVLSV (SEQID (SEQID NO:412) NO:403) Beta 478-497 catenin Beta catenin 489-506 YGLPVW- Hcit-LLHPPSH WPL 43.17- 61.12 43.17- PVVVKLLMP (SEQID NO:414) 4.66-4.96 VVKLLHPPS (SEQID NO:417) 16.56- 20.53 16.56- VVKLLHPPS (SEQID NO:417)
(SEQID 49.37 LPVVVKLLH 20.53 VKLLHPPSH NO:73) 61.92 (SEQID (SEQID NO:415) NO:418) VVVKLLHPP (SEQID NO:416) Beta catenin 503-522 HWPLI-Hcit- ATVGLIRNL 12.35- 30.05 IKATVGLIR (SEQID 10.06- 13.34 WPLIKATVG (SEQID 22.88- 24.83 LIKATVGLI (SEQID
ALCPA 12.35- NO:419) NO:421) 22.88 NO:420) (SEQID 30.05 LIKATVGLI HWPLIKATV NO:74) (SEQID (SEQID NO:420) NO:422) Beta catenin 607-625 LENIQRVAA GVLCELAQD- 39.59 VLCELAQDK (SEQID
Hcit- NO:423) (SEQID NO:75) Beta catenin 650-667 GVATYAAA VLFRMSED- 9.35- 12.15 VLFRMSEDK (SEQID
Hcit-P NO:424) (SEQID NO:76) HSP60 117-136 TVLARSIA- Hcit-EGFE- 8.56- 21.55 IAKEGFEKI (SEQID 21.85- 52.27 IAKEGFEKI (SEQID 45.06- 65.33 VLARSIAKE (SEQID
Hcit-IS- NO:425) 21.85- NO:425) 45.06- NO:428) Hcit-GAN 42.65 LARSIAKEG 71.85 LARSIAKEG (SEQID 31.74 (SEQID 71.85- (SEQID NO:78) NO:426) 83.97 NO:426) EGFEKISKG 71.85- SIAKEGFEK (SEQID 83.97 (SEQID NO:427) NO:429) RSIAKEGFE (SEQID NO:430) HSP60 280-299 GEALSTLV LNRL-Hcit- 10.52- 13.78 STLVLNRLK (SEQID 7-7.76 11.03- LVLNRLKVG (SEQID 19.9- 22.24 LNRLKVGLQ (SEQID
VGLQVVA 19.17- NO:431) 13.12 NO:433) 22.24 NO:435) (SEQID 27.64 RLKVGLQVV LNRLKVGLQ TLVLNRLKV NO:79) 19.17- (SEQID (SEQID (SEQID 27.64 NO:432) NO:435) NO:434) 23.62- LVLNRLKVG 27.64 (SEQID NO:433) TLVLNRLKV (SEQID NO:434) HSP60 381-398 TTSEYE-Hcit- E-Hcit- 18.89- 23.14 KLNERLAKL (SEQID 27.33- 36.43 YEKEKLNER (SEQID 51.23- 59.99 KLNERLAKL (SEQID
LNERLA-Hcit- 29.24 NO:436) 27.33 NO:438) 74.42- NO:436) LS 18.89- TTSEYEKEK KLNERLAKL 77.04 YEKEKLNER (SEQID 29.24 (SEQID (SEQID 51.23- (SEQID NO:80) NO:437) NO:436) 77.04 NO:438) YEKEKLNER KEKLNERLA (SEQID (SEQID NO:438) NO:439) HSP60 517-536 GIIDPTV- Hcit-VRTALLD 18.25- 30.13 KVVRTALLD (SEQID 14.63- 17.84 TKVVRTALL (SEQID 15.82- 21.8 PTKVVRTAL (SEQID
AAGVA 18.25- NO:440) 17.84 NO:441) 20.49- NO:443) (SEQID 30.13 TKVVRTALL IDPTKVVRT 21.8 TKVVRTALL NO:81) (SEQID (SEQID 21.19 (SEQID NO:441) NO:442) NO:441) IDPTKVVRT (SEQID NO:442) HSP60 84-103 IDL-Hcit-D- Hcit-Y-Hcit- 50.61- 63.16 YKNIGAKLV (SEQID 4.84-11.64 YKNIGAKLV (SEQID 0.42-2.37 0.42-2.37 KYKNIGAKL (SEQID
NIGA-Hcit- 50.61- NO:444) NO:444) NO:446) LVQDVAN 63.16 KNIGAKLVQ YKNIGAKLV (SEQID (SEQID (SEQID NO:77) NO:445) NO:444)
Example 4. Responses in Healthy Human Donors and Cancer Patients
(147) 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 (
(148) 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 (
(149) Analysis of cytokine expression was also performed on majority of donors (example plots shown
(150) 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 (
(151) 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 nave 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 and Chain Pairing of TCRs Recognising Peptides Containing Homocitrulline
(152) 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 V and V sequences among CD4+proliferating cells from donor BD00016 ALDOA 74 Hcit (
(153) To identify the correct pairing of the TCRV and TCRV chains, single proliferating cells were sorted into 96 individual wells and TCR and TCRB chains were sequenced using iPair technology. 76/92 wells contained both TCRV and TCRV 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%.
(154) TABLE-US-00015 TABLE7 iPairsequencingandanalysisoftheTCRandchainsALDOA74Hcit FRE- QUEN- CY (40 CDR1 CDR2 CDR3 Read Bulk Unique/ SEQ WELLS) WELLS Peptide Peptide Peptide VGene DGene JGene CGene Count Rank shared ID TCR 8 B11/D02/ ENHRY SYGVKD AISERRDQETQY hTR8V10-3 * hTR8J2- hTRBC 9659 1 S 3 9 G10/B801/ (SEQID (SEQID (SEQID hTRAV26-2 * 5 hTRBC 4355 1 S 4 DE12/DE NO:85) NO:86) NO:87) hTRAJ20 09/DF06/ TISGTDY GLTSN ILRDVYDYKLS DH01 (SEQID (SEQID (SEQID NO:88) NO:89) NO:90) 10 C04/E02/ DFQATT SNEGSKA SAPIHTDTQY hTR8V20-1 * hTR8J2- hTBDC 1503 5 S 15 10 F02/H98/ (SEQID (SEQID (SEQID hTRAV36/ * 3 hTRBC 12749 3 S 16 H11/DB10/ NO:91) NO:92) NO:93) DV7 hTRAJ39 DB12/ VTNFRS LTSSGIE AVHPAGNMLT DE02/ (SEQID (SEQID (SEQID DE03/DF08 NO:94) NO:95) NO:96) 3 G08/DF04/ SGHDY FNNNVP ASRGGLAS hTR8V12-4 hTR8D2 hTRBJ2- hTRBC 404 4 S 17 11 DG06 (SEQID (SEQID NEQF hTRAV8-6 * 1 hTRAC 15860 2 S 18 NO:97) NO:98) (SEQID SSVSVY YLSGST NO:99) hTRAJ6 (SEQID LV AVSEGGGSYIPT NO:100) (SEQID (SEQID NO:101) NO:102) 5 C02/DA04/ LNHDA SQIVND ASSLGTFYEQY hTR8V19 hTR8D1 hTR8J2- hTRBC 900 3 S 19 12 DC07/ (SEQID (SEQID (SEQID hTRAV13-1 * 7 hTRAC 5387 7 S 20 20 DE01/ NO:103) NO:104) NO:105) hTRAJ27 DH10 DSASNY IRSNVGE AASGNTNA (SEQID (SEQID GKST NO:106) NO:107) (SEQID NO:108) 7 C03/E05/ SGHRS YFSETQ ASSLGVMVVST hTRBVS-1 * hTRBJ2- hTRBC 1244 12 U 21 13 E08/F03/ (SEQID (SEQID DTQV 3 F08/ NO:110) NO:109) (SEQID hTRAV26-2 * hTRAJ4B hTRAC 11109 6 S 22 DF10/ TISGTDV GLTSN NO:111) DG07 (SEQID (SEQID ILRDRVSNF NO:88) NO:89) GNEKLT (SEQID NO:112) 1 A06 SGHAT FQNNGV ASSPTQGASYE hTRBV11-2 hTRBD1 hTRBJ2- hTRBC 4927 2 S 23 14 (SEQID (SEQID QY hTRAV3 * 7 hTRAC 3557 10 S 24 NO:113) NO:114) (SEQID hTRAJ11 VSGNPY YITGDNLV NO:115) (SEQID (SEQID AVRDAGYSTLT NO:116) NO:117) (SEQID NO:118) 1 DC02 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2- hTRBC 10415 1 S 3 15 (SEQID (SEQID (SEQID hTRAV13-1 * S hTRAC 52 7 S 20 NO:85) NO:86) NO:87) hTRAJ27 DSASNY IRSNVGE AASGNTNAGKST (SEQID (SEQID (SEQID NO:106) NO:107) NO:108) 1 G01 ENHRY SYGVKD AISERRDQETQY hTRBV10-3 * hTRBJ2- hTRBC 1437 1 S 3 16 (SEQID (SEQID (SEQID hTRAV13-1 * 5 hTRAC 9890 58 U 25 NO:85) NO:86) NO:87) hTRAJ30 DSASNY IRSNVGE AASIDRDDKII (SEQID (SEQID (SEQID NO:106) NO:107) NO:119) 1 E06 MDHEN SYDVKM ATTQGSYN hTR3V28 hTRBD1 hTRBJ2- hTRBC 5226 7 S 26 17 (SEQID (SEQID EQF hTRAV20 * 1 hTRAC 8047 8 S 27 NO:120) NO:121) (SEQID hTRAJG VSGLRG LYSAGEE NO:122) (SEQID (SEQID AVQAGSYIPT NO:123) NO:124) (SEQID NO:125)
(155) The full sequences of these TCRs are shown in
(156) 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 V and V sequences among CD4+proliferating cells from donor BD00016 ALDOA 140-157 Hcit (
(157) To identify the correct pairing of the TCRV and TCRV chains, single proliferating cells were sorted into 96 individual wells and TCR and TCRB chains were sequenced using iPair technology. 60/75 wells contained both TCRV and TCRV 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%.
(158) TABLE-US-00016 TABLE8 iPairsequencingandanalysisoftheTCRandchainsALDOA140Hcit FRE- QUEN- CY (34 CDR1 CDR2 Read Bulk Unique/ SEQ WELLS) WELLS Peptide Peptide CDR3Peptide VGene DGene JGene CGene Count Rank shared ID TCR 3 G07/DA04/ EMHRY SYGVKD AISERRDQETQV hTRBV10-3 * hTRBJ2- hTRBC 3159 1 S 8 18 DE06 (SEQID (SEQID (SEQID hTRAV26-2 * 5 hTRAC 12015 6 S 4 NO:85) NO:86) NO:87) hTRAJ20 TISGTDY GLTSN ILRDVYDVKLS (SEQID (SEQID (SEQID NO:88) NO:89) NO:90) 9 B01/B06/ DFQATT SNEGSKA SARTSGTNTQY Htrbv20-3 hTRBD2 hTRBJ2- hTRBC 3305 2 S 28 19 C02/E01/ (SEQID (SEQID (SEQID hTRAV8-4 * 3 hTRAC 12349 1 29 E04/E11/ NO:91) NO:92) NO:126) hTRAJ20 H08/DE09/ SSVPPY YTSAATLV AVSGRNDYKLS DG11 (SEQID (SEQID (SEQID NO:127) NO:128) NO:129) 6 B03/C01/ MNHEY SVGEGT ASSRSWTASGVT hTRBV6-3 hTRBD1 hTRBJ1- hTRBC 2389 3 S 30 20 C03/DB06/ (SEQID (SEQID (SEQID hTRAV26-2 * 2 HTRAC 13952 2 S 31 DC05/DC09 NO:130) NO:131) NO:132) hTRAJ48 TISGTDY GLTSN ILRDGSGNEKLT (SEQID (SEQID (SEQID NO:88) NO:89) NO:133) 2 B08/E12 SGHNS FNNNVP ASSVAQLAGKGEQF hTRBV12-3 hTRBD2 hTRBJ2- hTRBC 3352 5 S 32 21 (SEQID (SEQID (SEQID hTRAV13-1 * 1 hTRAC 8867 3 S 25 NO:134) NO:98) NO:135) hTRAJ30 DSASNY IRSNVGE AASIDRDDKII (SEQID (SEQID (SEQID NO:106) NO:107) NO:119) 3 A1G/D02/ SNHLY FYNNEI ASRRVMGYGVT hTRBV2 * hTRBJ1- hTRBC 5484 4 S 33 22 F09 (SEQID (SEQID (SEQID hTRAV21 * 2 hTRAC 8520 11 S 34 NO:136) NO:137) NO:138) hTRAJ53 DSAIVN IQSSQRE ALNSGGSNYKLT (SEQID (SEQID (SEQID NO:139) NO:140) NO:141) 1 F02 DFQATT SNEGSKA SAGRAGTSGTYEQY hTRBV20-3 hTRBD2 hTRBJ2- HTRBC 1569 11 U 35 23 (SEQID (SEQID (SEQID hTRAV26-2 * 7 hTRAC 12730 4 S 36 NO:91) NO:92) NO:142) hTRAJ48 TISGTDY GLTSN ILRSNPGNEKLT (SEQID (SEQID (SEQID NO:88) NO:89) NO:143) 1 E03 LNHDA SQIVND ASSGGQFNQPQH hTRBV10 hTRBD1 hTRBJ1- HTRBC 1303 9 S 37 24 (SEQID (SEQID (SEQID hTRAV6 * 5 hTRAC 10507 8 U 38 NO:103) NO:104) NO:144) hTRAJ36 NYSPAY IRENEKE ALGQTGANNLF (SEQID (SEQID (SEQID NO:145) NO:146) NO:147) 2 G11/DC02 KGHSH IQKENI ASSPEALANTGELF hTRBV18 * hTRBJ2- hTRBC 2473 8 S 39 25 (SEQID (SEQID (SEQID hTRAV26-1 * 2 hTRAC 4309 7 S 40 NO:148) NO:149) NO:150) hTRAJ43 TISGNEY GLKNN IVRVGYNNNDMR (SEQID (SEQID (SEQID NO:151) NO:152) NO:153) 2 DU/DA12 KGHDR SFDVKD ATSDPSGPPYEQY hTRBV24-1 hTRBD2 hTRBJ2- hTRBC 480 21 S 41 26 (SEQID (SEQID (SEQID hTRAV26-2 * 7 hTRAC 13807 10 S 42 NO:154) NO:155) NO:156) hTRAJ57 TISGTDY GLTSN ILRAQGGSEKLV (SEQID (SEQID (SEQID NO:88) NO:89) NO:157) 1 B07 SNHLV FYNNEI ASRAGTGIGGVT hTRBV2 hTRBD1 hTRBJ1- hTRBC 2294 10 S 43 27 (SEQID (SEQID (SEQID hTRAV21 * 2 hTRAC 12152 17 S 44 NO:136) NO:137) NO:158) hTRAJ53 DSAIYN IQSSQRE AVYSGGSNYRLT (SEQID (SEQID (SEQID NO:139) NO:140) NO:159)
(159) The full sequences of these TCRs are shown in
(160) 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 V and V sequences among CD4+proliferating cells from donor BD00016 ALDOA 140-157 Hcit (
(161) To identify the correct pairing of the TCRV and TCRV chains, single proliferating cells were sorted into 96 individual wells and TCR and TCRB chains were sequenced using iPair technology. 70/80 wells contained both TCRV and TCRV 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%.
(162) TABLE-US-00017 TABLE9 iPairsequencingandanalysisoftheTCRandchainsforvimentin116Hcit FRE- QUEN- CY (30 CDR1 CDR2 CDR3 Read Bulk Unique/ SEQ wells) WELSS Peptide Peptide Peptide VGene DGene JGene CGene Count Rank shared ID TCR 6 A01/ MDHEN SYDYKM ASSLLGSS hTRBV28 hTRBD2 hTRBJ1- hTRBC 236 7 S 1 1 A06/ (SEQID (SEQID PLH hTRAV38-2/ * 6 hTRAC 17129 1 S 2 B11/ NO:120) NO:121) (SEQID DV8 HTRAJ23 C05/ TSESDYY QEAYKQQN NO:160) E02/ (SEQID (SEQID AYRSYNQG H07 NO:161) NO:162) GKLI (SEQID NO:163) 1 D12 ENHRY SYGVKD AISERRDQ hTRBV10-3 * hTRBJ2- hTRBC 545 1 S 3 2 (SEQID (SEQID ETQY hTRAV26-2 * 5 hTRAC 12336 2 S 4 NO:85) NO:86) (SEQID HTRAJ20 TISGTDY GLTSN NO:87) (SEQID (SEQID ILRDVYDY NO:88) NO:89) KLS (SEQID NO:90) 1 G02 ENHRY SYGVKD AISERRDQ hTRBV10-3 * hTRBJ2- hTRBC 58 1 S 3 3 (SEQID (SEQID ETQY hTRAV38- * 5 hTRAC 3641 1 S 2 NO:85) NO:86) (SEQID 2/DV8 HTRAJ23 TISGTDY QEAYKQQN NO:87) (SEQID (SEQID AYRSYNQG NO:161) NO:162) GKLI (SEQID NO:163) 2 D11/F01 LGHDI YNNKEL ASSQEPSI hTRBV3-1 * hTRBJ2- hJRbC 822 8 S 5 4 (SEQID (SEQID HNEQF hTRAV26-2 * 1 hTRAC 10799 3 S 6 NO:164) NO:165) (SEQID hTRAJ42 TISGTDY GLTSN NO:166) (SEQID (SEQID ILKNYGGS NO:88) NO:89) QGNLI (SEQID NO:167) 1 D09 MNHNY SVGAGI ASSPGQPY hTRBV6-6 hTRBD1 hTRBJ1- hTRBC 972 4 U 7 5 (SEQID (SEQID GYT hTRAV16 2 hTRAC 6779 6 S 8 NO:168) NO:169) (SEQID HTRAJ26 YSGSPE HISR NO:170) (SEQID (SEQID ALSGPSYG NO:171) NO:172) QNFV (SEQID NO:173) 1 C03 MNHNS SASEGT ASEGLASY hTRBV6-1 hTRBD2 hTRBJ2- hTRBC 6119 6 S 9 6 (SEQID (SEQID NEQF hTRAV9-2 * 1 hTRAC 3386 169 U 10 NO:174) NO:175) (SEQID HTRAJ13 ATGYPS ATKADDK NO:176) (SEQID (SEQID ALTGGGYQ NO:177) NO:178) KVT (SEQID NO:179) 2 A05/H06 MNREY SMNVEV ASSFREGE hTRBV27 hTRBD2 hTRBJ1- hTRBC 516 61 U 11 7 (SEQID (SEQID KLF hTRAV17 * 4 hTRAC 12085 8 S 12 NO:130) NO:180) (SEQID hTRAJ8 TSINN IRSNERE NO:181) (SEQID (SEQID ATAMNTGF NO:182) NO:183) QKLV (SEQID NO:184) 3 B05/ LGHNA YNFKEQ ASSREGLA hTRBV4-2 hTRBD1 hTRBJ2- hTRBC 10614 26 S 13 8 E01/F10 (SEQID (SEQID GLNEQF hTRAV29/ * 1 hTRAC 2057 122 S 14 NO:185) NO:186) (SEQID DV5 HTRAJ32 NSMFDY ISISSIKDK NO:187) (SEQID (SEQID AASGWGDG NO:188) NO:189) GATNKLI (SEQID NO:190)
(163) The full sequences of these TCRs are shown in
Example 6. Immunisation with Homocitrulline Peptides Provides Efficient Therapy of the Aggressive B16 Melanoma
(164) 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.
(165) HHDII/DR1 (
(166) HHDII/DP4 mice were challenged with B16 HHDII/DP4 tumour cells and then immunised with ALDOA homocitrulline peptides (
(167) 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) (
(168) 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 IFN. To mimic the naturally occurring tumour, we engineered B16F1 cells to express HLA-DR4 under control of mouse IFN-inducible promoter. The HLA-DR4 expression level can be upregulated in the presence of mouse IFN (Brentville et al. 2016; Cook et al. 2018). HLA-DR4 mice were then implanted with this IFN-inducible B16 DR4 tumour followed by immunisation with vimentin 116-135Hcit peptide (
(169) In another similar model, HHDII/DP4 mice were implanted with B16F1 cells expressing HLA-DP4 under control of mouse IFN-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) (
(170) 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
(171) 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 (
(172) 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 (
(173) 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) (
(174) Next, antibodies were used to deplete the Ly6C+ population (
(175) 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 IL18. 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 (
(176) 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 (
Example 8. Tumour Therapy is Mainly Mediated by the Direct Action of CD4 Cells Upon Tumour Cells Presenting Peptide on MHC-II
(177) 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 (
(178) 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 (
(179) 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
(180) 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 (
(181) 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% (
(182) 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 (
(183) HLA-DP4 transgenic mice.
(184) 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
REFERENCES
(185) Alland, C., D. Moreews, D. Boens, M. Carpentier, S. Chiusa, M. Lonquety, N. Renault, Y. Wong, H. Cantalloube, J. Chomilier, J. Hochez, J Pothier, B.O. Villoutreix, J.F. Zagury, and P. Tuffery. 2005. RPBS: a web resource for structural bioinformatics, Nucleic Acids Res., 33: W44-9. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool, J Mol Biol, 215:403-10. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res, 25:3389-402. Andreatta, M., V. I. Jurtz, T. Kaever, A. Sette, B. Peters, and M. Nielsen. 2017. Machine learning reveals a non-canonical mode of peptide binding to MHC class II molecules, Immunology, 152:255-64. Andreatta, M., C. Schafer-Nielsen, O. Lund, S. Buus, and M. Nielsen. 2011. NNAlign: a web-based prediction method allowing non-expert end-user discovery of sequence motifs in quantitative peptide data, PLOS One, 6: e26781. Arap, M. A., J. Lahdenranta, P. J. Mintz, A. Hajitou, A. S. Sarkis, W. Arap, and R. Pasqualini. 2004. Cell surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands, Cancer Cell, 6:275-84. Arnold, P. Y., N. L. La Gruta, T. Miller, K. M. Vignali, P. S. Adams, D. L. Woodland, and D. A. Vignali. 2002. The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues, J Immunol, 169:739-49. Barra, C., B. Alvarez, S. Paul, A. Sette, B. Peters, M. Andreatta, S. Buus, and M. Nielsen. 2018. Footprints of antigen processing boost MHC class II natural ligand predictions, Genome Med, 10:84. Benfeitas, R., M. Uhlen, J. Nielsen, and A. Mardinoglu. 2017. New Challenges to Study Heterogeneity in Cancer Redox Metabolism, Front Cell Dev Biol, 5:65. Blass, S., A. Union, J. Raymackers, F. Schumann, U. Ungethum, S. Muller-Steinbach, F. De Keyser, J. M. Engel, and G. R. Burmester. 2001. The stress protein BiP is overexpressed and is a major B and T cell target in rheumatoid arthritis, Arthritis Rheum, 44:761-71. Blobel, Gerd A., Roland Moll, Werner W. Franke, and Ingolf Vogt-Moykopf. 1984. Cytokeratins in normal lung and lung carcinomas, Virchows Archiv B, 45:407-29. Bobisse, S., R. Genolet, A. Roberti, J. L. Tanyi, J. Racle, B. J. Stevenson, C. Iseli, A. Michel, M. A. Le Bitoux, P. Guillaume, J. Schmidt, V. Bianchi, D. Dangaj, C. Fenwick, L. Derre, I. Xenarios, O. Michielin, P. Romero, D. S. Monos, V. Zoete, D. Gfeller, L. E. Kandalaft, G. Coukos, and A. Harari. 2018. Sensitive and frequent identification of high avidity neo-epitope specific CD8 (+) T cells in immunotherapy-naive ovarian cancer, Nat Commun, 9:1092. Bodanzsky, Bodanzsky &. 1984. The practice of peptide synthesis (Springer Verlag: New York). Boulter, J. M., M. Glick, P. T. Todorov, E. Baston, M. Sami, P. Rizkallah, and B. K. Jakobsen. 2003. Stable, soluble T-cell receptor molecules for crystallization and therapeutics, Protein Eng, 16:707-11. Box, J. K., N. Paquet, M. N. Adams, D. Boucher, E. Bolderson, K. J. O'Byrne, and D. J. Richard. 2016. Nucleophosmin: from structure and function to disease development, BMC Mol Biol, 17:19. Brentville, V. A., R. L. Metheringham, B. Gunn, P. Symonds, I. Daniels, M. Gijon, K. Cook, W. Xue, and L. G. Durrant. 2016. Citrullinated Vimentin Presented on MHC-II in Tumor Cells Is a Target for CD4+ T-Cell-Mediated Antitumor Immunity, Cancer Res, 76:548-60. Brentville, V. A., P. Symonds, K. W. Cook, I. Daniels, T. Pitt, M. Gijon, P. Vaghela, W. Xue, S. Shah, R. L. Metheringham, and L. G. Durrant. 2019. T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; Targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy, Oncoimmunology, 8: e1576490. Bronte, V., S. Brandau, S. H. Chen, M. P. Colombo, A. B. Frey, T. F. Greten, S. Mandruzzato, P. J. Murray, A. Ochoa, S. Ostrand-Rosenberg, P. C. Rodriguez, A. Sica, V. Umansky, R. H. Vonderheide, and D. I. Gabrilovich. 2016. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards, Nat Commun, 7:12150. Brown, J. H., T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger, and D. C. Wiley. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1R1, Nature, 364:33-9. Burska, A. N., L. Hunt, M. Boissinot, R. Strollo, B. J. Ryan, E. Vital, A. Nissim, P. G. Winyard, P. Emery, and F. Ponchel. 2014. Autoantibodies to posttranslational modifications in rheumatoid arthritis, Mediators Inflamm, 2014:492873. Cappello, F., E. Conway de Macario, L. Marasa, G. Zummo, and A. J. Macario. 2008. Hsp60 expression, new locations, functions and perspectives for cancer diagnosis and therapy, Cancer Biol Ther, 7:801-9. Cappello, P., B. Tomaino, R. Chiarle, P. Ceruti, A. Novarino, C. Castagnoli, P. Migliorini, G. Perconti, A. Giallongo, M. Milella, V. Monsurro, S. Barbi, A. Scarpa, P. Nistico, M. Giovarelli, and F. Novelli. 2009. An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen, Int J Cancer, 125:639-48. Carson, R. T., K. M. Vignali, D. L. Woodland, and D. A. Vignali. 1997. T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage, Immunity, 7:387-99. Castillo-Tong, D. C., D. Pils, G. Heinze, I. Braicu, J. Sehouli, A. Reinthaller, E. Schuster, A. Wolf, R. Watrowski, R. A. Maki, R. Zeillinger, and W. F. Reynolds. 2014. Association of myeloperoxidase with ovarian cancer, Tumour Biol, 35:141-8. Cedervall, J., A. Hamidi, and A. K. Olsson. 2018. Platelets, NETs and cancer, Thromb Res, 164 Suppl 1: S148-S52. Chang, X., Y. Zhao, Y. Wang, Y. Chen, and X. Yan. 2013. Screening citrullinated proteins in synovial tissues of rheumatoid arthritis using 2-dimensional western blotting, J Rheumatol, 40:219-27. Chen, X., T. T. Yang, Y. Zhou, W. Wang, X. C. Qiu, J. Gao, C. X. Li, H. Long, B. A. Ma, Q. Ma, X. Z. Zhang, L. J. Yang, and Q. Y. Fan. 2014. Proteomic profiling of osteosarcoma cells identifies ALDOA and SULT1A3 as negative survival markers of human osteosarcoma, Mol Carcinog, 53:138-44. Chou, Y. H., O. Skalli, and R. D. Goldman. 1997. Intermediate filaments and cytoplasmic networking: new connections and more functions, Curr Opin Cell Biol, 9:49-53. Consogno, G., S. Manici, V. Facchinetti, A. Bachi, J. Hammer, B. M. Conti-Fine, C. Rugarli, C. Traversari, and M. P. Protti. 2003. Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3, Blood, 101:1038-44. Cook, K., I. Daniels, P. Symonds, T. Pitt, M. Gijon, W. Xue, R. Metheringham, L. Durrant, and V. Brentville. 2018. Citrullinated alpha-enolase is an effective target for anti-cancer immunity, Oncoimmunology, 7: e1390642. Corper, A. L., T. Stratmann, V. Apostolopoulos, C. A. Scott, K. C. Garcia, A. S. Kang, I. A. Wilson, and L. Teyton. 2000. A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes, Science, 288:505-11. Coulombe, P. A., and M. B. Omary. 2002. Hard and soft principles defining the structure, function and regulation of keratin intermediate filaments, Curr Opin Cell Biol, 14:110-22. Crooks, G. E., G. Hon, J. M. Chandonia, and S. E. Brenner. 2004. WebLogo: a sequence logo generator, Genome Res, 14:1188-90. Dai, L., G. Pan, X. Liu, J. Huang, Z. Jiang, X. Zhu, X. Gan, Q. Xu, and N. Tan. 2018. High expression of ALDOA and DDX5 are associated with poor prognosis in human colorectal cancer, Cancer Manag Res, 10:1799-806. Davis, M. M., J. J. Boniface, Z. Reich, D. Lyons, J. Hampl, B. Arden, and Y. Chien. 1998. Ligand recognition by alpha beta T cell receptors, Annu Rev Immunol, 16:523-44. Dessen, A., C. M. Lawrence, S. Cupo, D. M. Zaller, and D. C. Wiley. 1997. X-ray crystal structure of HLA-DR4 (DRA*0101, DRB1*0401) complexed with a peptide from human collagen Il, Immunity, 7:473-81. Dorner, A. J., L. C. Wasley, and R. J. Kaufman. 1992. Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster ovary cells, EMBO J, 11:1563-71. Droeser, R. A., C. Hirt, S. Eppenberger-Castori, I. Zlobec, C. T. Viehl, D. M. Frey, C. A. Nebiker, R. Rosso, M. Zuber, F. Amicarella, G. lezzi, G. Sconocchia, M. Heberer, A. Lugli, L. Tornillo, D. Oertli, L. Terracciano, and G. C. Spagnoli. 2013. High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor, PLOS One, 8: e64814. Du, S., Z. Guan, L. Hao, Y. Song, L. Wang, L. Gong, L. Liu, X. Qi, Z. Hou, and S. Shao. 2014. Fructose-bisphosphate aldolase a is a potential metastasis-associated marker of lung squamous cell carcinoma and promotes lung cell tumorigenesis and migration, PLOS One, 9: e85804. Eruslanov, E. B., P. S. Bhojnagarwala, J. G. Quatromoni, T. L. Stephen, A. Ranganathan, C. Deshpande, T. Akimova, A. Vachani, L. Litzky, W. W. Hancock, J. R. Conejo-Garcia, M. Feldman, S. M. Albelda, and S. Singhal. 2014. Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer, J Clin Invest, 124:5466-80. Falini, B., C. Mecucci, E. Tiacci, M. Alcalay, R. Rosati, L. Pasqualucci, R. La Starza, D. Diverio, E. Colombo, A. Santucci, B. Bigerna, R. Pacini, A. Pucciarini, A. Liso, M. Vignetti, P. Fazi, N. Meani, V. Pettirossi, G. Saglio, F. Mandelli, F. Lo-Coco, P. G. Pelicci, M. F. Martelli, and Gimema Acute Leukemia Working Party. 2005. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype, N Engl J Med, 352:254-66. Feng, H., Y. Zeng, L. Whitesell, and E. Katsanis. 2001. Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity, Blood, 97:3505-12. Franke, W. W., D. L. Schiller, R. Moll, S. Winter, E. Schmid, I. Engelbrecht, H. Denk, R. Krepler, and B. Platzer. 1981. Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues, J Mol Biol, 153:933-59. Fremont, D. H., W. A. Hendrickson, P. Marrack, and J. Kappler. 1996. Structures of an MHC class II molecule with covalently bound single peptides, Science, 272:1001-4. Fremont, D. H., D. Monnaie, C. A. Nelson, W. A. Hendrickson, and E. R. Unanue. 1998. Crystal structure of I-Ak in complex with a dominant epitope of lysozyme, Immunity, 8:305-17. Fu, Q. F., Y. Liu, Y. Fan, S. N. Hua, H. Y. Qu, S. W. Dong, R. L. Li, M. Y. Zhao, Y. Zhen, X. L. Yu, Y. Y. Chen, R. C. Luo, R. Li, L. B. Li, X. J. Deng, W. Y. Fang, Z. Liu, and X. Song. 2015. Alpha-enolase promotes cell glycolysis, growth, migration, and invasion in non-small cell lung cancer through FAK-mediated PI3K/AKT pathway, J Hematol Oncol, 8:22. Fuchs, E., and D. W. Cleveland. 1998. A structural scaffolding of intermediate filaments in health and disease, Science, 279:514-9. Fukunaga, Y., S. Bandoh, J. Fujita, Y. Yang, Y. Ueda, S. Hojo, K. Dohmoto, Y. Tojo, J. Takahara, and T. Ishida. 2002. Expression of cytokeratin 8 in lung cancer cell lines and measurement of serum cytokeratin 8 in lung cancer patients, Lung Cancer, 38:31-8. Gay, M. H., T. Valenta, P. Herr, L. Paratore-Hari, K. Basler, and L. Sommer. 2015. Distinct adhesion-independent functions of beta-catenin control stage-specific sensory neurogenesis and proliferation, BMC Biol, 13:24. Gerstner, C., A. Dubnovitsky, C. Sandin, G. Kozhukh, H. Uchtenhagen, E. A. James, J. Ronnelid, A. J. Ytterberg, J. Pieper, E. Reed, C. Tandre, M. Rieck, R. A. Zubarev, L. Ronnblom, T. Sandalova, J. H. Buckner, A. Achour, and V. Malmstrom. 2016. Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted alpha-Enolase T Cell Epitope in Rheumatoid Arthritis, Front Immunol, 7:494. Geyer, F. C., M. Lacroix-Triki, K. Savage, M. Arnedos, M. B. Lambros, A. Mackay, R. Natrajan, and J. S. Reis-Filho. 2011. beta-Catenin pathway activation in breast cancer is associated with triple-negative phenotype but not with CTNNB1 mutation, Mod Pathol, 24:209-31. Ghosh, P., M. Amaya, E. Mellins, and D. C. Wiley. 1995. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3, Nature, 378:457-62. Godkin, A. J., K. J. Smith, A. Willis, M. V. Tejada-Simon, J. Zhang, T. Elliott, and A. V. Hill. 2001. Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions, J Immunol, 166:6720-7. Goeb, V., M. Thomas-LOtellier, R. Daveau, R. Charlionet, P. Fardellone, X. Le Loet, F. Tron, D. Gilbert, and O. Vittecoq. 2009. Candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes, Arthritis Res Ther, 11: R38. Gonzalez-Gronow, M., M. Cuchacovich, C. Llanos, C. Urzua, G. Gawdi, and S. V. Pizzo. 2006. Prostate cancer cell proliferation in vitro is modulated by antibodies against glucose-regulated protein 78 isolated from patient serum, Cancer Res, 66:11424-31. Gonzalez-Gronow, M., M. A. Selim, J. Papalas, and S. V. Pizzo. 2009. GRP78: a multifunctional receptor on the cell surface, Antioxid Redox Signal, 11:2299-306. Hagiwara, T., K. Nakashima, H. Hirano, T. Senshu, and M. Yamada. 2002. Deimination of arginine residues in nucleophosmin/B23 and histones in HL-60 granulocytes, Biochem Biophys Res Commun, 290:979-83. Hamaguchi, T., N. lizuka, R. Tsunedomi, Y. Hamamoto, T. Miyamoto, M. lida, Y. Tokuhisa, K. Sakamoto, M. Takashima, T. Tamesa, and M. Oka. 2008. Glycolysis module activated by hypoxia-inducible factor 1alpha is related to the aggressive phenotype of hepatocellular carcinoma, Int J Oncol, 33:725-31. Holmberg Olausson, K., T. Elsir, K. Moazemi Goudarzi, M. Nister, and M. S. Lindstrom. 2015. NPM1 histone chaperone is upregulated in glioblastoma to promote cell survival and maintain nucleolar shape, Sci Rep, 5:16495. Holzer, M., K. Zangger, D. El-Gamal, V. Binder, S. Curcic, V. Konya, R. Schuligoi, A. Heinemann, and G. Marsche. 2012. Myeloperoxidase-derived chlorinating species induce protein carbamylation through decomposition of thiocyanate and urea: novel pathways generating dysfunctional high-density lipoprotein, Antioxid Redox Signal, 17:1043-52. Huang, Z., Y. Hua, Y. Tian, C. Qin, J. Qian, M. Bao, Y. Liu, S. Wang, Q. Cao, X. Ju, Z. Wang, and M. Gu. 2018. High expression of fructose-bisphosphate aldolase A induces progression of renal cell carcinoma, Oncol Rep, 39:2996-3006. Hung, K., R. Hayashi, A. Lafond-Walker, C. Lowenstein, D. Pardoll, and H. Levitsky. 1998. The Central Role of CD4+ T Cells in the Antitumor Immune Response, J. Exp. Med., 188:2357-68. Jaisson, S., C. Pietrement, and P. Gillery. 2011. Carbamylation-derived products: bioactive compounds and potential biomarkers in chronic renal failure and atherosclerosis, Clin Chem, 57:1499-505. Jakobsen, C. G., N. Rasmussen, A. V. Laenkholm, and H. J. Ditzel. 2007. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78, Cancer Res, 67:9507-17. Jang, B., J. K. Jin, Y. C. Jeon, H. J. Cho, A. Ishigami, K. C. Choi, R. I. Carp, N. Maruyama, Y. S. Kim, and E. K. Choi. 2010. Involvement of peptidylarginine deiminase-mediated post-translational citrullination in pathogenesis of sporadic Creutzfeldt-Jakob disease, Acta Neuropathol, 119:199-210. Jang, B., E. Kim, J. K. Choi, J. K. Jin, J. I. Kim, A. Ishigami, N. Maruyama, R. I. Carp, Y. S. Kim, and E. K. Choi. 2008. Accumulation of citrullinated proteins by upregulated peptidylarginine deiminase 2 in brains of scrapie-infected mice: a possible role in pathogenesis, Am J Pathol, 173:1129-42. Jiang, Z., Y. Cui, L. Wang, Y. Zhao, S. Yan, and X. Chang. 2013. Investigating citrullinated proteins in tumour cell lines, World J Surg Oncol, 11:260. June, C. H., M. V. Maus, G. Plesa, L. A. Johnson, Y. Zhao, B. L. Levine, S. A. Grupp, and D. L. Porter. 2014. Engineered T cells for cancer therapy, Cancer Immunol Immunother, 63:969-75. Jurtz, V., S. Paul, M. Andreatta, P. Marcatili, B. Peters, and M. Nielsen. 2017. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data, J Immunol, 199:3360-68. Karlin, S., and S. F. Altschul. 1993. Applications and statistics for multiple high-scoring segments in molecular sequences, Proc Natl Acad Sci USA, 90:5873-7. Kim, A., I. Z. Hartman, B. Poore, T. Boronina, R. N. Cole, N. Song, M. T. Ciudad, R. R. Caspi, D. Jaraquemada, and S. Sadegh-Nasseri. 2014. Divergent paths for the selection of immunodominant epitopes from distinct antigenic sources, Nat Commun, 5:5369. Kjellin, H., H. Johansson, A. Hoog, J. Lehtio, P. J. Jakobsson, and M. Kjellman. 2014. Differentially expressed proteins in malignant and benign adrenocortical tumors, PLOS One, 9: e87951. Kollipara, L., and R. P. Zahedi. 2013. Protein carbamylation: in vivo modification or in vitro artefact?, Proteomics, 13:941-4. Lac, P., S. Saunders, E. Tutunea-Fatan, L. Barra, D. A. Bell, and E. Cairns. 2018. Immune responses to peptides containing homocitrulline or citrulline in the DR4-transgenic mouse model of rheumatoid arthritis, J Autoimmun, 89:75-81. Latek, R. R., A. Suri, S. J. Petzold, C. A. Nelson, O. Kanagawa, E. R. Unanue, and D. H. Fremont. 2000. Structural basis of peptide binding and presentation by the type I diabetes-associated MHC class II molecule of NOD mice, Immunity, 12:699-710. Lee, A. S. 2014. Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential, Nat Rev Cancer, 14:263-76. Lee, K. H., K. W. Wucherpfennig, and D. C. Wiley. 2001. Structure of a human insulin peptide-HLA-DQ8 complex and susceptibility to type 1 diabetes, Nat Immunol, 2:501-7. Leotoing, L., L. Meunier, M. Manin, C. Mauduit, M. Decaussin, G. Verrijdt, F. Claessens, M. Benahmed, G. Veyssiere, L. Morel, and C. Beaudoin. 2008. Influence of nucleophosmin/B23 on DNA binding and transcriptional activity of the androgen receptor in prostate cancer cell, Oncogene, 27:2858-67. Lessa, R. C., A. H. Campos, C. E. Freitas, F. R. Silva, L. P. Kowalski, A. L. Carvalho, and A. L. Vettore. 2013. Identification of upregulated genes in oral squamous cell carcinomas, Head Neck, 35:1475-81. Lewy, T. G., J. M. Grabowski, and M. E. Bloom. 2017. BiP: Master Regulator of the Unfolded Protein Response and Crucial Factor in Flavivirus Biology, Yale J Biol Med, 90:291-300. Li, Y., H. Li, R. Martin, and R. A. Mariuzza. 2000. Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins, J Mol Biol, 304:177-88. Liddy, N., G. Bossi, K. J. Adams, A. Lissina, T. M. Mahon, N. J. Hassan, J. Gavarret, F. C. Bianchi, N. J. Pumphrey, K. Ladell, E. Gostick, A. K. Sewell, N. M. Lissin, N. E. Harwood, P. E. Molloy, Y. Li, B. J. Cameron, M. Sami, E. E. Baston, P. T. Todorov, S. J. Paston, R. E. Dennis, J. V. Harper, S. M. Dunn, R. Ashfield, A. Johnson, Y. McGrath, G. Plesa, C. H. June, M. Kalos, D. A. Price, A. Vuidepot, D. D. Williams, D. H. Sutton, and B. K. Jakobsen. 2012. Monoclonal TCR-redirected tumor cell killing, Nat Med, 18:980-7. Lissin, N. M., N. J. Hassan, and B. K. Jakobsen. 2013. High-Affinity Monoclonal T-Cell Receptor (mTCR) Fusions (Wiley Online Library: doi.org/10.1002/9781118354599.ch32). Liu, C. Y., H. H. Lin, M. J. Tang, and Y. K. Wang. 2015. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation, Oncotarget, 6:15966-83. Liu, S., J. Matsuzaki, L. Wei, T. Tsuji, S. Battaglia, Q. Hu, E. Cortes, L. Wong, L. Yan, M. Long, A. Miliotto, N. W. Bateman, S. B. Lele, T. Chodon, R. C. Koya, S. Yao, Q. Zhu, T. P. Conrads, J. Wang, G. L. Maxwell, A. A. Lugade, and K. Odunsi. 2019. Efficient identification of neoantigen-specific T-cell responses in advanced human ovarian cancer, J Immunother Cancer, 7:156. Lu, M. C., C. L. Yu, H. C. Yu, H. B. Huang, M. Koo, and N. S. Lai. 2016. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60, Immunobiology, 221:76-83. Lugli, E. B., R. E. Correia, R. Fischer, K. Lundberg, K. R. Bracke, A. B. Montgomery, B. M. Kessler, G. G. Brusselle, and P. J. Venables. 2015. Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis, Arthritis Res Ther, 17:9. Lundberg, K., A. Kinloch, B. A. Fisher, N. Wegner, R. Wait, P. Charles, T. R. Mikuls, and P. J. Venables. 2008. Antibodies to citrullinated alpha-enolase peptide 1 are specific for rheumatoid arthritis and cross-react with bacterial enolase, Arthritis Rheum, 58:3009-19. Lundkvist, A., A. Reichenbach, C. Betsholtz, P. Carmeliet, H. Wolburg, and M. Pekny. 2004. Under stress, the absence of intermediate filaments from Muller cells in the retina has structural and functional consequences, J Cell Sci, 117:3481-8. Luo, B., and A. S. Lee. 2013. The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies, Oncogene, 32:805-18. Lv, L. H., Y. L. Wan, Y. Lin, W. Zhang, M. Yang, G. L. Li, H. M. Lin, C. Z. Shang, Y. J. Chen, and J. Min. 2012. Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro, J Biol Chem, 287:15874-85. Makrygiannakis, D., M. Hermansson, A. K. Ulfgren, A. P. Nicholas, A. J. Zendman, A. Eklund, J. Grunewald, C. M. Skold, L. Klareskog, and A. I. Catrina. 2008. Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells, Ann Rheum Dis, 67:1488-92. Martinez, G., J. A. Gomez, H. Bang, L. Martinez-Gamboa, D. Roggenbuck, G. R. Burmester, B. Torres, D. Prada, and E. Feist. 2016. Carbamylated vimentin represents a relevant autoantigen in Latin American (Cuban) rheumatoid arthritis patients, Rheumatol Int, 36:781-91. Miao, C. G., Y. Y. Yang, X. He, X. F. Li, C. Huang, Y. Huang, L. Zhang, X. W. Lv, Y. Jin, and J. Li. 2013. Wnt signaling pathway in rheumatoid arthritis, with special emphasis on the different roles in synovial inflammation and bone remodeling, Cell Signal, 25:2069-78. Miles, L. A., C. M. Dahlberg, J. Plescia, J. Felez, K. Kato, and E. F. Plow. 1991. Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor, Biochemistry, 30:1682-91. Moll, R., W. W. Franke, D. L. Schiller, B. Geiger, and R. Krepler. 1982. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells, Cell, 31:11-24. Moon, R. T., B. Bowerman, M. Boutros, and N. Perrimon. 2002. The promise and perils of Wnt signaling through beta-catenin, Science, 296:1644-6. Mydel, P., Z. Wang, M. Brisslert, A. Hellvard, L. E. Dahlberg, S. L. Hazen, and M. Bokarewa. 2010. Carbamylation-dependent activation of T cells: a novel mechanism in the pathogenesis of autoimmune arthritis, J Immunol, 184:6882-90. Myers, E. W., and W. Miller. 1989. Approximate matching of regular expressions, Bull Math Biol, 51:5-37. Neron, B., H. Menager, C. Maufrais, N. Joly, J. Maupetit, S. Letort, S. Carrere, P. Tuffery, and C. Letondal. 2009. Mobyle: a new full web bioinformatics framework, Bioinformatics, 25:3005-11. Ni, M., Y. Zhang, and A. S. Lee. 2011. Beyond the endoplasmic reticulum: atypical GRP78 in cell viability, signalling and therapeutic targeting, Biochem J, 434:181-8. Nozawa, Y., N. Van Belzen, A. C. Van der Made, W. N. Dinjens, and F. T. Bosman. 1996. Expression of nucleophosmin/B23 in normal and neoplastic colorectal mucosa, J Pathol, 178:48-52. Oshima, R. G. 2002. Apoptosis and keratin intermediate filaments, Cell Death Differ, 9:486-92. Oshima, R. G., H. Baribault, and C. Caulin. 1996. Oncogenic regulation and function of keratins 8 and 18, Cancer Metastasis Rev, 15:445-71. Ospelt, C., H. Bang, E. Feist, G. Camici, S. Keller, J. Detert, A. Kramer, S. Gay, K. Ghannam, and G. R. Burmester. 2017. Carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis, Ann Rheum Dis, 76:1176-83. Otsuki, S., M. Inokuchi, M. Enjoji, T. Ishikawa, Y. Takagi, K. Kato, H. Yamada, K. Kojima, and K. Sugihara. 2011. Vimentin expression is associated with decreased survival in gastric cancer, Oncol Rep, 25:1235-42. Owens, D. W., and E. B. Lane. 2003. The quest for the function of simple epithelial keratins, Bioessays, 25:748-58. Paramio, J. M., and J. L. Jorcano. 2002. Beyond structure: do intermediate filaments modulate cell signalling?, Bioessays, 24:836-44. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison, Proc Natl Acad Sci USA, 85:2444-8. Peng, Y., X. Li, M. Wu, J. Yang, M. Liu, W. Zhang, B. Xiang, X. Wang, X. Li, G. Li, and S. Shen. 2012. New prognosis biomarkers identified by dynamic proteomic analysis of colorectal cancer, Mol Biosyst, 8:3077-88. Pianta, A., C. Puppin, A. Franzoni, D. Fabbro, C. Di Loreto, S. Bulotta, M. Deganuto, I. Paron, G. Tell, E. Puxeddu, S. Filetti, D. Russo, and G. Damante. 2010. Nucleophosmin is overexpressed in thyroid tumors, Biochem Biophys Res Commun, 397:499-504. Polioudaki, H., S. Agelaki, R. Chiotaki, E. Politaki, D. Mavroudis, A. Matikas, V. Georgoulias, and P. A. Theodoropoulos. 2015. Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer, BMC Cancer, 15:399. Principe, M., P. Ceruti, N. Y. Shih, M. S. Chattaragada, S. Rolla, L. Conti, M. Bestagno, L. Zentilin, S. H. Yang, P. Migliorini, P. Cappello, O. Burrone, and F. Novelli. 2015. Targeting of surface alpha-enolase inhibits the invasiveness of pancreatic cancer cells, Oncotarget, 6:11098-113. Remington, R P. 1980. Remington's pharmaceutical sciences (Mack Pub. Co). Ritterson Lew, C., and D. R. Tolan. 2012. Targeting of several glycolytic enzymes using RNA interference reveals aldolase affects cancer cell proliferation through a non-glycolytic mechanism, J Biol Chem, 287:42554-63. Roberts, J. M., P. R. Veres, A. K. Cochran, C. Warneke, I. R. Burling, R. J. Yokelson, B. Lerner, J. B. Gilman, W. C. Kuster, R. Fall, and J. de Gouw. 2011. Isocyanic acid in the atmosphere and its possible link to smoke-related health effects, Proc Natl Acad Sci USA, 108:8966-71. Rose, S., A. Misharin, and H. Perlman. 2012. A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment, Cytometry A, 81:343-50. Saini, J., and P. K. Sharma. 2017. Clinical, Prognostic and Therapeutic Significance of Heat Shock Proteins in Cancer, Curr Drug Targets. Scinocca, M., D. A. Bell, M. Racape, R. Joseph, G. Shaw, J. K. McCormick, D. D. Gladman, J. Pope, L. Barra, and E. Cairns. 2014. Antihomocitrullinated fibrinogen antibodies are specific to rheumatoid arthritis and frequently bind citrullinated proteins/peptides, J Rheumatol, 41:270-9. Scott, C. A., P. A. Peterson, L. Teyton, and I. A. Wilson. 1998. Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues, Immunity, 8:319-29. Semenza, G. L., B. H. Jiang, S. W. Leung, R. Passantino, J. P. Concordet, P. Maire, and A. Giallongo. 1996. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1, J Biol Chem, 271:32529-37. Sette, A., L. Adorini, S. M. Colon, S. Buus, and H. M. Grey. 1989. Capacity of intact proteins to bind to MHC class II molecules, J Immunol, 143:1265-7. Shi, J., R. Knevel, P. Suwannalai, M. P. van der Linden, G. M. Janssen, P. A. van Veelen, N. E. Levarht, A. H. van der Helm-van Mil, A. Cerami, T. W. Huizinga, R. E. Toes, and L. A. Trouw. 2011. Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage, Proc Natl Acad Sci USA, 108:17372-7. Shi, J., P. A. van Veelen, M. Mahler, G. M. Janssen, J. W. Drijfhout, T. W. Huizinga, R. E. Toes, and L. A. Trouw. 2014. Carbamylation and antibodies against carbamylated proteins in autoimmunity and other pathologies, Autoimmun Rev, 13:225-30. Shoda, H., K. Fujio, M. Shibuya, T. Okamura, S. Sumitomo, A. Okamoto, T. Sawada, and K. Yamamoto. 2011. Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis, Arthritis Res Ther, 13: R191. Smith, K. J., J. Pyrdol, L. Gauthier, D. C. Wiley, and K. W. Wucherpfennig. 1998. Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein, J Exp Med, 188:1511-20. Stern, L. J., J. H. Brown, T. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, and D. C. Wiley. 1994. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide, Nature, 368:215-21. Stewart. 1984. Solid phase peptide synthesis (Illinois Pierce Chemical Company: Rockford). Stoop, J. N., B. S. Liu, J. Shi, D. T. Jansen, M. Hegen, T. W. Huizinga, L. A. Trouw, and R. E. Toes. 2014. Antibodies specific for carbamylated proteins precede the onset of clinical symptoms in mice with collagen induced arthritis, PLOS One, 9: e102163. Tadokoro, A., N. Kanaji, D. Liu, H. Yokomise, R. Haba, T. Ishii, T. Takagi, N. Watanabe, N. Kita, N. Kadowaki, and S. Bandoh. 2016. Vimentin Regulates Invasiveness and Is a Poor Prognostic Marker in Non-small Cell Lung Cancer, Anticancer Res, 36:1545-51. Tanaka, M., H. Sasaki, I. Kino, T. Sugimura, and M. Terada. 1992. Genes preferentially expressed in embryo stomach are predominantly expressed in gastric cancer, Cancer Res, 52:3372-7. Tanikawa, C., K. Ueda, H. Nakagawa, N. Yoshida, Y. Nakamura, and K. Matsuda. 2009. Regulation of protein Citrullination through p53/PADI4 network in DNA damage response, Cancer Res, 69:8761-9. Thomas, P. A., D. A. Kirschmann, J. R. Cerhan, R. Folberg, E. A. Seftor, T. A. Sellers, and M. J. Hendrix. 1999. Association between keratin and vimentin expression, malignant phenotype, and survival in postmenopausal breast cancer patients, Clin Cancer Res, 5:2698-703. Thomsen, M. C., and M. Nielsen. 2012. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion, Nucleic Acids Res, 40: W281-7. Torelli, A., and C. A. Robotti. 1994. ADVANCE and ADAM: two algorithms for the analysis of global similarity between homologous informational sequences, Comput Appl Biosci, 10:3-5. Tsui, K. H., A. J. Cheng, Pe Chang, T. L. Pan, and B. Y. Yung. 2004. Association of nucleophosmin/B23 mRNA expression with clinical outcome in patients with bladder carcinoma, Urology, 64:839-44. Turunen, S., P. Hannonen, M. K. Koivula, L. Risteli, and J. Risteli. 2015. Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides, Arthritis Res Ther, 17:2. Turunen, S., J. Huhtakangas, T. Nousiainen, M. Valkealahti, J. Melkko, J. Risteli, and P. Lehenkari. 2016. Rheumatoid arthritis antigens homocitrulline and citrulline are generated by local myeloperoxidase and peptidyl arginine deiminases 2, 3 and 4 in rheumatoid nodule and synovial tissue, Arthritis Res Ther, 18:239. Vaidya, M. M., A. M. Borges, S. A. Pradhan, R. M. Rajpal, and A. N. Bhisey. 1989. Altered keratin expression in buccal mucosal squamous cell carcinoma, J Oral Pathol Med, 18:282-6. Wang, P., J. Sidney, C. Dow, B. Mothe, A. Sette, and B. Peters. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach, PLOS Comput Biol, 4: e1000048. Wang, P., J. Sidney, Y. Kim, A. Sette, O. Lund, M. Nielsen, and B. Peters. 2010. Peptide binding predictions for HLA DR, DP and DQ molecules, BMC Bioinformatics, 11:568. Wang, X., P. Chen, J. Cui, C. Yang, and H. Du. 2015. Keratin 8 is a novel autoantigen of rheumatoid arthritis, Biochem Biophys Res Commun, 465:665-9. Wang, Z., S. J. Nicholls, E. R. Rodriguez, O. Kummu, S. Horkko, J. Barnard, W. F. Reynolds, E. J. Topol, J. A. DiDonato, and S. L. Hazen. 2007. Protein carbamylation links inflammation, smoking, uremia and atherogenesis, Nat Med, 13:1176-84. Xue, W., R. L. Metheringham, V. A. Brentville, B. Gunn, P. Symonds, H. Yagita, J. M. Ramage, and L. G. Durrant. 2016. SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade, Oncoimmunology, 5: e1169353. Yamashita, N., E. Tokunaga, H. Kitao, Y. Hisamatsu, K. Taketani, S. Akiyoshi, S. Okada, S. Aishima, M. Morita, and Y. Maehara. 2013. Vimentin as a poor prognostic factor for triple-negative breast cancer, J Cancer Res Clin Oncol, 139:739-46. Ye, F., Y. Chen, L. Xia, J. Lian, and S. Yang. 2018. Aldolase A overexpression is associated with poor prognosis and promotes tumor progression by the epithelial-mesenchymal transition in colon cancer, Biochem Biophys Res Commun, 497:639-45. Youn, J. I., M. Collazo, I. N. Shalova, S. K. Biswas, and D. I. Gabrilovich. 2012. Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice, J Leukoc Biol, 91:167-81. Yun, J. P., J. Miao, G. G. Chen, Q. H. Tian, C. Q. Zhang, J. Xiang, J. Fu, and P. B. Lai. 2007. Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters, Br J Cancer, 96:477-84. Zhang, F., J. D. Lin, X. Y. Zuo, Y. X. Zhuang, C. Q. Hong, G. J. Zhang, X. J. Cui, and Y. K. Cui. 2017. Elevated transcriptional levels of aldolase A (ALDOA) associates with cell cycle-related genes in patients with NSCLC and several solid tumors, BioData Min, 10:6. Zhang, L., K. Udaka, H. Mamitsuka, and S. Zhu. 2012. Toward more accurate pan-specific MHC-peptide binding prediction: a review of current methods and tools, Brief Bioinform, 13:350-64. Zhang, Y., R. Liu, M. Ni, P. Gill, and A. S. Lee. 2010. Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP, J Biol Chem, 285:15065-75. Zhao, M., W. Fang, Y. Wang, S. Guo, L. Shu, L. Wang, Y. Chen, Q. Fu, Y. Liu, S. Hua, Y. Fan, Y. Liu, X. Deng, R. Luo, Z. Mei, Q. Jiang, and Z. Liu. 2015. Enolase-1 is a therapeutic target in endometrial carcinoma, Oncotarget, 6:15610-27. Zhao, Y., T. Wu, S. Shao, B. Shi, and Y. Zhao. 2016. Phenotype, development, and biological function of myeloid-derived suppressor cells, Oncoimmunology, 5: e1004983. Zhu, Y., M. Shi, H. Chen, J. Gu, J. Zhang, B. Shen, X. Deng, J. Xie, X. Zhan, and C. Peng. 2015. NPM1 activates metabolic changes by inhibiting FBP1 while promoting the tumorigenicity of pancreatic cancer cells, Oncotarget, 6:21443-51.