CANCER VACCINE

Abstract

The present disclosure relates to an immunogenic agent comprising Dna J heat shock protein family (Hsp40) member B7 or an immunogenic fragment thereof; a DNA vaccine comprising a nucleic acid encoding said protein or at least one immunogenic fragment thereof; a pharmaceutical composition or vector or DNA vaccine for use in the treatment of cancer; and a method of treating cancer comprising the use of said immunogenic agent or pharmaceutical composition or vector or DNA vaccine.

Claims

1. An immunogenic agent for use as a cancer vaccine comprising or consisting of DnaJ heat shock protein family (Hsp40) member B7 (termed DNAJB7) or at least one immunogenic fragment thereof.

2. The immunogenic agent wherein DNAJB7 is human DNAJB7.

3. The immunogenic agent according to claim 2 wherein DNAJB7 is represented by the amino acid sequence set forth in SEQ ID NO: 31 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith.

4. The immunogenic agent according to claim 1, wherein said at least one fragment is 5-30 amino acids in length.

5. The immunogenic agent according to claim 1, wherein said at least one DNAJB7 immunogenic fragment comprises or consists of an amino acid sequence selected from the group of sequence listing consisting of: TABLE-US-00007 a) (SEQ ID NO: 1) MVDYYEVLGLQRYASPEDIK; (SEQ ID NO: 2) QRYASPEDIKKAYHKVALKW; (SEQ ID NO: 3) KAYHKVALKWHPDKNPENKE; (SEQ ID NO: 4) HPDKNPENKEEAERKFKEVA; (SEQ ID NO: 5) EAERKFKEVAEAYEVLSNDE; (SEQ ID NO: 6) EAYEVLSNDEKRDIYDKYGT; (SEQ ID NO: 7) KRDIYDKYGTEGLNGGGSHF; (SEQ ID NO: 8) EGLNGGGSHFDDECEYGFTF; (SEQ ID NO: 9) DDECEYGFTFHKPDDVFKEI; (SEQ ID NO: 10) HKPDDVFKEIFHERDPFSFH; (SEQ ID NO: 11) FHERDPFSFHFFEDSLEDLL; (SEQ ID NO: 12) FFEDSLEDLLNRPGSSYGNR; (SEQ ID NO: 13) NRPGSSYGNRNRDAGYFFST; (SEQ ID NO: 14) NRDAGYFFSTASEYPIFEKF; (SEQ ID NO: 15) ASEYPIFEKFSSYDTGYTSQ; (SEQ ID NO: 16) SSYDTGYTSQGSLGHEGLTS; (SEQ ID NO: 17) GSLGHEGLTSFSSLAFDNSG; (SEQ ID NO: 18) FSSLAFDNSGMDNYISVTTS; (SEQ ID NO: 19) MDNYISVTTSDKIVNGRNIN; (SEQ ID NO: 20) DKIVNGRNINTKKIIESDQE; (SEQ ID NO: 21) TKKIIESDQEREAEDNGELT; (SEQ ID NO: 22) REAEDNGELTFFLVNSVANE; (SEQ ID NO: 23) FFLVNSVANEEGFAKECSWR; (SEQ ID NO: 24) EGFAKECSWRTQSFNNYSPN; (SEQ ID NO: 25) TQSFNNYSPNSHSSKHVSQY; (SEQ ID NO: 26) SHSSKHVSQYTFVDNDEGGI; (SEQ ID NO: 27) TFVDNDEGGISWVTSNRDPP; (SEQ ID NO: 28) SWVTSNRDPPIFSAGVKEGG; (SEQ ID NO: 29) IFSAGVKEGGKRKKKKRKEV; and (SEQ ID NO. 30) KRKKKKRKEVQKKSTKRNC; or; 1)) a fragment that has at least 85% sequence identity with any one or more of the sequences in group a) and, in ascending order of preference, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one or more of the sequences in group a); or c) a fragment that is a variant of any one or more of the sequences in group a) and/or group b) wherein said variant is modified by the addition, deletion or substitution of one or more amino acid residues in any one or more of the above sequences and/or wherein said variant fragment retains or has enhanced or comparable immunogenicity when compared to the immunogenicity of any one or more of the variants in group a) and/or group b).

6. The immunogenic agent according to claim wherein said at least one DNAJB7 fragment is an amino acid sequence selected from the group consisting of: TABLE-US-00008 a) (SEQ ID NO: 23) FFLVNSVANEEGFAKECSWR; (SEQ ID NO: 11) FHERDPFSFHFFEDSLEDLL; (SEQ ID NO: 30) KRKKKKRKEVQKKSTKRNC; (SEQ ID NO: 26) SHSSKHVSQYTFVDNDEGGI; ((SEQ ID NO: 8) EGLNGGGSHFDDECEYGFTF; (SEQ ID NO: 13) NRPGSSYGNRNRDAGYFFST; (SEQ ID NO: 14) NRDAGYFFSTASEYPIFEKF; (SEQ ID NO: 19) MDNYISVTTSDKIVNGRNIN; and (SEQ ID NO: 27) TFVDNDEGGISWVTSNRDPP; or b) a fragment that has at least 85% sequence identity with any one or more of the sequences in group a) and, in ascending order of preference, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one or more of the sequences in group a); or c) a fragment that is a variant of any one or more of the sequences in group a) and/or wherein said variant is modified by the addition, deletion or substitution of one or more amino acid residues in any one or more of the above sequences and/or wherein said variant fragment retains or has enhanced or comparable immunogenicity when compared to the immunogenicity of any one or more of the variants in group a) and/or group b).

7. A vector or DNA vaccine comprising a nucleic acid molecule encoding a DnaJ heat shock protein family (Hsp40) member B7 (termed DNAJB7), or at least one immunogenic fragment thereof, according to claim 1.

8. The vector or DNA vaccine according to claim 7 wherein said nucleic acid molecule is part of, or provided in, an expression vector adapted to express said DNAJB7, or at least one of said fragments thereof.

9. The vector or DNA vaccine according to claim 8 wherein said adaptation includes the provision of at least one transcription control sequences which mediate(s) said expression.

10. The vector or DNA vaccine according to claim 9 wherein the at least one transcription control sequence(s) is/are cell/tissue specific and adapted for inducible or constitutive expression of said DNAJB7, or at least one fragment thereof.

11. The vector or DNA vaccine according to claim 7, wherein said nucleic acid molecule encodes the whole of said DNAJB7 and/or a number of fragments thereof.

12. A pharmaceutical composition comprising the immunogenic agent according to claim 1.

13. (canceled)

14. (canceled)

15. A method of vaccinating a subject suffering from or having a predisposition for cancer comprising administering an effective amount of the immunogenic agent according to claim 1.

16. The method of claim 15, wherein said cancer is selected from any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, rectal cancer, esophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

17. The method according to claim 16, wherein said cancer is selected from the group comprising the following cancers: colorectal cancer, thyroid, lymphoma, lung, liver, pancreatic, carcinoid, head & neck, stomach, urothelial, prostate, testis, endometrial, glioma, breast, cervical, ovarian, melanoma, liver, and renal cancers.

18. A pharmaceutical composition comprising the vector or DNA vaccine according to claim 7.

19. A method of vaccinating a subject suffering from or having a predisposition for cancer, comprising administering the vector or DNA vaccine according to claim 7.

20. A method of vaccinating a subject suffering from or having a predisposition for cancer, comprising administering the pharmaceutical composition according to claim 12 to said subject.

21. The method of claim 19, wherein said cancer is selected from any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, rectal cancer, esophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

22. The method according to claim 21, wherein said cancer is selected from the group comprising the following cancers: colorectal cancer, thyroid, lymphoma, lung, liver, pancreatic, carcinoid, head & neck, stomach, urothelial, prostate, testis, endometrial, glioma, breast, cervical, ovarian, melanoma, liver, and renal cancers.

23. The method according to claim 20, wherein said cancer is selected from any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, rectal cancer, esophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

24. The method according to claim 23, wherein said cancer is selected from the group comprising the following cancers: colorectal cancer, thyroid, lymphoma, lung, liver, pancreatic, carcinoid, head & neck, stomach, urothelial, prostate, testis, endometrial, glioma, breast, cervical, ovarian, melanoma, liver, and renal cancers.

25. A method of vaccinating a subject suffering from or having a predisposition for cancer, comprising administering the pharmaceutical composition according to claim 18 to said subject.

26. The method according to claim 25, wherein said cancer is selected from any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, tonsil, spleen, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, glioma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, muscle cancer, Paget's disease, cervical cancer, rectal cancer, esophagus cancer, gall bladder cancer, cholangioma cancer, head cancer, eye cancer, nasopharynx cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, myeloma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

27. The method according to claim 26, wherein said cancer is selected from the group comprising the following cancers: colorectal cancer, thyroid, lymphoma, lung, liver, pancreatic, carcinoid, head & neck, stomach, urothelial, prostate, testis, endometrial, glioma, breast, cervical, ovarian, melanoma, liver, and renal cancers.

Description

DETAILED DESCRIPTION

[0063] Methods and Materials

[0064] Patient Treatment Schedule Orally administered 50 mg cyclophosphamide was taken twice-a-day on treatment days 1-7 and 15-21; no cyclophosphamide was taken on treatment days 8-14 or 22-106, or until patient relapsed. Peripheral blood samples (40 mL) were taken at regular intervals during therapy.

[0065] Excision of Colonic and Tumour Tissue

[0066] Colorectal tumour and paired background (unaffected) colon specimens were obtained from three patients undergoing primary tumour resection for colorectal adenocarcinoma at the University Hospital of Wales, Cardiff. Autologous colon samples were cut from macroscopically normal sections of the excised tissue, both “near” (within 2 cm) and “far” (at least 10 cm) from the tumour site (FIG. 1A). All fresh tumour samples were derived from the luminal aspect of the specimen, so as not to interfere with the deep part of the tumour required for histopathological staging. Informed consent was obtained from all participants. The Wales Research Ethics Committee granted ethical approval for this study.

[0067] Purification of Tissue Samples

[0068] Background colon and tumour specimens were transported and washed in extraction medium comprising RPMI supplemented with penicillin, streptomycin and L-glutamine (Gibco), 2% human AB serum (Welsh Blood Service), 20 μg/ml gentamicin (ThermoFisher) and 2 μg/ml Fungizone (ThermoFisher). Within 30 minutes of resection from a patient, samples were minced with blades in a Petri dish and forced through 70 μm cell strainers to collect a single cell suspension. In no instances were collagenase or DNase treatments used. Dissociated cell preparations of tumour, near and far healthy colonic tissue were initially stained with the amine-reactive viability dye Live/Dead fixable Aqua (ThermoFisher) followed by surface marker staining with CD3-APC (BioLegend) and EpCAM-PE (Miltenyi Biotec) antibodies. EpCAM was chosen as it would enable isolation of epithelial populations over stromal tissue and immune populations (Martowicz et al., 2016; Schnell et al., 2013), with CD3 used to ensure T cell populations were not included in downstream analysis. Samples were resuspended in FACS buffer (PBS, 2% BSA) prior to sorting into Live/Dead.sup.−EpCAM.sup.+CD3.sup.− populations on a FACS Aria III (BD); gating strategy is shown (FIG. 1C). Tumour tissue also stained with CD3 and EpCAM antibodies was additionally passed through the cell sorter without gating, and used as an unsorted control for RNA-seq. All samples were sorted directly into RLT buffer (Qiagen) with beta mercapto-ethanol (Sigma Aldrich) and frozen at −80° C. Frozen samples were thawed and RNA isolated using a RNA easy micro isolation kit (Qiagen).

[0069] RNA Sequencing

[0070] Library preparation and RNA sequencing was carried out by VGTI-FL (Florida, USA). Purified RNA was used to make libraries using a TruSeq kit (Illumina). Libraries were sequenced to a depth of 37-63M read pairs on an Illumina HiSeq platform. Paired end reads were processed on a Cardiff University pipeline, trimmed, mapped and quality control analysis performed. Reads were trimmed with Trimmomatic (Bolger et al., 2014) and assessed for quality using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) using the default parameters. Reads were mapped to Ensembl human genome build GRCh38.89 downloaded from the Ensembl FTP site (http:/www.ensembl.org./info/data/ftp/index.html/) using STAR (Dobin et al., 2013).

[0071] Differential Expression Analysis

[0072] Aligned reads were normalised using DESEQ2 in R (Love et al., 2014). Differentially expressed genes were identified between purified tumour samples, purified near, and purified far epithelium. Differential expression analysis was carried out using DESEQ2 between sample types for all donors in a paired analysis. Comparisons of tumour and near or far tissue were carried out, with reads standardised for comparison using fragments per kilobase of transcript per million mapped reads (FPKM values). For the three-donor expression analysis, genes with a log 2-fold change greater than 3.5, FPKM values in healthy tissue less than 3.5, and FPKM values in tumour greater than 4.0 in any two of three donors as well as an adjusted P-value less than 0.05 in three donors were taken forward for further analysis.

[0073] The analysis was expanded to genes which were significantly differentially expressed in separate comparisons for data in two of the three donors (this helped identify genes which were not expressed in one donor but were highly expressed in the remaining two donors). Higher expression cut off values were used with FPKM greater than 5.0 in both donor's tumour tissue, and less than 1.0 in healthy tissues, with a log 2-fold change greater than 6, and adjusted p-value of less than 0.05. Gene lists arising from these comparisons were taken forward for further analysis. Inspection of reads mapped was carried out using integrative genomic viewer software (Broad institute).

[0074] PBMC Culture

[0075] Blood samples were collected in 10 ml lithium heparin tubes (BD Biosciences) no more than 7 days prior to surgery. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of heparinised blood over Lymphoprep (Axis-Shield). Cells were then washed and re-suspended in CTL Test Plus media (CTL Europe), L-glutamine and penicillin/streptomycin. PBMC were plated in 96-well plates (Nunc) and cultured in triplicate wells with specific antigens for 14 days, supplemented with fresh media containing 20 IU/ml IL-2 on days 3, 7 and 10.

[0076] ELISpot Assays

[0077] IFN-γ ELISpot assays were performed to assess for novel tumor antigen-specific T cell responses, as previously described (Scurr et al. 2017). Briefly, PVDF 96-well filtration plates were coated with 50 μI IFN-γ antibody (Mabtech). Cells were washed, plated, and stimulated with 5 μg/mlantigen in duplicate wells. Plates were incubated at 37° C., 5% CO2 for 24 hours before removing cells and developing spots. Spot-forming cells (SFC), i.e. IFN-γ-producing T cells, were enumerated using Smart Count settings on an automated ELISpot plate reader (ImmunoSpot S6 Ultra; CTL Europe GmbH). Positive responses were identified as having at least 20 SFC/10.sup.5 cultured PBMCs, and at least double that of the negative (no antigen) control. Wells with spot counts >1000 were deemed too numerous to count and capped at this level.

[0078] FluoroSpot Assays

[0079] IFN-γ/Granzyme B FluoroSpot assays were performed to assess for novel tumour antigen-specific T cell responses, as previously described (Scurr et al. 2017). Briefly, PVDF 96-well filtration plates designed for low autofluorescence (IPFL; Millipore) were used for all FluoroSpot assays. Antibodies to IFN-γ and Granzyme B, and fluorescence enhancer kits were obtained from Mabtech. All antibody incubations were with 50 μl/well. Cells were then washed, plated, and stimulated with 5 μg/mL antigen in duplicate wells. Plates were incubated at 37° C., 5% CO.sub.2 for 24 hours. Cytokine-producing T cells were enumerated using Smart Count settings on an automated FluoroSpot plate reader (ImmunoSpot S6 Ultra; CTL Europe GmbH), allowing for an assessment of single and dual cytokine-producing cells. Positive responses were identified as having at least 5 SFC/10.sup.5 PBMCs, and at least double that of the negative (no antigen) control.

[0080] Antigens

[0081] 20mer peptides overlapping by 10 amino acids, covering the entire protein sequence of each identified TAA, were synthesised by Fmoc chemistry to >95% purity (GL Biochem, Shanghai, China), and divided into pools, as shown (Table 4). The recall antigens tuberculin purified protein derivative (PPD; Statens Serum Institut) and hemagglutinin (HA; gift from Dr. John Skehel, National Institute of Medical Research, London, United Kingdom), and the T cell mitogen PHA (Sigma) were used as positive controls. All antigens were used at a final concentration of 5 μg/ml.

[0082] Immunohistochemistry

[0083] The identified TAAs from this study were evaluated for protein expression characteristics on healthy tissue and a range of tumour samples by utilising the Human Protein Atlas resource (Uhlén M et al. 2015).

[0084] Results

[0085] Purification of Samples Prior to RNA-Seq Analysis Provided Enhanced Resolution Of Differentially Expressed Genes

[0086] RNA-seq datasets were comparable following several normalisation procedures. Differential expression comparisons were run using DESEQ2 of healthy tissues (“near” and “far”) against purified tumour tissue in all three patients, and then separate analyses for each combination of two patients. An additional comparison of non-purified tumour tissue against healthy tissues was run to investigate the impact of EpCAM sorting. To find relevant genes that could be targeted by immunotherapy, we applied criteria that specified low levels of expression in healthy tissue combined with high expression in tumour tissue (based on FPKM and log 2-fold change). Only genes assigned an adjusted P-value<0.05 were taken forward for further analysis.

[0087] Initial gene lists gave 83 significant genes showing differential expression between tumour and far colon tissue, while 92 genes between tumour and near colon tissue. Cross referencing of these gene lists resulted in 5 genes that satisfied significant criteria in both comparisons (including 4 of those taken forward; ARSJ, CENPQ, ZC3H12B and CEACAM3). To expand our analysis, we looked at DEGs which were significantly expressed in tumour tissue of two of three patients to a higher level (increased expression cut-offs and lower threshold of healthy tissue expression). These gene lists were combined with three donor lists, and then near and far tissue cross referenced (FIG. 2A). This gave an initial set of 54 genes which were cut to 23 based on levels of expression in healthy tissue of all three donors. Of these 23 genes, 18 were protein coding. We inspected these 18 genes and selected those which were most suitable for further analysis, eliminating those involved in the central nervous system, or which exhibited an inconsistent expression or read mapping profile in three donors gauged by visual curation of mapped reads in IGV.

[0088] The final genes selected were DNAJB7, CENPQ, ZC3H12B, ZSWIM1, CEACAM3, ARSJ and CYP2B6, based on their ideal expression profile for therapeutic exploitation (FIG. 2B). Inspection of relevant expression profiles in RNA-seq data from non-purified tumour tissue exemplifies how none of these genes were detected in non-purified tissue of all donors, with expression levels much lower than purified tissue. Those that were detected in non-purified tissue of two donors did not come up as significant in differential expression analysis by DESEQ2, again emphasising the power of purification prior to RNA-seq analysis.

[0089] Analysis of Protein Expression Across Multiple Healthy Tissues Highlights DNAJB7 as a Cancer-Testis Antigen and a Suitable Target for Immunotherapy

[0090] The protein expression level of each candidate TAA was evaluated using immunohistochemistry data publicly available in the Human Protein Atlas. Whilst each candidate exhibited significant upregulation on tumour tissue over healthy tissue, although more limited for ARSJ, DNAJB7 was unexpectedly identified as a novel cancer-testis antigen given its complete lack of expression on any healthy tissue bar the testis, an immune-privileged site (FIG. 3A-C). Furthermore, DNAJB7, a protein belonging to the evolutionarily conserved DNAJ heat shock family, was expressed on a very wide range of solid tumours, in particular on tumours of the gastrointestinal tract and accessory organs of digestion, including colorectal cancer and pancreatic ductal adenocarcinoma (FIG. 3B; example immunohistochemistry data also included FIG. 3C).

[0091] The expression profile of DNAJB7 was compared to six other well-defined cancer-testis antigens, including NY-ESO-1, MAGE-A1 and SSX2. High protein expression of all these antigens was confirmed to be confined to the testis, apart from SPAG9 (FIG. 4A). In comparison to the other cancer-testis antigens, DNAJB7 was expressed on the greatest range of tumour types, with more than 67% of all patients tested exhibiting positive (low, medium or high) protein expression on their tumour, except for lymphoma (FIG. 4B). This remarkable protein expression profile sets DNAJB7 aside as an excellent target for broadly applicable cancer immunotherapy.

[0092] Analysis of Candidate TAA T.sub.H1 Responses Reveal DNAJB7 to be Immunogenic

[0093] Following identification of relevant genes and confirmed protein expression, we assessed their immunogenicity using over lapping peptide pools and culture with PBMC of CRC patients and healthy donors. Analysis of cultured PBMC by IFN-γ/Granzyme-B FluoroSpot determined five of the seven proteins to demonstrate immunogenicity in the majority of donors (FIGS. 5A-B and 6A-B). As the size of peptide libraries was highly variable for each protein, we standardised the immunogenicity relevant to the number of peptides in each pool (FIGS. 5B and 6B). This analysis revealed CYP2B6, DNAJB7, and CEACAM3 to be comparably immunogenic across multiple individuals without stratification by HLA-type. Conversely, CENPQ and ARSJ were poorly immunogenic in most donors tested. DNAJB7 was also found to be immunogenic in patients with other tumor types, including hepatocellular carcinoma, cholangiocarcinoma and the non-gastrointestinal head and neck squamous cell cancer (FIGS. 5G and 6C).

[0094] Furthermore, our peptide pool design, as described before, allowed us to interrogate immunogenicity based on a matrix format to determine the peptides responsible for the positive T cell responses (example for DNAJB7, FIGS. 5C-F and 6). This type of analysis is important for isolation of T.sub.H1 stimulating regions of the TAA, which may be incorporated in vaccines based on immunogenic components of multiple antigens important in CRC, as well as being regions that can be targeted by epitope-based modifications and strategies for enhancement of the immune response. A representative example of one CRC patient revealed positive IFN-γ and granzyme B responses to DNAJB7 peptide pools 3, 6 and 10, indicative of T cell responses to epitopes contained within peptides 3 and 23 (specifically peptides 11, 30, 26, 8, 13, 14, 19 and 27; FIG. 5D and E). Notably, these immunogenic fragments encompass significant portions of the full length DNAJB7 peptide, spanning from close to the C-terminus to the N-terminus, which advantageously demonstrate fragments spanning the entire length of the protein can elicit a response. Peptide 23 was the most immunogenic region of the DNAJB7 protein, with responses discovered in 39% of CRC patient and healthy control donors tested (FIG. 5F). DNAJB7 was tested in hepatocellular carcinoma, cholangioma, and head and neck cancer, and was also found to be immunogenic in these patients (FIGS. 5G and 6C).

[0095] Anti-DNAJB7 T.sub.H1 Responses are Induced During Cyclophosphamide Treatment

[0096] We have previously demonstrated that anti-tumor T.sub.H1 effector responses are controlled by regulatory T cells (Tregs), and that targeting these Tregs either by depletion in vitro, or inhibition/depletion in vivo with low dose cyclophosphamide, increases the anti-tumor (5T4) immune response (Scurr et al. 2017). We sought to assess whether T cell responses were induced to the novel tumor antigens in a colorectal cancer (CRC) patient (FIG. 7) and a hepatocellular (HCC) patient (FIG. 8) receiving short-term metronomic cyclophosphamide. Anti-5T4 T.sub.H1 responses increased by >4-fold in both patients, an effect previously identified as associating with improved survival outcomes (Scurr et al. 2017); intriguingly, anti-DNAJB7 T.sub.H1 responses also mirrored this treatment response profile in both instances, whereas no responses were induced to ARSJ, CENPQ, ZSWIM1 and CYP2B6 (FIG. 7B and C, and FIG. 8A and B). This could suggest that responses to DNAJB7 and CEACAM3 are suppressed in CRC and HCC, given that responses were unmasked by efficient regulatory T cell depletion (FIG. 7D and FIG. 8C).

SUMMARY

[0097] Here, a panel of broadly expressed, novel TAAs were identified (CENPQ, CEACAM3, CYP2B6, DNAJB7, ZC3H12B, ZSWIM1, ARSJ) by performing RNA sequencing of highly purified EpCAM+colorectal tumour cells in comparison to patient-matched EpCAM+colonic epithelial cells, analysing for the most differentially expressed genes. Tumour cell purification was necessary to reveal the genes, demonstrating how prior methods that sequence whole tumour fractions (i.e. inclusive of dead cells, stromal cells, immune cells and other tumour infiltrating cells) for antigen identification are flawed. Protein expression of the candidate TAAs was confirmed by immunohistochemistry, and pre-existing T cell immunogenicity towards these antigens tested by IFN-γ/Granzyme-B FluoroSpot. Of these, DNAJB7 (DnaJ heat shock protein family member B7), was identified here as a novel cancer-testis antigen, given its exceptionally restricted expression to cells of the testis and a wide variety of tumours, including glioma, breast, melanoma, pancreatic, liver, colorectal and renal cancers, was highly immunogenic recognized by effector and memory T cells lending itself lends to use a vaccine candidate in the treatment of a variety of cancers.

TABLE-US-00003 TABLE 1 Ensembl Gene ID Gene Name Description ENSG00000031691 CENPQ * centromere protein Q [Source: HGNC Symbol; Acc: HGNC: 21347] ENSG00000073464 CLCN4 chloride voltage-gated channel 4 [Source: HGNC Symbol; Acc: HGNC: 2022] ENSG00000079482 OPHN1 oligophrenin 1 [Source: HGNC Symbol; Acc: HGNC: 8148] ENSG00000102053 ZC3H12B * zinc finger CCCH-type containing 12B [Source: HGNC Symbol; Acc: HGNC: 17407] ENSG00000103021 CCDC113 coiled-coil domain containing 113 [Source: HGNC Symbol; Acc: HGNC: 25002] ENSG00000103184 SEC14L5 SEC14 like lipid binding 5 [Source: HGNC Symbol; Acc: HGNC: 29032] ENSG00000151632 AKR1C2 aldo-keto reductase family 1 member C2 [Source: HGNC Symbol; Acc: HGNC: 385] ENSG00000159263 SIM2 single-minded family bHLH transcription factor 2 [Source: HGNC Symbol; Acc: HGNC: 10883] ENSG00000162929 KIAA1841 KIAA1841 [Source: HGNC Symbol; Acc: HGNC: 29387] ENSG00000166529 ZSCAN21 zinc finger and SCAN domain containing 21 [Source: HGNC Symbol; Acc: HGNC: 13104] ENSG00000166762 CATSPER2 cation channel sperm associated 2 [Source: HGNC Symbol; Acc: HGNC: 18810] ENSG00000168612 ZSWIM1 * zinc finger SWIM-type containing 1 [Source: HGNC Symbol; Acc: HGNC: 16155] ENSG00000170956 CEACAM3 * carcinoembryonic antigen related cell adhesion molecule 3 [Source: HGNC Symbol; Acc: HGNC: 1815] ENSG00000172404 DNAJB7 * DnaJ heat shock protein family (Hsp40) member B7 [Source: HGNC Symbol; Acc: HGNC: 24986] ENSG00000180801 ARSJ * arylsulfatase family member J [Source: HGNC Symbol; Acc: HGNC: 26286] ENSG00000181638 ZFP41 ZFP41 zinc finger protein [Source: HGNC Symbol; Acc: HGNC: 26786] ENSG00000197408 CYP2B6 * cytochrome P450 family 2 subfamily B member 6 [Source: HGNC Symbol; Acc: HGNC: 2615] ENSG00000214376 VSTM5 V-set and transmembrane domain containing 5 [Source: HGNC Symbol; Acc: HGNC: 34443] ENSG00000254343 AC091563.1 ** ENSG00000255104 AC005324.4 ** ENSG00000267506 AC021683.2 ** ENSG00000272791 AC073389.3 ** ENSG00000283799 MIR1182 ** microRNA 1182 [Source: HGNC Symbol; Acc: HGNC: 35263]

TABLE-US-00004 TABLE 2 1 mvdyyevlgl qryaspedik kayhkvalkw hpdknpenke eaerkfkeva eayevlsnde 61 krdiydkygt eglngggshf ddeceygfff hkpddvfkei fherdpfsfh ffedsledll 121 nrpgssygnr nrdagyffst aseypifekf ssydtgytsq gslgheglts fsslafdnsg 181 mdnyisvtts dkivngrnin tkkiiesdqe reaedngelt fflvnsvane egfakecswr 241 tqsfnnyspn shsskhvsqy tfvdndeggi swvtsnrdpp ifsagvkegg krkkkkrkev 301 qkkstkrnc

TABLE-US-00005 TABLE 3 1 taacattgtc tttaacataa aagtgtacct atttgcagtc actacctcta tcaccaccac 61 cagcagagcc tgagctgagg aaaccacggt tctcaatacc cagcacaccc acttccaact 121 atctgttaaa acatggtgga ttactatgaa gttctaggac tgcaaagata tgcttcacct 181 gaggacatta aaaaagctta tcataaagtg gcacttaaat ggcaccctga taaaaatcca 241 gaaaataaag aagaagcaga gagaaaattc aaagaagtag ctgaggcata cgaggtatta 301 tcaaatgatg agaaacggga catttatgat aaatatggca cagaaggatt aaacggaggt 361 ggaagtcatt ttgatgatga atgtgagtac ggcttcacat tccataagcc agatgatgtt 421 tttaaagaaa tttttcatga aagggatcca ttttcttttc acttctttga agactcgctt 481 gaggacctgt taaatcgtcc aggaagctcc tatggaaaca gaaacagaga tgcaggatac 541 tttttctcca ctgccagtga atatccaatt tttgagaaat tttcttcata tgatacagga 601 tatacatcac agggttcatt ggggcatgaa ggccttactt ctttctcttc cctggctttt 661 gataatagtg ggatggacaa ctacatatct gttacaactt cagacaaaat cgttaatggc 721 agaaatatta atacaaagaa aattattgaa agtgatcaag aaagagaagc tgaagataat 781 ggagagttga cattttttct tgtaaatagt gtggccaatg aagagggctt tgcaaaagaa 841 tgcagctgga gaacacagtc attcaacaac tattcaccaa attctcacag ctccaaacat 901 gtatctcaat atactttcgt ggacaatgat gagggaggta tatcttgggt taccagcaac 961 agagatcccc ctattttctc agcaggagtc aaagagggtg gtaagaggaa aaaaaagaag 1021 cgtaaagagg tgcaaaagaa gtctaccaaa aggaattgtt aaattgactc ttcaaatata 1081 taacatttga acacaattgt gtgtgttttg gttaatcaca aattttgtag ataacactta 1141 atactatact aagagctttt caacactttt agcaggattg tggacatttg gttagtagtt 1201 tttttgaatg ggtatgtcag aaaaggatga gtttgtggtg acagttggtg ctaataagaa 1261 tttgcctggg cagtatagtg agatcttttc tctacaaaaa atttaaaaat tagccaagtg 1321 tggtggtgtg cacctgtagt cccagctact cgggaagctg gtagaaagac tgcttgagcc 1381 tggaagacat aggttgcagt gtaatgggat cacgccactg cactccagct tgggtgacaa 1441 agtaagaccc tgtcttaaaa aaaaaaaaaa ggaatttgga aaactgcaat gagtttttta 1501 cttttgaatt tttctttagt agagtttata gcctattttt cttttttcta attaaaacca 1561 ctatacaaac ttacgaaaca tgcatagcat tagaagacaa gaatgtatca agacacacag 1621 gttcagacta cacaattatt catgaaatgg ctgttttcca atggaggaac agcatttgat 1681 gcattataac ttaaaggtac tacaaattaa aaattaaagt ataaagtatt ttaagttaga 1741 aatagactta gctgggtgca gtggctcaca cctgtaatcc tagcatcttg ggaggcccag 1801 gcaggagggt tgaagtcaga atttcacgac agtccgggta acataatgag acctgtcctc 1861 tatgaaaaaa cttttttaaa aatttagctg ggtgtggtgg tacgtgcctg taggcctagc 1921 tacttaggag gctgaggtgg gaggattgct tgagcccagg agtttgaggt tgtagggagc 1981 tatgatcttg ccattgcact ccagctgaga gtgtgaccct gtctctaaag aaaaactata 2041 gaaaatttaa aaaattaaaa aacgggtaac atggtaaatt ttatgtagta tattttacca 2101 catttttttt aaaggccagt cttatcctga attcctcaga gcagtctgta tggtaggcca 2161 tcagtgaatc actccagaca ggaaattttc tatttttgtt tagagtttgt acacaaaatc 2221 tacaaataca ttactctaaa atagcaccta tagccatttt aatttagtca tgttgggaga 2281 aaaatagatg ggttacaaat acatcagata tcgagcaaat ggtttgagtt acatctagca 2341 gcactaaaac ttattcaact ttggccaaat acaaatgcga aattagaaaa aaattataat 2401 atctgctatt tttaaaagac agctatagtc aagagcttgt gttttgtatc ggtatttgat 2461 ttttagtggg cagatgggag aaggaaaggt tatttattct atagaagaga ctgcggacaa 2521 ttagtattca aagtttattt tcagaaaata aaataataaa agcccagggt attttaaa

TABLE-US-00006 TABLE 4 29 20mers, 1 19mer, overlapping by 10aa sequences. Peptide Pool 1 = Peptides 1-15; Peptide Pool 2 = Peptides 16-30. 1) MVDYYEVLGLQRYASPEDIK (SEQ ID NO: 1); 2) QRYASPEDIKKAYHKVALKW (SEQ ID NO: 2); 3) KAYHKVALKWHPDKNPENKE (SEQ ID NO: 3); 4) HPDKNPENKEEAERKFKEVA (SEQ ID NO: 4); 5) EAERKFKEVAEAYEVLSNDE (SEQ ID NO: 5); 6) EAYEVLSNDEKRDIYDKYGT (SEQ ID NO: 6); 7) KRDIYDKYGTEGLNGGGSHF (SEQ ID NO: 7); 8) EGLNGGGSHFDDECEYGETF (SEQ ID NO: 8); 9) DDECEYGETFHKPDDVEKEI (SEQ ID NO: 9); 10) HKPDDVFKEIFHERDPFSFH (SEQ ID NO: 10); 11) FHERDPFSFHFFEDSLEDLL (SEQ ID NO: 11); 12) FFEDSLEDLLNRPGSSYGNR (SEQ ID NO: 12); 13) NRPGSSYGNRNRDAGYFFST (SEQ ID NO: 13); 14) NRDAGYFFSTASEYPIFEKE (SEQ ID NO: 14); 15) ASEYPIFEKFSSYDTGYTSQ (SEQ ID NO: 15); 16) SSYDTGYTSQGSLGHEGLTS (SEQ ID NO: 16); 17) SLGHEGLTSFSSLAFDNSG (SEQ ID NO: 17); 18) FSSLAFDNSGMDNYISVTTS (SEQ ID NO: 18); 19) MDNYISVTTSDKIVNGRNIN (SEQ ID NO: 19); 20) DKIVNGRNINTKKIIESDQE (SEQ ID NO: 20); 21) TKKIIESDQEREAEDNGELT (SEQ ID NO: 21); 22) REAEDNGELTFFLVNSVANE (SEQ ID NO: 22); 23) FFLVNSVANEEGFAKECSWR (SEQ ID NO: 23); 24) EGFAKECSWRTQSENNYSPN (SEQ ID NO: 24); 25) TQSENNYSPNSHSSKHVSQY (SEQ ID NO: 25); 26) SHSSKHVSQYTEVDNDEGGI (SEQ ID NO: 26); 27) TFVDNDEGGISWVTSNRDPP (SEQ ID NO: 27); 28) SWVTSNRDPPIFSAGVKEGG (SEQ ID NO: 28); 29) IFSAGVKEGGKRKKKKRKEV (SEQ ID NO: 29); 30) KRKKKKRKEVQKKSTKRNC (SEQ ID NO: 30);

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