Patent Application
20210196808
The present disclosure relates to an immunogenic agent comprising DnaJ 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.
Despite understandable excitement surrounding results from clinical studies of immunotherapies for cancer, actual outcomes are disappointing. Many upregulated tumour associated antigen (TAA) targets for immunotherapy are often expressed to some extent in or on healthy tissue, e.g. the autoantigen carcinoembryonic antigen (CEA), and so directing immune responses against such antigens can lead to unwanted side-effects. Indeed, we have found T cell responses to CEA are associated with early disease relapse in patients treated for colorectal cancer (CRC). Thus, identification of useful TAAs that can be targeted by immunotherapy is a balance between: i) tumour expression; ii) the levels of expression of the same antigen in healthy tissue; and iii) controlling antigen-specific immunosuppressive responses driven by the same antigens. The challenge is further complicated by T cell cross reactivity which can result in off-target effects in distant tissue with potentially fatal consequences.
Whilst immunotherapies targeting neoepitopes hold promise as they are likely to differ sufficiently from self-antigens to ensure no cross reactivity, they are highly focused at the level of the individual and so prohibitively expensive to develop. For therapies relevant to the wider population such as cancer vaccines, immune mobilising monoclonal T-cell receptors Against Cancer (ImmTACs) and Chimeric Antigen Receptor T (CAR-T) cells, antigens must be broadly expressed in the same tumour types of multiple individuals and present at minimal levels in healthy tissue. Ideally, discovery pipelines would involve the large-scale analysis of TAA candidates followed by selection based on immunogenicity and tissue specific expression. Candidates that fit these criteria could be explored further as suitable cancer vaccinations.
In the context of CRC, known TAAs include CEA, GUCY2C, 5T4, MAGE antigens and Her-2, with several large investigations into cancer-testis antigen expression panels that have resulted in the identification of novel antigens; but the problem with these antigens is that they are often expressed in only a limited proportion of tumours. Of these antigens, CEA, GUCY2C and 5T4 have progressed towards pre-clinical and clinical studies.
There is therefore a need for new cancer vaccines that can be administered to a broad, ideally HLA-disparate population. The ideal vaccine would induce or boost T cell responses to target antigens expressed only in transformed cancer cells, but not normal cells; this is important to avoid off-target toxicity. Genomic analysis of paired tumour and healthy tissue in theory facilitates TAA identification but in practice appears to be limited by the diversity of cellular input in each sequencing sample.
The increasing use of RNA sequencing (RNA-seq) in differential expression analysis provides a useful methodology to initiate TAA discovery pipelines. However, for the colon, a mixture of immune cells, epithelium and stroma tends to complicate investigated expression profiles, thus hindering the identification of significantly and differently expressed genes, especially when one also considers that tumour immune infiltrate varies between individuals and tumour location.
The purification of epithelial and tumour cells prior to RNA seq analysis is a novel way for overcoming tissue heterogeneity. In this study, we used Epithelial cell adhesion molecule (EpCAM), i.e. a transmembrane glycoprotein mediating Ca2+-independent homotypic cell-cell adhesion in epithelia to aid purification. EpCAM purification of tumour and healthy colonic epithelium at two sites improved the resolution between tumour and healthy cell expression profiles and thus aided the identification of differentially expressed genes (DEG). Gene lists were created based on expression profiles between all tissues, and significant expression levels were established in a DESEQ2 comparison analysis. These lists were analysed, and several genes selected for further investigation as vaccine candidates. Immunogenic analysis and tissue expression of the protein products of these genes in healthy tissues were used to select the best candidate for future CRC immunotherapy. Several targets exhibited significant immunogenicity with respect to control antigens; but of these, one marker, DnaJ heat shock protein family (Hsp40) member B7 (DNAJB7) demonstrated the most favourable expression profile based on immunohistochemistry data of healthy and cancerous tissue. Further, we identified that all donors have the capability to mount T cell responses against DNAJB7. This protein antigen therefore harbours the potential to be used as a vaccine target in in future cancer therapeutic and prophylactic treatment strategies.
According to a first aspect of the invention there is provided 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.
Reference herein to DNAJB7 is to a protein belonging to the evolutionarily conserved DNAJ/HSP40 family of proteins, which regulate molecular chaperone activity by stimulating ATPase activity. As is known by those skilled in the art, DNAJ proteins may have up to 3 distinct domains: a conserved 70-amino acid J domain, usually at the N terminus; a glycine/phenylalanine (G/F)-rich region; and a cysteine-rich domain containing 4 motifs resembling a zinc finger domain.
In a preferred embodiment of the invention said DNAJB7 is human DNAJB7.
Preferably said 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.
Reference herein to an immunogenic fragment is to a part of DNAJB7 that can elicit an immune response when used in vivo; this response may be measured or determined using known tests such as those described herein. One test which may be used, but not exclusively, is whether the fragment can cause a tumour to be recognised and acted upon by components of the immune system. Advantageously, it has been found that these immunogenic fragments from portions of the full length DNAJB7 peptide, spanning from close to the C-terminus to the N-terminus i.e. the entire length of peptide, can elicit a response.
In a preferred embodiment of the invention said at least one fragment is 5-30 amino acids in length; preferably said at least one fragment is 8-25 amino acids in length. Most preferably said at least one fragment is 19 amino acids in length.
In a preferred embodiment of the invention said at least one DNAJB7 fragment comprises or consists of an amino acid sequence selected from at least one of the following groups:
or
As will be appreciated by those skilled in the art, measurement and/or comparison of immunogenicity can be carried out by any means known to those skilled in the art such as, but not limited to, in vitro FluoroSpot assays whereby peripheral blood mononuclear cell(s) is/are cultured in the presence of DNAJB7 protein or peptides derived therefrom, and T cell cytokine production in response to these peptides is measured.
As is known in the art, a variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions or truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid for another amino acid of like characteristics. For example, charged amino acid residues include lysine (+), arginine (+), histidine (+), aspartate (−) and glutamate (−); polar amino acids include serine, threonine, asparagine, glutamine, and tyrosine whereas the hydrophobic amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine and methionine. Generally, glycine is often found at the surface of proteins, within a loop- or coil region, providing high flexibility to the polypeptide chain at these locations. This suggests that it is rather hydrophilic. Proline, on the other hand, is generally non-polar and is mostly found buried inside the protein, although similarly to glycine, it is often found in loop regions. In contrast to glycine, proline provides rigidity to the polypeptide chain by imposing certain torsion angles on the segment of the structure. Glycine and proline are often highly conserved within a protein family since they are essential for the conservation of a particular protein fold.
In addition, in the following non-limiting groups of amino acids, each amino acid within each group are considered conservative replacements for one another: a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine, histidine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or have enhanced biological function, or immunogenicity, having regard to the reference polypeptide from which it varies.
In a preferred embodiment of the invention said at least one DNAJB7 fragment is represented by an amino acid sequence selected from the group comprising or consisting of:
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 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, ideally but not always, 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).
According to a further aspect of the invention there is provided a vector or DNA vaccine comprising a nucleic acid molecule encoding said DnaJ heat shock protein family (Hsp40) member B7 (termed DNAJB7), or at least one fragment thereof, as herein disclosed.
In a preferred embodiment of the invention 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.
Typically said adaptation includes, the provision of at least one transcription control sequences (e.g. at least one promoter sequence) which mediate(s) said expression. Preferably, the promoter is/are cell/tissue specific and more ideally still adapted for inducible or constitutive expression of said DNAJB7, or at least one fragment thereof.
In certain embodiments, said nucleic acid molecule encodes the whole of said DNAJB7 and/or a number of fragments thereof.
Those skilled in the art will appreciate that the term promoter includes the following features, which are provided by example only, and not by way of limitation: at least one enhancer element which is a cis acting nucleic acid sequences often found 5′ to the transcription initiation site of a gene (enhancers can also be found 3′ to a gene sequence or even located in intronic sequences and is therefore position independent) that functions to increase the rate of transcription of the gene to which the enhancer is linked. Further, enhancer activity is responsive to trans acting transcription factors (such as polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues which include, by example and not by way of limitation, intermediary metabolites (e.g. glucose, lipids), environmental effectors (e.g. light, heat,).
Promoter elements also include a TATA box and an RNA polymerase initiation selection (RIS) sequence which function to provide a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
Adaptations to the vector also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either a eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors and are included within the scope of the invention.
Further adaptations to the vector included within the scope of the invention, which facilitate the expression of vector encoded genes, include transcription termination/polyadenylation sequences. This or these features also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes.
Further adaptations to the vector included within the scope of the invention are expression control sequences, such as Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs in mice. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51: 975-985.
The above adaptations are well known in the art. Indeed, there is a significant amount of published literature with respect to expression vector construction and recombinant DNA expression techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994). Any one or more of these known techniques may be used when working the invention, provided the final vector or construct contains a nucleic acid molecule encoding DNAJB7, or at least one fragment thereof, which can be expressed by said vector in culture or a host environment.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising said immunogenic agent or vector or DNA vaccine of the invention.
According to a further aspect of the invention there is provided at least one immunogenic agent or vector or DNA vaccine or pharmaceutical composition containing or encoding DNAJB7, or at least one fragment thereof, for use in the treatment of cancer.
According to a further aspect of the invention there is provided at least one immunogenic agent or vector or DNA vaccine or pharmaceutical composition containing or encoding DNAJB7, or at least one fragment thereof, for use in the manufacture of a medicament to treat cancer.
As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by uncontrolled cell proliferation. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
Most preferably the cancer referred to herein includes 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.
More preferably still, 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, pancreatic, liver, and renal cancers.
Yet more preferably still, said cancer is colorectal cancer, head and neck squamous cell carcinoma or liver cancer.
According to a further aspect of the invention there is provided a method of vaccinating a subject suffering from or having a predisposition for cancer comprising administering an effective amount of the immunogenic agent, vector, DNA vaccine or pharmaceutical composition according to the invention to said subject.
Most preferably the cancer referred to herein includes 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, colon 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, pancreatic cancer, 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.
More preferably still, 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, pancreatic, liver, and renal cancers.
Yet more preferably still, said cancer is colorectal cancer, head and neck squamous cell carcinoma or liver cancer.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.
Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
An embodiment of the present invention will now be described by way of example only with reference to the following wherein:
Table 1. Details of all 23 genes identified by final analysis. ‘*’ highlights those taken forward, ‘**’ denotes those which were not protein coding.
Table 2. Amino acid sequence of DNAJB7 (SEQ ID NO: 31; accession number NP_660157.1)
Table 3. Nucleic acid sequence of DNAJB7 (SEQ ID NO: 32; accession number NM_145174.1)
Table 4. DNAJB7 Peptide Sequences.
Methods and Materials
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.
Excision of Colonic and Tumour Tissue
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 (
Purification of Tissue Samples
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−EpCAM+CD3− populations on a FACS Aria III (BD); gating strategy is shown (
RNA Sequencing
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).
Differential Expression Analysis
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.
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).
PBMC Culture
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.
ELISpot Assays
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/105 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.
FluoroSpot Assays
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% CO2 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/105 PBMCs, and at least double that of the negative (no antigen) control.
Antigens
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.
Immunohistochemistry
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).
Results
Purification of Samples Prior to RNA-Seq Analysis Provided Enhanced Resolution Of Differentially Expressed Genes
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.
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 (
The final genes selected were DNAJB7, CENPQ, ZC3H12B, ZSWIM1, CEACAM3, ARSJ and CYP2B6, based on their ideal expression profile for therapeutic exploitation (
Analysis of Protein Expression Across Multiple Healthy Tissues Highlights DNAJB7 as a Cancer-Testis Antigen and a Suitable Target for Immunotherapy
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 (
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 (
Analysis of Candidate TAA TH1 Responses Reveal DNAJB7 to be Immunogenic
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 (
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,
Anti-DNAJB7 TH1 Responses are Induced During Cyclophosphamide Treatment
We have previously demonstrated that anti-tumor TH1 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 (
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.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 1807831.1 | May 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/051309 | 5/14/2019 | WO | 00 |
