Peptide Vaccine

Abstract

The present invention provides one or more immunogenic peptides or derivatives thereof derived from long non-coding RNA genes regulated by PRMT5-E2F1 axis; their use in therapy, including pharmaceutical compositions comprising one or more of said peptides, vaccines comprising one or more of said peptides, and methods of generating immune response in mammalian subjects by administering agents capable of presenting peptides to a host.The present invention also relates to the use of PRMT5 inhibitors for use in treating cancer by stimulating host immunity.

Description

[Technical field]

[0001] The present invention provides a method for generating an immune response in a mammalian subject by administering one or more immunogenic peptides and an agent capable of presenting the peptides to a host.In particular, the present invention relates to a vaccine for therapeutic use, such as in the treatment of cancer.The present invention also relates to the use of PRMT5 inhibitors to treat cancer by stimulating host immunity. [Background technology]

[0002] The retinoblastoma protein (pRb)-E2F pathway is a key control point in the cell cycle. This pathway is often deregulated in tumor cells, and deregulation of this pathway is widely considered a "hallmark" of cancer. Classically, the pRb tumor suppressor protein is a negative regulator of E2F transcription factors that function as a transcriptional hub through which pRb exerts its effect on cell cycle progression. Temporal regulation of cyclin / CDK complexes and sequential phosphorylation of pRb releases E2F at the G1 / S phase transition, allowing E2F to drive the transcription of genes required for cell cycle progression. Classical E2F target genes include well-characterized cell cycle, DNA synthesis and apoptosis targets, as well as others associated with metabolic control, differentiation, senescence and autophagy ( Blanchet et al., 2011 ; Denchi and Helin, 2005 ; Kent and Leone, 2019 ; Munro et al., 2012 ; Roworth et al., 2015 ; Wu et al., 2001 ; Yao et al., 2008 ).

[0003] It is becoming clear that the pRb-E2F pathway regulates a much larger network of genes than originally anticipated, reflecting an influential "reader-writer" event that occurs at the site of arginine methylation on the E2F1 subunit by protein arginine methyltransferase (PRMT)5 (Barczak et al., 2020; Cho et al., 2012; Zheng et al., 2013). Residue-specific methylation occurs within a central arginine (R)-rich cluster that promotes proliferation and cell growth at the biological level (Cho et al., 2012; Zheng et al., 2013). Importantly, genome-wide analyses have identified a large repertoire of genes under PRMT5-E2F1 control (Roworth et al., 2019). This occurs via p100 / TSN, which reads meR marks by its Tudor domain, thereby switching E2F1 from its primary role as a transcriptional regulator to one with broader effects on other levels of gene expression control, including alternative RNA splicing (Roworth et al., 2019). The frequent overexpression of PRMT5 in a wide range of human tumors and the critical role E2F plays in the cancer cell cycle strongly argue for the importance of the PRMT5-E2F1 axis in driving malignant disease. A large portion of the human genome is populated by non-classical genes that are composed of different structural and regulatory elements, including, for example, genes encoding microRNAs and long non-coding (lnc)RNAs (Gebert and MacRae, 2019; Statello et al., 2021).

[0004] lncRNA genes are the major source of transcription in mammalian cells and typically encode transcripts longer than 200 nucleotides, the majority of which are thought to exist as non-coding RNAs ( Statello et al., 2021 ).

[0005] Here, we describe a novel and unexpected role of the PRMT5-E2F1 axis in controlling the expression of long non-coding (lnc) genes in non-coding cancer genomes. Although widely considered to be a non-translated population of RNA, we found that a group of lncRNA transcripts can be translated and further processed into peptides. Importantly, many lncRNA-derived peptides contribute to the antigenic landscape of tumor cells and are presented by MHC class I protein complexes in tumor cells to the immune system. Both PRMT5 and E2F1 affect the expression of lncRNA genes, thereby controlling the repertoire of peptides presented by MHC class I antigens. Pharmacological control of the PRMT5-E2F1 axis changes the profile of tumor-associated antigens derived from lncRNA genes, thereby affecting effective adaptive immune responses.

[0006] As described herein, we have designed a stand-alone therapeutic vaccine composed of lncRNA-derived peptide antigens and found that it is immunogenic and promotes effective antitumor immune responses. These results show that PRMT5 links E2F activity to the non-coding genome and antigenic landscape of tumor cells. Thus, manipulating PRMT5 activity represents a therapeutic strategy for controlling the immunogenicity of tumor cells.

[0007] Cancer vaccines and long-term tumor-specific immunity may prove useful for managing human or animal treatment of various tumors and for preventing tumor recurrence. The present invention provides novel tools and methods for vaccine immunotherapy. [Brief description of the drawings]

[0008] [Figure 1A]Figure 14. Differential expression analysis of lncRNA transcripts present in HCT116 cells. Venn diagram demonstrating the crossover of lncRNA transcripts up- or downregulated at 30% levels (q<0.05) in WT or E2F1 Cr cell lines treated with T1-44 for 48 hours as indicated versus WT E2F1 DMSO treated cells. [Figure 1B] Figure 1 shows differential expression analysis of lncRNA transcripts present in HCT116 cells. Bar graphs to represent the total number of lncRNA transcripts that are differentially up-regulated and down-regulated at statistically significant levels (q<0.05) in each cell line and treatment relative to WT E2F1 DMSO-treated cells. No fold change cutoff was applied to the data. [Figure 1C] Figure 1 shows differential expression analysis of lncRNA transcripts present in HCT116 cells. (i) WT E2F1 or E2F1 Cr cell lines were treated with 1 μM T1-44 for 48 hours followed by RT-qPCR analysis to determine the expression of the indicated lncRNA transcripts (labeled by their ENSEMBL transcript names). (ii) Immunoblots were also included to display input protein levels of E2F1, and SDMe was used as a marker for T1-44 activity. [Figure 1D]Figure 1. Differential expression analysis of lncRNA transcripts present in HCT116 cells. (i) WT E2F1 or E2F1 Cr cell lines were treated with 1 μM T1-44 for 48 hours, followed by chromatin extraction and ChIP analysis using E2F1-specific or IgG control antibodies. ChIP-seq data from ENCODE was used to identify potential E2F1-binding sites (marked by smaller red rectangles [no arrows]) around the TSS of each lncRNA gene (marked by arrows), and primers were designed around these sites for use in qPCR. CDC6 and actin genes were included as positive and negative controls, respectively. (ii) Immunoblots were also included to show input protein levels of E2F1, and SDMe was used to show activity of T1-44. [Figure 2A] Figure 1. Differential expression analysis of lncRNA transcripts present in CT26 cells grown in vitro and in situ as tumors. Bar graph representation of the number of lncRNA transcripts that were differentially up- or down-regulated after T1-44 treatment in the CT26 (30% change, q<0.05) RNA-seq dataset. [Figure 2B] Figure 1 shows differential expression analysis of lncRNA transcripts present in CT26 cells grown in vitro and in situ as tumors. Two different siRNAs targeting E2F1 were transfected into CT26 cells for 72 hours. 8 hours after transfection, additional treatment with 1 μM T1-44 was performed, followed by RT-qPCR analysis to determine the expression of the indicated lncRNA transcripts (labeled by their ENSEMBL transcript names). Immunoblots were also included to display the input protein levels of E2F1, and SDMe was used as a marker for T1-44 activity. [Figure 2C]Figure 1 shows differential expression analysis of lncRNA transcripts present in CT26 cells grown in vitro and in situ as tumors. (i) Schematic representation of the experiment with T1-44 on colon26 tumors (i). Balb / c mice were treated with T1-44 administered orally at 100 mg / kg for 19 days compared to vehicle-only controls; n=7 per group; (ii) Absolute tumor growth volume in T1-44-treated (squares) and untreated (circles) Balb / c mice presented as mean values ​​(±SEM), n=7; (iii) Scatter plot of absolute tumor volume of individual mice at day 12 (t-test; *p<0.05), n=7; T1-44-treated (right) and untreated (left). (iv) Relative body weight representation of T1-44 treated (squares) and untreated (triangles) Balb / c mice presented as mean values, n=7; (v) Survival curves of treated (dotted line) and untreated mice (solid line - ended at d16) (Log-rank (Mantel-Cox) test; *p<0.05), n=7. [Figure 2D] Figure 1 shows differential expression analysis of lncRNA transcripts present in CT26 cells grown in vitro and in situ as tumors. RNA was isolated from colon26 tumors treated with DMSO or T1-44 as indicated, followed by RT-qPCR analysis to determine the expression of the indicated lncRNA transcripts (labeled by their ENSEMBL transcript names). [Figure 2E]Figure 2 shows differential expression analysis of lncRNA transcripts present in CT26 cells grown in vitro and in situ as tumors. (i) Representative examples of immunohistochemical staining of SDMe in colon26 tumors harvested on day 14 from Balb / c mice or untreated controls after 19 days of treatment with 100 mg / kg T1-44 (see experiment in Figure 2C). Original magnification: 20x, scale bar, 50 μm; and 63x; scale bar, 16 μm. n=4; (ii) as above, but immunohistochemical staining was performed with anti-CD8; (iii) as above, but immunohistochemical staining was performed with anti-CD4; (iv) as above, but immunohistochemical staining was performed with anti-CD163; (v) results were quantified using ImageJ Fiji software and normalized optical density was presented as mean ± SD. Statistical analysis was performed using unpaired two-tailed Student's t-test using GraphPad Prism8 software, n=4. Control is shown in the upper panel and T1-44 is shown in the lower panel. [Figure 3A] Figure 1. Immunopeptidomics analysis of CT26 cells. Diagram to illustrate the workflow of the immunopeptidomics platform. Peptides extracted from cell surface MHC class I protein complexes are detected by mass spectrometry and identified by comparison against an in-house lncRNA proteome database or a database of all screened mouse SwissProt entries. Candidate peptides are then ranked based on their predicted immunogenicity, MHC binding affinity and RNA expression profile, and then subjected to selection as part of a dendritic cell-based cancer vaccine. [Figure 3B]Immunopeptidomic analysis of CT26 cells. CT26 cells were treated with 1 μM T1-44 or DMSO (control) for 72 hours, followed by immunoprecipitation with antibodies specific for MHC class I alleles and extraction of MHC-bound peptides for mass spectrometry. Experiments were performed in biological replicates (rep1, rep2). The overlap of MHC-bound lncRNA-derived peptides identified from qualitative immunopeptidomic analysis between each treatment and biological replicate is shown in the Venn diagram. 328 peptides were detected using Peaks software. [Figure 3C] Figure 1 shows immunopeptidomic analysis of CT26 cells: (i) Predicted allele frequency for each MHC allele bound by identified lncRNA-derived peptides from CT26 immunopeptidomic analysis is displayed on a bar graph and expressed as a percentage of the total; (ii) Sequence logos demonstrating amino acid residue conservation in MHC class I-bound lncRNA-derived peptides derived from protein-coding genes identified from immunopeptidomic analysis for each MHC allele. [Figure 3D] Immunopeptidomic analysis of CT26 cells. The peptide length of each peptide identified as derived from the mouse lncRNA database is displayed on the graph. 195 peptides were detected in the quantitative analysis using Progenesis software. [Figure 3E] Immunopeptidomic analysis of CT26 cells. Partial sequences of Gm37283, Gm17173, and Gm37494 lncRNA transcripts are displayed (SEQ ID NOs: 177, 178, and 179, respectively) along with predicted ORFs (highlighted in grey) that give rise to identified MHC class I-binding peptides (boxes). Amino acid sequences are shown in SEQ ID NOs: 180-187. [Figure 3F]Figure 1 shows immunopeptidomic analysis of CT26 cells. (i) Exemplary polysome profiling assays performed on CT26 cells treated with 1 μM T1-44 or DMSO for 72 hours showing the amount of total RNA detected in each collected fraction (by absorbance readout at 254 nm). Fractions representing free unbound RNA (fractions 1-5); 80S ribosomal fraction (fraction 6); and polysome fractions with increasing polysome size (fractions 7-12) are shown. (ii) Polysome profiling assays for Gm37494 lncRNA from CT26 cells treated with DMSO or 1 μM T1-44 for 72 hours yielding MHC class I peptides are displayed. Data are expressed as a percentage of total RNA in each fraction; n=3; (iii) Quantification of polysome profiling assays by calculation of heavy (fractions 10-12) to light (fractions 6-9) polysome ratios from each of the lncRNA ribosome profiling assays shown; (iv) Immunoblots are included to demonstrate SDMe levels as a measure of T1-44 activity. [Figure 4A] Immunopeptidomic analysis of HCT116 cells. HCT116 cells were treated with 1 μM T1-44 or DMSO (control) for 48 hours, followed by immunoprecipitation with antibodies specific for MHC class I alleles and extraction of MHC-bound lncRNA-derived peptides for mass spectrometry. Experiments were performed in biological replicates (rep1, rep2). The overlap of MHC-bound lncRNA-derived peptides identified from qualitative immunopeptidomic analysis between each treatment and biological replicate is shown in the Venn diagram. 55 peptides were identified from the qualitative GENCODE annotated database. [Figure 4B]Figure 1 shows immunopeptidomic analysis of HCT116 cells: (i) Predicted allele frequency for each MHC allele bound by identified lncRNA-derived peptides from immunopeptidomic analysis is displayed on a bar graph and expressed as a percentage of the total; (ii) Sequence logos demonstrating amino acid residue conservation in MHC class I-bound peptides derived from lncRNA genes identified from immunopeptidomic analysis for each MHC allele. [Figure 4C] Figure 1 shows immunopeptidomic analysis of HCT116 cells. The peptide length of each peptide identified as derived from the lncRNA GENCODE database (PROGENESIS software analysis) is displayed on the graph. 76 peptides were identified from the quantitative GENCODE database. [Figure 4D] Figure 1 shows immunopeptidomic analysis of HCT116 cells. (i) Exemplary polysome profiling assays performed on HCT116 cells treated with 1 μM T1-44 or DMSO for 48 hours showing the amount of total RNA detected in each collected fraction (by absorbance readout at 254 nm). Fractions representing free unbound RNA (fractions 1-5); 80S ribosomal fraction (fraction 6); and polysome fractions with increasing polysome size (fractions 7-12) are shown; (ii) Polysome profiling assays for MALAT1 and AC079135.1 lncRNAs from HCT116 cells treated with DMSO or 1 μM T1-44 for 48 hours are displayed. Data are expressed as a percentage of total RNA in each fraction; n=3; (iii) Quantification of polysome profiling assays by calculation of heavy (fractions 10-12) to light (fractions 6-9) polysome ratios from each of the indicated lncRNA ribosome profiling assays from HCT116 WT and E2F1 Cr cells treated with DMSO or 1 μM T1-44 for 48 h; (iv) Immunoblots are included to demonstrate SDMe levels as a measure of T1-44 activity. [Figure 4E-1]Figure 1. Immunopeptidomic analysis of HCT116 cells. (i) Schematic representation of the pSF-CMV-NEO-COOH-FLAG plasmid used as a cloning vector for insertion of predicted ORFs from human lncRNA transcripts found to encode peptides presented on MHC class I. The predicted ORFs and short sections of upstream sequences (including any endogenous Kozak sequences) were ligated into the multiple cloning site (MCS) of the vector in frame with a C-terminal 3xFLAG tag. [Figure 4E-2] Immunopeptidomic analysis of HCT116 cells. (ii) Parts of the sequences of MALAT1 and AC079135.1 lncRNA transcripts are shown (SEQ ID NOs: 188, 189) with predicted ORFs (highlighted in grey) that give rise to identified MHC class I-binding peptides (boxes). The amino acid sequences are shown in SEQ ID NOs: 189-193. The ORFs were cloned upstream of a C-terminal FLAG tag into the pSF-CMV-NEO-COOH-3xFLAG vector. (iii) 4 μg of MALAT1 and AC079135.1 ORF-Flag plasmids were transfected into HCT116 cells for 48 hours, followed by immunofluorescence analysis using anti-Flag antibody. Cell nuclei were stained with DAPI. (iv) 4 μg of MALAT1 and AC079135.1 ORF-Flag plasmids were transfected into HCT116 cells, followed by immunoblot analysis using Flag antibody. [Figure 4F] Figure 1 shows immunopeptidomic analysis of HCT116 cells. 4 μg MALAT1 (MAL) or AC079135.1 (AC) ORF-Flag plasmid and 0.5 μg GFP plasmid were transfected into HCT116 WT E2F1 and HCT116 E2F1Cr cells for 72 hours. For the last 48 hours, cells were also treated with DMSO or 1 μM T1-44, followed by immunoblot analysis with the indicated antibodies. SDMe was included to demonstrate the activity of T1-44 compounds. [Figure 5A]Heatmap of peptides encoding lncRNA transcripts comparing expression in tumor versus normal tissues using TCGA and cancer cell line datasets. Blue heatmap (top) - expression represented as the average [log2(fpkm+0.001)] of all samples according to the anatomical location of the tumor relative to normal tissue; red / green heatmap (bottom left) - display of tumor / normal ratio [Log2(tumor / normal FPKM ratio)], red (enriched in MALAT1) represents higher expression in normal and green represents higher expression in tumor tissue; orange heatmap (bottom center) - expression levels in different colorectal cancer cell lines [Log2(fpkm+0.001)]; light blue (bottom right) - raw Z-score normalized expression levels in microsatellite stable and unstable patients. [Figure 5B] Kaplan-Meier curves of overall survival for patients with adrenocortical carcinoma (i), colorectal cancer (ii), and pancreatic cancer (iii) with respect to the expression of several lncRNA genes. Plots were generated using the Gepia2 tool. For each analysis, patients were divided into two groups, one with high expression (red line) or one with low expression (blue line). [Figure 5C]FIG. 1 shows characterization of 20 selected peptides encoded by mouse lncRNAs identified in immunopeptidomics experiments on CT26 cells treated with T1-44 versus DMSO control. From left to right, each column represents the sequence of the peptide, lncRNA gene name, transcript accession ID; peptide length; net MHCpan score and allele. The columns show the results from the binding affinity prediction analysis; peptide abundance fold change (T1-44 treatment vs. DMSO treatment) (derived using PROGENESIS software); expression levels in CT26 cells (based on our in-house RNA-seq database and other databases - GENEVESTIGATOR software) (low - log2TPM<7.5; medium - log2TPM 8.5-11.5; high - log2TPM>11.5); characterized expression in the thymus (EXPRESSION ATLAS - based on www.ebi.ac.uk / gxa / home and GENEVESTIGATOR software) (low - log2TPM<7.5; medium - log2TPM 8.5-11.5; high - log2TPM>11.5; below cutoff - no expression); the last column represents the results from the immunogenicity experiment. [Figure 6A]Figure 1 shows lncRNA-derived MHC class I peptides as cancer vaccines in colon26 tumor model. (i) Schematic representation of immunogenicity assay used to measure immune response to MHC class I-bound peptides identified as derived from lncRNA. Briefly, Balb / c mice were vaccinated on day 0 with 20 peptides (50 μg each) divided into 4 groups (4 mice in each group) containing a pool of 5 peptides each, and booster vaccination was performed 7 days later. CD40 antibody and poly:IC were used as adjuvants. AH1 peptide (SPSYVYHQF; SEQ ID NO: 156) vaccination was used as a positive control. Mice were sacrificed 7 days after booster, spleens were removed, and splenocytes were isolated for ELIspot assay. (ii) Splenocytes from each separate mouse were stimulated with the individual peptide (15 μL / mL) with which the group was vaccinated. Each peptide was tested in duplicate with the indicated peptides and activity was measured in an interferon-gamma based ELIspot assay. DMSO was used as a negative control. Spots were quantified on an ELISPOT counter. [Figure 6B] Figure 1: lncRNA-derived MHC class I peptides as cancer vaccines in colon26 tumor model. (i) Schematic representation of dendritic cell (DC)-based vaccine strategy used in colon26 tumor challenge experiment. Randomized Balb / c mice were vaccinated with dendritic cells (DC) pulsed with a pool of 15 lncRNA-derived peptides on day 0 and a second dose on day 7. Unpulsed DCs were used as control; n=6. (ii) Absolute tumor growth volume in unpulsed and pulsed DC-treated BALB / c mice presented as mean (±SEM), n=6. (iii) Scatter plot of absolute tumor volume of individual mice on day 12 (t-test; *p<0.05), n=6; (iv) Relative body weight of unpulsed and pulsed DC-treated BALB / c mice presented as mean, n=7. [Figure 6C]Figure 1 shows lncRNA-derived MHC class I peptides as cancer vaccines in colon26 tumor model. Model diagram to show the regulation of lncRNA-derived antigen presentation by E2F1-PRMT5 axis. It is proposed that PRMT5-targeted methylation of E2F1 affects its transcriptional activity on multiple genes, including the expression of multiple lncRNAs that are subsequently translated into polypeptides that can be processed to generate peptide epitopes for presentation to MHC class I protein complexes. Pharmacological manipulation of PRMT5 activity with compound T1-44 results in altered expression of several lncRNA transcripts that code for immunogenic peptides. We propose that the subsequent presentation of these immunogenic peptides by MHC class I complexes contributes to the increased immune cell infiltration of the tumor microenvironment (TME) observed. Summary of the Invention

[0009] According to a first aspect of the present invention, there is provided a peptide or a derivative thereof derived from a long non-coding RNA gene regulated by the PRMT5-E2F1 axis.

[0010] According to a second aspect of the invention there is provided a nucleic acid sequence encoding a peptide according to the first aspect of the invention.

[0011] According to a third aspect of the present invention, there is provided a vector comprising a nucleic acid of the second aspect of the present invention. Suitably, the vector is an expression vector capable of expressing the nucleic acid of the second aspect of the present invention. Expressing the nucleic acid of the present invention also includes producing the peptide encoded by the sequence.

[0012] According to a fourth aspect of the invention there is provided a host cell comprising a nucleic acid of the second aspect of the invention or a vector (eg an expression vector) of the third aspect of the invention.

[0013] According to a fifth aspect of the invention there is provided a vaccine comprising at least one peptide according to the first aspect of the invention or a nucleic acid according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0014] According to the sixth aspect of the present invention, there is provided a method for producing a tumor-specific vaccine, comprising: identifying tumor-expressed peptides that are encoded by long non-coding RNA genes regulated by PRMT5-E2F1 axis; and incorporating one or more of said peptides or the nucleic acid encoding said one or more peptides into a vaccine.Optionally, said peptides are also tested to confirm that they are immunogenic or bind to MHC class 1 molecules.

[0015] According to a seventh aspect of the invention there is provided a pharmaceutical composition comprising a peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, or a vaccine according to the fifth aspect of the invention and a pharma- ceutical acceptable excipient.

[0016] According to an eighth aspect of the present invention, there is provided a pharmaceutical composition according to the seventh aspect of the present invention or a peptide according to the first aspect of the present invention or a nucleic acid according to the second aspect of the present invention or a vaccine according to the fifth aspect of the present invention for use in therapy. Suitably, the therapy is the treatment of cancer. In a particular embodiment, the treatment of cancer comprises the administration of a pharmaceutical composition according to the seventh aspect of the present invention or a peptide according to the first aspect of the present invention or a nucleic acid according to the second aspect of the present invention or a vaccine according to the fifth aspect of the present invention in combination with a PRMT5 inhibitor.

[0017] According to the ninth aspect of the present invention, there is provided a method for selecting peptides for inclusion in tumor vaccine, comprising contacting a tumor cell sample with a PRMT5 inhibitor and determining the expression level of one or more peptides encoded by lncRNA genes by the contacted (treated) cells, and selecting one or more peptides that are deregulated and immunogenic for inclusion in tumor vaccine.Preferably, the tumor cell sample is obtained from a patient with cancer / tumor.Preferably, the peptide is upregulated in tumor cells.

[0018] Thus, in a variant of the ninth aspect of the present invention, there is provided a method for selecting peptides for inclusion in a tumor vaccine, comprising contacting cells of a tumor type with a PRMT5 inhibitor and determining the expression level of one or more peptides encoded by lncRNA genes by the contacted cells and selecting one or more peptides that are upregulated for inclusion in the tumor vaccine, optionally prior to selection, the peptides are also tested to determine or predict whether they are immunogenic, and one or more immunogenic peptides are selected for inclusion in the tumor vaccine.

[0019] According to a tenth aspect of the present invention there is provided a method for producing a vaccine comprising selecting a peptide for inclusion in a vaccine according to the ninth aspect of the invention and generating a vaccine capable of presenting said peptide.

[0020] According to an eleventh aspect of the present invention there is provided a PRMT5 inhibitor for use in the treatment of cancer by stimulating the immune response.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, but suitable methods and materials are described below.In case of conflict, the present specification, including definitions, will control.In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0022] Other features and advantages of the present invention will be apparent from the following. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The disclosed method can be more readily understood with reference to the following detailed description, which forms a part of this disclosure. It should be understood that the disclosed method is not limited to the specific methods described and / or shown herein, and that the terms used herein are for the purpose of describing specific embodiments by way of example only, and are not intended to be limitations of the claimed method.

[0024] The methods of the present disclosure will utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry that are within the skill of the art, unless otherwise indicated. Exemplary techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989); "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., Current Protocols of Molecular Biology, John Wiley and Sons (1987); and "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., Birhauser, Boston, 1994).

[0025] Unless otherwise indicated, each gene name used herein corresponds to the official symbol assigned to the gene and provided by Entrez Gene (URL: www.ncbi.nlm.nih.gov / sites / entrez) as of the filing date of this application.

[0026] [Definition] A reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. When a range of values ​​is expressed, another embodiment includes from the one particular value and / or to the other particular value. Further, reference to values ​​stated in a range includes each and every value within that range. All ranges are inclusive and combinable.

[0027] It should be understood that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0028] The articles "a," "an," and "the" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0029] The use of the alternative (eg, "or") should be understood to mean either one, both, or a combination thereof of the alternatives.

[0030] The term "and / or" should be understood to mean either one or both of the alternatives.

[0031] When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0032] As used herein and unless otherwise specified, the term "about" should be understood to be used synonymously with the term "approximately." Illustratively and unless otherwise specified, use of the term "about" indicates values ​​slightly outside the recited reference value, such as ±15%, ±10%, ±8%, ±5%, or simply ±2%. Such values ​​are therefore encompassed by the scope of the claims recited in the term "about" or "approximately."

[0033] As used herein, the term "in vitro" means carried out or performed in a test tube, culture dish, or other location outside a living organism. The term also includes ex vivo, since the analysis is performed outside of a living organism.

[0034] As used herein, the term "isolated" refers to material that is substantially or essentially free from components that normally accompany it in its native state. In the context of "isolated from a subject," this can mean removed from a subject. In certain embodiments, the terms "obtained" or "derived" are used synonymously with "isolated."

[0035] A "subject," "individual," or "patient," as used herein, includes any animal that can be tested using the present invention. Suitable subjects include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals (such as horses, cows, sheep, pigs, etc.), and domestic animals or pets (such as cats or dogs). In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate, and in certain embodiments, the subject is a human.

[0036] <Peptides of the Invention> The present invention is based in part on the recognition that tumor cells subjected to inhibition of PRMT5 result in increased expression of various peptides in tumor cells, including cell surface, which the present inventors have identified as being encoded by long non-coding (lnc)RNA genes.Such peptides are regulated by PRMT5-E2F1 axis.Bioinformatics analysis of differentially expressed lncRNAs found that most of them are associated with or retain E2F binding sites, implying that they are under the transcriptional control of E2F1.

[0037] The inventors found that many of these peptides were immunogenic, capable of binding to MHC class I molecules, and elicited a host immune response that resulted in slowing of tumor cell growth. Thus, these peptides represent a novel source of tumor-associated antigens that can be utilized in vaccine design.

[0038] One aspect of the present invention also relates to PRMT5 inhibitors for use in the treatment of cancer by stimulating host immune response against tumor cells.This novel mechanism of targeting tumor cells suggests new clinical applications, including the ability to treat cancers that have acquired resistance to the direct cancer-killing effect of PRMT5 inhibition.Furthermore, dendritic cells exposed to these peptides can slow down tumor cell growth, demonstrating that these peptides can function as suitable agents for stimulating host immune response and therefore for use in vaccines.Thus, the peptides of the present invention can be used in therapy, particularly in the treatment of cancer.

[0039] The majority of the human genome is occupied by non-classical genes composed of different structural and regulatory elements, including, for example, genes encoding microRNAs and long non-coding (lnc)RNAs (Gebert and MacRae, 2019; Statello et al., 2021). lncRNA genes (referred to herein as lncRNAs or lnc genes) are the main source of transcription in mammalian cells and typically encode transcripts with lengths greater than 200 nucleotides. However, while a small number of lncRNA transcripts have been shown to be processed in the same manner as mRNAs, suggesting that they play biological roles in rare cases, the majority of them are thought to exist as non-coding RNAs (Statello et al., 2021).

[0040] According to a first aspect of the present invention, there is provided a peptide or a derivative thereof derived from a long non-coding RNA gene regulated by the PRMT5-E2F1 axis.

[0041] Preferably, the peptide is immunogenic. Preferably, the peptide is an MHC class I associated peptide or a derivative thereof. Preferably, the peptide is presented by a human leukocyte antigen (HLA) class I molecule. Preferably, the peptide is isolated.

[0042] The term "isolated" as used herein generally refers to a biological component (such as a nucleic acid molecule, protein, or peptide) that has been substantially separated or purified from other biological components in the cells of the organism in which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins; and / or is a relatively short component of the naturally occurring molecule. "Isolated" nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids, proteins, and peptides.

[0043] Preferably, the lncRNA transcript has a predicted E2F binding site.

[0044] Preferably, the peptide has an amino acid sequence encoded by a portion of a lncRNA transcript that also has a predicted E2F binding site. The E2F binding site is a DNA sequence in the promoter of a gene that binds to E2F1. E2F1 is a transcription factor; once bound to a DNA sequence, E2F1 can then drive the transcription of the gene. The canonical E2F1 binding site sequence is TTTSSCGC, where S is guanine or cytosine.

[0045] Suitably the peptide is one disclosed in Table 1 or a derivative of a peptide disclosed therein.

[0046] JPEG2024543184000001.jpg255169JPEG2024543184000002.jpg172170

[0047] Peptides can be of any length greater than 5 amino acids. Typically, peptides are in the range of 6-30 amino acids in length, for example 8-20 amino acids in length. The MHC1-presented immunogenic peptides identified in the Examples had an average length of 9 amino acids. In certain embodiments, the immunogenic peptides are 8, 9 or 10 amino acids in length.

[0048] The peptide of the first aspect may be referred to herein as the peptide of the invention or the lncRNA gene peptide of the invention.

[0049] A "derivative" of one of the peptides refers to a mutant or variant peptide that can include amino acid deletions, additions or substitutions, subject to the requirement of maintaining the immunogenicity of the peptide. That is, conservative amino acid substitutions can be made without substantially changing the nature of the peptide, such as in the case of truncation from the 5' or 3' end. Furthermore, deletions and substitutions can be made to the peptides of the present invention. Substitution, deletion or insertion variants of the peptide can be prepared by recombinant methods and screened for cross-reactivity with the native peptide. Derivatives also encompass fusion proteins, where one part of the fusion protein is the peptide of the present invention and some other part is another peptide sequence. "Derived from" includes the situation where the peptide is a part of a polypeptide expressed from a lncRNA. That is, for the purpose of illustration, if a lncRNA gene encodes a polypeptide of 80 amino acids in length, the peptide derived therefrom can be a relatively small part thereof, such as a peptide of 8 or 9 amino acids or more but less than 80 amino acids.

[0050] Therefore, the peptide derived from the long non-coding RNA gene regulated by PRMT5-E2F1 axis can be the whole translated polypeptide or a part thereof, i.e., peptide.Preferably, the peptide is immunogenic.Preferably, the peptide can be presented on MHC class 1 molecule, for example, as antigen-MHC class 1 complex.

[0051] In certain embodiments, a derivative of a peptide comprises at least 80%, e.g., at least 85%, at least 90%, or at least 95% amino acid sequence identity with the peptide. With respect to a peptide encoded by a parent lncRNA gene, such a derivative peptide may be referred to as an engineered variant of the peptide encoded by the parent lncRNA gene.

[0052] <Nucleic acids and host cells> According to a second aspect of the invention there is provided a nucleic acid sequence encoding a peptide according to the first aspect of the invention.

[0053] Suitably, the nucleic acid is DNA, cDNA, PNA, RNA or a combination thereof.

[0054] According to the third aspect of the present invention, there is provided a vector comprising the nucleic acid of the second aspect of the present invention. Any vector can be used, such as a plasmid, a cosmid, or a viral vector. The vector can be used to amplify multiple copies of the insert sequence, for example, using standard approaches of bacterial transformation, cell culture, and vector purification. The vector can also facilitate the expression of the encoded protein.

[0055] In certain embodiments, the vector is an expression vector and is capable of expressing the nucleic acid of the second aspect of the invention. Expressing the nucleic acid of the second aspect of the invention also includes producing the peptide encoded by the sequence.

[0056] According to a fourth aspect of the invention there is provided a host cell comprising a nucleic acid of the second aspect of the invention or a vector of the third aspect of the invention.

[0057] Any suitable host cell can be used, for example, a prokaryotic or eukaryotic cell, and in certain embodiments, the host cell is selected from the group consisting of a bacterial, a fungal (including yeast), and a mammalian cell.

[0058] <Vaccine> Cancer vaccines generally fall into one of the following categories: protein antigen / adjuvant vaccines, DNA vaccines, viral vector-based vaccines, tumor cell vaccines, and dendritic cell vaccines. Any of these types of vaccines can be utilized in the present invention.

[0059] According to a fifth aspect of the invention there is provided a vaccine comprising at least one peptide according to the first aspect of the invention or a nucleic acid according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0060] Vaccine technology is well advanced, and any vaccine platform capable of presenting the peptide of the present invention to the body's immune cells can be used.Preferably, the vaccine is a protein antigen / adjuvant vaccine, a peptide vaccine, an RNA vaccine, a DNA vaccine, a vector vaccine or a dendritic cell vaccine.Preferably, the vector vaccine is a viral vector vaccine.

[0061] Peptide vaccines can be used in which selected antigenic peptides are included in the vaccine and presented on antigen-presenting cells to elicit an immune response. Peptide vaccines are often co-administered with an adjuvant to prime a local immune response. The peptides are targeted to CD8 + and / or CD4 + It is necessary to stimulate specific T cells, and therefore most peptide vaccines contain nested CD8 T cells that contain the immunogenic portion. + T cells and / or CD4 + A peptide of about 8-30 amino acids in length containing a T cell epitope is used. The immunogenic portion is a peptide that MHC1 binds to, and such immunogenic peptides will typically be 8, 9 or 10 amino acids in length, with 9 amino acids being the most common. Preferably, a multi-peptide vaccine is used so that multiple antigens can be targeted simultaneously to generate a polyclonal antigen T cell response, enhancing the immune response and mitigating any antigen deficiencies on the tumor cells.

[0062] The advantage of peptide vaccines is that they can be chemically synthesized and produced on a large scale and at reduced cost compared to other cancer treatments.

[0063] Administration of free adjuvants with peptide antigens in cancer vaccines may cause them to dissociate after injection.One option to deal with this is to link adjuvants and peptide antigens to each other, for example, by direct conjugation.Peptides can be linked to hydrophobic carriers such as lipids, fatty acids and TLR agonists for more efficient delivery to antigen-presenting cells.

[0064] In certain embodiments of the invention, one or more peptides of the invention are conjugated to an adjuvant.

[0065] Nucleic acid-based vaccines are designed to enter cells and transcribe, translate and process antigenic epitopes (eg, peptides of the invention) so that they can be presented to MHC and elicit a host immune response.

[0066] DNA vaccines are simple to design, relatively inexpensive to produce, and have reasonable stability (2-8°C) and solubility. Plasmid DNA vaccines can be designed to function as both antigens and adjuvants, and unmethylated DNA containing CG-rich regions can also function as adjuvants. Plasmid DNA can be designed to be taken up into antigen-presenting cells where it is transcribed, translated, and processed with an epitope (e.g., an immunogenic peptide of the invention) that is presented on the cell surface in combination with MHC.

[0067] RNA vaccines have the advantage that they are not incorporated into the host cell genome, thus avoiding the safety concerns associated with such integration. RNA vaccines are easy to design, can code multiple epitopes, and because they are single-stranded, they also stimulate TLP7 and 8, thus having adjuvant function. RNA vaccines also only need to be delivered to the cytoplasm for translation into protein, unlike DNA, which needs to be delivered to the nucleus for transcription. However, RNA vaccines are much more susceptible to nuclease (e.g., RNase) degradation, and therefore more vulnerable than DNA vectors.

[0068] Viral vectors have been designed and made from a number of viruses. The most commonly used viral vectors are derived from adenoviruses, poxviruses and alphaviruses. The majority are replication-defective or attenuated versions of these. The drawback of viral vectors is that they are recognized as foreign by the immune system, and therefore repeated immunization with the same or similar vectors may result in the immune system neutralizing these vectors, thus weakening their effectiveness and preventing effective repeated administration.

[0069] Suitably, the viral vector is derived from a virus such as an adenovirus, adeno-associated virus (AAV), herpes virus, pox virus, alphavirus (such as Semliki Forest virus, Sindbis virus, or Venezuelan equine encephalitis virus), arenavirus (such as Lassa fever virus, Machupo virus, or Junin virus), measles virus, vaccinia virus, retrovirus (including lentivirus), or influenza virus.

[0070] Some are double-stranded DNA viruses (such as adenoviruses); some are single-stranded RNA viruses (such as flaviviruses and alphaviruses), which require reverse transcription of the genome into DNA followed by expression of the encoded polypeptide.

[0071] ChAdOx1 vector is an example of a suitable adenoviral vector.More recently, this vector has been used as a platform for COVID-19 vaccine known as ChAdOx1 nCoV-19.Other suitable adenoviral vectors that can be utilized in the present invention include, but are not limited to, Ad5-S-nb2 and Ad26-S.

[0072] For a review of viral vectors for therapeutic use, including vaccine applications, see Lundstrom (2020), in which Table 2 provides a list of preclinical and clinical cancer vaccines as of 2020 and identifies the specific vaccine vectors being utilized.

[0073] In certain embodiments, the vaccine vector for use in the present invention is selected from the group consisting of ChAdOx1, Ad5-S-nb2, Ad26-S, Ad5 / 35, SFV (Semliki Forest virus), AAV (adeno-associated virus), KUN, VSVΔG, HSV-1 T-VEC and VEE (Venezuelan equine encephalitis virus).

[0074] Cell-based vaccines can also be used. Dendritic cells are particularly suitable for cancer vaccines, given their ability to take up and present tumor-associated antigens (TAA) through various mechanisms and prime effector responses against tumor cells. Besides direct antigen presentation, DCs can migrate between lymphoid and non-lymphoid tissues and regulate cytokine and chemokine gradients to control inflammation and lymphocyte homing, all of which are important for systemic and long-term antitumor effects. Although different DC populations can be used, a large body of evidence suggests that conventional type 1 DCs (cDC1) play an essential role in tumor immunity and represent a promising alternative cell type for vaccination purposes.

[0075] DCs derived from patients can be exposed to the antigen peptides of the present invention ex vivo using established protocols and re-implanted into patients. Cells can be allogeneic or autologous. Cells that are properly pulsed with peptides should then be capable of antigen presentation in vivo.

[0076] When using a dendritic cell vaccine approach, dendritic cells can be exposed to multiple immunogenic peptides, and thus the vaccine can be a mixture of the immunogenic peptides of the invention.

[0077] One approach that is particularly suitable for viral vaccines is to include multiple immunogenic lncRNA gene-derived peptides in a chimeric polypeptide that contains these peptides in series, and present it as part of the vaccine.That is, in certain embodiments, the vaccine contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more peptides of the present invention.Optionally, especially when multiple peptides are included, the peptides are provided in a chimeric polypeptide that contains multiple peptides.These peptides can be in series and separated by one or more spacer amino acids and / or protease cleavage sites.

[0078] Suitably, the vaccine comprises at least 5 peptides, at least 10 peptides or at least 15 peptides of the invention.

[0079] According to a sixth aspect of the present invention, there is provided a method for producing a tumor-specific vaccine, comprising the steps of identifying tumor-expressed immunogenic peptides encoded by long non-coding RNA genes regulated by the PRMT5-E2F1 axis; and incorporating one or more of said peptides or nucleic acids encoding said one or more peptides into the vaccine.

[0080] lncRNA expression is tumor type specific, therefore the vaccine will act preferentially against tumors expressing the lncRNA from which the peptide was derived.

[0081] <Vaccine adjuvants> To enhance the adaptive immune response, vaccines can be administered with adjuvants that help attract immune cells to the injection site and can promote cell-mediated transport of antigens to the draining lymph nodes and the induction of antigen-presenting cells.

[0082] A large number of vaccine adjuvants have been developed and tested over the years. water-in-oil emulsions such as Montanide ISA-51 and Montanide ISA-720; pathogen-associated molecular pattern molecules (PAMPs); Toll-like receptor agonists such as polyinosinic-polycytidylic acid and carboxymethylcellulose with polylysine (Pol-ICLC, a TLR3 agonist), monophosphoryl lipid A (MPLA) (TLR4 agonist), imiquimod (TLR7 agonist), resiquimod (TLR7 and TLR8 agonists), and CpG oligodeoxynucleotides (CpG ODN) (TLR9 ​​agonist); CD40 agonists; stimulator of interferon genes protein (STING) agonists; and cytokines such as IL-2, IL12, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (for a review, see Paston et al., 2021).

[0083] The majority of cancer vaccines currently use TLP agonists as adjuvants.

[0084] Preferably, the peptide vaccine is directly fused to an adjuvant. Peptides can be linked to hydrophobic carriers such as lipids, fatty acids and TLR agonists for more efficient delivery to antigen-presenting cells.

[0085] In certain embodiments, the vaccines of the present invention are administered with a suitable adjuvant, such as those disclosed above.

[0086] One of skill in the art would be able to select an appropriate adjuvant for use with a selected vaccine.

[0087] <Vaccine delivery system> Delivery of nucleic acid-based vaccines into antigen-presenting cells (APCs) can be enhanced using electroporation, where a small electric pulse induces the formation of temporary pores in the cell membrane through which the nucleic acid can pass more easily. By causing some localized tissue damage, this stimulates nearby inflammatory cytokines and therefore also has an adjuvant effect.

[0088] Various nanoparticle-based delivery systems, such as polymeric nanoparticles, liposomes, micelles, carbon nanotubes, gold nanoparticles, mesoporous silica nanoparticles, and viral nanoparticles, have also been used for vaccine delivery. Liposome- and lipid-based nanoparticle formulations, such as DOTMA, DOTE, DOTAP, cholesterol, and Lipolex, are particularly popular.

[0089] Antigens for stimulating immune responses can also be delivered as part of self-assembling peptides, and these systems have certain advantages over liposomes or nanoparticles, including high drug loading, biodegradability, and relatively small size. Gene vectors based on cell membrane-penetrating peptides (CPPs), such as glycosaminoglycan (GAG) binding-enhanced transduction (GT) delivery systems, are one such example.

[0090] <Combination> Preferably, the vaccine of the present invention is administered in combination with another therapeutic agent. Cancer vaccines have the potential to induce a strong immune response, but tumor cells have various immune evasion mechanisms that prevent T cell function and recognition by T cells. Immune checkpoint inhibitors are cell surface receptors that regulate immune responses. In the TME, the expression of checkpoint receptors can suppress T cell activation and thus evade immune responses. Cytotoxic T-lymphocyte protein 4 (CTLA4) and programmed cell death protein 1 (PD-1) are the best characterized checkpoint receptors. Antibodies that specifically block CTLA-4 or PD-1 / PD-L1 pathways have the potential for T cell immune suppression, allowing successful recognition of tumor antigens and killing of tumor cells.

[0091] Therefore, checkpoint inhibitor molecules are particularly useful for combination with vaccines.Tumor microenvironment (TME) can cause its immunosuppression.The use of checkpoint inhibitors can reduce this TME-driven immunosuppression and maximize vaccine efficacy.

[0092] Many cancer vaccines currently undergoing clinical trials are combined with checkpoint inhibitors, such as CTLA-4, PD-1 or PDL-1 inhibitors. Exemplary checkpoint inhibitor molecules for use in combination with the vaccines of the present invention include those that block CTLA-4, such as ipilimumab, those that block PD-1, such as pembrolizumab or nivolumab, and those that block PDL-1, such as atezolizumab or durvalumab.

[0093] <Pharmaceutical Composition> According to a seventh aspect of the invention there is provided a pharmaceutical composition comprising a peptide according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, or a vaccine according to the fifth aspect of the invention and a pharma- ceutical acceptable excipient.

[0094] In this context, the peptide of the first aspect of the invention, the nucleic acid of the second aspect of the invention or the vaccine of the fifth aspect of the invention may be referred to as an "agent".

[0095] The term "pharmaceutical acceptable excipient" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to humans. The term "excipient" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate application. Suitable excipient types are salts, buffering agents, wetting agents, emulsifying agents, preservatives, compatible carriers, diluents, carriers, vehicles, adjuvants and supplementary immune enhancing agents such as cytokines, which are well known in the art and available from commercial sources for use in pharmaceutical preparations (see, for example, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition, Mack Publishing; Kibbe et al., (2000) Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press; and Ansel et al., (2004) Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins). Optionally, the pharmaceutical composition contains one or more other therapeutic agents or compounds. Suitable pharma- ceutically acceptable excipients are relatively inert and can facilitate delivery of the active compound / agent into a preparation that is optimized, for example, for stabilization, administration, processing or delivery to the body, and preferably directly to the site of action.

[0096] Pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules containing liquids, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.

[0097] When administered, the agents are administered in pharma- ceutically acceptable preparations / compositions.

[0098] Administration can be enteral (e.g., oral), i.e., the substance is given via the gastrointestinal tract, or parenteral, i.e., the substance is given by a route other than the digestive tract, such as by injection. Large biological or nucleic acid molecules (such as certain vaccines) are typically administered parenterally by injection.

[0099] Pharmaceutical compositions for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient / agent is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain one or more pharma- ceutically acceptable carriers, such as antioxidants, buffers, stabilizers, preservatives, suspending agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended patient. In certain embodiments, the composition may be lyophilized to provide a powder form that is easily reconstituted when and where needed. When reconstituted from a lyophilized powder, the aqueous liquid may be further diluted prior to administration. For example, diluted into an infusion bag containing 0.9% Sodium Chloride Injection, USP, or equivalent to achieve the desired dose for administration. In certain embodiments, such administration may be via intravenous infusion using an intravenous (IV) device.

[0100] Suitably, the drug is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the active drug for IV administration is in a solution, for example, in a sterile isotonic aqueous buffer solution. If necessary, the composition can also include a solubilizing agent. The composition for IV administration can optionally include a local anesthetic, such as lidocaine, to reduce pain at the injection site. Generally, the components are supplied either separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule. If the drug is to be administered by infusion, it can be dispensed, for example, using an infusion bottle containing sterile pharmaceutical grade water or saline. If the drug is to be administered by infusion, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0101] Compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs prepared by conventional means with pharma- ceutically acceptable excipients, such as, for example, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents, as appropriate. Oral compositions can include standard vehicles such as mannitol, lactol, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0102] The compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable excipients.That is, the drug and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or nose) or oral, parenteral or mucosal (buccal, vaginal, rectal, sublingual, etc.) administration.In certain embodiments, local or systemic parenteral administration is used.

[0103] Pharmaceutical compositions for use in the therapeutic methods of the present invention are for administration in an effective amount, which is that amount of the composition that alone, or together with further doses, produces the desired response.

[0104] Suitably, the agent may be administered as a pharmaceutical composition, the pharmaceutical composition comprising 0.1-1 mg, 1-10 mg, 10-50 mg, 50-100 mg, 100-500 mg, or 500 mg-5 g of agent.

[0105] The preparation of suitable pharmaceutical compositions of a drug and the dosages to administer to a subject are within the capabilities of one skilled in the art.

[0106] <Medical use> According to an eighth aspect of the invention there is provided a pharmaceutical composition of the seventh aspect of the invention or a peptide of the first aspect of the invention, or a nucleic acid of the second aspect of the invention, or a vaccine of the fifth aspect of the invention for use in therapy.

[0107] According to a variant of this eighth aspect of the invention there is provided a method of treating cancer in a subject suffering from cancer, the method comprising the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition of the seventh aspect of the invention or a peptide of the first aspect of the invention, or a nucleic acid of the second aspect of the invention, or a vaccine of the fifth aspect of the invention.

[0108] According to a variant of this eighth aspect of the invention, there is provided a peptide of the first aspect of the invention, or a nucleic acid of the second aspect of the invention, or a vaccine of the fifth aspect of the invention, for use in the manufacture of a medicament for therapy, such as for treating cancer.

[0109] In certain embodiments, the therapy is a cancer treatment.In certain embodiments, the cancer treatment comprises administering the pharmaceutical composition of the seventh aspect of the present invention or the peptide of the first aspect of the present invention, or the nucleic acid of the second aspect of the present invention, or the vaccine of the fifth aspect of the present invention, in combination with a PRMT5 inhibitor.

[0110] Any therapeutic agent capable of inhibiting PRMT5 can be utilized in this combination aspect of the present invention. In certain embodiments, the PRMT5 inhibitor can be selected from the group consisting of an antibody, an siRNA, an antisense oligonucleotide (ASO) or a small molecule compound.

[0111] The PRMT5 inhibitor for use in this aspect of the invention can be formulated as a pharmaceutical composition as described above for "medicament". The dosage, route of administration and actual treatment regimen can be determined by one skilled in the art. PRNT5 inhibitors are further described below.

[0112] Suitably, the PRMT5 inhibitor is a small molecule compound selected from the group consisting of GSK3326595 (pemrametostat), PF-6939999, JVNJ-64619178 (onametostat), LLY-283 and PRT543.

[0113] The components of the combination treatment can be administered in combination or in combination with each other.The combination treatment can be in the form of a combined preparation, for example, a combined preparation of the isolated peptide or vaccine of the present invention and a PRMT5 inhibitor.The combination can include separate formulations of each of the components, for example, separate formulations of the vaccine and the PRMT5 inhibitor.

[0114] The components in the combination (e.g., separate formulations) can be administered sequentially, separately and / or simultaneously, as described herein for combination treatment methods, i.e., for example, a vaccine of the invention can be administered separately, sequentially or simultaneously with a PRMT5 inhibitor.

[0115] In one embodiment, the components are administered simultaneously (optionally repeatedly). In one embodiment, the components are administered sequentially (optionally repeatedly). In one embodiment, the components are administered separately (optionally repeatedly).

[0116] One skilled in the art will appreciate that when separate formulations of the agents in a combination are administered sequentially or sequentially, this can be administration of the agents in either order.

[0117] When the administration of separate formulations is sequential or separate, the delay in the administration of the second (or subsequent) formulation should not cause the beneficial therapeutic effect of the combined treatment to be lost.Ideally, the two drugs will be present in the subject being treated at substantially the same time.Preferably, the two (or more) drugs will be administered to the subject in the same treatment regimen.

[0118] <Cancer> The various aspects of the present invention that use cancer cell or target the method or use for treating cancer can be applied to any cancer.Preferably, cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, biliary tract cancer such as cystic duct cancer or bile duct cancer, brain cancer, pancreatic cancer, colon cancer, colorectal cancer and gynecological cancer including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer including intraepithelial neoplasia of the cervix. In a preferred embodiment, the cancer is a leukemia and can be selected from the group consisting of acute lymphoblastic leukemia, acute myelogenous leukaemia (also known as acute myeloid leukaemia or acute nonlymphocytic leukaemia), acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia (also known as chronic myeloid leukaemia, chronic myelocytic leukaemia or chronic granulocytic leukaemia), chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia. In a further preferred embodiment, the cancer is acute lymphoblastic leukaemia. In a preferred embodiment, the cancer is a lymphoma, and may be selected from the group consisting of Hodgkin's lymphoma; non-Hodgkin's lymphoma; Burkitt's lymphoma; and small lymphocytic lymphoma.

[0119] In certain embodiments, the methods and uses disclosed herein provide precision medical approaches, such as those targeting specific types of tumors, or subsets of patients with specific tumors, or specific stages of tumors, or even individual patients.

[0120] Suitably, such treatment of cancer can achieve effective treatment of cancer by preventing or treating the onset of cancer, by preventing or treating the progression of cancer, by preventing or treating the recurrence of cancer, or by preventing or treating the spread (including metastasis) of cancer.

[0121] Preferably, the treatment induces the host (the subject to whom the treatment is administered) to mount an immune response against the cancer cells, thereby slowing their growth or killing them. The immune response can be humoral or cell-mediated.

[0122] <Vaccine design and synthesis> According to a ninth aspect of the present invention, there is provided a method for selecting peptides for inclusion in a tumor vaccine, comprising contacting a tumor cell sample with a PRMT5 inhibitor and determining the expression level of one or more peptides encoded by lncRNA genes by the contacted (treated) cells and selecting one or more deregulated peptides for inclusion in the tumor vaccine. Preferably, the tumor cell sample is obtained or has been obtained from a patient with cancer / tumor. Deregulated means upregulated or downregulated compared to expression in the absence of contact with the PRMT5 inhibitor. Preferably, there is a deviation of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% from the expression level in the absence of contact with the PRMT5 inhibitor. In certain embodiments, the expression level (e.g., transcript level) is upregulated, such as by at least 30%.

[0123] Optionally, the deregulated transcripts are tested to see whether they encode immunogenic peptides, e.g., whether the encoded peptides are presented or likely to be presented on the cell surface via major histocompatibility complex (MHC), e.g., MHC class 1. Such testing can include the use of immunopeptidomic analysis approaches, such as those described in the Examples. Alternatively, predictions can be made of whether peptides bind to MHC class 1 molecules (e.g., HLA class I peptide predictions using the NetMHC4.0 online algorithm).

[0124] Suitably, peptides can be predicted to be immunogenic using one or more in silico algorithms, such as the NetMHC4.0 online algorithm or EpiQuest-B.

[0125] Peptides can be determined to be immunogenic in various ways, including injection into host animals and detection of antibodies, or by immunopeptidomic analysis, for example, by immunopeptidomic mass spectrometry (MS) technology used in the examples. Immunogenic peptides induced after contacting tumor cells with PRMT5 inhibitors will bind to MNC class I molecules. MHC-bound peptides can be determined by immunoprecipitation using antibodies specific for MHC class I alleles, followed by mass spectrometry.

[0126] In embodiments, the method includes determining the expression levels of peptides encoded by lncRNA genes from multiple sources of a single tumor type to select appropriate peptides for inclusion in a vaccine that can be used for any patient with that type of cancer.

[0127] The peptides identified as being differentially expressed when cells are contacted with PRMT5 inhibitors can be tested to determine whether they are presented or likely to be presented on the cell surface via MHC (such as MHC class I).Predicted to be presented means that they are predicted to be presented using a suitable algorithm (such as NetMHC4.0).The peptides predicted or demonstrated to be immunogenic can be selected for inclusion in vaccines.

[0128] Thus, in a variant of the ninth aspect of the present invention, a method for selecting peptides for inclusion in a tumor vaccine is provided, comprising contacting a cell of a tumor type with a PRMT5 inhibitor, determining the expression level of one or more peptides encoded by lncRNA genes by the contacted cell, and selecting one or more peptides that are deregulated, preferably upregulated, for inclusion in a tumor vaccine. The peptides can be any of those in the first aspect of the present invention. Optionally, the peptides that are determined to be deregulated are evaluated to determine whether they are presented or prone to be presented on the cell surface via MHC, and thus immunogenic. Those that are presented or prone to be presented on the cell surface via MHC, such as MHC class I, are selected for inclusion in a tumor vaccine.

[0129]

[0023] In another variation of the ninth aspect of the invention, there is provided a method for selecting one or more peptides for inclusion in a tumor vaccine, comprising the steps of: (i) contacting a tumor cell sample with a PRMT5 inhibitor and identifying lncRNA transcripts that are differentially upregulated and downregulated following contact with the PRMT5 inhibitor; (ii) determining whether the differentially regulated lncRNA gene transcripts identified in step (i) encode immunogenic peptides; (iii) selecting one or more peptides determined to be immunogenic in step (ii) for inclusion in a tumor vaccine. A method is provided that includes.

[0130] In another variation of the ninth aspect of the present invention, a method for selecting one or more peptides for inclusion in a tumor vaccine, (i) contacting a tumor cell sample with a PRMT5 inhibitor; (ii) preparing a tumor cell lysate from the PRMT5 - contacted tumor cell sample of (i); (iii) contacting the tumor cell lysate with an MHC class I - specific antibody or an antibody fragment thereof bound to a resin on a column; and (iv) selecting one or more peptide - bound peptides for inclusion in a tumor vaccine A method is provided that includes.

[0131] Preferably, the bound peptide in step (ii) is eluted from the column and subsequently analyzed and identified by mass spectrometry. After this method, a vaccine containing the selected peptide is produced.

[0132] That is, according to the tenth aspect of the present invention, a method for manufacturing a vaccine, including the step of selecting a peptide for inclusion in a vaccine according to the ninth aspect of the present invention and the step of generating a vaccine capable of presenting said peptide is provided.

[0133] One of ordinary skill in the art will understand that there are numerous suitable examples of tumor cell samples that can be utilized. Preferably, such samples can include cells from a cancer or pre - cancerous condition. Suitable biological samples can be tissue samples such as samples from a biopsy or surgical resection, or biological fluid samples containing tumor cells such as blood, plasma, serum, sputum, saliva, pleural effusion, ascites, urine, etc. The sample can be fresh, frozen, or paraffin - embedded.

[0134] <PRMT5 inhibitor treatment> Protein arginine methyltransferase (PRMT) 5 (PRMT5) is responsible for arginine monomethylation and symmetric dimethylation, and its expression level and methyltransferase activity have been demonstrated to have a close relationship with the tumorigenesis, development and poor clinical outcomes of human cancers. PRMT5 is overexpressed in a wide range of cancers, implying an important oncogenic role. E2F1 is a key target of PRMT5, and the methylation marks expand the gene network under E2F1 control.

[0135] We found that pharmacological inhibition of PRMT5 altered lncRNA gene expression and, consequently, antigen presentation by tumor cells. The delayed tumor growth evident upon PRMT5 inhibition reflected the influx of lncRNA-derived peptide-specific cytotoxic CD8 T cells and helper CD4 T lymphocytes into the tumor microenvironment.

[0136] According to an eleventh aspect of the present invention there is provided a PRMT5 inhibitor for use in the treatment of cancer by stimulating the immune response.

[0137] Thus, administration of a PRMT5 inhibitor induced a host immune response against tumor cells, representing a novel mechanism of action and a novel clinical approach for treating cancer.

[0138] The immune response may be a humoral response and / or a cell-mediated response, such as an adaptive cell-mediated response. Suitably, when a PRMT5 inhibitor is administered to a subject, it is directed against CD8 + The subject is stimulated for the production of T cells and helper CD4 T lymphocytes.

[0139] Any therapeutic agent capable of inhibiting PRMT5 can be utilized in this tenth aspect of the invention. In certain embodiments, the PRMT5 inhibitor can be selected from the group consisting of an antibody, an siRNA, an antisense oligonucleotide (ASO) or a small molecule compound.

[0140] Suitably, the PRMT5 inhibitor is a small molecule compound selected from the group consisting of GSK3326595 (pemurametostat), PF-6939999, JVNJ-64619178 (onamettostat), LLY-283 and PRT543.

[0141] PRMT5 inhibitor compounds with different chemophores are known. DeFreitas et al. (2019) review some of the PRMT5 inhibitors, present their structures, and outline their mechanism of action.

[0142] The following table lists some of the patent publications filed by various pharmaceutical companies and other publications that are directed to PRMT5 inhibitors that may be used in the present invention.

[0143] JPEG2024543184000003.jpg250170JPEG2024543184000004.jpg118170

[0144] Compound 208 in WO2014 / 100719 (Epizyme) is GSK3326595 (pemurametostat).

[0145] The compound of Example 2 in WO2016 / 178870 (Eli Lilly) is LLY-283.

[0146] Compound 80 in WO2017 / 032840 (Janssen Pharmaceuticals) is JNJ-64619178.

[0147] See also De Freitas et al. (2019).

[0148] Other suitable PRMT5 inhibitors include: (1) WO2018 / 167269 (Argonaut Therapeutics Limited), which discloses a compound of formula I, or a salt, solvate or hydrate thereof; [ka] (In the formula, R1, R3, R4, R5 and R6 are each independently hydrogen or C 1-3 alkyl; R2 is hydrogen and R 14 Selected from; X is O or NR9, where R9 is hydrogen or C 1-3 is alkyl; Y1 is a group selected from one of formulae A and B: [ka] where R'" is independently H and C 1-3 alkyl; Q is C or N; T is selected from fused phenyl groups and fused 5- or 6-membered heteroaryl groups, each of which is selected from halo and C 1-3 optionally substituted with one or more substituents selected from alkyl; R7 and R8 together with the intervening nitrogen atom form a 3- to 12-membered heterocycloalkyl ring, wherein the 3- to 12-membered heterocycloalkyl ring is optionally joined by one or more R 10 and / or one or more C 6-12 Aryl, C 5-12 Heteroaryl, C 3-8 Optionally fused to a cycloalkyl and a 3- to 12-membered heterocycloalkyl ring, wherein each fused C 6-12 Aryl, C 5-12 Heteroaryl, C 3-8 Cycloalkyl and 3- to 12-membered heterocycloalkyl rings may be joined by one or more R 14 is optionally substituted by; R 10 is the formula L1-L2-R 11 or L2-L1-R 11 wherein L1 is selected from the group of formula -[CR12 R 13 ] n -, where n is an integer from 0 to 3, and R 12 and R 13 is independently selected from H and C1-C2 alkyl, at each occurrence; In the formula, L2 is absent or is O, S, SO, SO2, N(R'), C(O), C(O)O, [O(CH2) r ] s , [(CH2) r O] s is a linker selected from OC(O), CH(OR'), C(O)N(R'), N(R')C(O), N(R')C(O)N(R'), SON(R') or N(R')SO, where R' and R" are each independently selected from hydrogen and C1-C2 alkyl, where r is 1 or 2 and s is 1-4; R 11 is hydrogen, CN, NO2, hydroxyl, =O, halogen, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Alkyl, OC 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-12 Aryl, C 5-12 Heteroaryl, 3-10 membered heterocycloalkyl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)R d , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d, -SR d , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NR e R d , -OS(=O)2NR e R d , and -S(=O)NR e R d wherein R 11 is C 3-6 Cycloalkyl, C 6-12 Aryl, C 5-12 heteroaryl and 3- to 10-membered heterocycloalkyl; 3-6 Cycloalkyl, C 6-12 Aryl, C 5-12 Heteroaryl and 3- to 10-membered heterocycloalkyl can be represented by one or more R 14 is optionally substituted by; Each R a and R b is hydrogen and C 1-6 independently selected from alkyl; Each R d is hydrogen, hydroxyl, halogen, CN, C 1-6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl, OC 1-6 Alkyl and C 6-11 aryl, wherein 1-6 Alkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl and C 3-6 Cycloalkyl is hydroxyl, =O, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC 1-6 optionally substituted with one or more groups selected from alkyl; Each R e is hydrogen, hydroxyl, halogen, CN, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl and OC 1-6 alkyl; or R e and R d When attached to the same atom, together with the atom to which they are attached, they are considered to be hydroxyl, =O, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC 1-6 forming a 3- to 7-membered heterocycloalkyl ring optionally substituted with one or more substituents selected from alkyl; and R 14 is halo, CN, NO2, hydroxyl, =O, halogen, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Alkyl, OC 1-6 Alkyl, C 3-6 Cycloalkyl, C 6-12 Aryl, 5-6 membered heteroaryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl C 6-12 Aryl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)Rd , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d , -SR d , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NR e R d , -OS(=O)2NR e R d , and -S(=O)NR e R d are independently selected from

[0149] (2) WO2018 / 167276 (Argonaut Therapeutics Limited), which discloses a compound of formula I, or a salt, solvate or hydrate thereof: [ka] (In the formula, Y 1 is a group selected from one of formulae A and B: [ka] X is O, S, CH, or NR 7 Selected from; X 1 is selected from C and N; Y is selected from fused aryl and fused heteroaryl groups, each of which may be selected from one or more R 11is optionally substituted by; n is 1 and L is -(CH2) p N(R a )C(O)-, -(CH2) p C(O)N(R a )-, -(CH2) p N(R a )S(O q )-, -(CH2) p S(O q )N(R a )-, -(CH2) p N(R b )C(O)N(R b )-, -(CH2) p N(R c )C(O)O-, and -(CH2) p O C (O) N (R c )--or n is 0 and L is R d (R e )NC(O)-, -R d (R e )NC(O)N(R b )-, R d (R e )NC(O)O-, R d (R e )NS(O q ) and R d (R e )N-; p is a number selected from 0, 1, 2 and 3; q is a number selected from 1 and 2; Z is one or more R 10 C optionally replaced by 6-11 Aryl, one or more R 10 Optionally replaced by (C 7-16 ) alkylaryl, one or more R 10 C optionally replaced by 3-11 Cycloalkyl, one or more R 10 Optionally replaced by (C 4-17 ) cycloalkylalkyl, one or more R 103 to 15 membered heterocycloalkyl optionally substituted with one or more R 10 4 to 21 membered alkylheterocycloalkyl optionally substituted with one or more R 10 and one or more R 10 selected from 6-21 membered alkylheteroaryl optionally substituted by; R 1 is hydrogen, halogen, -NR e R d , OR f , and one or more R 9 C optionally replaced by 1-6 alkyl; R 2 is hydrogen, halogen and one or more R 9 C optionally replaced by 1-6 alkyl; R 3 , R 4 , R 5 and R 6 is hydrogen, halogen and one or more R 9 C optionally replaced by 1-6 independently selected from alkyl; R 7 is hydrogen, hydroxyl, C 1-6 Alkyl, C 1-6 Haloalkyl, Phenyl and C 3-6 cycloalkyl, wherein said C 1-6 Alkyl, phenyl and C 3-6 Cycloalkyl is hydroxyl, halogen, =O, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC 1-6 optionally substituted with one or more substituents selected from alkyl; Each R 9 is hydrogen, hydroxyl, halogen, CN, C 1-6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl, OC 1-6 alkyl and phenyl, 1-6 Alkyl, phenyl, 3-7 membered heterocycloalkyl and C 3-6 Cycloalkyl is hydroxyl, =O, halogen, CN, NR a R b , C.O.R. a , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, phenyl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC 1- 6 alkyl; Each R 10 is hydrogen, hydroxyl, =O, halogen, CN, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 1-6 Alkyl, OC 1-6 Alkyl, C 3-6 Cycloalkyl, phenyl, 5-6 membered heteroaryl, 3-7 membered heterocycloalkyl, -C(=O)R d , -C(=O)OR d , -C(=O)NR e R d , -C(O)C(=O)R d , -NR e R d , -NR e C(=O)R d , -NR e C(=O)OR d , -NR e C(=O)NR e R d , -NR e S(=O)2R d , -NR e S(=O)2NR e R d , -OR d , -SRd , -OC(=O)R d , -OC(=O)NR e R d , -OC(=O)OR d , -S(=O)2R d , -S(=O)R d , -OS(=O)R d , -OS(=O)2R d , -OS(=O)2OR d , -S(=O)NR e R d , -OS(=O)2NR e R d , and -S(=O)NR e R d wherein said C 3-6 Cycloalkyl, C 1-6 Alkyl, phenyl, 5- to 6-membered heteroaryl and 3- to 7-membered heterocycloalkyl are not affected by hydroxyl, halogen, =O, CN, C 1-6 Haloalkyl, C 1-6 Haloalkoxy, C 3-6 Cycloalkyl, C 1-6 Alkyl and OC 1-6 optionally substituted with one or more groups selected from alkyl; R 11 is hydrogen, hydroxyl, halogen, CN, NR a R b , C 1-6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl, OC 1-6 alkyl and phenyl, wherein 1-6 Alkyl, phenyl, 3-7 membered heterocycloalkyl and C 3-6 Cycloalkyl is hydroxyl, =O, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC1-6 optionally substituted with one or more groups selected from alkyl; Each R a , R b and R c is hydrogen and C 1-6 independently selected from alkyl; Each R d is hydrogen, hydroxyl, halogen, CN, C 1-6 Haloalkyl, 3-7 membered heterocycloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl, OC 1-6 Alkyl and C 6-11 aryl, wherein 1-6 Alkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl and C 3-6 Cycloalkyl is hydroxyl, =O, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C 1-6 Alkyl and OC 1-6 optionally substituted with one or more groups selected from alkyl; Each R e is hydrogen, hydroxyl, halogen, CN, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkyl and OC 1-6 alkyl; or R e and R d When attached to the same atom, together with the atom to which they are attached, they are considered to be hydroxyl, =O, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 6-11 Aryl, 3-7 membered heterocycloalkyl, C1-6 Alkyl and OC 1-6 forming a 3- to 7-membered heterocycloalkyl ring optionally substituted with one or more substituents selected from alkyl; and R f is hydrogen as well as hydroxyl, halogen, CN, COR a , N.R. a R b , C 1-6 Haloalkyl, C 3-6 Cycloalkyl, phenyl, 3-7 membered heterocycloalkyl and OC 1-6 C optionally substituted with one or more substituents selected from alkyl 1-6 alkyl).

[0150] (3) GB2108383.7 (Argonaut Therapeutics Limited), which discloses a compound of formula I, or a deuterated form, salt, solvate, or hydrate thereof: [ka] (In the formula, R 1A is represented by formula (A1): [ka] Z is =O; T, together with the intervening carbon and nitrogen atoms (e.g., as shown in formula (A1)), is selected from a monocyclic 5- to 7-membered heterocycloalkyl group, a fused bicyclic 6- to 10-membered heterocycloalkyl group, and a bridged bicyclic 6- to 9-membered heterocycloalkyl group, wherein each of the monocyclic 5- to 7-membered heterocycloalkyl group, the fused bicyclic 6- to 10-membered heterocycloalkyl group, and the bridged bicyclic 6- to 9-membered heterocycloalkyl group is selected from one or more R S1 is optionally substituted by; R S1 is C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Alkoxy, C3-12 cycloalkyl, hydroxy, halo, CN, and nitro, wherein C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl and C 3-12 Cycloalkyl is one or more R S2 each optionally substituted by R S2 is selected from hydroxy, halo, CN and nitro.

[0151] Any of these PRMT5 compounds can be used in the present invention.

[0152] In certain embodiments, the PRMT5 inhibitor for use in the present invention is a small molecule compound selected from the group consisting of GSK3326595 (pemurametostat), PF-6939999, JVNJ-64619178 (onamettostat), LLY-283 and PRT543.

[0153] For example, effective anti-cancer treatment using chemotherapy or radiation therapy is hindered by the ability of cancer cells to develop resistance to these treatments. The finding that PRMT5 inhibitors can also stimulate host immune responses against tumor cells will allow the use of PRMT5 inhibitors to treat patients or patient groups whose cancers have developed resistance to the direct cell targeting action of PRMT5.

[0154] The following examples and accompanying figures serve to illustrate the present invention. These examples and figures are in no way intended to limit the scope of the invention, but rather as examples from which equivalents thereto will be recognized by those skilled in the art.

[0155] Unless otherwise clear from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention. EXAMPLES

[0156] Protein arginine methyltransferase (PRMT)5 is overexpressed in a wide range of cancers, implying an important oncogenic role. E2F1 is a key target of PRMT5, and methylation marks expand the gene network under E2F1 control. We show here that the PRMT5-E2F1 axis has an additional, unexpected role in controlling expression from the non-coding genome, where a large group of long non-coding (lnc) genes are direct transcriptional targets. Analysis of antigen presentation by tumor cells identified that a large number of MHC class I protein-associated peptides were derived from small open reading frames in lncRNA genes. Furthermore, pharmacological inhibition of PRMT5 and manipulation of E2F1 activity altered lncRNA gene expression and, consequently, antigen presentation by tumor cells. The delayed tumor growth evident upon PRMT5 inhibition reflected the influx of lncRNA-derived peptide-specific cytotoxic CD8 T cells into the tumor microenvironment. When presented to the immune system as an independent therapeutic vaccine, lncRNA-derived peptides were found to be immunogenic and, importantly, to drive strong antitumor immune responses. These results indicate that PRMT5 links the E2F pathway to the non-coding genome and antigenic landscape of tumor cells. Thus, pharmacological control of PRMT5 activity offers a therapeutic strategy to affect tumor cell immunogenicity.

[0157] Materials and Methods Cell line generation, culture, and compound treatment Human p53 - / -HCT116 E2F1 CRISPR and CAS9 control cells have been previously described (Barczak et al., 2020). Mouse CT26 cells were obtained from ATCC (CRL-2638). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, St. Louis, MO, US) supplemented with 10% fetal bovine serum (Labtech, Heathfield, UK) and 1% penicillin / streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA). All cell lines were tested for mycoplasma contamination before use. The selective PRMT5 inhibitor (T1-44) (synthesized by Argonaut Therapeutics Ltd, Oxford UK) has been previously described and characterized (Barczak et al., 2020) and was used at 1 μM final concentration for 48 h unless otherwise stated.

[0158] Plasmid / siRNA transfection Plasmid transfection was performed using GeneJuice transfection reagent (Novagen) for 48 hours according to the manufacturer's instructions. RNA interference was performed using 25nM siRNA for 72 hours using Oligofectamine transfection reagent (Invitrogen) according to the manufacturer's instructions. The sequences for siRNA are as follows: non-targeting control, 5'-AGCUGACCCUGAAGUUCUU-3' (SEQ ID NO: 195); E2F1 (human), 5'-CUCCUCGCAGAUCGUCAUCUU-3'; E2F1 (mouse) (catalog number: EMU075181, Merck) (SEQ ID NO: 196).

[0159] Immunoblots and antibodies For immunoblotting, cells were harvested in modified RIPA buffer (50 mM tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal CA-630 [v / v], 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM AEBSF, protease inhibitor cocktail) and incubated on ice for 30 min, followed by SDS-PAGE and transfer to nitrocellulose. Antibodies for β-actin (AC-74, Sigma-Aldrich), E2F1 (Cell Signalling, 3742S), symmetric dimethylarginine (SDMe) (Cell Signalling, 13222S), FLAG M2 (Sigma, F1804), GFP (D5.1; Cell Signalling, 2956S), and GAPDH (Bethyl Laboratories) were used in the immunoblotting.

[0160] RNA isolation and quantitative RT-PCR RNA was isolated from cells using TRIzol (Thermo Fisher Scientific) or Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer's instructions. One microgram of total RNA was used for complementary DNA (cDNA) synthesis. Reverse transcription using oligo(dT)20 primer (Invitrogen) was performed with SuperScriptIII reverse transcriptase (Invitrogen) as per the manufacturer's instructions. Quantitative PCR (qPCR) was then performed in triplicate using the indicated primer pairs and BrilliantIII SYBR Green qPCR Master Mix (Stratagene) on an AriaMx (Agilent) qPCR instrument. Results were expressed as the average fold change compared to the control treatment using the ΔΔCt method from three biological replicate samples. Glyceraldehyde phosphate dehydrogenase (GAPDH) primer set was used as an internal calibrator. Error bars represent SD unless otherwise indicated.

[0161] RNA sequencing WT E2F1, E2F1 Cr HCT116, and CT26 cells were treated with 1 μM concentration of PRMT5 inhibitor (T1-44) or DMSO as a negative control for 48 hours (HCT116) or 72 hours (CT26). Total RNA from WT E2F1, WT E2F1 T1-44, E2F1 Cr, E2F1 Cr T1-44, CT26, and CT26 T1-44 (triplicates) was isolated using Direct-zol RNA MiniPrep kit (Zymo Research) according to the manufacturer's instructions. Alternatively, RNA isolated from mouse tumors in situ was used for RNA-seq analysis. RNA sequencing was performed by BGI Genomics. Briefly, Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) was used for RNA sample quality control purposes (RNA concentration, RIN value, 28S / 18S, and fragment length distribution). mRNA was isolated from total RNA using the oligo(dT) method. Subsequently, mRNA was fragmented and first-strand / second-strand cDNA was synthesized. cDNA fragments were purified and split using EB buffer for end repair and single nucleotide A (adenine) addition. Then, adapters were ligated to the cDNA fragments. Those cDNA fragments with suitable sizes were selected for PCR amplification. Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used in the quantification and qualification of the libraries. RNA sequencing was performed using Illumina HiSeqPlatform, generating 5.12 Gb per sample.

[0162] Processing long noncoding RNA (lncRNA) expression data p53 treated with PRMT5 inhibitors or DMSO control - / - WT E2F1, p53 - / -FASTQ files for E2F1 Cr HCT116 and CT26 cells were generated from three biological replicates and trimmed to remove adapters and low quality bases using TrimGalore v.0.4.3 (http: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ).

[0163] lncRNA expression analysis was performed using kallisto (v.0.44.0) with k-mer length 31 and 100 bootstrap samples. GENCODE mouse lncRNA annotation version M22 and human lncRNA annotation version 34 were used as reference datasets to construct the kallisto index. Differential expression of lncRNAs was calculated using the sleuth package (v0.30.0). The log2 (fold change) of expression was calculated from putative count values ​​(provided by kallisto) averaged across all replicates for a given condition.

[0164] Significantly differentially expressed transcripts were identified using an FDR threshold (q value) of 0.05.

[0165] HCT116 p53 - / - and HCT116 p53 - / - The E2F1 Cr RNA-seq dataset has been deposited in Gene Expression Omnibus (GEO) under the accession code GSE142430. The CT26 and colon26 tumor sample RNA-seq datasets have been deposited in GEO under the accession code GSE181401.

[0166] Data processing for proteomics mass spectrometry (lncRNA-derived peptide database) The nucleotide sequences of all lncRNAs expressed at detectable levels in the inventors' HCT116 and CT26 RNA-seq datasets (mouse: GENCODE annotation; human: FANTOM5 and GENCODE annotation) were converted to peptide sequences using 3-frame translation. The peptide sequence data were divided into three groups according to the expression values of the corresponding lncRNAs: non-expression (TPM = 0); weak expression (0.5 < TPM < 1.0); expression (TPM > 1.0). The non-expression group was used as a decoy database for MS proteomics experiments (detailed below).

[0167] HLA class I immunoprecipitation Antibodies were obtained from hybridoma supernatants (ATCC® HB-95 and -79, respectively) using a standard purification procedure with Sepharose-Protein A beads (Expedeon). 0.5 mL / sample of Sepharose-Protein A beads (Expedeon) was incubated with 5 mg / sample of the W632 antibody (specific for HLA class I, for HCT116), or antibody clone 34.1.2s (recognizing H-2Kd, Dd, Ld, for CT26) for 30 minutes at room temperature. The resin was washed with 10 cv (column bed volume) of borate buffer (50 mM borate, 50 mM KCl, pH 8.0), and the antibody was cross-linked by adding 10 cv of 40 mM dimethyl pimelimidate in borate buffer (pH 8.3) for 30 minutes at room temperature. The reaction was stopped using 10 cv of ice-cold 0.2 M Tris, pH 8.0, followed by a wash step with 10 cv of 0.1 M citrate, pH 3.0 to remove any unbound antibody, and finally equilibrated with 10 cv of 50 mM Tris, pH 8.0.

[0168] The cell pellet was lysed in 3 mL of lysis buffer (1% IGEPAL630; 100 mM Tris, pH 8.0; 300 mM NaCl; complete protease inhibitor cocktail added, EDTA-free, Roche) by gentle agitation. Samples were incubated on ice for 45 min. Lysates were subsequently clarified by successive centrifugation steps at 500 g for 10 min and 20,000 g for 1 h at 4° C. Peptide-HLA class I complexes were captured on immunoresin by overnight incubation at 4° C. under gentle agitation. The lysate was then removed by gravity flow and the column was washed successively with 10 mL wash buffer 1 (0.005% IGEPAL, 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA), 10 mL wash buffer 2 (50 mM Tris pH 8.0, 150 mM NaCl), 10 mL wash buffer 3 (50 mM Tris pH 8.0, 450 mM NaCl) and 10 mL wash buffer 4 (50 mM Tris pH 8.0). Peptide-HLA complexes were eluted by the addition of 5 cv of 10% acetic acid.

[0169] HLA peptide purification strategy Samples were loaded onto an Ultimate3000 HPLC system (ThermoFisher Scientific) and peptides were separated from larger complex components using a monolithic column (4.6 × 50 mm ProSwift RP-1S, ThermoFisher Scientific) by applying a 10 min gradient of 2–35% buffer B (0.1% TFA in acetonitrile) at a flow rate of 1000 μL / min. Each sample was fractionated into 15 fractions, and alternating fractions containing HLA peptides but not β2-microglobulin were pooled into two final fractions. Samples were dried, resuspended in 20 μL of loading buffer (0.1% TFA, 1% ACN), and stored at -80 °C until MS analysis.

[0170] LC tandem mass spectrometry (LC-MS / MS) For HCT116 cell samples, HLA peptides were analyzed by either an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) or a Q Exactive HF-X mass spectrometer (Thermo Scientific). CT26 cell samples were measured on a Q Exactive HF-X (Thermo Scientific). Either mass spectrometer instrument was coupled to an Ultimate 3000 RSLCnano System supplemented with a PepMap C18 column, 2 μm particle size, 75 μm × 50 cm (Thermo Scientific). Peptides were eluted using a 60 min linear gradient of 3% to 25% acetonitrile in 5% DMSO, 0.1% formic acid in water at a flow rate of 250 nL / min and 40 °C, and introduced into the mass spectrometer using a nano EASY-Spray ion source (Thermo Scientific) at 2000 V. The ion transfer tube was set at 305 °C for both instruments.

[0171] For samples analyzed by Orbitrap Fusion Lumos, the resolution for full MS was set to 120,000 with an ACG target of 400,000 and a scan range of 300-1500 m / z. Precursor selection and isolation were performed using TopSpeed ​​with a cycle time of 2 seconds and a quadrupole isolation width of 1.2 amu. The MS resolution was set to 30,000 and peptide ions were accumulated with a maximum injection time of 120 ms with an AGC target of 300,000. Precursor ions were fragmented using higher energy collisional dissociation (HCD): collision energy was set to 28 for peptides with charge states of 2-4 and 32 for singly charged ions. For samples analyzed on the Q Exactive HFX, the full MS (320-1600 m / z scan range) resolution was set to 120,000 and an AGC target of 300,000. Peptide ions were separated with a 1.6 amu isolation width. MS2 resolution was set to 60,000 with an AGC target of 50,000, and collision energy was set to 28 for peptides with precursor ion fragmentation charge states of 2–4 and 25 for those with charge states of 1–4.

[0172] HLA binding prediction HLA class I peptide prediction was performed using the NetMHC4.0 online algorithm (http: / / www.cbs.dtu.dk / services / NetMHC / ) and Seq2Logo2.0 (http: / / www.cbs.dtu.dk / services / NetMHC / ) or WebLogo (https: / / weblogo.berkeley.edu / logo.cgi).

[0173] Chromatin immunoprecipitation (ChIP) E2F1 ChIP was performed as previously described (Carr et al., 2017) using 3 μg of the appropriate antibody (control rabbit IgG, anti-E2F1 [A300-766A, Bethyl Laboratories) and pre-blocked Protein A beads. Recovered DNA was purified and real-time PCR was performed in triplicate using BrilliantIII Ultra-Fast SYBRgreen QPCR master mix on an AriaMx QPCR instrument (Agilent) with primers flanking the proposed E2F sites in the gene promoter. DNA occupancy was investigated by calculating the enrichment percentage of input for both E2F1 ChIP and IgG control from triplicate biological replicates. In all cases, figures presented display SD unless otherwise stated. CDC6 and actin promoters were used as positive and negative controls for E2F1 occupancy, respectively.

[0174] Human and mouse lncRNA promoter analysis lncRNA gene promoter characterization was performed utilizing bioinformatics tools present in the UCSC genome browser (https: / / genome.ucsc.edu; GRCh37 / h19 assembly) and analyzing ChIP-seq data for E2F tracks from the ENCODE project (http: / / genome.ucsc.edu / ENCODE / ) for three cell lines (K562, MCF7, HeLa). The "Transcription factor ChIP-seq clusters from ENCODE3", "Transcription factor ChIP-seq clusters containing factorbook motifs from ENCODE", "Transcription factor ChIP-seq peaks from ENCODE3", "Transcription factor ChIP-seq uniform peaks from ENCODE3 analysis" and "Transcription factor binding sites by ChIP-seq from ENCODE / Stanford / Yale / USC / Harvard" track tools were used to display E2F1 ChIP-seq peaks or signals where appropriate. Genes were scored as potential E2F1 targets if ChIP-seq data was evident within a 1000bp wide region around an annotated promoter (annotated by GENCODE and FANTOM6). Using the GRCm38 / mm10 assembly for promoter characterization in mouse, mouse E2F1 ChIP-seq peak data were loaded as a custom track with data deposited in GEO (GSM288349). ChIP-seq peak candidates traversed a 1000bp wide region around the lncRNA transcription start site (GENCODE annotation).

[0175] Polysome profiling Cells were treated with 100 mg / mL cycloheximide for 10 min at 37° C., 1× trypsin-EDTA solution for 10 min, and washed twice with ice-cold 1× PBS containing 100 mg / mL cycloheximide. Polysome lysis buffer consisting of 20 mM TrisHCl pH 7.4, 5 mM MgCl2, 100 mM KCl, 100 μg / mL cycloheximide, 1% TritonX-100, 1× RNase inhibitor, and 1× protease inhibitor was used to resuspend cells, followed by incubation on ice for 30 min (with occasional inversion) and centrifugation at 12,000 g for 10 min at 4° C. Sucrose gradients were prepared in polypropylene 13.2 mL tubes (Beckman Coulter) using 10% and 50% sucrose solutions (sucrose diluted in polysome extraction buffer prepared without Triton X-100 and in RNase-free conditions). To allow for linearity, the gradients were left at 4 °C overnight. Cleared supernatants from lysed cells were loaded onto the 10–50% sucrose gradient (equal amounts of 300 μg RNA measured by Nanodrop) and centrifuged at 39,000 rpm (190,000 g) (SW40Ti rotor, Beckman Coulter OptimaXE) for 90 min at 4 °C. Twelve sucrose gradient fractions were separated by manual collection, absorbance was measured at 254 nm (NanoDrop (Thermo Fisher Scientific)), and polysome profiles were recorded.

[0176] lncRNA ORF cloning strategy For lncRNAs identified to generate peptides loaded onto MHC class I in HCT116 and CT26 cells, peptide sequences were first reverse translated back into the lncRNA transcripts. Potential open reading frames (ORFs) were then identified by translating the lncRNA transcripts in all three frames and highlighting the entire sequence contained between all ATG codons (encoding the initiating methionine) and the subsequent in-frame STOP codon. Any potential ORFs that would generate a polypeptide containing the identified MHC peptide were identified as sequences for cloning into a plasmid vector expressing a C-terminal FLAG tag (pSF-CMV-NEO-COOH-3xFLAG; OG629, OxGene). Primers were designed to amplify the ORFs (excluding the STOP codon) and 30 bp upstream sequences (to include any Kozak sequences present in the endogenous transcripts), and included restriction sites for NotI and XhoI / EcoRV where appropriate. PCR reactions were performed using Phusion High Fidelity DNA Polymerase (M0530S, New England Biolabs) and HCT116 or CT26 cell-derived cDNA (generated as described above for quantitative RT-PCR) as template. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and digested with NotI and XhoI / EcoRV (Promega) where appropriate. Digested products were gel purified using a QIAquick Gel Extraction Kit (Qiagen) and then ligated into the digested vector using T4 DNA ligase (New England Biolabs). All plasmids were sequenced to confirm correct cloning prior to use in transfection.

[0177] Functional Genomics Analysis-TCGA For the analysis of expression levels of peptide-coding lncRNA transcripts in human cancers, the Xena browser (University of California) was used (https: / / xena.ucsc.edu / ). The TCGA TARGET GTEx dataset, which contains transcript expression data from TCGA (cancer tissues; https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga) and Genotype-Tissue Expression (GTEx; healthy tissues; https: / / gtexportal.org / home / ) samples, was selected. For subsequent detailed analysis of microsatellite instability and stage determination, the TCGA-derived dataset was used. In addition, the Broad Institute Cancer Cell Line Encyclopedia (portals.broadinstitute.org>ccle) was used to analyze the expression of lncRNA genes in colorectal cancer cell lines. Heatmapper tool (http: / / heatmapper.ca / ) was used to generate heatmaps. For detailed survival analysis, the GEPIA2 tool (http: / / gepia2.cancer-pku.cn / #index) was used.

[0178] Colon26 mouse tumor model using T1-44 treatment All experiments and protocols were approved by the animal welfare organization and local authorities at Charles River Discovery Research Services Germany (where the experiments were performed) and were performed in accordance with all applicable international, national and local laws and guidelines. Sixteen female Balb / c mice (8 mice per control and treatment group) aged 6-8 weeks (Charles River Laboratories, Germany) were inoculated with 5 × 10 IgG in PBS in a total injection volume of 100 μL / mouse. 5 Colon26 cells were injected subcutaneously on one side of the mouse. The tumor volume ranged from 50 to 150 mm. 3Upon reaching 100 mg / kg, mice were assigned to treatment groups based on tumor volume, aiming for comparable group mean / median tumor volumes. Mice were treated daily by oral gavage with T1-44 at 100 mg / kg (dosing volume 10 mL / kg) using 0.5% Tween / PBS as vehicle within 24 hours of randomization. Body weight and caliper tumor volume [mm 3 Measurements were taken twice weekly on day 19 of the study or at 1000 mm in the case of tumor ulceration. 3 Individual mice were censored for weight loss of >70% of initial body weight or >70% of initial body weight. From each group, four snap-frozen tumors were collected for RNA isolation and four formalin-fixed samples were prepared for immunohistochemical staining.

[0179] Mice and vaccinations Groups of 8-week-old Balb / c mice (Charles River) were vaccinated iv with the peptide mix shown in Table 2 (50 μg / peptide with 30 μg polyIC and 25 μg anti-CD40 mAb (GenScript)). As a positive control, AH1 peptide (SPSYVYHQF; SEQ ID NO: 156) vaccination was used (Huang et al., 1996). Vehicle-only mice were vaccinated with DMSO / PBS+adjuvant as above. Mice were boosted iv 7 days later with the same formulation. Mice were sacrificed 7 days after boost and spleens removed (4 mice per group). All animals were kept in specific pathogen-free conditions in the Biomedical Services Building (University of Oxford). All procedures were carried out under UK Home Office license PPL PP3430109 in accordance with the UK Animals (Scientific Procedures) Act 1986. All procedures were carried out by trained and licensed individuals.

[0180] JPEG2024543184000011.jpg166170

[0181] ELIspot assay The day before sacrifice, Merk multiscreen 96-well filter plates (Merck) were incubated with primary antibody (anti-mouse INFy mAb clone AN18, Mabtech) diluted in sterile PBS (Gibco) at 4 °C. The following day, the antibody was removed and the plates were washed 4 times with PBS at 250 μL / well, followed by blocking with 200 μL / well of R10 (RPMI (Gibco) supplemented with 10% heat-inactivated FCS, non-essential amino acids, L-glutamine and penicillin / streptomycin (all from Sigma), 2 h at 37 °C). Mice were sacrificed, spleens removed, passed through a 40 μm cell strainer (Falcon) and the single cell suspension pelleted by centrifugation. Splenocytes were resuspended in 3 mL of ACK lysis buffer (Lonza) for 3–5 min to lyse red blood cells, followed by arrest with 20 mL of PBS, then centrifuged at 1500 rpm for 5 min at room temperature. The splenocyte pellet was resuspended in 5 mL of R10, counted, and diluted to 4 × 10 5The cell concentration was adjusted to 100 / mL. Blocking buffer was removed and replaced with 50 μL of cells stimulated with the individual peptide (50 μL of peptide at 15 μg / mL) for which the group was inoculated. Each peptide was tested in duplicate. Negative control wells contained DMSO only when cells were stimulated with concanavalin A as a positive control. Plates were incubated overnight (15-20 hours) in a 37°C (5% CO2) incubator. Cells and peptides were removed and wells were washed 7 times with sterile PBS. Secondary antibody (biotin-conjugated mouse anti-INFy, MabTech) diluted 1:2000 in assay diluent (AD) (25 mg / mL BSA in PBS) was added (50 μL / well) and incubated for 2 hours at room temperature. Plates were then washed 4 times with PBS, followed by the addition of 50 μL of Anti-Biotin-Alkaline Phosphotase (Mabtech) diluted 1:750 in AD and incubated for 2 h at room temperature. Plates were washed 4 times with PBS, followed by the addition of 50 μL BCP / NBT substrate to each well and allowed to develop for 5-10 min until spots were visible in the positive control wells. The reaction was stopped by rinsing the plates 3 times in DI water. The rubber bottom was removed and the membrane was rinsed on both sides with DI water and subsequently dried. Spots were quantified on an ELISPOT counter (AID, Germany).

[0182] Dendritic cell vaccine strategy in the Colon26 mouse tumor model All experiments and protocols were approved by the animal welfare organization and local authorities of WuXi AppTec (Hong Kong) Limited (where the experiments were performed) and were performed in accordance with all applicable international, national and local laws and guidelines. Sixteen female Balb / c mice (RRID:IMSR_CRL:547) aged 6-8 weeks (8 mice per group: control (unpulsed stimulated dendritic cells) and peptide-pulsed stimulated dendritic cells) (Vital River Laboratory Animal Technology Co., LTD) were inoculated with 3 × 10 dendritic cells in PBS in a total injection volume of 100 μL / mouse.5 Colon26 cells were injected subcutaneously on one side of the mouse. The tumor volume was 60–80 mm. 3 Upon reaching tumor volume 10, mice were assigned to treatment groups based on tumor volume to achieve equivalent group mean / median tumor volumes. 6 Mice were vaccinated intravenously with unpulsed or pulsed dendritic cells at 0.2 mL / cell. To prepare the vaccine, mouse bone marrow cells were harvested. The cells were then treated with GM-CSF (250 IU / mL) and IL-4 (5 IU / mL)-containing medium and incubated at 37°C in 5% CO2. On day 3, the medium was half-replaced. On day 6, the cells were treated with GM-CSF, IL-4 and LPS to mature dendritic cells. After 24 h of incubation, DC cells were harvested and phenotypes were analyzed by FACS (CD11c, CD80, CD86). Subsequently, 2 × 10 5 DC cells / mL were pulsed with peptide (tab.X) at 75 μg / mL (15 peptides, 5 μg / mL each) and incubated for 5 hours. After harvesting and washing the DC cells with medium, they were ready for injection. The second vaccination was performed on day 7. Body weight and tumor volume [mm 3 ] was performed twice weekly by caliper measurement. 3 Individual mice were censored for weight loss of >80% of initial body weight. From each group, formalin-fixed samples were prepared for immunohistochemical staining.

[0183] Immunohistochemical staining FFPE slides were washed with Histochoice (Sigma Aldrich) for 5 min, followed by two washes in 100% ethanol for 3 min, 70% ethanol for 3 min, and tap water for 5 min. Samples were then incubated with antigen retrieval solution (sodium citrate buffer or Tris / EDTA depending on the antibody used) for 20 min at 99°C in a water bath. After three washes with purified water, samples were incubated with freshly made 6% methanol / H202 for 15 min and washed in tap water. In the next step, slides were washed in 1% PBST for 5 min, blocked in blocking serum solution (Vectastatin ABC Kit) for 20 min, washed again in 1% PBST for 5 min, and incubated with primary antibodies SDMe (1:5000, Cell signaling), CD8 (1:11000, Abcam), CD4 (1:8000, Abcam), FoxP3 (1:4000, Cell signaling), CD163 (1:5000, Abcam) overnight at 4° C. The following day, slides were washed with 1% PBST for 5 min, followed by incubation with secondary antibodies (Vectastain ABC Kit) for 30 min at room temperature. In the next step, ABC solution (VECTASTAIN® ABC-HRP Kit, peroxidase, rabbit IgG, PK-4001) was added for 30 min, slides were washed in 1% PBST and incubated with DAB solution (Vector DAB) for 10 min. Slides were then washed in purified water and counterstained in hematoxylin (Sigma Aldrich). Results were analyzed using a Leica microscope and presented as semiquantitative data using ImageJ software (National Institutes of Health, USA).

[0184] statistical analysis Statistical analysis was performed using unpaired two-tailed Student's t-test and one-way ANOVA test with GraphPad Prism8 software (GraphPad Software). Data are presented as means with SE shown unless otherwise indicated. P values ​​of less than 0.05 were considered significant, and 95% confidence interval (CI) was used for p<0.05.* ), p<0.01 ( ** ), p<0.001 for ( *** ), and for p ≤ 0.0001 ( **** ) marked with an asterisk.

[0185] Example 1: Controlled expression of human lncRNA genes E2F1 and PRMT5 We investigated the effects of pharmacological inhibition of PRMT5 activity and CRISPR knockout (KO) of the E2F1 gene on the expression of lncRNA genes in HCT116 colorectal cancer (CRC) cells using the small molecule active site inhibitor T1-44 (Barczak et al., 2020). The RNA-seq dataset was mined for changes in lncRNA gene expression (>30% change), which revealed that lncRNA transcripts were differentially expressed between each condition. We identified lncRNAs that were either upregulated or downregulated (237 upregulated compared to 303 downregulated), with some overlap in lncRNA expression evident between the different conditions (Figure 1A and B). The differentially expressed lncRNAs represented approximately 3% of the total lncRNAs present at detectable levels within our RNA-seq dataset (18,377 total lncRNA transcripts). When the expression of a select group of lncRNA transcripts was measured individually by qPCR, a gene expression pattern was evident, with LNCOC1 and others increasing upon T1-44 treatment, whereas CERNA1, CCNT2-AS1, and UBL7-AS1 were decreased (Figure 1C). Similarly, lncRNA expression was affected by E2F1 activity; for example, LINC01128 and KCTD21-AS1 transcripts were expressed at relatively high levels in E2F1 KO cells (Figure 1C).

[0186] We selected a group of lncRNA genes to assess whether they were direct targets of E2F1 by examining annotated E2F1 ChIP-seq datasets (Dunham et al., 2012). Many of the lncRNA transcripts scored as differentially expressed upon manipulation of PRMT5 and E2F1 were derived from genes that had ChIP-seq reads in close proximity to the transcript start site (TSS) or within the body of the transcribed sequence. This information prompted us to design primers around the predicted E2F binding sites, which we then used in gene-specific ChIP experiments to confirm the association with E2F1. For example, E2F1 was chromatin-bound in the KCTD21-AS1, CERNA1, CCNT2-AS1, and UBL7-AS1 genes in WT E2F1-expressing HCT116 cells, whereas E2F1 KO cells showed no E2F1 enrichment as expected (Figure 1D). Treatment of cells with T1-44 had no significant effect on ChIP-bound E2F1 (Figure 1D).

[0187] We assessed the proportion of differentially expressed lncRNAs that harbor E2F-binding sites or are located in close proximity to established protein-coding E2F target genes at the genome-wide level.

[0188] Notably, the percentage of lncRNA genes differentially regulated at a statistically significant level (q<0.05) in the RNA-seq dataset that scored as potential direct E2F1 target genes (reads within 500bp of the TSS using ChIP-seq data from ENCODE) was 39.2%, and an additional 38.9% of lncRNA genes were located close to or within known E2F target genes at the genomic level (overlapping gene boundaries of E2F1 target genes on the same or opposite strands or contained within gene boundaries of E2F1 target genes on the same or opposite strands) but did not appear to be direct E2F targets themselves. 21.9% were non-E2F1 targets. Thus, the majority of lncRNA genes identified here have a close relationship with the E2F pathway.

[0189] [Example 2: E2F1, PRMT5 and the mouse genome] We pursued whole genome RNAseq analysis in mouse CRC CT26 cell line. RNAseq analysis was performed in in vitro grown CT26 cells treated with compound T1-44 compared to control treatment. We mined RNAseq dataset to evaluate the action of PRMT5 focusing on lncRNA transcripts. A set of lncRNAs was found to be significantly regulated (at 30% cutoff), with 109 up-regulated and 282 down-regulated upon PRMT5 inhibition compared to control treatment (Figure 2A). We confirmed that lncRNA transcripts were regulated when analyzed at single gene level; for example, Gm44148, Gm46565, Ptprv, Epb41l4aos and G630030J09Rik transcripts were differentially expressed upon PRMT5 manipulation (Figure 2B). Many of the mouse lncRNA genes were shown to be E2F1 targets according to E2F1 ChIP-seq data.Notably, 83.9% of the differentially expressed lncRNAs were derived from genes that contained E2F binding site ChIP-seq peaks close to their TSS, while an additional 8.9% of the lncRNA genes were located close to or within E2F target genes at the genomic level.We also evaluated expression in siE2F1-treated cells, where expression levels were often downregulated (Figure 2B), highlighting the regulation of mouse lncRNA genes by PRMT5 and E2F1.

[0190] Following the analysis of lncRNA gene expression in syngeneic colon26 tumors growing in vivo, we assessed the impact of T1-44 treatment. Treatment of tumor-bearing mice with T1-44 caused a significant delay in tumor growth:

[0191] The percentage of lncRNA genes that are differentially regulated at a statistically significant level (q<0.05) in the CT26 RNA-seq data that are scored as potential direct E2F1 target genes (reads within 500bp of the TSS using ChIP-seq data from GEO [GSM288349]) was 83.9%; however, 8.9% were found to be associated with other potential E2F1 target genes (overlapping gene boundaries of E2F1 target genes on the same or opposite strands or contained within gene boundaries of E2F1 target genes on the same or opposite strands) while not being direct targets themselves. Non-E2F1 targets represent 7.2%.

[0192] RNAseq performed on growing tumors compared to T1-44-treated tumors identified lncRNA transcripts that were differentially regulated (30% cutoff) between the two treatment conditions. At the single lncRNA gene level, 4930473A02Rik, Gm45441, Gm15156, Lncppara, Kcnmb4os1, Lncenc1 and Epb41l4aos transcripts were upregulated upon treatment with compound T1-44 (Figure 2C). Furthermore, some of the lncRNAs identified in CT26 RNA-seq, including Epb41l4aos, Gm44148 and Gm46565, followed the same trend in colon26 tumors. Therefore, we conclude that the PRMT5-E2F1 axis regulates lncRNA gene expression in mouse tumor cells, including tumors growing in situ, similar to that seen in human cancer cell lines.

[0193] Example 3: PRMT5 regulates immune responses in the tumor microenvironment (TME) Given the role of the TME in promoting effective antitumor responses, we tested whether the delayed tumor growth upon PRMT5 inhibition (Figure 2C) reflected events in the TME caused by deregulated gene expression. Inhibition of tumor growth upon T1-44 treatment was accompanied by reduced levels of SDMe marks in tumor biopsies (Figure 2E), thus confirming catalytic inhibition of PRMT5. Upon further investigation of the TME, we found that T1-44 treatment had a profound effect on infiltrating lymphocyte populations, most clearly evidenced by the influx of cytotoxic CD8 and a modest increase in helper CD4 T lymphocytes; the effect of T1-44 treatment on other relevant cell populations, such as tumor-associated macrophages, was minimal (Figure 2E). It is possible that the increased levels of CD8 T lymphocytes were due to an effect on the adaptive immune response, and since CD8 T lymphocytes primarily engage the MHC class I antigen complex via their T cell receptors, it is plausible that T1-44 treatment affected antigen presentation via the MHC class I protein complex.

[0194] To address whether the peptide antigen content of MHC class I complexes was altered upon treatment of CT26 cells with compound T1-44, we performed mass spectrometry (MS) immunopeptidomic analysis to assess the repertoire of peptides presented by MHC class I complexes in treated cells compared to untreated cells (Figure 3A and B). Results revealed a large group of MHC class I-bound peptides, many of which were derived from cellular proteins. Given the role of the PRMT5-E2F1 axis in regulating lncRNA gene expression, we set out to investigate whether lncRNAs contribute to the MHC class I peptide repertoire. We created an in-house proteomic database containing predicted transcripts from all three frames of lncRNA transcripts expressed at detectable levels in our CT26 RNA-seq dataset. Peptides detected from the immunopeptidomic analysis were then matched against either this in-house database or a standard proteomic database containing all screened mouse SwissProt protein entries (Figure 3A). In particular, we identified 382 peptides (with an average size of 9 residues) derived from lncRNA genes, representing 6.5% of the total peptides detected in the analysis (Figure 3B-D). The lncRNA-derived peptides predicted high affinity for mouse MHC class I alleles H-2-Kd, Dd, Ld, Qa1, and Qa2, displaying conserved residues necessary for effective MHC class I binding (Figure 3C). A selection of peptide sequences identified in immunopeptidomics was subsequently confirmed using mass spectrometry by comparison against synthetic peptide sequences. Most importantly, both qualitative and quantitative differences were evident within the population of lncRNA-derived peptides when data were compared between T1-44-treated and control cell peptides.

[0195] We investigated the role of PRMT5 and E2F1 in regulating peptide-encoding lncRNAs and found that many of them, such as Gm20621, 1110038B12Rik and 4933406J09Rik, were upregulated in CT26 cells grown in vitro and as tumors upon PRMT5 inhibition.Furthermore, the expression profiles of lncRNAs generally correlated with similar qualitative changes in peptides, for example, Gm37283 (peptide sequence HIFSLHHF; SEQ ID NO: 176) and Gm17173 (peptide sequence RLAQLQTTI; SEQ ID NO: 166) were upregulated and 4732463B04Rik (peptide sequence RGPLLEKLF; SEQ ID NO: 167) was downregulated upon T1-44 treatment. We also used siE2F1 silencing to evaluate the effect of E2F1 on the expression of these lncRNAs, and found that in the set tested, the lncRNAs were regulated in an E2F1-dependent manner.In fact, the majority of lncRNA genes encoding MHC class I-binding peptides were found to be E2F target genes by referring to ChIP-seq datasets; about 81% were direct E2F1 targets, and another 8% were associated with or overlapped with other E2F1 target genes.

[0196] MHC class I-associated peptides are usually generated from larger proteins that undergo proteolysis and are funneled into the lysosomal vesicle system (Rock et al., 2016). Although lncRNAs are generally considered to be non-coding (Hartford and Lal, 2020; Statello et al., 2021), since we identified peptides derived from lncRNA genes, we wanted to test whether they could code for proteins that could potentially be processed to deliver small peptides. For many of the lncRNAs that gave rise to peptides, we were able to identify the theoretical open reading frame (ORF) from which the peptide would have been derived (Figure 3E); the majority of the ORFs were small coding polypeptides with less than 100 residues, but another group of lncRNAs was evident that had larger ORFs. Furthermore, transcripts derived from lncRNA genes could be annotated with the transcribing polysome fraction of ribosomes, including, for example, Gm37494, Gm37283, and Gm17173 (Figure 3E). In some cases, such as Gm37283 and Gm47761, their annotation was enhanced upon PRMT5 inhibition (Figure 3Fiii), which in turn was accompanied by increased levels of the induced peptides (HIFSLHHF; SEQ ID NO: 176 and YYIPGLKGI; SEQ ID NO: 194, respectively).

[0197] We then tested whether lncRNA gene RNA could be directly translated into detectable protein, which we did by cloning predicted ORF cDNA into an expression vector tagged with FLAG epitope at the C-terminus. As an example, Gm29253 lncRNA had a theoretical ORF encoding a 26 kD polypeptide. The specific polypeptide derived from ectopic expression of Gm29253 ORF was detected in transfected cells by immunostaining and immunoblotting, with the predicted molecular weight for the predicted ORF. Therefore, we conclude that lncRNA that produces MHC class I-bound peptides can associate with ribosomes and be further translated into polypeptides, which are then processed to deliver peptides to MHC class I protein complexes.

[0198] Example 4: lncRNA-derived MHC class I-associated peptides in human tumor cells We performed a similar immunopeptidomic analysis for MHC class I-associated peptides in human HCT116 cells comparing T1-44-treated to untreated cells, again highlighting a significant set of peptides derived from human lncRNA genes (118 unique peptides identified from all in-house databases; Figure 4A-C); an additional larger set derived from cellular proteins was also evident. Individual lncRNA peptide sequences were confirmed by comparing mass spectrometry-derived peptide sequences to their synthetic peptide counterparts. Peptides were an average of nine residues in size, including predicted conserved residues required for human HLA MHC class I binding (Figure 4B and C), and were predicted to have high affinity for human HLA-A, -B, and -C allele MHC class I proteins (Figure 4B). Furthermore, quantitative analysis showed that 42% of the peptides (as identified from the lncRNA database using GENCODE annotation) were regulated upon T1-44 treatment with a change of 30% or more, 10% were upregulated, 32% were downregulated, and 58% were unchanged.

[0199] We measured the expression of human lncRNAs that generate MHC class I-bound peptides by qPCR and found that many were differentially expressed upon T1-44 treatment, and some of the expression changes reflected the similar relative changes seen in MHC class I-associated peptides.For example, the increased levels of peptides derived from HELLPAR (peptide sequence LSLSLSLQFS; SEQ ID NO: 127) and RP11-660L16.2 (peptide sequence RLATHIDGA; SEQ ID NO: 36) lncRNAs reflected increased lncRNA expression upon T1-44 treatment, while the decreased expression shown, including AC079135.1 (peptide sequence AEKPPGSVA; SEQ ID NO: 106), RP11-319G6.1 (peptide sequence EETYFHLF; SEQ ID NO: 20) and VPS9D1-AS1 (peptide sequence RLLQETHQA; SEQ ID NO: 38) lncRNAs, was coincident with the decreased levels of peptides bound to MHC class I antigens. The expression of many of the lncRNAs was also affected by E2F1, showing either increased (AC004943.2, PPM1F-AS1, AC018445.6) or decreased expression (C5orf34-AS1, RP11-319G6.1, AC079135.1) in E2F1 KO cell lines.

[0200] We confirmed that many of the peptide-encoding lncRNAs in human cancer cells represent E2F1 target genes. We used ChIPseq data to identify E2F1 binding sites, designed primers around these sites, and then confirmed the presence of chromatin-bound E2F1 by ChIP. We observed that E2F1 was enriched in the promoters of many of the lncRNA genes that produced peptides; about 39% and 77% of the lncRNAs from the GENCODE and FANTOM databases, respectively, appeared to be direct E2F1 target genes, while an additional 26% (GENCODE) and 14% (FANTOM) of the peptide-encoding lncRNA genes were associated with other E2F1 target genes, respectively. The effect of T1-44 treatment on E2F1 recruitment appeared to be mild.

[0201] We performed a similar analysis on ORFs in human lncRNAs expressed in HCT116 cells. The majority of lncRNA ORFs that produced MHC-binding peptides were less than 100 residues, produced polypeptides less than 12 kD, and weak translation initiation sequences were evident when comparing all ORFs. We tested whether lncRNAs could associate with the translating polysome fraction of ribosomes and found that many lncRNAs, such as MALAT1, AC079135.1, and VPS9D1-AS1, could do so (Figure 4D); in some cases, such as MALAT1 and VPS9D1-AS1, showed a sedimentation shift upon PRMT5 inhibition and E2F1 KO conditions (Figure 4D).

[0202] We then addressed whether human lncRNA transcripts could be directly translated into proteins by cloning the predicted ORFs as cDNAs into expression vectors. For the two examples tested, MALAT1 and A0079135.1, small proteins derived from the ORFs containing MHC-associated peptides could be detected by immunostaining and immunoblotting (Figure 4E and Figure 4F). In addition, the expression of these lncRNA polypeptides was induced after treatment with PRMT5 inhibitors, while the expression of GFP from a control plasmid was not affected, suggesting that the translation of lncRNA-derived polypeptides could be further regulated by PRMT5 (Figure 4F).

[0203] Example 5: Relevance to human cancer It is noteworthy that some of the PRMT5-E2F1-responsive lncRNAs that encode MHC-binding peptides, such as MALAT1 and DANCR, are already known to exhibit deregulated expression in human cancers (Zhao et al., 2021). Therefore, we evaluated some of the peptide-encoding lncRNAs (identified here) that are not well characterized for their expression patterns in malignant diseases. As part of these exercises, we confirmed MALAT1 and DANCR expression across a range of cancer and normal tissues (Figure 5A). The expression patterns of other lncRNAs were variable; for example, VPS9D1-AS1 showed heterogeneous expression, with high expression in some cancers and generally low expression in normal tissues. This was in contrast to CTC-459F4, which had uniformly low expression in cancer and normal tissues.

[0204] In a detailed analysis of expression in CRC cell lines, lncRNA expression was detected across a range of tumor cell lines, with some lncRNA genes showing high expression and others lower expression across a panel of cell lines (Figure 5A). Interestingly, when lncRNA expression was analyzed in RNA prepared from human colorectal, gastric and esophageal cancers, a clear distinction was found between expression in the microsatellite stable (MSS) and microsatellite instability (MSI) subgroups; for example, in colorectal cancer, the majority of lncRNA expression occurred in the MSS subgroup but not in the MSI subgroup, which was relatively unremarkable in gastric and esophageal cancers (Figure 5A). Thus, in general, lncRNA expression is disease-specific.

[0205] It was of further interest to evaluate the prognostic significance of lncRNAs in human disease, in which we focused on adrenal cortical (AC) cancer, CRC and pancreatic cancer (PC), measuring expression levels across a collection of tumor biopsies and relating this information to survival. PRMT5 levels have prognostic significance, as high levels correlated with poor prognosis; E2F1 expression followed a similar trend (Fig. 5Bi). Some lncRNAs behaved similarly, with high expression correlated with poor prognosis (e.g., DANCR and LINC00094; Fig. 5Bi). However, this correlation was less clear in CRC and PC (Fig. 5Bii and Fig. 5Biii). In general, the prognostic value of lncRNA expression appears to be very good in AC, but highly variable in CRC and PC.

[0206] Example 6: Immunogenicity and tumor growth inhibition by lncRNA-derived peptides We tested whether lncRNA-derived MHC class I-binding peptides were immunogenic in mice. Twenty peptides encoded by mouse lncRNA genes and identified in immunopeptidomic analysis were selected for immunological analysis based on their predicted high affinity for MHC class I, low expression of their counterpart lncRNA genes in normal mouse tissues, and differential regulation upon PRMT5 inhibition (Figure 5C). A pool of five peptides was used to immunize mice, and splenocytes were harvested on day 14 for IFNγ ELISpot by restimulating cells from each mouse with the relevant pool of peptides. Among the peptides used to immunize mice, we identified 15 peptides that were immunogenic, which led to a significant increase in IFNγ-activated T cells compared to unimmunized mice (Figure 6A). We included a positive immunogenic control peptide (AH1) and several lncRNA-derived peptides that failed to show further immunogenicity (Figure 6A). These results provide evidence that lncRNA-derived MHC class I-bound peptides are immunogenic and stimulate effective T cell responses.

[0207] We were interested in testing whether T cell responses against lncRNA-derived peptides could be translated into therapeutic benefit, i.e., enhanced antitumor responses, when delivered in the context of a cancer vaccine. The approach we took to address this question was to use an ex vivo dendritic cell delivery platform. Bone marrow dendritic cells were harvested from mice, matured, and then pulsed with pooled immunogenic peptides. After 7 days, peptide-pulsed and control dendritic cells were transferred into Balb / c mice bearing established syngeneic colon26 tumors, and any effect on tumor growth was monitored. Remarkably, transfer of peptide-pulsed dendritic cells enhanced the antitumor response, which was reflected as delayed growth of colon26 tumors compared to control-treated dendritic cells (Figure 6B). In the context of dendritic cell delivery, lncRNA-derived peptides can impede the growth of colon26 tumors (from which the peptides were originally derived), thus providing a therapeutic benefit.

[0208] [Consideration] The classical view of the pRb-E2F pathway as a regulator of cell growth and division via G1-to-S-phase control has been significantly developed in recent years. It is now clear that E2F action is atypical and that a much broader repertoire of target genes is subject to E2F pathway control, including genes that affect alternative splicing rather than canonical transcriptional action (Roworth et al., 2019). By determining which gene control mechanism predominates, arginine methylation mediated by PRMT5, which targets a small R-rich region in E2F1, is the major influence on gene expression control by E2F1 (Zheng et al., 2013). Given the frequent overexpression of PRMT5 in cancer (Bedford and Clarke, 2009; Bedford and Richard, 2005; Jansson et al., 2008; Yang and Bedford, 2013), its role in regulating E2F activity is likely to be of great importance. In this study, we disclosed a set of unexpected observations that extend the influence of the PRMT5-E2F1 axis to the regulation of lncRNA genes and further to their role in immune recognition. The main findings of our study show that a large number of lncRNA genes, directly under E2F pathway control, encode small peptides that assemble with MHC class I proteins and are subsequently recognized by the immune system.

[0209] Human genome analysis has suggested that there are over 17,000 lncRNA genes (GENCODE v38) (Statello et al., 2021) with others implying approximately 28,000 human lncRNA genes (FANTOM5 database) (Hon et al., 2017). lncRNAs are typically 200 nucleotides long, and a small number have been described as being processed and spliced ​​in a manner similar to mRNAs (Statello et al., 2021). Whether lncRNAs are biologically important remains a subject of debate; some have been attributed to cellular functions, for example, in chromatin biology (Schlackow et al., 2017; Vos et al., 2018). Other studies have suggested that lncRNAs can affect RNA biogenesis (Statello et al., 2021). Furthermore, lncRNA expression has been associated with cancer. For example, MALAT1 is a highly conserved lncRNA that is abundantly expressed in cells and tissues and may play a role in regulating genes at both transcriptional and post-transcriptional levels (Engreitz et al., 2014; Yang et al., 2013). MALAT1 was initially identified as exhibiting elevated expression in metastatic lung cancer, and the same has been described in different solid or lymphoid tumors. In a mouse metastatic cancer model, MALAT1 deficiency resulted in a marked reduction in primary tumor differentiation and metastasis (Arun et al., 2016).

[0210] Through detailed immunopeptidomic analysis of the peptide composition of MHC class I proteins on cancer cells (human and mouse), we identified peptides derived from a significant proportion of lncRNA genes that were then shown to be genes regulated by the PRMT5-E2F1 axis. Many of these peptides were regulated upon pharmacological inhibition of PRMT5 activity, and were also E2F1 target genes, indicating that PRMT5 and E2F1 together regulate the immunogenicity of cancer cells and, therefore, the immune response to tumors. Interestingly, MALAT1 was one lncRNA that we found to encode peptides derived from larger polypeptides encoded by ORFs in the coding sequence. Using bioinformatics approaches, it is noteworthy that tumor-associated antigens can be derived from non-canonical parts of the genome, but the biological basis and therapeutic significance remain to be elucidated for the majority of parts (Chong et al., 2020; Laumont et al., 2018; Zhang et al., 2018). The results described herein firmly establish a novel and important role for lncRNA genes in contributing to the peptide composition of the MHC class I antigen presentation machinery.

[0211] lncRNA genes may be aberrantly expressed in cancer, exhibiting increased or decreased expression (Huarte, 2015). In the context of the results described herein, altered expression levels could be translated into quantitative differences in the spectrum of peptides presented by cancer cells to the immune system and, therefore, effects on immune recognition. These observations, in turn, reveal a cancer-related pathway regulated by PRMT5; i.e., the widely reported increased levels of PRMT5 in many human cancers through the control of lncRNA expression affect the immunogenicity of tumor cells by altering the peptide repertoire presented to the immune system. It is noteworthy that peptides derived from lncRNA genes are self-antigens, and therefore, in theory, T cells targeting such peptide antigens should be eliminated during development by central and peripheral tolerance mechanisms. However, we found that many of the peptides tested were able to drive active T cell responses, suggesting that any tolerance present to this class of lncRNA-derived peptides is partial or can be overridden. The fact that we obtained evidence of T cell responses to synthetic lncRNA-derived peptides led us to test whether this could be translated into an anti-cancer response against tumors. In this regard, we used the colon26 model, from which the peptides were originally identified and, importantly, naturally expressed and presented in the context of MHC class I proteins. Our results suggested that this was indeed the case: in the colon26 syngeneic mouse model, lncRNA-derived peptides delivered via ex vivo peptide-loaded dendritic cells could stimulate effective anti-cancer immune responses.

[0212] The results highlight lncRNA genes as an unexpected and abundant source of tumor-associated antigens controlled by the PRMT5-E2F1 axis and presented to the immune system via the classical pathway as MHC class I-associated antigens (Figure 6C). The ability to affect their expression through pharmacological manipulation of PRMT5, an enzyme with high cancer relevance itself, allows a novel approach to control the immunogenicity of tumor cells and further develop cancer vaccines that can ultimately be tailored to specific types of cancer. Our work also integrates the pRb-E2F pathway, a fundamental regulator of cell cycle progression and often deregulated in cancer, with antigen presentation and immune responses, thus establishing an interplay between cancer cell proliferation and the immune system.

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Claims

1. Peptides or derivatives thereof derived from long non-coding RNA genes regulated by the PRMT5-E2F1 axis.

2. The peptide according to claim 1, which is transcribed from an lncRNA gene sequence by a promoter containing an E2F binding site.

3. The peptide according to claim 1, which is immunogenic.

4. The peptide according to claim 1, which is regulated in cells when PRMT5 is inhibited.

5. The peptide according to claim 4, which is upregulated in cells when PRMT5 is inhibited.

6. The peptide according to claim 1, which is capable of inducing an immune response.

7. A nucleic acid encoding the peptide described in claim 1.

8. The nucleic acid according to claim 7, which is DNA, cDNA, PNA, RNA, or a combination thereof.

9. An expression vector capable of expressing the nucleic acid described in claim 7.

10. A host cell comprising the nucleic acid according to claim 7 or 8 or the expression vector according to claim 9.

11. A host cell according to claim 10, selected from the group consisting of bacteria, fungi (including yeast), and mammalian cells.

12. A vaccine comprising at least one peptide according to claim 1 or a nucleic acid encoding the peptide.

13. The vaccine according to claim 12, comprising at least five types of lncRNA gene peptides or nucleic acids encoding those peptides.

14. The vaccine according to claim 12 or 13, which is a peptide vaccine, RNA vaccine, DNA vaccine, viral vector vaccine, or dendritic cell vaccine.

15. The vaccine according to claim 14, wherein the viral vector is derived from an adenovirus, adeno-associated virus (AAV), herpesvirus, poxvirus, alphavirus (such as Semryki forest virus, Sindbis virus, or Venezuelan encephalitis virus), arenavirus (such as Lassa virus, Machupo virus, or Junin virus), measles virus, vaccinia virus, retrovirus (including lentivirus), or influenza virus.

16. The vaccine according to claim 15, wherein the vector is a monkey adenovirus vaccine such as a ChAdOx vector.

17. The vaccine according to claim 12, wherein the immunogenic peptide is presented by a human leukocyte antigen (HLA) class I molecule comprising one selected from the group including HLA A, HLA A2, HLA24, HLA Al, or HLA A3.

18. The vaccine according to claim 12, comprising a plurality of lncRNA-encoded peptides within a chimeric polypeptide that are overexpressed by cancer cells when PRMT5 is inhibited.

19. The vaccine according to claim 18, wherein the plurality of peptides encoded by lncRNA are linked together in series.

20. The vaccine according to claim 18 or 19, wherein the plurality of peptides are encoded by a single nucleic acid molecule.

21. The vaccine according to claim 12, which stimulates an immune response in a host administered the vaccine.

22. The vaccine according to claim 21, wherein the host immune response is a humoral response or an adaptive cell-mediated immune response.

23. The vaccine according to claim 12, which stimulates the production of anti-cancer CD-8 T cells when administered to a target.

24. A method for producing a tumor-specific vaccine, comprising the steps of: identifying a peptide expressed by a tumor, which is encoded by a long non-coding RNA gene regulated by the PRMT5-E2F1 axis; and incorporating one or more of the peptides or nucleic acids encoding the one or more peptides into the vaccine.

25. A pharmaceutical composition comprising the peptide according to claim 1, the nucleic acid according to claim 7, or the vaccine according to claim 12, and a pharmaceutically acceptable excipient.

26. A pharmaceutical composition according to claim 25 for use in treatment.

27. The pharmaceutical composition according to claim 25 for use in the treatment of cancer.

28. The pharmaceutical composition according to claim 25, for use in the treatment of cancer, in combination with a PRMT5 inhibitor.

29. A group of cancers is selected from the following: leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, biliary tract cancer such as cystic duct cancer or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer, and gynecological cancers including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer, and cervical cancer including cervical epithelial carcinoma. The above is selected at will. The pharmaceutical composition according to claim 25 for the treatment of cancer which is a leukemia selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia (also known as acute myeloid leukemia or acute non-lymphoblastic leukemia), acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia (also known as chronic myeloid leukemia, chronic myeloid leukemia or chronic granulocytic leukemia), chronic lymphocytic leukemia, monoblastic leukemia, and hairy cell leukemia.

30. A method for selecting peptides to be included in a tumor vaccine, comprising: (i) contacting a patient-derived tumor cell sample with a PRMT5 inhibitor; (ii) determining the expression level of one or more lncRNA gene-encoded peptides in the contacted cells; and (iii) selecting one or more upregulated or downregulated peptides to be included in a tumor vaccine.

31. A method for selecting peptides to be included in a tumor vaccine, comprising the steps of: contacting tumor-type cells with a PRMT5 inhibitor; determining the expression level of one or more peptides encoded by an lncRNA gene in the contacted cells; and selecting one or more peptides to be upregulated or downregulated to be included in the tumor vaccine.

32. The method according to claim 30 or 31, wherein, prior to step (iii), the peptide to be upregulated or downregulated is evaluated to determine whether it is presented or readily presented on the cell surface via MHCs such as MHC class I.

33. The tumors are selected from leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, Leydig cell tumor, biliary tract cancer such as cystic duct cancer or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer, and gynecological cancers including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer, and cervical cancer including cervical epithelial carcinoma, and are selected at will. The method according to claim 30 or 31, wherein the cancer is a leukemia selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia (also known as acute myeloid leukemia or acute non-lymphoblastic leukemia), acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia (also known as chronic myeloid leukemia, chronic myeloid leukemia or chronic granulocytic leukemia), chronic lymphocytic leukemia, monoblastic leukemia, and hairy cell leukemia.

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