Methods of generating one or more consensus cancer epitopes derived from alternative translation control

Abstract

The present invention relates to a method for generating or identifying one or more common cancer epitopes, and peptides comprising or consisting of the epitopes identified or produced by the method, expression vectors encoding the peptides, cytotoxic T lymphocytes (CTLs) produced in vitro by stimulating T cells with the peptides or vectors, CTLs of a subject treated with the peptides or vectors, and engineered T cells expressing T cell receptors that recognize the peptides. The invention also relates to the use of said peptide, expression vector, CTL or engineered T cell as a vaccine or medicament, in particular the use of said peptide, expression vector, CTL or engineered T cell for the prevention or treatment of at least one cancer in a subject in need thereof.

Description

Field of the Invention

[0001] The present invention relates to methods for generating or identifying one or more common cancer epitopes, and peptides comprising or consisting of the epitopes identified or generated by said methods, expression vectors encoding said peptides, cytotoxic T lymphocytes (CTLs) generated in vitro by stimulating T cells with said peptides or vectors, CTLs of a subject treated with said peptides or vectors, and engineered T cells expressing a T cell receptor that recognizes said peptides. The present invention also relates to the use of said peptides, expression vectors, CTLs or engineered T cells as vaccines or medicaments, in particular the use of said peptides, expression vectors, CTLs or engineered T cells for preventing or treating at least one cancer in a subject in need thereof. Background of the Invention

[0003] Adaptive T cell immune responses in cancer rely on the recognition of tumor epitopes specifically expressed by tumor cells. In the past decade, the role of neoantigens generated by non-synonymous mutations specific to the tumor genome has been extensively studied, and many clinical trials testing combinations of neoantigens in personalized cancer vaccines have been initiated and have yielded encouraging preliminary results. However, determining the optimal combination of neoepitopes for each patient remains challenging. In addition, many tumors are characterized by low or intermediate tumor mutation burdens. Therefore, uncovering other families of tumor antigens, such as those that may be shared among different patients with the same cancer or different cancer subtypes and are derived from alternative translational control, is crucial for the development of off-the-shelf tumor therapies.

[0004] In the present application, the applicant has developed a new method for identifying common cancer epitopes derived from alternative translational control. Specifically, the applicant has discovered new epitopes derived from the c-myc gene that can induce specific T cell responses and has demonstrated that the induced T cells are capable of recognizing and killing tumor cells. Summary of the Invention

[0005] The present invention relates to a method for generating one or more common cancer epitopes, wherein the method comprises the following steps:

[0006] (a) identifying peptides derived from non-canonical translation initiation and / or termination of a given gene (preferably an oncogene), and selecting one or several common cancer epitopes from the identified peptides,

[0007] (b) generating the epitopes selected in step (a).

[0008] The present invention also relates to a method for identifying one or more common cancer epitopes, wherein the method comprises the following steps:

[0009] (a) Identify peptides derived from non-canonical translation initiation and / or termination of a given gene (preferably an oncogene), and select one or several consensus cancer epitopes from the identified peptides.

[0010] In one embodiment, step (a) comprises the following steps:

[0011] (a1) Predict peptides derived from non-canonical translation initiation and / or termination of a given gene.

[0012] (a2) Among the predicted peptides identified in step (a1), identify 8-mer to 15-mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules.

[0013] (a3) From the sequences of the 8-mer to 15-mer peptides (or epitopes) identified in step (a2), identify peptides (or epitopes) found in healthy subjects and / or healthy tissues, and exclude said peptides (or epitopes), and

[0014] (a4) From the remaining peptides (or epitopes) of step (a3), select one or more epitopes found in at least one cancer.

[0015] In one embodiment, the gene is an oncogene, preferably an oncogene selected from the group consisting of or composed of c-myc and IGF1R, and preferably, the oncogene is c-myc.

[0016] In one embodiment, the cancer is a c-myc or IGF1R-related cancer, preferably, the cancer is breast cancer or colon cancer.

[0017] In one embodiment, the method further comprises in vitro validation of the selected epitopes after step (a).

[0018] In one embodiment, the in vitro validation comprises at least one, preferably three, of the following steps:

[0019] (i) Evaluate the induction of CD8+ T cell responses by the selected epitopes.

[0020] (ii) Evaluate the function of CD8+ T cells specific for the selected epitopes, and / or

[0021] (iii) Evaluate the cytotoxicity of CD8+ T cells specific for the selected epitopes against tumor cells and non-tumor cells.

[0022] Optionally, wherein the in vitro validation further comprises step (iv) evaluating the expression of the selected epitopes in tumor cells, preferably wherein the expression is evaluated by ribosome profiling or mass spectrometry.

[0023] The present invention also relates to an epitope identified or generated by the method as described above or a peptide consisting of such an epitope.

[0024] The present invention further relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), IMTASNWTL (SEQ ID NO:3), AMSPQLHNI (SEQ ID NO:4), GLAAPAPKL (SEQ ID NO:5), GLPPHPAHL (SEQ ID NO:6), GMPWPIPAV (SEQ ID NO:7), SLQETSYAL (SEQ ID NO:8), SLYPIACSL (SEQ ID NO:9), SVLGHDFSV (SEQ ID NO:10), VQDMIQTQV (SEQ ID NO:11), ILDDWLRHL (SEQ ID NO:12) and SLPSQHWSL (SEQ ID NO:13), preferably, the peptide comprises an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2) and IMTASNWTL (SEQ ID NO:3).

[0025] The present invention further relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), AMSPQLHNI (SEQ ID NO:4), GLAAPAPKL (SEQ ID NO:5), GLPPHPAHL (SEQ ID NO:6), GMPWPIPAV (SEQ ID NO:7), SLQETSYAL (SEQ ID NO:8), SLYPIACSL (SEQ ID NO:9), SVLGHDFSV (SEQ ID NO:10), VQDMIQTQV (SEQ ID NO:11), ILDDWLRHL (SEQ ID NO:12) and SLPSQHWS L (SEQ ID NO:13), preferably, the peptide comprises an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1) and SLTDLYLRI (SEQ ID NO:2).

[0026] The present invention also relates to an expression vector for inducing the expression of one or more of the peptides as described above.

[0027] The present invention also relates to cytotoxic T lymphocytes of a subject treated with one or more of the peptides as described above or one or more of the expression vectors as described above.

[0028] The present invention also relates to cytotoxic T lymphocytes generated in vitro by stimulating T cells with one or more of the peptides as described above or one or more of the expression vectors as described above.

[0029] The present invention also relates to engineered T cells expressing a T cell receptor that recognizes the peptides as described above.

[0030] The present invention also relates to one or more of the peptides as described above, one or more of the expression vectors as described above, one or more of the cytotoxic T lymphocytes as described above, or one or more of the engineered T cells as described above, for use as a vaccine or a medicament.

[0031] The present invention also relates to one or more of the peptides as described above, one or more of the expression vectors as described above, one or more of the cytotoxic T lymphocytes as described above, or one or more of the engineered T cells as described above, for treating or preventing at least one cancer in a subject in need thereof.

[0032] Definitions

[0033] In the present invention, the following terms have the following meanings:

[0034] “C-myc” refers to the proto-oncogene myc, which encodes a nuclear protein involved in nucleic acid metabolism and mediating the cellular response to growth factors. The truncation of the first exon, which appears to regulate c-myc expression, is crucial for tumorigenesis. The human c-myc gene is located at 8q24 on the long arm of chromosome 8.

[0035] “Epitope” refers to the antigenic portion capable of stimulating an immune response.

[0036] “Shared cancer epitope” refers to an epitope that is not specific to a particular subject. Shared cancer epitopes can be shared among different patients with the same cancer histology or among different patients with different cancer histologies.

[0037] “Frameshift” refers to a change in one or more bases in the open reading frame in the 5' or 3' direction during the translation process.

[0038] "Insulin-like growth factor 1 receptor" or "IGFR1" refers to a gene located on chromosome 15q26.3 that encodes a tyrosine kinase receptor with high binding affinity for insulin-like growth factors, which play a key role in the transduction events of cell growth and survival.

[0039] "Internal ribosome entry site" or "IRES" is a sequence that can recruit ribosomes and allow translation.

[0040] "Mass spectrometry" refers to an analytical method that uses a mass spectrometer to determine the identity of a chemical substance based on its mass.

[0041] "Non-canonical initiation and / or termination of translation of a gene" refers to non-conventional translation initiation and / or termination mechanisms that can be induced under stress conditions such as hypoxia, apoptosis, starvation, and viral infection. Conventional translation initiation mechanisms involve recruiting the 40S ribosome to the cap structure at the 5' end of the mRNA and then linearly scanning the 5'-UTR until the start codon is reached. Non-canonical mechanisms of gene translation initiation and / or termination include, but are not limited to, the following events: frameshifting, readthrough, translation of regions on the 5'UTR and / or 3'UTR that are not normally translated (e.g., initiation of translation upstream of the start codon, termination of translation downstream of the stop codon, and / or initiation of translation downstream of the stop codon in the coding sequence), and IRES-dependent translation initiation.

[0042] "Oncogene" refers to a gene whose gain-of-function alteration leads to the transformation of tumor cells. For example, they include genes that are activators or stimulators of cell proliferation, such as genes for growth factors, growth factor receptors, protein kinases, signal transducers, nucleophosmin, and transcription factors.

[0043] "Open reading frame" or "ORF" refers to a nucleotide triplet sequence that encodes the amino acids between the start codon and the stop codon located in the same reading frame.

[0044] "Peptide" refers to a linear amino acid polymer of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids joined together by peptide bonds. The amino acid residues in the peptide are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; glycine is Gly or G. The peptide may contain non-standard amino acids, which refer to amino acids that are chemically modified (referred to as "post-translational modification") after incorporation into proteins and amino acids that exist in living organisms but not in proteins. Post-translational modifications include, for example, phosphorylation and glycosylation of amino acids. Examples of such non-standard amino acids include, but are not limited to, selenocysteine, cystine, desmosine, isodesmosine, hydroxyproline and hydroxylysine, γ-carboxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine and inositol.

[0045] "Prevent (prevent, preventing, prevention)" refers to prophylactic and preventive measures aimed at reducing the likelihood that a subject will develop a pathological condition or disease within a specific time period. This reduction can be manifested, for example, by delaying the appearance of at least one symptom of the pathological condition or disease in the subject.

[0046] "Readthrough" refers to the process in translation where a stop codon is interpreted as a sense codon.

[0047] "Ribosome profiling" or "Ribo-seq" is a method based on the deep sequencing of ribosome-protected mRNA fragments (also known as "footprints"). Ribosome footprints typically show the precise positioning between the start codon and the stop codon of a gene, enabling global and experimental identification of genomic coding regions. It can also show the precise positioning of ribosomes on mRNA.

[0048] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject can be a "patient", i.e., a warm-blooded animal, more preferably a human, who is awaiting medical care, or is receiving medical care, or has been / is being / will be the subject of a medical procedure, or is being monitored for the development of a disease. The term "mammal" as used herein refers to any mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0049] "Therapeutically effective amount" refers to the level or amount of one or more peptides, one or more expression vectors, one or more CTLs or one or more engineered T cells herein, which is intended to (1) delay or prevent the onset of a disease, disorder or condition, (2) slow or arrest the progression, aggravation or worsening of one or more symptoms of a disease, disorder or condition, (3) improve the symptoms of a disease, disorder or condition, (4) reduce the severity or incidence of a disease, disorder or condition; or (5) cure a disease, disorder or condition without causing significant negative or adverse side effects to the target. The therapeutically effective amount can be administered before the occurrence of a disease, disorder or condition to achieve a preventive or inhibitory effect. Alternatively, or in addition, the therapeutically effective amount can also be administered after the occurrence of a disease, disorder or condition to achieve a therapeutic effect.

[0050] "Treating" or "alleviation" refers to a therapeutic treatment that is intended to slow down (reduce) a target pathological condition or disorder. If, after a subject or mammal has received a therapeutically effective amount of one or more peptides, one or more expression vectors, one or more cytotoxic T lymphocytes or one or more engineered T cells of the present invention, the patient exhibits one or more of the following observable and / or measurable decreases or disappearances, then the cancer of the subject or mammal has been successfully "treated": a decrease in the number of cancer cells (or tumor size); a decrease in the percentage of the total number of cancerous cells; and / or a certain degree of alleviation of one or more symptoms associated with a specific disease or condition; a decrease in morbidity and mortality, and an improvement in quality of life issues. The above parameters for evaluating the successful treatment and improvement of a disease can be easily measured by conventional procedures familiar to a doctor.

[0051] "Vaccine" refers to a compound that can induce a humoral and / or cellular immune response after being administered to a subject and this immune response is protective.

[0052] "Vector" or "expression vector" refers to a vector that can introduce a DNA or RNA sequence (such as a foreign gene) into a host cell, thereby transforming the host and promoting the expression (such as transcription and translation) of the introduced sequence.

[0053] "uORF" refers to a short coding sequence located upstream of the main open reading frame (mORF) and flanked by a start codon and a stop codon. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention relates to a method for generating one or more consensus cancer epitopes, wherein the method comprises the following steps:

[0056] (a) Identifying peptides derived from non-canonical translation initiation and / or termination of a given gene (preferably an oncogene), and selecting one or several consensus cancer epitopes from the identified peptides, and

[0057] (b) Generating the epitopes selected in step (a).

[0058] The present invention also relates to a method for identifying one or more consensus cancer epitopes, wherein the method comprises the following steps:

[0059] (a) Identifying peptides derived from non-canonical translation initiation and / or termination of a given gene (preferably an oncogene), and selecting one or several consensus cancer epitopes from the identified peptides.

[0060] In one embodiment, step (a) is completed by a computer method.

[0061] In one embodiment, step (a) comprises step (a1), which is to predict peptides derived from non-canonical translation initiation and / or termination of a given gene.

[0062] In one embodiment, step (a1) comprises predicting peptides derived from at least one non-canonical translation initiation and / or termination mechanism of a given gene.

[0063] Examples of non-canonical translation initiation and / or termination mechanisms of a given gene include but are not limited to frameshifting of the open reading frame, readthrough of the stop codon, translation of the 5'UTR and / or 3'UTR, or IRES-dependent translation initiation.

[0064] "Translation of the 5'UTR and / or 3'UTR" or "translation of the 3' and / or 5' region" refers to the translation of the 5'UTR and / or 3'UTR of a specific mRNA that is not normally translated in canonical translation. Translation of the 5'UTR refers to the start of translation beginning upstream of the start codon. Translation of the 3'UTR refers to the termination of translation stopping downstream of the stop codon and / or the start of translation occurring downstream of the stop codon of the coding sequence. Translation of the 5'UTR and / or 3'UTR can be complete or partial translation of the 5'UTR and / or 3'UTR.

[0065] In one embodiment, at least one non-canonical translation initiation and / or termination mechanism for a given gene is selected from:

[0066] - Frame-shift of the open reading frame,

[0067] - Read-through of the stop codon,

[0068] - Translation of the 5'UTR and / or 3'UTR,

[0069] - IRES-dependent translation initiation, and / or

[0070] - Upstream ORF (uORF).

[0071] In one embodiment, at least one non-canonical translation initiation and / or termination mechanism for a given gene is selected from:

[0072] - Frame-shift of the open reading frame,

[0073] - Read-through of the stop codon,

[0074] - Translation of the 5'UTR and / or 3'UTR, and / or

[0075] - IRES-dependent translation initiation.

[0076] In one embodiment, step (a1) includes predicting peptides derived from one, two, or three non-canonical translation initiation and / or termination mechanisms of a given gene.

[0077] In one embodiment, one, two, or three non-canonical translation initiation and / or termination mechanisms for a given gene are selected from:

[0078] - Frame-shift of the open reading frame,

[0079] - Read-through of the stop codon,

[0080] - Translation of the 5'UTR and / or 3'UTR, and / or

[0081] - IRES-dependent translation initiation.

[0082] In one embodiment, step (a1) includes predicting peptides derived from a given gene that result from the following four non-canonical translation initiation and / or termination mechanisms:

[0083] - Frame-shifting of the open reading frame,

[0084] - Read-through of stop codons,

[0085] - Translation of the 5'UTR and / or 3'UTR, and

[0086] - IRES-dependent translation initiation.

[0087] In one embodiment, step (a1) includes predicting peptides derived from a given gene that result from one, two, three, or four non-canonical translation initiation and / or termination mechanisms selected from:

[0088] - Frame-shifting of the open reading frame,

[0089] - Read-through of stop codons,

[0090] - Translation of the 5'UTR and / or 3'UTR, and

[0091] - IRES-dependent translation initiation, and / or

[0092] - Upstream open reading frame (uORF).

[0093] In one embodiment, step (a) includes step (a2), which involves identifying 8-mer to 15-mer peptides that bind to MHC class I molecules from the predicted peptides identified in step (a1). As used, step (a2) is capable of selecting 8-mer to 15-mer peptides that bind to MHC class I molecules, i.e., epitopes that bind to MHC class I molecules, from the peptides identified in step (a1).

[0094] Analytical tools for predicting the binding of a sequence (i.e., an epitope) to an MHC molecule are well known to those skilled in the art and include, for example, MHCflurry (T.J. O’Donnell et al., MHCflurry: Open-Source Class I MHC Binding Affinity Prediction. Cell Systems. 7, 129-132.e4 (2018)) or NetMHCPan (Reynisson et al., NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data, Nucleic Acids Res, 2020 Jul 2; 48(W1):W449-W454).

[0095] In one embodiment, the MHC class I molecule is an HLA molecule. In one embodiment, the MHC class I molecule is selected from the group consisting of or comprising: HLA-A, HLA-B, and HLA-C molecules. In one embodiment, the MHC class I molecule is an HLA-A molecule, such as the HLA-A2 molecule.

[0096] In one embodiment, the 8-mer to 15-mer peptides (or epitopes) identified in step (a2) bind strongly to an MHC class I molecule, preferably the HLA-A2 molecule.

[0097] In one embodiment, a percentile rank equal to or lower than 0.5% is used to select strongly binding peptides (or epitopes), based on the likelihood of the peptide being presented compared to the pool of natural ligands.

[0098] In one embodiment, the peptides (or epitopes) identified in step (a2) are 8-mer to 15-mer peptides, i.e., peptides comprising 8 to 15 amino acids.

[0099] In one embodiment, the peptides (or epitopes) identified in step (a2) are 9-mer to 10-mer peptides, i.e., peptides comprising 9 to 10 amino acids.

[0100] In one embodiment, the peptide (or epitope) identified in step (a2) is an 8-, 9-, 10-, 11-, 12-, 13-, 14- or 15-mer peptide, i.e., a peptide comprising 8, 9, 10, 11, 12, 13, 14 or 15 amino acids, respectively. In one embodiment, the peptide (or epitope) identified in step (a2) is a 9-mer peptide.

[0101] In one embodiment, step (a) includes step (a3), i.e., identifying peptides (or epitopes) found in healthy subjects (i.e., subjects without cancer) in the 8-mer to 15-mer peptide (or epitope) sequences identified in step (a2), and excluding said peptides. This step can in particular confirm that the selected peptides (or epitopes) do not match any self-proteins of subjects without cancer.

[0102] In one embodiment, step (a3) includes aligning the sequences of the 8- to 15-mer peptides (or epitopes) identified in step (a2) with the normal human proteome (i.e., the human proteome from healthy subjects), and excluding those peptides (or epitopes) that have complete sequence homology with the normal human proteome. The alignment can be performed using the BLAST protein database or the refseq_protein database.

[0103] In one embodiment, step (a3) includes comparing the sequences of the 8-mer to 15-mer peptides (or epitopes) identified in step (a2) with a normal tissue proteome database (i.e., a tissue proteome database from healthy tissue), and excluding those peptides (or epitopes) found in healthy / normal tissue. The data of the normal tissue proteome database can be found in public databases, e.g., in the Genotype-Tissue Expression (GTEx) database.

[0104] As used herein, healthy or normal tissue refers to tissue not affected by cancer.

[0105] In one embodiment, step (a3) includes at least one of the above steps (i.e., alignment with the human proteome or comparison with the tissue proteome database).

[0106] In one embodiment, step (a3) includes the above two steps. Thus, in one embodiment, step (a3) includes the following steps:

[0107] - Aligning the sequences of the 8-mer to 15-mer peptides (or epitopes) identified in step (a2) with the normal human proteome, and excluding those peptides (or epitopes) that have complete sequence homology with the normal human proteome, and

[0108] - Compare the sequences of the remaining peptides (or epitopes) with a normal tissue proteome database and exclude those peptides (or epitopes) found in healthy / normal tissues.

[0109] In one embodiment, step (a) includes step (a4), which is to select one or more epitopes found in at least one cancer among the remaining peptides (or epitopes) of step (a3). Thus, step (a4) allows, for example, the selection of one or more epitopes that are commonly shared among different patients with the same cancer or different patients with different cancers.

[0110] In one embodiment, step (a4) includes selecting one or more epitopes found in the proteomic mass spectrometry database of at least one cancer among the remaining peptides (or epitopes) of step (a3).

[0111] Examples of proteomic mass spectrometry databases of cancers include, but are not limited to, data from The Cancer Genome Atlas (TCGA) and data from Clinical Proteomic Tumor Analysis Consortium (CPTAC).

[0112] In one embodiment, step (a4) includes selecting one or more epitopes found in at least one, at least two or more cancers among the remaining peptides (or epitopes) of step (a3).

[0113] In one embodiment, the at least one cancer is a c-myc related cancer. In one embodiment, the at least one cancer is an IGF1R related cancer.

[0114] As used herein, c-myc related cancer refers to a cancer in which c-myc expression is dysregulated. As used herein, IGF1R related cancer refers to a cancer in which IGF1R expression is dysregulated.

[0115] Examples of myc related cancers include, but are not limited to, colon cancer, breast cancer, lung cancer, prostate cancer, bladder cancer and lymphoma. Examples of IGF1R related cancers include, but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer and sarcoma.

[0116] In one embodiment, the at least one cancer is selected from the group consisting of or comprising: breast cancer (including triple negative breast cancer), ovarian cancer, melanoma, sarcoma, teratocarcinoma, colon cancer, prostate cancer, bladder cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), head and neck cancer, colorectal cancer, glioblastoma, leukemia, lymphoma and other solid tumors and hematological malignancies.

[0117] In one embodiment, the at least one cancer is colon cancer. In one embodiment, the at least one cancer is breast cancer.

[0118] In one embodiment, step (a4) comprises selecting, from the remaining peptides (or epitopes) of step (a3), one or more epitopes found in at least one cancer, wherein the cancer is breast cancer or colon cancer.

[0119] In one embodiment, step (a4) comprises selecting, from the remaining peptides (or epitopes) of step (a3), one or more epitopes found in both breast cancer and colon cancer.

[0120] In one embodiment, the common cancer epitope is an epitope found in at least one of the above cancers. In one embodiment, the common cancer epitope is an epitope found in breast cancer and / or colon cancer.

[0121] In one embodiment, step (a) comprises at least one, preferably four, of the above steps (a1), (a2), (a3) and (a4). 。

[0122] In one embodiment, step (a) comprises the following steps:

[0123] (a1) Predicting peptides derived from non-canonical translation start and / or termination of a given gene

[0124] (a2) Identifying, among the predicted peptides identified in step (a1), 8-mer to 15-mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules

[0125] (a3) Identifying, among the 8-mer to 15-mer peptide (or epitope) sequences identified in step (a2), peptides (or epitopes) found in healthy subjects (i.e., subjects without cancer) and / or healthy tissues, and excluding said peptides (or epitopes), and

[0126] (a4) Selecting, from the remaining peptides (or epitopes) of step (a3), one or more epitopes found in at least one cancer.

[0127] In one embodiment, step (a) comprises the following steps:

[0128] (a1) Predicting peptides derived from non-canonical translation start and / or termination of a given gene

[0129] (a2) Identifying, among the predicted peptides identified in step (a1), 8 to 15-mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules

[0130] (a3) From the 8-mer to 15-mer peptide (or epitope) sequences identified in step (a2), identify the peptides (or epitopes) found in healthy subjects and / or healthy tissues, and exclude said peptides (or epitopes), with the following steps:

[0131] - Align the sequences of the 8-mer to 15-mer peptides (or epitopes) identified in step (a2) with the normal human proteome, and exclude those peptides (or epitopes) that have complete sequence homology with the normal human proteome, and

[0132] - Compare the sequences of the remaining peptides (or epitopes) with a normal tissue proteome database, and exclude those peptides (or epitopes) found in healthy / normal tissues, and

[0133] (a4) Select one or more epitopes found in at least one cancer from the remaining peptides (or epitopes) of step (a3).

[0134] In one embodiment, the given gene is an oncogene.

[0135] In one embodiment, the given gene is an IRES (Internal Ribosome Entry Site)-dependent gene. Examples of IRES-dependent genes include but are not limited to c-myc and IGF1R.

[0136] In one embodiment, the gene is an oncogene, preferably selected from the group consisting of or comprising: c-myc and IGF1R.

[0137] In one embodiment, the gene is c-myc. In one embodiment, the gene is IGF1R.

[0138] In one embodiment, step (b) includes the step of generating the epitopes identified in step (a). The epitopes can be prepared by any technique known to those skilled in the art, including expressing proteins, polypeptides or peptides by standard molecular biology techniques (such as recombinant methods), isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides.

[0139] The chemical synthesis as used herein involves synthesizing the epitopes identified in step (a) by sequentially adding amino acids and / or epitope fragments to a mixture to react with the growing chain. The chemical synthesis can be carried out in the liquid phase or the solid phase.

[0140] As used herein, the recombinant method involves synthesizing the epitopes identified in step (a) by expressing an expression vector encoding the epitope in a host cell and collecting the epitopes produced by the host cell.

[0141] In one embodiment, the method of obtaining an epitope as described above comprises:

[0142] - introducing a vector encoding the epitope into competent host cells in vitro or ex vivo;

[0143] - culturing the host cells transformed with the expression vector in vitro or ex vivo under conditions suitable for epitope expression;

[0144] - optionally, selecting cells that express and / or secrete the epitope; and

[0145] - recovering the expressed epitope.

[0146] In one embodiment, the method as described above further comprises the step of in vitro validation of the selected epitope after step (a).

[0147] In one embodiment, the above method is combined with one or more steps described below. In one embodiment, the above method is combined with one, two, three or four steps described below.

[0148] In one embodiment, in vitro validation comprises step (i) assessing the induction of a T cell response (e.g., CD8+ T cell response) by the selected epitope.

[0149] In one embodiment, the induction of a T cell response is assessed by measuring the induction of T cells (e.g., CD8+ T cells) specific for the selected epitope.

[0150] Examples of methods for inducing CD8+ T cells include, for example, performing an in vitro or ex vivo priming assay using the selected epitope. For example, human monocyte-derived dendritic cells can be pulsed with the selected epitope to induce specific CD8+ T cells.

[0151] Examples of methods for assessing CD8+ T cell induction include, for example, dextramer-based quantification.

[0152] In one embodiment, in vitro validation comprises step (ii) assessing the functionality of T cells (e.g., CD8+ T cells or TCR-engineered T cells) specific for the selected epitope. As used herein, TCR-engineered T cells refer to engineered T cells that express a TCR that recognizes the selected epitope.

[0153] In one embodiment, the functionality of T cells (e.g., CD8+ T cells or TCR-engineered T cells) specific for the selected epitope is assessed by measuring the production of IFN-γ, TNFα or granzyme B in the presence of cells stimulated with the epitope. Examples of methods for measuring the production of such molecules include, for example, flow cytometry, ELISA or fluorescence spot assay.

[0154] In one embodiment, the functionality of T cells (such as CD8+ T cells or TCR-engineered T cells) specific to a selected epitope is evaluated by measuring extracellular staining of a marker (such as 4-1BB (CD137) or CD107a).

[0155] In one embodiment, in vitro validation includes step (iii) evaluating the cytotoxicity of T cells (such as CD8+ T cells or TCR-engineered T cells) specific to a selected epitope against tumor cells and non-tumor cells.

[0156] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably.

[0157] In one embodiment, the cytotoxicity of T cells specific to a selected epitope is evaluated by measuring cell death of cells presenting the cognate epitope on the cell surface in the presence of the epitope-specific T cells (such as CD8+ T cells or TCR-engineered T cells).

[0158] In one embodiment, the cells do not naturally express the selected epitope and can be pulsed with the cognate epitope. For example, T2 cells pulsed with the selected epitope can be co-cultured with CD8+ T cells or TCR-engineered T cells specific to the selected epitope, and the death of the T2 cells can be measured.

[0159] As used herein, “cells that do not naturally express the selected epitope” means that the cells do not naturally contain the molecular machinery necessary for expressing the selected epitope and its expression on the cell surface.

[0160] In one embodiment, the cells naturally express the selected epitope. For example, tumor cell lines (such as MDA-MB-231 or HCT116) can be co-cultured with CD8+ T cells or TCR-engineered T cells specific to the selected epitope, and the death of the tumor cells can be measured.

[0161] As used herein, “cells that naturally express the selected epitope” means that the cells naturally contain all of the molecular machinery necessary for expressing the selected epitope and its expression on the cell surface.

[0162] In one embodiment, in vitro validation includes step (iv) evaluating the expression of the selected epitope in tumor cells.

[0163] In one embodiment, the expression of the selected epitope in tumor cells is evaluated by ribosome profiling (or ribo-seq).

[0164] In one embodiment, the expression of the selected epitope in tumor cells is evaluated by mass spectrometry.

[0165] In one embodiment, the expression of the selected epitopes in tumor cells is evaluated by the valid-NEO method. As used herein, valid-NEO is a multi-omics platform for detecting and quantifying neoantigens from limited clinical samples.

[0166] In one embodiment, in vitro validation includes at least one, preferably three, of the following steps:

[0167] (i) Evaluating the induction of CD8+ T cell responses by the selected epitopes,

[0168] (ii) Evaluating the functionality of T cells specific for the selected epitopes (e.g., CD8+ T cells or TCR-engineered T cells), and / or

[0169] (iii) Evaluating the cytotoxicity of T cells specific for the selected epitopes (e.g., CD8+ T cells or TCR-engineered T cells) against tumor cells and non-tumor cells,

[0170] Optionally, wherein the in vitro validation further includes step (iv) evaluating the expression of the selected epitopes in tumor cells, preferably wherein the expression is evaluated by ribosome profiling or mass spectrometry.

[0171] Compared with prior art methods, the methods described herein may exhibit one or more of the following advantages. In some embodiments, compared with prior art methods, the methods described herein are capable of detecting cancer epitopes with higher sensitivity. In some embodiments, the methods described herein are capable of identifying cancer epitopes that are weakly expressed in tumors and that may not be identified by prior art methods. In some embodiments, the methods described herein, particularly when applied to oncogenes, are capable of detecting shared cancer epitopes, i.e., cancer epitopes that are shared between different subjects with the same type of cancer or between different subjects with different types of cancer. In some embodiments, the methods described herein, particularly when applied to oncogenes, are capable of detecting cancer epitopes with a low risk of escape due to deletion or mutation (i.e., cancer epitopes that have a low risk of being deleted or mutated during the patient's lifetime).

[0172] The invention also relates to peptides comprising or consisting of epitopes identified or generated by the methods described above.

[0173] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of the following: LLLEATANL (SEQ ID NO: 1), SLTDLYLRI (SEQ ID NO: 2), IMTASNWTL (SEQ ID NO: 3), AMSPQLHNI (SEQ ID NO: 4), GLAAPAPKL (SEQ ID NO: 5), GLPPHPAHL (SEQ ID NO: 6), GMPWPIPAV (SEQ ID NO: 7), SLQETSYAL (SEQ ID NO: 8), SLYPIACSL (SEQ ID NO: 9), SVLGHDFSV (SEQ ID NO: 10), VQDMIQTQV (SEQ ID NO: 11), ILDDWLRHL (SEQ ID NO: 12), SLPSQHWSL (SEQ ID NO: 13), FLLMPLSFL (SEQ ID NO: 14), IILGIVFLL (SEQ ID NO: 15), SLDHLLLEA (SEQ ID NO: 16), FLWKRGRLL (SEQ ID NO: 17), ALLDGVLPA (SEQ ID NO: 18), LLFKVDFFL (SEQ ID NO: 19), RLGAAVFLL (SEQ ID NO: 20), RLLAKGQSL (SEQ ID NO: 21), SQPPPSLFV (SEQ ID NO: 22), ALLRCGHTL (SEQ ID NO: 23), GQASVPLFL (SEQ ID NO: 24), KLQTLLASI (SEQ ID NO: 25), LLASILFYI (SEQ ID NO: 26), LLVSTGVTV (SEQ ID NO: 27), QMQPHNLGV (SEQ ID NO: 28), SLFKLQTLL (SEQ ID NO: 29), VMFFKSQHL (SEQ ID NO: 30), ALADEWRNL (SEQ ID NO: 31), ALMISLGSV (SEQ ID NO: 32), ALWQDHTEI (SEQ ID NO: 33), AQWPAPRLV (SEQ ID NO: 34), CLLSKPVRL (SEQ ID NO: 35), FLFSICKQL (SEQ ID NO: 36), FLLTPRNFL (SEQ ID NO: 37), FMMITAYTV (SEQ ID NO: 38), FSIELLFSV (SEQ ID NO: 39), GLFFSLMFL (SEQ IDNO:40), GLKPWTQYA (SEQ ID NO:41), KLFGFCFQL (SEQ ID NO:42), KLISELRRI (SEQ ID NO:43), KLSELLMSF (SEQ ID NO:44), LLFSVNREV (SEQ ID NO:45), LLLAGGPGL (SEQ ID NO:46), LLPGGLLLL (SEQ ID NO:47), LLPPAPLVV (SEQ ID NO:48), LLQALMISL (SEQ ID NO:49), LLVISLWSV (SEQ ID NO:50), LLWKLISEL (SEQ ID NO:51), QIIQLVIRV (SEQ ID NO:52), RLAPLFQQL (SEQ ID NO:53), SLKDGVFTT (SEQ ID NO:54), SLSWETPGV (SEQ ID NO:55), SLWPHPTTV (SEQ ID NO:56), SMMGRMPAA (SEQ ID NO:57), SVHPTAPAV (SEQ ID NO:58), SVPKHVWEA (SEQ ID NO:59), VLFKLSELL (SEQ ID NO:60), VLFSILVST (SEQ ID NO:61), ALLTFSLFL (SEQ ID NO:62), ALPGLVQRA (SEQ ID NO:63), FIFGLHLRL (SEQ ID NO:64), FITEKLPQV (SEQ ID NO:65), FLFSRWILL (SEQ ID NO:66), FLVKKKFFV (SEQ ID NO:67), GLCSLPPLL (SEQ ID NO:68), GLLRGMSRL (SEQ ID NO:69), LLHSLSTKV (SEQ ID NO:70), LLLERDPSL (SEQ ID NO:71), LLLGKCLGV (SEQ ID NO:72), RVTDVILFL (SEQ ID NO:73), SLAPDSRPV (SEQ ID NO:74), SLISVFNRA (SEQ ID NO:75), SLSHSVFPL (SEQ ID NO:76), SMLDHETFA (SEQ ID NO:77), SVDEKNFKM (SEQ ID NO:78), TLLSIPNYV (SEQ ID NO:79), TLSFFTLKL (SEQ ID NO:80), VQMEPTHFV (SEQ IDNO: 81), YLSPFGHEI (SEQ ID NO: 82), RLPPLGRTI (SEQ ID NO: 84), LPRGSSWTV (SEQ ID NO: 85), LPPLGRTIL (SEQ ID NO: 86), ATANLLTAH (SEQ ID NO: 87), TQRLPPLGR (SEQ ID NO: 88), ILLPRGSSW (SEQ ID NO: 89), LLEATANLL (SEQ ID NO: 90), KSMLFLWKR (SEQ ID NO: 91), MLFLWKRGR (SEQ ID NO: 92), KMRKKSMLF (SEQ ID NO: 93), HRPPPAATL (SEQ ID NO: 94), PPLGRTILL (SEQ ID NO: 95), SPHISTTTQ (SEQ ID NO: 96), GQSLDHLLL (SEQ ID NO: 97) and RPPPAATLR (SEQ ID NO: 98).

[0174] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of the following: FLLMPLSFL (SEQ ID NO: 14), IILGIVFLL (SEQ ID NO: 15), LLLEATANL (SEQ ID NO: 1), SLDHLLLEA (SEQ ID NO: 16), SLTDLYLRI (SEQ ID NO: 2), FLWKRGRLL (SEQ ID NO: 17), ALLDGVLPA (SEQ ID NO: 18), LLFKVDFFL (SEQ ID NO: 19), RLGAAVFLL (SEQ ID NO: 20), RLLAKGQSL (SEQ ID NO: 21), SQPPPSLFV (SEQ ID NO: 22), ALLRCGHTL (SEQ ID NO: 23), GQASVPLFL (SEQ ID NO: 24), KLQTLLASI (SEQ ID NO: 25), LLASILFYI (SEQ ID NO: 26), LLVSTGVTV (SEQ ID NO: 27) and VMFFKSQHL (SEQ ID NO: 30).

[0175] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of the following: ALADEWRNL (SEQ ID NO: 31), ALMISLGSV (SEQ ID NO: 32), ALWQDHTEI (SEQ ID NO: 33), AMSPQLHNI (SEQ ID NO: 4), AQWPAPRLV (SEQ ID NO: 34), CLLSKPVRL (SEQ ID NO: 35), FLFSICKQL (SEQ ID NO: 36), FLLTPRNFL (SEQ ID NO: 37), FMMITAYTV (SEQ ID NO: 38), FSIELLFSV (SEQ ID NO: 39), GLFFSLMFL (SEQ ID NO: 40), GLKPWTQYA (SEQ ID NO: 41), KLFGFCFQL (SEQ ID NO: 42), KLISELRRI (SEQ ID NO: 43), KLSELLMSF (SEQ ID NO: 44), LLFSVNREV (SEQ ID NO: 45), LLLAGGPGL (SEQ ID NO: 46), LLPGGLLLL (SEQ ID NO: 47), LLPPAPLVV (SEQ ID NO: 48), LLQALMISL (SEQ ID NO: 49), LLVISLWSV (SEQ ID NO: 50), LLWKLISEL (SEQ ID NO: 51), QIIQLVIRV (SEQ ID NO: 52), RLAPLFQQL (SEQ ID NO: 53), SLKDGVFTT (SEQ ID NO: 54), SLSWETPGV (SEQ ID NO: 55), SLWPHPTTV (SEQ ID NO: 56), SMMGRMPAA (SEQ ID NO: 57), SVHPTAPAV (SEQ ID NO: 58), SVPKHVWEA (SEQ ID NO: 59), VLFKLSELL (SEQ ID NO: 60), VLFSILVST (SEQ ID NO: 61), ALLTFSLFL (SEQ ID NO: 62), ALPGLVQRA (SEQ ID NO: 63), FIFGLHLRL (SEQ ID NO: 64), FITEKLPQV (SEQ ID NO: 65), FLFSRWILL (SEQ ID NO: 66), FLVKKKFFV (SEQ ID NO: 67), GLCSLPPLL (SEQ ID NO: 68), GLLRGMSRL (SEQ IDNO: 69), LLHSLSTKV (SEQ ID NO: 70), LLLERDPSL (SEQ ID NO: 71), LLLGKCLGV (SEQ ID NO: 72), RVTDVILFL (SEQ ID NO: 73), SLAPDSRPV (SEQ ID NO: 74), SLISVFNRA (SEQ ID NO: 75), SLSHSVFPL (SEQ ID NO: 76), SMLDHETFA (SEQ ID NO: 77), SVDEKNFKM (SEQ ID NO: 78), TLLSIPNYV (SEQ ID NO: 79), TLSFFTLKL (SEQ ID NO: 80), VQMEPTHFV (SEQ ID NO: 81) and YLSPFGHEI (SEQ ID NO: 82).

[0176] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of the following: LLLEATANL (SEQ ID NO: 1), SLDHLLLEA (SEQ ID NO: 16), RLLAKGQSL (SEQ ID NO: 21), FLWKRGRLL (SEQ ID NO: 17), RLPPLGRTI (SEQ ID NO: 84), LPRGSSWTV (SEQ ID NO: 85), LPPLGRTIL (SEQ ID NO: 86), ATANLLTAH (SEQ ID NO: 87), TQRLPPLGR (SEQ ID NO: 88) and ILLPRGSSW (SEQ ID NO: 89).

[0177] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of the following: LLEATANLL (SEQ ID NO: 90), KSMLFLWKR (SEQ ID NO: 91), MLFLWKRGR (SEQ ID NO: 92), KMRKKSMLF (SEQ ID NO: 93), HRPPPAATL (SEQ ID NO: 94), PPLGRTILL (SEQ ID NO: 95), SPHISTTTQ (SEQ ID NO: 96), GQSLDHLLL (SEQ ID NO: 97) and RPPPAATLR (SEQ ID NO: 98).

[0178] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), IMTASNWTL (SEQ ID NO:3), AMSPQLHNI (SEQ ID NO:4), GLAAPAPKL (SEQ ID NO:5), GLPPHPAHL (SEQ ID NO:6), GMPWPIPAV (SEQ ID NO:7), SLQETSYAL (SEQ ID NO:8), SLYPIACSL (SEQ ID NO:9), SVLGHDFSV (SEQ ID NO:10), VQDMIQTQV (SEQ ID NO:11), ILDDWLRHL (SEQ ID NO:12), and SLPSQHWSL (SEQ ID NO:13).

[0179] The present invention relates to a peptide comprising an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), AMSPQLHNI (SEQ ID NO:4), GLAAPAPKL (SEQ ID NO:5), GLPPHPAHL (SEQ ID NO:6), GMPWPIPAV (SEQ ID NO:7), SLQETSYAL (SEQ ID NO:8), SLYPIACSL (SEQ ID NO:9), SVLGHDFSV (SEQ ID NO:10), VQDMIQTQV (SEQ ID NO:11), ILDDWLRHL (SEQ ID NO:12), and SLPSQHWSL (SEQ ID NO:13).

[0180] In one embodiment, the peptide comprises an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), and IMTASNWTL (SEQ ID NO:3).

[0181] In one embodiment, the peptide comprises an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1) and SLTDLYLRI (SEQ ID NO:2).

[0182] In one embodiment, the peptide comprises or consists of an epitope of the sequence LLLEATANL (SEQ ID NO:1).

[0183] In one embodiment, the peptide comprises or consists of an epitope of the sequence SLTDLYLRI (SEQ ID NO:2).

[0184] In one embodiment, the peptide comprises or consists of an epitope of the sequence IMTASNWTL (SEQ ID NO:3).

[0185] The present invention also relates to an expression vector for inducing the expression of one or more of the above peptides.

[0186] The vector can in particular be an RNA vector, a DNA vector or plasmid, a viral vector or a bacterial vector. The expression cassette can be integrated into the host cell genome or not, depending on the nature of the vector, which is well known to those skilled in the art. The expression vector or expression cassette can also contain the elements necessary for expressing a nucleic acid (polynucleotide) in a subject. For example, this can consist of a start codon (ATG), a stop codon and a promoter, as well as a polyadenylation sequence for certain vectors (such as viral vectors other than plasmids and poxviruses). The ATG can be located at the 5' end of the reading frame and the stop codon can be located at the 3' end. It is well known that other elements enabling expression control can also be present, such as enhancer sequences, stabilization sequences and signal sequences allowing the secretion of the peptide.

[0187] For RNA vectors, the vector can use, for example, non-replicating mRNA or virus-derived self-amplifying RNA. Conventional mRNA-based vectors can encode the target peptide and can contain 5' and 3' untranslated regions (UTRs). Self-amplifying RNA can not only encode the target peptide but also encode the viral replication machinery, thus enabling intracellular RNA amplification and abundant protein expression.

[0188] Examples of viral vectors include but are not limited to lentiviruses and retroviruses.

[0189] The present invention also relates to cytotoxic T lymphocytes (CTLs) of a subject treated with one or more of the above peptides.

[0190] The present invention also relates to cytotoxic T lymphocytes (CTLs) of a subject treated with one or more of the above expression vectors.

[0191] The present invention also relates to cytotoxic T lymphocytes (CTLs) generated in vitro by stimulating T cells with one or more of the above peptides or one or more of the above expression vectors.

[0192] The present invention also relates to T cell receptors (TCRs) that recognize the peptides as described above.

[0193] The present invention also relates to engineered T cells that express TCRs that recognize the peptides as described above (i.e., TCR-engineered T cells).

[0194] Methods for preparing these T cells are known to those skilled in the art. It can be as follows: (i) isolating the TCR α-chain and β-chain from T cells that recognize the peptides as described above and inserting them into a vector; (ii) modifying T cells isolated from the peripheral blood of a patient or donor with the said vector so that they encode the desired TCR αβ sequence; (iii) subsequently amplifying these modified T cells in vitro to obtain a sufficient number for treatment and administering them to the patient. It should be noted that the TCR sequence can be modified to optimize TCR affinity.

[0195] The present invention also relates to one or more of the peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above, used as a vaccine.

[0196] The present invention also relates to one or more of the peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above, used as a drug.

[0197] The present invention also relates to one or more of the peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above, for treating or preventing at least one cancer in a subject in need thereof.

[0198] In one embodiment, one or more of the peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above are used for treating or preventing at least one, at least two or more cancers.

[0199] In one embodiment, the at least one cancer is a c-myc related cancer. In one embodiment, the at least one cancer is an IGF1R related cancer.

[0200] Examples of c-myc and IGF1R related cancers are provided above.

[0201] In one embodiment, the at least one cancer is selected from the group consisting of or comprising: breast cancer (including triple-negative breast cancer), ovarian cancer, melanoma, sarcoma, teratocarcinoma, colon cancer, prostate cancer, bladder cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), head and neck cancer, colorectal cancer, glioblastoma, leukemia, lymphoma and other solid tumors as well as hematological malignancies.

[0202] In one embodiment, the at least one cancer is colon cancer. In one embodiment, the at least one cancer is breast cancer.

[0203] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above are used for treating or preventing at least one cancer, wherein the cancer is breast cancer or colon cancer.

[0204] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above are used for treating or preventing breast cancer and colon cancer.

[0205] The present invention also relates to a method for treating or preventing at least one cancer in a subject in need thereof, wherein the method comprises administering to the subject one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above.

[0206] In one embodiment, the method of the present invention is used for treating or preventing at least one, at least two or more cancers as defined herein.

[0207] In one embodiment, the method of the present invention is used for treating or preventing breast cancer and / or colon cancer.

[0208] The present invention also relates to one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above, for use in the preparation of a medicament for treating or preventing at least one cancer in a subject in need thereof.

[0209] In one embodiment, the medicament is used for treating or preventing at least one, at least two or more cancers as defined herein.

[0210] In one embodiment, the medicament is used for treating or preventing breast cancer and / or colon cancer.

[0211] In one embodiment, one or more of the peptides, one or more of the expression vectors as described above induce an immune response, such as a T cell response.

[0212] One of ordinary skill in the art will be aware of various assays for determining whether an immune response against a tumor-associated epitope has been generated. A variety of B lymphocyte and T lymphocyte assays are known, such as ELISA, cytotoxic T lymphocyte (CTL) assays (such as chromium release assays), flow cytometry-based assays or real-time cytotoxicity assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays.

[0213] Accordingly, the present invention also relates to a method of inducing an immune response in a subject in need thereof, wherein the method comprises administering to the subject one or more of the peptides or one or more of the expression vectors as described above.

[0214] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs or one or more of the engineered T cells as described above reduces the number of tumor cells in vivo. Accordingly, the present invention also relates to an in vivo method for reducing the number of tumor cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the peptides, one or more of the expression vectors, one or more of the CTLs or one or more of the engineered T cells as described above.

[0215] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs or one or more of the engineered T cells as described above reduces the volume of a tumor in vivo. Accordingly, the present invention also relates to a method for reducing the volume of a tumor in vivo, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of the peptides, one or more of the expression vectors, one or more of the CTLs or one or more of the engineered T cells as described above.

[0216] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above has cytotoxic activity against tumor cells, but has no cytotoxic effect on normal cells (i.e., non-tumor cells).

[0217] In one embodiment, one or more of the peptides, one or more of the expression vectors, one or more of the CTLs, or one or more of the engineered T cells as described above is administered in a therapeutically effective amount.

[0218] However, it should be understood that the total daily usage of one or more of the peptides, one or more of the expression vectors, one or more of the CTLs or one or more of the engineered T cells described above will be determined by the attending physician within the scope of reasonable medical judgment.

[0219] The specific dosage for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed; and like factors well known in the medical arts.

[0220] For administration to a subject, the one or more peptides, the one or more expression vectors, the one or more CTLs or the one or more engineered T cells are formulated for administration to a subject.

[0221] The one or more peptides or the one or more expression vectors as described above can be administered by enteral or parenteral routes of administration.

[0222] The one or more CTLs or the one or more engineered T cells as described above can be administered by parenteral routes of administration.

[0223] Enteral routes are optionally selected from the group consisting of: the buccal route (including the perlingual route and the sublingual route), the oral route, and the rectal route.

[0224] Parenteral routes include any non-enteral route. Parenteral routes are optionally selected from the group consisting of: the epidermal route, the transdermal route, the intradermal route, the subcutaneous route, the nasal route, the intramuscular route, the intraocular route, the intravitreal route, and the intracameral route.

[0225] Sequence Listing

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] Brief Description of the Drawings

[0232] Figure 1A-1C is a combination of illustrations and tables. Figure 1A : Schematic diagram of a bioinformatics prediction process for identifying potential new epitopes derived from non-canonical translations of major oncogenes. All ≥9-mer peptides that can be generated by non-canonical translation have been predicted (1). Potential HLA-A*02:01 strong binder 9-mer epitopes have been identified from these predicted peptides (2). Epitopes with complete sequence homology to the human proteome have been removed from the selection (3). The predicted HLA-A*02:01 strong binder epitopes have been screened using proteomic mass spectrometry databases from tumor and normal tissues (4). Figure 1B : List of all predicted HLA-A*02-01 strong binder epitopes for MYC (left column) and IGF1R (right column) analysis, using netMHCpan. Figure 1C: List of predicted HLA-A*02-01 strong binder epitopes that are "confident" at least once in the tumor tissue proteome mass spectrometry database but "not confident" in the normal tissue database, for MYC (left column) and IGF1R (right column) analysis, using Pepquery. Figure 1D : List of predicted class I HLA strong binder epitopes of MYC non-canonical peptides containing PR3 epitopes, using netMHCpan and MHCflurry. Figure 1E : List of predicted class I HLA weak binder epitopes of MYC non-canonical peptides containing PR3 epitopes, using netMHCpan and MHCflurry. Figure 1F : Binding score distribution of predicted class I HLA strong (upper panel) and weak (lower panel) binder epitopes of MYC non-canonical peptides containing PR3 epitopes.

[0233] Figure 2A-2B Combination of multiple figures. Figure 2A : Representative images of dextramer staining of CD8+ T cells after MoDCs were pulsed with specific peptides for 12 days (upper row) or not pulsed (lower row): CD8+ naive T cell priming. Figure 2B : Schematic diagram of dextramer analysis of 12 healthy donors after 12-day MoDC:CD8+ naive T cell priming.

[0234] Figure 3 Schematic diagrams of dextramer analysis of CD8+ T cells after 12-day MoDC:CD8+ naive T cell priming (left panel) and after sorting and expansion of peptide-specific CD8+ T cells and non-specific counterparts (right panel).

[0235] Figure 4 A-4B is a combination of two histograms. Figure 4 A: Representative graphs of the percentage of 4-1BB expression of peptide-specific or non-specific CD8+ T cells after contact with T2 cells pulsed with irrelevant (non-specific) or specific peptides for 3 donors under PR3 conditions and 1 donor under PR5 conditions. Figure 4 B: Representative graphs of the percentage of CFSE+ T2 cell death after contact with peptide-specific or non-specific CD8+ T cells for 2 donors under PR3 conditions and 1 donor under PR5 conditions, pulsed with irrelevant (non-specific) or specific peptides.

[0236] Figure 5 A-5B is a combination of two histograms. Representative graphs of the concentration of IFNγ (A.) or TNFα (B.) produced after peptide-specific CD8+ T cells or non-specific CD8+ T cells contacted T2 cells pulsed with irrelevant peptides (non-specific) or specific peptides.

[0237] Figure 6 A-6B is a combination of two figures that represent the Incucyte quantification of real-time cell death of MDA-MB-231 (A.) or HCT116 (B.) cell lines (pulsed or not pulsed with PR3 peptide) co-cultured with PR3-specific CD8+ T cells or their negative counterparts (dextramer-neg T cells).

[0238] Figures 7A - 7C are a combination of figures showing the conversion of the PR3 (LLLEATANL) epitope in tumor cell lines (A. MDA-MB-231 and B. OVCAR-3) or a set of HLA-A2+ normal human primary cells (C.) as shown using the Valid-NEO method builder bioinformatics pipeline (Complete Omics Inc., MD, USA).

[0239] Figure 8 The histogram shows the IFNγ concentration produced by PR3-specific CD8+ T cells or their negative counterparts (dextramer-neg T cells) after 48 hours of co-culture with HLA-A2+ normal human primary cells (cardiomyocytes, bronchial epithelial cells, or keratinocytes) or a tumor cell line (MDA-MB-231). Examples

[0240] The present invention is further illustrated by the following examples.

[0241] Bioinformatics prediction pipeline for identifying potential new epitopes derived from non-canonical translations of major oncogenes Process

[0242] All transcripts of two major oncogenes, MYC and IGF1R, were identified using the genomic browser Ensembl. For each transcript of each oncogene, a bioinformatics pipeline was established to predict all peptide sequences of size greater than or equal to 9-mer that could be caused by translational defects (frameshift (+1) or (-1), translational termination defects such as stop codon readthrough) and / or any non-canonical initiation (uORF, IRES, translation in 5'UTR or 3'UTR). Among these sequences, 9-mer strong binder epitopes (% rank ≤ 0.5) for HLA-A*02:01 were predicted using an epitope prediction tool (netMHCpan v4.1). Any potential epitopes with complete sequence homology to the human proteome were removed from the previous selection (BLAST protein, refseq_protein database) ( Figure 1A) Through this analysis, 17 potential strong binder epitopes of HLA-A*02:01 were identified for MYC oncogene analysis, and 62 potential strong binder epitopes of HLA-A*02:01 were identified for IGF1R oncogene analysis ( Figure 1B ).

[0243] Proteomic mass spectrometry (MS) database filtering

[0244] Based on the mass spectrometry-based proteomics dataset (PepQuery V1.6.2), a targeted peptide retriever has been used to filter potential translated epitopes. Epitopes with at least 1 confidence in the proteomic mass spectrometry database of breast cancer or colon cancer (patient tumor datasets: TCGA and CPTAC) but no confidence in the normal tissue proteomic database (GTEx) were selected ( Figure 1A ). For MYC oncogene analysis, 2 epitopes (PR3 and PR5) were selected from the previously predicted 17 epitopes. For IGF1R oncogene analysis, 10 epitopes were selected from the previously predicted 62 epitopes ( Figure 1C ).

[0245] Predicting class I HLA epitopes of one of the MYC non-canonical predicted peptides containing the PR3 epitope

[0246] Using the previous bioinformatics prediction pipeline, we identified several non-canonical translated amino acid sequences derived from the MYC oncogene. One of them (the sequence with SEQ ID NO:83) was predicted to contain the PR3 sequence, which generates the PR3 epitope after processing.

[0247] SEQ ID NO:83

[0248] MRRHRPPPAATLRRNKKMRKKSMLFLWKRGRLLAKGQSLDHLLLEATANLLTAHWSSRGATS

[0249] PHISTTTQRLPPLGRTILLPRGSSWTVSES

[0250] Using this sequence and epitope prediction tools (netMHCpan v4.1 or MHCFlurry), we predicted all 9-mer strong and weak binder epitopes of common class I HLA alleles. Figure 1D-1F )

[0251] Peptide synthesis

[0252] The peptides were synthesized by the vendor (JPT peptide technology, Germany) and their identity was confirmed by mass spectrometry. The expected purity was >95%, determined by high performance liquid chromatography. The lyophilized peptides were dissolved in deionized water containing <5% DMSO, aliquoted and stored at -20 °C for later use.

[0253] PBMC stimulation assay of HLA-A*02:01 healthy donors

[0254] PBMCs were obtained by Ficoll density gradient centrifugation of blood from HLA-A*02:01 healthy donors (“Etablissement du Sang”, EFS, Lyon). For the stimulation assay, PBMCs were rapidly thawed at 37 °C, washed thoroughly and left at room temperature for 2 h, then their viability was assessed. Monocytes were isolated by positive selection of CD14+ cells (Miltenyi). CD14+ monocytes were cultured for 4 days in complete RPMI (RPMI medium containing 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (PS)) supplemented with recombinant human IL-4 (10 ng / ml) and recombinant human GM-CSF (800 UI / ml on day 1; 1600 UI / ml on day 3) to generate monocyte-derived dendritic cells (MoDCs). Then the MoDCs were matured and pulsed overnight (18 h) with peptides (10 μg / ml specific peptide, 2.5 μg / ml positive control), IL-4 (10 ng / ml), GM-CSF (800 UI / ml), TNFa (20 ng / ml) and Poly-IC (40 μg / ml) (negative control not pulsed). Meanwhile, CD8+ naive T cells were isolated from thawed autologous PBMCs (Miltenyi kit) and cultured overnight in AIM-V medium supplemented with recombinant human IL-7 (5 ng / ml) containing 5% human serum AB (sAB) and 1% PS. The next day, the pulsed MoDCs were co-cultured with CD8+ naive T cells in 48-well plates supplemented with human recombinant IL-21 (60 ng / ml) containing AIM-V + 5% sAB + 1% PS for 12 days (MoDC:CD8+ naive T cell ratio of 1:4). Within 12 days, the cells were expanded from 48-well plates to 12-well plates and then to 6-well plates, and AIM-V 5% sAB + 1% PS supplemented with IL-7 and IL-15 (10 ng / ml each on days 3 and 5; 20 ng / ml on days 7 and 10) was added. The optimized peptide MART1 (ELAGIGILTV) was used as a positive control for the stimulation assay.

[0255] Dextramer analysis

[0256] On day 12, peptide-specific CD8+ T cells were identified using dextramer staining (Immudex). For each peptide condition, cells pulsed with the respective peptide and unpulsed cells were stained with the corresponding dextramer. For staining, 3.10 6 cells were placed in polypropylene tubes and washed with FACS buffer. The cells were stained with 8 μL of dextramer for 10 minutes at room temperature in the dark, and then stained with 1 / 400 dilution of Zombie Near Infra-Red (NIR) for an additional 10 minutes in the dark to assess viability (Biolegend). Anti-CD3 BV421 and anti-CD8 FITC (Biolegend) antibodies were added for 20 minutes at 4 °C in the dark. The cells were then washed twice with FACS buffer and resuspended in FACS buffer for flow cytometry analysis (FACS Fortessa BD).

[0257] Figure 2A The results show CD8+ T cells stained with dextramer in unpulsed (bottom row) and peptide-specific pulsed (top row) stimulated cells for each peptide condition. For the MART1 positive control, up to 18.2% of CD8+ T cells were positive after stimulation with MART1 peptide, while only 0.1% were positive under the unpulsed condition. Interestingly, the conditions stimulated with specific epitopes (PR3 and PR5) produced 0.094% and 0.068% dextramer-positive CD8+ T cells for PR3 and PR5, respectively, compared to 0.001% and 0.002% under the unpulsed condition. Figure 2B Summarizes the results obtained in 12 different donors, with dark boxes indicating when peptide-specific CD8+ T cells were detected after stimulation and white boxes otherwise. Among them, the stimulation of peptide-specific CD8+ T cells was identified in 4 donors for PR3 and 2 donors for PR5.

[0258] Peptide-specific CD8+ T cell sorting

[0259] After 12 days of MoDC:CD8+ naive T cell stimulation and verification of peptide-specific CD8+ T cells by dextramer staining analysis, the cells were sorted using two different protocols.

[0260] For the first protocol, cells from MoDC:CD8+ naive T cell stimulation were stained using the same protocol as for dextramer analysis, but without the anti-CD3 antibody. After gating on live CD8+ T cells, BD FACSAria TMA cell sorter sorts dextramer-positive cells corresponding to peptide-specific CD8+ T cells and negative cells corresponding to non-specific counterparts.

[0261] For the second protocol, peptide-specific CD8+ T cells are sorted using a peptide-specific monomer conjugated to magnetic beads. In a 1.5 ml tube, 10 μL of a peptide-specific biotinylated monomer at a concentration of 100 μg / ml (P2R Facility, Nantes, France) is incubated with 10 μL of dynabead M-280 streptavidin in PBS1X 0.1% BSA (final volume of 100 μL) on a rotary shaker for 1 hour at room temperature. Then the formed magnetic peptide-specific monomer is washed 3 times with PBS1X 0.1% BSA using a DynaMag Spin Magnet. 5.10 6 cells from MoDC:CD8+ naive T cell stimulation are washed and resuspended in 500 μL of PBS1X 0.1% BSA. They are mixed with the previously formed magnetic monomer and incubated on a rotary shaker for 4 hours at room temperature. Then the cells are washed 8 to 10 times with PBS1X 0.1% BSA using a DynaMag Spin Magnet. The only sorted part in this protocol is the peptide-specific CD8+ T cells bound to the magnetic peptide-specific monomer.

[0262] Feeding protocol

[0263] The peptide-specific CD8+ sorted T cells and non-specific counterparts are expanded on a feeder layer composed of allogeneic PBMCs and a B lymphoblastoid cell line irradiated with 35 Gy (ratio 10:1). The feeder layer cells are seeded in a 96-well round-bottom plate at a concentration of 0.10x106 cells per well in RPMI 8% sAB 1% PS supplemented with PHA-L (1.5 μg / mL), human recombinant IL-2 (150 IU / mL), and human recombinant IL-7 (10 ng / ml). Up to 5x103 sorted cells are added per well. The cells are cultured for 14 days. Starting from day 5, according to the proliferation situation, half of the medium is replaced every two days (days 5, 7, 9, and 12) or each well is split in half and supplemented with RPMI 8% sAB 1% PS containing human recombinant IL-2 (300 UI / ml), human recombinant IL-7 (20 ng / ml), and human recombinant IL-15 (20 ng / ml). After 12 days, the purity of the specific and non-specific parts is evaluated. If there are ≥65% dextramer-positive CD8+ T cells in the positive part and <0.5% in the negative part, the cells are used for cytotoxicity experiments.

[0264] Figure 3The results in showed the dextramer staining results of CD8+ T cells after 12-day MoDC:CD8+ naive T cell priming, and after sorting and expansion of peptide-specific CD8+ T cells or non-specific counterparts. CD8+ T cell populations that were 98.6% specific for the PR 3 peptide and 69% specific for the PR5 peptide were sorted and expanded from cells generated by peptide-pulsed MoDC:CD8+ T cell priming.

[0265] Cytotoxicity and functional assays using T2 cells

[0266] T2 (SD cell line) is a lymphoblastoid cell line lacking transporter associated with antigen processing (TAP) protein, and thus unable to present endogenous peptides on class I MHC, but can be used to monitor cytotoxic T lymphocyte (CTL) responses to target foreign antigens in a non-competitive environment.

[0267] T2 cells were first stained with the CFSE Cell Division Tracking Kit (Biolegend) at 37 °C for 13 minutes and then washed 3 times. CFSE-stained T2 cells were pulsed with irrelevant (non-specific) or specific peptides at 37 °C for 2 hours. After thorough washing, the CFSE-pulsed T2 cells were resuspended in the corresponding T cell medium (RPMI + 8% sAB + 1% PS). The T cells were rested. First, CD8+ T cells were resuspended in RPMI 8% sAB + 1% PS supplemented with 50 UI / ml human recombinant IL-2 and incubated overnight at 37 °C. The next day, the CD8+ T cells were resuspended in their corresponding medium and incubated at 37 °C for 2 hours without any cytokines added. Then, the CFSE-pulsed T2 cells were co-cultured with the T cells (at a ratio of T2 cells to CD8+ T cells of 1:10) in 96-well U-bottom plates in duplicate. After 24 hours, the supernatants were collected for further ELISA analysis and the cells were pooled in V-bottom plates and washed with FACS buffer. For each condition, an antibody mixture containing Zombie NIR (diluted 1 / 400), anti-CD3 BV421, anti-CD8 APC, and anti-human CD137 (4-1BB) Pe-Dazzle594 (Biolegend) antibodies was added and incubated at 4 °C in the dark for 30 minutes. The cells were washed and resuspended in FACS buffer and then subjected to flow cytometry analysis (FACS Fortessa BD).

[0268] Figure 4 A shows the percentage of 4-1BB expression of peptide-specific or non-specific CD8+ T cells after contact with T2 cells pulsed with irrelevant or specific peptides. Figure 4Panel B shows the percentage of T2 cell death after contact with peptide-specific CD8+ T cells or non-specific counterparts. These results show the specific activation (A.) and specific killing (B.) of PR3- and PR5-specific CD8+ T cells after specific antigen stimulation.

[0269] For ELISA assays, the IFN gamma Human Uncoated ELISA kit (Invivogen) and TNF alpha Human Uncoated ELISA kit (Invivogen) were used according to the manufacturer's instructions. Supernatants at different dilutions (dilution 1 / 2, 1 / 5, 1 / 10, or 1 / 20) were assayed, and the diluted supernatants with absorbance results within the standard range were analyzed.

[0270] Figure 5 The results show the quantification of cytokine (A. IFNγ and B. TNFα) secretion by peptide-specific and non-specific CD8+ T cells after contact with T2 cells pulsed with irrelevant or specific peptides. As in previous results, after specific antigen stimulation, PR3- and PR5-specific CD8+ T cells have specific IFNγ and TNFα secretion.

[0271] Verification of epitope presentation by mass spectrometry

[0272] Epitope validation by mass spectrometry was performed by Complete Omics Inc. (MD, USA). Briefly, a total of 20 million cells were lysed, and peptide-HLA complexes were immunoprecipitated using a self-packed Valid-NEO neoantigen enrichment column preloaded with anti-human HLA-A, B, and C antibody clone W6 / 32 (BioXCell). After elution, dissociation, filtration, and purification, the peptides were lyophilized and then further analyzed. The conversion parameters of each epitope peptide were checked and collated by the Valid-NEO method builder bioinformatics pipeline to exclude ions with excessive noise due to co-elution with impurities, and the detectability was improved by recursive optimization of important ions.

[0273] The results in Figures 7A - 7B show the conversion parameters of the PR3 epitope in two tumor cell lines, MDA-MB-231 and OVCAR-3. Similar quantitative conversions were identified when analyzed using heavy peptides or peptides eluted from HLA of the MDA-MB-231 or OVCAR-3 cell lines, which confirmed the presentation of the PR3 epitope on the surface of both tumor cell lines.

[0274] In contrast, when analyzing a panel of HLA-A2+ normal human primary cells (cardiomyocytes, keratinocytes, astrocytes, renal proximal tubular cells, or bronchial epithelial cells,Figure 7C ) When no conversion was found, this means that the PR3 epitope was not presented by normal cells. These data indicate that the PR3 epitope is a tumor-specific and safe target.

[0275] Detection of in vitro cytotoxicity of PR3-specific T cells against tumor cell lines using InCucyte technology

[0276] To monitor tumor cell death in real time, we performed immune cell killing assays using the IncuCyte technology. The tumor cell lines used as targets were the breast cancer cell line MDA-MB-231 and the colon cancer cell line HCT116. For the positive control condition, the cell lines were pulsed with specific peptides to artificially present the target epitope on class I MHC. One day before the experiment, the tumor cell lines were seeded at 5,000 cells / well in 96-well flat-bottom plates. For the previous T cells, for cytotoxicity and functional analysis, T2 cells were rested, rested overnight in the presence of supplemented cytokines, and then rested for 2 hours the next day in the absence of cytokines. At the same time, the tumor cell lines were pulsed with 10 μg / ml specific peptides or not pulsed for 2 hours, and then washed three times. T cells were added to the corresponding wells (CD8+ T cell:tumor cell line ratio of 2:1). For the tumor cell death control condition, DMSO was added to the medium (final concentration of 20%). Finally, Cytotox Green (Sartorius) was added to a final concentration of 250 nM per well. When the cytoplasmic membrane integrity is damaged, this reagent enters the cell and increases the fluorescence intensity by 100 - 1000 times after binding to deoxyribonucleic acid (DNA). Live imaging was performed for 56 hours using Incucyte Zoom at 37°C and 5% CO2. For analysis, the number of dead tumor cells per well was calculated by evaluating the number of green fluorescent tumor cells per well.

[0277] Figure 6 The results show the kinetics of tumor cell death of the MDA-MB-231 cell line (A.) and the HCT116 cell line (B.) after co-culture with PR3-specific CD8+ T cells or non-specific counterparts (dextramer-neg T cells), as well as representative images at 24 hours for each condition. For both cell line conditions, these results show an increase in cell death when the tumor cells are co-cultured with PR3-specific CD8+ T cells compared to their negative counterparts. When the tumor cells are pulsed with PR3 and co-cultured with PR3-specific CD8+ T cells, the cell death further increases, which is consistent with an epitope-specific response.

[0278] Safety analysis of PR3-specific T cells against HLA-A2+ normal human primary cells

[0279] The safety of PR3-specific T cells was evaluated using HLA-A2+ normal human primary cells, including cardiomyocytes, bronchial epithelial cells, and keratinocytes (Promocell). Normal human primary cells and the tumor cell line MDA-MB-231, used as a cytotoxic positive control, were seeded at 5,000 cells per well in 96-well flat-bottom plates. For T cells, for cytotoxicity and functional analysis, T2 cells were rested, rested overnight in the presence of cytokines, and then rested for 2 hours the next day in the absence of cytokines. T cells were added to the corresponding wells (CD8+ T cell: tumor cell line ratio of 10:1). After 48 hours of co-culture, the supernatant was collected and further ELISA analysis was performed using the IFNγ Human Uncoated ELISA kit (Invivogen) described previously.

[0280] Figure 8 The results showed the quantification of IFNγ secretion by PR3-specific CD8+ T cells or non-specific counterparts (dextramer-neg T cells) after 48 hours of co-culture with normal human primary cells and the tumor cell line MDA-MB-231, used as a cytotoxic positive control. No IFNγ secretion was detected when PR3-specific CD8+ T cells were co-cultured with any of the tested normal human primary cells, indicating the safety of the product. When PR3-specific CD8+ T cells were co-cultured with the tumor cell line MDA-MB-231 (positive control), we observed the secretion of IFNγ.

[0281] In summary, these experiments demonstrated that PR3-specific CD8+ T cells specifically recognize and are functional against target cells (T2 cells) presenting the cognate peptide, and can specifically recognize and kill tumor cells (MDA-MB-231 and HCT116) expressing endogenous peptides derived from non-canonical translation, without any toxicity to normal human primary cells.

Claims

1. A method for generating one or more common cancer epitopes, wherein the method comprises the following steps: (a) Identifying peptides derived from non-canonical translation initiation and / or termination of a given gene, preferably an oncogene, and selecting one or more common cancer epitopes from the identified peptides, (b) Generating the epitopes selected in step (a).

2. A method for identifying one or more common cancer epitopes, wherein the method comprises the following steps: (a) Identifying peptides derived from non-canonical translation initiation and / or termination of a given gene, preferably an oncogene, and selecting one or more common cancer epitopes from the identified peptides.

3. The method according to any one of claims 1 to 2, wherein step (a) comprises the following steps: (a1) Predicting peptides derived from non-canonical translation initiation and / or termination of a given gene, (a2) Identifying 8-mer to 15-mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules, among the predicted peptides identified in step (a1), (a3) Identifying epitopes found in healthy subjects and / or healthy tissues from the sequences of the 8-mer to 15-mer epitopes identified in step (a2) and excluding said epitopes, and (a4) Selecting one or more epitopes found in at least one cancer from the remaining epitopes of step (a3).

4. The method according to any one of claims 1 to 3, wherein the gene is an oncogene, preferably an oncogene selected from the group consisting of or comprising c-myc and IGF1R, preferably wherein the oncogene is c-myc.

5. The method according to any one of claims 1 to 4, wherein the cancer is a c-myc or IGF1R-related cancer, preferably the cancer is breast cancer or colon cancer.

6. The method according to any one of claims 1 to 5, wherein the method further comprises in vitro validation of the selected epitopes after step (a).

7. The method according to claim 6, wherein the in vitro validation comprises at least one, preferably three, of the following steps: (i) Evaluating the induction of CD8+ T cell responses by the selected epitopes, (ii) Evaluating the functionality of CD8+ T cells specific for the selected epitopes, and / or (iii) Evaluating the cytotoxicity of CD8+ T cells specific for the selected epitopes in tumor cells and non-tumor cells, Optionally, wherein the in vitro validation further comprises step (iv) evaluating the expression of the selected epitopes in tumor cells, preferably wherein the expression is evaluated by ribosome profiling or mass spectrometry.

8. A peptide comprising or consisting of an epitope identified or generated by the method according to any one of claims 1 to 7.

9. A peptide, said peptide comprising or consisting of an epitope having a sequence selected from the group consisting of the following: LLLEATANL (SEQ ID NO:1), SLTDLYLRI (SEQ ID NO:2), AMSPQLHNI (SEQ ID NO:4), GLAAPAPKL (SEQ ID NO:5), GLPPHPAHL (SEQ ID NO:6), GMPWPIPAV (SEQ ID NO:7), SLQETSYAL (SEQ ID NO:8), SLY PIACSL (SEQ ID NO:9), SVLGHDFSV (SEQ ID NO:10), VQDMIQTQV (SEQ ID NO:11), ILDDWLRHL (SEQ ID NO:12) and SLPSQHWSL (SEQ ID NO:13), preferably, wherein the peptide comprises or consists of an epitope having a sequence selected from the group consisting of or consisting of: LLLEATANL (SEQ ID NO:1) and SLTDLYLRI (SEQ ID NO:2).

10. An expression vector that induces the expression of one or more of the peptides of claim 8 or claim 9.

11. A cytotoxic T lymphocyte of a subject, wherein the subject is treated with one or more of the peptides of claim 8 or claim 9, or one or more of the expression vectors of claim 10.

12. A cytotoxic T lymphocyte that is produced in vitro by stimulating T cells with one or more of the peptides of claim 8 or claim 9 or one or more of the expression vectors of claim 10.

13. An engineered T cell that expresses a T cell receptor that recognizes the peptide of claim 8 or claim 9.

14. One or more of the peptides of claim 8 or claim 9, one or more of the expression vectors of claim 10, one or more of the cytotoxic T lymphocytes of claim 11 or claim 12, or one or more of the engineered T cells of claim 13, for use as a vaccine or a medicament.

15. One or more of the peptides of claim 8 or claim 9, one or more of the expression vectors of claim 10, one or more of the cytotoxic T lymphocytes of claim 11 or claim 12, or one or more of the engineered T cells of claim 13, for the treatment or prevention of at least one cancer in a subject in need thereof.

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