Methods for generating one or several shared cancer epitopes derived from alternative translational control

Abstract

The present invention relates to methods for generating or identifying one or several shared cancer epitopes, as well as peptides comprising or consisting of the epitopes identified or generated by the methods, expression vectors encoding the peptides, cytotoxic T lymphocytes (CTLs) generated in vitro by stimulation of T cells with the peptides or vectors, CTLs from subjects treated with the peptides or vectors, and engineered T cells expressing a T cell receptor that recognizes the peptides. The present invention also relates to the use of the peptides, expression vectors, CTLs, or engineered T cells as vaccines or medicaments, particularly the use of the peptides, expression vectors, CTLs, or engineered T cells in a subject in need thereof to prevent or treat at least one cancer.

Description

[Technical Field]

[0001] The present invention relates to methods for generating or identifying one or several shared cancer epitopes, as well as peptides comprising or consisting of the epitopes identified or generated by the methods, expression vectors encoding the peptides, cytotoxic T lymphocytes (CTLs) generated in vitro by stimulation of T cells with the peptides or vectors, CTLs from subjects treated with the peptides or vectors, and engineered T cells expressing a T cell receptor that recognizes the peptides. The present invention also relates to the use of the peptides, expression vectors, CTLs, or engineered T cells as vaccines or medicaments, particularly the use of the peptides, expression vectors, CTLs, or engineered T cells in a subject in need thereof to prevent or treat at least one cancer. [Background technology]

[0002] Adaptive T cell immune responses in cancer depend on the recognition of tumor epitopes specifically expressed by tumor cells. The role of neoantigens generated by nonsynonymous mutations specific to tumor genomes has been extensively studied over the past decade, and numerous clinical trials testing neoantigen combinations in personalized cancer vaccines have been initiated, with promising preliminary results. However, determining the optimal combination of neoepitopes for each patient remains challenging. Furthermore, many tumors are characterized by low or moderate tumor mutation burden. Therefore, identifying other families of tumor antigens, such as those derived from alternative translational control, that are likely shared between different patients with the same cancer or different cancer subtypes is paramount for the development of off-the-shelf therapies in oncology.

[0003] In this application, we developed a new method for identifying shared cancer epitopes derived from alternative translational control. In particular, we discovered a new epitope derived from the c-myc gene that can induce specific T cell responses, and demonstrated that the induced T cells can recognize and kill tumor cells. Summary of the Invention

[0004] The present invention provides a method for generating one or several shared cancer epitopes, the method comprising the following steps: (a) identifying peptides derived from non-canonical translation initiation and / or translation termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among the identified peptides; (b) generating the epitopes selected in step (a).

[0005] The present invention also provides a method for identifying one or several shared cancer epitopes, the method comprising the following steps: (a) identifying peptides derived from non-canonical translation initiation and / or translation termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among the identified peptides.

[0006] In one embodiment, step (a) comprises the steps of: (a1) predicting peptides derived from non-canonical translation initiation and / or translation termination of a given gene; (a2) identifying 8-15mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules, from the predicted peptides identified in step (a1); (a3) identifying peptides (or epitopes) found in healthy subjects and / or healthy tissues from the sequences of 8-15 mer peptides (or epitopes) identified in step (a2) and excluding the peptides (or epitopes); (a4) selecting one or several epitopes found in at least one cancer from among the remaining peptides (or epitopes) of step (a3).

[0007] In one embodiment, the gene is an oncogene, preferably an oncogene selected from the group comprising or consisting of c-myc and IGF1R, preferably the oncogene is c-myc.

[0008] In one embodiment, the cancer is a c-myc or IGF1R associated cancer, preferably the cancer is breast cancer or colon cancer.

[0009] In one embodiment, the method further comprises in vitro validation of the selected epitopes after step (a).

[0010] In one embodiment, the in vitro validation comprises at least one, preferably three of the following steps: (i) assessing the induction of CD8+ T cell responses by the selected epitopes; (ii) assessing the functionality of CD8+ T cells specific for the selected epitopes; and / or (iii) assessing the cytotoxicity of CD8+ T cells specific for the selected epitope in tumor cells and non-tumor cells; Optionally, the in vitro validation further comprises step (iv) of assessing expression of the selected epitope in tumor cells, preferably where expression is assessed by ribosome profiling or mass spectrometry.

[0011] The present invention further relates to peptides comprising or consisting of epitopes identified or generated by the methods as described above.

[0012] The present invention further relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising 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 or consists of an epitope having a sequence selected from the group comprising or consisting of LLLEATANL (SEQ ID NO: 1), SLTDLYLRI (SEQ ID NO: 2) and IMTASNWTL (SEQ ID NO: 3).

[0013] The present invention further relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising 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), preferably the peptide comprises or consists of an epitope having a sequence selected from the group comprising or consisting of LLLEATANL (SEQ ID NO: 1) and SLTDLYLRI (SEQ ID NO: 2).

[0014] The present invention further relates to expression vectors that direct the expression of one or more peptides as described above.

[0015] The present invention further relates to cytotoxic T lymphocytes of a subject treated with one or more peptides as described above or one or more expression vectors as described above.

[0016] The present invention further relates to cytotoxic T lymphocytes generated in vitro by stimulation of T cells with one or more peptides as described above or one or more expression vectors as described above.

[0017] The present invention further relates to engineered T cells that express a T cell receptor that recognizes a peptide as described above.

[0018] The present invention further relates to one or more peptides as described above, one or more expression vectors as described above, one or more cytotoxic T lymphocytes as described above, or one or more engineered T cells as described above, for use as a vaccine or medicament.

[0019] The present invention further relates to one or more peptides as described above, one or more expression vectors as described above, one or more cytotoxic T lymphocytes as described above, or one or more engineered T cells as described above, for use in treating or preventing at least one cancer in a subject in need thereof.

[0020] definition In the present invention, the following terms have the following meanings:

[0021] "C-myc" refers to the proto-oncogene myc, which encodes a nuclear protein involved in nucleic acid metabolism and mediates cellular responses to growth factors. Cleavage of the first exon, which appears to regulate c-myc expression, is critical for tumorigenesis. The human c-myc gene is located at 8q24 on the long arm of chromosome 8.

[0022] "Epitope" refers to a part of an antigen that is capable of stimulating an immune response.

[0023] A "shared cancer epitope" refers to an epitope that is not specific to a given subject. A shared cancer epitope can be shared between different patients with the same cancer tissue type or different cancer tissue types.

[0024] "Frameshift" refers to a change in the open reading frame by one or more bases in either the 5' or 3' direction during translation.

[0025] "Insulin-like growth factor 1 receptor" or "IGFR1" refers to a gene on chromosome 15q26.3 that encodes a tyrosine kinase receptor with high binding affinity for insulin-like growth factors that play an important role in transforming events in cell growth and survival.

[0026] An "internal ribosome entry site" or "IRES" is a sequence that can recruit ribosomes and allow translation.

[0027] "Mass spectrometry" refers to an analytical method used to determine the identity of a chemical substance based on its mass using a mass analyzer / mass spectrometer.

[0028] "Non-canonical initiation and / or termination of gene translation" refers to non-conventional mechanisms of translation initiation and / or termination that can be induced under stress conditions, such as hypoxia, apoptosis, starvation, and viral infection. Conventional mechanisms for translation initiation include recruitment of 40S ribosomes to the cap structure at the 5' end of mRNA, followed by linear scanning of 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 of the coding sequence), and IRES-dependent translation initiation.

[0029] "Oncogene" refers to a gene whose gain-of-function alteration leads to neoplastic cell transformation. They include, for example, genes for activators or stimulators of cell proliferation, such as growth factors, growth factor receptors, protein kinases, signal transducers, nuclear phosphoproteins, and transcription factors.

[0030] "Open reading frame" or "ORF" refers to a sequence of nucleotide triplets encoding amino acids located between a start codon and a stop codon in the same reading frame.

[0031] "Peptide" refers to a linear 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. Amino acid residues in peptides 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 VaI 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 GIu or E; cysteine ​​is Cys or C; tryptophan is Trp or W; arginine is Arg or R; glycine is GIy or G, and peptides may contain non-standard amino acids, which refer to amino acids that have been chemically modified after being incorporated into a protein (called "post-translational modifications"), and amino acids that exist in vivo but are not found 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, gamma-carboxyglutamate, phosphoserine, phosphothreonine, phosphotyrosine, and inositol.

[0032] "Prevent," "preventing," and "prevention" refer to prophylactic and preventative measures aimed at reducing the likelihood that a subject will develop a pathological condition or disorder over a given period of time. Such reduction may be reflected, for example, in a delay in the onset of at least one symptom of the pathological condition or disorder in the subject.

[0033] "Read-through" refers to the process in translation where a stop codon is interpreted as a sense codon.

[0034] "Ribosome profiling" or "ribosequencing" refers to a method based on deep sequencing of ribosome-protected mRNA fragments (also called "footprints"). Ribosome footprints typically reveal the precise location between the start and stop codons of a gene, enabling global and experimental identification of genomic coding regions. It also allows for the precise positioning of ribosomes on mRNA.

[0035] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting medical treatment, receiving medical treatment, has been / is / will be the subject of medical treatment, or is being monitored for the development of 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, and the like. Preferably, the mammal is a primate, more preferably a human.

[0036] A "therapeutically effective amount" refers to a 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 intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or halt the progression, progression, or worsening of one or more symptoms of a disease, disorder, or condition; (3) result in an improvement in 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 harmful side effects to the target. A therapeutically effective amount can be administered prior to the onset of a disease, disorder, or condition for prophylactic or preventative purposes. Alternatively, or additionally, a therapeutically effective amount can be administered after the onset of a disease, disorder, or condition for therapeutic purposes.

[0037] "Treating" or "treatment" or "palliative" refers to therapeutic treatment. The goal is to slow (reduce) the targeted pathological condition or disorder. A subject or mammal is successfully "treated" for cancer if, after receiving a therapeutic amount of one or more peptides, one or more expression vectors, one or more cytotoxic T lymphocytes, or one or more engineered T cells according to the present invention, the patient exhibits an observable and / or measurable reduction in one or more of the following: a reduction in the number of cancer cells (or tumor size); a reduction in the percentage of total cells that are cancerous; and / or some alleviation of one or more symptoms associated with a particular disease or condition; a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing successful treatment and improvement of disease are readily measurable by routine procedures familiar to physicians.

[0038] "Vaccine" refers to a compound that, when administered to a subject, is capable of inducing a humoral and / or cellular immune response, which immune response is protective.

[0039] "Vector" or "expression vector" means a vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell in order to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences.

[0040] "uORF" refers to a short coding sequence adjacent to the start and stop codons upstream of the main open reading frame (mORF). DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides a method for generating one or several shared cancer epitopes, the method comprising the following steps: (a) identifying peptides derived from non-canonical translation initiation and / or translation termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among the identified peptides; (b) generating the epitopes selected in step (a).

[0042] The present invention also provides a method for identifying one or several shared cancer epitopes, the method comprising the following steps: (a) identifying peptides derived from non-canonical translation initiation and / or translation termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among the identified peptides.

[0043] In one embodiment, step (a) is performed by in silico techniques.

[0044] In one embodiment, step (a) comprises the step (a1) of predicting peptides derived from non-canonical translation initiation and / or translation termination of a given gene.

[0045] In one embodiment, step (a1) comprises predicting peptides derived from at least one mechanism of non-canonical translation initiation and / or translation termination of a given gene.

[0046] Examples of non-canonical translation initiation and / or translation termination mechanisms for a given gene include, but are not limited to, frameshifting of the open reading frame, stop codon readthrough, translation of the 5' UTR and / or 3' UTR, or IRES-dependent translation initiation.

[0047] "Translation of the 5'UTR and / or 3'UTR" or "translation of the 3' and / or 5' region" means that the 5'UTR and / or 3'UTR of a given mRNA, which are not normally translated by standard translation, are translated. Translation of the 5'UTR means that translation initiation begins upstream of the start codon. Translation of the 3'UTR means that translation termination stops downstream of the stop codon and / or that translation initiation occurs 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.

[0048] In one embodiment, at least one mechanism of non-canonical translation initiation and / or translation termination of a given gene is selected from the following: Frameshift of the open reading frame, Stop codon readthrough, Translation of the 5'UTR and / or 3'UTR, IRES-dependent translation initiation, and / or · Upstream ORF (uORF).

[0049] In one embodiment, at least one mechanism of non-canonical translation initiation and / or translation termination of a given gene is selected from the following: Frameshift of the open reading frame, Stop codon readthrough, Translation of the 5'UTR and / or 3'UTR, and / or ·IRES-dependent translation initiation.

[0050] In one embodiment, step (a1) comprises predicting peptides derived from one, two or three mechanisms of non-canonical translation initiation and / or translation termination of a given gene.

[0051] In one embodiment, the one, two, or three mechanisms of non-canonical translation initiation and / or translation termination for a given gene are selected from among the following: Frameshift of the open reading frame, Stop codon readthrough, Translation of the 5'UTR and / or 3'UTR, and / or ·IRES-dependent translation initiation.

[0052] In one embodiment, step (a1) comprises predicting peptides derived from four mechanisms of non-canonical translation initiation and / or translation termination for a given gene: Frameshift of the open reading frame, Stop codon readthrough, Translation of the 5'UTR and / or 3'UTR, and ·IRES-dependent translation initiation.

[0053] In one embodiment, step (a1) comprises predicting peptides derived from one, two, three or four mechanisms of non-canonical translation initiation and / or translation termination for a given gene selected from the following: Frameshift of the open reading frame, Stop codon readthrough, Translation of the 5'UTR and / or 3'UTR, and IRES-dependent translation initiation, and / or · Upstream ORF (uORF).

[0054] In one embodiment, step (a) comprises step (a2) of identifying 8- to 15-mer peptides that bind to MHC class I molecules from among the predicted peptides identified in step (a1). When used, step (a2) allows for the selection of 8- to 15-mer peptides that bind to MHC class I molecules, i.e., epitopes that bind to MHC class I molecules, from among the peptides identified in step (a1).

[0055] Analytical tools for predicting sequence (i.e., epitope) binding to MHC molecules 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).

[0056] In one embodiment, the MCH class I molecule is an HLA molecule. In one embodiment, the MCH class I molecule is selected from the group comprising or consisting of HLA-A, HLA-B and HLA-C molecules. In one embodiment, the MCH class I molecule is an HLA-A molecule, for example, an HLA-A2 molecule.

[0057] In one embodiment, the 8-15 mer peptide (or epitope) identified in step (a2) binds strongly to an MHC class I molecule, preferably an HLA-A2 molecule.

[0058] In one embodiment, strong binding peptides (or epitopes) are selected using a percentile rank of 0.5% or less, which is based on the likelihood of the peptide being presented when compared to a pool of natural ligands.

[0059] In one embodiment, the peptides (or epitopes) identified in step (a2) are 8-15mer peptides, ie peptides comprising 8-15 amino acids.

[0060] In one embodiment, the peptides (or epitopes) identified in step (a2) are 9-10mer peptides, ie peptides comprising 9-10 amino acids.

[0061] In one embodiment, the peptides (or epitopes) identified in step (a2) are 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-mer peptides, i.e., peptides comprising 8, 9, 10, 11-, 12-, 13-, 14-, or 15-amino acids, respectively. In one embodiment, the peptides (or epitopes) identified in step (a2) are 9-mer peptides.

[0062] In one embodiment, step (a) includes step (a3) ​​of identifying and excluding peptides (or epitopes) found in healthy subjects (i.e., subjects not affected by cancer) from the sequence of 8-15 mer peptides (or epitopes) identified in step (a2). This step particularly makes it possible to confirm that the selected peptides (or epitopes) do not match any self-proteins in subjects not affected by cancer.

[0063] In one embodiment, step (a3) ​​comprises aligning the sequences of the 8- to 15-mer peptides (or epitopes) identified in step (a2) with a normal human proteome (i.e., a human proteome from a healthy subject) and excluding those with complete sequence identity to the normal human proteome. Alignment can be performed using the BLAST protein database or the refseq_protein database.

[0064] In one embodiment, step (a3) ​​comprises comparing the sequences of the 8-15 mer peptides (or epitopes) identified in step (a2) with a normal tissue proteomics database (i.e., a tissue proteomics database from healthy tissues) and excluding those found in healthy / normal tissues. Data from the normal tissue proteomics database can be found, for example, in public databases such as the Genotype-Tissue Expression (GTEx) database.

[0065] As used herein, healthy or normal tissue refers to tissue that is not affected by cancer.

[0066] In one embodiment, step (a3) ​​comprises at least one of the steps described above (ie, alignment with the human proteome or comparison with a tissue proteomics database).

[0067] In one embodiment, step (a3) ​​comprises the above two steps. Thus, in one embodiment, step (a3) ​​comprises the following steps: aligning the sequences of the 8-15 mer peptides (or epitopes) identified in step (a2) with the normal human proteome and excluding those that have complete sequence homology with the normal human proteome; comparing the sequences of the remaining peptides (or epitopes) with a normal tissue proteomics database and excluding those found in healthy / normal tissues; Includes:

[0068] In one embodiment, step (a) comprises step (a4) of selecting one or several epitopes found in at least one cancer from among the remaining peptides (or epitopes) of step (a3). Thus, step (a4) allows for the selection of one or several epitopes that are commonly shared by, for example, different patients with the same cancer or different cancers.

[0069] In one embodiment, step (a4) comprises selecting, from among the remaining peptides (or epitopes) from step (a3), one or several epitopes found in at least one cancer proteomic mass spectrometry database.

[0070] Examples of cancer proteomic mass spectrometry databases include, but are not limited to, data from The Cancer Genome Atlas (TCGA) and data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC).

[0071] In one embodiment, step (a4) comprises selecting, from among the remaining peptides (or epitopes) of step (a3), one or several epitopes found in at least one, at least two, or more cancers.

[0072] In one embodiment, at least one cancer is a c-myc-associated cancer. In one embodiment, at least one cancer is an IGF1R-associated cancer.

[0073] As used herein, a c-myc-associated cancer is a cancer in which deregulation of c-myc expression occurs. As used herein, an IGF1R-associated cancer is a cancer in which deregulation of IGF1R expression occurs.

[0074] Examples of c-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.

[0075] In one embodiment, the at least one cancer is selected from the group comprising or consisting of 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.

[0076] In one embodiment, the at least one cancer is colon cancer. In one embodiment, the at least one cancer is breast cancer.

[0077] In one embodiment, step (a4) comprises selecting one or several epitopes found in at least one cancer from among the remaining peptides (or epitopes) of step (a3), wherein the cancer is breast cancer or colon cancer.

[0078] In one embodiment, step (a4) comprises selecting one or several epitopes found in breast and colon cancer from among the remaining peptides (or epitopes) of step (a3).

[0079] In one embodiment, the shared cancer epitope is an epitope found in at least one of the cancers described above. In one embodiment, the shared cancer epitope is an epitope found in breast cancer and / or colon cancer.

[0080] In one embodiment, step (a) comprises at least one, preferably four steps (a1), (a2), (a3), and (a4), as described herein above.

[0081] In one embodiment, step (a) comprises the steps of: (a1) predicting peptides derived from non-canonical translation initiation and / or translation termination of a given gene; (a2) identifying 8-15mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules, from the predicted peptides identified in step (a1); (a3) identifying peptides (or epitopes) found in healthy subjects (i.e., subjects not affected by cancer) and / or healthy tissues from the sequences of 8-15 mer peptides (or epitopes) identified in step (a2) and excluding the peptides (or epitopes); (a4) selecting one or several epitopes found in at least one cancer from among the remaining peptides (or epitopes) of step (a3).

[0082] In one embodiment, step (a) comprises the steps of: (a1) predicting peptides derived from non-canonical translation initiation and / or translation termination of a given gene; (a2) identifying 8-15mer peptides (i.e., epitopes) that bind to MHC class I molecules, preferably HLA-A molecules, more preferably HLA-A2 molecules, from the predicted peptides identified in step (a1); (a3) identifying peptides (or epitopes) found in healthy subjects and / or healthy tissues from the sequences of 8-15 mer peptides (or epitopes) identified in step (a2) and excluding peptides (or epitopes), comprising the steps of: aligning the sequences of the 8-15 mer peptides (or epitopes) identified in step (a2) with the normal human proteome and excluding those that have complete sequence homology with the normal human proteome; and Comparing the sequences of the remaining peptides (or epitopes) with normal tissue proteomics databases and excluding those found in healthy / normal tissues and the steps to be performed in (a4) selecting one or several epitopes found in at least one cancer from among the remaining peptides (or epitopes) of step (a3).

[0083] In one embodiment, the given gene is an oncogene.

[0084] 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.

[0085] In one embodiment, the gene is an oncogene, preferably selected from the group including or consisting of c-myc and IGF1R.

[0086] In one embodiment, the gene is c-myc. In one embodiment, the gene is IGF1R.

[0087] In one embodiment, step (b) comprises generating the epitope identified in step (a). The epitope may be produced by any technique known to those skilled in the art, including expression of a protein, polypeptide, or peptide by standard molecular biology techniques, such as, for example, recombinant approaches, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide.

[0088] As used herein, chemical synthesis refers to the synthesis of the epitope identified in step (a) by sequentially adding amino acids and / or fragments of the epitope to the mixture for reaction with the growing chain. Chemical synthesis can be performed in liquid phase or solid phase.

[0089] As used herein, a recombinant approach relates to the synthesis of an epitope identified in step (a) by expressing an expression vector encoding the epitope in a host cell and collecting the epitope produced by the host cell.

[0090] In one embodiment, the method for obtaining such an epitope comprises: introducing the epitope-encoding vector into a competent host cell in vitro or ex vivo; culturing in vitro or ex vivo host cells transformed with the expression vector under conditions suitable for expression of the epitope; optionally selecting cells that express and / or secrete the epitope; and recovering the expressed epitope.

[0091] In one embodiment, the method as described above further comprises a step of in vitro validation of the selected epitopes after step (a).

[0092] In one embodiment, the above method is combined with one or several of the steps described below: In one embodiment, the above method is combined with one, two, three or four of the steps described below:

[0093] In one embodiment, the in vitro validation comprises step (i) assessing the induction of a T cell response, such as a CD8+ T cell response, by the selected epitope.

[0094] In one embodiment, induction of a T cell response is assessed by measuring the induction of T cells, eg, CD8+ T cells, specific for a selected epitope.

[0095] Examples of methods for inducing CD8+ T cells include in vitro or ex vivo priming assays using selected epitopes. For example, human monocyte-derived dendritic cells can be pulsed with a selected epitope to induce specific CD8+ T cells.

[0096] Examples of methods for assessing CD8+ T cell induction include, for example, dextramer-based assays.

[0097] In one embodiment, the 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.

[0098] In one embodiment, the functionality of T cells, e.g., CD8+ T cells or TCR-engineered T cells, specific for a selected epitope is assessed by measuring the production of IFN-γ, TNFα, or Granzyme B in the presence of epitope-stimulating cells. Exemplary methods for measuring the production of such molecules include, for example, flow cytometry, ELISA, or fluorospot assays.

[0099] In one embodiment, the functionality of T cells, such as T cells specific for a selected epitope, e.g., CD8+ T cells or TCR-engineered T cells, is assessed by measuring extracellular staining of markers such as 4-1BB (CD137) or CD107a.

[0100] In one embodiment, the in vitro validation comprises step (iii) evaluating the cytotoxicity of T cells, such as CD8+ T cells or TCR-engineered T cells, specific for the selected epitope in tumor cells and non-tumor cells.

[0101] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably.

[0102] In one embodiment, the cytotoxicity of T cells specific for a selected epitope is assessed by measuring cell death of cells displaying the cognate epitope on their cell surface in the presence of epitope-specific T cells, e.g., CD8+ T cells or TCR-engineered T cells.

[0103] In one embodiment, cells do not naturally express the selected epitope and can be pulsed with the cognate epitope. As an example, T2 cells pulsed with the selected epitope can be co-cultured with CD8+ T cells or TCR-engineered T cells specific for the selected epitope and T2 cell death measured.

[0104] As used herein, a "cell that does not naturally express a selected epitope" means that the cell does not naturally contain the molecular machinery required for expression of the selected epitope and its expression on the cell surface.

[0105] In one embodiment, the cells naturally express the selected epitope. As an example, tumor cell lines such as MDA-MB-231 or HCT116 may be co-cultured with CD8+ T cells or TCR-engineered T cells specific for the selected epitope, and tumor cell death may be measured.

[0106] As used herein, a "cell that naturally expresses a selected epitope" means that the cell naturally contains all of the molecular machinery necessary for the expression of the selected epitope and its expression on the cell surface.

[0107] In one embodiment, the in vitro validation comprises step (iv) of assessing the expression of the selected epitope in tumor cells.

[0108] In one embodiment, expression of selected epitopes in tumor cells is assessed by ribosome profiling (or ribo-sequencing).

[0109] In one embodiment, expression of selected epitopes in tumor cells is assessed by mass spectrometry.

[0110] In one embodiment, expression of selected epitopes in tumor cells is assessed by the valid-NEO method. As used herein, valid-NEO is a multi-omics platform for the detection and quantification of neoantigens from limited clinical samples.

[0111] In one embodiment, the in vitro validation comprises at least one, preferably three, of the following steps: (i) assessing the induction of CD8+ T cell responses by the selected epitopes; (ii) assessing the functionality of T cells, e.g., CD8+ T cells or TCR-engineered T cells, specific for the selected epitope; and / or (iii) assessing the cytotoxicity of T cells (e.g., CD8+ T cells or TCR-engineered T cells) specific for the selected epitope in tumor cells and non-tumor cells; Optionally, the in vitro validation further comprises step (iv) of assessing expression of the selected epitope in tumor cells, preferably where expression is assessed by ribosome profiling or mass spectrometry.

[0112] Compared to prior art methods, the methods described herein may offer one or more of the following advantages: In some embodiments, the methods described herein enable detection of cancer epitopes with higher sensitivity compared to prior art methods; In some embodiments, the methods described herein enable identification of cancer epitopes that are weakly expressed in tumors and cannot be identified by prior art methods; In some embodiments, the methods described herein, particularly when applied to cancer genes, enable detection of shared cancer epitopes, i.e., shared by different subjects suffering from the same type of cancer or different subjects suffering from different types of cancer; In some embodiments, the methods described herein, particularly when applied to cancer genes, enable detection of cancer epitopes with a low risk of escape due to deletion or mutation (i.e., cancer epitopes with a low risk of being deleted or mutated during a patient's lifetime).

[0113] The present invention further relates to peptides comprising or consisting of epitopes identified or generated by the methods as described above.

[0114] The present invention relates to the following genes: 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), SLDHLLEA (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), Sequence number 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), GLFSLMFL (SEQ ID NO: 40), GLKPWTQYA (SEQ ID NO: 41), KLFGF CFQL (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), FLVKK KFFV (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 ( The present invention relates to a peptide comprising or consisting of an epitope having a sequence selected from the group consisting of: SEQ ID NO: 78), TLLSIPNYV (SEQ ID NO: 79), TLSFFTLKL (SEQ ID NO: 80), VQMEPTHFV (SEQ ID NO: 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), HRPPPPAATL (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).

[0115] The present invention relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising or consisting of FLLMPLSFL (SEQ ID NO: 14), IILGIVFLL (SEQ ID NO: 15), LLLEATANL (SEQ ID NO: 1), SLDHLLLLEA (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), or VMFFKSQHL (SEQ ID NO: 30),

[0116] The present invention relates to 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), and GLKPWTQYA (SEQ ID NO: 43). 2), 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 ID NO: 81),and YLSPFGHEI (SEQ ID NO: 82).

[0117] The present invention relates to peptides comprising or consisting of an epitope having a sequence selected from the group comprising or consisting of 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).

[0118] The present invention relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising or consisting of LLEATANLL (SEQ ID NO: 90), KSMLFLWKR (SEQ ID NO: 91), MLFLWKRGR (SEQ ID NO: 92), KMRKKSMLF (SEQ ID NO: 93), HRPPPPAATL (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).

[0119] The present invention relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising 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).

[0120] The present invention relates to a peptide comprising or consisting of an epitope having a sequence selected from the group comprising 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).

[0121] In one embodiment, the peptide comprises or consists of an epitope having a sequence selected from the group comprising or consisting of LLLEATANL (SEQ ID NO: 1), SLTDLYLRI (SEQ ID NO: 2) and IMTASNWTL (SEQ ID NO: 3).

[0122] In one embodiment, the peptide comprises or consists of an epitope having a sequence selected from the group comprising or consisting of LLLEATANL (SEQ ID NO: 1) and SLTDLYLRI (SEQ ID NO: 2).

[0123] In one embodiment, the peptide comprises or consists of an epitope of the sequence LLLEATANL (SEQ ID NO: 1).

[0124] In one embodiment, the peptide comprises or consists of an epitope of the sequence SLTDLYLRI (SEQ ID NO: 2).

[0125] In one embodiment, the peptide comprises or consists of an epitope of the sequence IMTASNWTL (SEQ ID NO: 3).

[0126] The present invention also relates to expression vectors that direct the expression of one or more peptides as described above.

[0127] The vector may be, in particular, an RNA vector, a DNA vector, or a plasmid, a viral vector, or a bacterial vector. Depending on the nature of the vector, and as is well known to those skilled in the art, the expression cassette may or may not be integrated into the host cell genome. The expression vector or expression cassette may further comprise elements necessary for the in vivo expression of a nucleic acid (polynucleotide) in a subject. For example, this may consist of an initiation codon (ATG), a stop codon, a promoter, and, for certain vectors, such as plasmids and viral vectors other than poxviruses, a polyadenylation sequence. The ATG may be located 5' of the reading frame, and the stop codon may be located 3'. As is well known, other elements that allow expression to be controlled may be present, such as enhancer sequences, stabilizing sequences, and signal sequences that allow the secretion of the peptide.

[0128] Regarding RNA vectors, the vector may use, for example, non-replicating mRNA or self-amplifying RNA derived from a virus. Conventional mRNA-based vectors may encode a peptide of interest and may contain 5' and 3' untranslated regions (UTRs). Self-amplifying RNA may not only encode the peptide of interest, but also the viral replication mechanism that allows intracellular RNA amplification and abundant protein expression.

[0129] Examples of viral vectors include, but are not limited to, lentiviruses and retroviruses.

[0130] The present invention also relates to cytotoxic T lymphocytes (CTLs) of a subject treated with one or more peptides as described above.

[0131] The present invention also relates to cytotoxic T lymphocytes (CTLs) of a subject treated with one or more expression vectors as described above.

[0132] The present invention also relates to cytotoxic T lymphocytes (CTLs) generated in vitro by stimulation of T cells with one or more peptides or one or more expression vectors as described above.

[0133] The present invention also relates to a T cell receptor (TCR) that recognizes such a peptide.

[0134] The present invention also relates to engineered T cells that express a TCR that recognizes such peptides (ie, TCR-engineered T cells).

[0135] The process of preparing these T cells is known to those skilled in the art. It can be as follows: (i) TCR α chain and β chain are isolated from T cells that recognize the above-mentioned peptide and inserted into a vector; (ii) T cells isolated from the peripheral blood of a patient or donor are modified with such a vector to encode the desired TCR αβ sequence; (iii) These modified T cells are then expanded in vitro to obtain a sufficient number for treatment and administered to patients. Of note, the TCR sequence can be modified to optimize TCR affinity.

[0136] The present invention also relates to one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above for use as a vaccine.

[0137] The present invention also relates to one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above for use as a medicament.

[0138] The present invention also relates to one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above for use in treating or preventing at least one cancer in a subject in need thereof.

[0139] In one embodiment, the one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above are for use in treating or preventing at least one, at least two, or more cancers.

[0140] In one embodiment, at least one cancer is a c-myc-associated cancer. In one embodiment, at least one cancer is an IGF1R-associated cancer.

[0141] Examples of c-myc and IGF1R associated cancers are provided hereinabove.

[0142] In one embodiment, the at least one cancer is selected from the group comprising or consisting of 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.

[0143] In one embodiment, the at least one cancer is colon cancer. In one embodiment, the at least one cancer is breast cancer.

[0144] In one embodiment, the one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above are for use in treating or preventing at least one cancer, wherein the cancer is breast cancer or colon cancer.

[0145] In one embodiment, the one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above are for use in treating or preventing breast and colon cancer.

[0146] The present invention also relates to a method for treating or preventing at least one cancer in a subject in need thereof, comprising administering to the subject one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above.

[0147] In one embodiment, the method of the present invention is for treating or preventing at least one, at least two or more cancers as defined herein.

[0148] In one embodiment, the method of the present invention is for treating or preventing breast and / or colon cancer.

[0149] The present invention also relates to one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above for the manufacture of a medicament for treating or preventing at least one cancer in a subject in need thereof.

[0150] In one embodiment, the medicament is for treating or preventing at least one, at least two or more cancers as defined herein.

[0151] In one embodiment, the medicament is for treating or preventing breast cancer and / or colon cancer.

[0152] In one embodiment, the one or more peptides, one or more expression vectors as described above induce an immune response, such as a T cell response.

[0153] Those skilled in the art will be familiar with various assays for determining whether an immune response to a tumor-associated epitope has been generated. Various B- and T-lymphocyte assays are well known, such as cytotoxic T-lymphocyte (CTL) assays such as ELISA, chromium release assays, cytometry-based assays or real-time cytotoxicity assays, proliferation assays using peripheral blood lymphocytes (PBLs), tetramer assays, and cytokine production assays.

[0154] Therefore, the present invention also relates to a method for inducing an immune response in a subject in need thereof, comprising administering to the subject one or more peptides or one or more expression vectors as described above.

[0155] In one embodiment, the one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above reduce 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, comprising administering to a subject in need thereof a therapeutically effective amount of one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above.

[0156] In one embodiment, the one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above reduce tumor volume in vivo. Accordingly, the present invention also relates to a method for reducing tumor volume in vivo, comprising administering to a subject in need thereof a therapeutically effective amount of one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above.

[0157] In one embodiment, one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above have cytotoxic activity against tumor cells but do not have a cytotoxic effect on normal cells (i.e., non-tumor cells).

[0158] In one embodiment, one or more peptides, one or more expression vectors, one or more CTLs, or one or more engineered T cells as described above are administered in a therapeutically effective amount.

[0159] However, it will be understood that the total daily usage of the one or more peptides, one or more expression vectors, one or more CTLs or one or more engineered T cells as described above will be determined by the attending physician within the scope of sound medical judgment.

[0160] The specific dose for any particular subject will depend upon a variety of factors, including the condition being treated and the severity of the condition; the activity of the particular compound used; the particular composition used, the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed; the duration of treatment; drugs used in combination or concomitantly with the particular compound employed; and similar factors well known in the medical arts.

[0161] For use in administration to a subject, one or more peptides, one or more expression vectors, one or more CTLs or one or more engineered T cells should be formulated for administration to a subject.

[0162] One or more peptides or one or more expression vectors as described above may be administered by enteral or parenteral administration routes.

[0163] One or more CTLs or one or more engineered T cells as described above can be administered by a parenteral route of administration.

[0164] The enteral route may be selected from the group consisting of the buccal route (including the lingual and sublingual route), the oral route and the rectal route.

[0165] Parenteral includes any route that is not enteral. The parenteral route can be selected from the group consisting of epicutaneous, transdermal, intradermal, subcutaneous, nasal, intramuscular, intraocular, intravitreal and intravitreal routes. Sequence Listing [Table 1] JPEG2025531430000002.jpg253167JPEG2025531430000003.jpg14168 [Brief explanation of the drawings]

[0166] [Figure 1A]Figure 1A is a combination diagram and table. A schematic of the bioinformatic prediction pipeline for identifying potential neoepitopes derived from noncanonical translations of key cancer genes. (1) Predict all peptides of 9-mer length or greater that could result from noncanonical translation. (2) From these predicted peptides, potential HLA-A*02:01 strong-binding 9-mer epitopes are identified. (3) Epitopes with complete sequence homology to the human proteome are removed from the selection. (4) A proteomic mass spectrometry database from tumor and normal tissues is used to filter the predicted HLA-A*02:01 strong-binding epitopes. [Figure 1B] Figure 1B is a combined diagram and table listing all predicted HLA-A*02-01 strong binding epitopes for MYC (left column) and IGF1R (right column) analysis using netMHCpan. [Figure 1C] Figure 1C is a combined diagram and table showing the list of predicted HLA-A*02-01 strong binding epitopes that were found to be "confident" at least once in the tumor tissue proteomics mass spectrometry database but "unconfident" in the normal tissue database for MYC (left column) and IGF1R (right column) analyses using Pepquery. [Figure 1D] List of predicted class I HLA strong binding epitopes for MYC non-canonical peptides containing PR3 epitopes using netMHCpan and MHCflurry. [Figure 1E] List of predicted class I HLA weak binding epitopes for MYC non-canonical peptides containing PR3 epitopes using netMHCpan and MHCflurry. [Figure 1F] Distribution of binding scores for predicted class I HLA strong (upper panel) and weak (lower panel) binding epitopes for MYC non-canonical peptide-containing PR3 epitopes.

[0167] [Figure 2A]Figure 2A is a composite graph showing representative plots of dextramer staining of CD8+ T cells after 12 days of specific peptide-pulsed (top row) or non-pulsed (bottom row) MoDC:CD8+ naive T cell priming. [Figure 2B] Figure 2B is a composite graph of the dextramer analysis after 12 days of MoDC:CD8+ naive T cell priming in 12 healthy donors.

[0168] [Figure 3] Representative plots of dextramers staining of CD8+ T cells after 12 days of 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).

[0169] [Figure 4A] Figure 4A is a combination of two histograms: representative graphs of the percentage of 4-1BB expression on peptide-specific or nonspecific CD8+ T cells after contact with irrelevant (nonspecific) or specific peptide-pulsed T2 cells for three donors for PR3 status and one donor for PR5 status. [Figure 4B] Figure 4B is a combination of two histograms: representative graphs of the percentage of CFSE+ T2 cell death pulsed with irrelevant (nonspecific) or specific peptides after contact with peptide-specific or nonspecific CD8+ T cells for two donors for PR3 status and one donor for PR5 status.

[0170] [Figure 5A] Figure 5A is a combination of two histograms: a representative graph of IFN-gamma produced by peptide-specific or non-specific CD8+ T cells after contact with T2 cells pulsed with irrelevant (non-specific) or specific peptides. [Figure 5B]Figure 5B is a combination of two histograms: a representative graph of TNF-alpha concentrations produced by peptide-specific or non-specific CD8+ T cells after contact with irrelevant (non-specific) or specific peptide-pulsed T2 cells.

[0171] [Figure 6A] Figure 6A is a combination of two graphs showing real-time cell death quantification by Incucyte of MDA-MB-231 cell lines (pulsed or not with PR3 peptide) co-cultured with PR3-specific CD8+ T cells or their negative counterparts (dextramer-neg T cells). [Figure 6B] Figure 6B is a combination of two graphs showing real-time cell death quantification by Incucyte of HCT116 cell lines (pulsed or not with PR3 peptide) co-cultured with PR3-specific CD8+ T cells or their negative counterparts (dextramer-neg T cells).

[0172] [Figure 7A] Figure 7A is a composite graph showing the evolution of the PR3 (LLLEATANL) epitope in a panel of tumor cell lines (MDA-MB-231) using the Valid-NEO bioinformatics pipeline (Complete Omics Inc., Maryland, USA). [Figure 7B] Figure 7B is a composite graph showing the evolution of the PR3 (LLLEATANL) epitope in a panel of tumor cell lines (B.OVCAR-3) using the Valid-NEO bioinformatics pipeline (Complete Omics Inc., Maryland, USA). [Figure 7C]Figure 7C is a composite graph showing the evolution of the PR3 (LLLEATANL) epitope in a panel of HLA-A2+ normal human primary cells using the Valid-NEO bioinformatics pipeline (Complete Omics Inc., Maryland, USA).

[0173] [Figure 8] Histogram showing IFN-gamma concentrations produced by PR3-specific CD8+ T cells or their negative counterparts (dextramer-neg T cells) after 48 hours of coculture with either HLA-A2+ normal human primary cells (cardiomyocytes, bronchial epithelial cells, or keratinocytes) or a tumor cell line (MDA-MB-231). [Example]

[0174] The present invention is further illustrated by the following examples.

[0175] A bioinformatic prediction pipeline to identify potential neoepitopes derived from non-canonical translations of major cancer genes All transcripts of two major oncogenes, MYC and IGF1R, have been identified using the genome browser Ensembl. For each transcript of each oncogene, a bioinformatics pipeline was set up to predict all peptide sequences with a size of 9 mers or greater that could result from translation defects (frameshifts (+1) or (-1), translation termination defects, e.g., stop codon readthrough) and / or any non-canonical initiation (translation in uORFs, IRESs, 5'UTRs, or 3'UTRs). Among these sequences, strong 9-mer binding epitopes (% rank ≤ 0.5) for HLA-A*02:01 were predicted using an epitope prediction tool (netMHCpan v4.1). Potential epitopes with complete sequence homology to the human proteome were removed from previous selections (BLAST protein, refseq_protein database) (Figure 1A). This analysis identified 17 potential strong binding epitopes for HLA-A*02:01 for the MYC oncogene analysis and 62 for the IGF1R oncogene analysis (Fig. 1B).

[0176] Filtering proteomics mass spectrometry (MS) databases A targeted peptide search engine was used to filter potential translational epitopes based on mass spectrometry-based proteomics datasets (PepQuery v1.6.2). Epitopes that were found to be reliable at least once in the breast or colon cancer proteomics mass spectrometry databases (patient tumor datasets: TCGA and CPTAC) but not in the normal tissue proteomics database (GTEx) were selected (Figure 1A). For MYC oncogene analysis, two epitopes (PR3 and PR5) were selected from among 17 previously predicted epitopes. For IGF1R oncogene analysis, 10 epitopes were selected from among 62 previously predicted epitopes (Figure 1C).

[0177] Prediction of class I HLA epitopes derived from one of the MYC non-canonical predicted peptides containing the PR3 epitope Using a previous bioinformatic prediction pipeline, we identified several amino acid sequences derived from non-canonical translations of the MYC oncogene. One of them (the sequence of SEQ ID NO: 83) was predicted to contain the PR3 sequence and yield the epitope PR3 after processing. SEQ ID NO: 83 MRRHRPPPAATLRRNKKMRKKSMLFLWKRGRLLAKGQSLDHLLLEATANLLTAHWSSRGATSPHISTTTQRLPPLGRTILLPRGSSWTVSES

[0178] Using this sequence, we predicted all 9-mer strong and weak binding epitopes for frequent HLA class I alleles using epitope prediction tools (netMHCpan v4.1 or MHCFlurry) (Figures 1D-F).

[0179] Peptide synthesis Peptides were synthesized (JPT Peptide Technology, Germany) and their identity was confirmed by mass spectrometry analysis by the vendor. Purity >95% was expected and determined by high-performance liquid chromatography. Lyophilized peptides were dissolved in deionized water <5% DMSO, aliquoted, and stored at -20°C until use.

[0180] PBMC priming assay for HLA-A*02:01 healthy donors PBMCs were obtained by Ficoll density gradient centrifugation of blood from HLA-A*02:01 healthy donors ("Etablissement Français du Sang", EFS, Lyon). For the priming assay, PBMCs were rapidly thawed at 37°C, extensively washed, and left at room temperature for 2 hours before their viability was assessed. Monocytes were isolated by positive selection of CD14+ cells (Miltenyi). Monocyte-derived dendritic cells (MoDCs) were generated from a 4-day culture of CD14+ monocytes 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). MoDCs were then matured and pulsed overnight (18 hours) with peptides (10 μg / ml for specific peptides, 2.5 μg / ml for positive control), IL-4 (10 ng / ml), GM-CSF (800 UI / ml), TNFα (20 ng / ml), and Poly-IC (40 μg / ml) (or not pulsed as a negative control). Meanwhile, CD8+ naive T cells were isolated from thawed autologous PBMCs (Miltenyi kit) and cultured overnight in AIM-V medium containing 5% human serum albumin (sAB) and 1% PS supplemented with recombinant human IL-7 (5 ng / ml). The next day, pulsed moDCs were co-cultured with CD8+ naive T cells (moDC:CD8+ naive T cell ratio 1:4) in 48-well plates containing AIM-V + 5% sAB + 1% PS supplemented with human recombinant IL-21 (60 ng / ml) for 12 days. Over a 12-day period, cells were expanded from 48-well plates to 12-well and then 6-well plates in AIM-V 5% sAB + 1% PS supplemented with IL-7 and IL-15 (10 ng / ml on days 3 and 5, respectively; 20 ng / ml on days 7 and 10). The optimized peptide MART1 (ELAGIGILTV) was used as a positive control for the priming assay.

[0181] Dextramer analysis On day 12, peptide-specific CD8+ T cells were identified using dextramer staining (Immudex). For each peptide condition, peptide-pulsed and non-pulsed cells were stained with the corresponding dextramer. For staining, 3x10 cells were used. 6 Cells were placed in polypropylene tubes and washed with FACS buffer. Cells were stained with 8 μL of Dextramar for 10 minutes at room temperature in the dark, and then stained with 1 / 400 diluted Zombi Near Infra-Red (NIR) for another 10 minutes in the dark to assess viability (Biolegend). Anti-CD3 BV421 and anti-CD8 FITC (Biolegend) antibodies were added for 20 minutes in the dark at 4°C. Cells were then washed twice with FACS buffer and resuspended in FACS buffer for flow cytometry analysis (FACS Fortessa BD).

[0182] Figure 2A shows the CD8+ T cells stained with dextramer in peptide-specific pulsed cells (top row) versus unpulsed cells (bottom row) for each peptide condition. For the MART1-positive control, up to 18.2% of CD8+ T cells were positive after stimulation with the MART1 peptide, compared with 0.1% in the unpulsed condition. Interestingly, stimulation with specific epitopes (PR3 and PR5) resulted in 0.094% and 0.068% of dextramer-positive CD8+ T cells for PR3 and PR5, respectively, compared with 0.001% and 0.002% in the unpulsed condition. Figure 2B summarizes the results obtained in 12 different donors, with dark boxes indicating peptide-specific CD8+ T cells were detected after priming, and white boxes indicating otherwise. Among these, peptide-specific CD8+ T cell priming was identified in four donors for PR3 and two donors for PR5.

[0183] Peptide-specific CD8+ T cell sorting After 12 days of MoDC:CD8+ naive T cell priming and validation of peptide-specific CD8+ T cells by dextramer staining analysis, cells were sorted using two different protocols.

[0184] In the first, cells from MoDC:CD8+ naive T cell priming were stained using the same protocol as for the dextramer analysis, but without anti-CD3 antibody. After gating on viable CD8+ T cells, dextramer-positive cells, corresponding to peptide-specific CD8+ T cells, and dextramer-negative cells, corresponding to their nonspecific counterparts, were sorted using a BD FACSAria™ Cell Sorter.

[0185] For the second protocol, peptide-specific CD8+ T cells were selected using peptide-specific monomers coupled to magnetic beads. In a 1.5 ml tube, 10 μL of peptide-specific biotinylated monomers at 100 μg / ml (P2R facility, Nantes, France) were incubated with 10 μL of dynabeads M-280 streptavidin in PBS 1X 0.1% BSA (100 μL final volume) on a rotating shaker at room temperature for 1 hour. The formed magnetic peptide-specific monomers were then washed three times with PBS 1X 0.1% BSA using a DynaMag Spin Magnet. MoDCs: 5.10 from CD8+ naive T cell priming. 6 The cells are washed and resuspended in 500 µL of PBS 1X 0.1% BSA. They are mixed with preformed magnetic monomers and incubated for 4 hours on a rotating shaker at room temperature. The cells are then washed 8-10 times with PBS 1X 0.1% BSA using a DynaMag Spin Magnet. The only fraction sorted in this protocol is peptide-specific CD8+ T cells bound to magnetic peptide-specific monomers.

[0186] Supply Protocol Peptide-specific CD8+ sorted T cells and nonspecific counterparts were expanded at a 10:1 ratio on feeders composed of 35 Gy-irradiated allogeneic PBMCs and a B lymphoblastoid cell line. Feeder cells were seeded into 96-well round-bottom plates at a concentration of 0.10 × 10 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). A maximum of 5 × 10 sorted cells were added per well. Cells were cultured for 14 days. Starting from day 5, every two days (days 5, 7, 9, and 12), half of the medium was replaced with RPMI 8% sAB 1% PS supplemented with human recombinant IL-2 (300 UI / ml), human recombinant IL-7 (20 ng / ml), and human recombinant IL-15 (20 ng / ml), or each well was split in half depending on the growth. After 12 days, the purity of the specific fraction versus the nonspecific fraction was assessed. If 65% or more of the dextramer-positive CD8+ T cells were present in the positive fraction and less than 0.5% of the dextramer-positive CD8+ T cells were present in the negative fraction, the cells were used for cytotoxicity experiments.

[0187] The results in Figure 3 show dextramer staining of CD8+ T cells after 12 days of moDC:CD8+ naive T cell priming, as well as after sorting and expansion of peptide-specific CD8+ T cells or nonspecific counterparts. CD8+ T cell populations that were 98.6% specific for the PR3 peptide and 69% specific for the PR5 peptide were sorted and expanded from cells obtained from peptide-pulsed moDC:CD8+ T cell priming.

[0188] Cytotoxicity and functional assays using T2 cells T2 (SD cell line) is a lymphoblastoid cell line deficient in transporter-associated antigen processing (TAP) protein and therefore unable to present endogenous peptides on class I MHC, but can be used to monitor cytotoxic T lymphocyte (CTL) responses to exogenous antigens of interest in a non-competitive environment.

[0189] T2 cells were first stained with a CFSE cell division tracking kit (Biolegend) for 13 minutes at 37°C and then washed three times. CFSE-stained T2 cells were pulsed with an irrelevant (nonspecific) or specific peptide for 2 hours at 37°C. After extensive washing, CFSE-pulsed T2 cells were resuspended in the corresponding T cell medium, RPMI + 8% sAB + 1% PS. T cells were then rested. First, CD8+ T cells were resuspended overnight in RPMI 8% sAB + 1% PS supplemented with 50 UI / ml of human recombinant IL-2 at 37°C. The next day, CD8+ T cells were resuspended in their corresponding medium without cytokine supplementation for 2 hours at 37°C. CFSE-pulsed T2 cells and T cells were then co-cultured in duplicate in 96-well U-bottom plates (T2:CD8+ T cell ratio 1:10). After 24 hours, supernatants were collected for further ELISA analysis, and cells were pooled into V-well plates and washed with FACS buffer. For each condition, a mixture of antibodies containing Zombi NIR (diluted at 1 / 400), anti-CD3 BV421, anti-CD8 APC, and anti-human CD137 (4-1BB) Pe-Dazzle594 (Biolegend) antibodies is added for 30 min at 4 °C in the dark. Cells are washed and resuspended in FACS buffer before flow cytometry analysis (FACS Fortessa BD).

[0190] Figure 4A shows the percentage of 4-1BB expression in peptide-specific or nonspecific CD8+ T cells after contact with irrelevant or specific peptide-pulsed T2 cells. Figure 4B shows the percentage of T2 cell death after contact with peptide-specific CD8+ T cells or nonspecific counterparts. These results demonstrate the specific activation (A) and specific killing (B) of PR3- and PR5-specific CD8+ T cells upon specific antigen stimulation.

[0191] For ELISA analysis, the IFN gamma Human Uncoated ELISA Kit (Invivogen) and the TNF alpha Human Uncoated ELISA Kit (Invivogen) were used according to the manufacturer's instructions. Different dilutions of supernatant were tested (diol 1 / 2, 1 / 5, 1 / 10, or 1 / 20), and absorbance results within the standard range were analyzed.

[0192] The results in Figure 5 show quantification of cytokine (A. IFN-gamma and B. TNF-alpha) secretion by peptide-specific and non-specific CD8+ T cells after contact with irrelevant or specific peptide-pulsed T2 cells. Previous results indicate specific IFN-gamma and TNF-alpha secretion by PR3- and PR5-specific CD8+ T cells upon specific antigen stimulation.

[0193] Validation of epitope presentation by mass spectrometry Mass spectrometry-based epitope validation was performed by Complete Omics Inc. (Maryland, 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, peptides were lyophilized before further analysis. To eliminate excessively noisy ions due to coelution with impurities and increase detectability through recursive optimization of significant ions, transition parameters for each epitope peptide were examined and curated through the Valid-NEO method development bioinformatics pipeline.

[0194] The results in Figures 7A-7B show the transition parameters of the PR3 epitope in two tumor cell lines, MDA-MB-231 and OVCAR-3. Similar quantitative changes were identified when analysis was performed using one or more heavy peptides eluted from HLA from either the MDA-MB-231 or OVCAR-3 cell lines, confirming the presentation of the PR3 epitope on the surface of both tumor cell lines.

[0195] In contrast, when a panel of HLA-A2+ normal human primary cells (cardiomyocytes, keratinocytes, astrocytes, kidney proximal tubule cells, or bronchial epithelial cells, Figure 7C) was analyzed, no alterations were identified, implying that the PR3 epitope was not presented by normal cells. These data suggest that the PR3 epitope is a tumor-specific and safe target.

[0196] In vitro cytotoxicity of PR3-specific T cells against tumor cell lines using InCucyte technology To monitor tumor cell death in real time, we performed an immune cell killing assay using 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. In the positive control condition, the cell lines were pulsed with specific peptides to artificially present the target epitope on class I MHC. The day before the experiment, tumor cell lines were seeded at 5,000 cells / well in a 96-well flat-bottom plate. For the previous T cells, which were used for cytotoxicity and functional analysis against T2 cells, they were rested overnight with cytokine supplementation and then for 2 hours the following day without cytokines. Meanwhile, tumor cell lines were pulsed with or without 10 μg / ml of specific peptide for 2 hours, followed by three washes. T cells were added to the corresponding wells (CD8+ T cell:tumor cell line ratio 2:1). In the tumor cell death control condition, DMSO (final 20%) was added to the medium. Finally, add Incucyte® Cytotox Green for Counting Dead Cells (Sartorius) to reach a final concentration of 250 nM per well. This reagent enters cells when plasma membrane integrity is compromised and increases fluorescence 100-1000-fold upon binding to deoxyribonucleic acid (DNA). Live imaging was performed for 56 hours at 37°C and 5% CO2 using an Incucyte Zoom. For analysis, calculate the number of dead tumor cells per well by assessing the number of green fluorescent tumor cells per well.

[0197] The results in Figure 6 show the kinetics of tumor cell death after co-culture with PR3-specific CD8+ T cells or non-specific counterparts (dextramer-neg T cells) for the MDA-MB-231 cell line (A) or HCT116 cell line (B), as well as representative images for each condition at 24 h. For both cell line conditions, these results demonstrate increased cell death when tumor cells were co-cultured with PR3-specific CD8+ T cells compared to their negative counterparts. Consistent with epitope-specific reactivity, a further increase in cell death was observed when tumor cells were pulsed with PR3 and co-cultured with PR3-specific CD8+ T cells.

[0198] Safety analysis of PR3-specific T cells against HLA-A2+ normal human primary cells 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, a positive control for cytotoxicity, were seeded at 5,000 cells per well in a 96-well flat-bottom plate. For cytotoxicity and functional analysis against T2 cells, T cells were rested overnight with cytokine supplementation and then rested for 2 hours the following day without cytokines. T cells were added to the corresponding wells (CD8+ T cell:tumor cell line ratio 10:1). After 48 hours of coculture, supernatants were collected for further ELISA analysis using an IFN gamma human uncoated ELISA kit (Invivogen) as previously described.

[0199] The results in Figure 8 show quantification of IFN-gamma secretion between PR3-specific CD8+ T cells or nonspecific counterparts (dextramer-neg T cells) after 48 hours of coculture with normal human primary cells and the tumor cell line MDA-MB-231 as a positive control for cytotoxicity. When PR3-specific CD8+ T cells were cocultured with any of the normal human primary cells tested, no IFN-g secretion was detected, suggesting the safety of the product. We observed IFN-gamma secretion when PR3-specific CD8+ T cells were cocultured with the tumor cell line MDA-MB-231 (positive control).

[0200] Collectively, these experiments demonstrate that PR3-specific CD8+ T cells specifically recognize and are functional on target cells (T2 cells) presenting the cognate peptide, without any toxicity to normal human primary cells, and specifically recognize and kill tumor cells (MDA-MB-231 and HCT116) expressing endogenous peptides derived from noncanonical translation.

Claims

1. 1. A method for generating one or several shared cancer epitopes, said method comprising the steps of: (a) identifying peptides derived from non-canonical translation initiation and / or termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among said identified peptides; (b) generating the epitopes selected in step (a).

2. 1. A method for identifying one or several shared cancer epitopes, said method comprising the following steps: (a) identifying peptides derived from non-canonical translation initiation and / or translation termination of a given gene, preferably an oncogene, and selecting one or several shared cancer epitopes from among said identified peptides.

3. The step (a) comprises the steps of: (a １ a.) predicting said peptides derived from non-canonical translation initiation and / or translation termination of a given gene; (a ２ ) Step (a １ identifying 8-15mer 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 the above step; (a ３ ) Step (a ２ Identifying epitopes found in healthy subjects and / or healthy tissues from the sequences of the 8-15 mer epitopes identified in step (1) and excluding said epitopes; (a ４ ) Step (a ３ 3. The method of claim 1, further comprising the step of selecting one or several epitopes found in at least one cancer from among the remaining epitopes of said method.

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

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

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

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

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

9. 1. A peptide comprising or consisting of an epitope having a sequence selected from the group comprising 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), preferably said peptide comprising or consisting of an epitope having a sequence selected from the group comprising or consisting of LLLEATANL (SEQ ID NO: 1) and SLTDLYLRI (SEQ ID NO: 2).

10. 10. An expression vector directing the expression of one or more peptides according to claim 8 or claim 9.

11. Cytotoxic T lymphocytes of a subject treated with one or more peptides according to claim 8 or claim 9 or one or more expression vectors according to claim 10.

12. Cytotoxic T lymphocytes generated in vitro by stimulation of T cells with one or more peptides according to claim 8 or claim 9 or one or more expression vectors according to claim 10.

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

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

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

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