A tumor-specific HLA-bound neoantigenic peptide encoded by EWSR1::FLI1-induced neogenes

Abstract

The present invention provides tumor-specific HLA-bound neoantigenic peptides encoded by EWSR1::FLI1-Induced neogenes, vaccinal compositions comprising such tumor-specific neoantigenic peptides, nucleic acids, antibodies or fragments thereof and immune cells that can be used in cancer therapy, particularly in Ewing sarcoma.

Description

[0001] A TUMOR-SPECIFIC HLA-BOUND NEOANTIGENIC PEPTIDE ENCODED BY E W SRI ::FLI1 -INDUCED NEOGENES

[0002] FIELD OF THE INVENTION:

[0003] The present disclosure provides tumor-specific HLA-bound neoantigenic peptides encoded by EWSR1::FLI1 -Induced neogenes, vaccinal compositions comprising such tumorspecific neoantigenic peptides, nucleic acids, antibodies or fragments thereof and immune cells that can be used in cancer therapy, particularly in Ewing sarcoma.

[0004] BACKGROUND OF THE INVENTION:

[0005] Ewing sarcoma (EwS) is the second most frequent bone and soft tissue cancer of childhood and adolescence. EwS is a highly aggressive cancer with a 5-year overall survival of only 30% for patients with metastases. These metastatic diseases are often resistant to intensive therapy and associated with acute and chronic adverse effects. EwS possesses one of the lowest mutation rates among cancer with immune tolerance and low immunogenicity (Morales et al., 2021). However, preliminary results from the Ewing cell vaccination Vigil study suggest that Ewing cells can generate an immune response to yet uncharacterized tumour-associated antigens (Ghisoli et al., 2016).

[0006] EwS is characterized by specific gene fusions between members of FET family of RNA-binding proteins and the ETS (E-twenty-six) family of transcription factors, the most frequent fusion being between EWSR1 and FLU in 85% of cases (Delattre et al., 1992; Grunewald et al., 2018). EWSR1::FLI1 acts as an aberrant tumour-specific transcriptional factor reprogramming the genome through binding DNA at two types of DNA motives, i.e. bona fide ETS family sites centered on a single GGAA / T motif and GGAA microsatellite repeats. As compared to wild-type ETS transcription factors, EWSR1::FLI1 has the neomorphic activity to generate neo-enhancers upon binding GGAA microsatellite sequences (Grunewald et al., 2018).

[0007] The inventors recently showed that EWSR1::FLI1 can drive transcription and processing of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome upon binding specific GGAA microsatellite sequences (Vibert et al., 2022). These novel transcribed genomic regions were termed Ewingspecific neogenes (Ew_NG). The inventors further showed that peptides encoded by some ofthese neogenes could be detected by whole cell proteomics (Vibert et al., 2022). The exquisite tumour specificity of these neogenes and of encoded peptides make them attractive as a source of neo-epitopes.

[0008] Identifying neoantigenic peptides that are specifically expressed by tumor cells and not by healthy cells is crucial for eliciting a targeted immune response against cancer. Therefore, there is a need to and it is essential to detect these tumor-specific neoantigenic peptides, which are expressed in tumor cells and recognized by the immune cells of a patient with cancer or at risk of developing cancer. Identifying these tumor-specific neoantigenic antigens enables the administration of the corresponding peptides as a vaccine to prevent the growth of tumor cells expressing these antigens. This allows the patient's immune cells to specifically target the tumor cells. By specific identification and selection of antigens that are not present in healthy cells, the risk of autoimmune recognition is minimized.

[0009] The inventors investigated the possibility that open reading frames (ORFs) derived from these neogenes represent a source of immunogenic tumor-specific neoantigens. To explore this unexpected ability, the inventors performed MHC-I immunopeptidomics on EwS cell lines and patients-derived xenografts (PDXs) models. The inventors identified a set of neopeptides derived from Ew NGs which was specifically identified in EwS samples. The inventors demonstrated that tumour-specific peptides can be presented to T cells by Ewing cell MHC-I molecules and are able to functionally activate T cells through HLA- restricted recognition. The inventors demonstrated the proof of concept of immunogenic function of tumor-specific neoantigenic peptides encoded by EWSR1: ELI 1 -Induced neogenes which highlight an unexpected potential of these Ew NGs to generate HLA class Lassociated peptides in EwS.

[0010] There is no disclosure in the art of the tumor-specific HLA-bound neoantigenic peptides encoded by EWSR1: ELI 1 -Induced neogenes, and their use in the treatment of cancer, particularly in Ewing sarcoma.

[0011] SUMMARY OF THE INVENTION:

[0012] The invention relates to methods and pharmaceutical compositions for the treatment of cancer, particularly Ewing sarcoma. In particular, the invention is defined by the claims.DETAILED DESCRIPTION OF THE INVENTION:

[0013] The inventors investigated the role and specific contribution of EWSR1::FLI1 -Induced neogenes in providing tumor-specific HLA-bound neoantigenic peptides. The EWSR1::FLI1 chimeric transcription factor of Ewing sarcoma (EwS) induces transcription of EwS-specific neogenes (Ew NGs) through binding and subsequent transcription activation at GGAA microsatellites in genomic regions that are transcriptionally silent in normal tissues. The inventors showed that these Ew NGs encode proteins thus raising their potential for immunotherapies. Here the inventors demonstrated that peptides encoded by Ew NGs can be detected by HLA- 1 immunopeptidomics in EwS samples. The inventors demonstrated that CD8 T cells isolated from healthy donors and specific for Ew_NG-derived HLA-1 -bound peptides can be activated by HLA-l-matched EwS cells but not by non-EwS cells. The inventors also demonstrated that these T cells kill EwS cells in an HLA-1 restricted specific manner. This cytotoxicity depends on the expression of EWSR1::FLH and of the corresponding Ew_NG. This finding can be fully reproduced by CD8 T cells transduced with a TCR specific for an Ew_NG-derived peptide.

[0014] The chimeric transcription factor EWSR1 ::FLH generates tumor-specific neogenes that encode tumor-associated neoepitopes presented by the MHC-I molecules of Ewing cells. CD8+ T-cell clones and engineered TCR-T cells specific for the neoepitope can selectively recognize and kill EwS tumor cells in vitro, supporting the potential of neogene-derived neoantigens as therapeutic targets. Altogether, the present invention highlights the role of this specific tumorspecific neoantigenic peptide, its use in the treatment of cancer, in particular in the treatment of Ewing sarcoma.

[0015] Accordingly, in a first aspect, the invention relates to a tumor-specific neoantigenic peptide comprising at least one amino acid sequence set forth in SEQ ID No. 1 to SEQ ID No.

[0016] 16, or a conservative variant thereof.

[0017] The amino acid sequences of the tumor-specific neoantigenic peptides are disclosed in table 1.

[0018]

[0019] Table 1: Sequences of the tumor-specific neoantigenic peptides.

[0020] The term “tumor-specific neoantigenic peptide” refers to antigens generated by tumor cells as a result of various tumor-specific alterations. In the present invention, the tumorspecific neoantigenic peptides are encoded by EW SR 1::FLI1 -Induced neogenes. A tumorspecific neoantigenic peptide comprises or consists of at least one amino acid sequence set forth in SEQ ID No.l to SEQ ID No.16 or a conservative variant thereof. These tumor-specific neoantigenic peptides are expressed in EWSR1::FLI1 tumors but not in healthy cells. Any one of these tumor-specific neoantigenic peptides being specifically expressed in tumor cells, theiruse in human medicine allows the provision of a vaccine composition that leads to the production of immune cells that target tumor cells with a low risk of auto-immune recognition.

[0021] As used herein, the term “conservative variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to any one of amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 16.

[0022] As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al, 1997; Altschul et al., 2005). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman- Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

[0023] Preferably, the term "conservative variant” refers to a polypeptide having an amino acid sequence that differs from any one of sequences of SEQ ID NO: 1 to 16 by less than 3 or 2 substitutions, insertions and / or deletions. In a preferred embodiment, the conservative variantdiffers from any one of the amino acid sequences of SEQ ID NO: 1 to 16 by one or more conservative substitutions, preferably by less than 3, or 2 conservative substitutions.

[0024] By "substituted" or "modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids. The term "conservative substitution" as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and function of the peptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like).

[0025] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine).

[0026] The term “fusion gene” as used herein is a hybrid or chimeric gene formed from two previously independent genes. It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Such fusion genes have been found to be prevalent in all main types of human tumors (Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer 7, 233-245 (2007)) and can thus also be named oncogenic gene fusion. For example, the Ewing sarcoma is characterized by the reciprocal chromosomal translocation generating a fusion oncogene between the EWS gene (also named EWSR1) involved in various cellular processes, including gene expression, cell signaling, and RNA processing and transport, and an Ets family transcription factor, most commonly FLI-1. As used herein a transcription factor fusion is encoded by a fusion gene involving a gene coding for a transcription factor.

[0027] A “Neogene” or a “new gene”, as herein intended, corresponds to a region of the genome which is transcriptionally silent in normal cell or tissue, but which transcription is induced by a transcription factor fusion typically in a cancer cell wherein the cancer is driven by said transcription factor fusion. Neogenes, or new genes typically correspond to intergenic or intronic regions of the genome. A Neogene from a cancer cell as above defined thereforeencodes a “neotranscript”, which is unannotated in a database referencing the transcriptome data of the corresponding normal cell from the same organism.

[0028] In contrast to the wild-type FLU, the chimeric EWS-FLI1 has gain-of-function activities and, in particular, the ability to bind GGAA microsatellite sequences in the genome. It thus creates neomorphic enhancer regions that can interact with neighboring promoters and activate corresponding genes. EWSR1::FLI1 acts as an aberrant tumour-specific transcriptional factor reprogramming the genome through binding DNA at two types of DNA motives, i.e. bona fide ETS family sites centered on a single GGAA / T motif and GGAA microsatellite repeats. As compared to wild-type ETS transcription factors, EWSR1::FLI1 has the neomorphic activity to generate neo-enhancers upon binding GGAA microsatellite sequences. EWSR1::FLI1 can drive transcription and processing of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome upon binding specific GGAA microsatellite sequences. These novel transcribed genomic regions were termed Ewingspecific neogenes (Ew_NG).

[0029] In some embodiments, the present invention relates to a plurality or combination of different neoantigenic peptides, wherein each different neoantigenic peptide comprising or consisting of an amino acid sequence as set forth in any sequence from SEQ ID No. 1 to SEQ ID No. 16 or of a single amino acid sequence set forth in SEQ ID No. 1 to SEQ ID No. 16.

[0030] In an embodiment of the present invention, it is provided 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 different neoantigenic peptides, each different neoantigenic peptide comprising or consisting of an amino acid sequence as set forth in any sequence from SEQ ID No. 1 to SEQ ID No. 16.

[0031] In a second aspect, the invention relates to a vaccine or immunogenic composition comprising at least one tumor-specific neoantigenic peptide selected from the list consisting of amino acid sequences SEQ ID No. 1 to SEQ ID No. 16.

[0032] Typically, a vaccine or immunogenic composition comprises a pharmaceutically acceptable carrier or vehicle, a carrier substance, and / or one or more adjuvants. The pharmaceutically acceptable carriers, the carrier substances, and the adjuvants are those conventionally used. Additionally, stabilizers, diluents, excipients, and / or any other materialswell-known to those skilled in the art may be used. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.

[0033] The carrier, or vehicle, is preferably an aqueous carrier but the precise nature of the carrier or other material will depend on the route of administration. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution before administration.

[0034] The carrier substances are advantageously selected from the group consisting of unilamellar or multilamellar liposomes, ISCOMs, virosomes, viral pseudoparticles, saponin micelles, solid microspheres which are saccharide (poly(lactide-co-glycolide)) or gold-bearing in nature, and nanoparticles.

[0035] The adjuvants typically increase or expand the immune response of a host to an antigenic compound. Example adjuvants include emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, saponins, oils, Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides, cytokines, squalene and combinations thereof. Emulsifiers include, for example, potassium, sodium, and ammonium salts of lauric and oleic acid, calcium, magnesium, and aluminum salts of fatty acids, organic sulfonates such as sodium lauryl sulfate, cetyltrimethylammonium bromide, glycerylesters, polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters and their polyoxyethylene, acacia, gelatin, lecithin and / or cholesterol. Adjuvants that comprise an oil component include mineral oil, vegetable oil, or animal oil. Other adjuvants include Freund's Complete Adjuvant (FCA) or Freund's Incomplete Adjuvant (FIA). Cytokines useful as additional immunostimulatory agents include interferon alpha, interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF), or combinations thereof.

[0036] The compositions may further contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. See, for example, Butterfield, BMJ. 2015 22;350 for a discussion of cancer vaccines.In one embodiment of the present disclosure, the vaccine or immunogenic composition comprises one or more tumor-specific neoantigenic peptides as defined previously.

[0037] In another embodiment of the present disclosure, the vaccine or immunogenic composition comprises a plurality of tumor-specific neoantigenic peptides as defined previously.

[0038] In another embodiment of the present disclosure, the vaccine or immunogenic composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 tumor-specific neoantigenic peptides as defined previously.

[0039] In a particular aspect, it is provided a vaccine or immunogenic composition comprising a plurality of different tumor-specific neoantigenic peptides, each different tumor-specific neoantigenic peptide comprising or consisting of an amino acid sequence as set forth in sequences SEQ ID No. 1 to SEQ ID No. 16.

[0040] In a particular aspect, it is provided a vaccine or immunogenic composition comprising at least one copy, in particular a plurality of copies, of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 different tumor-specific neoantigenic peptides, each different tumor-specific neoantigenic peptide comprising or consisting of a single amino acid sequence as set forth in sequences SEQ ID No. 1 to SEQ ID No. 16.

[0041] In a particular aspect, it is provided a vaccine or immunogenic composition comprising at least one copy, in particular a plurality of copies, of at least one tumor-specific neoantigenic peptide comprising or consisting of an amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 16.

[0042] In a particular aspect, it is provided a vaccine or immunogenic composition comprising at least one copy, in particular a plurality of copies, of 16 different tumor-specific neoantigenic peptides, each different neoantigenic peptide comprising or consisting of a single amino acid sequence as set forth in sequence SEQ ID No. 1 to SEQ ID No. 16.

[0043] An “immunogenic composition” is to be understood as a composition that comprises or generates antigen(s) and is capable of eliciting an antigen-specific humoral or cellular immune response, e.g. T-cell response.

[0044] In one embodiment of the present disclosure, the said vaccinal or immunogenic composition comprises a plurality of tumor-specific neoantigenic peptides. Preferably, at least one copy, in particular a plurality of copies, of the 16 different tumor-specific neoantigenicpeptides, each different tumor-specific neoantigenic peptides comprising or consisting of a single amino acid sequence set forth in sequences SEQ ID No. 1 to SEQ ID No. 16.

[0045] For purposes of the present disclosure, the terms "cancer" and "cancer disease" are used interchangeably with the term "tumor" or "tumor disease".

[0046] A cancer is a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. According to the invention, the cancer may be Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

[0047] In a particular embodiment of the present invention, it is provided a vaccinal or immunogenic composition according to any embodiment disclosed herein, the tumor-specific neoantigenic peptide being encoded from a transcript associated with a EWSR1 : ELI 1 -Induced neogenes Ewing sarcoma.

[0048] As used herein, the terms “subject”, “individual” or “patient” are interchangeable and refer to a mammal. Typically, a subject according to the invention refers to any subject, preferably human. In some embodiment, the subject is afflicted or at risk to be afflicted with cancer. In some embodiments, the term “subject” refers to a subject afflicted or at risk to be afflicted with Ewing sarcoma. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with Ewing sarcoma with EWSR1 : ELIl-Induced neogenes.

[0049] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used forthe initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0050] As used herein, the term "tumor" refers to an abnormal growth of cells (called neoplastic cells, tumorigenic cells, or tumor cells) preferably forming a swelling or lesion. By "tumor cell" it means an abnormal cell that grows by a rapid, uncontrolled cellular proliferation that continues to grow after the stimuli that initiated the new growth ceases. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue and usually form a distinct mass of tissue, which may be either benign, pre-malignant, or malignant.

[0051] A malignant tumor is essentially synonymous with cancer. Malignancy, malignant neoplasm, and malignant tumor are essentially synonymous with cancer.

[0052] A benign tumor is a tumor that lacks all three of the malignant properties of a cancer. Thus, by definition, a benign tumor does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not spread to non-adjacent tissues (metastasize).

[0053] A neoplasm is an abnormal mass of tissue as a result of neoplasia. Neoplasia is the abnormal proliferation of cells. The growth of the cells exceeds and is uncoordinated with that of the normal tissues around them. The growth persists in the same excessive manner even after cessation of the stimuli. It usually causes a lump or tumor. Neoplasms may be benign, pre-malignant, or malignant.

[0054] "Growth of a tumor" or "tumor growth" according to the present disclosure relates to the tendency of a tumor to increase its size and / or to the tendency of tumor cells to proliferate."Metastasis" means the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then after being transported by blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis" which relates to a metastasis that is remote from the primary tumor and the regional lymph node system.

[0055] A relapse or recurrence occurs when a person is affected again by a condition that affects them. For example, if a patient has suffered from a tumor disease, has received a successful treatment of said disease, and again develops said disease, the newly developed disease may be considered as a relapse or recurrence. However, according to the present disclosure, a relapse or recurrence of a tumor disease may but does not necessarily occur at the site of the original tumor disease. Thus, for example, if a patient has suffered from an ovarian tumor and has received a successful treatment a relapse or recurrence may be the occurrence of an ovarian tumor or the occurrence of a tumor at a site different to the ovary. Relapse or recurrence of a tumor also includes situations wherein a tumor occurs at a site different from the site of the original tumor as well as at the site of the original tumor. Preferably, the original tumor for which the patient has received a treatment is a primary tumor and the tumor at a site different to the site of the original tumor is a secondary or metastatic tumor.

[0056] The present disclosure also encompasses a population of dendritic cells or antigen-presenting cells (APCs) that have been pulsed with one or more of the tumor-specific neoantigenic peptide or the vaccine or immunogenic composition as previously described. The present invention also encompasses a population of dendritic cells or antigen-presenting cells (APCs) transfected with a polynucleotide encoding one or more tumor-specific neoantigenic peptides as defined previously or the vaccinal or immunogenic composition hereabove. In a preferred embodiment, the dendritic cells are autologous dendritic cells.

[0057] Preferably, the antigen-presenting cells are dendritic cells (DCs) or artificial antigen-presenting cells (aAPCs). Dendritic cells (DC) are professional antigen-presenting cells (APC)that have an extraordinary capacity to stimulate naive T-cells and initiate primary immune responses to pathogens. Indeed, the main role of mature DCs is to sense antigens and produce mediators that activate other immune cells, particularly T cells. DCs are potent stimulators for lymphocyte activation as they express MHC molecules that trigger TCRs (signal 1) and costimulatory molecules (signal 2) on T cells. Additionally, DCs also secrete cytokines that support T-cell expansion. T cells require presented antigens in the form of a processed peptide to recognize foreign pathogens or tumors. Presentation of peptide epitopes derived from pathogen / tumor proteins is achieved through MHC molecules. MHC class I (MHC -I) and MHC class II (MHC -II) molecules present processed peptides to CD8+ T cells and CD4+ T cells, respectively. Importantly, DCs home to inflammatory sites containing abundant T cell populations to foster an immune response. Thus, DCs can be a crucial component of any immunotherapeutic approach, as they are intimately involved with the activation of the adaptive immune response. In the context of vaccines, DC therapy can enhance T cell immune responses to a desired target in healthy volunteers or patients with infectious disease or cancer. In one embodiment, APCS are artificial APCs, which are genetically modified to express the desired T-cell co-stimulatory molecules, human HLA alleles, and / or cytokines.

[0058] Such artificial antigen-presenting cells (aAPC) can provide the requirements for adequate T-cell engagement, co-stimulation, as well as sustained release of cytokines that allow for controlled T-cell expansion. These cells are not subject to the constraints of time and limited availability and can be stored in small aliquots for subsequent use in generating T-cell lines from different donors, thus representing an off-the-shelf reagent for immunotherapy applications. Expression of potent co-stimulatory signals on these aAPC endows this system with higher efficiency lending to increased efficacy of adoptive immunotherapy. Furthermore, aAPC can be engineered to express genes directing the release of specific cytokines to facilitate the preferential expansion of desirable T-cell subsets for adoptive transfer; such as long-lived memory T-cells (Hasan et al., 2015; Kim et al., 2004; Wang et al., 2017).

[0059] Typically, the dendritic cells are autologous dendritic cells that are pulsed with one or more tumor-specific neoantigenic peptide as defined previously or vaccinal or immunogenic composition or transfected with a polynucleotide encoding one or more tumor-specific neoantigenic peptide as defined previously, or vaccine or immunogenic composition. The antigen-presenting cell (or stimulator cell) typically has MHC class I or II molecules on its surface, and in one embodiment is substantially incapable of loading the MHC class I or IImolecules with the selected antigen. The MHC class I or II molecules may readily be loaded with the selected tumor-specific neoantigenic peptide in vitro.

[0060] In an embodiment, it is provided an antibody, or an antigen-binding fragment thereof, or a T cell receptor (TCR), or a chimeric antigen receptor (CAR) that specifically binds to at least one tumor-specific neoantigenic peptide as defined herein.

[0061] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., VHH antibodies, sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise variants modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibodies and fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and subclasses thereof, IgGl, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. In some embodiments, the antibody comprises a light chain variable domain and a heavy chain variable domain, e.g. in a scFv format.

[0062] Antibodies include variant polypeptide species that have one or more amino acid substitutions, insertions, or deletions in the native amino acid sequence, provided that the antibody retains or substantially retains its specific binding function.

[0063] In one embodiment, antibodies may be chimeric, humanized, or human antibodies. Humanized antibodies contain rodent-sequence-derived CDR regions; typically, the rodent CDRs are engrafted into a human framework, and some of the human framework residues may be back-mutated to the original rodent framework residue to preserve affinity, and / or one or a few of the CDR residues may be mutated to increase affinity. Fully human antibodies have no murine sequence and are typically produced via phage display technologies of human antibodylibraries, or immunization of transgenic mice whose native immunoglobin loci have been replaced with segments of human immunoglobulin loci.

[0064] The present disclosure also encompasses a T cell receptor (TCR), in particular in association with an MHC class I molecule, that specifically binds to the tumor-specific neoantigenic peptide. A “T cell receptor” or “TCR” refers to a complex consisting of two TCR chains and six clusters of differentiation 3 (CD3) chains. This complex is triggered by MHC class I and class II molecules with the presentation of an antigen. In the present disclosure, the complex is triggered by the MHC class I molecule specifically bound to the tumor-specific neoantigenic peptide described previously. TCR molecule contains two distinct heterodimers of two variable chains: TCRa / TCRp and TCRy / TCRS. These heterodimers are associated with CD3 complexes and expressed either on the T cell surface or in a soluble form. Usually, T cells express TCRa / TCRp on their surface. TCR is usually responsible for recognizing antigen peptides bound to major histocompatibility complex (MHC) molecules. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments or variants thereof. The term also encompasses intact or full-length TCRs, including TCRs in the aP form or y6 form. In some embodiments, the TCR contains variable a and P chains.

[0065] In some embodiments, said TCRs are made soluble and are fused to an antibody fragment directed to a T cell antigen, optionally the targeted antigen is CD3 or CD 16.

[0066] The term “fused to an antibody fragment directed to a T cell antigen” refers herein to a TCR with, on the N-term of the variable a and P chains portion, the presentation of the tumorspecific neoantigenic peptide by MHC molecules, preferentially MHC class I molecule, and on the C-terminal of the constant a and P chains portion, the fusion to an antibody fragment directed to a T cell antigen. By “T cell antigen”, it means that it targets a substance expressed on the surface of a T cell, in order to trigger an immune response. Preferentially, the targeted antigen is CD3 or CD 16.

[0067] “CD3” is a multi-protein complex that contains 6-8 and y-e heterodimers that contain extracellular and intracellular domains, and a

[0068]

[0069] homodimer that has a very short extracellular domain and a long intracellular domain. By targeting the CD3 complex, it triggers the activation of TCR of the lymphocyte. The complex contains a cryptic proline-rich sequence exposed on the cytoplasmic tail of the CD3s chain that is necessary for downstream signaling leading to T cell activation (Menon elal.. 2023).“CD16” or “FcyRIIIa” is an Immunoglobulin G (IgG) Fc receptor expressed on natural killer (NK) cells as well as phagocytes. By targeting CD16, ittriggers cytotoxicity and cytokine production. CD16 binds to the Fc portion of IgG antibodies. There are two types of CD16: CD16a and CD16b. CD16a is localized on NK cells. Upon ligation to the Fc portion of IgG antibodies, it induces a series of signals resulting in cytokine production and cytotoxic effector activity via Antibody-dependent cell-mediated cytotoxicity (ADCC). CD 16b is localized on neutrophils (Romee et al., 2013).

[0070] As enclosed above, “Chimeric antigen receptor” or “CAR” refers to an engineered molecule expressed on the cell surface that can recognize specific proteins and deliver an activation signal to the cells. CAR design can be introduced in various immune cells such as NK cells, y5 T cells, mucosal-associated invariant T (MAIT) cells, dendritic cells (DC), macrophages, regulatory T cells (Treg), B cells, or T cells. In some embodiments, CAR molecules specifically bind to tumor-specific neoantigenic peptides as defined previously.

[0071] In an embodiment of the invention, it is provided an antibody, an antigen-binding fragment thereof, a CAR or a TCR as defined herein, wherein said antibody, antigen-binding fragment thereof, CAR or T-cell receptor is a multispecific antibody or antigen-binding fragment thereof or multispecific CAR or multispecific receptor, that further targets at least an immune cell, an immune cell antigen or a tumor cell antigen, optionally wherein the immune cell is a T cell, an NK cell, or a dendritic cell, optionally wherein the immune cell antigen is CD3, CD 16, CD30 or a TCR.

[0072] In some embodiments, the invention relates to a TCR comprising a TCR a chain of SEQ ID NO: 17 (CAMQQGGSEKLVF) and a TCR 0 chain of SEQ ID NO: 18 (CATSRARPSEQYF) or a functional TCR fragments or variants thereof.

[0073] As used herein, the term “functional variant” refers to a polypeptide having the activity of the native sequence. The activity of a variant may be assessed using methods well-known by the skilled person.

[0074] As used herein, the term “functional variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95 or 99% sequence identity to amino acid sequence SEQ ID NO: 17 or 18. Preferably, the term "functional variant” refers to a polypeptide havingan amino acid sequence that differs from SEQ ID NO: 17 or 18 by less than 3 or 2 substitutions, insertions and / or deletions.

[0075] As used herein, the term “functional fragment” with respect to a TCR a or P chain polypeptide having a sequence of at least 10 consecutive amino acids and having the activity of the native sequence.

[0076] In some embodiments, the invention relates to a viral vector, particularly lentiviral vector comprising the TCR of the invention.

[0077] In some embodiments, the invention relates to an immune cell comprising the TCR of the invention. Particularly, a T cell comprising the TCR of the invention.

[0078] The term “immune cell antigen" refers to a polypeptide produced in immune cells. It can either be expressed on the surface of the cell, or inside. Preferably, the polypeptide is found on the surface of the immune cell.

[0079] The term “tumor cell antigen" refers to a polypeptide produced in tumor cells. It can either be expressed on the surface of the cell, or inside. Preferably, the polypeptide is found on the surface of the tumor cell. Typically, this tumor cell antigen triggers an immune response in the host. Tumor cell antigens are separated into two categories: Tumor-Specific Antigens (TSA), and Tumor-Associated Antigens (TAA). They differentiate by their presence only on tumor cells for TSA and their presence on some tumor cells and some normal cells for TAA. They can also be classified based on their molecular structure and source.

[0080] The term “target” or “recognize” refers to the ability of an antibody or antigen-binding fragment thereof to specifically bind to an antigen or tumor cell antigen thereof.

[0081] The term “multispecific” refers herein to an antibody or an antigen-binding fragment thereof or CAR or T-cell receptor that may bind to at least one antigen. It may bind to more than one antigen and still trigger an immune response. In the preferred embodiment, it targets at least an immune cell antigen or a tumor cell antigen. In some embodiments, it targets two or more immune or tumor cell antigens. In an embodiment, it targets a plurality of immunogenic or tumor cell antigens.In a further aspect, the present disclosure provides a polynucleotide encoding a tumorspecific neoantigenic peptide as defined previously, or an antibody or an antigen-binding fragment thereof, a CAR, or a TCR as defined in previous embodiments.

[0082] The term “polynucleotide” refers herein to a nucleic acid sequence encoding a tumorspecific neoantigenic peptide as herein disclosed. The nucleic acid may be selected from DNA, cDNA, PNA, CAN, RNA, either single- and / or double-stranded, or native or stabilized forms of nucleic acids, such as nucleic acids with a phosphorothiate backbone, or combinations thereof and it may or may not contain introns so long as it codes for the peptide. Typically, the sequence of said nucleic acid is that of the cDNA encoding said peptide polypeptide or said fusion protein.

[0083] In a further aspect, the present disclosure provides a vector comprising a polynucleotide encoding a tumor-specific neoantigenic peptide as defined previously, or an antibody or an antigen-binding fragment thereof, a CAR, or a TCR as defined in previous embodiments.

[0084] In some embodiments, the vector comprises the polynucleotide as previously defined. In some embodiments, the vector comprises one or more polynucleotide sequences encoding a tumor-specific neoantigenic peptide as previously defined. In some embodiments, the polynucleotide may be linked to a heterologous regulatory control sequence (e.g., heterologous transcriptional and / or translational regulatory control nucleotide sequences as well-known in the field). In some embodiments, the vector comprises a nucleic acid sequence encoding a tumor-specific neoantigenic peptide as previously defined.

[0085] According to the present disclosure, the term “vector” is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector that can be used in the present invention includes, in a non-limiting manner, a linear or circular DNA or RNA molecule consisting of chromosomal, non-chromosomal, synthetic or semisynthetic nucleic acids, such as in particular a viral vector, a plasmid or an RNA vector.

[0086] Numerous vectors into which a nucleic acid molecule of interest can be inserted in order to introduce it into and maintain it in a eukaryotic or prokaryotic host cell are known in themselves; the choice of an appropriate vector depends on the use envisioned for this vector (for example, replication of the sequence of interest, expression of this sequence, maintaining of this sequence in extrachromosomal form, or else integration into the chromosomal material of the host), and also on the nature of the host cell. For example, naked nucleic acids (DNA orRNA) or viral vectors such as adenoviruses, retroviruses, lentiviruses, and AAVs, into which the sequence of interest has been previously inserted may be used; said sequence (isolated or inserted into a plasmid vector) can also be combined with a substance which allows it to cross the host cell membrane, such as a transporter, for instance, a nanotransporter or a preparation of liposomes, or cationic polymers, or else makes it possible to introduce it into a said host cell using physical methods such as electroporation or microinjection. In addition, these methods can advantageously be combined, for example using electroporation combined with liposomes.

[0087] Preferably, said vector is an expression vector comprising all the elements required for the expression of a neoantigenic peptide as herein disclosed. For example, said vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate heterologous regulatory sequences for transcription and optionally for translation (promoter, enhancer, intron, start codon (ATG), stop codon, polyadenylation signal, splice site) recognized by the desired host. The polynucleotide encoding the tumor neoantigenic peptide may be linked to such heterologous regulatory control nucleotide sequences or may be nonadj acent yet operably linked to such heterologous regulatory control nucleotide sequences. The vector is then introduced into the host through standard techniques. Guidance can be found for example in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0088] In an embodiment, it is provided an immune cell that specifically binds to one or more tumor-specific neoantigenic peptides as defined herein. In some embodiments, the immune cell specifically binds to at least two tumor-specific neoantigenic peptides as defined herein. In some embodiments, the immune cell binds to several tumor-specific neoantigenic peptides as defined herein.

[0089] In an embodiment, the immune cell is allogeneic or autologous. In a preferred embodiment, the immune cell is selected from T cell, NK cell, CD4+ / CD8+, tumour infiltrating lymphocyte (TILs) / tumor-derived CD8 T cells, central memory CD8+ T cells, Regulatory T cells (Treg), Mucosal-Associated Invariant T cell (MAIT), and Y5 T cell.

[0090] In the present disclosure, an “immune cell” is a cell involved in the immune response. They are of hematopoietic origin. Immune cells include lymphocytes such as B cells and T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes. Said immune cells may originate from a healthy donor or from a subject suffering from cancer.

[0091] Immune cells can be extracted from blood or derived from stem cells. The stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human cells are CD34+ cells.

[0092] In another embodiment, said cell can be derived from a healthy donor, from a subject diagnosed with cancer. The cell can be autologous or allogeneic.

[0093] In allogeneic immune cell therapy, the cells are collected from a donor other than the patient. Usually, the donor and the patient have similar HLA to reduce the likelihood of graft vs. host disease. Once the cells are collected, the immune cells as herein disclosed can be expanded in vivo or ex vivo. The immune cells, in particular T-cells, can be activated and expanded generally using methods known in the art. Generally, the T-cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In some embodiments of the present disclosure, the immune cell can be modified to target tumor-specific neoantigenic peptides as previously defined.

[0094] In autologous immune cell therapy, the cells are collected from the individual itself. These cells are cultured and expanded outside the body using methods known in the art. In some embodiments of the present disclosure, the immune cell can be modified to target tumorspecific neoantigenic peptides as previously defined.

[0095] In some embodiments of the present disclosure, it is provided a T cell which comprises:

[0096] a T cell receptor that specifically binds to one or more tumor-specific neoantigenic peptides as defined previously, or

[0097] a CAR that specifically that specifically binds to one or more tumor-specific neoantigenic peptides as defined previously.

[0098] In an embodiment, the tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell asdefined as defined herein, for use the treatment or the prevention of cancer, or for reducing or inhibiting tumor growth, for reducing or preventing tumor metastasis, in particular for use in inhibiting cancer cell proliferation, more particularly for treating cancer in a subject in need thereof or for use in cancer vaccination therapy of a subject, optionally wherein the subject is suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes or wherein the subject is at risk of suffering from with Ewing sarcoma, particularly Ewing sarcoma with EWSR1 : ELI 1 -Induced neogenes.

[0099] In an embodiment, it is provided a method for the treatment or the prevention of cancer, or for reducing or inhibiting tumor growth, for reducing or preventing tumor metastasis, in particular for use in inhibiting cancer cell proliferation, more particularly for treating cancer in a subject in need thereof or for use in cancer vaccination therapy of a subject, optionally wherein the subject is suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSREELIl-Induced neogenes, or wherein the patient is at risk of suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSR1 : ELI 1 -Induced neogenes, the method comprising the administration of an effective amount of tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein, to a patient in need thereof.

[0100] In an embodiment, it is provided a method for treatment or the prevention of cancer, or for reducing or inhibiting tumor growth, for reducing or preventing tumor metastasis, in particular for use in inhibiting cancer cell proliferation, more particularly for treating cancer in a subject in need thereof or for use in cancer vaccination therapy of a subject, optionally wherein the subject is suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSREELIl-Induced neogenes, or wherein the patient is at risk of suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes, the method comprising:

[0101] detecting at least one tumor-specific neoantigenic peptide as defined herein in a biological sample previously obtained from a subject, the subject suffering from a cancer, in particular Ewing sarcoma with EWSR1 : ELI 1 -Induced neogenes,when at least one tumor-specific neoantigenic peptide as defined herein is detected in the biological sample, administering to the subject an effective amount of tumorspecific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein.

[0102] In an embodiment of the invention, it is provided an effective amount of tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein, for treating or preventing Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

[0103] In some embodiments, the invention relates to pharmaceutical composition, for example a therapeutic or a vaccine composition, comprising the tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined as defined herein.

[0104] In some embodiments, the tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein or the compounds of the invention may be used or prepared in a pharmaceutical composition.In one embodiment, the invention relates to the pharmaceutical composition comprising the tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein or the compounds of the invention and a pharmaceutical acceptable carrier for use in the treatment of Ewing sarcoma in a subject of need thereof, particularly Ewing sarcoma with EWSR1::FLI1-Induced neogenes.

[0105] Typically, said pharmaceutical compositions are formulations for administration, preferably sterile compositions and formulations, such as for adoptive cell therapy. The pharmaceutical composition of the invention generally comprises a sterile pharmaceutically acceptable carrier.

[0106] As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can further be incorporated into the compositions. In some aspects, the choice of carrier in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by the particular method used to administer the vector or host cells expressing the CAR. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001 to about 2% by weight of the total composition.

[0107] A pharmaceutical composition is formulated to be compatible with its intended route of administration. The pharmaceutical composition can be formulated for any conventional route of administration including a parenteral, intravenous, intramuscular, subcutaneous administration and the like.Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of cancer.

[0108] In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

[0109] In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of Ewing sarcoma in a subject of need thereof, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

[0110] In some embodiments, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of Ewing sarcoma in a subject of need thereof, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

[0111] In an embodiment of the invention, it is provided an effective amount of tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs of as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein, for use in combination with at least one further therapeutic agent, or in combination with targeted therapy, immunotherapy such as immune checkpoint therapy and immune checkpoint inhibitor, co-stimulatory antibodies, chemotherapy and / or radiotherapy.

[0112] As used herein, the term “immunotherapy” refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.

[0113] Immune checkpoint therapy such as checkpoint inhibitors include, but are not limited to programmed death- 1 (PD-1) inhibitors, programmed death ligand- 1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte-activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyteattenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) inhibitors. In particular, checkpoint inhibitors include antibodies anti-PDl, anti-PD-L1, anti-CTLA-4, anti-TIM-3, anti-LAG3. Immune checkpoint therapy also includes costimulatory antibodies delivering positive signals through immune-regulatory receptors including but not limited to ICOS, CD 137, CD27, OX-40 and GITR.

[0114] Example of anti-PDl antibodies include, but are not limited to, nivolumab, cemiplimab (REGN2810 orREGN-2810), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708, W02009114335, WO2013043569 and W02014047350. Example of anti-PD-Ll antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab and avelumab. Example of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti-CTLA4 antibodies (see, e.g., International patent applications WO 1997020574 and WO2007123737) and antibodies described in US patent US8,491,895. Example of anti-VISTA antibodies are described in US patent application US20130177557. Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578. Example of KIR inhibitor is IPH4102 targeting KIR3DL2.

[0115] In some embodiments, the compound and / or pharmaceutical composition of the invention may be used in combination with targeted therapy. As used herein, the term “targeted therapy” refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression. For example, targeted therapy agents such as therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed tointerfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin-dependent kinase inhibitor, histone deacetylase inhibitor. Targeted therapy may also use cytokines. Examples of such targeted therapy include with no limitations: Ado-trastuzumab emtansine (HER2), Afatinib (EGFR (HER1 / ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1 / 2 / 3), Belimumab (BAFF), Belinostat (HDAC), Bevacizumab (VEGF ligand), Blinatumomab (CD19 / CD3), bortezomib (proteasome), Brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK), Cetuximab (EGFR), cofimetinib (MEK), Crizotinib (ALK, MET, ROS1), Dabrafenib (BRAF), Daratumumab (CD38), Dasatinib (ABL), Denosumab (RANKL), Dinutuximab (B4GALNT1 (GD2)), Elotuzumab (SLAMF7), Enasidenib (IDH2), Erlotinib (EGFR), Everolimus (mTOR), Gefitinib (EGFR), Ibritumomab tiuxetan (CD20), Sonidegib (Smoothened), Sipuleucel-T, Siltuximab (IL-6), Sorafenib (VEGFR, PDGFR, KIT, RAF),(Tocilizumab (IL-6R), Temsirolimus (mTOR), Tofacitinib (JAK3), Trametinib (MEK), Tositumomab (CD20), Trastuzumab (HER2), Vandetanib (EGFR), Vemurafenib (BRAF), Venetoclax (BCL2), Vismodegib (PTCH, Smoothened), Vorinostat (HDAC), Ziv-aflibercept (PIGF, VEGFA / B), Olaparib (PARP inhibitor).

[0116] In some embodiments, the compound and / or pharmaceutical composition of the invention may be used in combination with chemotherapy. As used herein, the term “antitumor chemotherapy” or “chemotherapy” has its general meaning in the art and refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents. Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the syntheticanalogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-trichlorotri ethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel and doxetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophylotoxins, enzymes such as L-asparaginase; anthracenediones; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0117] In some embodiments, the compound and / or pharmaceutical composition of the invention is administered to the patient in combination with radiotherapy. Suitable examples of radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3 -dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like. Gamma rays are another form of photons used in radiotherapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, radiotherapy may be proton radiotherapy or proton minibeam radiation therapy. Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. 2019 Jun l;104(2):266-271. doi: 10.1016 / j .ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C,Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7;8(1):16479. doi: 10.1038 / s41598-018-34796-8). Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation. FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31 : 121-123. DOI: 10.1051 / medsci / 20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j ijrobp.2018.06.403. Epub 2018 Jul 11).

[0118] The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

[0119] In an embodiment, it is provided a method of selecting an tumor-specific neoantigenic peptide as defined herein, wherein the tumor-specific neoantigenic peptide obtained is for use in therapy or prevention of cancer in a subject in need thereof, optionally wherein the subject is suffering from Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

[0120] It is also provided a method for treating a subject suffering from cancer, in particular Ewing sarcoma, particularly Ewing sarcoma with EW SR 1 : ELI 1 -Induced neogenes, or wherein the patient is at risk of suffering from a cancer, in particular Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes, said method comprising:

[0121] a) Obtaining a biological sample, in particular a blood sample, more particularly a plasma sample, from a human patient;

[0122] b) Detecting in the biological sample if memory and / or CD8+ T cells specifically bind to tumor-specific neoantigenic peptide as defined herein;

[0123] c) Diagnosing the patient with a cancer, in particular Ewing sarcoma, particularly Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes, when memory and / or CD8+ T cells specifically bind to tumor-specific neoantigenic peptide as defined herein; andd) Administering to the patient suffering from a cancer an effective amount of tumor-specific neoantigenic peptide as defined herein, the vaccine or immunogenic composition as defined herein, the population of dendritic cells or APCs as defined herein, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) as defined herein, the polynucleotide as defined herein, the vector as defined herein, the immune cell as defined herein, or the T-cell as defined herein.

[0124] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0125] FIGURES:

[0126] Figure 1: Identification of MHC-I bound peptides encoded by the EwS neogenes.

[0127] (A) Workflow for the identification of EwS neogene-derived peptides by MS-based immunopeptidomics. (B) Summary of EwS neogene-derived peptides found by immunopeptidomics in each EwS sample analyzed.

[0128] Figure 2: Identification of T cells specific for Ew_NG-derived peptides.

[0129] (A) HLA-I in vitro monomer binding assay of Ew_NG-derived peptides. Relative binding compared to positive peptide controls is displayed for each HLA. Above 50% of binding relative to positive control is considered good binding. (B) Workflow for the identification of T cell clones against Ew_NG-derived peptides. (C) Clonal expansion and purity analysis. (D, E) (D) TNFa and (E) fFNy cytokine secretion after stimulation of T cell clones with increasing concentration of the peptide NeoE_4, NeoE_7 and NeoE_9.

[0130] Figure 3: Recognition and killing of EwS cells by Ew-NG-derived peptides specific T Cells. E, effector cells; T, target cells. Killing assays by NeoE_4-specific 127E10 CD8+ T cells of (A) HL A- A* 02:01 -positive (dark blue) or HL A- A* 02:01 -negative (light blue) EwS cells, HLA-A*02:01 restriction is indicated in the table on the right.

[0131] IFN-y cytokine secretion after incubation of (B) NeoE_4-specific 127E10 T cells or (C) CMV-specific pp65 T cells with EwS cells, non- EwS cells without or with anti-HLA-I blocking antibody. Results are represented as mean ± SEM, n=2.Figure 4: Activation and cytotoxicity of TCR127E10transduced T cells on Ewing cells. Killing assay by TCR127E10-T cells specific for NeoE_4 with EwS HLA-A*02:01 positive target cells or EwS HLA-A*02:01 negative target cells. Results are represented as mean ± SEM, n=2.

[0132] Figure 5: Therapeutic efficacy and safety of this TCR127E10. (A) Development of a standardized TCR activation assay using flow cytometry. (B) Peptide fingerprinting with a library of 171 peptides, each containing a single amino acid substitution of the target peptide NeoE_4. Heatmap indicate the percentage of TCR activation in response to each variant. (C) Positional scan matrix for TCR127E10. (D) Test of Uniprot peptides (SEQ ID NO: 9 (NEO 4); SEQ ID NO: 27-33 (PL1-7)) encoded by the positional scan matrix. (E) Evaluation of TCR127E10 alloreactivity using a panel of 21 lymphoblastic cell lines.

[0133] Figure 6: in vivo efficacy of TCR127E10CD8 T cells. (A) Experimental plan. (B) Individual tumor growth curves of EW-7 engrafted mice, either untreated, treated with untransduced TCR127E10CD8 T cells. (C) Representative mice per treatment group at day 18 after T-cells infusion.

[0134] EXAMPLE:

[0135] HLA-bound peptides encoded by EWSR1::FLI1-Induced neogenes constitute shared and specific immune targets in Ewing sarcoma

[0136] Materials and methods

[0137] Cell lines and patient-derived xenografts (PDXs)

[0138] A673 and A673 / TR / shEFl clone (also called ASP14) cell lines were cultured at 37°C, in 5% CO2 with Dulbecco’s Modified Eagle Medium (DMEM) with High Glucose, 4 mM of L-Glutamine (Gibco), 4,500 mg / L Glucose and sodium 5-pyruvate (HyClone) supplemented with 10% FBS (Eurobio) and 1% antibiotics (v / v) (penicillin and streptomycin (Gibco). Blasticidin (10 pg / mL; Merk, Darmstadt, Germany) and Zeocin (100 pg / mL; Invitrogen, Waltham, MA, USA) were added to A673 / TR / EF1 at 37 °C with 5% CO2. TC71, EW7, EW16, STA-ET-1, POE, MHH-ES1, Mel202, MP41 and MON cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma), 10% FBS (Eurobio), 1% antibiotics (v / v)(penicillin and streptomycin (Gibco). Culture cells were tested monthly for mycoplasma contamination.

[0139] EwS patient-derived xenograft (PDX) models (IC-pPDX-5, IC-pPDX-8, IC-pPDX-141, IC- pPDX-164 and IC-pPDX-179) were generated at Institut Curie from patients under an Institutional-Review-Board-approved protocol (OBS170323CPP ref3272; dossier No. 2015-A00464-45). All PDX tumors exhibited a fusion transcript of EWSR1::FLI1 except for IC-pPDX- 164, showing EWSR1::FEV fusion (Datas not shown).

[0140] EWSR1::FLI1 downregulation and CRISPR interference (CRISPRi) Inhibition of EWSR1::FLI1 in the A673 / TR / shEFl clone (ASP 14) was induced by the expression of EWSR1 : :FLI1 specific shRNA by adding 1 pg / mL of doxycycline in the medium ex-tempo. After seven days of treatment, doxycycline was removed and cells were washed three times to stop the shRNA induction, thus enabling re-expression of EWSR1::FLI1.

[0141] Specific NG8 inhibition was obtained by CRISPRi method targeted regulatory EWSR1::FLI1- bound microsatellites as previously described (5). The A673 cell line was previously transduced using Lenti-dCas9-KRAB-blast plasmid (Addgene) with a MOI (Multiplicity Of Infection) 3, and cultured in DMEM High Glucose (Gibco), supplemented with 10% FBS (Eurobio), 1% Penicillin / Streptomycin (Gibco) and blasticidin (20 pg / mL; Merk, Darmstadt, Germany), at 37°C in 5% CO2. Cells were plated in 6-well plates (50,000 cells per well) and cultured overnight to allow cell adhesion to wells. Cells were transfected using lipofectamine RNAiMAX transfection Reagent (ThermoFisher) an equimolar mix of NG8 or control guide RNAs and trackRNA (IDT) at a final concentration of 1 pM, diluted in OptiMeM medium (Gibco), for four days. Efficacy of CRISPRi was assessed by RT-qPCR.

[0142] RT-qPCR

[0143] RNA was extracted with the RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCRs were performed using PowerSYBR green Mastermix (Applied Biosystems). Oligonucleotides were purchased from Eurofins Genomics. Reactions were run on CFX384 Touch Real-Time PCR instrument (Bio-Rad) and analyzed using the CFX Manager Software (Bio-Rad). Relative expression level was assessed with the AACt method using RPLP0 as a housekeeping gene.Ribosome profiling

[0144] Ribosome profiling on 3 EwS cell lines (EW16, STA-ET-1 and TC71) and 5 EwS PDXs (IC- pPDX-5, IC-pPDX-8, IC-pPDX-141, IC-pPDX-164 and IC-pPDX-179) was performing by EIRNA Bio (https: / / eirnabio.com / ). Briefly, samples were flash frozen and transferred into ice- cold polysome isolation buffer supplemented with cycloheximide (Millipore Sigma, #C1988). The library was sequenced using NovaSeq 6000 (Illumina). The per base sequencing quality of each three independent replicas passed the quality threshold. Ribo-seq data analysis was conducted using RiboTricer, including filter fastq files by quality, followed by removal of the adaptor sequence, alignment with GENCODE vl 9 reference transcripts and annotation of the Ew NGs, using a minimal ORF length of 30 nucleotides. Translating ORF predictions were then performed using default parameters on Ribotricer QC data.

[0145] Immunopeptidomics

[0146] Five Ewing sarcoma cell lines (A673, EW7, EW16, STA-ET-1, TC71) and five PDX (IC-pPDX- 5, -8, -179, -164, -141) were used to search peptides associated to HLA-ABC. Cell suspensions were lysed in 50 mM Tris- HC1 pH 8, 150 mM NaCl, IX complete protease inhibitor (Roche), 5 mM EDTA, and 1% n-dodecyl-P-D-maltoside (Thermo Fisher Scientific # 89902), sonicated twice and incubated for 30 minutes at 4 °C with rotation. Samples were centrifuged at 20000 g for 1 hour at 4°C to remove insoluble fraction. Supernatants containing stabilized MHC-I-peptide complexes were quantified by BCA and incubated for 18h with CNBr activated Sepharose beads (GE Healthcare Life Sciences) coupled to anti-HLA-ABC W6 / 32 antibody (Bio X Cell). After immunoprecipitation samples were washed (i) three times with 50 mM Tris- HC1 (pH 8.0), 150 mM NaCl, and 0.5% n-dodecyl-P-D-maltoside, (ii) three times with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, (iii) one time with high salt concentration (50 mM Tris-HCl pH 8.0 and 0.5 M NaCl, (iv) three times with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, (v) one time with 20 mM Tris-HCl pH 8.0. Then MHC-I-peptide complexes were eluted with 0.25% trifluoroacetic acid (TFA).

[0147] Samples were evaporated and resuspended in 30% acetonitrile (ACN), 0.1% formic acid (FA) and injected into an HPLC system (Agilent HP 1100) for fractionation by strong cation exchange using a PolyLC sulfoethyl A column. Fractions were evaporated to dryness and reconstituted in 5% methanol / 0.5% TFA for mass spectrometry (MS) analysis. Samples wereanalyzed by liquid chromatography coupled to mass spectrometry (LC-MS / MS) using the high-resolution mass spectrometer 480 Exploris (Thermo Scientific) coupled to a Thermo Scientific Dionex Ultimate 3,000 chromatographic system. The separation was done at 300 nL / min, gradient 0- 30% ACN (0.1% FA) in 60 min. The spectrometric analysis was performed in a data dependent mode and HCD fragmentation. The range acquired was 350-900 m / z.

[0148] LC-MS / MS spectra were searched using ProteomeDiscoverer 2.5 (ThermoFisher) and MSFragger (22) against predictive EwS neoprotein database (4,938 Ew_NG ORFs) (5), EwS fusion protein (6 fusions proteins) and Swissprot Human Reference Proteome. Peptides matching with annotated proteins were discarded. FDR was set to 1. HLA compliance between class I HLA alleles of the sample and amino acid motifs of the peptides was predicted using NetMHCpan 4.0.

[0149] Class I HLA-peptide binding assays and tetramers

[0150] Biotinylated recombinant class I HLA molecules were purchased from immunAware (Copenhagen, Denmark) as easYmers (catalog #1002-1). Peptides were synthetized at >95% purity (GeneCust). Monomers were loaded with peptides by incubation at 18 °C for 48 hours (easYmers). Binding of peptide to class I HLA was measured by flow cytometry following the manufacturer’s instructions. HLA / peptide monomers were incubated with streptavidin-coated beads, stained with anti-human b2-microglobulin antibody coupled to phycoerythrin and analyzed by flow cytometry. Peptides used as positive controls (100%) of HLA binding were provided by the manufacturer: HLA-A*01:01 (YFV NS5 286-295, KSEYMTSWFY, SEQ ID NO: 21), HLA-A*02:01 (CMVpp65 495-503, NLVPMVATV, SEQ ID NO: 22), HLA-A*03:01 (CMVIE1 99-107, RIKEHMLKK, SEQ ID NO: 23), HLA-A*68:01 (CMV IE1 33-41, TTFLQTMLR, SEQ ID NO: 24), HLA-B*07:02 (CMV pp65 417-426, TPRVTGGGAM, SEQ ID NO: 25), HLA-B*51:01 (Cryptosporidium parvum, VPFVSVNPI, SEQ ID NO: 26). For each tetramer, HLA / peptide complex (100 pM) was combined for 1 hour at room temperature with fluorescent streptavidin (BioLegend). Tetramers were stored at 4 °C for a maximum of 3 months.

[0151] Tetramer & antibody staining

[0152] Peripheral blood mononuclear cells (PBMCs) were isolated using standard Ficoll-gradient procedures and either studied fresh for phenotyping or frozen. PBMC were thawed inRPMI medium (GIBCO) containing 10% FBS. Cell suspensions were incubated for 30 minutes in culture medium containing 50 nmol / L dasatinib (Lissina el al., 2009) to improve tetramer staining. To isolate and clone CD8+ T cells specific for a given HLA-peptide, healthy donors bearing the corresponding HLA allele were selected. 2xl08PBMC were first stained with live / dead aqua (Invitrogen) followed by the peptide-loaded tetramers associated to PE and APC for 20 minutes at room temperature. Then cells were washed and incubated with a mix of anti-PE and anti-APC microbeads (Miltenyi) for 20 minutes at 4°C. After washing in PBS+ (PBS 1% human male AB serum (Biowest), 2 mM EDTA (Invitrogen)) tetramer positive cells were enriched in a MS column (Miltenyi). Enriched cells were stained with anti-CD45RA FITC (BD 566349), CCR7 BV421 (BioLegend 353208), CD8 BUV395 (BD 563795) and CD3 BUV737 (BD 612750) for 20 minutes at 4°C. Cells were then washed and analyzed in an ARIA Fusion cell sorter (BD). Data were analyzed using FlowJo VI 0.10 software (BD).

[0153] T cell clone generation

[0154] Double tetramer positive CD8+ single cells were sorted into 96 well plates containing 1:1 AIM- V / RPMI medium supplemented with 5% human serum, 100 U / mL penicillin, 100 pg / mL streptomycin in the presence of 2xl05irradiated (25 Gy) feeder cells. Cells were stimulated with 300UI / ml human IL-2 (Proleukin, Novartis) and 30 ng / ml anti-CD3 (OKT3, eBioscience). Growing clones were cultured for 3 weeks in the same medium without addition of anti-CD3 and tested for tetramer staining. Tetramer positive clones were stimulated every 3 weeks using the same media containing IL-2, anti-CD3 and irradiated feeders. After each cycle of amplification, the clones were tested for tetramer binding by cytometry. cDNA from each clone was amplified by PCR using primers for TRAV, TRBV, and constant regions (23), the PCR products sequenced and the resulting sequences analyzed using IMGT / V-QUEST (24).

[0155] T cell clone activation

[0156] The avidity of the clones for HLA-A2 restricted peptides was assessed after coculture of with K562-HLA-A*02:01 cell line loaded with peptide. Briefly, the cells were washed in RPMI without serum and incubated for 2 hours at 37°C at increasing doses of peptide (0.3 pM to 14 pM), then washed and co-cultured with clone cells at 1:1 ratio for 18 hours. The supernatants were collected and the secreted IFN-y, TNF-a, Granzyme B and Perforin quantified using LEGENDplex (BioLegend) and a Cytoflex cytometer (Beckman).TCR-T cell generation

[0157] To generate TCR-T cells bearing the 127E10 TCR specific for neoE-4 peptide the TCR alpha and beta chains (TRAV12-3 TRAJ57 CAMQQGGSEKLVF (SEQ ID NO: 17), TRBV15 TRBD2 TRBJ2-7 CATSRARPSEQYF (SEQ ID NO: 18)) were cloned into a lentiviral vector bearing murinized TRA and TRB constant regions (10). The same was performed for a CMV pp65 (NLVPMVATV, SEQ ID NO: 22) specific TCR (TRAV26-2 TRAJ43 CILDNNNDMRF (SEQ ID NO: 19), TRBV7-6 TRBJ1-4 TRBD1 CASSLAPGTTNEKLFF (SEQ ID NO:20)).

[0158] Lentivirus were prepared by VectorBuilder. Human CD8+ T cells from HLA-A*02:01 negative healthy donors were isolated from PBMC using human CD8+ T cell enrichment kit (StemCell #19053) and frozen or used fresh. CD8+ T cells were activated for 2 days in Xvivo 15 (Lonza) media supplemented with 5% human AB serum, 50 pM b-mercaptoethanol, 300 Ul / ml IL-2, 5 ng / ml IL-7, 5 ng / ml IL-15 and Dynabeads human T-activator CD3 / CD28. The cells were then transfected with the TCR encoding lentivirus at MOI 5 in the presence of polybrene 5 ng / ml. Lentivirus were spinoculated for 45 minutes at 1000 g and 32°C. At day 6 the Dynabeads were removed and the exogenous TCR expression tested by flow cytometry using anti-mouse TCRP BV711 (BioLegend 109243), anti-human CD8 BUV395 (BD 563795) and CD3 BUV737 (BD 612751). When indicated IFN-y and TNF-a release was detected using the kit ELISA MAX Deluxe (BioLegend), following the manufacturer’s instructions.

[0159] Tumor cell killing

[0160] The capacity of the cloned T cells and engineered TCR-T cells to kill Ewing sarcoma cell lines was assessed using a killing assay in an IncuCyte system. Tumors cells were plated in 96-well plates (50,000 cells per well) and cultured overnight to allow cell adhesion. RPMI media was complemented with 1 mM CaCh. Then, Annexin V Red dye (Sartorious) was added following manufacturer’s instructions. As a control, in some wells the interaction between TCR and peptide MHC was blocked by the addition of anti-HLA (W6 / 32) antibody (10 pM). Effector T cells (clone or TCR-T) were added at different effectortarget (E / T) ratios. As a positive control the cognate peptides (1 pM) were added. Cell killing was monitored and analyzed by an IncuCyte ZOOM live cell analysis system (Sartorius) for 24 hours. Four images per well at 40x zoom were collected at each time point. Tumor growth was quantified by measuring total integrated Annexin V Red intensity per well every 4h. Annexin V Red signal was normalizedto the time 0 signal. Supernatants were collected at 24 hours, and TNF-a and IFN-y were determined by ELISA as above. Duplicates were plated for each condition, and t-test analysis was applied.

[0161] Flow Cytometry

[0162] TCR-T cells activation by tumor cells was assessed by flow cytometry to evaluate CD137 expression. To this end, tumors cells were plated in 96-well plates (density of 50,000 cells per well in lOOul of media) and cultured overnight to allow cell adhesion. Effector T cells (TCR-T or unstransduced) were added at an effectortarget (E / T) ratio of 1 : 1 and cultured for 24h. As control, cultured TCR-T cells without tumor cells were used. Then TCR-T cells were stained with various antibodies using anti-CD8 BUV395 (BD563795), anti-CD3 BUV 737 (BD 612751), anti-mTCR BV711 (Biolegend 109243) and anti-4-lBB BV650 (Biolegend 309828) for 20 minutes at 4°C. All antibodies were diluted using the BD Horizon Brillant Stain Buffer (BD Bioscience). Samples acquisition was performed with the BD LSRFortessa™ and FACSDiva software. Results were generated using FlowJo software (TreeStar, Inc) and Graphpad Prims 10.

[0163] Results

[0164] Ew NGs-encoded peptides contribute to the EwS-specific MHC-I immunopeptidome

[0165] The inventors previously showed that some EWSR1: ELI 1 -specific neogenes (Ew NGs) transcripts are associated with ribosomes and may encode proteins that can be identified by whole-cell proteomics (5). To investigate whether peptides encoded by these neogenes can be processed and presented by HLA-I molecules on the surface of Ewing cells, the inventors performed immunoprecipitation of HLA-I molecules in five Ewing cell lines and five PDX followed by analysis of the eluted peptides using LC-MS / MS. The inventors thus generated a database of EwS immunopeptidomics MS spectra. Two search tools, Proteome Discoverer and MSFragger, were used to identify matches with the human reference proteome and with a database of Ew_NGs (5). Proteome Discoverer identified 25 Ew_NGs-derived peptides, 17 of which being compliant with the HLA-I alleles of the corresponding sample. MSFragger identified 22 Ew_NG-derived peptides, with 17 being HLA-I compliant. Sixteen peptides were common to both analyses (Fig. 1 A and B, Table 2).The Ew_NG-encoded peptides length distribution peaked at 9 amino acids consistent with the expected length for HLA-I presented peptides (Table 2) and similar to that of peptides from annotated genes (Data not shown). Additionally, for both Ew_NG-derived peptides and annotated peptides the ratio of calculated hydrophobicity to measured HPLC retention time were similar (Data not shown). The robustness of Ew_NG-derived peptide detection was further corroborated by similar MS / MS spectrum scores with annotated peptides (Data not shown).

[0166] To further validate EwS peptide detection, the inventors compared immunopeptidomics MS / MS spectra to corresponding predicted and synthetic peptide spectra (Table 2). Some peptides were found in several samples with identical HLA-I restriction (Fig. IB). These sixteen peptides were encoded by eight distinct Ew_NGs (Fig. IB). Ew_NG8 was the most represented, with a total of eight peptides encoded by its various isoforms. Five of these isoforms include an ORF encoding five peptides (Data not shown). Further reinforcing the Ewing specificity of these peptides, none of them was detected across 407 immunopeptidomic samples from normal tissues available in public databases (6).

[0167] The inventors compared immunopeptidomic results with Ribosome profiling from the same samples. In addition to the two previously analyzed cell lines (5), the inventors also investigated the three additional EwS cell lines and the five EwS PDX models analyzed by immunopeptidomics. Following removal of rRNA and tRNA, ribosome-protected fragments (RPFs) were aligned with a database comprising the Gencode vl9 transcriptome and Ew NGs, enabling the assignment of RPFs to the EwS transcriptome (Data not shown). As expected, the mean length of Ew_NG ORFs with RPFs was shorter than that of annotated genes (Data not shown). The quality of the RiboSeq data was validated by observing a trinucleotide periodicity consistent with the known reading frames of coding genes (Data not shown). Given the variation in start codons for noncanonical ORFs, the inventors considered 13 alternative start codons (7). Only ORFs detected in all three replicates of a given sample were retained. The detected ORFs were classified into categories: canonical (coding sequences), extension, readthrough, upstream (uORF), downstream (dORF) or Ew_NG (Datas not shown).Further processing of the 663 Ew_NG ORFs detected by ribosome profiling, by grouping overlapping ORFs and filtering to retain the most 5’ ones, yielded 91 unique Ew_NG ORFs associated with RPFs corresponding to 12 Ew NGs (Data not shown). The ORFs detected by RiboSeq on Ew_NG8 included the five peptides found by immunopeptidomics MS / MS. They had various potential start codons including one methionine (Data not shown). With some variation from one cell line or PDX to the other, the ribosome protection of this ORF was observed in most samples (Data not shown). Similarly, the number of ORFs and their TPM values for other Ew NGs varied across samples (Data not shown). As indicated above, the sixteen peptides found by immunopeptidomics were encoded by 11 distinct Ew NGs ORFs from 8 Ew NGs. Riboseq data indicates that two of these (Ew_NG 3 and 8) were unambiguously associated with RPFs detected by Riboseq (Data not shown). The consistency of the findings between two orthogonal approaches, Riboseq and immunopeptidomics, and the number of detected peptides points out Ew_NG8 as the strongest candidate for subsequent experiments. Sensitivity issues of both approaches may account for the only partial overlap between Riboseq and immunopeptidomics data.

[0168] Altogether, this data indicates that the chimeric transcription factor EWSR1::FLI1 induces the expression of EwS NGs that can be further translated into Ew_NG-derived neoproteins which are subsequently processed into neopeptides and presented by HLA-I molecules.

[0169]

[0170]

[0171] Table 2: Ew_NG-encoded peptides identified by immunopeptidomics.

[0172] *, Part of an ORF predicted by Ribosome profiling; #, this gene corresponds to an expressed sequenced tag (EST) identified in Ewing cells (hCG1821234); ND, Not Done; PCC, Pearson Coefficient Correlation; NetMHCPan 4.0 predictions: Strong Binder (SB) Rank lower than 0.05; Weak Binder (WB) between 0.05 and 5.

[0173] Identification of CD8+ T cells specific for Ew_NG-derived peptides

[0174] Next, the inventors addressed whether the Ew_NG-derived peptides presented by HLA-I molecules can be detected by CD8+ T cells and generate effective anti-Ewing cell responses. First, the inventors confirmed peptide binding predictions to HLA-I molecules using an HLA monomer refolding assay. The HLA-I complex stabilization by the candidate peptides was compared to control peptides. All thirteen tested EwS peptides stabilized (more than 50% of control) at least one HLA-I allele of the source EwS samples (Fig. 2A).

[0175] The inventors focused on the four peptides able to strongly bind HLA-A*02:01, the most frequent allele in populations of European descent. The inventors generated HLA-A*02:01 tetramers by associating peptides with biotinylated HLA and fluorescent streptavidin. These tetramers were used as tools to detect peptide-specific T cells. As it is challenging to obtain large amounts of Peripheral Blood Mononuclear Cells (PBMCs) from Ewing sarcoma patients due to their young age, the inventors decided to explore the potential of naive T cells from healthy donors to recognize Ew_NG- derived peptides at the surface of Ewing cells (8).

[0176] Given the expected low frequency of specific T cells in the naive pool, PBMCs from HLA- A*02:01+ healthy donors were obtained by leukapheresis to start with a large number of CD8+ T cells. After staining with tetramers combined with PE and APC, CD8+ T cells recognizing the peptides were enriched with anti-PE and APC microbeads and magnetic columns (Fig. 2B). Tetramer-positive CD8+ T cells were isolated by fluorescence-activated cell sorting (FACS) as single cells on feeder cells with anti-CD3 stimulation and IL-2 and expanded to obtain CD8+ T cell clones (Data not shown).

[0177] The inventors successfully cloned CD8+ T cells specific for NeoE_4 (clone 127E10), NeoE_7 (clone 125D3) and NeoE_9 (clone 126B2) but were unable to detect a significant number of NeoE_3- specific CD8+ T cells despite multiple attempts. These clones were specifically stained with the corresponding tetramers (Fig. 2C) and were activated by the cognate peptides loaded on the K562-HLA-A*02:01 antigen presenting cell line, resulting in the secretion of TNF-a and IFN-y (Fig. 2D-E). When comparing the functional avidity of the three clones, clone 127E10 demonstrated the highest sensitivity, detecting the lowest amount of peptide (around 20 nM). This clone was thus selected for further analysis.

[0178] These data show that a naive immune system can recognize and be activated by peptides encoded by Ew_NGs.

[0179] T cell clone reactivity and killing ability toward Ewing cells

[0180] To determine whether endogenously processed and presented NeoE_4 peptide is recognized by the 127E10 CD8+ T cell clone, the inventors utilized a panel of both HLA-A* 02:01 positive and negative EwS cell lines (Data not shown). Each cell line was co-cultured with 127E10 clone T cells and tumor cell killing was monitored using an Annexin V assay using the Incucyte system. Presentation of the peptide NeoE_4 by HLA-A*02:01 molecules was required, as clone 127E10 efficiently killed the five EwS cell lines expressing HLA-A*02:01 but not the three EwS cell lines lacking this allele (Fig. 3A). Moreover, the absence of killing of non-EwS cell lines regardless of HLA-A*02:01 status (Data not shown) was consistent with the exclusive expression of Ew_NG8 in EwS cells (5). Finally, blocking HLA-I presentation with a specific antibody completely abrogated EwS cell killing (Data not shown) while adding the NeoE_4 peptide in the co- culture was sufficient to induce cell killing of HLA-A* 02:01 non EwS cells (Data not shown).

[0181] Notably, none of the EwS cell lines was killed by anti-pp65 CMV control T cell clone generated in the same conditions (Data not shown) whereas addition of the cognate pp65 peptide to HLA- A*02:01 non-EwS cells induced killing by this CMV specific T clone (Data not shown). IFN-y secretion in the supernatant of the cell cultures were fully consistent with the killing assay (Fig. 3B, C).

[0182] In EwS cells, the expression of Ew_NG is strictly dependent on EWSR1::FLI1 (5). To determine whether activation of clone 127E10 was abrogated by EWSR1::FLI1 knockdown, the inventors used the A673 / TR / shEF EwS cell line (9), in which doxycycline (DOX) induces the expression of a EWSR1: ELI 1 -specific shRNA (Data not shown). Killing of A673 / TR / shEF EwS cells was dramatically reduced by DOX treatment, as was IFN-y secretion (Data not shown). The NeoE_4 peptide is encoded by Ew_NG8 which expression is induced by EWSR1::FLI1 binding at a GGAA microsatellite within its promoter region. The inventors previously showed that CRISPR interference targeting sequences in the vicinity of this microsatellite was sufficient to abolish expression of this neogene (5). The inventors therefore used the same two single guides (sgRNA) to silence Ew_NG8 in the A673 cell line stably expressing a dCas9-KRAB (Data not shown). These guides did not influence EWSR1::FLI1 expression (Data not shown). Again, the killing was abolished by the two Ew_NG8 sgRNAs to a level similar to preincubation of this cell line with an anti-MHC-I antibody as was IFN-y secretion (Data not shown). In contrast, a control sgRNA did not influence cell killing (Data not shown). Altogether, these data show that the killing of EwS cell lines by 127E10-specific T cells is strictly dependent upon the HLA- A*02:01 restriction and upon the expression of EWSR1 : :FLI1 as well as of its target Ew_NG8 which encodes the NeoE_4 peptide.

[0183] NeoE_4 activates engineered TCR-T cells in vitroThe next objective was to confirm that the detection of NeoE_4 on the surface of EwS cells was mediated by the T cell receptor (TCR) of clone 127E10 (TCR127E10). To achieve this, the inventors sequenced the TCRs of the 127E10 clone and of the control pp65 CMV T cell clone. The inventors then expressed the TRAV-TRAJ and TRBV-TRAJ regions associated with mouse constant chains (10) in a lentiviral vector and transduced CD8+ T cells from an HLA-A*02:01- healthy donor to obtain TCR-T cells (Fig. 4A). The TCRs indeed conferred the expected specificity to the cognate peptides, as the TCR-T cells were only stained and activated by tetramers loaded with the specific peptides (Fig. 4B and C).

[0184] The inventors then reproduced the functional assays previously done with the T cell clones by performing co-cultures of EwS cell lines with the TCR-T cells. The capacity of tumor cell lines to activate TCR-T cells was assessed by the expression of CD137 and by IFN-y production. TCR127E10T cells indeed was activated and produced IFN-y cytokine when cocultured with HL A- A* 02:01 EwS cell lines but neither with the non-HLA-A*02:01 EwS cell lines nor with non-EwS cell lines (Data not shown). Neither the CMV specific TCRpp65-T cells nor the untransduced T cells were activated or produced IFN-y secretion after stimulation by any cell lines (Data not shown). Finally, and most importantly, TCR127E10-T cells specifically killed HLA-A*02:01 EwS cell lines (Fig. 4D & E), an effect fully inhibited by an anti- HLA-I antibody (Fig. 4F). In contrast TCRpp65-T cells did not kill any cells in our panel (Fig. 4G).

[0185] These data indicates that TCR127E10is sufficient to promote specific killing of HLA-A*02:01 EwS cell lines.

[0186] Translational preclinical studies of TCR127E10

[0187] TCR127E10 specificity was assessed applying a highly stringent method of peptide fingerprinting (25) aiming to identify all possible off-target events. A positional scanning library of 172 peptides where each amino acid residue of NeoE_4 was substituted by the 19 other natural amino acids was used. An HLA-A*02:01 lymphoblastic cell line was pulsed with these individual peptides and co-cultured with reporter cells expressing TCR127E10 (Fig. 5A). The crucial and less permissive positions were 5, 7 and 8 (Fig. 5 B-C).

[0188] The 42 peptides able to activate TCR127E10 were not encoded by the human proteome. The positional matrix including amino acids positions enabling TCR127E10 activation was searched in Uniprot and yielded seven matches. In the activation tests only one of them showedsome minimal activation and 1000-fold less than NeoE_4 and under high, non-physiological peptide concentration (Fig. 5D). To rule out the allogeneic activation the reporter T cells were co-cultured with a panel of lymphoblastic cell lines. Notably, TCR127E10 did not elicit any allogeneic activation in the presence of these cell lines (Fig. 5E).

[0189] TCR127E10CD8 T cells eliminate Ewing sarcoma cells in vivo

[0190] To investigate the in vivo killing efficacy of TCR127E10T cells, NOD / SCTD / IL Ry- / -(NSG) mice were engrafted intraperitoneally (IP) with 106HLA-A*02:01 EW-7 Ewing sarcoma cells transduced with a firefly luciferase-GFP (ffluc-GFP) fusion gene. Tumor engraftment was monitored by bioluminescence imaging (BLI) to assign mice to treatment groups. Seven days after tumor injection (day 0), mice were either left untreated (n = 3) or treated IP with 10* 106untransduced CD8 T cells (n = 6) or TCR127E10CD8 T cells (n = 8) (Figure 6A). All mice received daily intraperitoneal injections of 2,500 IU human IL-2 until day 25 after T-cell injection. By day 8 post-treatment, 7 of 8 mice receiving TCR127E10CD8 T cells were tumor-free, whereas only 1 of 6 mice treated with untransduced T cells achieved a complete response (Fig. 6 B and C). TCR127E10CD8 T cells, but not untransduced CD8 T cells, mediated sustained tumor control and significantly improved survival in NSG mice.

[0191] Recent evidence from ribosome profiling and proteomic experiments suggests that peptides encoded by non-canonical ORFs (ORFs that are not reported in protein-coding sequence database), may encode microproteins with important biological functions (11-14). These non- canonical ORFs may be derived from alternative reading frames of protein-coding genes but also from long non-coding RNA (IncRNA) that may therefore not be as non-coding as their name suggests and encode microproteins with important biological functions. The estimated abundance of such noncanonical ORFs in the genome is a matter of intense debate (H).

[0192] The role of such microproteins in cancer is emerging. A growing number of reports indicate that HLA-I-bound peptides, derived from such non-canonical ORFs, constitute a significant fraction of the immunopeptidome raising a strong interest for immunotherapy approaches given the tumor-specific expression of some of these ORFs (15-18). However, tothe best of our knowledge a clear demonstration that the unconventional immunopeptidome may constitute a target for immunotherapy has not been provided.

[0193] The inventors recently discovered a novel class of IncRNAs that are uniquely regulated by oncogenic transcription factors in different sarcomas (5). In Ewing sarcoma, these IncRNA, also termed EwS-specific neogenes (Ew NGs) are regulated by direct binding of EWSR1::FLI1, or related EwS-ETS fusion oncogenes, to microsatellite sequences leading to subsequent activation of transcription of intergenic regions. Their exclusive regulation by the disease-specific chimeric transcription factor gives them exquisite tumor specificity, as none of the normal tissues express EWSR1::FLI1. This makes them - if protein coding - extremely attractive candidates for immunotherapy. Our prior results showed that some of these Ew NGs demonstrate ribosome protected fragments and that corresponding microproteins could be detected by whole cell proteomics (5).

[0194] Here, the inventors further investigated Ewing cell lines and PDXs by riboseq and immunopeptidomics. The inventors employed multiple layers of validation. RiboSeq proved to be a valuable tool for prioritizing candidate neogenes, with Ew_NG8 being the most robustly detected by this approach. Immunopeptidomics further corroborated Ew_NG8 as a strong candidate through the detection of eight different HLA-I-bound peptides encoded by this neogene. T cells isolated from an HLA-A*02:01 healthy donor, specific for a peptide derived from Ew_NG8, demonstrated, through cell activation and functional assays, the specific detection of the peptide on the surface of EwS cell with the same HLA-I restriction. Strikingly, this T cell clone could kill all HLA-A*02:01 EwS cell lines tested, even those where the corresponding peptide was not detected by immunopeptidomics. This indicates that T cell recognition is more sensitive in peptide detection than immunopeptidomics.

[0195] In addition to the HLA-I restriction, the killing was strictly dependent upon the expression of EWSR1::FLI1 and of the source Ew_NG8. In addition, this cytotoxic property could be fully reconstituted by transduction of the TCR into recipient T cells. These results demonstrate the ability of these Ew_NG-encoded peptides to be naturally processed in tumor cells as well as to be presented to T cells by HLA-I on Ewing cells and their potential to functionally activate T cells.In any case, the presence of exquisitely Ewing specific neo-peptides presented by HLA molecules at the surface of EwS cells opens multiple therapeutic possibilities. Importantly, these peptides represent shared epitopes across EwS patients with a given HLA-I restriction. Accordingly the invention relates to therapeutic vaccination targeting different ORFs encoded by the Ew NGs, following similar schemes developed for mutation-associated neopeptides (19) facilitated by the shared nature of the antigens across patients. The invention also relates to an adoptive TCR-T cell therapy with autologous T cells genetically engineered to express a TCR able to recognize Ew_NG- derived antigens, especially in the context of the recent FDA approval of the first TCR-T cell therapy, which targets MAGE-A4-HLA-I complex in aggressive synovial sarcoma (20). The invention also relates to a Bi-specific T cell engagers targeting EwS specific HLA-I peptides complexes. Such a bi-specific reagent (tebentafusp) was recently successfully developed for the treatment of uveal melanoma (21).

[0196] REFERENCES:

[0197] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS1. A tumor-specific neoantigenic peptide comprising at least one amino acid sequence set forth in SEQ ID No. 1 to SEQ ID No. 16, or a conservative variant thereof.

2. A vaccinal or immunogenic composition comprising at least one tumor-specific neoantigenic peptide according to claim 1.

3. The vaccine or immunogenic composition according to claim 2, which comprises a plurality of tumor-specific neoantigenic peptides according to claim 1.

4. The vaccine or immunogenic composition according to claim 2 or 3, comprising the 16 tumor-specific neoantigenic peptides of claim 1 comprising the amino acid sequence set forth in SEQ ID NO. 1 to SEQ ID No. 16.

5. The vaccine or immunogenic composition according to any one of claims 2 to 4, wherein the tumor-specific neoantigenic peptide is encoded by EWSR1: ELI 1 -Induced neogenes in Ewing sarcoma.

6. A population of dendritic cells or antigen-presenting cells (APCs) that have been pulsed with one or more tumor-specific neoantigenic peptides as defined in claim 1 or the vaccine or immunogenic composition according to any one of claims 2 to 5, or transfected with a polynucleotide encoding one or more tumor-specific neoantigenic peptide as defined in claim 1 or comprised in the vaccine or immunogenic composition according to any one of claims 2 to 5.

7. An antibody, an antigen-binding fragment thereof, a T cell receptor (TCR), or a chimeric antigen receptor (CAR), that specifically binds a tumor-specific neoantigenic peptide defined in claim 1.

8. The TCR according to claim 7 comprising a TCR a chain of SEQ ID NO: 17 and a TCR P chain of SEQ ID NO: 18 or a functional TCR fragments or variants thereof.

9. The antibody, the antigen-binding fragment thereof, the CAR or the TCR as defined according to claim 7 or 8, wherein said antibody, antigen-binding fragment thereof, CAR or T-cell receptor is a multispecific antibody or antigen-binding fragment thereof or multispecific CAR or multispecific T-cell receptor, that further targets at least an immune cell, an immune cell antigen or a tumor cell antigen, optionally wherein the immune cell is a T cell, an NK cell, or a dendritic cell, optionally wherein the immune cell antigen is CD3, CD 16, CD30 or a TCR.

10. An immune cell that specifically binds to one or more tumor-specific neoantigenic peptides as defined in claim 1 optionally wherein the immune cell is an allogenic or autologous cell selected from T cell, NK cell, CD4+ / CD8+, TILs / tumor-derived CD8 T cells, central memory CD8+ T cells, Treg, MAIT, and Y5 T cell.

11. A T cell according to claim 10, which comprises:a T cell receptor that specifically binds one or more tumor-specific neoantigenic peptides as defined in claim 1, ora CAR that specifically binds one or more tumor-specific neoantigenic peptides as defined in claim 1.

12. The tumor-specific neoantigenic peptide of claim 1, the vaccine or immunogenic composition according to any one of claims 2 to 5, the population of dendritic cells or APCs of claim 6, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) according to claim 7 to 9, the immune cell according to claim 10, or the T-cell according to claim 11, for use the treatment of cancer in a subject in need thereof, in particular for use in inhibiting cancer cell proliferation, for reducing or inhibiting tumor growth, for reducing or preventing tumor metastasis, or foruse in cancer vaccination therapy of a subject, particularly wherein the subject is suffering from Ewing sarcoma with EWSR1: ELI 1 -Induced neogenes.

13. The tumor-specific neoantigenic peptide of claim 1, the vaccine or immunogenic composition according to any one of claims 2 to 5, the population of dendritic cells or APCs of claim 6, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) according to claim 7 to 9, the immune cell according to claim 10, or the T-cell according to claim 11, for use according to claim 12, for treating Ewing sarcoma with EWSR1 : ELI 1 -Induced neogenes.

14. The tumor-specific neoantigenic peptide of claim 1, the vaccine or immunogenic composition according to any one of claims 2 to 5, the population of dendritic cells or APCs of claim 6, the antibody, or an antigen-binding fragment thereof or T cell receptor (TCR) or chimeric antigen receptor (CAR) according to claim 7 to 9, the immune cell according to claim 10, or the T-cell according to claim 11, for use in combination with at least one further therapeutic agent, optionally wherein the therapeutic agent is a chemotherapeutic agent of an immunotherapeutic agent.