RNA vaccines containing RNA pools generated from double-stranded DNA pools

Abstract

1. A method for producing an RNA vaccine comprising multiple epitopes specifically derived from a target, comprising the steps of: (i) obtaining a plurality of synthetic DNA constructs in a pool, the synthetic DNA constructs encoding a plurality of different epitopes derived from the target; and in vitro transcribing said plurality of synthetic DNAs into a corresponding plurality of RNAs, The target is a peptide derived from an infectious agent or a cancer neoepitope that has an amino acid sequence that is specifically identified in a patient and that differs by at least one amino acid from an amino acid sequence that naturally occurs in normal cells of the patient. method.

Description

[Technical field]

[0001] The present invention relates to the use of information extracted from a patient's genome to generate ribonucleic acid RNA vaccines, and in particular to the discovery of methods for producing RNA vaccines that contain multiple epitopes specifically derived from targets derived from infectious agents or cancer neoepitopes.

[0002] More specifically, the present invention relates to an mRNA cancer vaccine for a patient with a tumor. The present invention also relates to an ex vivo method for selecting neoantigens for a patient, comprising providing a blood sample from a patient administered the mRNA cancer vaccine. In oncology, personalized vaccines for treating tumors, such as personalized therapeutic vaccines that contain tumor-specific antigens (neoantigens) of a patient's tumor, are very promising as the next generation of personalized cancer immunotherapy. [Background technology]

[0003] The concept of personalized cancer vaccination is based on the identification of neo-antigens capable of eliciting an appropriate immunological response, which makes it possible to generate optimal personalized vaccines that specifically boost the patient's immune system in attacking the tumor.

[0004] RNA vaccines have been developed in personalized cancer therapy, but also to treat and / or prevent different infectious diseases. RNA vaccines can be used to induce a balanced immune response against cancer or against infectious diseases, including both cellular and humoral immunity, without risking the possibility of insertional mutagenesis. RNA vaccines can be utilized to treat and / or prevent diseases, cancers at various stages or metastases.

[0005] Currently, a significant limitation in RNA (mRNA) vaccines is the proper identification of specific epitopes, i.e., tumor neoepitopes, that induce an appropriate immunological response in the patient, leading to sufficient activation of T cells in the patient to efficiently attack the tumor and / or disease, which is particularly true for personalized mRNA cancer vaccines.

[0006] The document EP3400005 relates to an anti-cancer vaccine directed against multiple neoepitopes derived from tumor neoantigens, thereby resulting in a personalized neoantigen vaccine that specifically targets the identified tumor antigens.

[0007] In one aspect, the invention relates to a therapeutic anti-cancer vaccine comprising an immunologically effective amount of a polynucleotide comprising a nucleotide sequence encoding: (i) 2 to 50 antigenic subunits, each subunit comprising at least a portion of a cancer neoepitope sequence and a linker, and (ii) a final antigenic unit comprising a cancer neoepitope sequence.

[0008] Currently, the production of mRNA vaccines is based on the in vitro transcription of a DNA template, which can be a plasmid or result from multiple ligation steps of multiple DNA fragments, generally encoding concatemeric epitopes or neoepitopes.

[0009] For example, WO2018 / 144082 describes an RNA cancer vaccine and a method for its preparation. In particular, this specification describes a concatemer-based mRNA vaccine in which multiple cancer epitopes are present on a single mRNA.

[0010] Document WO2020 / 097291 describes a method for treating cancer in patients with an mRNA vaccine, which, at a given length, exhibits maximum anti-cancer efficacy.

[0011] Kreiter Sebastian et al. (The Journal of Immunology, Vol 180, 2008, pp 309-318) describes the efficiency of antigen presentation by coupling the antigen to an MHC molecule, where the vector has a promoter, a polyA tail and a 3' UTR sequence.

[0012] Unfortunately, although the state of the art literature describes the advantages of these techniques, currently no methods exist that allow the development of mRNA vaccines that induce immunological responses in patients with high efficiency. In mRNA cancer vaccines, only 4-20% of the neoepitopes encoded by the mRNA vaccine are able to activate T cell responses. Tumors can be characterized by a large number, for example more than 100, of different neoepitopes. Thus, a key challenge is to select the appropriate neoepitopes, or epitopes of a specific disease, to be incorporated into the mRNA vaccine in order to induce an appropriate immunological response in patients.

[0013] Current research and development is attempting to improve predictive algorithms that aim to identify the "best" neo-antigen candidates of a patient's tumor that will elicit an optimal immunological response in the patient, characterized by activation of the patient's specific T cells.

[0014] The inventors consider that the methods known in the state of the art for identifying the best neo-antigen candidates are based on affinity data between neo-antigen peptides and HLA receptors, which are only available for a specific type of HLA receptor. Thus, while the current methods for identifying the best neo-antigen candidates may reach a certain degree of efficacy for patients with said specific type of HLA receptor, this method is ineffective for the majority of patients with different specific types of HLA receptor. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] EP3400005 [Patent Document 2] WO2018 / 144082 [Patent Document 3] WO2020 / 097291 [Non-patent literature]

[0016] [Non-Patent Document 1] The Journal of Immunology, Vol 180, 2008, pp 309-318) Summary of the Invention [Problem to be solved by the invention]

[0017] Thus, there is a need to develop processes for producing RNA vaccines that contain multiple epitopes specifically derived from a patient's target, where the multiple epitopes have affinity for the patient's specific HLA receptors. [Means for solving the problem]

[0018] The present invention provides a method for producing an RNA vaccine that contains multiple epitopes specifically derived from a target, comprising: - encoding a plurality of different epitopes derived from said target adjacent to a sequence that allows translation of the RNA sequence into peptides by a eukaryotic cell; and Obtaining in a pool a plurality of different synthetic DNA constructs having a DNA construct encoding said epitope and a promoter for the transcription of said sequence into an mRNA sequence allowing its translation into a peptide; and - in vitro transcribing said plurality of synthetic DNAs into a corresponding plurality of RNAs; Here, the target is a peptide derived from an infectious agent or a cancer neoepitope specifically identified in one patient as having at least one amino acid difference at the peptide level.

[0019] In other words, this equates to a peptide that has at least one amino acid difference compared to a peptide derived from normal cells of said patient.

[0020] Indeed, the method according to the invention, whereby multiple synthetic DNA constructs are obtained in a pool, makes it possible to have a large, potentially infinite, number of different DNA constructs. As a result, the RNA vaccine according to the invention can incorporate a very large number of epitopes of infectious diseases or tumor-derived epitopes, i.e. neoepitopes, some of which lead to T cell activation in the patient.

[0021] According to the present invention, the immunological response in a patient refers to the production of specific T cells, preferably cytotoxic T cells, that target the neoantigen(s) or infectious agent. Alternatively, the immunological response is tested in vitro by measuring the production of one or more immunological markers, such as IFN-γ, by T cells of the patient, where said T cells of the patient are contacted with specific epitopes of the mRNA vaccine.

[0022] Alternatively, or in addition, the immunological response is measured by identification of CD8+ CD107a+ T cells from blood samples of patients administered the mRNA vaccine of the present invention.

[0023] According to the present invention, the term "at least one amino acid difference compared to a peptide derived from normal cells" refers to an amino acid sequence encoded from an open reading frame that is present in the DNA or RNA of tumor cells from a patient, or in the DNA or RNA derived from an infectious agent, and that is not present in the patient's normal cells, i.e., peripheral blood mononuclear cells (PBMCs), or in matched non-cancerous tissue.

[0024] The present invention also relates to an mRNA cancer vaccine for a tumor-bearing patient comprising a plurality of mRNAs encoding a plurality of neo-antigens, said plurality of neo-antigens being derived from the tumor, said plurality of mRNAs encoding at least 1%, preferably at least 2%, more preferably at least 5%, and even more preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of all tumor neo-antigens identified in the tumor.

[0025] The present invention also relates to an ex vivo method for selecting neoantigens for a patient, comprising the steps of providing a blood sample from a patient administered an mRNA cancer vaccine according to the present invention or an RNA vaccine obtained by the method according to the present invention with the corresponding synthetic peptide neoepitopes or one or several truncated forms thereof, and identifying which synthetic peptide neoepitopes induce an immune response.

[0026] Other characteristics and advantages of the invention emerge from the following non-limiting description, with reference to the drawings and examples.

[0027] According to the present invention, the term "neoantigen" refers to a tumor-specific antigen of a patient. In the framework of the present invention, a "neoantigen" comprises an amino acid sequence that differs in at least one amino acid compared to a peptide naturally occurring in normal cells of a patient.

[0028] According to the present invention, the term "neoepitope" refers to a tumor-specific peptide derived from a neoantigen that is capable of binding to the major histocompatibility complex (MHC) of a patient.

[0029] According to the present invention, the term "amino acid sequence which differs by at least one amino acid compared to a peptide naturally occurring in the patient's normal cells" means (i) a peptide having an amino acid sequence which differs by at least one amino acid at the corresponding position of the amino acid sequence naturally occurring in the patient's normal cells, and / or (ii) a de novo amino acid sequence which is not present in the patient's normal cells, i.e. which is present in the tumor and not in the patient's normal cells (this situation shares more similarity with vaccination with the amino acid sequence of an infectious agent), and thus is advantageous.

[0030] In addition, the term "amino acid sequence that differs by at least one amino acid compared to a peptide naturally occurring in normal cells of a patient" encompasses neoantigens that are generated after translocation events or mutations at splice sites; these events generate peptides that have a segment of high homology to a peptide from normal tissue and a different C-terminal segment, or a peptide that has high homology to another peptide from normal tissue.

[0031] In other words, any genetic event that produces a peptide that is not 100% identical to a peptide found in normal tissue corresponds to the above definition.

[0032] In the present invention (the process for producing the mRNA, the (starting) DNA construct, the resulting mRNA or the mRNA as such), preferably the DNA, and thus the mRNA, comprises a 3' polyA segment.

[0033] Preferably, the 3' poly A segment consists of 100% adenosines or consists essentially of adenosines; For example, a stretch of at least 10 consecutive adenosine residues, preferably at least 20, 30, 40 consecutive adenosine residues, separated by other nucleotides (less than 20, preferably a stretch of 10 residues that do not form said stretch), and followed by a second stretch of at least 10 consecutive adenosine residues (at least 20, 30, 40 consecutive adenosine residues, regardless of the length of the first stretch); Possibly, subsequent and subsequent adenosine stretches separated by less than 20 nucleotides, preferably 10 residues not forming a stretch, are also included in the term "poly A segment", provided that at least 90 adenosine residues are present, preferably at least 120 adenosine residues are present in these stretches, forming a "poly A segment".

[0034] The present inventors have developed a method for producing an mRNA vaccine that contains multiple epitopes specifically derived from a target, making it possible to ensure the administration of epitopes that are recognized by the patient's immune system.

[0035] The inventors have shown that such an approach provides a more robust immune response than methods based on vaccination in which one or a few different epitopes are administered to a patient, where the epitopes are selected and / or designed using modern techniques, including bioinformatics tools, developed to increase the predictability of the immune response.

[0036] Such positive results are surprising considering that the potent epitopes are diluted by the abundant non-potent epitopes.

[0037] The method includes the steps of: - obtaining a plurality of different synthetic DNA constructs in a pool, encoding a plurality of different epitopes derived from said target, adjacent to a sequence allowing the translation of an RNA sequence into a peptide by a eukaryotic cell, and having a promoter for the transcription of said DNA constructs encoding said epitopes and said sequence allowing said translation into a peptide, And, - in vitro transcription of said plurality of synthetic DNAs into a corresponding plurality of RNAs; Where: The target is a peptide derived from an infectious agent or cancer neoepitope specifically identified in a patient, the peptide having an amino acid sequence that differs by at least one amino acid compared to a peptide naturally occurring in normal cells of the patient.

[0038] Preferably, the transcription of DNA into an RNA sequence is carried out in the presence of pseudouridine and / or N1-methylpseudouridine, 15-methyluridine (m5U), and / or 2-thiouridine (s2U).

[0039] Preferably, the process for obtaining an RNA sequence comprises the further step of adding a 5'Cap element, which comprises two nucleotides (linked 5'-3') separated by a triphosphate stretch.

[0040] The two nucleotides may be natural or modified, for example by methylation of -OH residues at the 2' position (2-O-Me nucleotides) at one or two residues forming the 5' Cap element.

[0041] The 5' Cap element can contain additional groups, including additional nucleotides, provided that the two nucleotides are separated by a triphosphate group (linked 5'-3') instead of a monophosphate.

[0042] Preferably, the DNA construct (and thus the encoded mRNA as well) further comprises a number of sequence elements encoding a peptide sequence for endosomal targeting of the encoded peptide, for example a signal sequence of a lysosome-associated membrane protein (LAMP1 or LAMP2), such as a dendritic cell (DC-LAMP), and / or a sequence targeting an epitope to the MHC-I receptor, such as SEQ ID NO: 3.

[0043] Advantageously, the DNA construct (and thus the encoded mRNA as well) further comprises a number of sequence elements encoding peptide sequences for loading of the encoded peptide for presentation to T lymphocytes.

[0044] More preferably, the plurality of sequence elements encoding a peptide sequence for loading of the encoded peptide for presentation to T lymphocytes is a dendritic cell lysosomal-associated membrane glycoprotein signal sequence, such as SEQ ID NO:5.

[0045] Preferably, the different DNA constructs share the same sequence elements that encode peptide sequences for endosomal targeting of the encoded peptide and / or the same sequence elements that encode peptide sequences for loading of the encoded peptide for presentation to T lymphocytes.

[0046] Preferably, the sequence enabling translation of the RNA sequence into a peptide by a eukaryotic cell is a translation enhancer / promoter sequence located in the 5'UTR region (upstream of the coding region) of the DNA construct. More preferably, the sequence enabling translation is a translation enhancer / promoter sequence selected from the group of translation enhancer / promoter sequences including human cytomegalovirus (CMV) enhancer, alpha or beta globin enhancer (such as SEQ ID NO: 2), EF-1alpha enhancer, simian virus 40 (SV40) enhancer, PGK1 promoter, ubiquitin C promoter, HSD17B4 and beta-actin promoter.

[0047] Preferably, the DNA construct (and thus the encoded mRNA as well) further comprises at least one sequence element encoding a 3'-UTR and / or a 3' poly-A tail segment. More preferably, the 3' poly-A tail segment(s) is / are at least 68 nucleotides, preferably at least 100 nucleotides (see definition above).

[0048] Advantageously, the DNA constructs share the same promoter sequence (for transcription into an RNA molecule).

[0049] Preferably, the promoter sequence is a bacteriophage RNA polymerase promoter. More preferably, the promoter sequence belongs to a group of promoter sequences including promoter sequences of T7 RNA polymerase (SEQ ID NO: 1), SP6 RNA polymerase, T3 RNA polymerase, Syn5 RNA polymerase, and KP34 RNA polymerase. Preferably, the promoter sequence according to the present invention is a promoter sequence of T7 RNA polymerase.

[0050] A detailed description of the DNA construct and the DNA sequence of the DNA construct can be found in EP21196390.5, filed on September 13, 2021. Preferably, a transcription factor that recognizes the shared promoter sequence is added during the transcription describing step.

[0051] According to the invention, a plurality of synthetic mRNA constructs in a pool can be obtained by a method for producing a plurality of synthetic DNA constructs, comprising the steps of synthesizing: (i) a first single-stranded DNA molecule comprising: A promoter for transcription into RNA, a segment for its translation and a targeting sequence for endosomial targeting fused in-frame with an epitope derived from a tumor neoantigen or an infectious agent, and a first portion of a genetic element encoding a sequence for loading of an epitome from an infectious agent or the encoded tumor neoantigen for presentation to T lymphocytes; and (ii) a second single-stranded DNA molecule comprising: a second single-stranded DNA molecule encoding a second portion of the genetic element encoding a sequence for loading an epitope from an infectious agent or the encoded tumor neoantigen for presentation to T lymphocytes, wherein the second single-stranded DNA molecule is antisense, and wherein the first and second portions form a complete sequence for loading an epitope from an infectious agent or the encoded tumor neoantigen for presentation to T lymphocytes, and wherein the first and second portions present an overlap (of at least 15 consecutive nucleotides) that allows for specific base pairing. The process further includes: hybridizing a DNA molecule under (i) with a DNA molecule under (ii); and extending the two molecules to generate a double stranded DNA molecule encoding a genetic element encoding a promoter, a translation initiator, a targeting sequence for endosomal targeting fused in frame with an epitope from an infectious agent or a tumor neoantigen, and a sequence for loading of the epitope from the infectious agent or the encoded tumor neoantigen for presentation to T lymphocytes; and then transcribing the multiple DNA molecules in vitro to form multiple mRNA constructs.

[0052] According to the present invention, the synthesis of the first and possibly the second single-stranded DNA molecule is preferably synthesised using silicon-based DNA writing technology.

[0053] Alternatively, the second single-stranded molecule is a "master stock" that is synthesized once and is enzymatically amplified and stored to increase the efficiency of the overall process since only one molecule needs to undergo chemical synthesis.

[0054] Preferably, the method according to the invention further comprises a preliminary step of optimizing the sequences of the multiple DNA, suitably by selecting more abundant codons and / or by avoiding deleterious secondary structures.

[0055] Deleterious secondary structures can be predicted using available in silico tools and therefore avoided, if possible.

[0056] Preferably, pseudouridine is added to the RNA during the transcription step. Preferably, pseudouridine is added during the transcription step into the RNA, either in addition to the uridine or replacing all the uridine.

[0057] Alternatively, or in addition to pseudouridine and / or N1-methylpseudouridine, 15-methyluridine (m5U) and / or 2-thiouridine (s2U) can be incorporated into the mRNA. Preferably, the RNA sequence of each mRNA of the plurality of mRNAs is enriched with pseudouridine, possibly by using codon degeneracy, while maintaining the sequence of the corresponding encoded peptide.

[0058] Preferably, as mentioned, the RNA sequence comprises a 5' Cap element, which comprises two nucleotides (linked 5'-3') separated by a triphosphate stretch.

[0059] The two nucleotides may be natural or modified, for example by methylation of the -OH residue at the 2' position (2-O-Me nucleotides).

[0060] The 5' Cap element can include an additional group that includes an additional nucleotide, except that the two nucleotides are separated by a triphosphate group instead of a monophosphate (linked 5'-3').

[0061] Preferably, the plurality of different DNA molecules is at least 40 different DNA molecules encoding at least 40 different epitopes, preferably at least 50, or at least 60, 80, 100, or even 120 different epitopes.

[0062] Preferably, the method according to the invention further comprises the step of amplifying a plurality of the synthetic DNA constructs to generate an amplified pool of DNA constructs, which is used as a template for the in vitro transcription step.

[0063] Preferably, the method for producing an mRNA vaccine further comprises the step of checking the quality and / or quantity of each of the plurality of mRNAs after the in vitro transcription step. Preferably, the quality and / or quantity of each of the plurality of mRNAs is performed by sequencing the plurality of mRNAs. Preferably, the process for producing an mRNA vaccine also further comprises the step of formulating the plurality of mRNAs, optionally with one or more adjuvants, into a vector that can be administered to a patient.

[0064] However, adjuvants are not required for such RNA vaccinations, especially if the uridine residues are low in abundance or completely replaced by pseudouridine.

[0065] The present invention also relates to an mRNA cancer vaccine for a patient having a tumor comprising a plurality of mRNAs encoding a plurality of neo-antigens, said plurality of neo-antigens being derived from the tumor, said plurality of mRNAs encoding at least 1%, preferably at least 2%, more preferably at least 5%, and even more preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of all tumor neo-antigens identified in the tumor.

[0066] Preferably, the mRNA vaccine according to the present invention further comprises (i) a segment encoding a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen, and / or (ii) a segment encoding a sequence directing the encoded tumor neoantigen to an MHC molecule, and / or (iii) a segment encoding a translation enhancer, and / or (iv) a 3'-UTR and / or (v) a 3' polyA tail segment(s) and / or (vi) a 5'-UTR, as described above.

[0067] Preferably, the RNA sequence comprises a 5' Cap element, which comprises two nucleotides (linked 5'-3') separated by a triphosphate stretch.

[0068] The two nucleotides may be natural or modified, for example by methylation of the -OH residue at the 2' position (2-O-Me nucleotides).

[0069] The 5' Cap element can include an additional group that includes an additional nucleotide, except that the two nucleotides are separated by a triphosphate group instead of a monophosphate (linked 5'-3').

[0070] Preferably, the plurality of mRNAs encodes at least 2%, preferably at least 5%, more preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of all tumor neo-antigens identified in the tumor.

[0071] Advantageously, the mRNAs are incorporated into one or more lipid nanoparticles.Preferably, the mRNAs are complexed with positively charged lipids.Preferably, the mRNAs and the positively charged lipids are mixed in a microfluidic chip.

[0072] Optionally, one or more type 1 adjuvants are incorporated with the plurality of mRNAs to form an mRNA cancer vaccine, where the one or more adjuvants are preferably selected from the group of type 1 adjuvants having amorphous aluminum hydroxyphosphate (AAHS), aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (Alum), or monophosphoryl lipid A. However, such adjuvants are not necessary, particularly when pseudouridine is incorporated into the mRNA vaccine.

[0073] Optionally, the plurality of mRNAs further comprises a type 2 adjuvant in the form of DNA or mRNA, such as a type 2 adjuvant encoding constitutively active TLR4 (caTLR4) and / or CD70 and / or CD40L and / or interferon-γ and / or a decoy interleukin receptor, i.e., interleukin 1 receptor type II.

[0074] Advantageously, type 2 adjuvant DNA or mRNA, when incorporated into an mRNA vaccine, promotes stimulation of the patient's dendritic cells and increases the effectiveness of presentation of multiple neoepitopes to the patient's T cells.

[0075] Such adjuvants are particularly useful for ex vivo maturation of a patient's dendritic cells.Preferably, the plurality of mRNAs further comprises a type 3 adjuvant in the form of DNA or mRNA, such as a type 3 adjuvant encoding an RNA-dependent RNA polymerase (RdRp), e.g., DNA or RNA encoding the nonstructural proteins of the alphavirus nsP1-4.

[0076] Indeed, it is useful that the multiple mRNAs of an mRNA vaccine are capable of trans-amplification, such as trans-amplification based on the encoded RdRp, such that RNA trans-amplification allows for a reduction in the dose of the mRNA vaccine administered to a patient.

[0077] Preferably, at least 25%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the uridines present in the RNA sequence of each mRNA of the plurality of mRNAs are substituted by pseudouridine or N1-methylpseudouridine or 15-methyluridine or 2-thiouridine.

[0078] More preferably, at least 25%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the uridines present in the RNA sequence of each mRNA of the plurality of mRNAs are substituted by pseudouridine.

[0079] The present invention also relates to an ex vivo method for selecting neoantigens for a patient, comprising the steps of providing a blood sample from a patient administered an mRNA cancer vaccine according to the present invention or an RNA vaccine obtained by the method according to the present invention to the corresponding synthetic peptide neoepitopes or one or several truncated forms thereof, and identifying which synthetic peptide neoepitopes induce an immune response.

[0080] Indeed, this method for selecting neoantigens of a patient's tumor comprises providing an ex vivo blood sample from said patient, previously administered a number of tumor neoantigens as described herein above, and testing which neoepitopes react with said blood sample. This method allows to rapidly generate a more purified pool of neoantigens to treat said patient.

[0081] In practice, peptides corresponding to the identified neo-antigens are synthesized and then prepared in aqueous solution in different compartments, and one peptide is reacted with the ex vivo sample in a given compartment.

[0082] For neoantigens that are predicted to be too long or have too low water solubility, deletion peptides can be generated with sequences derived from the neoantigen, e.g., peptides starting at the N-terminus of the neoantigen or starting at an amino acid downstream to the N-terminus (e.g., 1, 2, 3, 4 or 5 amino acids from the N-terminus) and ending at or 1, 2, 3, 4 or 5 amino acids after the first identified mutation.

[0083] Alternatively, or preferably, in addition, a second overlapping peptide is synthesized, in which the first identified mutation is flanked by 3, 4, 5, 6, 7, 8, 9 amino acids upstream of the first mutation and by 3, 4, 5, 6, 7, 8, 9 amino acids downstream of the first mutation.

[0084] Alternatively, or preferably, in addition, a (second or third) overlapping peptide is synthesized starting at the first identified mutation or 1, 2, 3, 4, 5 amino acids upstream of this first identified mutation and ending at the C-terminus of the neoantigen or 1, 2, 3, 4, 5 amino acids upstream of this C-terminus of the neoantigen.

[0085] These (two or three) overlapping peptides can be placed in the same compartment or in two or three different compartments of an mRNA cancer vaccine according to the invention for testing or obtained by the method according to the invention for the in vitro identification of a set of neo-antigens of a patient's tumor. The present invention also relates to neo-antigens selected according to the ex vivo method of the invention for use in the vaccination of a patient suffering from a tumor.

[0086] The present invention also relates to a method for expanding specific in vitro cytotoxic T cells against a tumor affecting a patient, comprising the step of conditioning a blood sample from said patient with a selected neoantigen of the present invention. EXAMPLES

[0087] Example 1: Preparation of 97 mRNAs corresponding to neoepitopes identified in a patient's lung tumor We isolated DNA and RNA from a sample of the patient's lung tumor. We also isolated DNA from the patient's normal cells (peripheral blood mononuclear cells, PBMCs). Then, we performed the following steps: - Sequencing the DNA from the tumor, the DNA and the RNA from PBMC cells. - identifying the DNA variants of the tumor that encode neo-antigens of the tumor by comparing the DNA sequencing data obtained from the DNA of the tumor and the DNA from PBMC cells, and identifying the open reading frames of the tumor that have DNA variants that result in detection of mRNA transcripts. - Identifying the neo-antigens of the tumor, a plurality of 97 different neo-antigens were identified. - 97 double-stranded DNA templates were generated, each containing a DNA sequence encoding one different neo-epitope, with an amino acid sequence of 27 amino acids, where each neo-epitope sequence is part of the sequence of one neo-antigen. - The 97 double-stranded DNA templates are transcribed in vitro to produce the 97 mRNAs.

[0088] Example 2: Composition of an mRNA vaccine according to the present invention The inventors have developed an mRNA vaccine comprising: - 97 different mRNAs, each encoding one of the 27 amino acid neoepitopes, identified from the patient's lung tumor. The total amount of these 97 different mRNAs is 1000 μg per dose of the mRNA vaccine. - Positively charged lipids.

[0089] Example 3: Quality and quantity of the 97 different mRNAs contained in an mRNA vaccine Before incorporating multiple mRNAs (see 97 different mRNAs in Example 1) into an mRNA vaccine composition (see Example 2), we checked the quality and quantity of each mRNA of the multiple mRNAs by performing RNA sequencing. To do so, we prepared double-stranded cDNA synthesis using reverse transcriptase, and performed Illumina adaptor ligation and PCR cDNA library amplification. Then, RNA sequencing was performed using a high-throughput device, here an Illumina NovaSeq 6000, in paired-end 100b mode.

[0090] Example 4: Use of mRNA vaccines as diagnostic tools to generate more efficient personalized mRNA vaccines by detecting T cell activation The inventors used the mRNA vaccine of Example 2 as a diagnostic tool to identify the 97 different mRNAs, each encoding a different neoepitope, a neoepitope that stimulates the patient's T cells.

[0091] To do so, the mRNA vaccine according to Example 2 was administered to the patient with lung tumor. After 10 days, the patient's blood was taken and peripheral blood mononuclear cells and T cells were isolated. T cells from PBMCs of blood were isolated by anti-CD3 magnetic beads (Milteny). In parallel, 3 mAbs corresponding to the neoepitope amino acid sequences of 97 different mRNAs were isolated. * Ninety-seven peptides were synthesized in amounts of 1–4 micrograms of each peptide; In each case, one peptide starting from the N-terminus of the neoantigen and ending three amino acids after the first identified difference (mutation); said second peptide beginning 7 amino acids before and ending 7 amino acids after the first identified difference (mutation); and A third peptide starting three amino acids before the first difference (mutation) and ending at the C-terminus of the neoepitope. 3 * Each peptide of the 97 neoepitopes was tested by ELISpot assay, where T cells isolated from the patient's blood were contacted with each peptide (three peptides from one neoepitope were present and combined in one well).Then, we detected whether the patient's T cells were activated by a given neoepitope (peptide) encoded by the mRNA vaccine by detecting the production of IFN-γ in the well corresponding to the given neoepitope (peptide).From the 97 neoantigens, we identified 63 neoantigens that activate the patient's T cells.

[0092] Example 5: Use of mRNA vaccines as a tool for generating second generation mRNA vaccines by detecting CD8 cell activation As in Example 4, the mRNA vaccine according to Example 2 was administered to the patient with lung tumor. After 10 days, a blood sample was taken from the patient, and peripheral blood mononuclear cells (PBMCs) and T cells were isolated. T cells from the PBMCs of blood were isolated by anti-CD3 magnetic beads (Milteny). From the isolated fraction of CD3+ cells (T cells), CD8+ and CD4+ cells were isolated by anti-CD8 and anti-CD4 magnetic beads, respectively. The fraction of CD3+ cells depleted from the CD8+ and CD4+ cells represents a fraction of antigen-presenting cells (APCs). In parallel, 3 subunits corresponding to the neoepitope amino acid sequences of the 97 different mRNAs were * 97 peptides were synthesized using the same approach as in Example 4, in amounts of 1-4 micrograms of each peptide. T cells isolated from the patient's blood were contacted with each neoantigen, and each peptide of the 97 neoantigens was tested by ELISpot assay (three peptides from one neoantigen are present and combined in one well). The antigen presenting cell (APC) fraction was then electroporated with each peptide in the presence of CD8+ cells. IFN-γ production was then measured. From the 97 neoantigens, the inventors identified 24 neoantigens that specifically activate the patient's CD8+ cells.

[0093] Example 6: Use of mRNA vaccines as a tool to generate more efficient personalized mRNA vaccines by detection of CD107a The same process as in Example 5 was carried out. In addition, anti-CD107a marker was used to measure CD107a expression in each well. CD107a is a marker of degranulation (an indicator of functional cytotoxicity of CD8+ cells). From 24 neoantigens that specifically activate CD8+ cells of patients (see Example 5), 7 neoantigens were identified as functionally cytotoxic by measuring the expression of CD107a.

[0094] Example 7: Optimized mRNA vaccine Two months after injection of the mRNA vaccine in the patient's lung tumor (see Example 5), the patient was administered 300 μg of the optimized mRNA vaccine containing the seven identified neo-antigens.

[0095] Example 8: Second Generation Cell Therapy For some patients, a cell therapy approach is preferred; therefore, the inventors have taken advantage of the broad and sophisticated system described above to generate in vitro T cells capable of attacking tumors directly. To do so, as in Examples 4-7, two months after injection of the mRNA vaccine in the patient's lung tumor and the identification of the optimized neo-epitopes, cell therapy of the tumor was performed by isolating the patient's seven CD8+ CD107a+ cell lines. To do so, the patient's DC cells were matured with the seven optimized neo-epitopes administered as an mRNA vaccine, together with administration by transfection of plasmids encoding active TLR4 (caTLR4), CD70 and CD40L. The seven CD8+ CD107a+ cell lines of the patient were then expanded ex vivo and re-injected into the patient with a lung tumor. It should be understood that the invention is not limited to the described embodiments, but modifications can be applied without departing from the scope of the claims.

Claims

1. 1. A method for producing an RNA vaccine comprising multiple epitopes specifically derived from a target, comprising the steps of: - obtaining a plurality of different synthetic DNA constructs encoding a plurality of different epitopes derived from said target, said different epitopes being adjacent to sequences that allow transcription of said different epitopes encoded by said different synthetic DNA constructs into RNA sequences and sequences for translation of said transcribed RNA into peptides by a eukaryotic cell; and - in vitro transcribing a plurality of said synthetic DNAs into a corresponding plurality of RNAs; Here, the target is a peptide derived from an infectious agent or a cancer neoepitope(s) specifically identified in a patient as having at least one amino acid difference compared to a peptide derived from normal cells of the patient.

2. The method of claim 1 , wherein the DNA construct further comprises a plurality of sequence elements encoding peptide sequences for endosomal targeting of the encoded peptide.

3. The DNA construct further comprises a plurality of sequence elements encoding peptide sequences for loading the encoded peptides into T lymphocytes for presentation, and / or the DNA construct further comprises at least one sequence element encoding a translation enhancer, and / or the DNA construct further comprises at least one sequence element encoding a 3'-UTR and / or a 3' polyA tail segment. Or, the different DNA constructs share the same sequence elements encoding peptide sequences for endosomal targeting of the encoded peptide and / or loading of the encoded peptide for presentation to T lymphocytes. The method according to claim 1 or 2.

4. The method of claim 1 , wherein the multiple DNA constructs share the same promoter sequence.

5. The method of claim 4, wherein the transcription factor that recognizes the shared promoter sequence is added during the transcription step.

6. 2. The method of claim 1, wherein pseudouridine is added to the RNA during the transcription step.

7. 2. The method of claim 1, further comprising a preliminary step of optimizing the sequences of said plurality of DNAs by selecting more abundant codons and / or by avoiding deleterious secondary structures.

8. 2. The method of claim 1, wherein the plurality of different DNA molecules is at least 40 different DNA molecules encoding at least 40 different epitopes, (or) preferably at least 50, or at least 60, 80, or 100, or even 120 different epitopes.

9. 2. The method of claim 1, further comprising adding a 5' Cap element to the different epitopes, wherein the Cap element comprises two nucleotides separated by a triphosphate stretch.

10. 1. An mRNA cancer vaccine for a patient having a tumor comprising a plurality of mRNAs encoding a plurality of neoantigens, wherein the plurality of neoantigens are derived from the tumor, and the plurality of mRNAs encodes at least 1%, at least 2%, at least 5%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of all tumor neoantigens identified in the tumor.

11. The mRNA cancer vaccine of claim 10, wherein at least 25%, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the uridines present in the RNA sequence of each mRNA of the plurality of mRNAs are substituted with pseudouridine, N1-methylpseudouridine, 15-methyluridine, or 2-thiouridine.

12. 1. An ex vivo method for selecting neoantigens for a patient, comprising the steps of: Providing a blood sample from a patient who has been administered an mRNA cancer vaccine according to claim 10 or 11, or an RNA vaccine obtained by the method according to any one of claims 1 to 9 to the corresponding synthetic peptide neoepitope or one or several truncated forms thereof; Identifying which synthetic peptide neoepitopes elicit an immune response.

13. The method of claim 12, wherein the immune response is a cytotoxic immune response.

14. 14. A medicament for use in vaccinating a patient suffering from a tumor, comprising a neoantigen selected by the method of claim 12 or claim 13.

15. 14. A method for expanding specific, in vitro, cytotoxic T cells against a tumor affecting a patient, comprising conditioning a blood sample from said patient with a selected neoantigen according to claim 12 or 13.