Methods for generating pools of double-stranded DNA encoding neoantigens from a patient's tumor

Abstract

A synthetic DNA molecule comprising one segment encoding a tumor neoantigen or an epitope derived from an infectious agent under the control of a promoter for transcription into a corresponding RNA molecule and a segment for translating the translated RNA molecule into a peptide.

Description

[Technical field]

[0001] The present disclosure relates to the use of information extracted from a patient's genome to generate tumor neoantigens. In particular, it relates to the discovery of synthetic DNA molecules that contain a segment encoding a tumor neoantigen or an epitope derived from an infectious agent. Such synthetic DNA molecules can be used as templates for RNA vaccines, such as personalized vaccines that contain tumor neoantigens of the patient's tumor, or vaccines that contain epitopes derived from infectious agents.

[0002] More specifically, but not by way of limitation, the present disclosure relates to methods for producing DNA molecules that can be transcribed into RNA vaccines.

[0003] The present disclosure also relates to a pool of DNA molecules according to the invention.

[0004] The present disclosure also relates to a method for producing an RNA vaccine against a cancer affecting a patient, comprising the step of producing a DNA molecule according to the invention or a pool of DNA molecules according to the invention.

[0005] The present disclosure also relates to a vaccine obtainable by the method for producing an RNA vaccine according to the present invention. [Background technology]

[0006] Patent Document 1 provides a method for de novo gene synthesis. In particular, this document provides a method, an apparatus, and a system that can efficiently and rapidly synthesize large gene libraries and relatively long oligonucleotide fragments with fewer errors. The substrate used for de novo gene synthesis is silicon.

[0007] Patent document 2 provides a method for nucleic acid assembly, comprising: providing a predetermined nucleic acid sequence; and providing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, each double strand comprising a sticky end sequence of 5'-A(Nx)T-3' or 5'-G(Nx)T-3', where N is a nucleotide, x is the number of nucleotides between nucleotides A and T, or between nucleotides G and C, x is 1 to 10, and no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence.

[0008] Patent Document 3 provides an apparatus for synthesizing oligonucleic acids, the apparatus comprising: a plate; a main channel, the main channel extending vertically into the plate from an opening on the top surface of the plate, the width of the main channel being 0.5 to 2 mm; and a plurality of microchannels connected to the main channel, each of the plurality of microchannels extending vertically into the main channel from an opening on the bottom surface of the plate.

[0009] Patent Document 4 provides systems, methods, and compositions for efficient de novo synthesis of highly accurate and uniform polynucleotide libraries. In particular, Patent Document 4 provides a method for polynucleotide synthesis comprising the steps of: a) providing a structure including a surface; b) attaching at least one nucleoside to a polynucleotide attached to the surface; c) depositing an oxidizing solution onto the surface; d) attaching a washing solvent to the surface, the washing solvent comprising a ketone, an ester, an ether, a hydrocarbon, or a functional equivalent thereof; and e) repeating steps b to d to synthesize a plurality of polynucleotides.

[0010] Patent Document 5 provides a ribonucleic acid (RNA) cancer vaccine that can safely instruct the body's cellular machinery to produce almost any cancer protein of interest or fragments thereof. This document exemplifies cancer vaccines with 20 epitopes or 52 epitopes. This document describes an mRNA cancer vaccine with an open reading frame that codes for a concatemer of epitopes.

[0011] US Patent No. 6,399,633 describes a method of treating cancer in a patient with an mRNA vaccine, in which the vaccine nucleic acid has maximum anti-cancer effect for a given length.

[0012] Non-Patent Document 1 refers to the efficiency of antigen presentation by binding an antigen to an MHC molecule, and the vector described therein has a promoter, a polyA tail, and a 3'UTR sequence.

[0013] Non-Patent Document 2 describes in vitro transcription of a polyepitope mRNA construct encoding 32 epitopes of CMV, EBV and influenza, the mRNA construct including a DC-LAMP signal sequence, a 3'-UTR sequence and a polyA tail.

[0014] US Patent No. 5,399,633 describes methods and compositions for a vaccine against malaria, which may include a cocktail of several antigens produced using plasmid or adenovirus expression vectors.

[0015] Unfortunately, although these documents describe the advantages of these techniques, there is currently no de novo synthesis of DNA molecules, preferably long DNA molecules with a length of more than 200 nucleotides, that code for tumor-specific antigens (neoantigens) or epitopes derived from infectious agents identified from a patient's tumor. In particular, there is no method of generating said DNA molecules in a pool that allows for the generation in a pool of multiple DNA molecules that code for multiple different tumor neoantigens or epitopes derived from infectious agents. In fact, diseases, such as cancer, may be characterized by a large number of disease-specific antigens. The production of efficient personalized RNA vaccines depends on the development of vaccines that contain a sufficiently large number of disease-specific antigens, such as tumor neoantigens, that can induce an appropriate immunological response in patients. Currently, personalized vaccines are not highly effective, partly due to the fact that there is a limited number of personalized epitopes or neoantigens that can be incorporated into the vaccine. In fact, the state of the art personalized vaccines are based on DNA or RNA that encodes an open reading frame with a concatemer of epitopes. The size of the DNA or RNA is limited by the maximum size of the corresponding plasmid. The limited size does not allow the incorporation of a large number of different epitopes, which is necessary to induce a large immune response characterized by the production of specific T cells targeting different epitopes. The state of the art also mentions the de novo production of DNA fragments containing concatemeric epitopes that can be used as vaccine templates, but such de novo production is limited by the synthesis of short DNA fragments with a length of 200 nucleotides or less, which are further assembled by multiple ligations. Therefore, another important limitation of the current methods according to the state of the art is the presence of multiple ligation steps that can introduce errors into the resulting DNA fragments. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] US2015038373A1 [Patent Document 2] WO2016126987A1 [Patent Document 3] WO2016172377A1 [Patent Document 4] US2020222875 [Patent Document 5] EP3576751 [Patent Document 6] W02020 / 097291 [Patent Document 7] WO2018 / 183922 [Non-patent literature]

[0017] [Non-Patent Document 1] Kreiter Sebastian et al., The Journal of Immunology, Vol180, 2008, pp 309-318 [Non-Patent Document 2] Nielsen et al., Journal of Immunological Methods, Vol 360, 2010, pp 149-156) Summary of the Invention [Problem to be solved by the invention]

[0018] Therefore, there is a need to prepare synthetic DNA molecules that contain a segment encoding a tumor neoantigen or an epitope derived from an infectious agent, preferably the DNA molecule has a length of 200 nucleotides or more, and that can be generated in a pool of DNA molecules without any limit on the number of different DNA molecules present in the pool. [Means for solving the problem]

[0019] According to the present invention, synthetic DNA molecules are provided that contain a segment encoding a tumor neoantigen or an epitope derived from an infectious agent under the control of a promoter for transcription into a corresponding RNA molecule, and a segment for translation of the translated RNA molecule into a peptide.

[0020] According to the invention there is also provided a (complementary) DNA molecule comprising (i) a segment encoding a (second) portion of a peptide for addressing an antigen to an MHC molecule, (ii) a segment for increasing the stability of the transcribed RNA or increasing the translation of the transcribed RNA into a protein, and (iii) a segment encoding a polyA tail. As further explained in the detailed description, said (complementary) DNA molecule according to the invention is used as a reverse primer, allowing the generation of multiple double-stranded DNA molecules in a pool using only one reverse primer.

[0021] The present invention also relates to a method for producing a DNA molecule according to the invention, comprising a step of chemically synthesizing a DNA molecule encoding a tumor neoantigen under the control of a promoter.

[0022] Another subject of the present invention is a pool of DNA molecules according to the invention or a pool of DNA molecules obtained by the method according to the invention, said pool comprising epitopes from a plurality of different tumor neoantigens or infectious agents. In fact, a pool of DNA molecules according to the invention comprising different epitopes from a plurality of different tumor neoantigens or infectious agents is not limited by the number of different neoantigens or epitopes present in said pool. As a result, a pool of DNA molecules according to the present invention can serve as a template for a personalized vaccine for a patient that can incorporate an unlimited number of neoantigens of the patient, thus inducing an optimal immunological response and ensuring a high efficacy of the personalized vaccine.

[0023] The present invention also relates to a method for the production of an RNA vaccine against a cancer affecting a patient, comprising the steps of producing a DNA molecule according to the invention or a pool of DNA molecules according to the invention, and in vitro transcription of the DNA molecule into an RNA molecule.

[0024] The present invention also relates to a vaccine obtainable by a process according to the invention.

[0025] The dependent claims refer to further advantageous embodiments. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 is a schematic diagram of a DNA molecule according to the invention, including different fragments along with the corresponding transcribed RNA and translated polynucleotide. [Diagram 2] FIG. 2 shows various variations of the DNA molecule according to the invention. [Diagram 3] FIG. 3 shows a DNA molecule according to the invention with an overlapping region of two single-stranded DNA molecules. [Figure 4] FIG. 4 shows a schematic diagram illustrating overlap hybridization between a first and a second single-stranded DNA molecule. [Diagram 5] FIG. 5 shows a pool of DNA molecules according to the invention. [Figure 6] FIG. 6 shows an example of the sequence of a DNA molecule according to the invention. [Figure 7] FIG. 7 shows an example of a DNA molecule sequence and the corresponding protein sequence according to the invention. [Figure 8] FIG. 8 shows an example of a DNA molecule sequence and corresponding protein sequence according to the invention, along with forward and reverse primers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In the drawings, the same reference numbers are assigned to the same or similar elements.

[0028] Other characteristics and advantages of the invention will emerge from the non-limiting description that follows and with reference to the drawings and examples.

[0029] Figure 1 shows a schematic diagram illustrating that a synthetic DNA molecule 1.10 according to the invention comprises one segment 1.13 encoding a tumor neoantigen or an epitope derived from an infectious agent under the control of a promoter 1.11 for transcription into a corresponding RNA molecule 1.20 and a segment for translation 1.12 of said transcribed RNA molecule 1.20 into a peptide 1.30. Preferably, the synthetic DNA molecule 1.10 according to the invention comprises a segment 1.13 encoding only one tumor neoantigen or only one epitope derived from an infectious agent.

[0030] Preferably, DNA molecule 1.10 is synthesized as a single stranded DNA molecule.

[0031] Preferably, segment 1.13, encoding a tumor neoantigen or an epitope derived from an infectious agent, is a sequence comprising 21 to 504 nucleotides, preferably 51 to 201 nucleotides, more preferably 63 to 84 nucleotides, advantageously 81 nucleotides, wherein segment 1.13 advantageously encodes a tumor neoantigen of the patient's tumor.

[0032] Figure 2 shows that the DNA molecule 1.10 according to the invention preferably further comprises a segment 2.11 encoding a sequence for addressing and / or stabilizing and / or targeting and / or transporting the tumor neoantigen or the epitope derived from the infectious agent to the vesicle region or the cell surface. Preferably, the DNA molecule 1.10 comprises a segment 2.21 encoding a sequence for representing the encoded tumor neoantigen or the epitope derived from the infectious agent to the MHC molecule. Advantageously, the DNA molecule 1.10 further comprises a segment 2.31 encoding a translation enhancer.

[0033] Preferably, the DNA molecule 1.10 comprises a 3'-UTR and / or a 3' polyA tail segment 2.41 and / or a 5'-UTR. Advantageously, the DNA molecule according to the invention is not limited to the DNA molecule shown in Figures 1 and 2. Indeed, the DNA molecule according to the invention comprises at least a promoter 1.11, a segment for translation 1.12, and a segment 1.13 encoding a tumor neoantigen or an epitope derived from an infectious agent. Preferably, the segment 2.11 may further comprise a sequence for stabilizing the tumor neoantigen or infectious agent derived epitope and / or for targeting and / or transporting it to the vesicle region and / or the cell surface, and / or a segment 2.21 may further comprise a sequence for addressing the encoded tumor neoantigen or infectious agent derived epitope to MHC molecules, and / or a translation enhancer and / or a 3'-UTR and / or a 3' polyA tail segment 2.41 and / or a segment 2.31 may further comprise a 5'-UTR encoding segment 2.11 and / or 2.21 and / or 2.31 and / or 2.41. According to the present invention, the sequential order from 5' to 3' of the different segments 1.11 and / or 1.12 and / or 2.11 and / or 1.13 and / or 2.21 and / or 2.31 and / or 2.41 of the DNA molecule may vary and is not limited to the sequential order shown in the DNA molecules shown in figures 1 to 3. Advantageously, the inventors found that each DNA segment 2.11 and / or 2.21 and / or 2.31 and / or 2.41 added to DNA molecule 1.10 increases peptide expression of tumor neoantigens or infectious pathogen-derived epitope 1.13 by approximately 5-fold, respectively.

[0034] Advantageously, a synthetic DNA molecule comprising a segment encoding an epitope derived from a tumor neoantigen or an infectious pathogen under the control of a promoter for transcription into a corresponding RNA molecule and a segment for translating the transcribed RNA molecule into a peptide further comprises a segment encoding a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen or epitope derived from an infectious pathogen in the vesicle region, and preferably further comprises a segment encoding a sequence for addressing the encoded tumor neoantigen or epitope derived from an infectious pathogen to an MHC molecule, a segment encoding a translation enhancer, and a 3'-UTR and / or a 3' polyA tail segment and / or a 5'-UTR.

[0035] Alternatively, a synthetic DNA molecule comprising a segment encoding an epitope derived from a tumor neoantigen or an infectious pathogen under the control of a promoter for transcription into a corresponding RNA molecule and a segment for translating said transcribed RNA molecule into a peptide further comprises a segment encoding a sequence for addressing the encoded tumor neoantigen or epitope derived from an infectious pathogen to an MHC molecule, and preferably further comprises a segment encoding a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen or epitope derived from an infectious pathogen in the vesicle region, a segment encoding a translation enhancer, and a 3'-UTR and / or a 3' polyA tail segment and / or a 5'-UTR.

[0036] Alternatively, a synthetic DNA molecule comprising a segment encoding an epitope derived from a tumor neoantigen or an infectious pathogen under the control of a promoter for transcription into a corresponding RNA molecule and a segment for translating said transcribed RNA molecule into a peptide further comprises a segment encoding a translation enhancer and preferably further comprises a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen or epitope derived from an infectious pathogen in the vesicle region, a segment encoding a sequence for addressing the encoded tumor neoantigen or epitope derived from an infectious pathogen to an MHC molecule and a 3'-UTR and / or a 3' polyA tail segment and / or a 5'-UTR.

[0037] Alternatively, a synthetic DNA molecule comprising a segment encoding an epitope derived from a tumor neoantigen or an infectious pathogen under the control of a promoter for transcription into a corresponding RNA molecule and a segment for translating said transcribed RNA molecule into a peptide further comprises a 3'-UTR and / or a 3' polyA tail segment and / or a 5'-UTR, and further comprises sequences for stabilizing and / or targeting and / or transporting the tumor neoantigen or epitope derived from an infectious pathogen, preferably in the vesicle region, sequences for addressing the encoded tumor neoantigen or epitope derived from an infectious pathogen to an MHC molecule, and a segment encoding a translation enhancer.

[0038] Preferably, promoter 1.11 is a bacteriophage RNA polymerase promoter. More preferably, promoter 1.11 belongs to the group of promoters comprising the promoter sequences of T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, Syn5 RNA polymerase, KP34 RNA polymerase. Even more preferably, according to the present invention, the promoter sequence of T7 RNA polymerase is a binding sequence for T7 RNA polymerase having at least 95% identity to SEQ ID NO: 1 over its entire sequence, the promoter sequence of SP6 RNA polymerase is a binding sequence for SP6 RNA polymerase having at least 95% identity to SEQ ID NO: 73 over its entire sequence, the promoter sequence of T3 RNA polymerase is a binding sequence for T3 RNA polymerase having at least 95% identity to SEQ ID NO: 74 over its entire sequence, the promoter sequence of Syn5 RNA polymerase is a binding sequence for Syn5 RNA polymerase having at least 95% identity to SEQ ID NO: 75 over its entire sequence, and the promoter sequence of KP34 RNA polymerase is a binding sequence for KP34 RNA polymerase having at least 95% identity to SEQ ID NO: 76 (strong promoter 1) over its entire sequence, or to SEQ ID NO: 77 (strong promoter 2) over its entire sequence, or to SEQ ID NO: 78 (weak promoter) over its entire sequence. Preferably, promoter 1.11 according to the invention is a promoter sequence of the T7 RNA polymerase which has at least 95% identity to SEQ ID NO: 1 over its entire sequence.

[0039] Advantageously, the translational segment 1.12 is a translational enhancer sequence located in the 5'UTR region (upstream of the coding region) of the DNA molecule 1.10. The coding region, the coding portion of the DNA molecule 1.10 is represented by a grey box in Figure 1, Figure 2 and Figure 3. Preferably, the translational 1.12 segment is a translational enhancer / promoter sequence selected from the group of translational enhancer / promoter sequences comprising the human cytomegalovirus (CMV) enhancer, the alpha or beta globin enhancer, the EF-1 alpha enhancer, the simian virus 40 (SV40) enhancer, the PGK1 promoter, the HSD17B4 promoter, the ubiquitin C promoter and the beta actin promoter. More preferably, the translational 1.12 segment is a beta globin translational enhancer sequence. Preferably, said beta globin translational enhancer sequence has at least 95% identity to SEQ ID NO: 2 over its entire sequence length.

[0040] Preferably, segment 2.11, encoding a sequence for addressing and / or stabilizing and / or targeting and / or transporting tumor neoantigens or epitopes from infectious agents to vesicular regions, is a signal sequence for endosomal processing, such as a signal sequence of a lysosomal associated membrane protein (LAMP1 or LAMP2; such as SEQ ID NO: 3), or a signal sequence of dendritic cells (DC-LAMP), or a sequence for addressing to vesicular compartments for binding to MHC-I. Preferably, a signal sequence for endosomal processing, said signal sequence preferably having at least 95% identity to SEQ ID NO: 3 over its entire length.

[0041] Preferably, segment 2.21 encodes a sequence for addressing the encoded tumor neoantigen or infectious agent derived epitope to MHC molecules and immune processing for presentation to T lymphocytes.More preferably, segment 2.21 is a dendritic cell lysosomal associated membrane glycoprotein signal sequence, preferably having at least 95% identity to SEQ ID NO:5 over the entire length of the sequence.

[0042] Advantageously, segment 2.31, which codes for a translation enhancer, is a translation enhancer and / or translation stabilizing sequence preferentially located in the 3'UTR region (downstream of the coding region) of DNA molecule 1.10. Preferably, segment 2.31 is a beta-globin translation enhancer sequence, which preferably has at least 95% identity to SEQ ID NO:6 over its entire length.

[0043] Preferably, segment 2.41 is a 3' polyA tail sequence of at least 68 nucleotides, preferably at least 100 nucleotides.

[0044] Preferably, the DNA molecule may further comprise 3' and / or 5' segments corresponding to conserved sequence elements (3' and / or 5' CSE segments), which are advantageously recognized by an RNA-dependent RNA polymerase (RdRp) to allow RNA amplification of the transcribed DNA molecule.

[0045] Advantageously, the DNA molecules according to the invention have a size of from 100 nucleotides per strand to about 2500 nucleotides per strand, preferably from 300 nucleotides per strand to about 800 nucleotides per strand, more preferably from about 400 nucleotides per strand to about 700 nucleotides per strand, and even more preferably from about 500 (or 550) nucleotides per strand to about 650 (or 600) nucleotides per strand.

[0046] FIG. 3 shows that, in accordance with the present invention, the (complementary) DNA molecule 3.20 comprises (i) a segment encoding a (second) portion of segment 2.21 of a peptide for addressing the antigen (epitope 1.13) to an MHC molecule, (ii) segment 2.31 for increasing the stability of the transcribed RNA or for increasing the translation of the transcribed RNA into a protein, and (iii) segment 2.41 encoding a polyA tail.

[0047] Advantageously, the DNA molecule 3.20, which is antisense according to the invention, is preferably for base pairing with a sense DNA molecule 3.10, said DNA molecule 3.10 comprising, under the control of a promoter for transcription into a corresponding RNA molecule, a segment 1.13 encoding an antigen or epitope, a segment 1.12 for its translation, a segment 2.11 encoding a sequence for addressing and / or stabilizing and / or targeting and / or transporting a tumor neoantigen or an epitope from an infectious agent to a vesicle region and a segment encoding a (first) part of a segment 2.21 of a peptide for loading an antigen for presentation to T lymphocytes, wherein said first and second parts form a complete sequence 2.21 for loading an antigen for presentation to T lymphocytes, and said first and second parts are sense and antisense, and there is an overlapping sequence 3.30 allowing base pairing of the DNA molecule 3.10 with the DNA molecule 3.20.

[0048] Advantageously, the overlapping sequence 3.30 is a contiguous sequence of at least 5 nucleotides, preferably at least 10 nucleotides, more preferably at least 15 nucleotides, advantageously at least 20 nucleotides and / or preferably at most 100 nucleotides, more preferably at most 75 nucleotides, advantageously at most 40 nucleotides. Preferably, the overlapping sequence 3.30 is located in a constant sequence region. According to the present invention, a constant sequence region refers to a sequence region present in any DNA molecule 1.10 according to the present invention.

[0049] Preferably, the overlapping sequence is a sequence that has at least 95% identity over its entire length to the sequence corresponding to nucleotides 30 to 52 of SEQ ID NO:5.

[0050] Preferably, the method for producing a DNA molecule according to the invention comprises a step of chemically synthesizing a DNA molecule encoding a tumor neoantigen under the control of a promoter.

[0051] Preferably, the chemical synthesis is single-stranded oligonucleotide synthesis using silicon-based DNA writing technology (i.e., US2018104664A1).

[0052] Preferably, the method according to the invention comprises: (i) a first single-stranded DNA molecule comprising a promoter 1.11 for transcription into RNA, a segment 1.12 for its translation and a targeting sequence 2.11 for addressing to endosomes fused in frame with a tumor neoantigen or infectious agent-derived epitope 1.13 and a first portion of a genetic element 2.21 encoding a sequence for loading the encoded tumor neoantigen or infectious agent-derived epitope for presentation to T lymphocytes; (ii) a second single-stranded DNA molecule 3.20 encoding a second portion of the genetic element 2.21 encoding a sequence for loading an encoded tumor neoantigen or an epitope derived from an infectious agent for presentation to T lymphocytes, said second single-stranded DNA molecule being antisense, and said first portion and said second portion forming a complete sequence 2.21 for loading an encoded tumor neoantigen or an epitope derived from an infectious agent for presentation to T lymphocytes, said first portion and said second portion being overlapped (by at least 15 consecutive nucleotides) 3.30 to allow specific base pairing; A method comprising the steps of: The method further comprises the steps of hybridizing the DNA molecule of (i) with the DNA molecule of (ii) and extending the two molecules to generate a double stranded DNA molecule encoding a genetic element encoding a promoter 1.11, a translation initiator 1.12, a targeting sequence 2.11 for addressing to endosomes fused in frame with a tumor neoantigen or an infectious pathogen derived epitope 1.13, and a sequence 2.21 for loading of the encoded tumor neoantigen or infectious pathogen derived epitope for presentation to T lymphocytes.

[0053] Preferably, in the method according to the present invention, the DNA molecule (ii) further comprises a 3'-UTR 2.31 and / or a polyA tail 2.41.

[0054] FIG. 4 further shows that at 4.10, the first single-stranded DNA molecule 3.10 comprises a sequence 4.11 located in a first portion of the genetic element 2.21 that overlaps with a sequence 4.12 located in a second portion of the genetic element 2.21 of the second single-stranded DNA molecule 3.20. The first single-stranded DNA molecule 3.10 comprises a segment 1.13 of an epitope from a tumor neoantigen or infectious agent. The first single-stranded DNA molecule 3.10 hybridized with the second single-stranded DNA molecule 3.20 via the overlap 3.30 (see 4.20 in FIG. 4). An extension starting from the overlap region 3.30 then allows the generation of the double-stranded DNA molecule 4.30. Preferably, the extension is an enzymatic process carried out by a DNA polymerase using both single-stranded DNA molecules 3.10 and 3.20 as templates.

[0055] Advantageously, the present invention also relates to a pool of DNA molecules according to the invention or a pool obtained by the method according to the invention, said pool comprising a plurality of different tumor neoantigens or epitopes from infectious agents, preferably each tumor neoantigen of the plurality being a specific tumor antigen identified in a patient's tumor.

[0056] Advantageously, molecules 3.20 are identical and complementary to a pool of molecules 3.10.

[0057] Figure 5 shows a pool 5.10 of DNA molecules according to the invention, or a pool obtained by a method according to the invention. Advantageously, the pool comprises a first single-stranded DNA molecule 3.10 comprising a sequence 4.11 located in a first part of the genetic element 2.21, which overlaps with a sequence 4.12 located in a second part of the genetic element 2.21 of a second single-stranded DNA molecule 3.20. The first single-stranded DNA molecule 3.10 has a sequence 1.13 encoding a tumor neoantigen or an epitope derived from an infectious agent. Preferably, the pool also comprises a plurality of different first single-stranded DNA molecules, for example 3.10a and 3.10b, each of which has a respective specific sequence encoding a second and a third specific tumor neoantigen (or an epitope derived from a second and a third infectious agent) 1.13a and 1.13b. Preferably, the amino acid sequences of the tumor neoantigens encoded by sequences 1.13, 1.13a and 1.13b differ by at least one amino acid each from the corresponding "reference" sequence, or said amino acid sequences are de novo amino acid sequences encoded from an open reading frame that is present only in the patient's tumor and not in the patient's normal cells, i.e. the patient's peripheral blood mononuclear cells (PBMCs). The plurality of different first single-stranded DNA molecules 3.10a and 3.10b also comprises a sequence 4.11 located in a first portion of genetic element 2.21 that overlaps with a sequence 4.12 located in a second portion of genetic element 2.21 of the second single-stranded DNA molecule 3.20. The pool may also comprise a plurality of hybridized molecules between a first single-stranded DNA molecule (e.g. 3.10, 3.10a and / or 3.10b) and a second single-stranded DNA molecule 3.20 (e.g. hybridized molecule 4.20 between the first single-stranded DNA molecule 3.10 and the second single-stranded DNA molecule 3.20), and a plurality of hybridized molecules between the first single-stranded DNA molecule 3.10b and the second single-stranded DNA molecule 3.20 5.30. Advantageously, the pool also comprises a plurality of double-stranded DNA molecules, each double-stranded DNA molecule comprising a portion of the sequence corresponding to the first single-stranded DNA molecule (e.g. 3.10, 3.10a or 3.10b) and its complementary sequence, and a second portion corresponding to the second single-stranded DNA molecule 3.20 and its complementary sequence.In figure 5, a double-stranded DNA molecule 4.30 is shown, which comprises a first single-stranded DNA molecule 3.10 and a second single-stranded DNA molecule 3.20, and a double-stranded DNA molecule 5.40, which comprises a first single-stranded DNA molecule 3.10b and a second single-stranded DNA molecule 3.20. Preferably, in a pool of DNA molecules according to the invention, a plurality of DNA molecules of the pool have the same promoter for transcription of a tumor neoantigen or an epitope from an infectious agent into RNA.

[0058] According to the present invention, the corresponding "reference" sequence corresponds to the predicted peptide sequence of DNA sequence data derived from normal cells of the patient, i.e. PBMCs.

[0059] More preferably, in a pool of DNA molecules according to the invention, multiple DNA molecules of said pool have identical genetic elements encoding sequences for targeting tumor neoantigens or epitopes derived from infectious agents to endosomes and / or identical translation enhancers and / or identical genetic elements encoding sequences for loading the encoded tumor neoantigens or the encoded epitopes derived from infectious agents for presentation to T lymphocytes and / or identical 3'-UTRs.

[0060] Advantageously, the pool of DNA molecules according to the present invention comprises a plurality of second single-stranded DNA molecules 3.20, each second single-stranded DNA molecule 3.20 of the plurality of second single-stranded DNA molecules 3.20 having an identical DNA sequence.

[0061] Preferably, each second single stranded DNA molecule 3.20 of the plurality of second single stranded DNA molecules 3.20 is defined by a sequence having at least 95% identity to SEQ ID NO:8 over its entire length.

[0062] Preferably, the pool of DNA molecules according to the present invention comprises a plurality of first single-stranded DNA molecules comprising epitopes from a plurality of different tumor neoantigens or a plurality of different infectious pathogens.

[0063] Advantageously, the overlapping sequence 3.30 between the first single-stranded DNA molecule 3.10 and the second single-stranded DNA molecule 3.20 is a continuous sequence within the constant sequence region, thus allowing the generation of a pool of double-stranded DNA molecules by extension of the two partially overlapping and paired strands 3.10 and 3.20.

[0064] Advantageously, the first single-stranded DNA molecules 3.10, 3.10a, and 3.10b are DNA molecules each having a sequence that differs from each other only for the segment 1.13 of the tumor neoantigen or infectious agent-derived epitope that can code for a plurality of different tumor neoantigens or a plurality of different infectious agent-derived epitopes, i.e., a first specific tumor neoantigen 1.13, a second specific tumor neoantigen 1.13a, and / or a third specific tumor neoantigen 1.13b. With the exception of the segment 1.13 of the tumor neoantigen or infectious agent-derived epitope, other segments of each first single-stranded DNA molecule 3.10 of the plurality of first single-stranded DNA molecules are substantially identical, e.g. the promoter 1.11 for transcription into RNA, the segment 1.12 for its translation, and the targeting sequence 2.11 for addressing to endosomes and the first part of the genetic element 2.21 encoding a sequence for loading the encoded tumor neoantigen or infectious agent-derived epitope for presentation to T lymphocytes.

[0065] Advantageously, the tumor neoantigen-encoding segments, i.e. 1.13, 1.13a or 1.13b, generate tumor neoantigens that have a qualitative difference in amino acid sequence compared to the corresponding peptide and / or novel amino acid sequence encoded in the open reading frame that is present only in the tumor and not in normal cells of the patient. Preferably, each tumor neoantigen of the plurality of different tumor neoantigens is defined by an amino acid sequence that differs from each other by at least one amino acid.

[0066] According to the invention, a pool of double-stranded DNA molecules counts at least 10, preferably at least 20, more preferably at least 40, even more preferably at least 60, advantageously at least 100 different double-stranded DNA molecules, each encoding a different tumor neoantigen (or different epitopes from an infectious agent), and a double-stranded DNA molecule is different from another double-stranded DNA molecule of the pool of double-stranded DNA molecules if the double-stranded DNA molecule is defined by a sequence encoding a tumor neoantigen having an amino acid sequence that differs by at least one amino acid sequence from the amino acid sequence of another neoantigen encoded by another double-stranded DNA molecule. Of course, the pool may contain multiple copies of the same double-stranded DNA molecule.

[0067] Advantageously, each tumor neoantigen in the plurality of different tumor neoantigens is defined by an amino acid sequence having an identical mutation compared to the amino acid sequence of the corresponding reference sequence, or by a de novo amino acid sequence encoded from a de novo open reading frame that is present only in the DNA of the patient's tumor and not in the DNA derived from the patient's normal cells (i.e. PBMCs), where the identical mutation is located at a different position in the amino acid sequence for each neoantigen in the plurality of different tumor neoantigens. For example, if for a first tumor neoantigen, the first mutation is located substantially in the middle of the amino acid sequence of the first tumor neoantigen, then for a second tumor neoantigen, the same first mutation is located substantially in the first half or first quarter, or in the second half or last quarter of the amino acid sequence of the second tumor neoantigen, and the first amino acid of the tumor neoantigen corresponds to the first 5' codon of segment 1.13 of the tumor neoantigen.

[0068] Preferably, the pool of double-stranded DNA molecules is amplified by PCR amplification using forward primer 5.50 and reverse primer 5.60. Preferably, the reverse primer is an oligonucleotide poly-T capable of extending to the 3' end of the DNA molecule, such as SEQ ID NO: 79 or SEQ ID NO: 80 according to the present invention.

[0069] Preferably, the generation of the pool of double-stranded DNA molecules and the amplification of the pool of double-stranded DNA molecules are carried out using the same tube, and therefore the same reactants (primer pair, polymerase, dNTPs, reaction medium) are used, and the two strands are first extended, each serving as a template for extending the other strand, and then the two extended strands are separated and amplified in a simultaneous or subsequent manner while base pairing with specific primers (5.50, 5.60) at low temperature and then extended at high temperature. Alternatively, the primers are added to the reaction medium after the first extension, i.e. the first extension step is completed.

[0070] Preferably, the pool of double-stranded DNA molecules also includes a segment recognized by an RNA-dependent polymerase, such as the nonstructural proteins of alphavirus nsP1-4. In fact, when the pool of double-stranded DNA molecules is used as a template for in vitro transcription of DNA molecules into RNA molecules to generate an RNA vaccine, it is useful that the generated mRNA is capable of transamplification, for example, transamplification based on the encoded RdRp. Such RNA transamplification can reduce the dose of the mRNA vaccine administered to the patient.

[0071] Preferably, the present invention also relates to a method for producing an RNA vaccine against a cancer affecting a patient, comprising the steps of producing a DNA molecule according to the present invention or a pool of DNA molecules according to the present invention, and performing in vitro transcription of the DNA molecule into an RNA molecule.

[0072] Preferably, the method for producing an RNA vaccine according to the present invention further comprises the step of formulating the RNA molecule into a vaccine.

[0073] Advantageously, the present invention also relates to a vaccine obtained by the method for producing an RNA vaccine according to the invention. Preferably, the present invention also relates to a vaccine obtainable according to the present invention for use in the treatment of a cancer suffered by a patient. EXAMPLES

[0074] Example 1: Identification of tumor neoantigens in patient tumors.

[0075] The inventors performed whole genome sequencing of DNA from the patient's tumor samples and DNA from the patient's peripheral blood mononuclear cells (PBMCs). RNASeq of the tumor samples was also performed. Identification of tumor neoantigens was realized based on open reading frames (ORFs) from which mRNAs were identified from the tumor RNASeq, the mRNAs having predicted peptide sequences with at least one confirmed difference compared to a reference peptide sequence. The reference peptide sequence is a predicted peptide sequence derived from an open reading frame identified from whole genome sequencing data from the DNA of the patient's PBMCs. The identified differences could be the result of point mutations with different amino acids compared to the reference peptide sequence, or translocations, deletions and / or indels, or fusions. A detailed protocol for identifying tumor neoantigens in the patient's tumors is described in patent application EP21 196366.5.

[0076] Example 2: Identification of amino acid and DNA sequences of neoantigens.

[0077] The present inventors have identified a neoantigen peptide sequence of 27 amino acids, where the difference identified compared to a reference peptide sequence is located substantially in the middle of the 27 amino acid sequence length of the neoantigen peptide sequence.

[0078] The peptide sequences of the neoantigens are converted to nucleic acid sequences using online tools that optimize codon usage for optimal expression in humans, reduce the formation of RNA secondary structures, and increase desirable immune responses or reduce unwanted immune responses when the nucleic acid sequences are used as templates for RNA vaccines.

[0079] Example 3: Synthesis of a pool of DNA molecules according to the invention containing multiple tumor neoantigens.

[0080] The inventors used silicon-based DNA writing technology (see Twist Oligo Pools technology from TWIST Bioscience) to synthesize single-stranded DNA molecules in a pool containing DNA sequences of multiple tumor neoantigens obtained in Example 2. In the pool of DNA molecules according to the present invention, multiple second single-stranded DNA molecules are synthesized, each having substantially the same sequence SEQ ID NO: 8, which is 297 nucleotides long. Multiple 60 different first single-stranded DNA molecules are also synthesized in the pool, the 60 different first single-stranded DNA molecules having substantially the sequences SEQ ID NO: 13 to SEQ ID NO: 72, each sequence of SEQ ID NO: 13 to SEQ ID NO: 72 being a sequence of 291 nucleotides long. The second single-stranded DNA molecule, which is antisense, can hybridize with each first single-stranded DNA molecule having an overlap with a sequence corresponding to nucleotides 30 to 52 of SEQ ID NO: 5 (sequence for sense / antisense pairing).

[0081] Example 4: DNA molecules according to the invention

[0082] An example of the sequence of a DNA molecule obtained according to the invention is shown in FIG. 6, which shows a DNA sequence of 600 nucleotides. Starting from the first nucleotide 1, the DNA molecule contains an optimized T7 RNA polymerase promoter segment (bold lowercase letters in the sequence of the DNA molecule in FIG. 6), followed by the sequence of a β-globin translation enhancer segment (lowercase letters). The next segment is a segment encoding the human LAMP-1 signal peptide (bold lowercase letters), followed by a segment encoding a 27 amino acid tumor neoantigen (uppercase letters). The next segment is a segment encoding DC-Lamp (lowercase letters). Within the DC-Lamp sequence, there is an overlapping sequence (highlighted uppercase letters) that represents the overlapping sequence between the first single-stranded DNA molecule and the second single-stranded DNA molecule. After the DC-Lamp sequence, there is a 3'UTR β-globin segment (β-globin UTR) (bold lowercase letters) for optimization of translation and stabilization, followed by a polyA tail of 70 nucleotides up to 105 nucleotides.

[0083] FIG. 7 shows the same sequence of a DNA molecule with the amino acid sequence of the coded portion of the DNA sequence juxtaposed to the DNA sequence.

[0084] Figure 8 is similar to Figure 7, in which the 5' to 3' sequence of a first single-stranded DNA molecule is represented (Forward Pr.), while the 5' to 3' sequence of a second single-stranded DNA molecule that is antisense to the first single-stranded DNA molecule is also represented (Rev.Pr.). The forward primer (PCR prim.) and reverse primer (gc-T150) used in the subsequent PCR amplification are also shown.

[0085] Example 5: Creation of a pool of double-stranded DNA molecules

[0086] The hybridized DNA molecules between the first single-stranded DNA molecule and the second single-stranded DNA molecule are obtained in Example 3. From these hybridized DNA molecules, double-stranded DNA molecules can be generated at femtomolar concentration levels by extending each strand using DNA polymerase.

[0087] Example 6: Amplification of a pool of double-stranded DNA molecules

[0088] To allow the generation of RNA molecules to be incorporated into an RNA vaccine according to the invention and to generate sufficiently large amounts of double-stranded DNA molecules necessary for the subsequent in vitro transcription, the pool of double-stranded DNA molecules obtained in Example 5 was amplified. To do so, the inventors performed 13 cycles of PCR amplification of the double-stranded DNA molecules using a forward primer with the sequence corresponding to SEQ ID NO: 7 and a reverse primer with the sequence 5'(T100)-GC-3' corresponding to SEQ ID NO: 79.

[0089] The PCR reaction components are: - 5x KAPA HiFi Fidelity Buffer (1x), - 10mM each dNTP mix, - 10μM forward primer, - 10 μM reverse primer, - the pool of DNA molecules obtained in Example 4 (20 ng / ul), - KAPA HiFi HotStart DNA Polymerase (1 U / ul) and, - PCR grade water.

[0090] PCR reaction conditions: - Initial denaturation (95°C for 3 min) - Denaturation (98°C for 20 seconds) - Annealing (15 seconds) - Extension (15 seconds at 72°C) - Final extension (1 min at 72°C).

[0091] Example 7: In vitro transcription of pools of double-stranded DNA molecules

[0092] The amplified pool of double-stranded DNA molecules obtained in Example 6 is in vitro transcribed to produce a corresponding pool of RNA molecules.

[0093] The inventors have shown that a pool of double-stranded DNA molecules and a pool of corresponding mRNA molecules amplified after IVT are correctly generated, with the sizes of the DNA fragments in the DNA pool and the RNA fragments in the RNA pool matching the expected theoretical sizes of the fragments in the DNA pool and the RNA pool. Furthermore, the inventors have shown that the read count ratio of each DNA molecule before IVT is positively correlated with the read count ratio of each corresponding RNA molecule obtained after IVT. Finally, the inventors have shown that all DNA molecules in the pool of DNA molecules are correctly transcribed into mRNA molecules.

[0094] It is to be understood that the invention is not limited to the described embodiments but is capable of variations without departing from the scope of the claims.

[0095] Drawing Terminology promoter translation epitope addressing stability and / or enhancer poly A tail Optimized T7RNApol prom. Optimized T7RNA polymerase promoter β-globin transl. enhancer Human LAMP-1 Sig. peptide Human LAMP-1 signal peptide NEOANTIGEN Neoantigen OVERLAP Overlap 3' β-globin UTR 3' β-globin UTR

Claims

1. a synthetic DNA molecule comprising a segment encoding a tumor neoantigen or an epitope derived from an infectious agent, under the control of a promoter for transcription into a corresponding RNA molecule, and a segment for translation of the transcribed RNA molecule into a peptide; wherein the synthetic DNA molecule further comprises a segment encoding a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen or the epitope derived from the infectious agent in the vesicle region and / or a sequence for addressing the encoded tumor neoantigen or the epitope derived from the infectious agent to an MHC molecule; and / or The synthetic DNA molecule further comprises a segment encoding a translation enhancer and / or a 3'-UTR and / or a 3' poly A tail segment and / or a 5'-UTR.

2. 2. The DNA molecule of claim 1, wherein the DNA molecule has a size comprising from 100 nucleotides per strand to about 2500 nucleotides per strand, or from 300 nucleotides per strand to about 800 nucleotides per strand, or from about 400 nucleotides per strand to about 700 nucleotides per strand, or from about 500 nucleotides per strand to about 650 nucleotides per strand, or from about 550 nucleotides per strand to 600 nucleotides per strand and is double-stranded.

3. A DNA molecule comprising: (i) a segment encoding a (second) portion of a peptide for addressing an antigen to an MHC molecule; (ii) a segment for increasing the stability of the transcribed RNA or for increasing the translation of the transcribed RNA into a protein; and (iii) a segment encoding a polyA tail.

4. 4. The DNA molecule according to claim 3, which is an antisense for base pairing with the DNA molecule according to claim 1 or 2 as a sense DNA molecule.

5. A method for producing a synthetic DNA molecule comprising a segment encoding an epitope derived from a tumor neoantigen or an infectious agent, under the control of a promoter for transcription into a corresponding RNA molecule, and a segment for translation of the transcribed RNA molecule into a peptide, comprising the steps of: a step of chemically synthesizing the DNA molecule encoding the tumor neoantigen or the epitope derived from the infectious pathogen under the control of a promoter, wherein the chemical synthesis is single-stranded oligonucleotide synthesis using silicon-based DNA writing technology.

6. 6. The method of claim 5, wherein the synthetic DNA molecule further comprises a segment encoding a sequence for stabilizing and / or targeting and / or transporting the tumor neoantigen or the epitope derived from the infectious agent in the vesicle region and / or a sequence for addressing the encoded tumor neoantigen or the epitope derived from the infectious agent to an MHC molecule; and / or The synthetic DNA molecule further comprises a segment encoding a translation enhancer and / or a 3'-UTR and / or a 3' poly A tail segment and / or a 5'-UTR.

7. 7. The method of claim 6, (i) a first single-stranded DNA molecule comprising a first portion of a genetic element encoding a promoter for transcription into RNA, a segment for its translation and a targeting sequence for addressing to endosomes fused in-frame with the epitope derived from the tumor neoantigen or infectious pathogen, and a sequence for loading the encoded tumor neoantigen or infectious pathogen-derived epitope for presentation to T lymphocytes; and (ii) a second single-stranded DNA molecule encoding a second portion of the genetic element encoding a sequence for loading the encoded tumor neoantigen or the epitope from an infectious agent for presentation to T lymphocytes, wherein the second single-stranded DNA molecule is antisense, and the first portion and the second portion form a complete sequence for loading the tumor neoantigen or the epitope from an infectious agent for presentation to T lymphocytes, wherein there is an overlap (of at least 15 contiguous nucleotides) between the first portion and the second portion to allow specific base pairing. comprising a step of chemically synthesizing The method further comprises hybridizing the DNA molecule of (i) with the DNA molecule of (ii) and extending the two molecules to generate a double-stranded DNA molecule encoding genetic elements encoding a promoter, the translation enhancer, a targeting sequence for addressing the tumor neoantigen or the epitope derived from an infectious pathogen to an endosome to which the tumor neoantigen or the epitope derived from an infectious pathogen is fused in frame, and the sequence for loading the encoded tumor neoantigen or the epitope derived from an infectious pathogen for presentation to T lymphocytes.

8. The method of claim 7, wherein the DNA molecule of (ii) further comprises a 3'-UTR and / or a polyA tail.

9. A pool of at least 10 different synthetic DNA molecules comprising multiple different epitopes derived from multiple different tumor neoantigens or infectious pathogens, each of the synthetic DNA molecules comprising a segment encoding one epitope derived from one tumor neoantigen or infectious pathogen under the control of a promoter for transcription into a corresponding RNA molecule, and a segment for translation of the transcribed RNA molecule into a peptide.

10. 10. The pool of DNA molecules of claim 9, wherein multiple of the synthetic DNA molecules of the pool have the same promoter for transcription of the epitope derived from the tumor neoantigen or infectious pathogen into RNA.

11. 11. The pool according to claim 9 or 10, wherein the synthetic DNA molecules further comprise a segment encoding a sequence for stabilizing and / or targeting and / or transporting the epitope derived from the tumor neoantigen or infectious pathogen in the vesicle region and / or a sequence for addressing the encoded tumor neoantigen or infectious pathogen-derived epitope to an MHC molecule; and / or The synthetic DNA molecule further comprises a segment encoding a translation enhancer and / or a 3'-UTR and / or a 3' poly A tail segment and / or a 5'-UTR.

12. 12. The pool of DNA molecules according to claim 11, wherein a plurality of DNA molecules of the pool of DNA molecules have the same genetic element encoding a sequence for targeting the epitope derived from the tumor neoantigen or infectious pathogen to endosomes, and / or the same translational enhancer and / or the same genetic element encoding a sequence for loading the encoded tumor neoantigen or the epitope derived from the infectious pathogen for presentation to T lymphocytes and / or the same 3'-UTR.

13. A method for producing an RNA vaccine against a cancer affecting a patient, comprising the steps of producing a pool of DNA molecules according to claim 1 or 2 or DNA molecules according to any one of claims 9 to 12, and performing in vitro transcription of the DNA molecules into RNA molecules.

14. 14. The method of claim 13, further comprising formulating the RNA molecule into a vaccine.

15. 15. A vaccine obtainable by the method of claim 14 for use in the treatment of cancer or an infectious disease affecting a patient. (Other) Amended claim 1 combines original claims 1 to 3. Original claims 4 to 6 are amended claims 2 to 4. Amendment Subsequent claims 5 to 8 are manufacturing process inventions and are based on original claims 7 to 9. Amended claims 9 to 12 are Poole's inventions and are based on original claims 10 to 12.

Images