Cancer vaccine

Abstract

The invention relates to nucleic acid molecules comprising an IL-12 sequence and a neoantigen cassette. The invention also relates to vaccines comprising the nucleic acid molecule(s). In addition, the invention relates to methods of producing the vaccines and uses of the nucleic acid molecules or vaccines in treating cancer.

Description

[0001] CANCER VACCINE

[0002] FIELD

[0003] The invention relates to nucleic acid molecules comprising an IL-12 sequence and a neoantigen cassette. The invention also relates to vaccines comprising the nucleic acid molecule(s). In addition, the invention relates to methods of producing the vaccines and uses of the nucleic acid molecules or vaccines in treating cancer.

[0004] BACKGROUND

[0005] Cancer is a fatal disease globally. Around 9.6 million people died worldwide due to cancer in 2018 and there were 18.1 million cancer cases around the world in 2020 (worldwide cancer data). Each patient's cancer is unique at the genetic level, which means that every patient’s cancer is different and, ideally, should be treated differently. Likewise, every patient responds to treatment differently. Hence, there is a need for customised treatments to fit each patient's needs. Until now the cancer treatments have been focussed on either removing or stopping cells from growing abnormally either with chemotherapy, radiotherapy, or surgery. All these treatments are painful, costly, time-consuming, and put burden on patient’s recovery.

[0006] Personalised cancer therapies are non-toxic and can have fewer side effects than other types of treatment. The majority of personalized cancer vaccine therapies are allogeneic. Allogeneic cancer vaccines are composed of neoantigens isolated from the tumour of one patient, processed and administered to another patient to stimulate cytotoxic immune responses to a similar tumour type. There are hundreds of allogeneic cancer vaccines in clinical trials but with limited success. Currently, there are no FDA approved allogeneic cancer vaccines. On the other hand, autologous vaccine therapies are patient-specific, involving the isolation and selection of neoantigens and their subsequent re-introduction into the same individual. Only a percent of neoantigens is shared by any two individuals within similar cancer types. Hence, cancer mutations and neoantigens are truly patient specific.

[0007] Current cancer vaccine delivery platforms in development include cell-based vaccines, viruses-based vaccines, peptide-based vaccines, and nucleic acids-based vaccines. Cell-based vaccines are the main form of primeval cancer vaccines, where dendritic cells (DCs) vaccine has achieved significant results in clinical trials. However, cell-based vaccines are typically very complex to prepare and have potential for HLA-restriction. Virus-based cancer vaccines mainly use viruses as vectors to treat and prevent tumours. This, however, is often associated with unwanted immune responses. Peptide- based vaccines are composed of known or predicted neoantigens. However, peptide-based vaccines are often less immunogenic, requiring a combination with adjuvants to enhance their immunogenicity. Nucleic acid vaccines include DNA and RNA vaccines, composed of the encoding gene and carrier group of pathogen antigens. DNA cancer vaccines are typically closed circular DNA plasmids encoding tumour-associated antigens to induce tumour-specific responses. However, production of DNA plasmids in the context of personalised cancer treatment is very time consuming. mRNA vaccines are synthesized in vitro, they could encode antigens and express proteins following internalization to stimulate an immune response in a timely manner.

[0008] None of the current cancer vaccine delivery platforms are easy to manufacture, include a very good safety profile and trigger a good immune response in patents. Accordingly, a need exists for an improved cancer vaccine.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the present invention provides a nucleic acid molecule comprising: a. an interleukin 12 (IL-12) sequence; and b. a neoantigen cassette.

[0011] Preferably, the neoantigen cassette encodes at least two neoantigens.

[0012] The nucleic acid molecule may be single or double stranded. The IL-12 sequence may be encoded 5’ (i.e. upstream) of the neoantigen cassette. Alternatively, the IL-12 sequence may be encoded 3’ (i.e. downstream) of the neoantigen cassette. The nucleic acid molecule may further comprise a poly-A sequence. The nucleic acid molecule may be a DNA molecule or an mRNA molecule. In all aspects and embodiments described herein, preferably, the nucleic acid molecule is an mRNA molecule.

[0013] In one aspect, the invention provides a nucleic acid molecule, which is an mRNA molecule, which comprises (optionally in the 5’ to 3’ direction): a. an IL-12 sequence; b. a neoantigen cassette; and c. a poly-A sequence.

[0014] The mRNA molecule may be derivable from the DNA molecule as described herein.

[0015] In one aspect, the invention provides a nucleic acid molecule, which is a DNA molecule, which comprises (optionally in the 5’ to 3’ direction): a. a promoter; b. an IL-12 sequence; and c. a neoantigen cassette.

[0016] The DNA molecule may be transcribed into the mRNA molecule as described herein. That is to say that the DNA molecule may be used as a template for the production of the mRNA molecule as described herein.

[0017] In a second aspect, the present invention provides a neoantigen cassette comprising or consisting of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I. In a third aspect, the present invention provides a neoantigen cassette comprising or consisting of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0018] In a fourth aspect, the invention provides a vaccine composition comprising the nucleic acid molecule as described herein. Preferably, the vaccine composition comprises at least 2, at least 3, at least 4, or at least 5 nucleic acid molecules as described herein.

[0019] In a fifth aspect, the invention provides the nucleic acid molecule as described herein, or the vaccine composition as described herein for use in therapy.

[0020] In a sixth aspect, the invention provides the nucleic acid molecule as described herein, or the vaccine as described herein for use in a method of treating or preventing cancer.

[0021] In a further aspect, the invention provides a method of selecting a personalized therapy for a patient suffering from cancer comprising: a. obtaining sequencing data from the patient suffering from cancer; and b. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule as described herein based on the obtained sequencing data.

[0022] In a further aspect, the invention provides a method of manufacturing a cancer vaccine comprising (a) performing the steps of the method of selecting a personalized therapy as described herein; and (b) producing a cancer vaccine comprising the selected sequence(s) of neoantigen(s).

[0023] In a further aspect, the invention provides a use of the nucleic acid molecule as described herein or the neoantigen cassette as described herein in the preparation of a vaccine.

[0024] In a further aspect, the invention provides a use of the nucleic acid molecule as described herein or the neoantigen cassette as described herein, or the vaccine composition as described herein in in vitro or in vivo translation.

[0025] The invention also relates to a nucleic acid molecule comprising an interleukin 12 (IL-12) sequence; and a cassette encoding at least one antigen. The nucleic acid molecule may be comprised in a vaccine. The invention provides the nucleic acid molecule as described herein, or the vaccine as described herein for use in a method of treating or preventing a viral disease.

[0026] Each aspect or embodiment as defined herein may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0027] The present inventor has surprisingly discovered a nucleic acid molecule that is stable, easy to manufacture and enables high protein expression. The present invention has additional advantageous properties, such as a lack of bacterial contamination and no endotoxicity. In addition, the present invention is able to further stimulate an immune response by activating T cells and NK cells. These properties make the nucleic acid molecule of the present invention particularly suitable for use therapy, for example, in a vaccine composition for treating or preventing cancer in patients, or a vaccine composition for treating or preventing a viral disease in patients. The inventor has also developed a method for the production of a nucleic acid molecule and a vaccine using a cell-free system. This has an added advantage over conventional plasmid-based cloning methods of developing a vaccine in that it requires less regulatory steps, and is faster and more efficient.

[0028] DETAILED DESCRIPTION

[0029] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0030] It should be understood that singular prepositions such as “a,” “an,” and “the,” are often used for convenience, however, all instances of the singular are intended to encompass the plural unless otherwise indicated either explicitly or from context. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Further, it should be understood that all references, including journal articles, books, patents, technical documents, and the like, mentioned in this disclosure are hereby incorporated by reference in their entirety and for all purposes.

[0031] The term “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to vaccine composition, is intended to encompass a product comprising the nucleic acid molecule(s) and, optionally, the additional ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the vaccine compositions of the present invention encompass any composition comprising the nucleic acid molecule(s) of the present invention, and optionally a pharmaceutically acceptable carrier. The vaccine compositions of the present invention encompass any composition comprising the mRNA molecule(s) described herein, and optionally a pharmaceutically acceptable carrier. It is to be understood that the composition may comprise at least 1 , at least 2, at least 3, at least 4, or at least 5 mRNA molecules described herein. Preferably, the composition comprises at least 1 , at least 2, at least 3, at least 4, or at least 5 different mRNA molecules described herein. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Preferably, however, the nucleic acid molecule described herein is not formulated with a carrier and is, instead, delivered to a patient in a “naked” form.

[0032] The term “therapeutically effective amount” means the amount of a nucleic acid molecule or a vaccine composition that, when administered to a patient for treating or preventing a disease, is sufficient to effect such treatment or prevention for the disease. The “therapeutically effective amount” will vary depending on the disease and its severity, and the age and weight of the patient to be treated. The term “patient” (or “subject”) includes, but is not limited to, animals such as, for example, mammals. Preferably, the patient is a human. The term “therapeutically effective amount” may also refer to the amount of a nucleic acid molecule or a vaccine composition that, when administered to a patient, is prophylactic, for example, in patients who do not have cancer but who are at high risk of developing cancer.

[0033] The terms “neoantigen” or “neoantigen sequence” as used herein are intended to encompass peptides (or the corresponding nucleic acid sequences encoding the peptides) that are produced by tumour or cancer cells. Cancer cells accumulate many DNA mutations that can alter the structure of proteins, which result in the formation of neoantigens. Therefore, neoantigens are a class of tumourspecific antigens that are absent from normal (i.e. healthy) tissue. The resulting neoantigens are displayed by human leukocyte antigens (HLA) on the surface of cancer cells, helping to stimulate immune responses when immune cells - such as T cells - recognize the neoantigens as “non-self.

[0034] The term “Major Histocompatibility Complexes” (MHC) (class I and II) refers to proteins found on the surfaces of cells that help the immune system recognize foreign substances. MHC proteins are found in all higher vertebrates. In human beings the complex is also called the human leukocyte antigen (HLA) system. There are two major types of MHC protein molecules — class I and class II. Class I MHC molecules span the membrane of almost every cell in an organism, while class II molecules are generally restricted macrophages and lymphocytes.

[0035] The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.

[0036] The invention provides a nucleic acid molecule comprising: a. an interleukin 12 (IL-12) sequence; and b. a neoantigen cassette. Preferably, the neoantigen cassette encodes at least two, at least three, at least four, or at least five neoantigens.

[0037] The nucleic acid molecule may be single or double stranded. The IL-12 sequence may be encoded 5’ (i.e. upstream) of the neoantigen cassette. Alternatively, the IL-12 sequence may be encoded 3’ (i.e. downstream) of the neoantigen cassette. The nucleic acid molecule may further comprise a poly-A sequence. The nucleic acid molecule may be a DNA molecule or an mRNA molecule. The DNA molecule may be a plasmid or a DNA cassette.

[0038] In the embodiments in which the nucleic acid molecule is a DNA molecule, the nucleic acid molecule may comprise (optionally, in the 5’ to 3’ direction): a. a promoter; b. an interleukin 12 (IL-12) sequence; and c. a neoantigen cassette.

[0039] The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, an IL-12 sequence, and a neoantigen cassette. The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, a ribosomal recruitment sequence, an IL-12 sequence, and a neoantigen cassette. The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, a protein translation initiation site sequence, an IL-12 sequence, and a neoantigen cassette. The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, and a neoantigen cassette. The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, and a nucleic acid localisation sequence. The DNA molecule may comprise (optionally, in the 5’ to 3’ direction) a promoter, a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, a stop codon, and a nucleic acid localisation sequence.

[0040] In some embodiments, for example, in which the DNA molecule is a plasmid, the DNA molecule also comprises a poly-A sequence. In some embodiments, for example, in which the DNA molecule is a plasmid, the DNA molecule may not include a neoantigen cassette.

[0041] The DNA molecule may be transcribed into the mRNA molecule as described herein. That is to say that the DNA molecule may be used as a template for the production of the mRNA molecule as described herein. Thus, the invention provides an mRNA molecule derived from the nucleic acid as described herein, wherein the mRNA molecule encodes an IL-12 sequence and a neoantigen cassette. Preferably, as used herein, the term “mRNA molecule” is intended to encompass mRNA molecules derivable (e.g. transcribed) from the nucleic acid molecules as described herein. In some embodiments, the term “nucleic acid molecule” may also encompass the mRNA molecule as described herein.

[0042] In the embodiments in which the nucleic acid molecule is an mRNA molecule, the nucleic acid molecule may comprise (optionally, in the 5’ to 3’ direction): a. an interleukin 12 (IL-12) sequence; and b. a neoantigen cassette.

[0043] Preferably, the neoantigen cassette encodes at least two neoantigens (i.e. neoantigen sequences). The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, an IL-12 sequence, and a neoantigen cassette. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, an IL-12 sequence, and a neoantigen cassette. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a protein translation initiation site sequence, an IL-12 sequence, and a neoantigen cassette. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, and a neoantigen cassette. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, and a nucleic acid localisation sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, a stop codon, and a nucleic acid localisation sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) an IL-12 sequence, a neoantigen cassette and a poly-A sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, an IL-12 sequence, a neoantigen cassette and a poly-A sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, an IL-12 sequence, a neoantigen cassette and a poly-A sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, a stop codon, a nucleic acid localisation sequence, and a poly-A sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, a nucleic acid localisation sequence, and a poly-A sequence. The mRNA molecule may comprise (optionally, in the 5’ to 3’ direction) a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, an IL-12 sequence, a neoantigen cassette, and a poly-A sequence. Preferably, the mRNA molecule comprises (optionally, in the 5’ to 3’ direction) a ribosomal recruitment sequence, an IL-12 sequence, a neoantigen cassette encoding at least two neoantigens, and a poly-A sequence.

[0044] The IL-12 sequence may be encoded 5’ (i.e. upstream) of the neoantigen cassette. Alternatively, the IL-12 sequence may be encoded 3’ (i.e. downstream) of the neoantigen cassette. That is to say that the nucleic acid molecule may comprise, in a 5’ to 3’ direction, a promoter, an IL-12 sequence; and a neoantigen cassette. The mRNA molecule may comprise, in a 5’ to 3’ direction, an IL-12 sequence and a neoantigen cassette. Alternatively, the nucleic acid molecule may comprise, in a 5’ to 3’ direction, a promoter, a neoantigen cassette and an IL-12 sequence. The mRNA molecule may comprise, in a 5’ to 3’ direction, a neoantigen cassette and an IL-12 sequence.

[0045] The term “IL-12 sequence” in the context of a nucleic acid molecule intends to encompass a nucleic acid sequence which encodes IL-12. IL-12 is a dimeric protein consisting of the p35 and p40 subunits (also known as alpha and beta subunits) and is classically produced by antigen-presenting cells (APCs) including macrophages, dendritic cells (DCs), and Langerhans' cells. IL-12 signals through the IL-12R, which in turn, activates a STAT4 dependent intracellular pathway. This results in the production of IFNy but also directly leads to IL-18 synthesis. IL-12 acts to stimulate the proliferation and cytotoxicity of both NK cells and CTL. The IL-12 sequence may comprise the nucleic acids encoding the p35 and / or p40 subunits in any order. Preferably, the IL-12 sequence comprises, in a 5’ to 3’ direction, the nucleic acids encoding the p40 subunit followed by the nucleic acid encoding the p35 subunit. That is to say that, preferably, the IL-12 sequence encodes both the p40 subunit and the p35 subunit of IL-12. The IL-12 sequence may be of animal origin, for example human or mouse. Preferably, the IL-12 sequence is of human origin. Thus, the inclusion of the IL-12 sequence in the nucleic acid molecule improves the immune cell response to neoantigens encoded by the nucleic acid molecule (e.g. the mRNA molecule). The inclusion of the IL-12 sequence in the nucleic acid molecule (e.g. the mRNA molecule) also improves the transfection efficiency.

[0046] The IL-12 sequence may comprise of consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2. The IL-12 sequence may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotide substitutions as compared to the sequence of SEQ ID NO: 2. The IL-12 sequence may comprise of consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 164. The IL-12 sequence may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or at least 30 nucleotide substitutions as compared to the sequence of SEQ ID NO: 164. Preferably, the IL-12 sequence encodes cysteine at position 177. The cytosine and guanine nucleotides of the IL-12 sequence may constitute between 30-70% of the total nucleotide content of the IL-12 sequence. Preferably, the cytosine and guanine nucleotides of the IL-12 sequence constitute between 50%-60% of the total nucleotide content of the IL-12 sequence. More preferably still, the cytosine and guanine nucleotides of the IL-12 sequence constitute between 53%-58% of the total nucleotide content of the IL-12 sequence. This specific cytosine and guanine nucleotides content in the IL-12 sequence prolongs the half-life of the nucleic acid molecule and improves the efficiency of translation. The IL-12 sequence may encode IL-12 protein of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 98. It would be understood to the skilled person that, in the context of the mRNA molecule as described herein, the sequences encoding the IL-12 protein comprise an uracil base instead of the thymine base. The IL-12 sequence may encode the p40 and p35 subunits that are fused together (when translated). The IL-12 sequence may encode (optionally N-term to C-term) an IL-12 protein of sequences of UniProt ID: P29460 (i.e. the p40 subunit) and P29459. The IL-12 sequence which encodes both the p40 subunit and the p35 subunit is particularly advantageous because it aids in neoantigen binding to either or both MHC class I and MHC class II proteins.

[0047] The promoter may be a T7 promoter or a CMV promoter. The T7 promoter may comprise or consist of a sequence of SEQ ID NO: 1 . The T7 promoter may comprise or consist of a sequence having at least 1 , at least 2 or at least 3 nucleotide substitutions in the sequence of SEQ ID NO: 1.

[0048] The nucleic acid molecule may further comprise a poly-A (i.e. polyadenylation) sequence. Thus, the nucleic acid molecule (e.g. a DNA molecule) may comprise, optionally in the 5’ to 3’ direction: (a) a promoter, (b) an IL-12 sequence, (c) a neoantigen cassette; and (d) a poly-A sequence. The nucleic acid molecule (e.g. an mRNA molecule) may comprise, optionally in the 5’ to 3’ direction: an IL-12 sequence, a neoantigen cassette; and a poly-A sequence. The poly-A sequence may comprise or consist of at least 100 adenine nucleotides. Preferably, the poly-A sequence comprises or consists of 128 adenine nucleotides. The presence of at least 100 (preferably 128) adenine nucleotides improves the stability of the nucleic acid molecule.

[0049] The neoantigen cassette typically comprises or consists of neoantigen sequence(s) identified from a patient suffering from cancer. That is to say that the neoantigen cassette may encode neoantigen(s) identified from a patient suffering from cancer. Typically, a sample (e.g. biopsy) from a patient suffering from cancer is collected and the mRNA content of the sample (e.g. comprising cancer cells) is sequenced. The sequencing data is compared to a reference data set (e.g. obtained from a sample of a patient not suffering from cancer or from the patient before the patient suffered from cancer or compared with human genome Ref Seq GRCh38.p14, Homo sapiens (human) genome assembly GRCh38.p14 (hg38) from Genome Reference Consortium [GCA_000001405.29

[0050] GCF_000001405.40])) to identify neoantigens specific to the patient suffering from cancer. The identified neoantigens are computationally scored for their likelihood and / or efficacy of binding to MHC I or MHC II complexes and / or for their known association with a specific cancer type. The neoantigens for MHC I may also be predicted to elicit immunogenicity using Class I Immunogenicity prediction algorithm. Based on that scoring, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 neoantigens are formulated in the neoantigen cassette. The neoantigens may be formulated on separate nucleic acid molecules. For example, 3 neoantigens may be encoded by a first nucleic acid molecule and 5 further neoantigens may be encoded by a second nucleic acid molecule. The first and second nucleic acid molecules may be introduced to a patient in a single vaccine composition or separate vaccine compositions. Preferably, the first and second nucleic acid molecules are introduced to a patient in a single vaccine composition. The neoantigen cassette may be located downstream or upstream of the IL-12 sequence. The neoantigen cassette is preferably located downstream of the promoter (if present).

[0051] Preferably, the neoantigen cassette comprises or consists of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I (i.e. the neoantigens binding to MHC class I). Alternatively, preferably, the neoantigen cassette comprises or consists of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II (i.e. the neoantigens binding to MHC class II). That is to say that the neoantigen cassette preferably does not combine sequences encoding the amino acid sequence of peptides which bind to MHC class I and MHC class II.

[0052] The peptides binding to the Major Histocompatibility Complex (MHC) class I may be the peptides of any one of SEQ ID NOs: 3-23, 28-43, 49-71 , 119-128, or 156-163. The peptides binding to the Major Histocompatibility Complex (MHC) class II may be the peptides of any one of SEQ ID NOs: 24-27, 44- 48, 72-75, or 136-139.

[0053] The neoantigen cassette may comprise or consist of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 3-28, 136-139, or 156-162. The neoantigen cassette may comprise or consist of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 29- 53, or 119-128, or 163. The neoantigen cassette may comprise or consist of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 54- 75. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 3-7, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 3, 4, 5, 6, and 7. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 8-12, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11 , and 12. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 13-17, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, and 17. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 18-22, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 18, 19, 20, 21 , and 22. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequence of SEQ ID NOs: 23. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequence of SEQ ID NOs: 28. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 44-48, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 44, 45, 46, 48 and 47. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 29, and 31-34, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 29, 31 , 32, 33, and 34. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 35, 36, and 39-41 , preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 35, 36, 39, 40, and 41 .

[0054] The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 49-53, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 49, 50, 51 , 52, and 53. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 72-75, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 72, 73, 74, and 75. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 54, 55, and 57-59, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 54, 55, 57, 58 and 59. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 61-65, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 61 , 62, 63, 64, and 65. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequences of SEQ ID NOs: 66-70, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 66, 67, 68, 69, and 70. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequence of SEQ ID NO: 60. The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequence of SEQ ID NO: 71.

[0055] The neoantigen cassette may comprise a sequence encoding at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker sequence(s) (i.e. spacers). For example, the linker sequence (i.e. spacer) may be linking different neoantigen sequences in the neoantigen cassette. The linker sequence (i.e. spacer) may link the neoantigen cassette with other elements of the nucleic acid molecule. For example, the linker sequence (i.e. spacer) may link the neoantigen cassette with the IL- 12 sequence. The linker sequence(s) (i.e. spacer) may comprise a sequence encoding glycine and / or serine. The linker sequence(s) (i.e. spacer) may comprise a sequence of SEQ ID NOs: 76 and / or 77. Preferably, if the neoantigen cassette comprises a sequence encoding one neoantigen sequence, the linker of SEQ ID NO: 77 links the neoantigen cassette to the other elements in the nucleic acid molecule (e.g. to the IL-12 sequence and / or the stop codon). Preferably, if the neoantigen cassette comprises a sequence encoding more than one (e.g. at least 2, at least 3, at least 4, or at least 5) neoantigen sequences, the linker of SEQ ID NO: 76 links the neoantigen cassette to the other elements in the nucleic acid molecule (e.g. to the IL-12 sequence and the stop codon). Preferably, if the neoantigen cassette comprises a sequence encoding more than one (e.g. at least 2, at least 3, at least 4, or at least 5) neoantigen sequences, the linker (or linkers) of SEQ ID NO: 76 links the different neoantigen sequences in the neoantigen cassette. Preferably, each neoantigen in the nucleic acid molecule is separated with a spacer (i.e. a linker). That is to say that the neoantigens encoded by the neoantigen cassette are preferably separated (from each other) by a spacer (i.e. a linker).

[0056] The neoantigen cassette may comprise or consist of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHO) class I. The neoantigen cassette may comprise or consist of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II .

[0057] Figure 2 shows exemplary elements of the neoantigen cassette. The top panel shows a single neoantigen sequence linked to other elements of the nucleic acid molecule with a longer linker (i.e. spacer) than the linker (i.e. spacer) of the two bottom panels. The middle panel shows four neoantigen sequences linked with linker sequences (i.e. spacer sequences). The bottom panel shows five neoantigen sequences linked with linker (i.e. spacer) sequences. The neoantigen sequences encoded by the neoantigen cassette are preferably different.

[0058] The neoantigen cassette may comprise or consist of a sequence encoding the amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 78-95 or 165-172.

[0059] The nucleic acid molecule may further comprise a cap sequence. The cap sequence may comprise or consist of a sequence AGGG or GGG. The cap sequence may be located downstream of the promoter and upstream of the IL-12 sequence and / or the neoantigen cassette. In the context of the mRNA molecule as described herein, the cap sequence may be located at the 5’ end of the mRNA molecule and / or upstream of the IL-12 sequence and / or the neoantigen cassette.

[0060] The nucleic acid molecule may comprise a ribosomal recruitment sequence. The ribosomal recruitment sequence may be a 5’-UTR sequence (e.g. a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence). The 5 -UTR sequence may be derived from the haemoglobin subunit alpha (HBA) gene. The ribosomal recruitment sequence may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 96. The ribosomal recruitment sequence may be located downstream of the promoter and / or upstream of the IL-12 sequence and / or the neoantigen cassette. In the context of the mRNA molecule as described herein, the ribosomal recruitment sequence may be located downstream of the cap sequence and / or upstream of the IL-12 sequence and / or the neoantigen cassette. The ribosomal recruitment sequence (e.g. a 5’-UTR sequence derived from the HBA gene) may improve transfection efficiency as compared to a nucleic acid molecule without a 5’- UTR sequence and / or a nucleic acid molecule comprising 5’-UTR sequences other than the 5’-UTR sequence derived from the haemoglobin subunit alpha 1 (HBA 1) sequence (SEQ ID NO: 96).

[0061] The nucleic acid molecule may comprise a protein translation initiation site sequence. The protein translation initiation site sequence may be a Kozak sequence or a Shine-Dalgarno sequence. The protein translation initiation site sequence may comprise or consist of a sequence GCCACC, or a sequence having one or two nucleotide substitution(s) in the sequence GCCACC. It is understood that in the context of an mRNA molecule, the sequence may be AGGAGG, or a sequence having one or two nucleotide substitution(s) in the sequence AGGAGG. The protein translation initiation site sequence may comprise or consist of a sequence ACCATGG, or a sequence having one or two nucleotide substitution(s) in the sequence ACCATGG. The protein translation initiation site sequence may comprise or consist of a sequence GCCACCATGG (SEQ ID NO: 173), or a sequence having one or two nucleotide substitution(s) in the sequence GCCACCATGG. Preferably, the protein translation initiation site sequence is GCCACCATGG (in a DNA molecule), or GCCACCAUGG (in the context of an mRNA molecule; SEQ ID NO: 173). As used herein the expression “Kozak sequence” may refer to the sequence GCCACCATGG (in a DNA molecule), or GCCACCAUGG (in the context of an mRNA molecule). Preferably, the Kozak sequence has the sequence GCCACCATGG (in a DNA molecule), or GCCACCAUGG (in the context of an mRNA molecule). The protein translation initiation site sequence may be located downstream of the promoter and upstream of the IL-12 sequence and / or the neoantigen cassette. In the context of the mRNA molecule as described herein, the protein translation initiation site sequence may be located downstream of the cap sequence and / or the ribosomal recruitment sequence and / or upstream of the IL-12 sequence and / or the neoantigen cassette.

[0062] The nucleic acid molecule may comprise a stop codon downstream of the neoantigen cassette or downstream ofthe IL-12 sequence (if the IL-12 sequence is located downstream of the neoantigen cassette). The stop codon may have a sequence TAA, TAG or TGA (or UAA, UAG or UGA if it is an mRNA molecule).

[0063] The nucleic acid molecule may comprise a nucleic acid localisation sequence. The nucleic acid localisation sequence may be a 3 -UTR sequence (e.g. a 3 -UTR-HBA sequence). The 3 -UTR sequence may be derived from the HBA gene. The nucleic acid localisation sequence may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 97. The nucleic acid localisation sequence may be located downstream of the promoter, the IL-12 sequence and the neoantigen cassette. In the context of the mRNA molecule as described herein, the nucleic acid localisation sequence may be located downstream of the IL-12 sequence and the neoantigen cassette.

[0064] The nucleic acid molecule may comprise a transcription termination sequence at the 3’ end of the molecule.

[0065] It is to be understood that the “T” (thymine) nucleotide as included in the sequences described herein may be a “U” (uracil) nucleotide if described in the context of an RNA (e.g. mRNA) molecule as the nucleic acid molecule, or the mRNA molecule derived or derivable from the nucleic acid (e.g. DNA) molecule as described herein.

[0066] The nucleic acid molecule may be a DNA molecule. The DNA molecule may be or form part of a plasmid. The DNA molecule may be or comprise a synthetic DNA cassette. The synthetic DNA cassette may not comprise a poly-A sequence. The synthetic DNA cassette may comprise different elements of the nucleic acid molecule as described herein. The synthetic DNA cassette may be free of bacterial plasmid contaminants, such as antibiotic resistance genes of bacterial replication initiation sites.

[0067] The nucleic acid molecule (e.g. a DNA molecule, such as a plasmid) may comprise, optionally in the 5’ to 3’ direction: a. a promoter; b. a ribosomal recruitment sequence; c. an interleukin 12 (IL-12) sequence; d. a neoantigen cassette; e. a stop codon; f. a nucleic acid localisation sequence; and g. a poly-A sequence.

[0068] The nucleic acid molecule (e.g. a DNA molecule, such as a plasmid) may comprise, optionally in the 5’ to 3’ direction: a. a promoter; b. a ribosomal recruitment sequence; c. an interleukin 12 (IL-12) sequence; d. a stop codon; e. a nucleic acid localisation sequence; and f. a poly-A sequence.

[0069] The nucleic acid molecule (e.g. a DNA molecule, such as a plasmid) may comprise, optionally in the 5’ to 3’ direction: a. a promoter; b. a cap sequence; c. a ribosomal recruitment sequence; d. a protein translation initiation site sequence; e. an interleukin 12 (IL-12) sequence; f. a stop codon; g. a nucleic acid localisation sequence; and h. a poly-A sequence.

[0070] The nucleic acid molecule (e.g. a DNA molecule, such as a plasmid) may comprise, optionally in the 5’ to 3’ direction: a. a promoter; b. a cap sequence; c. a ribosomal recruitment sequence; d. a protein translation initiation site sequence; e. an interleukin 12 (IL-12) sequence; f. a stop codon; g. a nucleic acid localisation sequence; and h. a poly-A sequence.

[0071] The nucleic acid molecule (e.g. a DNA molecule, such as a synthetic DNA cassette) may comprise, optionally in the 5’ to 3’ direction: a. a promoter, for example, a T7 promoter or a CMV promoter; b. a ribosomal recruitment sequence, for example, a 5 -UTR- haemoglobin subunit alpha 1 (HBA) sequence; c. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; d. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; e. a stop codon; and f. a nucleic acid localisation sequence, for example, a 3 -UTR-HBA sequence.

[0072] The nucleic acid molecule (e.g. a DNA molecule, such as a synthetic DNA cassette) may comprise, optionally in the 5’ to 3’ direction: a. a promoter, for example, a T7 promoter or a CMV promoter; b. a ribosomal recruitment sequence, for example, a 5 -UTR- haemoglobin subunit alpha 1 (HBA) sequence; c. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; d. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; e. a stop codon; and f. a nucleic acid localisation sequence, for example, a 3 -UTR-HBA sequence.

[0073] The nucleic acid molecule (e.g. a DNA molecule, such as a synthetic DNA cassette) may comprise, optionally in the 5’ to 3’ direction: a. a promoter, for example, a T7 promoter or a CMV promoter; b. a cap sequence; c. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence; d. a protein translation initiation site sequence, for example, a Kozak sequence; e. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; f. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; g. a stop codon; and h. a nucleic acid localisation sequence, for example, a 3’-UTR-HBA sequence.

[0074] The nucleic acid molecule (e.g. a DNA molecule, such as a synthetic DNA cassette) may comprise, optionally in the 5’ to 3’ direction: a. a promoter, for example, a T7 promoter or a CMV promoter;; b. a cap sequence; c. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence; d. a protein translation initiation site sequence, for example, a Kozak sequence; e. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; f. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; g. a stop codon; and h. a nucleic acid localisation sequence for example, a 3’-UTR-HBA sequence.

[0075] The nucleic acid molecule may be an mRNA molecule. The mRNA molecule may comprise: a. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA-1) sequence; b. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; c. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; and d. a poly-A sequence.

[0076] The mRNA molecule may comprise: a. a ribosomal recruitment sequence, for example, a 5 -UTR- haemoglobin subunit alpha 1 (HBA) sequence; b. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; c. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; d. a stop codon; e. a nucleic acid localisation sequence, for example, a 3’-UTR-HBA sequence; and f. a poly-A sequence.

[0077] The mRNA molecule, optionally in the 5’ to 3’ direction, may comprise: a. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence; b. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; c. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; d. a stop codon; e. a nucleic acid localisation sequence, for example, a 3’-UTR-HBA sequence; and f. a poly-A sequence.

[0078] The mRNA molecule may comprise, optionally in the 5’ to 3’ direction: a. a cap sequence; b. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence; c. a protein translation initiation site sequence, for example, a Kozak sequence; d. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; e. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; f. a stop codon; g. a nucleic acid localisation sequence, for example, a 3’-UTR-HBA sequence; and h. a poly-A sequence.

[0079] The mRNA molecule may comprise, optionally in the 5’ to 3’ direction: a. a cap sequence; b. a ribosomal recruitment sequence, for example, a 5 -UTR- haemoglobin subunit alpha 1 (HBA) sequence; c. a protein translation initiation site sequence, for example, a Kozak sequence; d. a neoantigen cassette, preferably encoding at least two neoantigens, which may be separated by at least one linker; e. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; f. a stop codon; g. a nucleic acid localisation sequence, for example, a 3 -UTR-HBA sequence; and h. a poly-A sequence.

[0080] Figure 1 A illustrates one example of the nucleic acid molecule (here a DNA molecule) of the present invention. The nucleic acid molecule may be used to produce an mRNA molecule.

[0081] Figure 1 B illustrates another example of the nucleic acid molecule (here an mRNA molecule) of the present invention. The nucleic acid molecule may be a cell-free neoantigen delivery platform.

[0082] Figure 1 C illustrates one example of the nucleic acid molecule (here a plasmid DNA molecule) of the present invention. The nucleic acid molecule may be used to produce an mRNA molecule or in the process of incorporation of a neoantigen cassette.

[0083] The nucleic acid molecule may comprise modified nucleotide(s). For example, the modified nucleotide(s) may be methylated or phosphorylated nucleotide(s). The modified nucleotide(s) may be 2-Thiouridine-5'-Triphosphate (2-Thio-UTP). The modified nucleotide(s) may be 5-Methylcytidine-5'- Triphosphate (5-Methyl-CTP). The modified nucleotide(s) may be phosphorothioated nucleotide(s).

[0084] Provided herein is also a (e.g. circular) vector comprising the nucleic acid molecule described herein. That is to say that the nucleic acid molecule (e.g. a DNA molecule) described herein may form part of a vector (e.g. plasmid). The vector is particularly useful during in vitro transcription (e.g. cell free in vitro transcription) for generation of the nucleic acid molecule for therapy. In some embodiments, in the vector, the nucleic acid molecule may not comprise a poly-A sequence. The poly-A sequence may be added to the nucleic acid molecule after the in vitro transcription. Thus, the vector may comprise: (a) a promoter, (b) an IL-12 sequence, and (c) a neoantigen cassette. In some embodiments, the nucleic acid molecule (e.g. a plasmid) may comprise a poly-A sequence.

[0085] The vector may include the pVAX1 vector backbone and the nucleic acid molecule as described herein. The nucleic acid molecule may be flanked by restriction enzyme sites.

[0086] The invention also provides a neoantigen cassette comprising or consisting of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I. The invention also provides a neoantigen cassette comprising or consisting of at least 1 , at least 2, at least 3, at least 4, or at least 5 sequence(s) encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0087] The peptides binding to the Major Histocompatibility Complex (MHC) class I may be the peptides of any one of SEQ ID NOs: 3-23, 28-43, 49-71 , 119-128, or 156-163. The peptides binding to the Major Histocompatibility Complex (MHC) class II may be the peptides of any one of SEQ ID NOs: 24-27, 44- 48, 72-75, or 136-139.

[0088] The neoantigen cassette may comprise or encode at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker (i.e. spacer) sequence(s). The linker sequence(s) may comprise a sequence encoding glycine and / or serine. The linker sequence(s) may comprise a sequence of SEQ ID NOs: 76 and / or 77. Preferably, if the neoantigen cassette comprises or encodes more than one (e.g. at least 2, at least 3, at least 4, or at least 5) neoantigen sequences, the linker (or linkers) of SEQ ID NO: 76 links the different neoantigen sequences in the neoantigen cassette. The neoantigen cassette may comprise or encode the neoantigen sequences as defined in the context of the nucleic acid molecule.

[0089] The invention also provides a vaccine composition comprising the nucleic acid molecule (e.g. an mRNA molecule) as described herein or the neoantigen cassette as described herein. Preferably, the vaccine composition comprises at least 2, at least 3, at least 4, or at least 5 nucleic acid molecules as described herein.

[0090] To improve stereochemistry of binding of the nucleic acid molecule to MHC class I and / or MHC class II in a cell, preferably, the nucleic acid molecules included in the vaccine composition comprise sequences encoding neoantigens that bind to either MHC class I or MHC class I. That is to say that a single nucleic acid molecule in the vaccine composition may include at least 1 , at least 2, at least 3, at least 4 or at least 5 sequences encoding neoantigens that bind to MHC class I. Similarly, a different nucleic acid molecule in the vaccine composition may include at least 1 , at least 2, at least 3, at least 4 or at least 5 sequences encoding neoantigens that bind to MHC class II.

[0091] For example, if the vaccine composition comprises at least 2 nucleic acid molecules, the first of the at least 2 nucleic acid molecules may comprise at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I and the second of the at least 2 nucleic acid molecules may comprise at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0092] For example, if the vaccine composition comprises at least 3 nucleic acid molecules, the first and second of the at least 3 nucleic acid molecules may (each) comprise at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I and the third of the at least 3 nucleic acid molecules may comprise at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0093] For example, if the vaccine composition comprises at least 5 nucleic acid molecules, the first, second, third and fourth of the at least 5 nucleic acid molecules may (each) comprise at least 1 , at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I and the fifth of the at least 5 nucleic acid molecules may comprise at least 1 , at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II. That is to say that, preferably, the nucleic acid molecules in the vaccine composition encode neoantigen sequences which bind either to MHC class I or MHC class II. However, the vaccine composition may comprise multiple nucleic acid molecules which separately target either MHC class I or MHC class II. Thus, the vaccine composition may comprise neoantigen sequences targeting both MHC class I and MHC class II, preferably, on separate nucleic acid molecules. This distribution of neoantigen sequences improves stereochemistry of binding to MHC complexes.

[0094] The invention also relates to a nucleic acid molecule comprising an interleukin 12 (IL-12) sequence; and a cassette encoding at least one antigen. The nucleic acid molecule may comprise other components, for example, a cap sequence, a ribosomal recruitment sequence, a protein translation initiation site sequence, a stop codon, a nucleic acid localisation sequence, and / or a poly-A sequence. The cap sequence, the ribosomal recruitment sequence, the protein translation initiation site sequence, the stop codon, the nucleic acid localisation sequence, and / or a poly-A sequence are described herein in the context of the nucleic acid molecule comprising a neoantigen cassette. The description of these component made in the context of a nucleic acid molecule comprising a neoantigen cassette applies mutatis mutandis to the nucleic acid molecule comprising at least one antigen. Thus, for example, the nucleic acid molecule (e.g. an mRNA molecule) may comprise, optionally in the 5’ to 3’ direction: a. a ribosomal recruitment sequence, for example, a 5 -UTR- haemoglobin subunit alpha 1 (HBA) sequence; b. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; c. a cassette encoding at least one antigen; and d. a poly-A sequence.

[0095] The nucleic acid molecule (e.g. an mRNA molecule) may comprise, optionally in the 5’ to 3’ direction: a. a cap sequence; b. a ribosomal recruitment sequence, for example, a 5’-UTR- haemoglobin subunit alpha 1 (HBA) sequence; c. a protein translation initiation site sequence, for example, a Kozak sequence; d. an interleukin 12 (IL-12) sequence, for example, an IL-12 sequence encoding the p40 and p35 subunits of IL-12; e. a cassette encoding at least one antigen; f. a stop codon; g. a nucleic acid localisation sequence, for example, a 3 -UTR-HBA sequence; and h. a poly-A sequence.

[0096] The at least one antigen may be a protein or a part thereof triggering an immune response in a subject. The subject may be an animal subject, such as mice, or a human subject. The at least one antigen may be a protein or a part thereof found in a capsid or an envelope of a virus. The virus may be, for example, Influenza, COVID-19, HIV, Rabies, or Zika. The antigen may be hemagglutinin (HA) protein, neuraminidase (NA) protein, the spike protein (S protein), gp120 protein, gp41 protein, Gag (Group-Specific-Antigen) protein, Polymerase protein, Nef protein, rabies virus glycoprotein, Zika virus envelope (E) protein and / or Zika pre-membrane / envelope (prM-E) protein.

[0097] The invention also provides the nucleic acid molecule (e.g. an mRNA molecule) as described herein, or the vaccine as described herein for use in therapy.

[0098] The nucleic acid molecule as described herein, or the vaccine as described herein may be used in a method of treating or preventing cancer. The cancer may be a lung cancer, a skin cancer, a colorectal (i.e. colon) cancer, a cervix cancer, a liver cancer, a uterus cancer, a prostate cancer, a head and neck cancer or leukaemia (e.g. acute lymphoblastic leukaemia).

[0099] The nucleic acid molecule comprising at least one antigen (rather than the neoantigen cassette) may be used in a method of treating or preventing a viral disease. The viral disease may be flu, COVID-19 infection, HIV (or AIDS), rabies, or an infection caused by a Zika virus.

[0100] For example, to treat or prevent colon cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of a sequence encoding the amino acid sequences of any one of SEQ ID NOs: 3-28, 136-139, or 156-162.

[0101] For example, to treat or prevent colon cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of:

[0102] - a sequence encoding the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24;

[0103] - a sequence encoding the amino acid sequences of SEQ ID NOs: 3-7, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 3, 4, 5, 6, and 7; - a sequence encoding the amino acid sequences of SEQ ID NOs: 8-12, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11 , and 12;

[0104] - a sequence encoding the amino acid sequences of SEQ ID NOs: 13-17, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, and 17;

[0105] - a sequence encoding the amino acid sequences of SEQ ID NOs: 18-22, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 18, 19, 20, 21 , and 22;

[0106] - a sequence encoding the amino acid sequence of SEQ ID NO: 23; and / or

[0107] - a sequence encoding the amino acid sequence of SEQ ID NO: 28

[0108] For example, to treat or prevent skin cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of a sequence encoding the amino acid sequences of any one of SEQ ID NOs: 29-53, 119-128, or 163.

[0109] For example, to treat or prevent skin cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of:

[0110] - a sequence encoding the amino acid sequences of SEQ ID NOs: 44-48, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 44, 45, 46, 48 and 47;

[0111] - a sequence encoding the amino acid sequences of SEQ ID NOs: 29, and 31-34, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 29, 31 , 32, 33, and 34;

[0112] - a sequence encoding the amino acid sequences of SEQ ID NOs: 35, 36, and 39-41 , preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 35, 36, 39, 40, and 41 ;

[0113] - a sequence encoding the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30; and / or

[0114] - a sequence encoding the amino acid sequences of SEQ ID NOs: 49-53, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 49, 50, 51 , 52, and 53;

[0115] - a sequence encoding the amino acid sequences of SEQ ID NOs: 49 and 50; preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 49, and 50;

[0116] - a sequence encoding the amino acid sequences of SEQ ID NOs: 51-53; preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 51 , 52, and 53; - a sequence encoding the amino acid sequences of SEQ ID NOs: 29 and 31 ; preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 29 and 31 ; and / or

[0117] - a sequence encoding the amino acid sequences of SEQ ID NOs: 32-34; preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 32, 33, and 34.

[0118] For example, to treat or prevent lung cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of a sequence encoding the amino acid sequences of any one of SEQ ID NOs: 54-75.

[0119] For example, to treat or prevent lung cancer, the neoantigen cassette (of the nucleic acid molecule or molecules) may comprise or consist of:

[0120] - a sequence encoding the amino acid sequences of SEQ ID NOs: 72-75, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 72, 73, 74, and 75;

[0121] - a sequence encoding the amino acid sequences of SEQ ID NOs: 54, 55, and 57-59, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 54, 55, 57, 58 and 59;

[0122] - a sequence encoding the amino acid sequences of SEQ ID NOs: 54 and 55, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 54and 55;

[0123] - a sequence encoding the amino acid sequences of SEQ ID NOs: 57-59, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 57, 58 and 59;

[0124] - a sequence encoding the amino acid sequences of SEQ ID NOs: 61-65, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 61 , 62, 63, 64, and 65;

[0125] - a sequence encoding the amino acid sequences of SEQ ID NOs: 66-70, preferably the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 66, 67, 68, 69, and 70;

[0126] - a sequence encoding the amino acid sequence of SEQ ID NO: 60; and / or

[0127] - a sequence encoding the amino acid sequence of SEQ ID NO: 71 .

[0128] A method of treating cancer is also provided, the method comprising administering to a patient in need thereof a therapeutically effective amount of the nucleic acid molecule (e.g. an mRNA molecule) as described herein or the vaccine composition as described herein.

[0129] Typically, the nucleic acid molecule (e.g. the mRNA molecule) or the vaccine composition may be administered to the patient by any acceptable route of administration including, but not limited to, injection. The injection may be intradermal, subcutaneous, intramuscular, intranodal, or intravenous. Other routes, such as intranasal injection, intravaginal injection and intratumoral injection are also within the scope of the invention.

[0130] Injection may include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solvents include, for example, distilled water for injection, physiological saline and / or nuclease-free phosphate-buffered saline (PBS) buffer. Examples of non-aqueous solvents include alcohols such as ethanol.

[0131] The vaccine composition may comprise one or more further pharmaceutically active agents. This combination therapy involves using a nucleic acid molecule (e.g. the mRNA molecule) combined with one or more of the further pharmaceutically active agents, either formulated together (for example, packaged together in a single formulation) or formulated separately (for example, packaged as separate unit dosage forms).

[0132] The nucleic acid molecule (e.g. the mRNA molecule) is preferably delivered or administered to the patient in a “naked” form; i.e. without the use of carries (e.g. lipids). The use of a naked form reduces the costs of manufacture, making it more affordable in the context of personalized cancer treatment.

[0133] A method of selecting a personalized therapy for a patient suffering from cancer is also provided. The method comprises: a. obtaining sequencing data from the patient suffering from cancer; and b. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule as described herein based on the obtained sequencing data.

[0134] The step of selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette may comprise comparing the sequencing data to a reference data. The step of selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette may comprise selecting genes which show somatic mutations. The reference data may be data obtained from a subject who does not suffer from cancer, from the patient before the patient was suffering from cancer, or from RefSeq GRCh38.p14 (hg38) from Genome Reference Consortium as a control sample. The sequencing data may be obtained from mRNA sequencing. The sequencing of just mRNA speeds up the sequencing process as mRNA constitute only able 5% of the genetic material obtained from a sample.

[0135] The method of selecting a personalized therapy for a patient suffering from cancer may comprise the steps: a. obtaining a sample from the patient suffering from cancer; b. obtaining sequencing data from the sample; and c. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule as described herein based on the obtained sequencing data.

[0136] The sample may be a biopsy sample.

[0137] A method of manufacturing a cancer vaccine is also provided. The method may comprise:

[0138] (a) performing the steps of the method of selecting a personalized therapy as described herein; and

[0139] (b) producing a cancer vaccine, wherein the cancer vaccine comprises the nucleic acid molecule(s) (e.g. the mRNA molecule(s)) (encoding the selected sequence(s) of neoantigen(s)).

[0140] Provided herein is also a use of the nucleic acid molecule as described herein or the neoantigen cassette as described herein in the preparation of a vaccine.

[0141] Provided here is also a use of the nucleic acid molecule as described herein or the neoantigen cassette as described herein or the vaccine composition as described herein in in vitro or in vivo translation.

[0142] Provided here is also a use of the vector described herein in in vitro transcription.

[0143] The invention is further disclosed in the following clauses:

[0144] 1 . A nucleic acid molecule comprising, optionally in the 5’ to 3’ direction: a. a promoter; b. an interleukin 12 (IL-12) sequence; c. a neoantigen cassette; and d. a poly-A sequence.

[0145] 2. The nucleic acid molecule of clause 1 , wherein the nucleic acid molecule is a DNA molecule.

[0146] 3. The nucleic acid molecule of clause 1 or clause 2, wherein the nucleic acid molecule further comprises a cap sequence.

[0147] 4. The nucleic acid molecule of any one of clauses 1-3, wherein the nucleic acid molecule comprises a ribosomal recruitment sequence.

[0148] 5. The nucleic acid molecule of any one of clauses 1-4, wherein the nucleic acid molecule comprises a protein translation initiation site sequence.

[0149] 6. The nucleic acid molecule of any one of clauses 1-5, wherein the nucleic acid molecule comprises a stop codon after the neoantigen cassette.

[0150] 7. The nucleic acid molecule of any one of clauses 1-6, wherein the nucleic acid molecule comprises a nucleic acid localisation sequence. The nucleic acid molecule of any one of clauses 1-7, wherein the nucleic acid molecule comprises, optionally in the 5’ to 3’ direction: a. a promoter; b. a ribosomal recruitment sequence; c. an interleukin 12 (IL-12) sequence; d. a neoantigen cassette; e. a stop codon; f. a nucleic acid localisation sequence; and g. a poly-A sequence. The nucleic acid molecule of any one of clauses 1-8, wherein the nucleic acid molecule comprises, optionally in the 5’ to 3’ direction: a. a promoter; b. a cap sequence; c. a ribosomal recruitment sequence; d. a protein translation initiation site sequence; e. an interleukin 12 (IL-12) sequence; f. a neoantigen cassette; g. a stop codon; h. a nucleic acid localisation sequence; and i. a poly-A sequence. The nucleic acid molecule of any one of clauses 1-9, wherein the promoter sequence is a T7 promoter sequence, optionally wherein the T7 promoter comprises or consists of a sequence of SEQ ID NO: 1. The nucleic acid molecule of any one of clauses 1-10, wherein the IL-12 sequence comprises or consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 164. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 3-28, 136-139, or 156-162. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 29-53, 119-128, or 163. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid sequence(s) of any one of SEQ ID NOs: 54-75. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 26, 27, 25, and 24. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 3-7, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 3, 4, 5, 6, and 7. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 8-12, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 8, 9, 10, 11 , and 12. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 13-17, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 13, 14, 15, 16, and 17. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 18-22, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 18, 19, 20, 21 , and 22. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequence of SEQ ID NOs: 23. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequence of SEQ ID NOs: 28. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 44-48, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 44, 45, 46, 48 and 47. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 29, and 31-34, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 29, 31 , 32, 33, and 34. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 35, 36, and 39-41 , preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 35, 36, 39, 40, and 41. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 38, 37, 43, 42, and 30. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 49-53, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 49, 50, 51 , 52, and 53. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 72-75, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 72, 73, 74, and 75. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 54, 55, and 57-59, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 54, 55, 57, 58 and 59. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 61-65, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 61 , 62, 63, 64, and 65. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequences of SEQ ID NOs: 66-70, preferably wherein the neoantigen cassette comprises or consists of a sequence encoding in the 5’ to 3’ direction the amino acid sequences of SEQ ID NOs: 66, 67, 68, 69, and 70. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequence of SEQ ID NO: 60. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequence of SEQ ID NO: 71 . The nucleic acid molecule of any one of clauses 1-32, wherein the neoantigen cassette comprises at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker sequence(s), optionally wherein the linker sequence(s) comprise a sequence encoding glycine and / or serine. The nucleic acid molecule of clause 33, wherein the linker sequence(s) comprise a sequence of SEQ ID NOs: 76 and / or 77. The nucleic acid molecule of any one of clauses 1-11 , wherein the neoantigen cassette comprises or consists of a sequence encoding the amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 78-95 or 165-172. The nucleic acid molecule of any one of clauses 1-35, wherein the poly-A sequence consists of at least 100, preferably 128 adenine nucleotides. The nucleic acid molecule of clause 3 or clause 9, wherein the cap comprises or consists of a sequence AGGG or GGG. The nucleic acid molecule of clause 4, clause 8 or clause 9, wherein the ribosomal recruitment sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 96. The nucleic acid molecule of clause 5 or clause 9, wherein the protein translation initiation site sequence comprises or consists of a sequence GCCACC, or a sequence having one or two nucleotide substitution(s) in the sequence GCCACC. The nucleic acid molecule of any one of clauses 7-9, wherein the nucleic acid localisation sequence comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 97. The nucleic acid molecule of any one of clauses 1-40, wherein the neoantigen cassette comprises or consists of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I. The nucleic acid molecule of any one of clauses 1-40, wherein the neoantigen cassette comprises or consists of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II. A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I. A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II. The neoantigen cassette of clause 41 or clause 42, wherein the neoantigen cassette comprises at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker sequence(s), optionally wherein the linker sequence(s) comprise a sequence encoding glycine and / or serine. The neoantigen cassette of claim 45, wherein the linker sequence(s) comprise a sequence of SEQ ID NOs: 76 and / or 77. An mRNA molecule derived from the nucleic acid molecule of any one of clauses 1-42. The mRNA molecule of clause 47, wherein the mRNA molecule comprises: a. an interleukin 12 (IL-12) sequence; b. a neoantigen cassette; and c. a poly-A sequence. A vaccine composition comprising the nucleic acid molecule of any one of clauses 1-42 or the mRNA molecule of clause 47 or clause 48. The vaccine composition of clause 49 comprising at least 2, at least 3, at least 4, or at least 5 nucleic acid molecules of any one of clauses 1-42 or at least 2, at least 3, at least 4, or at least 5 mRNA molecules of clause 47 or clause 48. The vaccine composition of clause 50, wherein the first of the at least 2 nucleic acid molecules or mRNA molecules comprises at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I and the second of the at least 2 nucleic acid molecules or mRNA molecules comprises at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II. The vaccine composition of clause 50, wherein the first and second of the at least 3 nucleic acid molecules or mRNA molecules comprise at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I and the third of the at least 3 nucleic acid molecules or mRNA molecules comprises at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0151] 53. The nucleic acid molecule of any one of clauses 1-42, the mRNA molecule of clause 47 or clause 48, or the vaccine any one of clauses 49-52 for use in therapy.

[0152] 54. The nucleic acid molecule of any one of clauses 1-42, the mRNA molecule of clause 47 or clause 48, or the vaccine of any one of clauses 49-52 for use in a method of treating or preventing cancer.

[0153] 55. The nucleic acid molecule of any one of clauses 1-12, or 15-21 for use in a method of treating or preventing colon cancer.

[0154] 56. The nucleic acid molecule of any one of clauses 1-11 , 13 or 22-26 for use in a method of treating or preventing skin cancer.

[0155] 57. The nucleic acid molecule of any one of clauses 1-11 , 14 or 27-32 for use in a method of treating or preventing lung cancer.

[0156] 58. A method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of the nucleic acid molecule of any one of clauses 1-42, the mRNA molecule of clause 47 or clause 48, or the vaccine composition of any one of clauses 49-52.

[0157] 59. The method of clause 55, wherein the cancer is colon cancer, skin cancer or lung cancer.

[0158] 60. A method of selecting a personalized therapy for a patient suffering from cancer comprising: a. obtaining sequencing data from the patient suffering from cancer; and b. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule of any one of clauses 1-11 based on the obtained sequencing data.

[0159] 61 .The method of clause 60, wherein the step of selecting comprises comparing the sequencing data to a reference data.

[0160] 62. The method of clause 60 or clause 61 , wherein the sequencing data is obtained from mRNA sequencing.

[0161] 63. A method of manufacturing a cancer vaccine comprising the method of any one of clauses 60-62 and producing a cancer vaccine comprising the nucleic acid molecule(s) comprising the selected sequence(s) of neoantigen(s).

[0162] 64. Use of the nucleic acid molecule of any one of clauses 1-42, the neoantigen cassette of any one of clauses 43-46, or the mRNA molecule of clause 47 or clause 48 in the preparation of a vaccine.

[0163] 65. Use of the nucleic acid molecule of any one of clauses 1-42, the mRNA molecule of clause 47 or clause 48. or the vaccine composition of any one of clauses 49-52 in in vitro or in vivo translation.

[0164] The invention is disclosed in the further following clauses:

[0165] 1 . A nucleic acid molecule comprising, optionally in the 5’ to 3’ direction: a. an interleukin 12 (IL-12) sequence; and b. a neoantigen cassette encoding at least two neoantigens. 2. The nucleic acid molecule of clause 1 , wherein the nucleic acid molecule is an mRNA molecule.

[0166] 3. The nucleic acid molecule of clause 2, wherein the mRNA molecule further comprises a poly- A sequence downstream of the neoantigen cassette.

[0167] 4. The nucleic acid molecule of clause 1 , wherein the nucleic molecule is a DNA molecule.

[0168] 5. The nucleic acid molecule of clause 4, wherein the DNA molecule comprises a promoter upstream of the IL-12 sequence.

[0169] 6. The nucleic acid molecule of any one of clauses 1-5, wherein the nucleic acid molecule further comprises a cap sequence.

[0170] 7. The nucleic acid molecule of any one of clauses 1-6, wherein the nucleic acid molecule comprises a ribosomal recruitment sequence.

[0171] 8. The nucleic acid molecule of any one of clauses 1-7, wherein the nucleic acid molecule comprises a protein translation initiation site sequence.

[0172] 9. The nucleic acid molecule of any one of clauses 1-8, wherein the nucleic acid molecule comprises a stop codon downstream of the neoantigen cassette and the IL-12 sequence.

[0173] 10. The nucleic acid molecule of any one of clauses 1-9, wherein the nucleic acid molecule comprises a nucleic acid localisation sequence.

[0174] 11 . The nucleic acid molecule of clause 5, wherein the promoter is a T7 promoter, optionally wherein the T7 promoter comprises or consists of a sequence of SEQ ID NO: 1 .

[0175] 12. The nucleic acid molecule of any one of clauses 1-11 , wherein the IL-12 sequence encodes the p40 and p35 subunits of IL-12.

[0176] 13. The nucleic acid molecule of any one of clauses 1-12, wherein the IL-12 sequence comprises or consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 164.

[0177] 14. The nucleic acid molecule of any one of clauses 1-13, wherein the neoantigen cassette encodes at least 2, at least 3, at least 4, or at least 5 sequences of neoantigens binding to the Major Histocompatibility Complex (MHO) class I.

[0178] 15. The nucleic acid molecule of any one of clauses 1-13, wherein the neoantigen cassette encodes at least 2, at least 3, at least 4, or at least 5 sequences of neoantigens binding to the Major Histocompatibility Complex (MHC) class II .

[0179] 16. The nucleic acid molecule of any one of clauses 1-15, wherein the neoantigen cassette encodes at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker sequence(s), optionally wherein the linker sequence(s) comprise a sequence encoding glycine and / or serine, and / or optionally wherein the linker sequence is located between the sequences of neoantigens.

[0180] 17. The nucleic acid molecule of clause 16, wherein the linker sequence(s) comprise a sequence of SEQ ID NOs: 76 and / or 77.

[0181] 18. The nucleic acid molecule of clause 3, wherein the poly-A sequence consists of at least 100, preferably 128 adenine nucleotides.

[0182] 19. An mRNA molecule comprising, optionally in the 5’ to 3’ direction: a. a ribosomal recruitment sequence, optionally, wherein the ribosomal recruitment sequence is a 5 -UTR sequence derived from a haemoglobin subunit alpha 1 (HBA) sequence; b. an interleukin 12 (IL-12) sequence, wherein the IL-12 sequence encodes the p40 and p35 subunits of IL-12; c. a neoantigen cassette encoding at least two neoantigens, which are, optionally, separated by at least one linker; and d. a poly-A sequence.

[0183] 20. A DNA molecule, comprising, optionally in the 5’ to 3’ direction: a. a promoter, optionally wherein the promoter is a T7 promoter; b. a ribosomal recruitment sequence, optionally, wherein the ribosomal recruitment sequence is a 5 -UTR sequence derived from a haemoglobin subunit alpha 1 (HBA) sequence; c. an interleukin 12 (IL-12) sequence, wherein the IL-12 sequence encodes the p40 and p35 subunits of IL-12; and d. a neoantigen cassette encoding at least two neoantigens, which are, optionally, separated by at least one linker.

[0184] 21 . A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I.

[0185] 22. A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

[0186] 23. A vaccine composition comprising at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleic acid molecule(s) of any one of clauses 1-18, or the mRNA molecule of clause 19.

[0187] 24. The nucleic acid molecule of any one of clauses 1-18, the vaccine composition of clause 23, or the mRNA molecule of clause 19 for use in therapy.

[0188] 25. The nucleic acid molecule of any one of clauses 1-18, the vaccine composition of clause 23, or the mRNA molecule of clause 19 for use in a method of treating or preventing cancer.

[0189] 26. A method of selecting a personalized therapy for a patient suffering from cancer comprising: a. obtaining sequencing data from the patient suffering from cancer; and b. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule of any one of clauses 1-18 based on the obtained sequencing data.

[0190] 27. A method of manufacturing a cancer vaccine comprising: a. performing the method of clause 26; and b. producing a cancer vaccine comprising the selected sequence(s) of neoantigen(s).

[0191] 28. Use of the nucleic acid molecule of any one of clauses 1-18, the mRNA molecule of clause

[0192] 19, the DNA molecule of clause 20, or the neoantigen cassette of clause 21 or clause 22 in the preparation of a vaccine.

[0193] The sequences described in this application are identified in Table 1 .

[0194] Table 1. Sequences referenced in the application. BRIEF DESCRIPTION OF THE DRAWINGS

[0195] Figure 1 A illustrates one example of the nucleic acid molecule (here a DNA molecule) of the present invention.

[0196] Figure 1 B illustrates another example of the nucleic acid molecule (here an mRNA molecule) of the present invention.

[0197] Figure 1 C illustrates one example of the nucleic acid molecule (here a plasmid DNA molecule) of the present invention.

[0198] Figure 2 shows exemplary elements of the neoantigen cassette.

[0199] Figure 3 shows a comparison of performance of neoantigens identified as part of the present invention and BioNTech neoantigens in the context of skin and colon cancers.

[0200] Figure 4 illustrates an exemplary vector design (A), the restriction enzyme digest of the exemplary vector (B) and an agarose gel showing different mRNA products produced from the exemplary vectors (C).

[0201] Figure 5 (top panel) illustrates nucleic acid molecule constructs used in the examples, and (bottom panel) the results of EGFP mRNA transfection efficiency in macrophages.

[0202] Figure 6 illustrates a vaccination schedule for B16F10 / C57BL / 6 mice. The figure shows the timeline of the tumour implantation and vaccine dosing in B16F10 / C57BL / 6 mice, for both prevention (prophylactic) and treatment (therapeutic) studies.

[0203] Figure 7A shows the average tumour growth in C57BL / 6 mice treated with either the control (vaccine backbone), prophylactic vaccine, or therapeutic vaccine after being injected with B16F10 cancer cells. Figure 7B shows how the tumours shrank over time.

[0204] Figure 7C displays the survival rates of the mice.

[0205] Figure 8 illustrates a vaccination schedule for BALB / c mice suffering from CT-26 tumour. The figure shows the timeline of the tumour implantation and vaccine dosing in CT-26 tumour model in BALB / c mice, for both prevention (prophylactic) and treatment (therapeutic) studies.

[0206] Figures 9A and B show the average tumour growth in BALB / c mice treated with either the control (vaccine backbone), prophylactic vaccine, or therapeutic vaccine after being injected with CT-26 cancer cells. Figure 9C shows how the tumours shrank over time. Figure 9D displays the survival rates of the mice.

[0207] Figure 10 shows a vaccination schedule for C57BL / 6 mice with Lung tumour. The figure shows the timeline of the tumour implantation and vaccine dosing in LL-2 tumour model in C57BL / 6 mice, for both prevention (prophylactic) and treatment (therapeutic) studies.

[0208] Figure 11 A shows how the tumours regressed over time and Figure 11 B shows the average tumour growth in C57BL / 6 mice treated with either the control (vaccine backbone) or prophylactic vaccine. Figure 11 C shows the average tumour growth in C57BL / 6 mice treated with the therapeutic vaccine after being injected with LL-2 cancer cells. Figure 11 D illustrates the survival rates of the mice with the vaccines.

[0209] Figure 12 shows the results of the transfection efficiency assay for nucleic acid molecules comprising either a c-Myc UTR (1stcolumn), a 5' HBA UTR (2nd column), no UTR sequence (3rd column), or for control (cells only - 4thcolumn). EXAMPLES

[0210] The following non-limiting examples further illustrate the present invention.

[0211] Example 1 - Methods for injecting tumour cells in mice and obtaining biopsy samples

[0212] The method for injecting the tumour cells includes the steps:

[0213] 1) Keep syringes and needles, and cells, on ice to prevent Matrigel from solidifying.

[0214] 2) Draw up cells into syringe and prime immediately prior to injection into the animal.

[0215] 3) Anaesthetise the animals using gaseous isoflurane.

[0216] 4) Once anaesthetised, using an ice-cold primed syringe and needle, inject the cells subcutaneously into the back.

[0217] 5) Twist the needle to dislodge cells and keep hold of the skin as you withdraw the needle to reduce the likelihood of cells tracking along the injection site.

[0218] 6) Place animals back in their home cage and observe until they have fully recovered from the anaesthetic.

[0219] 7) Monitor the tumour implantation site daily for signs of tumour growth (this is mainly visual with Matrigel, with a gentle rub over the area if it looks to be developing into a tumour)

[0220] 8) Once there is a measurable tumour (the whole of the administration pocket is no longer flexible but a solid mass can be felt instead) they are measured 3x a week. Length and width are measured and volume calculated.

[0221] The mRNA is then collected from the mice biopsy sample. Specifically, half of the tumour samples were grinded in the presence of Liquid N2 and grinded with mortar and pestle. 500ul of lysis buffer was used for each samples and were passed through 23G syringe several times to obtain a uniform solution. mRNA was obtained using standard protocols.

[0222] Example 2 - Sequencing mRNA sequencing was performed using the standard protocol for library preparation by Illumina and sequencing was carried out using the NovaSeq 6000 Sequencing System guide [https: / / emea.support.illumina.com / content / dam / illumina- support / documents / documentation / system_documentation / novaseq / 1000000019358_17_novaseq- 6000-system-guide.pdf; last accessed: 17 April 2024].

[0223] Libraries were sequenced on S2 flow cell 200 cycles. The run time for that flow cell is 25h. However, the run in the present case took less as only 150 cycles (2*75pb) performed. Dragen Analysis was completed in 1 hour 46 minutes 23 seconds.

[0224] The VCFs were generated from the whole transcriptome and later the virtual panel of neoantigens was applied.

[0225] Example 3 - Neoantigen analysis and identification # Neoantigen Prediction Analysis Pipeline

[0226] The neoantigen prediction analysis pipeline was primarily inspired by the NeoPredPipe pipeline (https: / / github.com / MathOnco / NeoPredPipe). In this pipeline, neoantigens are analyzed by annotating the mutated sequences and predicting their binding ability to the given HLAs.

[0227] Below are the detailed steps involved in our analysis:

[0228] ## 01. Annotate VCF

[0229] The variants present in the VCF files were annotated using Annovar, based on the RefGene database of the GRCm39 reference genome. Focus on exon variations as they are more likely to impact protein sequences. The variants are identified by their line numbers in the VCF input file (including comments).

[0230] For instance, the variant "11SMCLID1_7173166" at "Iine141" corresponds to the variant at "chr1 :4846929" in row No.141 of "11SMCLID1_7173166.hard-filtered.vcf.

[0231] ## 02. Mutated Sequence

[0232] Both the peptides before and after the exon variation are recorded in "sample.fasta". 27aa_pep sequences

[0233] (flanking 13aa around the nonsynonymous variant aa) are extracted from the mutated peptide sequences.

[0234] This step prepares the input for the subsequent MHC binding prediction analysis.

[0235] ## 03. MHC Binding Prediction

[0236] For MHC (class I and class II) binding prediction, we utilized NetMHCpan-4.1 and NetMHCIIpan-4.3 with a parameter setting of 10mer peptides. The MHC Allele is referenced from the standard MHC typing of each mouse sample's strain. These tools predict peptide binding to any MHC molecule of known sequence using artificial neural networks (ANNs). The comprehensive results of Strong Binder / Weak Binder Neoantigen prediction are listed in the Summary XLS table, and each prediction includes annotated SNV information as well as the mutated sequence. For further insights on the output format see: https: / / services.healthtech.dtu. dk / services / NetMHCpan-4.1 / and https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.3 / .

[0237] For MHC class I, IEDB.org was used to predict immunogenicity for the Strong Binder Neoantigens as predicted above. The Immunogenicity score of 1 or close to 1 were used. While for class II MHC all the weak and strong binders were passed through MHC II binding predictor and score close to or 1 was selected as strong binders. Moreover, the Neoantigens which were selected as vaccine candidates, were looked for their relevant mutations in specific cancer using ICGC data portal. Only those genes which have relevant mutations in specific cancer type was chosen over other candidate genes.

[0238] Figure 3 shows the results. The neoantigens identified as part of the present invention have ranked better than BioNTech and other pharma companies.

[0239] Example 4 - Design of vector

[0240] Exemplary vector elements and neoantigens cassettes are illustrated in Figures 1 and 2.

[0241] For example, for MHC I complex the neoantigen cassette may comprise a nucleic acid sequence encoding:

[0242] Linker-27 amino acids neoantigen (27AA)-Linker-27AA-Linker-27AA-Linker-27AA-Linker- 27AA-Linker

[0243] The linker may comprise the glycine serine amino acid sequence: GGGGSGGGGS (SEQ ID NO: 76).

[0244] If only a single neoantigen sequence is included in the cassette, the neoantigen cassette may comprise a nucleic acid sequence encoding:

[0245] Linker-27AA-Linker

[0246] The GGGGSGGGGSGGGGS (SEQ ID NO: 77) linkers were used.

[0247] For example, for MHC II complex the neoantigen cassette may comprise a nucleic acid sequence encoding:

[0248] Linker-27AA-Linker-27AA-Linker-27AA-Linker-27AA-Linker

[0249] The linker may comprise the glycine serine amino acid sequence: GGGGSGGGGS (SEQ ID NO: 76).

[0250] A vector was designed incorporating the nucleic acid molecule as described herein (and shown, for example, in Figure 1). Exemplary vector included pVAX1 vector backbone and the nucleic acid molecule as described herein. The nucleic acid molecule was flanked by restriction enzyme sites. Figure 4 shows (A) the vector design; and (B) Gel showing in Lane M: KB ladder, in Lane 1 : undigested vector and in Lane 2: vector digested with Mlul and XhoL

[0251] Figure 4C shows four purified mRNA products produced from the exemplary vector of Figure 4A. In this example, the neoantigen cassette sequences expressed by the mRNA products have the sequences:

[0252] SK5-MH-1-1 :

[0253] GGGGSGGGGSQALGGFFTYFVILVENGFLPFHLLGIRGGGGSGGGGSPFLIFIIANIPLPMGTVTILCID LGTDGGGGSGGGG [SEQ ID NO: 167] SK5-MH1-2:

[0254] GGGGSGGGGSAYTLTSNIPEITPVLIFIIANIPLPLGGGGGSGGGGSGQTPIAEEIEHFILLITGVAVFLG VSFGGGGSGGGGSLEGGQTPIAEEIELFIHLITGVAVFLGGGGSGGGGS [SEQ ID NO: 168]

[0255] SK2-MH1-1 :

[0256] GGGGSGGGGSCGTAFFINFIAIYHHASRAIPFGTMVAGGGGSGGGGSIAYTLTSNIPEITTFLIFIIANIP LPLGGGGSGGGGS [SEQ ID NO: 165]

[0257] SK2-MH1-2:

[0258] GGGGSGGGGSPSVEDEPLLRENPHRFVVFPIEYHDIWGGGGSGGGGSSKEGTEAWEAAMKIYDER IDRVETRITGGGGSGGGGSAGVINRERIPTFEHMLWRVCRGNVFLRGGGGSGGGGS [SEQ ID NO: 166]

[0259] In which, the linker sequences have been underlined and the neoantigen sequences have not been underlined.

[0260] The IL-12 sequence included in the plasmid is of the human origin (SEQ ID NO: 2).

[0261] Example 5- In vitro transcription (IVT)

[0262] Reagents: mMESSAGE mMACHINE™ T7 mRNA Kit with CleanCap™ Reagent AG Catalog number: A57620, Invitrogen

[0263] Poly(A) Tailing Kit Catalog number: AM1350, Invitrogen

[0264] MEGAclear™ Transcription Clean-Up Kit Catalog number: AM1908, Invitrogen

[0265] After IVT reaction using T7 mRNA kit, the mRNA is subjected to poly A tail and the mRNA is cleaned up either using transcription clean up kit. The mRNA is then captured and eluted using standard protocols. mRNA can be quantified on Nanodrop reading the absorbance at 260 nm (A260) ( A260 x 40 = pg / mL mRNA) and further on agarose gel.

[0266] IVT reaction reagents are provided in Table 2.

[0267] Table 2. IVT reagents.

[0268] Pseudo -UTP was replaced with thio-UTP in some reactions.

[0269] Capping was performed according to NEB cap-1 (NEB m2081). 50 pg of mRNA was generated per reaction.

[0270] Poly-A tailing procedure:

[0271] 1) Anneal oligos of 100T and 50 A at equimolar ratio (1 :2) in 100 pl annealing buffer (6 mM Tris- HCI, pH 7.5, 7 mM MgCI2, 50 mM NaCI, 1 mM DTT)

[0272] 2) heated to 100°C for 5 min followed by incubation at 25°C for 3-4 h or cooling down overnight to room temperature.

[0273] 3) Oligos were treated with T4 polymerase, (NEB M0203S) DNA Polymerase I, Large (Klenow) Fragment, T4 PNK ( NEB M0201S , to fill gaps and create phosphorylated ends) and ligated using T4 DNA Ligase (M0202), NEB. All these are done following instruction from the supplier.

[0274] 4) Gel and column purified and ligated into G-blocks.

[0275] Or as an alternate protocol use, Poly (A) tailing Kit available commercially example AM1350, Invitrogen.

[0276] Example 6 - The quantity of in vitro transcribed (IVT) mRNA product produced from the nucleic acid molecules of the present invention

[0277] The constructs were designed as shown in Figure 5 (top panel) with the nucleic acid molecules comprising T7 promoter, a cap sequence, 5’UTR derived from HBA, Kozak sequence, an IL-12 sequence (comprising both p40 and p35 subunits) in the “Gene X” construct (and no IL-12 sequence in the “Mut-Gene X” construct), EGFP (instead of the neoantigen cassette), stop codon, 3’UTR derived from the HBA gene, poly-A tail, and the TT sequence. The abbreviations shown in the Figure are as follows:

[0278] T7-PS- T7 promoter sequence

[0279] CAP-0 / 1 - The Cap-0 / 1 structure. The Cap-0 is a N7-methyl guanosine connected to the 5' nucleotide through a 5' to 5' triphosphate linkage (also known as m7G cap or m7Gppp). An additional methylation on the 2'0 position of the initiating nucleotide generates Cap-1 (also known as m7GpppNm-, where Nm denotes any nucleotide with a 2'0 methylation).

[0280] 5-UTR-HBA- Human alpha globin gene ( in this case, mouse alpha globin gene was used) KS- Kozak Sequence - specifically sequence: GCCACCAATGG

[0281] Mut-Gene X-construct without the IL-12 sequence , Gene X - IL-12 gene (SEQ ID NO: 164).

[0282] EGFP -for the purpose of this example, the neoantigen cassette was substituted with the sequence of Enhanced Green Fluorescent Protein (EGFP) to examine the quantity of IVT mRNA.

[0283] STOP- Stop codon

[0284] 3-UTR-HBA1- Hemoglobin Subunit Alpha 1 gene UTR sequence

[0285] TT-seq - Transcription Termination sequence. This prevents continuous transcription.

[0286] The control (i.e. “EGFP”) construct is a plasmid comprising the EGFP.

[0287] Methods:

[0288] Primary macrophages were cultures using the conventional methods. IVT-mRNA was made as described in Examples1-5 and was transfected in 100 mm culture dishes with macrophages performed using Lipofectamine RNAIMAX (Invitrogen) according to the manufacturer’s instructions. The GFP images were generated with Invitrogen™ FLoid™ Cell Imaging Station after 24 hrs of transfection.

[0289] Results:

[0290] Figure 5 (bottom panel) shows the results of EGFP mRNA transfection efficiency in macrophages.

[0291] The bottom, left panel shows that the construct with GeneX-EGFP has the higher quality of the IVT mRNA. The bottom, right panel shows that EGFP mRNA expression level in macrophages transfected with low doses and high doses of the EGFP / IL-12 and Mut-IL-12 constructs. The mut- Gene -X construct (Figure 5 (top) has all elements of the nucleic acid molecule other than the IL-12 sequence as compared to the GeneX comprising construct.

[0292] The IL-12 - deficient mutant construct (i.e. “Mut-Gene X”) was tested alongside the wild-type IL-12 construct (i.e. “Gene X”) for mRNA transfection efficiency. Results showed that the mutant did not replicate the expression or transfection efficiency of the wild-type, instead behaving similarly to a GFP-only control construct.

[0293] Example 7 - In vivo testing of the constructs in melanoma mice model

[0294] Materials and Methods

[0295] Mice

[0296] Female C57BL / 6 mice (C57BL / 6NCrl Charles River) were eight weeks old, with body weights (BWs) ranging from 16.7 to 21 .1 grams (g), on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’Cobs™ bedding in static microisolators on 14- hour light / 10-hour dark cycles at 20-22 °C (68-72 °F) and 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

[0297] Tumour Cell Culture

[0298] B16F10 murine melanoma cells (Lineage 3) were maintained in DMEM medium containing 10% fetal bovine serum, 2 mM glutamine, 100 units / mL sodium penicillin G, 25 pg / mL gentamicin, 100 pg / mL streptomycin sulfate, and starting at the second passage, 1 pg / mL puromycin. The tumor cells were cultured in tissue culture flasks in an incubator set at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

[0299] Dosing, In Vivo Tumour Implantation, and Tumour Growth

[0300] On Day 1 , mice were pair-matched according to body weight (BW) into three groups (n=6 per group) with group mean BWs between 18.9 and 19.2 g. Animals in Groups 1 and 2 were isoflurane anesthetized and received intranodal vaccines (20 pg in a fixed volume of 15 pL per animal) injected in the right inguinal lymph node (or left inguinal node if necessary) followed by intra-incisional bupivacaine at 0.1 mg / kg administered subcutaneously (s.c ) QID x 6 . On Day 2, B16F10 cells were harvested during the exponential growth phase and resuspended at a concentration of 5 x 105cells / mL in PBS containing 50% Matrigel™ (BD Biosciences). Each mouse was injected subcutaneously in the right flank with 5 x 104B16F10 cells (in a 0.1 mL suspension). Tumours were measured with calipers to monitor growth three times a week. Tumour volume, in mm3, was calculated from the following equation:

[0301] Tumour Volume= w2x | / 2 where w = width and I = length, in mm, of the tumour. Tumour weight was estimated based on the assumption that 1 mg is equivalent to 1 mm3of tumour volume.

[0302] Treatment

[0303] Figure 6 illustrates a vaccination schedule for B16F10 / C57BL / 6 mice.

[0304] On Day 1 , vaccine dosing was initiated according to the treatment plan, with tumour implant and group sorting according to BW on Day 2. Animals received two doses of either control (i.e, nucleic acid molecule without the neoantigen cassette) or personalized B16F10 vaccine (i.e. vaccine composition comprising the nucleic acid molecules comprising neoantigen cassettes, which encode peptides of, for example, SEQ ID NOs: 165- 168, 85, 87, and 88) three days apart on the days indicated below. Vaccines were administered twice into the right inguinal lymph node (intranodal). Intranodal doses were injected in the right inguinal node of isoflurane anesthetized animals in a fixed volume of 15 pL per animal.

[0305] Group 1 received intranodal 18 pg control vaccine on Day 1 , tumour implant on Day 2, intranodal 18 pg control vaccine on Day 4.

[0306] Group 2 received intranodal 18 pg personalized B16F10 vaccine on Day 1 , tumour implant on Day 2, intranodal 18 pg personalized B16F10 vaccine on Day 4.

[0307] Group 3 received tumour implant on Day 2, intranodal 18 pg personalized B16F10 vaccine on Days 4 and 7. Results:

[0308] The results are shown in Figure 7 A, B and C. Tumour volumes were reduced by 58% for Therapeutic vaccine and by 45% for prophylactic vaccines when compared with the control vaccines. The lifespan of the treated animal was extended by six days compared to controls in a 23-day cycle.

[0309] Immunogenicity: Significant increase in tumour-specific CD8+ T-cells was observed. Other markers include CD 45+, CD4, CD3, TIM3, Ki67, PD1 and PDL1.

[0310] Predictive Model: using 42pg of vaccine for 4 doses the tumour growth can be abolished.

[0311] Example 8 - In vivo testing of the constructs in colon cancer mice model

[0312] Materials and Methods

[0313] Mice

[0314] Female BALB / c mice (BALB / cAnNcr1 , Charles River) were eight weeks old with body weights (BWs) range from 14.6 to 19.6 grams (g) on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’cobs™ Laboratory Animal Bedding in static microisolators on 14-hour light / 10-hour dark cycles at 20-22 °C (68-72 °F) and 40-60% humidity. Charles River Discovery Services in North Carolina (CR Discovery Services) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

[0315] Tumour Cell Culture

[0316] CT26 murine colon carcinoma cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 4.5 g / L glucose, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.075% sodium bicarbonate, 100 units / mL penicillin G sodium, 100 pg / mL streptomycin sulfate, 25 pg / mL gentamicin, and 50 pM p-mercaptoethanol. The cells were cultured in tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 5% CO2 and 95% air.

[0317] Dosing, In Vivo Tumour Implantation, and Tumour Growth

[0318] On Day 1 , isoflurane anesthetized animals received intranodal vaccines (18 pg in a fixed volume of 17 pL per animal) injected in the right inguinal lymph node. On Day 1 , mice were pair-matched according to body weight (BW) into three groups (n=6 per group) with group mean BWs between 16.6 and 17.1 g. Also on Day 2, CT26 cells used for implantation were harvested during log phase growth and resuspended in cold phosphate- buffered saline (PBS). Tumour growth was initiated by subcutaneous (s.c.) injection of 2 x 106CT26 cells (in a 0.1 mL cell suspension) into the right flank of each mouse. Tumours were calipered in two dimensions three times per week. Tumour volume, in mm3, was calculated from the following equation Tumour Volume= w2x | / 2 where w = width and I = length, in mm, of the tumour. Tumour weight was estimated with the assumption that 1 mg is equivalent to 1 mm3of tumour volume.

[0319] Treatment

[0320] Figure 8 illustrates a vaccination schedule for BALB / c mice suffering from CT-26 tumour.

[0321] On Day 1 , vaccine dosing was initiated according to the treatment plan, with tumour implant and group sorting according to individual animal body weights on Day 2. Animals received two doses of either control (i.e, nucleic acid molecule without the neoantigen cassette) or personalized CT-26 vaccine (i.e. vaccine composition comprising the nucleic acid molecules comprising neoantigen cassettes, which encode peptides of, for example, SEQ ID NOs: 169, 170, 78, 79, 81-84) three days apart on the days indicated below. Vaccines were administered twice into the right inguinal lymph node (intranodal). Intranodal doses were injected in the right inguinal node of isoflurane anesthetized animals in a fixed volume of 17 pL per animal, Group 1 received intranodal 18 pg control vaccine on Day 1 , tumour implant on Day 2, intranodal 18 pg control vaccine on Day 4.

[0322] Group 2 received intranodal 18 pg personalized CT-26 vaccine on Day 1 , tumour implant on Day 2, intranodal 18 pg personalized CT-26 vaccine on Day 4. Group 3 received tumour implant on Day 2, intranodal 18 pg personalized CT-26 vaccine on Days 4 and 7.

[0323] Results:

[0324] The results are shown in Figures 9 A, B, C and D. A 60% reduction in tumour size for Therapeutic vaccine and 45% for Prophylactic vaccine was observed when compared with the control vaccine. Median survival of the animal was extended by 50%, 4 days for therapeutic and 2 days for prophylactic vaccine in 25-day cycle. No significant adverse effects observed.

[0325] Immunogenicity: Significant increase in tumour-specific CD8+ T-cells was observed. Other markers include CD 45+, CD4, CD3, TIM3, Ki67, PD1 and PDL1.

[0326] Predictive Model: using 42pg of vaccine for 4 doses the tumour growth can be abolished.

[0327] Example 9 - In vivo testing of the constructs in lung cancer mice model

[0328] Materials and Methods

[0329] Mice

[0330] Female C57BL / 6 mice (C57BL / 6NCrl Charles River) were ten weeks old, with body weights (BWs) ranging from 19.1 to 22.6 grams (g), on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o’Cobs™ bedding in static microisolators on 14- hour light / 10-hour dark cycles at 20-22 °C (68-72 °F) and 40-60% humidity. Charles River Discovery Services North Carolina (CR Discovery Services) specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

[0331] Tumour Cell Culture

[0332] The LL / 2(LLc1) (ECACC 90020104) murine lung carcinoma cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC). LL / 2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units / mL sodium penicillin G, 100 pg / mL streptomycin sulfate and 25 pg / mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.

[0333] Dosing, In Vivo Tumour Implantation, and Tumour Growth

[0334] Figure 10 shows a vaccination schedule for C57BL / 6 mice with Lung tumour.

[0335] On Day 1 , mice were pair-matched according to body weight (BW) into three groups (n=6 per group) with group mean BWs between 20.8 and 21.6 g. Following sorting, the animals in Groups 1 and 2 were anesthetized with isoflurane and received intranodal vaccine therapies (20 pg in a fixed volume of 15 pL per animal) injected in the right inguinal lymph node (or left inguinal node if necessary) followed by intra-incisional bupivacaine at 0.1 mg / kg administered subcutaneously (s.c ) QID x 6. On Day 2 (24 hours post the Day 1 treatments), LL-2 cells were harvested during the exponential growth phase and resuspended in PBS. Each mouse on study (Groups 1 , 2 and 3) was injected subcutaneously in the right flank with 2 x 106LL-2 cells (in a 0.1 mL suspension). Tumours were measured with calipers to monitor growth three times a week. Tumour volume, in mm3, was calculated from the following equation:

[0336] Tumour Volume= w2x / / 2

[0337] Treatment

[0338] Three groups of female C57BL / c mice (n=6 per group) were sorted according to BW on Day 1 . Staggered vaccine doses and tumour implants were initiated according to the treatment plan. All groups received LL-2 tumour cells at 2 x 106cells per animal administered subcutaneously (s.c.) on Day 2 in a fixed volume of 0.1 mL.

[0339] On Day 1 (24 hours prior to LL-2 cell implant) and Day 4, Group 1 received 18 pg intranodal control vaccine (i.e, nucleic acid molecule without the neoantigen cassette).

[0340] Group 2 received 18 pg intranodal personalized LL-2 vaccine (i.e. vaccine composition comprising the nucleic acid molecules comprising neoantigen cassettes, which encode peptides of, for example, SEQ ID NOs: 171 , 172, 90, 92-95) on Day -7 (7 days prior to LL-2 cell implant) and Day 4.

[0341] Group 3 received 20 pg intranodal personalized LL-2 vaccine on Days 4 and 7.

[0342] Results: The results are shown in Figures 11 A, B, C, and D. Tumour volumes were reduced by 48% compared to controls for Therapeutic vaccines. For prophylactic vaccines the tumour stopped growing until 11 days and then growth was reduced to 40% compared with the control. Median survival was extended by 40%, over 5 days in a 29 days cycle.

[0343] Immunogenicity: Significant increase in tumour-specific CD8+ T-cells was observed. Other markers include CD 45+, CD4, CD3, TIM3, Ki67, PD1 and PDL1.

[0344] Predictive Model: using 42pg of vaccine for 4 doses the tumour growth can be abolished.

[0345] Example 10 - The incorporation of the 5' HBA UTR (Hemoglobin Alpha Untranslated Region) in the nucleic acid molecule

[0346] Computation predictions performed during the formulation of the codon-optimised construct used to produce the mRNA molecule show that the 5' HBA UTR offers several advantages in mRNA vaccine development, particularly in enhancing stability, translation efficiency, and overall immunogenicity.

[0347] The key benefits:

[0348] 1. Increased mRNA Stability

[0349] - The 5' HBA UTR enhances mRNA half-life, reducing degradation and ensuring prolonged antigen expression.

[0350] - Protects against exonuclease activity, leading to sustained immune stimulation.

[0351] 2. Enhanced Translation Efficiency

[0352] - Optimized ribosome binding increases protein translation, leading to higher antigen production.

[0353] - More efficient cap-dependent translation ensures strong immune responses with lower mRNA doses.

[0354] 3. Improved Immunogenicity

[0355] - Higher and sustained antigen expression leads to stronger and longer-lasting immune responses.

[0356] - Supports efficient activation of CD8+ T-cells, crucial for targeting tumours.

[0357] 4. Compatibility with Non-LNP Delivery

[0358] - Enhances mRNA function even in non-lipid nanoparticle (LNP) delivery methods, such as intranodal administration, improving vaccine stability.

[0359] 5. Proven Success in Preclinical Models

[0360] - superior antigen expression and tumour regression when using 5' HBA UTR.

[0361] Computation studies show that by incorporating the 5' HBA UTR, the nucleic acid molecule shows the best effectiveness, durability, and clinical translation.

[0362] Cells (HeLa) were transfected with different UTRs to measure the transfection efficiency using various UTR sequences. The transfection was carried out using luciferase assay to determine efficiency of IVT. Figure 12 shows that by incorporating 5' HBA UTR in the nucleic acid molecule (2ndcolumn), the transfection efficiency is higher as compared to other sequences (1stcolumn - c-Myc), and nucleic acid without a UTR sequence (3rdcolumn). Column 4 represents control (cells only).

[0363] Example 11 -Toxicity

[0364] Comprehensive organ histopathology analysis was conducted in mouse models to assess potential toxicity associated with the mRNA-based cancer vaccines described herein (as in Examples above). Key organs, including the liver, spleen, and kidneys, were closely monitored for any signs of inflammation, structural damage, or adverse immune responses. The results demonstrated no significant toxicity, indicating a favourable safety profile. Additionally, biomarker analysis and liver enzyme assessments further confirmed the absence of hepatotoxicity or systemic toxicity. These findings support the translational potential of our vaccine and provide a strong foundation for advancing into clinical trials with a high degree of confidence in its safety. There was less than 10% Body weight loss and no necrosis. In addition, there were no treatment related deaths.

Claims

CLAIMS1 . A nucleic acid molecule comprising, optionally in the 5’ to 3’ direction: a. an interleukin 12 (IL-12) sequence; and b. a neoantigen cassette encoding at least two neoantigens.

2. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is an mRNA molecule.

3. The nucleic acid molecule of claim 2, wherein the mRNA molecule further comprises a poly-A sequence downstream of the neoantigen cassette.

4. The nucleic acid molecule of claim 1 , wherein the nucleic molecule is a DNA molecule.

5. The nucleic acid molecule of claim 4, wherein the DNA molecule comprises a promoter upstream of the IL-12 sequence.

6. The nucleic acid molecule of any one of claims 1-5, wherein the nucleic acid molecule further comprises a cap sequence.

7. The nucleic acid molecule of any one of claims 1-6, wherein the nucleic acid molecule comprises a ribosomal recruitment sequence, optionally wherein the ribosomal recruitment sequence has a sequence having at least 95% identity to SEQ ID NO: 96.

8. The nucleic acid molecule of any one of claims 1-7, wherein the nucleic acid molecule comprises a protein translation initiation site sequence, optionally wherein the protein translation initiation site sequence has a sequence of SEQ ID NO: 173.

9. The nucleic acid molecule of any one of claims 1-8, wherein the nucleic acid molecule comprises a stop codon downstream of the neoantigen cassette and the IL-12 sequence.

10. The nucleic acid molecule of any one of claims 1-9, wherein the nucleic acid molecule comprises a nucleic acid localisation sequence, optionally wherein the nucleic acid localisation sequence has a sequence having at least 95% identity to SEQ ID NO: 97.11 . The nucleic acid molecule of claim 5, wherein the promoter is a T7 promoter, optionally wherein the T7 promoter comprises or consists of a sequence of SEQ ID NO: 1 .

12. The nucleic acid molecule of any one of claims 1-11 , wherein the IL-12 sequence encodes (optionally in the 5’ to 3’ direction) the p40 and p35 subunits of IL-12.

13. The nucleic acid molecule of any one of claims 1-12, wherein the IL-12 sequence comprises or consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 164.

14. The nucleic acid molecule of any one of claims 1-13, wherein the neoantigen cassette encodes at least 2, at least 3, at least 4, or at least 5 sequences of neoantigens binding to the Major Histocompatibility Complex (MHO) class I.

15. The nucleic acid molecule of any one of claims 1-13, wherein the neoantigen cassette encodes at least 2, at least 3, at least 4, or at least 5 sequences of neoantigens binding to the Major Histocompatibility Complex (MHC) class II.

16. The nucleic acid molecule of any one of claims 1-15, wherein the neoantigen cassette encodes at least 1 , at least 2, at least 3, at least 4, at least 5, or at least 6 linker sequence(s), optionally wherein the linker sequence(s) comprise a sequence encoding glycine and / or serine, and / or optionally wherein the linker sequence is located between the sequences of neoantigens.

17. The nucleic acid molecule of claim 16, wherein the linker sequence(s) comprise a sequence of SEQ ID NOs: 76 and / or 77.

18. The nucleic acid molecule of claim 3, wherein the poly-A sequence consists of at least 100, preferably 128 adenine nucleotides.

19. An mRNA molecule comprising, optionally in the 5’ to 3’ direction: a. a ribosomal recruitment sequence, optionally, wherein the ribosomal recruitment sequence is a 5 -UTR sequence derived from a haemoglobin subunit alpha 1 (HBA) sequence; b. an interleukin 12 (IL-12) sequence, wherein the IL-12 sequence encodes the p40 and p35 subunits of IL-12; c. a neoantigen cassette encoding at least two neoantigens, which are, optionally, separated by at least one linker; and d. a poly-A sequence.

20. A DNA molecule, comprising, optionally in the 5’ to 3’ direction: a. a promoter, optionally wherein the promoter is a T7 promoter; b. a ribosomal recruitment sequence, optionally, wherein the ribosomal recruitment sequence is a 5’-UTR sequence derived from a haemoglobin subunit alpha 1 (HBA) sequence;c. an interleukin 12 (IL-12) sequence, wherein the IL-12 sequence encodes the p40 and p35 subunits of IL-12; and d. a neoantigen cassette encoding at least two neoantigens, which are, optionally, separated by at least one linker.21 . A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class I.

22. A neoantigen cassette comprising or consisting of at least 2, at least 3, at least 4, or at least 5 sequences encoding the amino acid sequence of peptides binding to the Major Histocompatibility Complex (MHC) class II.

23. A vaccine composition comprising at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleic acid molecule(s) of any one of claims 1-18, or the mRNA molecule of claim 19.

24. The nucleic acid molecule of any one of claims 1-18, the vaccine composition of claim 23, or the mRNA molecule of claim 19 for use in therapy.

25. The nucleic acid molecule of any one of claims 1-18, the vaccine composition of claim 23, or the mRNA molecule of claim 19 for use in a method of treating or preventing cancer.

26. A method of selecting a personalized therapy for a patient suffering from cancer comprising: a. obtaining sequencing data from the patient suffering from cancer; and b. selecting the sequence(s) of neoantigen(s) to be incorporated into the neoantigen cassette of the nucleic acid molecule of any one of claims 1-18 based on the obtained sequencing data.

27. A method of manufacturing a cancer vaccine comprising: a. performing the method of claim 26; and b. producing a cancer vaccine comprising the selected sequence(s) of neoantigen(s).

28. Use of the nucleic acid molecule of any one of claims 1-18, the mRNA molecule of claim 19, the DNA molecule of claim 20, or the neoantigen cassette of claim 21 or claim 22 in the preparation of a vaccine.

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