KRAS immunogenic non-mutated epitope mRNA anticancer vaccine

Abstract

The present invention relates to a KRAS immunogenic non-mutated epitope mRNA vaccine and a use thereof. The present invention provides an mRNA vaccine encoding a KRAS immunogenic non-mutated epitope peptide, and thus may exhibit excellent tumor therapeutic and recurrence-preventive effects against KRAS-expressing cancers.

Description

KRAS immunogenic non-mutagenic epitope mRNA anticancer vaccine

[0001] The present invention relates to an mRNA anticancer vaccine using a KRAS immunogenic non-mutant epitope and the use thereof.

[0002] Cancer vaccines are designed to activate cytotoxic T cells so that the activated T cells recognize specific types of cancer and act against them, or to induce the production of antibodies that bind to antigens present on the surface of cancer cells. The basic therapeutic goal of cancer vaccines is to reduce the size of existing cancer tissue in patients or to suppress cancer recurrence after the cancer tissue has been removed through surgery or chemotherapy.

[0003] KRAS is a member of the RAS protein family, which plays a crucial role in the development and progression of tumors, and it regulates the excessive growth and division of tumor cells. The importance of KRAS lies in the fact that abnormal activation or mutations in this protein can promote the development and growth of tumors. Gene mutations in KRAS account for 85–90% of all RAS gene mutations; structural studies indicate that most KRAS mutations maintain the active state of KRAS-GTP by interfering with the ability to hydrolyze GTP. These KRAS mutations are closely associated with various types of cancer, such as colorectal and lung cancer, and can influence the development and metastasis of cancer.

[0004] KRAS is considered one of the key topics in tumor biology and anticancer therapy research, and efforts to develop therapies targeting it are still ongoing. In particular, research on developing therapies targeting KRAS mutations is predominant; however, due to the existence of various mutations, there is a challenge in developing individual treatments for each specific mutation. Some groups are conducting vaccine research using mutated KRAS as an antigen, and although they have attempted to verify vaccine efficacy using MHC class I restricted peptides that directly stimulate CD8+ cytotoxic T cells, they do not appear to have achieved the intended success. Recently, attempts have been made to include as many steps as possible, such as binding affinity with MHC class II, TCR binding affinity, and antigen processing, into predictive models to identify neoantigens. Following this research trend, the development of peptide vaccines capable of inducing Th1 immune responses and blocking cancer development using relatively long peptides expected to possess binding affinity for MHC class II is underway, but these efforts have not yet shown remarkable results. Despite extensive efforts, it is still difficult to find a clinically viable cancer treatment that effectively blocks the function of KRAS by targeting it.

[0005] Meanwhile, gene therapy and gene vaccination are among the most promising and rapidly developing methods in modern medicine. Gene therapy and gene vaccination can provide highly specific and individualized options for treating various diseases. These gene vaccines are generally understood as "third-generation" vaccines. Gene vaccines typically consist of genetically engineered nucleic acid molecules that enable the expression of peptide or protein (antigen) fragments characteristic of pathogens or tumor antigens within the body. When administered to a patient, a gene vaccine is expressed following absorption by target cells. The expression of the administered nucleic acid leads to the production of the encoded protein. If this protein is recognized as a foreign substance by the patient's immune system, an immune response is triggered.

[0006] In gene vaccines, not only DNA but also mRNA can be used as nucleic acid molecules for administration. By using mRNA instead of DNA, the risk of unwanted genomic integration and the generation of anti-DNA antibodies is minimized or prevented.

[0007] There are many challenges regarding nucleic acid delivery to induce desired responses in biological systems. While nucleic acid-based therapeutics, such as vaccines, hold tremendous potential, there remains a need to deliver nucleic acids more effectively to appropriate sites within cells or organisms to realize this potential.

[0008] Under this technical background, the inventors manufactured a KRAS immunogenic non-mutant epitope mRNA vaccine and confirmed its tumor growth inhibitory effect to complete the present invention.

[0009] One object of the present invention is to provide a nucleic acid molecule comprising: an expression regulatory element having expression initiation activity; and a coding region operably connected to said expression regulatory element; wherein the coding region comprises a base sequence encoding the peptide sequence of SEQ ID NO. 1.

[0010] Another objective of the present invention is to provide a recombinant expression vector comprising the nucleic acid molecule.

[0011] Another objective of the present invention is to provide an in vitro transcription (IVT) mRNA comprising the nucleic acid molecule.

[0012] Another objective of the present invention is to provide a vaccine composition for treating cancer comprising mRNA expressed from the vector or in vitro transcription (IVT) mRNA.

[0013] Another objective of the present invention is to provide a method for preventing, suppressing, or treating cancer, comprising the step of administering a nucleic acid molecule containing a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 mentioned above, a vector containing the same, or IVT mRNA to an individual in need thereof.

[0014] Another objective of the present invention is to provide a composition comprising the aforementioned nucleic acid molecule, a vector containing the same, or an IVT mRNA for use in the prevention, suppression of recurrence, or treatment of cancer.

[0015] Another objective of the present invention is to provide the use of the aforementioned nucleic acid molecule, a vector containing it, or IVT mRNA in the manufacture of a drug for the treatment of cancer.

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0017] The present invention provides a nucleic acid molecule comprising: an expression regulatory element having expression initiation activity; and a coding region operably connected to the expression regulatory element; wherein the coding region comprises a base sequence encoding the peptide sequence of SEQ ID NO. 1.

[0018] In the present invention, "nucleic acids" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified.

[0019] In the present invention, the terms "expression control / regulation sequence" or "expression control / regulation element" may refer to a nucleic acid sequence that regulates the transcription of nucleic acids and / or translation from nucleic acids in transcript form. Additionally, "transcription control / regulation sequence" or "transcription control / regulation element" refers to a nucleic acid sequence that regulates the transcription of nucleic acids. Transcription control sequences include promoters such as constructive promoters or inducible promoters, or enhancers.

[0020] Additionally, the terms "translation control / regulation sequence" or "translation control / regulation element" may be used for nucleic acid sequences that regulate translation from a nucleic acid in transcript form into a protein or peptide. These expression control sequences / elements, transcription control sequences / elements, and / or translation control sequences / elements are operatively linked to the sequence to be expressed, e.g., the nucleic acid sequence to be transcribed or translated.

[0021] In this specification, the term "operatively linked" refers to a functional linkage between an expression regulatory sequence (element) of a nucleic acid (e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, etc.) and another nucleic acid sequence, thereby allowing the regulatory sequence (element) to regulate the transcription and / or translation of the other nucleic acid sequence.

[0022] In terms of selection, nucleotide sequences that can increase the expression efficiency of coding regions connected in the form of open reading frames (ORFs) can be further inserted into the nucleic acid molecule. For example, the nucleic acid molecule may have a transcription control element (TCCE) that promotes the transcription of the nucleic acid molecule adjacent to a translation control element, such as an upstream-translation control element (U-TLCE).

[0023] For example, a transcriptional regulatory element (TCCE) can be located upstream of an upstream translational regulatory element (U-TLCE). Such a transcriptional regulatory element (TCCE) is not specifically limited.

[0024] In an exemplary aspect, a transcriptional regulatory element (TCCE) may be a promoter that promotes the transcription of a peptide encoded in the form of an ORF in the coding region. The transcriptional regulatory element (TCCE) may operate in animal cells, more specifically mammalian cells, to regulate the transcription of a gene or a fragment thereof encoded in the coding region.

[0025] For example, transcriptional regulatory elements (TCCEs) include promoters derived from mammalian viruses, promoters derived from the genome of mammalian cells, or promoters derived from bacteriophages. For example, transcriptional regulatory elements (TCCEs) include the CMV (cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, T7 bacteriophage promoter, T3 bacteriophage promoter, SP6 promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, cancer cell-specific promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter), and tissue-specific promoters (e.g., albumin promoter). Includes, but is not limited to.

[0026] In an exemplary aspect, any transcription regulatory element (TCCE) capable of transcribing mRNA from a linearized DNA template, such as the T7 bacteriophage promoter, T3 bacteriophage promoter, and SP6 bacteriophage promoter, may be located near an upstream translation regulatory element (U-TLCE), specifically upstream of the upstream translation regulatory element (U-TLCE).

[0027] In addition, the translation regulatory element may include an element having cap-dependent translation initiation activity or an expression regulatory element having internal ribosome entry site (IRES) activity.

[0028] In an exemplary sense, an upstream translation regulatory element (U-TLCE) may be all or part of a 5'-untranslated region (5'-UTR) that possesses cap-dependent translation initiation activity. Most eukaryotic mRNAs have a 7-methyl-guanosine (cap, m7G) at the 5' end, and the translation initiation complex recognizes the cap located at the 5' end and proceeds to the initiation codon AUG to start protein synthesis. In other words, the cap structure located at the 5' end of the mRNA not only initiates protein translation but also prevents the destruction of the mRNA by the action of nucleases.

[0029] In vitro transcription can be performed by first linearizing pDNA (plasmid DNA) with restriction enzymes, and then using an appropriate RNA polymerase to attach m7G(5')-ppp(5')G (regular cap analog) to the prepared mRNA to create capped mRNA. Alternatively, transcription can be performed in vitro without a cap analog, and a commercially available vaccinia virus capping enzyme can be used to perform the capping reaction. An 'anti-reverse' cap analog (ARCA) can also be used to block the reverse reaction of this cap. When ARCA is introduced, only the 3'-O-methylation of the methylated guanosine can bind to the nucleotide of the unmethylated guanosine.

[0030] For example, modified 5'-Cap structures are Cap1 (methylation of ribose in adjacent nucleotides of m7G), Cap2 (methylation of ribose in the nucleotide located 2nd downstream of m7G), Cap3 (methylation of ribose in the nucleotide located 3rd downstream of m7G), Cap4 (methylation of ribose in the nucleotide located 4th downstream of m7G), ARCA (anti-reverse Cap analogs, modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-diaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine).

[0031] For example, the upstream translation control element (U-TLCE) and / or the downstream translation control element (D-TLCE) may each consist of a nucleotide sequence or a transcript sequence having cap-dependent translation initiation activity derived from an animal, e.g., mammal, specifically primates, more specifically humans. The upstream translation control element (U-TLCE) is a region to which a translation initiation complex binds during the translation process of a peptide and / or protein expressed from a coding region, and may be a cis-acting nucleotide sequence that induces translation of the coding region.

[0032] In an exemplary aspect, a downstream translation regulatory element (D-TLCE) may be located relative to an upstream translation regulatory element (U-TLCE). For example, if the upstream translation regulatory element (U-TLCE) is all or part of a nucleotide sequence having cap-dependent translation initiation activity, it may include a downstream translation regulatory element (D-TLCE) that may be located downstream of the coding region. A nucleic acid molecule containing the upstream translation regulatory element (U-TLCE) and the downstream translation regulatory element (D-TLCE) can further enhance the expression efficiency of the open frame constituting the coding region.

[0033] Additionally, in terms of selection, the upstream translation regulatory element (U-TLCE) may be all or part of a 5'-UTR having internal ribosome binding site (IRES) activity. The upstream translation regulatory element (U-TLCE) may be all or part of a 5'-UTR having viral IRES activity. The upstream translation regulatory element (U-TLCE) is a region where the translation initiation complex binds during the translation process of a peptide and / or protein expressed from the coding region, and the IRES is a cis-acting nucleotide sequence that induces translation of a target gene by forming complex secondary and tertiary structures.

[0034] In addition, nucleotide sequences that can induce the expression of an open-reading frame consisting of a gene or its transcript sequence that constitutes the coding region can be inserted into the nucleic acid molecule. In one exemplary aspect, a Kozak sequence can be inserted between an upstream translation regulatory element (U-TLCE) and the start codon of the coding region.

[0035] Additionally, to insert a coding region into the nucleic acid molecule, the nucleic acid molecule may include one or more cloning sites, preferably multiple cloning sites (MCS). One or more cloning sites may include one or more restriction endonuclease recognition sites and / or sequences that are cleaved by restriction endonucleases. Restriction endonucleases may include natural restriction endonucleases found in bacteria or archaea, as well as artificially produced restriction endonucleases (e.g., Zinc finger nucleases, restriction endonucleases based on DNA binding sites of TAL effectors, or PNA-based PNAznymes).

[0036] For example, naturally occurring restriction enzymes can be classified into 1) Type I restriction enzymes (cleave a site away from the recognition site and require ATP, S-adenosyl-L-methionine, and magnesium ions), 2) Type II restriction enzymes (cleave a specific site within or slightly away from the recognition site and mostly require magnesium ions), 3) Type III restriction enzymes (cleave a site slightly away from the recognition site and require ATP, but hydrolysis of ATP is not required), 4) Type IV restriction enzymes (target modified sites such as methylation, hydroxymethylation, or glucosyl-hydroxymethylation), and 5) Type V restriction enzymes (CRISPR cas9-gRNA complex).

[0037] For example, a multiple cloning site (MCS) may include, but is not limited to, a site recognized by the following restriction enzymes and / or a restriction enzyme cleavage site: AngⅠ, AatⅠ, AbaⅠ, BamHⅠ, BbvⅠ, BcgⅠ, BplⅠ, BsmAⅠ, Alw26Ⅰ, BsrⅠ, ClaⅠ, EarⅠ, Eco57Ⅰ, EcoRⅠ, EcoRⅡ, EcoRⅤ, FokⅠ, HaeⅢ, HindⅢ, HpaⅢ, HphⅠ, KpnⅠ, MboⅠ, MluⅠ, NaeⅠ, NdeⅡ, NgoMⅣ, NlaⅢ, NotⅠ, PacⅠ, PstⅠ, SacⅠ, SacⅡ, SalⅠ, SfaNⅠ, SmaⅠ, TaqⅠ, XbaⅠ, XhoⅠ, PvuⅠ, and combinations thereof.

[0038] Additionally, the nucleic acid molecule may optionally further include at least one nucleotide sequence among a polyadenosine sequence or a polyadenosine sequence (PA), which can further improve the translation efficiency of an ORF consisting of a gene or its transcription sequence present in the coding region while stabilizing the transcribed nucleic acid molecule downstream of the coding region, more specifically downstream of a downstream translation regulatory element (D-TLCE).

[0039] In an exemplary aspect, the polyadenylation signal sequence and / or polyadenosine sequence (PA) may be located downstream of the coding region, for example, between the downstream translation regulatory element (D-TLCE) and the stem-loop element (SL1) described below. In another exemplary aspect, the polyadenylation signal sequence and / or polyadenosine sequence (PA) may be located downstream of the stem-loop element (SL1). For example, if the nucleic acid molecule of the present invention consists of a transcription sequence in the form of RNA, the polyadenosine sequence (PA) may be a nucleic acid sequence consisting of approximately 25 to approximately 400, preferably 30 to 400, more preferably 50 to 250, and most preferably 50 to 120 adenosines.

[0040] Additionally, the nucleic acid molecule may have a nucleotide encoding a signal sequence located upstream of the coding region. The signal sequence may be a fragment directing the secretion of a biologically active molecule drug and / or peptide, and may be cleaved after translation in a host cell. For example, the signal sequence may be a polynucleotide encoding an amino acid sequence that initiates the movement of a peptide through the endoplasmic reticulum (ER) membrane.

[0041] The above signal sequence is well known in the art for its characteristics and is typically known to contain 16 to 30 amino acid residues, and may contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer while the immature polypeptide moves. After initiation, the signal peptide is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. For example, the signal sequence may include, but is not limited to, a polynucleotide encoding a human albumin signal peptide.

[0042] In the present invention, the "coding region" is a part of mRNA composed of codons. Codons are translated into proteins according to information provided by the "genetic code." The coding region typically begins with a start codon and ends with a stop codon. Generally, the start codon is an AUG triplet, and the stop codon is UAA, UAG, or UGA. In addition to protein coding, parts of the coding region can serve as regulatory sequences in pre-mRNA as exonic splicing enhancers or exonic splicing silencers.

[0043] In addition, the coding region of the present invention may include a sequence transcribed from the nucleic acid sequence of various foreign genes, and since there are variations in nucleotides that do not cause changes in the protein, even an mRNA sequence for obtaining the same target protein may include a coding region having variations in various nucleotides. The aforementioned variations in nucleotides that do not cause changes in the protein include nucleic acid molecules containing functionally equivalent codons or codons coding for the same amino acid (for example, due to the degeneracy of codons, there are six codons for arginine or serine), or codons coding for biologically equivalent amino acids, and considering the variations having the aforementioned biologically equivalent activity, the nucleic acid molecules used in the present invention are interpreted to include sequences that exhibit substantial identity with the sequences listed in the sequence list. By using an mRNA molecule in which the 5'UTR and 3'UTR of the present invention or a fragment thereof are bound to the coding region of an mRNA sequence capable of translating a protein to be introduced into a subject, the translation level of the protein can be significantly enhanced.

[0044] The coding region of the present invention may include a nucleotide sequence encoding a KRAS immunogenic peptide.

[0045] In the present invention, "immunogenic peptide" is a composition capable of inducing an immune response in animals, including humans, when administered to said animals.

[0046] Immunogenic peptides are peptides that contain antigens and can induce an immune response. The induced immune response can be a cellular (T-cell mediated) or humoral (B-cell mediated, antibody production) immune response. Immunogenic peptides can induce, for example, both cellular and humoral immune responses.

[0047] Cellular immune response is CD8 + T lymphocyte-mediated response (i.e., cytotoxic response), or CD4 + It may be a T lymphocyte-mediated response (i.e., a helper response). It may also be a combination of cytotoxic and helper cell immune responses. The helper response may involve Th1, Th2, or Th17 lymphocytes (such lymphocytes may induce different cytokine responses as known in the art). CD8 + T cells are stimulated and become active by binding to cells expressing MHC class I via immunogenic peptides, and CD4 + T cells are stimulated and activated by binding to antigen-presenting cells expressing MHC class II via immunogenic peptides. The immunogenic peptide-MHC class II complex is presented on the surface of antigen-presenting cells (APCs).

[0048] In this specification, “immunogenic peptide,” “epitope (antigenic determinant),” and “antigenic determinant peptide” are used interchangeably.

[0049] KRAS (Kirsten rat sarcoma virus, hereinafter referred to as KRAS) is a protein composed of 189 amino acid residues. Mutations in the KRAS gene are mainly reported as single mutations in which the 12th amino acid changes to aspartic acid (G12D), valine (G12V), cysteine ​​(G12C), arginine (G12R), or aspartic acid (G13D), while only a few cases of multiple mutations have been reported.

[0050] The coding region of the present invention may include a nucleotide sequence encoding the KRAS immunogenic peptide of SEQ ID NO. 1.

[0051] In addition, the nucleotide sequence encoding the KRAS immunogenic peptide of SEQ ID NO. 1 may further include a nucleotide sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs. 2 to 6.

[0052] Specific sequence information for sequence numbers 1 to 6 is as follows.

[0053] non-mutant epitope: TKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIR (Sequence No. 1)

[0054] The peptide sequence of SEQ ID NO. 1 according to the present invention may be an amino acid sequence located at positions 127–164 based on the full length sequence of KRAS (SEQ ID NO. 22).

[0055] G12C mutant epitope: KLVVVGACGVGKSAL (Sequence No. 2)

[0056] G12D mutant epitope: KLVVVGADGVGKSAL (Sequence No. 3)

[0057] G12V mutant epitope: KLVVVGAVGVGKSAL (Sequence No. 4)

[0058] G12R mutant epitope: KLVVVGARGVGKSAL (Sequence No. 5)

[0059] G13D mutant epitope: KLVVVGAGDVGKSAL (Sequence No. 6)

[0060] The peptide sequences of SEQ ID NOs 2 to 6 according to the present invention may be amino acid sequences located at positions 5 through 19 based on the full length sequence of KRAS (SEQ ID NO 22).

[0061] The coding region of the present invention is included within the scope of the present invention if it is any nucleic acid (DNA or RNA) sequence encoding the aforementioned amino acid.

[0062] According to one example, the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 may be any one nucleotide sequence selected from the group consisting of SEQ ID NOs 11 to 14. This nucleotide sequence corresponds to different nucleotide sequences that translate the same epitope through codon optimization for producing mRNA suitable for anticancer vaccines.

[0063] ACCAAGCAGGCACAGGATCTGGCCAGATCCTATGGTATCCCATTTATCGAGACCAGCGCCAAGACCAGACAGAGAGTGGAGGACGCTTTCTACACCCTGGTCAGGGAGATCAGG (SEQ ID NO: 11)

[0064] ACCAAACAGGGCCCAGGACCTGGCCAGATCCTATGGAATCCCTTTCATCGAGACCTCAGCCAAGACCAGACAGCGGGTGGAAGACGCCTTCTACACACTGGTGCGTGAGATTCGG (SEQ ID NO: 12)

[0065] ACCAAGCAGGCCAGGACCTGGCCAGATCTTATGGCATCCCATTTATCGAGACCAGTGCCAAGACCAGGCAGAGGGTGGAAGACGCCTTTTACACCCTGGTGAGAGAGATCAGG (SEQ ID NO: 13)

[0066] ACAAAACAGGCCCAGGACCTGGGCCCGATCTTACGGCATCCCATTTATAGAGACCTCTGCCAAAACACGGCAGAGGGTGGAGGATGCATTTTACACCCTGGTCAGAGAAATCAGA (SEQ ID NO: 14)

[0067] In the nucleic acid molecule mentioned in the present invention, the coding region operably connected to the expression regulatory element may include at least one copy of the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1, thereby increasing the production amount of the KRAS immunogenic peptide of SEQ ID NO. 1. Specifically, the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 may include 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies or more of repetitions.

[0068] More specifically, the coding region may include one or more nucleotide sequences selected from SEQ ID NOs 11 to 14 repeated in 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies, 10 copies or more.

[0069] According to one embodiment of the present invention, the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 mentioned above may be repeated three times. According to a specific embodiment of the present invention, the sequence may be the nucleotide sequence of SEQ ID NO. 11 repeated three times.

[0070] The base sequence encoding the peptide of the repeating sequence number 1 and / or the base sequence encoding the peptides of sequence numbers 2 to 6 may be connected by any linker.

[0071] The linker applied to the nucleic acid molecule may be any known linker and is not limited thereto, but the linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include hydrophilic amino acids such as glycine and / or serine, but is not limited thereto. Specifically, the linker may be a self-cleavage 2A linker known in expired published patents and papers, e.g., a P2A linker, a T2A linker, an F2A linker, or an E2A linker, and may be a (GS)n, (GGS)n, (GSGGS)n, or (GnS)m linker (where n and m are each 1 to 10). Preferably, it may be a P2A linker or a T2A linker.

[0072] P2A linker: ATNFSLLKQAGDVEENPGP (Sequence No. 7)

[0073] T2A linker: EGRGSLLTCGDVEENPGP (Sequence No. 8)

[0074] The nucleotide sequences for each of these P2A linkers or T2A linkers are shown in sequence numbers 23 to 29.

[0075] In addition, the coding region of the present invention may further include one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 15 to 19.

[0076] AAGCTGGTGGTGGTTGGGGCCTGCGGCGTGGGCAAGTCCGCCCTG (SEQ ID NO: 15)

[0077] Specifically, the above sequence number 15 may be a nucleotide sequence encoding a G12C mutant epitope (sequence number 2).

[0078] AAGTTGGTGGTGGTGGGTGCCGACGGAGTGGGCAAGTCAGCCCTG (SEQ ID NO: 16)

[0079] The sequence of SEQ ID NO. 16 above may be a nucleotide sequence encoding a G12D mutant epitope (SEQ ID NO. 3).

[0080] AAACTGGTGGTGGTGGCGCCGTCGGCGTGGGAAAGTCAGCTCTG (SEQ ID NO: 17)

[0081] The sequence of SEQ ID NO. 17 above may be a nucleotide sequence encoding a G12V mutant epitope (SEQ ID NO. 4).

[0082] AAGCTGTGGTGGTGGGAGCCAGGGGAGTGGGTAAGTCTGCACTC (SEQ ID NO: 18)

[0083] The sequence of SEQ ID NO 18 above may be a nucleotide sequence encoding a G12R mutant epitope (SEQ ID NO 5).

[0084] AAACTGGTGGTGGTGGGAGCCGGCGACGTGGGTAAGTCAGCTCTG (SEQ ID NO: 19)

[0085] The sequence of SEQ ID NO 19 above may be a nucleotide sequence encoding a G13D mutant epitope (SEQ ID NO 6).

[0086] Each of the above base sequences may be connected by the previously mentioned linker.

[0087] According to one embodiment of the present invention, an expression regulatory element having expression initiation activity; and

[0088] A coding region operably connected to the above-mentioned expression regulatory element; comprising,

[0089] The above coding region may be a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 and a nucleotide sequence encoding one or more peptide sequences selected from the group consisting of SEQ ID NOs 2 to 6.

[0090] The present invention provides a nucleic acid molecule comprising at least one copy of a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 (including a specific mRNA sequence thereof mentioned in the present specification) and a nucleotide sequence encoding any one or more peptide sequences selected from the group consisting of SEQ ID NOs. 2 to 6 (including a specific mRNA sequence thereof mentioned in the present specification), wherein any combination of the sequences mentioned above falls within the scope of the present invention.

[0091] According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention may include a base sequence encoding the peptide sequence of SEQ ID NO. 1 in three repetitions and may include a P2A / T2A cleavage sequence connecting the base sequences. Specifically, the nucleic acid molecule may have the sequence of SEQ ID NO. 20.

[0092] According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention may comprise a base sequence encoding the peptide sequence of SEQ ID NO. 1, a base sequence encoding the peptide sequences of SEQ ID NOs. 2 to 6, and a P2A / T2A cleavage sequence connecting the respective base sequences. Specifically, the nucleic acid molecule may have the sequence of SEQ ID NO. 21.

[0093] In the present invention, by using a nucleic acid molecule containing a sequence encoding the peptide of SEQ ID NO. 1, which is a non-mutant epitope, the advantages of a universal KRAS epitope that does not have a specific dependence on a specific mutant KRAS are obtained.

[0094] In addition, by further including nucleic acid molecules encoding peptides of KRAS variant antigen regions such as G12C, G12D, G12V, G12R, and G13D, it has the advantage of an epitope that targets cancer-specific mutant KRAS.

[0095] (1) an expression regulatory element having expression initiation activity; and

[0096] A coding region operably connected to the above-mentioned expression regulatory element; comprising,

[0097] A nucleic acid molecule wherein the above coding region comprises a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1, and

[0098] (2) an expression regulatory element having expression initiation activity; and

[0099] A coding region operably connected to the above-mentioned expression regulatory element; comprising,

[0100] The present invention also provides a combination of nucleic acid molecules in which the coding region comprises a base sequence encoding one or more peptide sequences selected from the group consisting of SEQ ID NOs 2 to 6.

[0101] The nucleic acid molecule according to the present invention preferably has an RNA form. Accordingly, it may have superior effects compared to using a nucleic acid molecule in the form of DNA. In particular, it is excellent for use in in vitro transcription (IVT) mRNA expression systems.

[0102] First, unlike DNA, RNA-based nucleic acid molecules do not need to enter the host cell nucleus for transcription into mRNA due to their inherent characteristics. Since mRNA molecules can synthesize desired peptides or proteins within the cytoplasm, they are more advantageous to the human body compared to DNA-based nucleic acid molecules. Specifically, mRNA molecules have no possibility of intruding into the host chromosomes from the nucleus; antibiotic resistance genes, which serve as selection markers for selective production in host cells, are unnecessary for the production of mRNA; and because mRNA has a shorter half-life than DNA, it does not induce persistence genetic transformation. In other words, while general-form nucleic acid molecules are delivered into cells, activated for only a short period to express target peptides / proteins, and destroyed by enzymatic reactions within a few days, the specific immune response to the initially expressed target peptides / proteins remains.

[0103] Second, nucleic acid molecules in the form of mRNA can induce a desired in vivo immune response even when using a relatively smaller amount compared to nucleic acid molecules in the form of DNA. As mentioned above, unlike nucleic acid molecules in the form of DNA, nucleic acid molecules in the form of mRNA do not need to enter the nucleus and only need to pass through the cell membrane. Therefore, by using nucleic acid molecules in the form of mRNA, it is possible to express target peptides / proteins that exhibit the same efficacy even when using a smaller amount than nucleic acid molecules in the form of DNA.

[0104] Third, because all production processes can be artificially controlled, vaccines can be safely produced in small-scale GMP (good manufacturing practice) facilities without the risk of biological contamination. In other words, nucleic acid molecules in the form of mRNA require only facilities at the level of a small-scale GMP laboratory, and since there is no need to handle direct infectious agents (viruses or pathogenic microorganisms), a wide variety of vaccines can be manufactured and produced rapidly.

[0105] Fourth, nucleic acid molecules in the form of mRNA can induce a stronger immune response compared to naked DNA nucleic acid molecules. This is thought to be due to the mutual cooperation between the innate immune response induced by the stem-loop structure of mRNA nucleic acid molecules and the inherent characteristics of mRNA, and the adaptive immune response induced by target peptides / proteins expressed in immunized host cells.

[0106] Fifth, mRNA nucleic acid molecules are easy to produce. As mentioned above, large quantities of mRNA nucleic acid molecules can be produced using only in vitro transcription (IVT). Recently, reagents related to the IVT reaction, particularly DNA-dependent RNA polymerase, have been improved, allowing for the rapid production of large quantities of mRNA within 1 to 2 weeks using a small amount of DNA template. Furthermore, multiple antigens intended to induce an immune response can be produced simultaneously, mixed, and then immunized. Since there are no specific restrictions on the gene length of the antigens to be expressed, the applicability and convenience of producing RNA nucleic acid molecules can be increased.

[0107] In cases where mRNA-based nucleic acid molecules are utilized, an appropriate transcriptional regulatory element (TCCE) enabling IVT can be located upstream of the upstream translational regulatory element (U-TLCE). Since mRNA-based nucleic acid molecules can be synthesized through the IVT process, there is no need to directly handle live viruses or pathogenic microorganisms used in the production of conventional live or inactivated vaccines, nor is the cultivation of host cells—such as yeast, E. coli, or insect cells—essential for producing recombinant proteins or peptides, unnecessary.

[0108] The present invention provides a recombinant expression vector comprising the above nucleic acid molecule.

[0109] In the present invention, the term "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferable to use a plasmid vector.

[0110] That is, vectors that can be used as gene carriers can be produced in various forms and include viral vectors, DNA or mRNA expression vectors, plasmids, cosmids or phage vectors, DNA or mRNA expression vectors linked with CCAs (cationic condensing agents), for example, packaged DNA or mRNA expression vectors packaged in liposomes or niosomes containing plasmids, and specific eukaryotic cells such as producer cells.

[0111] One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector into which additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episosomal mammalian vectors) can be incorporated into the host cell's genome upon introduction, thereby replicating along with the host's genome. Additionally, expression vectors useful for recombinant DNA technology often exist in the form of plasmids.

[0112] The nucleic acid molecules of the present invention can be formulated to enter and be expressed within mammalian cells. Such compositions may be particularly useful for use in the treatment of diseases. There are many methods for expressing nucleic acid molecules within host cells, and any suitable method may be used. For example, the nucleic acid molecules according to the present invention may be applied to any expression construct or gene delivery system.

[0113] For example, nucleic acid molecules can be inserted into host cells using a viral gene delivery system. Viral vectors into which nucleic acid molecules can be inserted may include vectors derived from adenoviruses, adeno-associated viruses (AAVs), retroviruses, vaccinia or other poxviruses (e.g., avian pox virus), lentiviruses, and herpes simplex virus. For example, viral vectors include, but are not limited to, vectors derived from lentiviruses such as human immunodeficiency virus (HIV) and monkey immunodeficiency virus (SIV); and retrovirus vectors derived from murine retroviruses, gibbon ape leukemia virus, AAVs (adeno-associated viruses), and adenoviruses. In addition, retroviral vectors derived from rodent leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, etc., are widely used. The vector system according to the present invention can be constructed through various methods known in the art.

[0114] A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication origin that enables efficient replication to include hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that allows host cells transformed with the plasmid vector to be selected, and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to conventional methods.

[0115] In the present invention, the term "recombinant expression vector" typically refers to a recombinant carrier into which a fragment of heterogeneous DNA is inserted, generally meaning a fragment of double-stranded DNA. Here, heterogeneous DNA refers to atypical DNA that is not naturally found in host cells. Once inside a host cell, the recombinant vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterogeneous) DNA can be produced.

[0116] As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be activatorily linked to transcription and translation expression regulatory sequences that are functional within the selected expression host.

[0117] The present invention provides an in vitro transcription (IVT) mRNA comprising the above nucleic acid molecule.

[0118] In the present invention, "in vitro transcription (IVT)" refers to DNA-dependent mRNA synthesis outside of a cell and inside a test tube. Template DNA may be linearized with a suitable restriction enzyme prior to in vitro transcription. Reagents used for mRNA in vitro transcription are typically polymerases such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); and rNTPs (ribonucleotide triphosphates) for four bases (adenine, cytosine, guanine, and uracil); optionally may include, but are not limited to, cap analogs; modified nucleotides; RNase inhibitors, etc.

[0119] The present invention provides a vaccine composition for treating cancer comprising mRNA expressed from the vector or in vitro transcription (IVT) mRNA.

[0120] In the present invention, "cancer" refers to a disease in which abnormally changed cells proliferate rapidly and uncontrollably due to changes in the genes of cells caused by various reasons. Cancer cells can spread to various parts of the body through the bloodstream and lymphatic system.

[0121] The above cancer may be any one selected from the group consisting of brain cancer, neurocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, biliary tract cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head cancer, cervical cancer, head and neck cancer, skin cancer, kidney cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, ovarian mucinous cancer, rectal cancer, prostatic cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. It may be, preferably, pancreatic cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, endometrial carcinoma, gastric cancer, ovarian mucinous cancer, cervical cancer, head and neck cancer, kidney cancer, or skin cancer.

[0122] Specifically, the cancer may be a cancer expressing KRAS. For example, the cancer may have one or more cancer cells expressing mutant KRASG12C, G12D, G12V, G12R, and / or G13D proteins. Any of the aforementioned cancer types having such mutations are included in the cancer types mentioned in the present invention.

[0123] In this invention, the term "treatment" refers to any act of improving or beneficially altering the symptoms of cancer through the administration of a composition according to this invention.

[0124] The above mRNA may be encapsulated in lipid nanoparticles. mRNA encapsulated in LNPs has been commercialized as a COVID-19 vaccine after receiving FDA approval, and in addition to preventive vaccines, mRNA-LNPs are being researched and clinically evaluated in the fields of protein replacement therapy and antibodies as a protein expression platform.

[0125] In the present invention, lipid nanoparticles (LNPs) can protect nucleic acid molecules and enhance bio-injection activity. Lipid nanoparticles comprise a plurality of lipid molecules physically associated with one another, including microspheres (including monolayer and multilayer vesicles, e.g., liposomes), phases dispersed in emulsions, micelles, or internal phases of suspensions. Lipid nanoparticles can be used to encapsulate nucleic acid molecules of the present invention or peptides (proteins) expressed from nucleic acid molecules for delivery.

[0126] The above lipid nanoparticles may include cationic lipids, and formulations containing cationic lipids are useful for delivering polyvalent anions such as nucleic acid molecules. Other lipids that may be included include neutral lipids (i.e., uncharged or cationic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time the nanoparticles can exist in vivo.

[0127] For example, the lipids for encapsulation may be cationic lipids or biodegradable lipids. For example, these lipids include (9Z,12Z)-3((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca9,12-dienoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octan-8,1-diyl)bis(decanoate), 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), Examples include 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate, heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (also known as Dlin-MC3-DMA (MC3)).

[0128] Suitable neutral lipids may include neutral, non-charged, or amphoteric lipids. Neutral phospholipids include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), nanphosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), It may include, but is not limited to, 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-diecosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidyl ethanolamine (DOPE).

[0129] Helper lipids can enhance transfection and / or enhance membrane fusogenicity. Helper lipids include steroids, sterols, and alkyl resorcinols. Specifically, helper lipids may include cholesterol, 5-heptadecyl resorcinol, and cholesterol hemisuccinate.

[0130] Stealth lipids can assist the formulation process or control the pharmacokinetic properties of LNPs by reducing particle aggregation and controlling particle size. For example, stealth lipids may include polymers having hydrophilic heads such as PEG (polyethylene glycol or polyethylene oxide), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and polyN-(2-hydroxypropyl)methacrylamide. Specifically, stealth lipids may include, but are not limited to, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), etc.

[0131] Therapeutic antibodies can destroy viruses by targeting specific antigens on the surface of the virus or by engaging the immune system to eliminate infected cells. Due to these characteristics, therapeutic antibodies exhibit significant antiviral activity and excellent specificity.

[0132] Recently, mRNA technology has achieved great success, particularly in vaccine development. Unlike conventional protein-based therapies, mRNA therapies have the advantage of rapid development and production. Additionally, synthesis and purification can be simplified without the need for cell culture procedures. Complexity and associated costs are drastically reduced, significantly decreasing the time and resources required for mass production and leading to a dramatic reduction in overall development costs.

[0133] According to one embodiment, a cancer treatment vaccine composition comprising mRNA of the present invention is administered in an LNP-encapsulated form and exhibits a high tumor growth inhibition rate, thereby having excellent tumor treatment, prevention, or recurrence inhibition effects.

[0134] Specifically, the mRNA vaccine of the KRAS immunogenic non-mutant epitope peptide of the present invention can effectively inhibit tumor growth and exhibit excellent efficacy in treating tumors. In addition, when an mRNA vaccine (e.g., including a separate mRNA form or a form merged into a single mRNA) further comprising an immunogenic epitope peptide containing a KRAS mutation site in the peptide is administered, the tumor treatment effect can be further maximized.

[0135] The cancer treatment vaccine composition of the present invention comprises a pharmaceutically effective amount of the aforementioned nucleic acid molecule and, if necessary, may comprise a pharmaceutically acceptable carrier. In this case, "pharmaceutical effective amount" means an amount sufficient to achieve the efficacy or activity of the nucleic acid molecule according to the present invention.

[0136] The composition according to the present invention may include a carrier, a diluent, and / or an excipient. Examples of the excipient include aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, etc. The carrier may be a pharmaceutically acceptable carrier, and examples of the pharmaceutically acceptable carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc.

[0137] The nucleic acid molecule, which is the active ingredient of the composition according to the present invention, can be formulated by mixing it with a physiologically acceptable carrier—that is, a carrier that is non-toxic to the recipient at the dosage and concentration used in the form of herbal medicine administration—at ambient temperature and an appropriate pH to a desired degree of purity. The pH of the formulation depends mainly on the specific use and concentration of the compound, but is preferably in the range of about 3 to about 8. In another embodiment, the compound is sterilized. The compound may be stored, for example, as a solid or amorphous composition, a lyophilized formulation, or an aqueous solution.

[0138] Additionally, the nucleic acid molecule may be stabilized in the vaccine composition using a cationic polymer, a cationic peptide, or a cationic polypeptide. The cationic (poly)peptide that can be used as a stabilizer may be a multiple cationic polymer such as polylysine or polyarginine, a cationic lipid, or a lipofectant. More specifically, the stabilizer may be histone, nucleolin, protamine, oligofectamine, spermine or spermidine, and cationic polysaccharides, in particular chitosan, TDM, MDP, muramyl dipeptide, Pluronic, and / or derivatives thereof. Histones and protamines are cationic proteins that naturally compact DNA.

[0139] For example, histones capable of forming complexes with nucleic acid molecules include histones H1, H2a, H3, and H4, and protamines include protamine P1 or P2, in particular, the cationic subsequence of protamine is preferred.

[0140] If necessary, the above composition can be used to produce a sustained-release formulation. Suitable examples of sustained-release formulations include a semipermeable matrix of a solid hydrophobic polymer containing nucleic acid molecules or gene delivery vehicles, and this matrix is ​​in the form of a molded article, e.g., a film or a microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid.

[0141] The composition according to the present invention may further include preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. The preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B, etc. The vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may include a protective agent, and protective agents known in the art may be used without limitation, which may include, but are not limited to, lactose (LPGG) or trehalose (TPGG).

[0142] The vaccine composition according to the present invention may appropriately further include, if necessary depending on the administration method or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, an antioxidant, etc.

[0143] The above composition may be administered to a subject in a therapeutically effective amount. In this case, "therapeutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the individual, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The dosage of the vaccine composition of the present invention is in the range of 0.0001 to 1,000 mg / kg (body weight) per day, but may vary depending on the type of individual and is not limited thereto. Meanwhile, the dosage may be appropriately adjusted according to the patient's age, sex, and condition.

[0144] The term "subject" of the present invention includes animals or humans whose symptoms may be improved by the administration of the composition according to the present invention.

[0145] The term "administration" in the present invention means introducing a specific substance into a human or animal by any appropriate method, and said administration may be performed via routes such as intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation, oral, intralymph nodes, and tumor tissues.

[0146] The present invention provides a method for preventing, suppressing recurrence of, or treating cancer, comprising the step of administering a nucleic acid molecule containing a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 mentioned above, a vector containing the same, or IVT mRNA to an individual in need thereof.

[0147] In addition, the present invention provides a method for preventing, suppressing, or treating cancer, comprising the step of administering a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 and a nucleotide sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs. 2 to 6, a vector containing the same, or an IVT mRNA to an individual in need thereof.

[0148] In the present invention, "prevention" refers to any act of suppressing a disease or delaying its onset through the administration of a composition. In the present invention, "suppression of recurrence" refers to any act of reducing the frequency or likelihood of the disease recurring after primary treatment, or extending the period until recurrence, through the administration of a composition. In the present invention, "treatment" refers to any act of improving or beneficially altering the symptoms of the disease through the administration of a composition. In the present invention, "improvement" refers to any act of at least reducing parameters related to the treated state, such as the severity of symptoms.

[0149] That is, the present invention provides a method for increasing the production of KRAS immunogenic peptide in an individual to treat, prevent, alleviate, or suppress recurrence of cancer in an individual. The method comprises the step of administering to an individual requiring this a therapeutically effective amount of a nucleic acid molecule containing a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1, a vector containing the same, or IVT mRNA.

[0150] Alternatively, the nucleic acid molecule according to the present invention, the vector containing it, or the IVT mRNA may be used in addition to or in combination with known treatments for treating, preventing, alleviating, or suppressing the recurrence of a disease.

[0151] The present invention provides a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 for use in the prevention, recurrence suppression, or treatment of cancer, a vector comprising the same, or an IVT mRNA.

[0152] In addition, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 and further comprising a nucleotide sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs. 2 to 6, a vector comprising the same, or a composition comprising an IVT mRNA.

[0153] The present invention provides a use of a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1, a vector comprising the same, or an IVT mRNA in the manufacture of a drug for the treatment of cancer.

[0154] In addition, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 and further comprising a nucleotide sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs. 2 to 6, a vector comprising the same, or an IVT mRNA for use.

[0155] All characteristics described in this application (including all attached claims, abstract, and drawings) and / or all steps of all methods or processes disclosed herein may be combined with any of the above-mentioned embodiments in any combination, except for mutually exclusive combinations of at least some of these characteristics and / or steps.

[0156] The present invention provides an mRNA vaccine encoding a KRAS immunogenic non-mutant epitope peptide, which can exhibit excellent tumor treatment and prevention of recurrence of the same cancer against cancers expressing KRAS.

[0157] Figure 1 shows the tumor growth inhibitory effect confirmed by administering a KRAS immunogenic mRNA vaccine (AST-11X) to a KRAS mutant syngeneic cancer mouse model.

[0158] Figure 2 is a figure showing the process of administering KRAS immunogenic mRNA vaccines (AST-11X and AST-1138) to a KRAS mutant syngeneic cancer mouse model.

[0159] Figure 3 shows the results of administering KRAS immunogenic mRNA vaccines (AST-11X and AST-1138) to a KRAS mutant syngeneic cancer mouse model and confirming the tumor growth inhibitory effect.

[0160] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0161] Experimental Example 1. Design of KRAS immunogenic mRNA vaccine

[0162] To design the mRNA vaccine of the present invention, E. coli was cultured to produce plasmid DNA (pDNA), and the pDNA was purified using a TFF (Tangential Flow Ultrafiltration / Diafiltration) system and FPLC (Fast Protein Liquid Chromatography) after undergoing an appropriate pretreatment process. The purified pDNA was linearized using restriction enzymes, and then buffer exchange and purification were performed again through the TFF process to be used as a template suitable for in vitro transcription (IVT).

[0163] Using the above linearized DNA as a template, an IVT reaction was performed using a reaction mixture containing rNTPs (ribonucleotide triphosphates, ATP, CTP, GTP, UTP), T7 RNA polymerase, capping enzyme, etc. After the reaction was completed, the generated mRNA was purified using an oligo dT column. In addition, high-purity mRNA was obtained by performing buffer exchange and impurity removal using TFF.

[0164] The mRNA (AST-11X mRNA) for the epitope peptide of SEQ ID NO. 1 described in the above process, and the epitope peptide containing the epitope peptide of SEQ ID NO. 1 and KRAS mutation sites (G12C, G12D, G12V, G12R, G13D) and the mRNA sequences thereof are listed in Tables 1 and 2 below.

[0165]

[0166] * The P2A / T2A cleavage sequence is underlined, and the KRAS mutation site is in bold. In addition, the nucleotide sequences for the expression of the above epitopes and the nucleotide sequences of the mRNA are shown in Table 2.

[0167]

[0168] * Kozak sequences are italicized, P2A / T2A cleavage sequences are underlined, translation stop codons are italicized and underlined, and base sequences coding for epitopes are in bold.

[0169] To encapsulate each mRNA into lipid nanoparticles (LNPs), all lipid components, including cholesterol, PEG lipids, phospholipids (DSPC), and ionic lipids, were dissolved using a mixed solvent of ethanol. Based on published patents and papers, ALC-0315, an ionic lipid used by Pfizer and BioNTech in COVID-19 mRNA vaccines, was used for the composition of the lipid nanoparticles. The molar ratio of the composition was 46.3% ALC-0315, 9.4% phospholipids (DSPC), 42.7% cholesterol, and 1.6% PEG lipids. After mixing the above lipid mixture with the mRNA, LNP-encapsulated mRNA was prepared by injecting it into a nanoparticle formulation system. The LNP-encapsulated mRNA, prepared through pH neutralization and purification processes, was concentrated using a Tangential Flow Ultrafiltration (TFF) process.

[0170] Experimental Example 2. Cell and Material Information

[0171] (1) Cells

[0172] Specific information on the cell lines used in this experiment is shown in Table 3.

[0173] Item Details Cell Name: LL / 2 (LLC1) Manufacturer: ATCC Supplier: Coram Biogen Co., Ltd. ATCC No.: CRL-1642 Lot No.: 70040227 Format: Adherent, round Disease: Lewis Lung Carcinoma

[0174] Various media and reagents were used to culture the aforementioned cells and perform related experiments. Specifically, DMEM (Dulbecco's Modified Eagle Medium) (Cytiva, Cat No. SH30243.01) was used as the basic medium and served as the basal medium for cell culture. Additionally, FBS (fetal bovine serum) (Gibco, Cat No. 16000044) was added to promote cell growth. Penicillin / streptomycin (Hyclone, Cat No. SV30010) was used as an antibiotic to prevent cell contamination, and trypsin-EDTA (Hyclone, Cat No. SH30236.01) was used for cell subculture and isolation. PBS (phosphate buffer saline) (Hyclone, Cat No. SH30256.FS) was used for cell washing and reagent dilution.

[0175] In addition, Miltenyi Biotec’s MACS cell storage solution (Cat No. 130-130-263) was used for cell storage, and Matrigel Matrix (CORNING, Cat No. 354248) was used to induce cell adhesion.

[0176] Meanwhile, RPMI 1640 Medium (Hyclone, Cat No. SH30255.01) was used for the composition of the medium for mouse T cell culture.

[0177] (2) Materials

[0178] The AST-11X mRNA used as the administered substance in this experiment was provided in the form of a white to off-white suspension, with an RNA content of 0.2 mg / mL. Additionally, the mRNA was encapsulated in lipid nanoparticles, and the encapsulation efficiency was confirmed to be 89.6%. Particle characterization results showed a Z-average of 83.3 nm and a polydispersity index (PDI) of 0.072, indicating a uniform particle distribution. The AST-11X mRNA preparation was stored at -80 ℃.

[0179] In addition, phosphate buffered saline (PBS) with a pH of 7.4 was used for the dilution and administration of the mRNA preparation. The PBS was a colorless, transparent liquid and was stored under refrigerated conditions.

[0180] Example 1. Confirmation of the efficacy of the KRAS immunogenic mRNA vaccine (AST-11X) in a Syngeneic lung cancer mouse model

[0181] (1) Experimental method

[0182] 1) Preparation of a Syngeneic lung cancer mouse model

[0183] To prepare the Syngeneic lung cancer mouse model, 5-week-old C57BL / 6J mice (CLEA Japan, Inc. / Saeron Bio Co., Ltd.) with a body weight of 13.48 to 16.40 g were used, and all mice were acclimatized for at least 3 to 7 days prior to the start of the experiment.

[0184] LLC-1 cells, a mouse-derived lung cancer cell line containing the G12C mutation, were used for tumor induction. Specifically, 5 × 10⁶ cells per individual 6 Cells were suspended in 100 μL of culture medium, mixed with Matrigel in a 1:1 ratio, and then administered subcutaneously to the right flank of a mouse.

[0185] Subsequently, among individuals that reached approximately 7 weeks of age (body weight 15.79 to 19.81 g), the average tumor size was approximately 150 mm 3 Mice that had reached the required level were selected and used in the experiment. The selected mice were divided into two groups of eight, and each group was configured as follows.

[0186] Group 1 (G1): PBS

[0187] Group 2 (G2): AST-11X mRNA

[0188] The above AST-11X mRNA was set at a dose of 20 μg / head per animal and administered intramuscularly (IM). The administration was repeated a total of 6 times over days 0, 4, 8, 11, 15, and 21 after tumor formation.

[0189] General symptoms of all mice were observed once a day during the experimental period. In addition, to evaluate tumor size and body weight changes, the 9th day after tumor transplantation was set as the reference (Day 0), and individual tumor sizes were measured on days 0, 3, 6, 10, 13, 17, 20, and 25. Tumor size was determined by the tumor volume (mm²) of each individual. 3 ) was calculated and evaluated, and the tumor growth inhibition rate of each test group was calculated by comparing it with the control group.

[0190] 2) Experiment Termination and Sample Separation

[0191] All experiments were terminated on the 25th day from the start of drug administration. After removing tumor tissue from each mouse, tumor-infiltrating leukocytes were isolated, suspended in a cell banker (amsbio), and then frozen.

[0192] In addition, the spleen was excised from each mouse, and total splenocytes were isolated after removing red blood cells and suspended in a cell preservative for frozen storage.

[0193] Autopsy was performed immediately on individuals that died during the experiment. For surviving individuals, blood was drawn from the abdominal aorta under Isoflurane (Hana Pharmaceutical, I-Fran Solution) anesthesia, followed by lethal hemorrhage. Subsequently, a gross examination was performed on the external surface and all orifices, the cranial cavity, thoracic and abdominal cavities, and their internal organs. If abnormalities were identified in an organ, histopathological examination was performed after fixation in 10% neutral buffered formalin, as necessary.

[0194] 3) Statistical analysis

[0195] Data such as body weight, tumor volume, and tumor weight measured after the experiment were analyzed using the SPSS statistical program.

[0196] First, Levene's test was conducted to evaluate the homogeneity of variance among the groups. Subsequently, one-way ANOVA was performed to confirm statistical significance between the test groups.

[0197] In cases where no significance was confirmed between test groups in the above one-way ANOVA analysis, no additional post-hoc analysis was performed. Conversely, when significance was confirmed between test groups, post-hoc tests were conducted based on the homogeneity of variances. Specifically, Scheffe's multiple comparison test was applied when the variances were homogeneous, while Dunnett's T3 test was applied when the variances were unequal.

[0198] (2) Confirmation of antitumor effect of KRAS immunogenic mRNA vaccine (AST-11X)

[0199] The tumor growth inhibition rate (TGI) of each group was compared and analyzed from the start of drug administration until the end of the experiment.

[0200] In a Syngeneic lung cancer (LL / 2) mouse model, the AST-11X mRNA monotherapy group showed a significant tumor growth inhibitory effect compared to the negative control group, PBS. Specifically, a maximum tumor growth inhibition rate of 62.2% was confirmed on day 13 after drug administration, and a very high tumor growth inhibition rate of 59.1% was confirmed even at the end of the experiment (Fig. 1).

[0201] That is, it was confirmed that the mRNA vaccine of the KRAS immunogenic non-mutant epitope peptide (AST-11X) of the present invention is effective in treating, preventing, or suppressing the recurrence of tumors.

[0202] Example 2. Confirmation of the efficacy of KRAS immunogenic mRNA vaccines (AST-11X and AST-1138) in a Syngeneic lung cancer mouse model

[0203] (1) Preparation of a KRAS mutant syngeneic cancer mouse model

[0204] To construct a KRAS-mutated syngeneic cancer mouse model, 7-week-old mice weighing 19–21 g were purchased and acclimatized for at least 3–7 days. The mouse-derived lung cancer cell line LL / 2 (LLC1), containing the G12C mutation, was used in the experiments. Specifically, 5 x 10⁶ per individual 5 Canine cells were suspended in 50 µl of serum-free culture medium and injected subcutaneously into the area between the left scapula and the chest wall of the mouse (Fig. 2). On day 9 after transplanting the LL / 2 cells in this manner, the tumor size was approximately 70 mm 3 Twenty-four mice that reached [the target] were selected and divided into three groups of eight, and each group was configured as follows.

[0205] Group 1 (G1): Vehicle (Saline)

[0206] Group 2 (G2): AST-11X mRNA + LNP

[0207] Group 3 (G3): AST-1138 mRNA + LNP

[0208] (2) Confirmation of antitumor effects of KRAS immunogenic mRNA vaccines (AST-11X and AST-1138)

[0209] Saline and the test substance for each mouse model group (G1 to G3) were administered intramuscularly a total of two times on days 0 and 7 (day 9 after transplanting LL / 2 cells was designated as day 0) based on a dose of 10 μg per mouse. Subsequently, tumor size was measured individually a total of five times (days 0, 3, 6, 10, and 13) from day 0 to day 13, and the tumor growth inhibition rate (%) of each test group was calculated by comparing it with the control group.

[0210]

[0211] The results of confirming the tumor growth inhibition rates of the control group and the test group according to the above calculation method are as shown in Table 4.

[0212] Group Treatment TGI rate (%) Maximum inhibition rate Last measurement inhibition rate G1 Vehicle (Saline) -- G2 AST-11X + LNP2 4.9 24.9 G3 AST-11 38 + LNP3 5.5 35.5

[0213] That is, it was confirmed that tumor growth was effectively inhibited when the mRNA vaccine of the epitope peptide discovered in the present invention (AST-11X mRNA, SEQ ID NO. 20) was administered alone after being encapsulated in LNP. Furthermore, when the mRNA vaccine of the epitope peptide discovered in the present invention and the epitope peptide containing a KRAS mutation site (AST-1138 mRNA, SEQ ID NO. 21) was administered after being encapsulated in LNP, an even more superior tumor growth inhibitory effect was observed (Fig. 3). Accordingly, it was confirmed that the mRNA vaccine of the KRAS immunogenic non-mutant epitope peptide (AST-11X) of the present invention is effective for the treatment, prevention, or inhibition of recurrence of tumors, and it was confirmed that the treatment, prevention, or inhibition of recurrence of tumors is further maximized when the mRNA vaccine of the form (AST-1138) combined with the immunogenic epitope peptide containing a KRAS mutation site is administered.

[0214] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An expression regulatory element having expression initiation activity; and A coding region operably connected to the above-mentioned expression regulatory element; comprising, A nucleic acid molecule wherein the above coding region comprises a base sequence encoding the peptide sequence of SEQ ID NO.

1.

2. The nucleic acid molecule according to claim 1, wherein the nucleotide sequence encoding the peptide sequence of SEQ ID NO. 1 is repeated at least one copy or more.

3. A nucleic acid molecule according to paragraph 2, wherein the base sequence encoding the repeating peptide sequence of SEQ ID NO. 1 is connected by a linker.

4. A nucleic acid molecule according to claim 1, wherein the coding region further comprises a base sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs 2 to 6.

5. A nucleic acid molecule according to paragraph 4, wherein each of the above base sequences is connected by a linker.

6. A nucleic acid molecule according to any one of claims 1 to 5, wherein the expression regulatory element comprises at least one transcription regulatory element and at least one translation regulatory element.

7. A nucleic acid molecule according to claim 6, wherein the translation control element comprises an upstream translation control element located upstream of the coding region or a downstream translation control element located downstream of the coding region.

8. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule further comprises at least one nucleotide sequence among a polyadenylation signal sequence or a polyadenosine sequence located downstream of the coding region.

9. A nucleic acid molecule according to any one of claims 1 to 8, wherein the nucleic acid molecule is RNA.

10. A recombinant expression vector comprising a nucleic acid molecule according to any one of claims 1 to 9.

11. In vitro transcription (IBT) mRNA comprising a nucleic acid molecule according to any one of claims 1 to 9.

12. A vaccine composition for treating cancer comprising mRNA according to claim 11.

13. A composition according to claim 12, wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

14. In paragraph 12, the above cancers are brain cancer, neurocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, biliary tract cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head cancer, cervical cancer, head and neck cancer, skin cancer, kidney cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, ovarian mucinous cancer, rectal cancer, proanal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary A composition, which is one selected from a group consisting of adenomas.

15. A composition according to claim 14, wherein the cancer is any one selected from the group consisting of pancreatic cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, endometrial carcinoma, gastric cancer, ovarian mucinous cancer, cervical cancer, head and neck cancer, kidney cancer and skin cancer.

16. A method for preventing, suppressing, or treating cancer, comprising the step of administering a therapeutically effective amount of a nucleic acid molecule containing a base sequence encoding the peptide sequence of SEQ ID NO. 1 to an individual in need thereof.

17. A method for preventing, inhibiting recurrence of, or treating cancer, wherein the nucleic acid molecule further comprises a base sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs 2 to 6.

18. In paragraph 16, the above cancers are brain cancer, neurocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, biliary tract cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head cancer, cervical cancer, head and neck cancer, skin cancer, kidney cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, ovarian mucinous cancer, rectal cancer, proanal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary A method for preventing, suppressing recurrence of, or treating cancer, selected from a group consisting of adenomas.

19. A method for preventing, suppressing recurrence of, or treating cancer, wherein the cancer is any one selected from the group consisting of pancreatic cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, endometrial carcinoma, gastric cancer, ovarian mucinous cancer, cervical cancer, head and neck cancer, kidney cancer, and skin cancer.

20. Use of a nucleic acid molecule comprising a base sequence encoding the peptide sequence of SEQ ID NO. 1 for manufacturing a medicine for the prevention, suppression of recurrence, or treatment of cancer.

21. The use according to claim 20, wherein the nucleic acid molecule further comprises a base sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs 2 to 6.

22. In paragraph 20, the above cancers are brain cancer, neurocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, biliary tract cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head cancer, cervical cancer, head and neck cancer, skin cancer, kidney cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, ovarian mucinous cancer, rectal cancer, proanal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary A use that is one of the selected from the group of Zen sects.

23. In paragraph 22, the above cancer is any one selected from the group consisting of pancreatic cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, endometrial carcinoma, gastric cancer, ovarian mucinous cancer, cervical cancer, head and neck cancer, kidney cancer and skin cancer.

24. A pharmaceutical composition comprising a nucleic acid molecule comprising a base sequence encoding the peptide sequence of SEQ ID NO. 1 for use in the prevention, inhibition of recurrence, or treatment of cancer.

25. A pharmaceutical composition according to claim 24, wherein the nucleic acid molecule further comprises a base sequence encoding one or more peptides selected from the group consisting of SEQ ID NOs 2 to 6.

26. In paragraph 24, the above cancers are brain cancer, neurocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, biliary tract cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, head cancer, cervical cancer, head and neck cancer, skin cancer, kidney cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, ovarian mucinous cancer, rectal cancer, proanal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary A pharmaceutical composition selected from a group consisting of adenomas.

27. A pharmaceutical composition according to claim 26, wherein the cancer is any one selected from the group consisting of pancreatic cancer, colorectal cancer, non-small cell lung cancer, biliary tract cancer, endometrial carcinoma, gastric cancer, ovarian mucinous cancer, cervical cancer, head and neck cancer, kidney cancer, and skin cancer.

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