Antigen peptides containing multiple KRAS mutant peptides linked together, nucleic acids encoding the same, and uses thereof

Abstract

The present invention provides an antigenic peptide to which multiple KRAS mutant peptides are linked, mRNA encoding the same, DNA encoding the same, an immunogenic composition comprising the antigenic peptide to which multiple KRAS mutant peptides are linked or mRNA encoding the same, and a method for inducing an immune response against the KRAS mutant peptide in an individual.

Description

[Technical Field]

[0001] The present invention relates to an antigen peptide in which multiple KRAS mutant peptides are linked, a nucleic acid encoding the same, and uses thereof. [Background technology]

[0002] Nucleic acid vaccines rely on antigens encoded by DNA or RNA. DNA vaccines generally contain an antigen-encoding gene inserted into a bacterial plasmid under the control of a eukaryotic promoter, while RNA vaccines use messenger RNA (mRNA) or other antigen-encoding RNA. Similar to protein vaccines, nucleic acid vaccines can be delivered via a variety of routes, such as intramuscular, subcutaneous, mucosal, or transdermal delivery.

[0003] DNA vaccines are known to induce less immune responses than peptide vaccines, cellular vaccines, viral vector vaccines, and RNA vaccines. Furthermore, DNA vaccines not only have low immunogenicity, but also have the potential to induce oncogenesis by being inserted into the host genome. Therefore, there is a demand for RNA-based vaccines. Summary of the Invention [Problem to be solved by the invention]

[0004] One aspect provides an antigenic peptide in which two or more KRAS mutant peptides selected from the group consisting of KRAS mutant peptides (variant peptides) are linked together, including G12C, G12D, and G13D mutant peptides.

[0005] Another embodiment provides an mRNA encoding the antigenic peptide.

[0006] Another embodiment provides DNA encoding the antigenic peptide.

[0007] Another embodiment provides an immunogenic composition comprising the antigenic peptide or the mRNA encoding it.

[0008] Another embodiment provides a method of inducing an immune response to a KRAS mutant peptide in an individual, comprising administering to the individual the antigenic peptide, or mRNA encoding it. [Means for solving the problem]

[0009] As used herein, the term "identity" in the context of polypeptides or polynucleotides refers to the relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. Identity indicates the degree of sequence relatedness, as determined by the number of matches between two or more strings of amino acid residues or nucleotide residues. The identity of related polypeptides or polynucleotides is calculated by well-known methods. "Percent identity," as applied to polypeptides or polynucleotides, is defined as the percentage of residues in a candidate sequence that are identical to those in a second sequence, after aligning the amino acid or nucleotide sequence of the candidate with the second sequence and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and programs for such alignments are well known. Such programs may be, for example, BLAST, the Smith-Waterman algorithm, or the Needleman-Wunsch algorithm.

[0010] As used herein, the term "5'-untranslated region (5'-UTR)" refers to the region of an mRNA that is immediately upstream (i.e., 5') from the start codon, i.e., the first codon of the mRNA transcript that is translated by the ribosome, that does not code for a polypeptide.

[0011] As used herein, the term "3'-untranslated region (3'-UTR)" refers to the region of an mRNA that is immediately downstream (i.e., 3') from a stop codon that does not code for a polypeptide, i.e., a codon in the mRNA transcript that signals translation termination.

[0012] As used herein, "open reading frame (ORF)" refers to a contiguous region of nucleic acid that begins with a start codon, e.g., a methionine codon (ATG), ends with a stop codon, e.g., TAA, TAG, or TGA, and encodes a polypeptide.

[0013] As used herein, the term "polyadenylate sequence" or "polyA tail" refers to a region of an mRNA downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple consecutive adenosine monophosphates. The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. The polyA tail can contain, for example, 50 to 250 adenosine monophosphates. In living organisms such as cells or individuals, the poly(A) tail can protect mRNA from attack by enzymes, e.g., enzymes in the cytoplasm, and can play a role in terminating transcription, exporting mRNA from the nucleus, and aiding in translation.

[0014] As used herein, the term "operatively linked" refers to nucleotide sequences linked on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., an ORF) if it affects the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

[0015] In this specification, unless otherwise specified, the position of a nucleotide sequence is indicated as being linked or located in the direction from the 5' end to the 3' end.

[0016] As used herein, the term "vector" or "nucleic acid construct" refers to any nucleic acid capable of carrying a gene, ORF, or DNA fragment into a cell. The vector is also, for example, replicable in the cell. The vector can be a virus, bacteriophage, provirus, plasmid, phagemid, transposon, or artificial chromosome, such as a yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or plant artificial chromosome (PLAC).

[0017] A first aspect provides an antigenic peptide in which two or more KRAS mutant peptides selected from the group consisting of KRAS mutant peptides (variant peptides) are linked together, the antigenic peptide including G12C, G12D, and G13D.

[0018] The KRAS mutant peptide may further include G12A, G12V, G12R, G12S, G13C, G13D, G13R, Q61H, Q61R, A146T, or a combination thereof. The KRAS mutant peptide may have 7 to 50 amino acid residues. The mutant peptide may include a mutant residue compared to the wild-type sequence, and may include an extension sequence 1 extending 3 to 23 amino acids from the mutant residue to the N-terminus, and an extension sequence 2 extending 3 to 23 amino acids from the mutant residue to the C-terminus. The mutant peptide may be, for example, antigens 1 to 22 listed in Table 1. The extension sequence 1 is 3 to 12 amino acids in length, and the extension sequence 2 is 3 to 23, e.g., 3 to 19, amino acids in length. The extension sequence 1 may be 3, 5, 7, 8, 9, 11, or 12 amino acids in length, and the extension sequence 2 may be 3, 4, 5, 8, 9, 11, 18, or 19 amino acids in length.

[0019] The antigenic peptide may also be formed by linking multiple mutant peptides via peptide bonds. For example, the antigenic peptide may be formed by linking 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 mutant peptides via peptide bonds. The mutant peptides may be directly linked or linked via a peptide linker. The peptide linker may be a G4S (SEQ ID NO: 552) or LLSVGG (SEQ ID NO: 553) linker. Each mutant peptide included in the antigenic peptide may contain mutated residues at the same or different positions. For example, the antigenic peptide may contain mutated residues at one, two, three, or four mutated positions. The mutated positions may be C12, C13, Q61, or A146.

[0020] In one embodiment, the mutant peptide is 8, 17, or 31 aa in length. The antigenic peptide may further comprise one or more of the following mutant peptides: G12V, G12R, G13C, G13R, and Q61H.

[0021] In one embodiment, the antigenic peptide comprises G12C, G12D, G12V, G12R, G13C, G13D, and Q61H mutant peptides, each mutant peptide also being 17 aa in length.

[0022] In one embodiment, the antigenic peptide may include only mutant peptides 8 aa or 31 aa in length. The antigenic peptide may include a combination of mutant peptides 8 aa and 31 aa in length with the same mutant residues. The antigenic peptide may include G12C, G12D, G12V, G12R, G13C, G13D, G13R, and Q61H mutant peptides.

[0023] The antigen peptide may have a signal peptide linked to its N-terminus. The signal peptide may also be a secretory signal peptide. The signal peptide may include an IgK signal peptide, an AZU1 signal peptide, a LAMP1 signal peptide, or an MHC-1 signal peptide.

[0024] The antigenic peptide may be linked to an AMP1 transmembrane domain, an MHC-1 trafficking domain, a diphtheria toxin (DTT), a diphtheria toxin (DTT) domain (1-87aa), or a diphtheria toxin (DTT) domain (96-177aa). The antigenic peptide is linked to an AMP1 transmembrane domain, a diphtheria toxin (DTT), or a diphtheria toxin (DTT) domain (1-87aa) at the N-terminus, and to an MHC-1 trafficking domain or a diphtheria toxin (DTT) domain (96-177aa) at the N-terminus.

[0025] The antigen peptide may be any one of the amino acid sequences of SEQ ID NOs: 23 to 44.

[0026] A second aspect provides an mRNA encoding the antigenic peptide.

[0027] The mRNA may include an expression regulatory sequence or a sequence that affects stability. The expression regulatory sequence or a sequence that affects stability is a 5'-UTR, a 3'-UTR, and / or a polyA. The 5'-UTR, the 3'-UTR, and the polyA may be natural or non-natural sequences. These sequences may be ORFs or operably linked to each other.

[0028] The 5'-UTR can have 70% or more, e.g., 80% or more, 90% or more, or 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 553. The 5'-UTR can comprise SEQ ID NO:553.

[0029] The 5'-UTR may be derived from an mRNA encoding a protein selected from the group consisting of p53, OX40L, albumin, serum amyloid A, apolipoproteins A / B / E, transferrin, alpha-fetoprotein, erythropoietin, Factor VIII, MyoD, myosin, myoglobin, myogenin, herculin, Tie-1, CD36, C / EBP, AML1, G-CSF, GM-CSF, CD1 1b, MSR, Fr-1, i-NOS, CD45, CD18, CD36, GLUT4, ACRP30, adiponectin, PA / B / C / D, ORM1, HPX, FGA, CYP2E1, C3, ASC, APOA2, ALB, AGXT, α-globin, β-globin, tyrosine hydroxylase, and collagen.

[0030] The 3'-UTR can have 70% or more, eg, 80% or more, 90% or more, or 95% or more sequence identity with the nucleotide sequence of SEQ ID NO:554. The 3'-UTR may be derived from an mRNA encoding a protein selected from the group consisting of p53, OX40L, albumin, serum amyloid A, apolipoproteins A / B / E, transferrin, alpha-fetoprotein, erythropoietin, Factor VIII, MyoD, myosin, myoglobin, myogenin, herculin, Tie-1, CD36, C / EBP, AML1, G-CSF, GM-CSF, CD1 1b, MSR, Fr-1, i-NOS, CD45, CD18, CD36, GLUT4, ACRP30, adiponectin, PA / B / C / D, ORM1, HPX, FGA, CYP2E1, C3, ASC, APOA2, ALB, AGXT, α-globin, β-globin, tyrosine hydroxylase, and collagen.

[0031] The mRNA may contain one or more modified nucleotides. The modified polynucleotide, when introduced into a cell or individual, may exhibit reduced degradation in the cell or individual compared to the unmodified polynucleotide. The modified polynucleotide, when introduced into a cell or individual, may also exhibit reduced immunogenicity (e.g., reduced innate immune response) in the cell or individual compared to the unmodified polynucleotide.

[0032] Nucleotides having modified cytosines can include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methylpseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, or combinations thereof.

[0033] Nucleotides having modified adenines can include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), or combinations thereof.

[0034] Nucleotides having modified guanosine can include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, or combinations thereof.

[0035] The nucleotide having the chemical modification is pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2'-O-methyluridine, or a combination thereof. The chemical modification may be at the 5' position of uracil. The chemical modification may also be a modification of uracil to N1-methylpseudouridine. The chemical modification is also the modification of uracil to N1-ethylpseudouridine.

[0036] The mRNA may include a 5' cap. The term "5'-cap" refers to the cap structure found at the 5' end of an mRNA molecule. The 5'-cap may include a guanosine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate linkage. The guanosine may be methylated at the 7-position, e.g., m 7 G or 3'-O-Me-m 7 The term "conventional 5' cap" refers to naturally occurring RNA 5' caps, such as 7-methylguanosine (m7G). As used herein, the term "5' cap" includes 5' cap analogs that resemble RNA cap structures and have been modified to have the ability to stabilize RNA, e.g., in vivo and / or in cells, when attached to RNA. A 5' cap or 5' cap analog can be provided to an RNA by in vitro transcription of a DNA template in the presence of a 5' cap or 5' cap analog, followed by co-transcriptional insertion of the 5' cap or 5' cap analog into the resulting RNA strand, or the 5' cap or 5' cap analog can be generated post-transcriptionally using a capping enzyme, e.g., vaccinia virus capping enzyme. The 5'-end cap structure can be, for example, 7mG(5')ppp(5')ImpNp, 3'-O-Me-m7G(5')ppp(5')G, m7(3'OMeG)(5')ppp(5')(2'OMeA), or m7G(5')ppp(5')(2'OMeA)pG.

[0037] The mRNA may also have one or more U's substituted with N1-methyl-pseudouridine. In the mRNA, the 5'-terminal GA may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA). In the mRNA, all U's may be substituted with N1-methyl-pseudouridine. In the mRNA, all U's may be substituted with N1-methyl-pseudouridine, and the 5'-terminal GA may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA).

[0038] The mRNA is also codon-optimized for the host cell. The codon optimization can vary depending on the host cell in which the mRNA is produced.

[0039] The mRNA may include a nucleotide sequence of any one of SEQ ID NOs: 1 to 22. The mRNA may also include a nucleotide sequence of any one of SEQ ID NOs: 1 to 23. The mRNA may also include a nucleotide sequence of any one of SEQ ID NOs: 296 to 300.

[0040] A third aspect provides DNA encoding the antigenic peptide.

[0041] The DNA may further comprise expression control elements and restriction enzyme sites. The expression control elements are promoters. The expression control elements are operably linked to ORFs or other expression control elements.

[0042] The 3'-UTR coding sequence may include a recognition sequence for a restriction enzyme. The restriction enzyme may be XhoI, NheI, or XhoI and NheI. The promoter is operably linked to the 5'-UTR coding sequence. The 5'-UTR, the antigenic peptide coding sequence, and the 3'-UTR are linked to form a common transcript when transcribed in vitro or in vivo. The 3'-UTR coding sequence is operably linked to a poly(A) sequence.

[0043] The 5'-UTR-encoding sequence is a sequence encoding the sequence of SEQ ID NO: 553, and the 3'-UTR-encoding sequence is a sequence including a sequence encoding the sequence of SEQ ID NO: 554. In this case, the KRAS antigen peptide-encoding sequence may have any one of the nucleotide sequences of SEQ ID NOs: 1 to 22 (including only the coding region sequence). In addition, the polyA sequence may include the sequence of SEQ ID NO: 556.

[0044] As used herein, the term "nucleic acids capable of being transcribed to produce a common transcript" refers to nucleic acid sequences that are functionally or operably linked to each other such that, when transcribed, an RNA molecule is formed that comprises a transcript in which the nucleic acid sequences are covalently linked to each other. The transcription occurs under promoter control, if appropriate, after linearization of the nucleic acid molecule, e.g., a closed circular nucleic acid molecule, e.g., after restriction enzyme cleavage of the nucleic acid molecule. The common transcript may be separated, if appropriate, by sequences located between the nucleic acid sequences.

[0045] The DNA molecule can be a closed circular molecule or a linear molecule.

[0046] The DNA molecule may further comprise one or more selected from the group consisting of (i) a reporter gene, (ii) a selectable marker, and (iii) a proliferation origin. The DNA molecule may also be a vector. The vector may be a cloning vector or an expression vector. After linearization, the DNA molecule is suitable for in vitro transcription of mRNA.

[0047] A fourth aspect provides an immunogenic composition comprising the antigenic peptide or mRNA encoding the antigenic peptide as an active ingredient.

[0048] The antigen peptide and the mRNA encoding it are as described in the first and second embodiments.

[0049] The composition is for inducing an immune response to a KRAS mutant peptide. The composition is for preventing or treating cancer. The individual to be prevented or treated has a KRAS mutation. The mutation can be G12C, G12D, G12V, G12R, G13C, G13D, G12R, Q61H, Q61R, A147R, or a combination thereof.

[0050] The composition may also be a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0051] The composition may be for treating cancer. The cancer may be a cancer containing a KRAS mutation. The cancer may be a blood cancer or a solid cancer. The blood cancer may be leukemia, malignant lymphoma, multiple myeloma, or aplastic anemia. The solid tumor may be a brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male reproductive organ tumor, penile cancer, prostate cancer, female reproductive organ tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital organ cancer, female urethral cancer, or skin cancer. The lung cancer may be small cell lung cancer or non-small cell lung cancer.

[0052] The composition is administered to an individual and the mRNA can be translated in vivo to produce the antigenic peptide.

[0053] The composition may be contacted with a cell, tissue, or individual in an "effective amount" to induce an immune response.

[0054] The effective amount can be determined based on the target tissue, target cell, means of administration, characteristics of the mRNA, such as size and degree of content of modified nucleotides, and other components of the composition.

[0055] The composition may be administered together with other prophylactic or therapeutic compounds, such as anti-cancer agents. As used herein, the anti-cancer agents may be any known anti-cancer agents.

[0056] The composition may be administered systemically or locally. Local administration refers to administration to a tissue containing cancer. The tissue containing cancer may be intramuscular, subcutaneous, intradermal, nasal, or pulmonary.

[0057] The composition may include one or more pharmaceutically acceptable carriers, diluents, or excipients, in which the mRNA is formulated or complexed with the excipients. The carriers, diluents, or excipients may be those well known in the art.

[0058] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or other additional ingredients contained in the composition may vary depending on the characteristics, size, and / or condition of the individual being treated, and the route of administration. For example, the composition may contain 0.1% to 100%, e.g., 0.5% to 50%, 1.0% to 30%, 5.0% to 80%, or 80% or more (w / w) active ingredient.

[0059] The composition may also be formulated as nanoparticles. Nanoparticles are known for delivering mRNA to cells. For example, the nanoparticles are lipid nanoparticles (LNPs). The composition may be formulated as a lipid nanoparticle (LNP) or bound to an LNP. The binding includes binding within or to the surface of an LNP. For example, the composition may be formulated within a lipid polycation complex or bound to a lipid polycation complex. The lipid polycation complex is also called a cationic lipid nanoparticle. The polycation can include cationic polypeptides such as MC3, Lipid 319, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, SM-102, ALC-0315, Arcturus Lipid 2,2(8,8)4C CH3, Genevant CL1, polylysine, polyornithine, and / or polyarginine. The composition can also be formulated in or conjugated to lipid nanoparticles containing a sterol such as distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), cholesterol, or a non-cationic lipid such as dioleoylphosphatidylethanolamine (DOPE). The composition may also be formulated in or conjugated to lipid nanoparticles containing PEG lipids such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), and polyethylene glycol dimethacrylate (PEG-DMA).

[0060] The lipid nanoparticle formulation may contain a cationic lipid, a phospholipid, a sterol such as cholesterol, a PEG-lipid, or a combination thereof. For example, the lipid nanoparticle formulation may contain a cationic lipid, a phospholipid, a sterol such as cholesterol, and a PEG-lipid. The lipid nanoparticle may also contain a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable lipid, or a combination thereof. The lipid nanoparticle may contain 0.5-15 mol% PEG-modified lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 20-60 mol% ionizable lipid. The PEG-modified lipid may be 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG2000-DMG), the non-cationic lipid may be 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), the sterol may be cholesterol, and the ionizable cationic lipid may be Compound 1, having the following structure:

[0061] (Compound 1) [ka]

[0062] The PEG-modified lipid may be Compound 2 (ALC-0159) of the following structure, the non-cationic lipid may be 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), the sterol may be cholesterol, and the ionizable cationic lipid may be Compound 3 (ALC-0315) of the following structure:

[0063] (Compound 2) [ka]

[0064] (Compound 3) [ka]

[0065] A fifth aspect provides a method for inducing an immune response in an individual, comprising administering the antigenic peptide or mRNA to the individual. The method may be for preventing or treating cancer.

[0066] The administration can be in an amount effective to induce an immune response in the individual. The administration can be in an amount effective to prevent or treat cancer in the individual. The administration can be systemic or local. The local administration is localized administration to the tissue where the cancer is present. The tissue where the cancer is present can be intramuscular, subcutaneous, intradermal, nasal, or pulmonary. The administration can be parenteral or oral.

[0067] The individual is a mammal, including a human. The administration can be performed by locally administering the mRNA molecule intracellularly or extracellularly to a tissue in which cells with a high level of KRAS mutation are present.

[0068] The method can produce the antigenic peptide in cells of an individual to suppress cancer cell growth, treat disease in the individual, and induce an immune response against cancer in the individual. [Effects of the Invention]

[0069] According to one embodiment, an antigenic peptide in which two or more KRAS mutant peptides are linked can be used to efficiently induce an immune response in an individual.

[0070] According to one embodiment, the mRNA encoding said antigenic peptide can be used to efficiently induce an immune response in an individual.

[0071] According to one embodiment, DNA encoding the antigenic peptide can be used to produce the antigenic peptide.

[0072] The immunogenic composition according to one embodiment can be used to induce an immune response in an individual.

[0073] According to one embodiment of the method for inducing an immune response against a KRAS mutant peptide in an individual, an immune response against a KRAS mutant peptide can be efficiently induced in an individual. [Brief explanation of the drawings]

[0074] [Figure 1] 1 is a diagram showing vectors obtained through the processes of Examples 1 and 2. [Figure 2] 1 is a graph showing changes in tumor size and body weight after administration of an anti-cancer vaccine containing KRAS mRNA encoding antigen 2 to a Lewis lung cancer mouse model. [Figure 3] 1 is a graph showing changes in immune cell distribution in tumor tissue cells after an anti-cancer vaccine containing KRAS mRNA encoding antigen 2 is administered to a Lewis lung cancer mouse model. [Figure 4] 1 is a graph showing changes in tumor size and body weight after administration of an anti-cancer vaccine containing KRAS mRNA encoding antigen 6 to a Lewis lung cancer mouse model. [Figure 5] 1 is a graph showing changes in the frequency of immune CD8 T cells in tumor tissue cells after administration of an anti-cancer vaccine containing KRAS mRNA encoding antigen 6 to a Lewis lung cancer mouse model. [Figure 6] 1 is a graph showing changes in tumor size and body weight after administration of an anti-cancer vaccine containing KRAS mRNA encoding antigen 14 or antigen 15 to a Lewis lung cancer mouse model. [Figure 7A] 1 shows photographs of the results of co-culturing PBMCs expressing antigens 2 and 6 linked to KRAS mutant peptides with target cells. [Figure 7B] 1 shows the results of measuring the viability of target cells when the target cells were co-cultured with PBMCs expressing antigens 2 and 6 linked to KRAS mutant peptides, respectively. [Figure 8A]1 shows photographs of the results of co-culturing PBMCs expressing antigens 15 and 17 linked to KRAS mutant peptides with target cells. [Figure 8B] 1 shows the results of measuring the viability of target cells when PBMCs expressing antigens 15 and 17 linked to KRAS mutant peptides were co-cultured with the target cells. [Figure 9] This figure shows the results of Western blotting using an anti-RAS (G12D mutant) monoclonal antibody against proteins expressed in LL / 2 cells, a Lewis lung carcinoma cell line, into which mRNA encoding antigens 6, 14, and 15 linked to KRAS mutant peptides was introduced. DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0076] Example 1: Anticancer substance sequences aimed at increasing T cell activation against KRAS mutant sequences An antigen comprising multiple KRAS mutant peptides was designed for the treatment of cancer patients with KRAS mutations. The mutant peptides comprise a mutant residue in the KRAS wild-type sequence (SEQ ID NO: 557), and include extension sequence 1 extending from the mutant residue to the N-terminus and extension sequence 2 extending from the mutant residue to the C-terminus. The mutant residues include G12A, G12C, G12D, G12V, G12R, G12S, G13C, G13D, G13R, Q61H, Q61R, or A146T. Extension sequence 1 can be 3 to 23 amino acids in length. Extension sequence 2 can be 3 to 23 amino acids in length.

[0077] The mutant peptide is designated E1-M-E2, where M represents the mutated residue, and E1 and E2 represent extension sequence 1 extending from the mutated residue to the N-terminus and extension sequence 2 extending from the mutated residue to the C-terminus, respectively. In the mutant peptide E1-M-E2, M is G12A, G12C, G12D, G12V, G12R, G12S, G13C, G13D, Q61H, Q61R, or A146T, E1 is 3 to 12 amino acids in length, and E2 is 3 to 23, e.g., 3 to 19, amino acids in length. E1 can be 3, 5, 7, 8, 9, 11, or 12 amino acids in length, and E2 can be 3, 4, 5, 8, 9, 11, 18, or 19 amino acids in length.

[0078] The antigenic peptide may also be formed by linking multiple mutant peptides via peptide bonds. For example, the antigenic peptide may be formed by linking 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 mutant peptides via peptide bonds. The mutant peptides may be directly linked or linked via a peptide linker. The peptide linker may be a G4S (SEQ ID NO: 552) or LLSVGG (SEQ ID NO: 553) linker. Each mutant peptide included in the antigenic peptide may contain mutated residues at the same or different positions. For example, the antigenic peptide may contain mutated residues at one, two, three, or four mutated positions. The mutated positions may be C12, C13, Q61, or A146.

[0079] Furthermore, the antigen peptides may all contain the same mutated residue at the same mutation position, or may contain different mutated residues at the same mutation position.

[0080] The antigenic peptide may be linked to a secretory signal peptide. The secretory signal peptide may be removed during secretion. The antigenic peptide may be linked to a secretory signal peptide at its N-terminus. The signal peptide may be an IgK signal peptide, an AZU1 signal peptide, a LAMP1 signal peptide, or an MHC-1 signal peptide. The antigenic peptide may also be linked to a LAMP1 transmembrane domain, an MHC-1 trafficking domain, a diphtheria toxin (DTT) domain (aa 1-87), or a diphtheria toxin (DTT) domain (aa 96-177). The antigenic peptide may be linked to a LAMP1 transmembrane domain, an MHC-1 trafficking domain, or a diphtheria toxin (DTT) domain (aa 1-87) at its N-terminus. The antigenic peptide may be linked to a diphtheria toxin (DTT) domain (aa 96-177) at its C-terminus.

[0081] Table 1 shows the KRAS mutant peptide structures and their amino acid sequences (SEQ ID NOS: 23 to 44) of antigen peptides 1 to 22 prepared in this example. In Table 1, SEQ ID NOS: 1 to 22 listed in the "KRAS mutant peptide structure" column indicate the nucleotide sequences of the ORF mRNA of each antigen peptide.

[0082] [Table 1-1]

[0083] [Table 1-2]

[0084] [Table 1-3]

[0085] [Table 1-4]

[0086] [Table 1-5]

[0087] [Table 1-6]

[0088] Sequences were synthesized in which the 5'UTR and / or signal sequence were linked to the open reading frame (ORF) encoding the antigen peptides listed in Table 1 (SEQ ID NOS: 23 to 44). The synthesized sequences contained HindIII and XhoI restriction enzyme sequences at the 5' and 3' ends, respectively, for cloning. The synthesized sequences encoding each antigen were cleaved with HindIII and XhoI restriction enzymes, respectively, and ligated using DNA ligase to prepare vectors containing sequences encoding mRNA. The structures of the KRAS mutant peptides for antigens 1 to 22 are shown in Table 1. Each mutant peptide is separated by parentheses, and the presence or absence of a linker between the mutant peptides is indicated by "-". The linkers indicated by "-" are all cathepsin cleavage linkers with the amino acid sequence "LLSVGG (SEQ ID NOS: 553)". In cases where the antigen sequence is blanked between KRAS mutant peptides, this means that the mutant peptides were directly linked in order without a linker.

[0089] The full-length mRNAs encoding antigens 1 to 22 shown in Table 1 each have a 5'UTR-ORF-interrupt codon-3'UTR-polyA structure. Table 2 shows the mutant peptides contained in antigen 1 and their mRNA sequences.

[0090] [Table 2]

[0091] Table 3 shows the mutant peptides contained in antigen 2 and their mRNA sequences.

[0092] [Table 3]

[0093] Table 4 shows the mutant peptides contained in antigen 3 and their mRNA sequences.

[0094] [Table 4]

[0095] Table 5 shows the mutant peptides contained in antigen 4 and their mRNA sequences.

[0096] [Table 5]

[0097] Table 6 shows the mutant peptides contained in antigen 5 and their mRNA sequences.

[0098] [Table 6]

[0099] Table 7 shows the mutant peptides contained in antigen 6 and their mRNA sequences.

[0100] [Table 7]

[0101] Table 8 shows the mutant peptides contained in antigen 7 and their mRNA sequences.

[0102] [Table 8]

[0103] Table 9 shows the mutant peptides contained in antigen 8 and their mRNA sequences.

[0104] [Table 9]

[0105] Table 10 shows the N-terminal domain contained in antigen 9, the mutant peptides, and its mRNA sequence.

[0106] [Table 10-1]

[0107] [Table 10-2]

[0108] Antigen 9 is a peptide in which the DTT domain (aa 1-177) and six mutant peptides listed in Table 10 are linked in order with linkers. Table 11 shows the mutant peptides, C-terminal domains, and mRNA sequences contained in Antigen 10.

[0109] [Table 11-1]

[0110] [Table 11-2]

[0111] Antigen 10 is a peptide in which the six mutant peptides listed in Table 11 and a DTT domain (aa 1-177) are linked in order with a linker. Table 12 shows the N-terminal domain, mutant peptides, C-terminal domain, and mRNA sequence contained in Antigen 11.

[0112] [Table 12-1]

[0113] [Table 12-2]

[0114] Antigen 11 is a peptide in which the DTT domain (aa 1-87), six mutant peptides, and the DTT domain (aa 96-177) are linked in this order with linkers, as shown in Table 12. Table 13 shows the mutant peptides contained in antigen 12 and their mRNA sequences.

[0115] [Table 13]

[0116] Table 14 shows the mutant peptides contained in antigen 13 and their mRNA sequences.

[0117] [Table 14-1]

[0118] [Table 14-2]

[0119] Table 15 shows the mutant peptides contained in antigen 14 and their mRNA sequences.

[0120] [Table 15-1]

[0121] [Table 15-2]

[0122] Table 16 shows the mutant peptides contained in antigen 15 and their mRNA sequences.

[0123] [Table 16-1]

[0124] [Table 16-2]

[0125] Table 17 shows the mutant peptides contained in antigen 16 and their mRNA sequences.

[0126] [Table 17-1]

[0127] [Table 17-2]

[0128] [Table 17-3]

[0129] Table 18 shows the mutant peptides contained in antigen 17 and their mRNA sequences.

[0130] [Table 18-1]

[0131] [Table 18-2]

[0132] Table 19 shows the mutant peptides contained in antigen 18 and their mRNA sequences.

[0133] [Table 19-1]

[0134] [Table 19-2]

[0135] [Table 19-3]

[0136] Table 20 shows the mutant peptides contained in antigen 19 and their mRNA sequences.

[0137] [Table 20-1]

[0138] [Table 20-2]

[0139] Table 21 shows the mutant peptides contained in antigen 20 and their mRNA sequences.

[0140] [Table 21-1]

[0141] [Table 21-2]

[0142] Table 22 shows the mutant peptides contained in antigen 21 and their mRNA sequences.

[0143] [Table 22-1]

[0144] [Table 22-2]

[0145] Table 23 shows the mutant peptides contained in antigen 22 and their mRNA sequences.

[0146] [Table 23-1]

[0147] [Table 23-2]

[0148] Example 2: Cloning of a sequence encoding an antigen peptide linked to multiple KRAS mutant peptides To clone the nucleotide sequence encoding the antigen peptide to which multiple KRAS mutant peptides described in Example 1 are linked into an open reading frame (ORF), a T7 promoter and a 5'-UTR sequence were added to the 5' portion of the sequence, and HindIII and XhoI restriction enzyme sequences were inserted into the 5' and 3' portions, respectively, to obtain the ORF through gene synthesis.

[0149] The synthesized sequences contained HindIII and XhoI restriction enzyme sequences at the 5' and 3' ends, respectively, for use in cloning. The synthesized antigen-encoding sequences and the template vector were cleaved with HindIII and XhoI restriction enzymes, respectively, and ligated using DNA ligase to prepare vectors containing sequences encoding "mRNA."

[0150] The full-length mRNAs of antigens 1 to 22 shown in Table 1 each have the following structure: 5'UTR (SEQ ID NO: 554)-ORF-interruption codon-3'UTR (SEQ ID NO: 555)-polyA (SEQ ID NO: 556).

[0151] Table 24 shows the full-length mRNA sequences of antigens 2, 6, 14, 15, and 17 as KRAS mutant anti-cancer vaccine candidates used in efficacy evaluation.

[0152] [Table 24-1]

[0153] [Table 24-2]

[0154] [Table 24-3]

[0155] [Table 24-4]

[0156] Example 3: Evaluation of the anti-cancer efficacy of mRNA encoding antigen 2 linked to KRAS mutant peptide in a Lewis lung carcinoma mouse model The anti-cancer efficacy of an mRNA-containing vaccine encoding antigen 2 linked to a KRAS mutant peptide according to the present invention was evaluated. The mRNA-containing vaccine was administered to a Lewis lung carcinoma (LL / 2) tumor syngeneic mouse model, and the anti-tumor efficacy was evaluated by observing changes in tumor size. LL / 2 cells are a mouse lung cancer cell line established from the lung of a C57BL mouse bearing a tumor induced by the implantation of a primary Lewis lung carcinoma.

[0157] The treatment group received the vaccine at a dose of 30 μg per head per week for 5 weeks. The mRNA-containing vaccine was administered in a lipid nanoparticle (LNP) formulation loaded in PBS. The LNP contained ionizable lipid (ALC0315, 46.3 mol%), phospholipid (DSPC, 9.4 mol%), cholesterol (42.7 mol%), and PEG-lipid (ALC0159, 1.6 mol%) in PBS. The loaded mRNA concentration was 0.3 mg / mL. In the following examples, the same mRNA-containing vaccine was used. Specifically, after the first administration, LL / 2 cells (ATCC) cultured in DMEM medium in a cell culture flask were subcutaneously injected into the dorsal side of C57BL / 6 mice (female, 6 weeks old, average weight 18 g) (Daehan Bio). If tumors were observed with the naked eye several days later, tumor size was monitored. Each group consisted of seven mice with similar body weights. The administration group received the vaccine at 15 μg / head per dose, 50 μl, administered intramuscularly twice a week.

[0158] Tumor size in each group was monitored from the day of tumor injection through day 26. Figure 2 shows the changes in tumor size and body weight after administration of an anticancer vaccine containing mRNA encoding antigen 2 to a Lewis lung cancer mouse model. As shown in Figure 2, the anticancer vaccine containing mRNA encoding antigen 2 demonstrated significant tumor growth suppression efficacy compared to the vehicle-treated group in the Lewis lung cancer mouse model (Figure 2A). Furthermore, no weight loss was observed in the mice after 5 weeks of continuous vaccine administration, demonstrating safety in terms of side effects (Figure 2B). Previous reports have shown that Lewis lung tumors are a representative immunosuppressive tumor model, primarily populated by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) (Viitala et al., 2019, Clin Cancer Res (2019) 25 (11): 3289-3303).

[0159] Tumors were then isolated from the mice, and tumor tissue cells were analyzed by flow cytometry (FACS). Through flow cytometry, the percentages of CD8+ T cells (CD3+CD8+ cells), memory / effector CD8+ T cells (CD3+CD8+CD44+ cells), and regulatory T cells (CD3+CD8+Foxp3+ cells) in the tumor tissue were determined.

[0160] After administering an mRNA-containing anticancer vaccine to a Lewis lung carcinoma mouse model, we examined changes in the distribution of immune cells in tumor tissue. Administration of an mRNA-containing anticancer vaccine encoding antigen 2 increased the proportion of CD8+ T cells (CD3+CD8+ cells) and memory / effector CD8+ T cells (CD3+CD8+CD44+ cells) in tumor tissue (Figures 3A and 3B), while decreasing the proportion of regulatory T cells (Treg cells) (CD3+CD8+Foxp3+ cells), which are known to suppress immune responses (Figure 3C).

[0161] These results indicate that the KRAS mRNA anti-cancer vaccine exerts its anti-cancer efficacy by inducing the proliferation of cells involved in tumor killing, such as CD8+ T cells (CD3+CD8+ cells) and memory / effector CD8+ T cells (CD3+CD8+CD44+ cells), and suppressing the proliferation of regulatory T cells, Treg cells (CD3+CD8+Foxp3+ cells), which suppress immune responses. Furthermore, the anti-cancer effect was also observed in Lewis lung tumors, a well-known immunosuppressive tumor model, suggesting that the KRAS mRNA anti-cancer vaccine can exhibit excellent anti-cancer efficacy regardless of the immune environment of the cancer.

[0162] This indicates that the KRAS mRNA anti-cancer vaccine is effective in immunosuppressive tumors.

[0163] Example 4: Evaluation of the anti-cancer efficacy of mRNA encoding antigen 6 linked to KRAS mutant peptide in a Lewis lung cancer mouse model The anti-cancer efficacy of an mRNA-containing anti-cancer vaccine encoding antigen 6 linked to a KRAS mutant peptide according to the present invention was evaluated. The mRNA-containing vaccine was administered to a Lewis lung cancer mouse model (LL / 2 tumor syngeneic mouse model) and tumor size was monitored to evaluate its anti-tumor efficacy. Each treatment group received the vaccine at a dose of 20 μg / head / week for five weeks. The mRNA-containing vaccine had the same composition as described in Example 3. Specifically, after the first administration, LL / 2 cells (ATCC) cultured in DMEM medium in a cell culture flask were subcutaneously injected into the dorsal side of C57BL / 6 mice (female, 6 weeks old, average weight 18 g) (Dahan Bio). After several days, tumors were observed with the naked eye and tumor size was monitored. Seven mice of similar weight were assigned to each group. Each treatment group received 50 μl of the vaccine intramuscularly at 10 μg / head twice weekly.

[0164] Tumor size in each group was monitored from the day of tumor injection until day 26. After administering the anticancer vaccine containing mRNA encoding antigen 6 to a Lewis lung carcinoma mouse model, the results showed that the anticancer vaccine containing mRNA encoding antigen 6 exhibited significant tumor growth suppression efficacy compared to the vehicle-administered group (Figure 4A). Furthermore, even after continuous administration of the vaccine for 5 weeks, mice did not experience weight loss, confirming its safety in terms of side effects (Figure 4B).

[0165] Furthermore, the tumors isolated from the mice were subjected to flow cytometry (FACS analysis) to confirm the frequency of CD8+ T cells (CD3+CD8+ cells) in the tumor tissue cells.

[0166] After administering the mRNA-containing anticancer vaccine to a Lewis lung cancer mouse model, we observed changes in the distribution of immune cells in tumor tissue cells. Administration of the mRNA-containing anticancer vaccine encoding antigen 6 increased the proportion of CD8+ T cells (CD3+CD8+ cells) in tumor tissue cells (Figure 5).

[0167] These results indicate that the KRAS mRNA anti-cancer vaccine exerts its anti-cancer efficacy by inducing the proliferation of cells involved in tumor killing, such as CD8+ T cells (CD3+CD8+ cells). Furthermore, the anti-cancer effect was also observed in Lewis lung tumors, a well-known immunosuppressive tumor model, suggesting that the KRAS mRNA anti-cancer vaccine can exhibit excellent anti-cancer efficacy regardless of the immune environment of the cancer.

[0168] This indicates that the KRAS mRNA anti-cancer vaccine is effective in immunosuppressive tumors.

[0169] Example 5: Evaluation of the anti-cancer efficacy of mRNA encoding antigen 14 or antigen 15 linked to KRAS mutant peptide in a Lewis lung carcinoma mouse model The anti-cancer efficacy of an mRNA-containing anti-cancer vaccine encoding antigen 14 or antigen 15 linked to a KRAS mutant peptide according to the present invention was evaluated. The mRNA-containing vaccine was administered to a Lewis lung cancer mouse model (LL / 2 tumor syngeneic mouse model) and tumor size was monitored to evaluate its anti-tumor efficacy. Each treatment group received the vaccine at a dose of 30 μg / head / week for five weeks. The mRNA-containing vaccine was the same as that described in Example 3. Specifically, after the first administration, LL / 2 cells (ATCC) cultured in DMEM medium in a cell culture flask were subcutaneously injected into the dorsal side of C57BL / 6 mice (female, 6 weeks old, average weight 18 g) (Dahan Bio). After several days, tumors were observed with the naked eye and tumor size was monitored. Each group consisted of seven mice of similar weight. Each treatment group received 50 μl of the vaccine intramuscularly at 15 μg / head twice weekly.

[0170] The tumor size of each group was observed from the day of tumor injection until day 26. As a result, the anti-cancer vaccine containing mRNA of SEQ ID NO: 14 or 15 showed significant tumor growth suppression efficacy compared to the vehicle-administered group (Figure 6A). Furthermore, mRNA encoding antigen 14 or antigen 15 did not cause weight loss in mice even after continuous administration for 5 weeks, confirming its safety in terms of side effects (Figure 6B).

[0171] These results indicate that an anti-cancer vaccine containing mRNA encoding antigens 14 or 15 induces proliferation of cells involved in tumor killing and exerts anti-cancer efficacy.

[0172] Furthermore, the anti-cancer effect was also observed in Lewis lung tumors, which are known as a representative immunosuppressive tumor model, suggesting that the KRAS mRNA anti-cancer vaccine can exhibit excellent anti-cancer efficacy regardless of the immune environment of the cancer.

[0173] This indicates that the KRAS mRNA anti-cancer vaccine is effective in immunosuppressive tumors.

[0174] Example 6: Confirmation of cell viability when human peripheral blood mononuclear cells transformed with antigen-encoding mRNA are co-cultured with target cells In this example, we evaluated whether immune cells expressing mRNA encoding antigens 2, 6, 15, and 17 linked to KRAS mutant peptides specifically react with target cells. To this end, we performed co-culture experiments between human peripheral blood mononuclear cells (PBMCs) transfected with the mRNA and target cells. The target cells used were cancer cell lines expressing wild-type KRAS and mutant KRAS, respectively. The mRNA was synthesized by in vitro transcription. For animal experiments, mRNA delivery was administered loaded onto LNPs, and for cell experiments, mRNA delivery was performed via transfection using lipofectamine.

[0175] First, PBMCs were isolated from human blood collected from healthy donors by density gradient centrifugation using Histopaque®-1077 (Sigma, Cat. No. 10771). The isolated PBMCs were cultured for 2 hours in RPMI-1640 medium containing 10% FBS and 1% P / S, and then 1.5 × 10 6 Cells were seeded into 6-well plates at 1.5 mL / well in medium. mRNA encoding each antigen linked to a KRAS mutant peptide was mixed with Lipofectamine® MessengerMAX® Transfection Reagent (Thermo Scientific®, Cat. No. LMRNA001) at a ratio of 0.5 μl per 1 μg of mRNA and incubated for 20 minutes to form Lipofectamine / mRNA complexes. The reaction product equivalent to 5 μg of mRNA was added to the plated PBMCs and cultured for 36 hours at 37°C and 5% CO2. PBMCs transfected with mRNA encoding each antigen were obtained.

[0176] Target cells were MRC-5 (0.5 × 10 4 cells / well, 10% FBS DMEM), H1299 (1 × 10 4 cells / well, 10% FBS RPMI-1460), and AsPC-1 (1 × 10 4 Cells (6 × 10 cells / well, 10% FBS RPMI-1460) were seeded into the wells of a 96-well plate and cultured to adhere to the surface. PBMCs (6 × 10 cells / well, 10% FBS RPMI-1460) transfected with mRNA were added to the cells attached to the plate. 4 cells / well, 10% FBS RPMI-1460) were added to each target cell at a ratio of 1:12 or 1:6, and cultured at 37°C, 5% CO2 for 48 hours.

[0177] The cells were then stained with sulforhodamine B (SRB) (Sigma, Cat. No. S1402) and images were captured using a Cytation 5 Imaging system (Agilent, Cat. No. 132BR022520). Tris (Sigma, Cat. No. 10708976001) was added to the wells to lyse the cells, and the absorbance at 540 nm was measured. The 540 nm absorbance was used to calculate the cell viability (%) shown in Figures 7B and 8B.

[0178] In Figure 7A, the control shows the results of culturing MRC-5, H1299, and AsPC-1 cells alone without target cells. MRC-5 cells are human embryonic lung fibroblasts and represent a normal cell line. H1299 cells are a human lymph node-derived non-small cell lung carcinoma cell line that expresses wild-type KRAS protein. AsPC-1 cells are a nude mouse xenograft-derived cell line initiated from ascites cells of a human pancreatic cancer patient and express G12D mutant KRAS.

[0179] In addition, in the negative control groups, Ctrl and Lipo represent PBMCs not transfected with mRNA, and PBMCs co-cultured with PBMCs transfected in the absence of mRNA or in the presence of Lipofectamine, respectively. For candidate substance 2 and candidate substance 6, 2.5 μg and 5.0 μg represent cases where 2.5 μg and 5.0 μg of mRNA encoding antigens 2 and 6, respectively, were used for transfection.

[0180] As shown in Figure 7A, when MRC-5 and H1299 cell lines expressing wild-type KRAS were co-cultured with PBMCs transfected with mRNA encoding antigen 2 or 6 linked to a KRAS mutant peptide, the level of sulforhodamine B staining was unchanged compared to the negative control group. On the other hand, when AsPC-1 cell lines expressing the G12D mutant protein were co-cultured with PBMCs transfected with mRNA encoding antigen 2 or 6 linked to a KRAS mutant peptide, the level of sulforhodamine B staining was reduced compared to the negative control group. The cell viability was determined by staining viable cells with sulforhodamine B, measuring the absorbance at a wavelength of 540 nm, and quantifying the results.

[0181] As shown in Figure 7B, when the MRC-5 and H1299 cell lines, which express wild-type KRAS, were cocultured with hPBMCs transfected with mRNA encoding antigen 2 or 6 linked to a KRAS mutant peptide, there was no difference in target cell viability compared to the negative control group. On the other hand, when the AsPC-1 cell line, which expresses the KRAS G12D mutant protein, was cocultured with PBMCs transfected with mRNA encoding antigen 2 or 6 linked to a KRAS mutant peptide, there was a decrease in target cell viability compared to the negative control group.

[0182] In FIG. 7B, the survival rate of target cells was calculated by the following formula:

[0183] Target cell viability = Ti / Tc × 100 Tc: Absorbance of 100% growth control group of target cells alone Ti: Absorbance of target cells in co-culture with PBMC

[0184] These results indicate that PBMCs transfected with mRNA encoding antigen 2 or 6 linked to a KRAS mutant peptide selectively suppress the growth of only cancer cells expressing G12D mutant KRAS, i.e., AsPC-1 cells.

[0185] In Figure 8A, the control group consisted of 1 × 10 BEAS-2B, MIA PaCa-2, AsPC-1, Capan-2, and H460 cells without target cells. 4 cells / well, 10% FBS After seeding under RPMI-1460 conditions, PBMC (6 × 10 4 The results are shown for co-culture with 1:6 cells / well in 10% FBS RPMI-1460.

[0186] BEAS-2B cells are epithelial cells isolated from human bronchial epithelium derived from biopsies of noncancerous individuals and represent a normal cell line. MIA PaCa-2 cells are an epithelial cell line derived from human pancreatic tumor tissue and express the G12C mutant form of KRAS.

[0187] AsPC-1 cells are a nude mouse xenograft-derived cell line initiated from ascites-derived cells of a human pancreatic cancer patient, and are a pancreatic cancer cell line that expresses G12D mutant KRAS.

[0188] Capan-2 cells are a cell line with polygonal morphology isolated from the pancreas of a human pancreatic cancer patient, and express G12V mutant KRAS.

[0189] H460 cells are a cell line isolated from the pleural fluid of a patient with large cell lung cancer, and express the Q61H mutant KRAS.

[0190] In addition, in the negative control groups, Control and Lipofectamine represent PBMCs not transfected with mRNA and PBMCs co-cultured with transfected PBMCs in the absence of mRNA or in the presence of Lipofectamine, respectively. For candidate substance 15 and candidate substance 17, 2.5 μg and 5.0 μg represent the cases where 2.5 μg and 5.0 μg of mRNA encoding antigens 15 and 17, respectively, were used for transfection.

[0191] As shown in Figure 8A, when BEAS-2B, a normal cell line expressing wild-type KRAS, was co-cultured with PBMCs transfected with mRNA encoding antigen 15 or 17 linked to a KRAS mutant peptide, the degree of sulforhodamine B staining was not different from that of the negative control group. On the other hand, when MIA PaCa-2, AsPC-1, Capan-2, and H460 cell lines expressing KRAS G12C, G12D, G12V, and Q61H mutant proteins, respectively, were co-cultured with PBMCs transfected with mRNA encoding antigen 15 or 17 linked to a KRAS mutant peptide, the degree of sulforhodamine B staining was reduced compared to the negative control group.

[0192] As shown in Figure 8B, when the normal cell line BEAS-2B, which expresses wild-type KRAS, was cocultured with PBMCs transfected with mRNA encoding antigen 15 or 17 linked to KRAS mutant peptides, target cell viability was not different from that of the negative control group. On the other hand, when the MIA PaCa-2, AsPC-1, Capan-2, and H460 cell lines expressing KRAS G12C, G12D, G12V, and Q61H mutant proteins, respectively, were cocultured with PBMCs transfected with mRNA encoding antigen 15 or 17 linked to KRAS mutant peptides, target cell viability was reduced compared to the negative control group.

[0193] In FIG. 8B, the survival rate of target cells was calculated by the following formula:

[0194] Target cell viability = Ti / Tc × 100 Tc: Absorbance of 100% growth control group of target cells alone Ti: Absorbance of target cells in co-culture with PBMC

[0195] These results indicate that PBMCs transfected with mRNA encoding antigen 15 or 17 linked to KRAS mutant peptides selectively suppress the growth of only cancer cells expressing KRAS G12C, G12D, G12V, and Q61H mutant proteins, namely, MIA PaCa-2, AsPC-1, Capan-2, and H460 cells.

[0196] The results of Figures 7A, 7B, 8A, and 8B show that immune cells such as PBMCs transfected with mRNA encoding an antigen linked to a KRAS mutant peptide selectively suppress only cancer cells expressing the KRAS mutant peptide. Therefore, the KRAS mutant peptide-linked antigen can be used to specifically suppress cancer cells without acting on normal cells.

[0197] Example 7: Confirmation of the expression of KRAS mutant mRNA anticancer substances in LL / 2 cells The mRNA encoding each antigen linked to the KRAS mutant peptide prepared in this example was transfected into LL / 2 cells using Lipofectamine® MessengerMAX® Transfection Reagent (Thermo Scientific®, Cat. No. LMRNA001), and the expression of each antigen was confirmed. To this end, an in vitro experiment was performed as follows. LL / 2 cells were cultured in a 6-well plate at 4 x 10 5The cells were seeded at 2 mL / well and then incubated for 24 hours. Each mRNA was mixed with Lipofectamine® MessengerMAX® Transfection Reagent at a ratio of 0.5 μL per 1 μg of mRNA and incubated for 20 minutes. The reaction mixture was then added to wells containing cultured LL / 2 cells in an amount equivalent to 5 μg of mRNA and incubated at 37°C for 24 hours. DMEM medium was used.

[0198] After incubation, 100 μl / well of lysis buffer and M-PER® (Mammalian Protein Extraction Reagent) (Thermo Scientific®, Cat. No. 78501) were added to each well to rupture the cells. Proteins were then extracted and subjected to BCA protein quantification using the Pierce® BCA Protein Assay Kit (Thermo Scientific®, Cat. No. 23225). The data obtained were used to determine the amount of protein used in the experiment. The resulting samples were diluted with lysis buffer and 5x reducing dye (10% SDS, 0.5% bromophenol blue, 50% glycerol, 0.5% 2-mercaptoethanol, and 250 mM Tris pH 6.8) to achieve the same protein concentration.

[0199] The diluted samples were subjected to Western blot experiments using anti-RAS (G12D mutant) monoclonal antibody (CST, Cat No. #14429S) and anti-β-actin antibody (Abcam, Cat No. ab8227).

[0200] Figure 9 shows the results of Western blotting using an anti-RAS (G12D mutant) monoclonal antibody against proteins expressed in LL / 2 cells, a Lewis lung carcinoma cell line, into which mRNA encoding antigens 6, 14, and 15 linked to KRAS mutant peptides had been introduced.

[0201] As shown in Figure 9, antigens 6, 14, and 15 linked to KRAS mutant peptides differ in size and intracellular expression. When the expression levels of the antigens were compared using antigen 6 as a control, antigens 14 and 15 showed relatively high expression levels, but were not as high as antigen 6.

[0202] This suggests that the expression level may vary depending on the amino acid sequence, composition, length, combination of KRAS mutant peptides, total length, etc. of the KRAS mutant peptides contained in the antigen. For example, even among antigens containing the G12D mutant peptide, the expression level of G12D may vary depending on the sequence design, such as the sequence, length, and composition between peptides.

Claims

1. An antigenic peptide in which two or more KRAS variant peptides selected from the group consisting of KRAS variant peptides are linked together, including G12C, G12D, and G13D variant peptides.

2. The antigenic peptide of claim 1 , wherein the mutant peptide is 8, 17, or 31 aa in length.

3. The antigenic peptide of claim 1, further comprising one or more of the following mutant peptides: G12V, G12R, G13C, G13R, and Q61H.

4. 2. The antigenic peptide of claim 1, comprising G12C, G12D, G12V, G12R, G13C, G13D, and Q61H mutant peptides, each mutant peptide being 17 aa in length.

5. The antigenic peptide of claim 3, wherein the mutant peptide is 8 aa or 31 aa in length.

6. The antigen peptide according to claim 5, comprising a combination of mutant peptides having the same mutated residues and lengths of 8 aa and 31 aa.

7. The antigenic peptide of claim 6, comprising G12C, G12D, G12V, G12R, G13C, G13D, G13R, and Q61H mutant peptides.

8. The antigenic peptide of claim 1 , wherein one or more of the mutant peptides are linked to each other via a linker.

9. The antigenic peptide of claim 6, wherein the linker is G4S (SEQ ID NO: 552) or LLSVGG (SEQ ID NO: 553).

10. The antigenic peptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 24, 28, 36, or 37.

11. An mRNA encoding an antigenic peptide according to any one of claims 1 to 10.

12. The mRNA of claim 11, comprising the nucleotide sequence of SEQ ID NO: 2, 6, 14, 15, 296, 297, 298, 299, or 300.

13. An immunogenic composition comprising an antigenic peptide according to any one of claims 1 to 10, or an mRNA encoding said antigenic peptide.

14. The immunogenic composition of claim 13 for preventing or treating cancer.

15. The immunogenic composition of claim 13, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2, 6, 14, 15, 296, 297, 298, 299, or 300.

16. The immunogenic composition of claim 15, wherein the mRNA is bound to a lipid nanoparticle.

17. The immunogenic composition of claim 14, wherein the cancer is pancreatic cancer or lung cancer.

18. A method for inducing an immune response against a KRAS mutant peptide in an individual, comprising administering to the individual an antigenic peptide according to any one of claims 1 to 10, or an mRNA encoding the antigenic peptide.

19. 19. The method of claim 18 for preventing or treating cancer.

20. 20. The method of claim 19, wherein the cancer is pancreatic cancer or lung cancer.

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