Antigenic peptide having multiple KRAS variant peptides linked thereto, nucleic acid encoding same, and use thereof

Abstract

Provided are an antigenic peptide having multiple KRAS variant peptides linked thereto, mRNA encoding same, DNA encoding same, an immunogenic composition comprising an antigenic peptide having multiple KRAS variant peptides linked thereto or an mRNA encoding same, and a method for inducing an immune response to the KRAS variant peptides in a subject.

Description

Antigenic peptides comprising multiple KRAS variant peptides linked thereto, nucleic acids encoding the same, and uses thereof

[0001] The present invention relates to an antigenic peptide comprising multiple KRAS mutant peptides linked thereto, a nucleic acid encoding the same, and uses thereof.

[0002] Nucleic acid vaccines rely on antigens encoded by DNA or RNA. DNA vaccines typically contain antigen-coding genes inserted into bacterial plasmids under the control of eukaryotic promoters. RNA vaccines, on the other hand, utilize messenger RNA (mRNA) or other antigen-coding RNA. Similar to protein vaccines, nucleic acid vaccines can be delivered via various routes, including intramuscular, subcutaneous, mucosal, or transdermal delivery.

[0003] DNA vaccines are known to induce less immune responses than peptide, cell, viral vector, and RNA vaccines. Furthermore, DNA vaccines not only have low immunogenicity, but also have the potential to integrate into the host genome, leading to oncogenesis. Consequently, there is a growing demand for RNA-based vaccines.

[0004] One aspect provides an antigenic peptide comprising two or more KRAS variant peptides linked together, wherein the KRAS variant peptides are selected from the group consisting of G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L and A146T variant peptides.

[0005] Another aspect provides mRNA encoding the above antigenic peptide.

[0006] Another aspect provides DNA encoding the above antigenic peptide.

[0007] Another aspect provides an immunogenic composition comprising the antigenic peptide, or mRNA encoding the same.

[0008] Another aspect provides a method of inducing an immune response to a KRAS mutant peptide in a subject, comprising administering to the subject the antigenic peptide, or mRNA encoding the same.

[0009] As used herein, with respect to polypeptides or polynucleotides, the term "identity" refers to the relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. The identity refers to the degree of sequence relatedness determined by the number of matches between strings of two or more amino acid residues or nucleotide residues. The identity of related polypeptides or polynucleotides can be calculated by 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 residues in a second sequence after aligning the candidate amino acid or nucleotide sequences with the second sequence to obtain the maximum percent identity, introducing gaps if necessary. Methods and programs for such alignment are known. Such programs can be, for example, BLAST, the Smith-Waterman algorithm, or the Needleman-Wunsch algorithm.

[0010] As used herein, “5’-untranslated region (5’UTR)” refers to a region of an mRNA that is directly upstream (i.e., 5’) from the initiation codon, i.e., the first codon of an mRNA transcript translated by the ribosome, that does not encode a polypeptide.

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

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

[0013] As used herein, a "polyadenylate sequence" or "polyA tail" refers to a region of an mRNA downstream, for example directly downstream (i.e., 3') from a 3' UTR, that contains multiple consecutive adenosine monophosphates. The polyA tail may contain from 10 to 300 adenosine monophosphates. For example, the polyA tail can comprise 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 comprise, for example, 50 to 250 adenosine monophosphates. In a living organism, such as a cell or an organism, the polyA tail can protect mRNA from attack by enzymes, for example, enzymes in the cytoplasm, and assist in transcription termination, export of mRNA from the nucleus, and translation.

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

[0015] In this specification, unless otherwise stated with respect to the position of the nucleotide sequence, it refers to being connected or positioned 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 transporting a gene or ORF or DNA fragment into a cell. The vector may, for example, be capable of replicating in a cell. The vector may be a virus, a bacteriophage, a provirus, a plasmid, a phagemid, a transposon, or an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a plant artificial chromosome (PLAC), etc.

[0017] The first aspect provides an antigenic peptide comprising two or more KRAS variant peptides linked together, wherein the KRAS variant peptides are selected from the group consisting of G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L and A146T variant peptides.

[0018] As used herein, the term "specific substitution" variant peptide refers to a variant peptide having a "specific substitution." For example, "G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, and A146T variant peptide" refers to a KRAS variant peptide having "G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, and A146T" substitutions, respectively. In other words, it refers to a contiguous fragment of KRAS containing the "specific substitution."

[0019] The above antigen peptide may include G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, and A146T mutant peptides. The above KRAS mutant peptide may further include one or more of G12S, G13C, L19F, and K117N mutant peptides.

[0020] The above antigen peptide may include G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, K117N and A146T mutant peptides.

[0021] The KRAS mutant peptide may have 7 to 50 amino acid residues. The mutant peptide may be represented as E1-M-E2, where M represents a mutant residue relative to the wild-type sequence, and E1 and E2 represent an extension sequence 1 extending from the mutant residue to the N-terminus and an extension sequence 2 extending from the mutant residue to the C-terminus, respectively. In the mutant peptide, M may be G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S, or L19F. The E1 may be 0 to 23 amino acids in length, for example, 0 to 11 amino acids. The E2 may be 0 to 23 amino acids in length, for example, 0 to 11 amino acids. For example, the extended sequence 1 and the extended sequence 2 may be 0 to 11, 1 to 11, 0 to 8, 3 to 11, 1 to 5, 3 to 6, 3 to 7, 4 to 7, 8 to 11, or 8 to 12 amino acids. When the mutant peptide is 9 aa in length, E1 and E2 may be 0 to 8 and 0 to 8; 1 to 5 and 3 to 7; 1 to 5 and 3 to 7; or 4 and 4, respectively. When the mutant peptide is 15 aa in length, E1 and E2 may be 3 to 11 and 3 to 8; 8 to 11 and 3 to 6; Or it can be 7 and 7. The extended sequence 1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids in length, and the extended sequence 2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids in length.

[0022] The antigen peptide may be a plurality of the mutant peptides linked via peptide bonds. For example, the antigen peptide may be a plurality of the mutant peptides linked via peptide bonds, 3 to 50, 5 to 40, 5 to 35, 7 to 33, 9 to 33, 10 to 31, or 11 to 30. The antigen peptide may be a plurality of the mutant peptides linked via peptide bonds, 11, 22, or 30. The linkage may be a direct linkage of the mutant peptides, or a linkage via a peptide linker. The peptide linker may not affect or increase the expression thereof from mRNA, and / or may not affect or increase the immunogenicity of the antigen peptide. The linker may comprise a hydrophilic amino acid. The linker may be hydrophilic. The above linker may be GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK.

[0023] Each mutant peptide included in the above antigen peptide may include mutant residues at the same or different positions. For example, the antigen peptide may include mutant residues at 1, 2, 3, 4, 5, or 6 mutant positions.

[0024] In one specific embodiment, the variant peptide may be 9, 15, or 21 aa in length. The antigenic peptide may comprise a variant peptide having a length of 9 aa, a variant peptide having a length of 15 aa, and / or a variant peptide having a length of 21 aa.

[0025] In one specific embodiment, the antigen peptide may further comprise one or more of the G12S, G13C, L19F, and K117 variant peptides. The antigen peptide may comprise the G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S, and L19F variant peptides, wherein the variant peptides may be 9 or 15 amino acids in length. The above antigen peptide comprises a first portion and a second portion, each portion comprising at least two mutant peptides selected from the group consisting of G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S, and L19F mutant peptides, wherein each mutant peptide of the first portion may be 9 amino acids in length, and each mutant peptide of the second portion may be 15 amino acids in length. The above antigen peptide comprises a first portion and a second portion including G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S, and L19F mutant peptides, wherein each mutant peptide of the first portion may have a length of 9 amino acids, and each mutant peptide of the second portion may have a length of 15 amino acids. The first portion may be located closer to the N-terminus than the second portion. In addition, the first portion may be located closer to the C-terminus than the second portion. When the mutant peptide has a length of 9 amino acids, E1 and E2 may be 0 to 8 and 0 to 8, respectively. When the above mutant peptide has an amino acid length of 15, E1 and E2 may be 3 to 11 and 3 to 8, respectively.

[0026] In one specific example, the antigen peptide comprises G12R, G12D, Q61H, G12C, A146T, G12V, Q61R, G13D, Q61K, G12A, and Q61L variant peptides, each variant peptide having a length of 9 amino acids, and E1 and E2 having a length of 1 to 5 amino acids and 3 to 7 amino acids, respectively.

[0027] In one specific example, the antigen peptide comprises a first portion and a second portion comprising G12R, G12D, Q61H, G12C, A146T, G12V, Q61R, G13D, Q61K, G12A, and Q61L mutant peptides, wherein each mutant peptide of the first portion may be 9 amino acids in length, and each mutant peptide of the second portion may be 9 amino acids in length. The first portion may be located closer to the N-terminus than the second portion. Additionally, the first portion may be located closer to the C-terminus than the second portion. In the mutant peptides of the first portion and the second portion, E1 and E2 may be 1 to 5 and 3 to 7 amino acids in length, respectively.

[0028] In one specific example, the antigen peptide comprises a first portion and a second portion comprising G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L and A146T variant peptides, wherein each variant peptide of the first portion may have a length of 9 amino acids and each variant peptide of the second portion may have a length of 15 amino acids. The first portion may be located closer to the N-terminus than the second portion. Furthermore, the first portion may be located closer to the C-terminus than the second portion. When the variant peptide has a length of 9 amino acids, E1 and E2 may have a length of 1 to 5 amino acids and 3 to 7 amino acids; or 4 and 4 amino acids, respectively. When the variant peptide has a length of 15 amino acids, E1 and E2 may have a length of 8 to 11 amino acids and 3 to 6 amino acids, respectively. Or it could be 7 and 7.

[0029] In one specific embodiment, the antigen peptide comprises a first portion and a second portion comprising G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, K117N and A146T mutant peptides, wherein each mutant peptide of the first portion may have a length of 21 amino acids, and each mutant peptide of the second portion may have a length of 21 amino acids. The first portion may be positioned closer to the N-terminus than the second portion. Additionally, the first portion may be positioned closer to the C-terminus than the second portion. When the mutant peptide has a length of 21 amino acids, E1 and E2 may have a length of 8 to 12 and 8 to 12 amino acids; 9 to 11 and 9 to 11 amino acids; or 10 and 10 to 10 amino acids, respectively.

[0030] The antigen peptide may have a signal peptide linked to the N-terminus. The signal peptide may be a secretory signal peptide. The signal peptide may include an IgK signal peptide, a tPA signal peptide, an AZU1 signal peptide, a LAMP1 signal peptide, or an MHC-1 signal peptide. The antigen peptide may be linked to the signal peptide via a linker. The linker may be a peptide linker. The linker may include a hydrophilic amino acid. The linker may be hydrophilic. The linker may be GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK.

[0031] The above antigen peptide may be any one of the amino acid sequences of SEQ ID NOs: 8 to 14, 122 to 129, 289, and 312.

[0032] The second aspect provides mRNA encoding the antigenic peptide.

[0033] The mRNA may include an expression regulatory sequence or a stabilizing sequence. The expression regulatory sequence or stabilizing sequence may be a 5'UTR, a 3'UTR, and / or a polyA. The 5'UTR, 3'UTR, and polyA may be natural or non-natural sequences. These sequences may be an open reading frame (ORF) or operably linked to each other.

[0034] The above 5'UTR may have a sequence identity of 70% or more, for example, 80% or more, 90% or more, or 95% or more, with the nucleotide sequence of SEQ ID NO: 1. The above 5'UTR may include SEQ ID NO: 1.

[0035] The above 5'UTR includes p53, OX40L, albumin, serum amyloid A, apolipoprotein 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 lb, 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 It may be derived from mRNA encoding a protein selected from the group consisting of hydroxylase, and collagen.

[0036] The above 3'UTR may have a sequence identity of 70% or more, for example, 80% or more, 90% or more, or 95% or more, with the nucleotide sequence of SEQ ID NO: 2. The above 3'UTR is from an mRNA encoding a protein selected from the group consisting of p53, OX40L, albumin, serum amyloid A, apolipoprotein 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 lb, 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. It may have originated from.

[0037] The mRNA may comprise one or more modified nucleotides. The modified polynucleotide, when introduced into a cell or organism, may exhibit reduced degradation in the cell or organism compared to an unmodified polynucleotide. Furthermore, the modified polynucleotide, when introduced into a cell or organism, may exhibit reduced immunogenicity (e.g., a reduced innate immune response) in the cell or organism compared to an unmodified polynucleotide.

[0038] Nucleotides having a modified cytosine may include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, or combinations thereof.

[0039] Nucleotides having a modified adenine may include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), or combinations thereof.

[0040] Nucleotides having a modified guanine 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 a combination thereof.

[0041] Nucleotides having the above chemical modifications are 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-o-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dehydropseudouridine, 5-methoxyuridine, 2'-O-methyluridine, Or a combination thereof. The chemical modification may be at the 5' position of uracil. The chemical modification may be a modification of uracil to N1-methylpseudouridine. The chemical modification may be a modification of uracil to N1-ethylpseudouridine.

[0042] The mRNA may comprise a 5' cap. The term "5'-cap" refers to a cap structure found at the 5'-end of an mRNA molecule. The 5'-cap may comprise a guanosine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate linkage. The guanosine may be methylated at the 7-position, for example, m 7 G, or 3'-O-Me-m 7G may be. The term "conventional 5' cap" refers to a naturally occurring RNA 5' cap, for example, 7-methylguanosine (m7G). As used herein, the term "5' cap" includes 5' cap analogs that have been modified to resemble the RNA cap structure and have the ability to stabilize RNA when attached to RNA, for example, in vivo and / or in cells. The provision of a 5' cap or 5' cap analog to RNA can be accomplished by in vitro transcription of a DNA template in the presence of a 5' cap or 5' cap analog, such that the 5' cap or 5' cap analog is co-transcriptionally incorporated into the resulting RNA strand, or the 5' cap or 5' cap analog can be generated post-transcriptionally using a capping enzyme, for example, the capping enzyme of vaccinia virus. The above 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.

[0043] The mRNA may have one or more Us substituted with N1-methyl-pseudouridine. In the mRNA, the GA at the 5' end may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA). In the mRNA, all Us may be substituted with N1-methyl-pseudouridine. The mRNA may have all Us substituted with N1-methyl-pseudouridine, and the GA at the 5' end may have a cap structure of m7(3'OMeG)(5')ppp(5')(2'OMeA).

[0044] The mRNA may be codon optimized for the host cell. The codon optimization may vary depending on the host cell in which the mRNA is produced.

[0045] The mRNA may comprise any one of the nucleotide sequences of SEQ ID NOs: 281 to 288 and 313.

[0046] The third aspect provides DNA encoding the antigenic peptide.

[0047] The DNA may further comprise expression control and restriction enzyme sites. The expression control may be a promoter. The expression control may be operably linked to an open reading frame (ORF) or other expression control.

[0048] The 3'UTR coding sequence may include a recognition sequence of a restriction enzyme. The restriction enzyme may be HindIII, XhoI, NheI, or a combination thereof. The promoter may be operably linked to a sequence encoding the 5'UTR. The 5'UTR, the antigen peptide coding sequence, and the 3'UTR may be linked to form a common transcript when transcribed in vitro or in vivo. The sequence encoding the 3'UTR may be operably linked to a polyA sequence.

[0049] The sequence encoding the above 5'UTR may be a sequence encoding the sequence of SEQ ID NO: 1, and the sequence encoding the above 3'UTR may be a sequence including a sequence encoding the sequence of SEQ ID NO: 2. In this case, the sequence encoding the KRAS antigen peptide may have a nucleotide sequence of any one of the sequences of SEQ ID NOs: 281 to 288. In addition, the polyA sequence may include the sequence of SEQ ID NO: 3.

[0050] As used herein, the term "nucleic acid capable of being transcribed to produce a common transcript" refers to nucleic acid sequences that are functionally linked or operatively linked to each other such that, when transcribed, an RNA molecule is formed comprising a transcript in which the nucleic acid sequences are covalently linked to each other. The transcription may be performed under the control of a promoter, where appropriate, after linearization of the nucleic acid molecule, for example, a closed circular nucleic acid molecule, for example, after restriction enzyme digestion of the nucleic acid molecule. The common transcript may be separated, where appropriate, by a sequence located between the nucleic acid sequences.

[0051] The above DNA molecule may be a closed circular molecule or a linear molecule.

[0052] 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 be a vector. The vector may be a cloning vector or an expression vector. The DNA molecule may be suitable for in vitro transcription of mRNA after linearization.

[0053] A fourth aspect provides a composition comprising an mRNA encoding the antigenic peptide or a combination of the antigenic peptides as an active ingredient. The composition may be an immunogenic composition.

[0054] The antigen peptide and the mRNA encoding it are as described in the first aspect and the second aspect.

[0055] The composition may be for inducing an immune response to a KRAS mutant peptide. The composition may be for preventing or treating cancer. The subject to be prevented or treated may have a KRAS mutation. The mutation may be a mutation in one or more of G12, G13, L19, Q61, K117, and A146. The mutation may be one or more of G12D, G12R, G12C, G12V, G12A, G12S, G13D, G13C, L19F, Q61H, Q61R, Q61K, Q61L, K117N, and A146T. The mutation may be one or more of G12D, G12R, G12C, G12V, G12A, and G12S; one or more of G13D and G13C; L19F; It may have one or more of Q61H, Q61R, Q61K, and Q61L; K117N, and A146T.

[0056] The above composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0057] The composition may be for treating cancer. The cancer may be a cancer comprising a KRAS mutation. The mutation may have a mutation at one or more of positions G12, G13, L19, Q61, K117, and A146. The mutation may have one or more G12 mutations of G12D, G12R, G12C, G12V, G12A, and G12S, one or more G13 mutations of G13D and G13C, an L19F mutation, one or more Q61 mutations of Q61H, Q61R, Q61K, and Q61L, a K117N mutation, and an A146T. The mutation may have a mutation at one or more of positions G12, G13, Q61, and A146. The above mutation may have one or more G12 mutations among G12D, G12R, G12C, G12V, G12A, and G12S, one or more G13 mutations among G13D and G13C, one or more Q61 mutations among Q61H, Q61R, Q61K, and Q61L, and A146T.

[0058] The composition can be administered to a subject and the mRNA can be translated in vivo to produce the antigenic peptide.

[0059] The composition may be contacted with a cell, tissue or organism in an “effective amount” to induce an immune response.

[0060] The effective amount can be determined based on the target tissue, target cells, means of administration, properties of the mRNA, such as size and content of modified nucleosides, and other components of the composition.

[0061] The composition may be administered in combination with other prophylactic or therapeutic compounds. The prophylactic or therapeutic compound may be, for example, an anticancer agent. As used herein, the anticancer agent may be any known agent.

[0062] The composition may be administered systemically or locally. Local administration may involve administering the composition locally to the tissue where the cancer is present. The tissue where the cancer is present may be intramuscular, subcutaneous, intradermal, nasal, or pulmonary.

[0063] The composition may include one or more pharmaceutically acceptable carriers, diluents, or excipients. The mRNA in the composition may be formulated or complexed with the excipients. The carriers, diluents, or excipients may be those known in the art.

[0064] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or other additional ingredients included in the composition may vary depending on the nature, size, and / or condition of the subject to be treated and the route of administration. For example, the composition may comprise from 0.1% to 100%, for example, from 0.5% to 50%, from 1.0% to 30%, from 5.0% to 80%, or greater than 80% (w / w) of the active ingredient.

[0065] The composition may be formulated as a nanoparticle. The nanoparticle may be known to be capable of delivering mRNA to cells. For example, the nanoparticle may be a lipid nanoparticle (LNP). The composition may be formulated as a lipid nanoparticle (LNP) or may be bound to an LNP. The binding may include binding within or to the surface of an LNP. For example, the composition may be formulated within a lipid polycation complex or may be bound to a lipid polycation complex. The lipid polycation complex is also referred to as a cationic lipid nanoparticle. The polycation may include a cationic polypeptide 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. Additionally, the composition may be formulated or bound to lipid nanoparticles comprising a sterol such as distearoylphosphatidylcholine (DSPC), 1,2 distearoyl-sn-glycerol-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol, or a non-cationic lipid such as dioleoyl phosphatidylethanolamine (DOPE). Additionally, the composition may be formulated or bound to lipid nanoparticles comprising 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).

[0066] The lipid nanoparticle formulation can include a cationic lipid, a phospholipid, a sterol such as cholesterol, a PEG-lipid, or a combination thereof. The lipid nanoparticle formulation can include, for example, a cationic lipid, a phospholipid, a sterol such as cholesterol, and a PEG-lipid. Additionally, the lipid nanoparticle can include a PEG-modified lipid, a non-cationic lipid, a sterol, and an ionizable lipid, or a combination thereof. The lipid nanoparticle can include 0.5 to 15 mol % PEG-modified lipid, 5 to 25 mol % non-cationic lipid, 25 to 55 mol % sterol, and 20 to 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.

[0067]

[0068] (Compound 1)

[0069] The PEG-modified lipid may be compound 2 (ALC-0159) having 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) having the following structure.

[0070]

[0071] (Compound 2)

[0072]

[0073] (Compound 3)

[0074] A fifth aspect provides a method for inducing an immune response in a subject, comprising administering to the subject an antigenic peptide or mRNA. The method may be for preventing or treating cancer.

[0075] The above administration may be administered in an amount effective to induce an immune response in the subject. The above administration may be administered in an amount effective to prevent or treat cancer in the subject. The above administration may be systemic or local. The above local administration may be administered locally to the tissue where the cancer exists. The tissue where the cancer exists may be intramuscular, subcutaneous, intradermal, nasal, or pulmonary. The above administration may be parenteral or oral.

[0076] The subject may be a mammal, including a human. The administration may be by locally administering the mRNA molecule intracellularly or extracellularly to a tissue where cells with a high level of KRAS mutations are present.

[0077] The method may involve producing the antigen peptide in cells of a subject to inhibit cancer cell proliferation. Furthermore, the method may involve treating a disease in the subject. The antigen peptide may induce an immune response against cancer in the subject.

[0078] Antigenic peptides comprising two or more KRAS variant peptides linked according to one aspect can be used to efficiently induce an immune response in an individual.

[0079] mRNA encoding the above antigenic peptide according to one aspect can be used to efficiently induce an immune response in an individual.

[0080] DNA encoding the antigen peptide according to one aspect can be used to produce the antigen peptide.

[0081] An immunogenic composition according to one aspect can be used to induce an immune response in a subject.

[0082] According to a method for inducing an immune response to a KRAS mutant peptide in an individual according to one aspect, an immune response to a KRAS mutant peptide can be efficiently induced in an individual.

[0083] Figure 1 is a diagram showing a vector encoding an antigen to which multiple KRAS mutant peptides are linked.

[0084] Figure 2 shows the results of Western blot analysis confirming that KRAS antigen protein is expressed when KRAS mutant antigen mRNA is introduced into lung cancer cells.

[0085] Figure 3 is a drawing showing the results of Western blot analysis confirming that KRAS antigen protein is expressed when the produced KRAS mutant antigen mRNA is introduced into lung cancer cells.

[0086] Figure 4a is an image showing the results of SRB staining after co-culturing hPBMCs with mRNA introduced and target cells.

[0087] Figure 4b is a diagram showing the results of measuring absorbance after co-culturing mRNA-introduced hPBMCs and target cells and then staining with SRB.

[0088] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0089] Example 1: Anticancer substance sequence for increasing T cell activation against KRAS mutant sequence

[0090] An antigen comprising multiple KRAS mutant peptides for the treatment of cancer patients with KRAS mutations was designed. The mutant peptide comprises a mutant residue from the KRAS wild-type sequence (SEQ ID NO: 4), and comprises an extension sequence 1 extending N-terminally from the mutant residue, and an extension sequence 2 extending C-terminally from the mutant residue. The mutant residue comprises G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S, or L19F. Extension sequence 1 or extension sequence 2 may be 0 to 11 amino acids in length.

[0091] The above mutant peptide may be represented as E1-M-E2, where M represents a mutant residue, and E1 and E2 represent an extension sequence 1 extending from the mutant residue to the N-terminus and an extension sequence 2 extending from the mutant residue to the C-terminus, respectively. The above mutant peptide E1-M-E2 may be one in which M is G12D, G12R, G12C, Q61H, G12V, A146T, G13D, Q61R, G12A, Q61K, G13C, Q61L, K117N, G12S or L19F, E1 may be 0 to 11 amino acids in length, and E2 may be 0 to 11 amino acids in length.

[0092] The antigen peptide may be a plurality of mutant peptides linked via peptide bonds. The linkage may be a direct linkage of the mutant peptides or a linkage via a peptide linker. The peptide linker may be a GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK linker. Each epitope peptide included in the antigen peptide may include mutant residues at the same or different positions. For example, the antigen peptide may include mutant residues at 1, 2, 3, 4, 5, or 6 mutation positions. The antigen peptide may include the same mutant residue at the same mutation position, or may include different mutant residues at the same mutation position.

[0093] The antigen peptide may be linked to a secretory signal peptide. The signal peptide may be an IgK signal peptide, an AZU1 signal peptide, a LAMP1 signal peptide, an MHC-1 signal peptide, or a tPA signal peptide. The antigen peptide may be linked to the signal peptide via a linker. The linker may be a peptide linker. The peptide linker may be a GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK linker.

[0094] Table 1 shows the epitope composition of the antigen peptides prepared in this example and their amino acid sequences (SEQ ID NOs: 8 to 14).

[0095] [Table 1]

[0096]

[0097]

[0098]

[0099] The sequences in which the open reading frames (ORFs) encoding the antigen peptides (SEQ ID NOs: 8 to 14, and 289) described in Table 1 were linked to the 5'UTR and signal sequence were synthesized. The 5' and 3' ends of the synthesized sequences contained HindIII and XhoI restriction enzyme sequences, respectively, so that they could be used for cloning. The sequences encoding each of the synthesized antigens and the template vector were each cleaved with HindIII and XhoI restriction enzymes, and linked using DNA ligase to produce a vector containing a sequence encoding "mRNA". The composition of each antigen peptide is as shown in Table 1. Each mutant peptide was separated by parentheses, and the presence or absence of a linker between the mutant peptides was indicated by '-'. The linkers indicated by '-' are GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK linkers. The full-length mRNA encoding the antigen peptides shown in Table 1 each has the structure 5'UTR-ORF-stop codon-3'UTR-polyA. Table 2 shows the mutant peptides included in epitope structure 1 and their mRNA sequences.

[0100] [Table 2]

[0101]

[0102]

[0103] Table 3 shows the mutant peptides and their mRNA sequences included in epitope composition 2.

[0104] [Table 3]

[0105]

[0106] Table 4 shows the mutant peptides included in epitope configuration 3 and their mRNA sequences. The antigen peptides of epitope configuration 3 have the same mutant peptide configuration, but each linker is different. In the columns showing the mRNA sequences of the mutant peptides, for mutant peptides 1 to 22, the sequences in the top, second from the top, and third rows represent the mRNA sequences of the antigen peptides of SEQ ID NOs: 10, 11, and 12, respectively.

[0107] [Table 4]

[0108]

[0109]

[0110]

[0111]

[0112] Table 5 shows the mutant peptides and their mRNA sequences included in epitope composition 4.

[0113] [Table 5]

[0114]

[0115]

[0116] Table 6 shows the mutant peptides included in epitope composition 5 and their mRNA sequences.

[0117] [Table 6]

[0118]

[0119]

[0120] Table 7 shows the mutant peptides included in epitope composition 6 and their mRNA sequences.

[0121] [Table 7]

[0122]

[0123] Example 2: Cloning of a sequence encoding an antigenic peptide linked to multiple KRAS mutant peptides.

[0124] In order to clone a nucleotide sequence having a DNA sequence encoding an antigen peptide linked to multiple KRAS mutant peptides described in Example 1 as 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' terminal portions, respectively, to synthesize the gene.

[0125] The synthesized coding sequence and template vector were cut with HindIII and XhoI restriction enzymes, and connected using DNA ligase to produce a vector containing a sequence encoding "mRNA."

[0126] Figure 1 illustrates a vector encoding an antigen to which multiple KRAS mutant peptides are linked.

[0127] Table 8 shows the full-length amino acid and nucleotide sequences of antigenic peptides and mRNA encoding them, including signal sequences and linker sequences. The mRNA has the structure 5'UTR (SEQ ID NO: 1)-ORF-stop codon-3'UTR (SEQ ID NO: 2)-polyA (SEQ ID NO: 3).

[0128] [Table 8]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] In Table 8, candidates 1 to 3 and 5 to 9 each contain an IgK signal sequence, and candidate 4 contains a tPA signal sequence. In Table 8, the sequences in bold among the sequences of candidates 1 to 9 (SEQ ID NOs: 122 to 129 and 312) represent the sequences of the signal peptides.

[0140] Example 3: Confirmation of expression of antigen mRNA linked to KRAS mutant peptide in lung cancer cells.

[0141] mRNA encoding an antigen linked to a KRAS mutant peptide according to the present invention was introduced into lung cancer cells, and the level of expression was confirmed. Lewis lung carcinoma cells (LL / 2; ATCC, #CRL-1642) were used as lung cancer cells.

[0142] In Example 2, mRNA produced by in vitro transcription (IVT) was transfected with Lipofectamine TM MessengerMAX TM Transfection Reagents (Thermo Scientific TM , Cat No. LMRNA001) was used to deliver the RNA to LL / 2 cells and expression was confirmed.

[0143] Cells were seeded at 4x10 per well in a 6-well plate. 5 After seeding at 2 mL / cells, the cells were cultured for 24 hours. The mRNA and the reagent were mixed at a ratio of 0.5 μL per 1 μg of mRNA and reacted for 20 minutes. 10 μg of each reactant was added to the well and cultured for 24 hours in DMEM medium containing 10% FBS at 37°C and 5% CO2. After completion of the culture, lysis buffer, M-PER TM (Mammalian Protein Extraction Reagent) (Thermo Scientific TM, Cat No. 78501) was added to the well to rupture the cells and extract the proteins, then Pierce TM BCA Protein Assay Kit (Thermo Scientific TM , Cat No. 23225) was used to quantify BCA protein. The obtained samples were diluted with rupture 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 equalize the protein concentration. Western blot experiments were performed on the obtained diluted samples using anti-RAS (G12D mutant) monoclonal antibody (CST, Cat No. #14429S) and anti-GAPDH (Santa Cruz, Cat No. 32223).

[0144] Figure 2 shows the results of Western blot analysis of KRAS antigen protein expression after introducing the produced KRAS mutant antigen mRNA into lung cancer cells. The KRAS protein showed different sizes and intracellular expression levels for each candidate antigen, with high expression levels observed for candidate substances 1 and 3 to 8. In Figure 2, "short" and "long" indicate exposure times during the Western blot experiment, with exposure times of approximately 20 seconds and approximately 3 minutes, respectively.

[0145] Example 4: Confirmation of expression of antigen mRNA linked to KRAS mutant peptide in lung cancer cells.

[0146] In this example, mRNA candidate substances 1 and 9 encoding antigens linked to KRAS mutant peptides according to the present invention were delivered to lung cancer cells, and the level of their expression was confirmed. Lewis lung carcinoma cells (LL / 2; ATCC, #CRL-1642) were used as lung cancer cells. LL / 2 cells are a mouse lung cancer cell line established from the lungs of C57BL / 6 mice bearing tumors induced by implantation of primary Lewis lung carcinoma.

[0147] The mRNA prepared in Example 2 was used with Lipofectamine TM MessengerMAX TM Transfection Reagents (Thermo Scientific TM , Cat No. LMRNA001) was used to transfect LL / 2 cells and confirm expression. For this purpose, in vitro expression experiments were conducted as follows. KRAS mRNA was produced by in vitro transcription (IVT) as described above.

[0148] 4x10 cells in a 6-well plate 5 Cells were seeded at 2 mL / well and cultured for 24 hours. mRNA and the Lipofectamine reagent were mixed at a ratio of 0.5 μL per 1 μg of mRNA and reacted for 20 minutes. 5 μg of the reactants were added to each well and cultured for 24 hours. DMEM medium containing 10% FBS was used as the medium, and culture was performed at 37°C and 5% CO2. After completion of culture, lysis buffer, M-PER TM (Mammalian Protein Extraction Reagent) (Thermo Scientific TM , Cat No. 78501) was added to each well to rupture the cells and extract the proteins, then PierceTM BCA Protein Assay Kit (Thermo Scientific TM , Cat No. 23225) was used to quantify BCA protein. The obtained samples were diluted with rupture 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 equalize the protein concentration. Western blot experiments were performed on the obtained diluted samples using anti-RAS (G12D mutant) monoclonal antibody (CST, Cat No. #14429S) and anti-GAPDH (Santa Cruz, Cat No. 32223).

[0149] Figure 3 is a drawing showing the results of Western blot analysis confirming that KRAS antigen protein is expressed when the produced KRAS mutant antigen mRNA is introduced into lung cancer cells.

[0150] As shown in Fig. 3, candidate proteins 1 and 9 had molecular weights of 50 kDa and 32 kDa, respectively, as designed, and it was confirmed that both proteins were expressed at similar levels.

[0151] The above results demonstrate that the KRAS vaccine mRNA can be introduced into lung cancer cells and stably express KRAS protein. This demonstrates that the KRAS vaccine mRNA is sufficiently stable during the transfection process and within the cellular environment, and is fully functional enough to enable the intracellular transcription, translation, and secretion machinery to operate.

[0152] Example 5: Evaluation of target cell survival rate in co-culture of immune cells and target cells introduced with anticancer vaccine mRNA.

[0153] In this example, immune cells introduced with KRAS anticancer vaccine mRNA were co-cultured with target cells expressing KRAS wild type or mutant type, and it was confirmed whether the immune cells suppressed the survival of the target cells.

[0154] The KRAS anticancer vaccine used candidates 1 and 9, and human peripheral blood mononuclear cells (hPBMCs) were used as immune cells. Additionally, normal lung cells, lung cancer cells, colon cancer cells, and pancreatic cancer cells were used as target cells. The target cells and their KRAS types are shown in Table 9 below.

[0155] [Table 9]

[0156]

[0157] First, to select hPBMCs that effectively bind to candidate substances 1 and 9, we analyzed the human leukocyte antigen (HLA) types that effectively bind to the long candidate substance 9. HLA is a member of the major histocompatibility complex (MHC), has various genotypes, and plays a crucial role in the immune response. The analysis was performed using a program (NetMHCpan. Ver. 4.1) that predicts peptide binding affinity.

[0158] As a result, hPBMCs (StemCell Technologies, Cat No. 70025.3) with HLA-A types HLA-A*02:01 and HLA-A*11:01 that can bind to candidate substance 9 were used.

[0159] hPBMCs were seeded at 1.8 x 10 in a well of a 6-well cell culture plate containing 2 mL of medium. 6Cells were seeded at 2 mL / well and cultured for 2 hours to stabilize. The medium used was DMEM (Gibco, Cat No. 10569010) containing 10% FBS and 1% P / S (Penicillin-Streptomycin). KRAS anticancer vaccine mRNA candidates 1 or 9 and Lipofectamine TM MessengerMax TM The transfection reagent was mixed at a ratio of 1.25 μL per 1 μg of mRNA and reacted for 20 minutes. After that, 5 μg of the reaction mixture was added to each well and cultured for 30 hours to obtain hPBMCs introduced with mRNA.

[0160] Target cells expressing KRAS wild-type and KRAS proteins with G12C, G12D, G12V, or G13D mutations were seeded in a 96-well cell culture plate 24 hours before co-culture and allowed to attach. hPBMCs with mRNA introduced were collected in a single 50 mL tube, and the cell number was analyzed. Then, they were inoculated into a 96-well plate containing the DMEM medium with target cells attached at a ratio of 1:6 to 1:25, and co-cultured for 48 hours. In this example, all cultures were performed in a 37°C, 5% CO2 incubator.

[0161] After co-culture, sulforhodamine B (SRB) staining was performed to analyze target cell viability. SRB staining is a method for quantifying cell viability by staining intracellular proteins. First, the medium was removed to remove hPBMCs. The cells were fixed in a 10% TCA solution at 4°C for 30 minutes and then washed with distilled water.

[0162] Afterwards, 0.4% SRB solution was added to the 96-well plate containing the target cells and stained for 30 minutes at room temperature. After staining, images were acquired using a Canon scanner (Canon, Cat No. DADF-AV1).

[0163] Finally, the solid SRB was dissolved in 10 mM Tris (Trizma base solution, Sigma, Cat No. 10708976001), and the absorbance was measured at 540 nm using a microplate reader.

[0164] Figure 4a is an image showing the results of SRB staining after co-culture of mRNA-introduced hPBMCs and target cells. In Figure 4a, in the PBMC(-) column, the control represents the group treated only with serum-free media, and in the PBMC(+) column, the control and Lipo represent the groups treated only with serum-free media and Lipofectamine, respectively. TM MessengerMax TM Indicates the group that was processed.

[0165] Figure 4b is a diagram showing the results of measuring the absorbance after co-culture of mRNA-introduced hPBMCs and target cells, followed by SRB staining. In Figure 4b, control and Lipo represent the groups treated with serum-free medium only and Lipofectamine, respectively. TM MessengerMax TM Indicates the group that was processed.

[0166] As shown in Figures 4a and 4b, when hPBMCs and target cells were co-cultured with KRAS anticancer vaccine candidate 1 or 9 mRNA, the cell viability was analyzed, and the results showed that the cell viability was not affected for normal cells MRC5 and cancer cells H1299 expressing KRAS wild type. This indicates that the KRAS anticancer vaccine mRNA of the present invention does not inhibit the growth of cells that do not express the antigen included in the KRAS anticancer vaccine, regardless of whether they are cancer cells or normal cells.

[0167] On the other hand, target cells expressing KRAS mutants, such as HCT15 (G13D), MIAPaCa-2 (G12C), AsPC-1 (G12D), and CaPan-2 (G12V), were confirmed to have their survival inhibited by the candidate substance. This indicates that the KRAS anticancer vaccine mRNA of this invention specifically inhibits the growth of cancer cells expressing the antigen included in the KRAS anticancer vaccine. Table 10 tabulates the results of Fig. 4b.

[0168] [Table 10]

[0169]

[0170] These results suggest that the KRAS anticancer vaccine is a drug that shows excellent on-target therapeutic effects by activating immune cells according to the intended mechanism, specifically inhibiting only cancer cells expressing the antigen contained in the KRAS anticancer vaccine candidate while not acting on normal cells.

Claims

An antigenic peptide having 1.2 or more KRAS variant peptides linked thereto, wherein the KRAS variant peptides are selected from the group consisting of a G12R variant peptide, a G12D variant peptide, a G12C variant peptide, a G12V variant peptide, a G12A variant peptide, a G13D variant peptide, a Q61H variant peptide, a Q61R variant peptide, a Q61K variant peptide, a Q61L variant peptide, and an A146T variant peptide.

2. In claim 1, the mutant peptide is an antigen peptide having a length of 9, 15, or 21 amino acids.

3. An antigen peptide according to claim 1, further comprising at least one of a G12S mutant peptide, a G13C mutant peptide, an L19F mutant peptide, and a K117N mutant peptide.

4. In claim 3, a G12D mutant peptide, a G12R mutant peptide, a G12C mutant peptide, a Q61H mutant peptide, a G12V mutant peptide, an A146T mutant peptide, a G13D mutant peptide, a Q61R mutant peptide, a G12A mutant peptide, a Q61K mutant peptide, a G13C mutant peptide, a Q61L mutant peptide, a K117N mutant peptide, a G12S mutant peptide, and a L19F mutant peptide; or An antigenic peptide comprising a G12D mutant peptide, a G12C mutant peptide, a G12V mutant peptide, a G12A mutant peptide, a G13D mutant peptide, a Q61H mutant peptide, a Q61R mutant peptide, a Q61K mutant peptide, a Q61L mutant peptide, a K117N mutant peptide, and an A146T mutant peptide, wherein the mutant peptide is 9, 15, or 21 amino acids in length.

5. In claim 3, a first part and a second part each including a G12D mutant peptide, a G12R mutant peptide, a G12C mutant peptide, a Q61H mutant peptide, a G12V mutant peptide, an A146T mutant peptide, a G13D mutant peptide, a Q61R mutant peptide, a G12A mutant peptide, a Q61K mutant peptide, a G13C mutant peptide, a Q61L mutant peptide, a K117N mutant peptide, a G12S mutant peptide, and a L19F mutant peptide, wherein each mutant peptide of the first part has a length of 9 amino acids and each mutant peptide of the second part has a length of 15 amino acids; or An antigenic peptide comprising a first portion and a second portion, each portion comprising a G12D mutant peptide, a G12C mutant peptide, a G12V mutant peptide, a G12A mutant peptide, a G13D mutant peptide, a Q61H mutant peptide, a Q61R mutant peptide, a Q61K mutant peptide, a Q61L mutant peptide, a K117N mutant peptide, and an A146T mutant peptide, wherein each mutant peptide of the first portion is 10 amino acids in length, and each mutant peptide of the second portion is 10 amino acids in length.

6. An antigenic peptide according to claim 1, comprising a first part and a second part, each part comprising two or more mutant peptides selected from the group consisting of G12R, G12D, G12C, G12V, G12A, G13D, Q61H, Q61R, Q61K, Q61L, and A146T mutant peptides, wherein each mutant peptide of the first part has a length of 9 amino acids, and each mutant peptide of the second part has a length of 15 amino acids.

7. An antigen peptide according to claim 1, wherein the mutant peptides are linked to each other via a linker.

8. An antigen peptide according to claim 7, wherein the linker comprises at least one hydrophilic amino acid.

9. An antigen peptide according to claim 8, wherein the linker is GPGPG (SEQ ID NO: 5), HEYGAEALERAG (SEQ ID NO: 6), AAY, EAAAK (SEQ ID NO: 7), or KK.

10. An antigenic peptide comprising an amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 289, 122, 123, 124, 125, 126, 127, 128, 129, or 312 in claim 1.

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

12. An mRNA comprising a nucleotide sequence of SEQ ID NO: 281, 282, 283, 284, 285, 286, 287, 288, or 313 according to claim 11.

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

14. An immunogenic composition for preventing or treating cancer according to claim 13.

15. A composition according to claim 13, wherein the mRNA comprises a nucleotide sequence of SEQ ID NO: 281, 282, 283, 284, 285, 286, 287, 288, or 313.

16. A composition according to claim 13, wherein the mRNA is bound to a lipid nanoparticle.

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