KRAS immunogenic peptides

Abstract

The present invention relates to a KRAS immunogenic peptide and a use thereof. The KRAS immunogenic peptide according to the present invention selectively binds to MHC class II in a preferential mode to selectively enhance the immunogenicity of specific immune cells capable of killing cancer cells and thus can be advantageously used as an excellent cancer vaccine for the prevention and / or treatment of cancer by minimizing the immune escape mechanism of cancer cells.

Description

KRAS immunogenic peptide

[0001] The present invention relates to novel KRAS immunogenic peptides and uses thereof.

[0002] Cancer vaccines are designed to activate cytotoxic T cells, allowing them to recognize and fight specific types of cancer, or to induce the production of antibodies that bind to antigens on the surface of cancer cells. The primary therapeutic goal of cancer vaccines is to reduce the size of existing cancerous tissue in patients with cancer, or to prevent cancer recurrence after surgical or chemotherapy removal of cancerous tissue.

[0003] Among these cancer vaccines, the one that is being studied the most and is undergoing the most active clinical trials is known to be the HER2 cancer vaccine.

[0004] Although cancer vaccines using immunogenic peptides are being developed, one of the main reasons for the lack of significant results is that previous therapeutic vaccines target CD8 + Cytotoxic T cells (CD8 + It can be considered that the focus was solely on the activation of cytotoxic T cells. This development direction is based on the idea that cytotoxic immunity plays a major role in anticancer responses. However, CD4 + CD8 when not accompanied by activation of type 1 helper T cells (T-helper 1 cells; Th1 cells) + It is known that T cell activation alone cannot induce a sufficient immune response. Therefore, it is crucial to select an epitope that can induce antigen-specific Th1 immunity in the early stages of vaccine development.

[0005] KRAS is a member of the RAS protein family, a family of proteins that play a crucial role in tumor initiation and progression, regulating the excessive growth and division of tumor cells. The importance of KRAS lies in the fact that abnormal activation or mutation of this protein can promote tumor initiation and growth. KRAS mutations account for 86% of all RAS gene mutations, and structural studies have shown that most of these mutations interfere with KRAS's ability to hydrolyze GTP. These KRAS mutations are closely associated with various types of cancer, including colon and lung cancer, and can affect the development and metastasis of cancer.

[0006] KRAS is considered a key topic in tumor biology and anticancer therapy research, and efforts to develop therapies targeting it are ongoing. While research into the development of therapies targeting KRAS mutations is primarily ongoing, the presence of diverse mutations complicates the development of individual therapies for each mutation. Several groups are conducting vaccine research targeting mutant KRAS as an antigen, and attempts to confirm vaccine efficacy using MHC class I restricted peptides that directly stimulate CD8+ cytotoxic T cells have not achieved the desired success. Recently, efforts have been made to incorporate as many steps as possible into predictive models, including binding affinity for MHC class II, TCR binding affinity, and antigen processing, to identify neoantigens. Following this trend, peptide vaccines using relatively long peptides expected to have binding affinity for MHC class II are being developed to induce Th1 immune responses and block cancer development. However, significant progress has been made to date. Despite extensive efforts, clinically viable cancer therapies that effectively target and block KRAS function remain elusive.

[0007] Under this technical background, the present inventors confirmed that the immunogenicity of specific T cells recognizing the KRAS protein as an antigen is induced by using a non-mutant KRAS peptide alone or a KRAS peptide in which a mutant and non-mutant region are combined as an immunogenic peptide of the KRAS protein (T cell mediated immunity), and completed the present invention for the purpose of utilizing a cancer vaccine that overcomes the shortcomings of existing vaccines by using the above partial sequence.

[0008] The present invention provides a novel KRAS immunogenic peptide, a polynucleotide encoding the peptide, a composition comprising the same, uses thereof, and methods of use thereof.

[0009] The present invention provides a KRAS immunogenic peptide.

[0010] The present invention provides a polynucleotide encoding a KRAS immunogenic peptide, a vector comprising the same, and / or a transformant modified to express the same.

[0011] The present invention provides an immunogenic composition comprising a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0012] The present invention provides a pharmaceutical composition comprising a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; and optionally one or more adjuvants, pharmaceutically acceptable carriers, preservatives, and / or excipients.

[0013] The present invention provides a method for treating or preventing a disease by administering to a subject a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof.

[0014] The present invention provides a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof, for use in medicine.

[0015] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0016] KRAS immunogenic peptide

[0017] One aspect of the present invention for achieving the above purpose provides a peptide having an amino acid sequence of 50 or fewer amino acids and having at least 90% homology with any one sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18.

[0018] Preferably, it may be a peptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to any one sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18, and more preferably, it may be a peptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to the sequence of SEQ ID NO: 18.

[0019] As used herein, the term "peptide" refers to a molecule formed by amino acid residues linked together by amide bonds (or peptide bonds). The peptide may be synthesized using genetic recombination and a protein expression system, and preferably, may be synthesized in vitro using a peptide synthesizer or the like.

[0020] KRAS (Kirsten rat sarcoma virus, hereinafter referred to as KRAS, sequence number 1) is a protein consisting of 189 amino acid residues. The sequence number 1 is derived from Uniprot No. P01116. Most mutations in the KRAS gene are single mutations, such as a change in the 12th amino acid to aspartic acid (G12D), a change in the 12th amino acid to valine (G12V), a change in the 12th amino acid to cysteine ​​(G12C), and a change in the 13th amino acid to aspartic acid (G13D). Only a few cases of multiple mutations have been reported.

[0021] A peptide having a sequence of 50 or fewer amino acids according to the present invention and having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to any one sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18 corresponds to a fragment sequence derived from or modified from the full-length sequence of KRAS.

[0022] In particular, the above sequence according to the present invention can be effective as an immunogenic peptide.

[0023] The peptide according to the present invention may be characterized by stimulating type 1 helper T cells (Th1 cells).

[0024] In addition, the peptide according to the present invention may be characterized by specifically binding to Major Histocompatibility Complex II (MHC class II).

[0025] In addition, the peptide according to the present invention may be characterized in that it does not stimulate immune cells with immune inhibitory function that secrete IL-10.

[0026] An “immunogenic peptide” is a composition capable of inducing an immune response in an animal, including a human, when administered to said animal.

[0027] Immunogenic peptides are peptides that contain antigens and can induce an immune response. The induced immune response can be cellular (T-cell-mediated) or humoral (B-cell-mediated, antibody-producing) immune response. For example, immunogenic peptides can induce both cellular and humoral immune responses.

[0028] Cellular immune response is CD8 + T lymphocyte-mediated response (i.e. cytotoxic response), or CD4 + It may be a T lymphocyte-mediated response (helper response). It may also be a combination of cytotoxic and helper cell immune responses. The helper response may include Th1, Th2, or Th17 lymphocytes (which can induce different cytokine responses, as is known in the art). CD8 + T cells are stimulated and activated by binding to cells expressing MHC class I via immunogenic peptides, and CD4 + T cells are stimulated and activated by binding to antigen-presenting cells expressing MHC class II via immunogenic peptides.

[0029] Immunogenic peptide-MHC class II complexes are presented on the surface of antigen-presenting cells (APCs).

[0030] The peptide according to the present invention may have a peptide length of at least 10, 11, 12, 13, 14, 15, 16, 17, or 18 units, as the minimum unit for presenting the peptide to an MHC class II molecule.

[0031] If desired, the immunogenic peptide may be a combination of multiple immunogenic peptides that can be segmented to present multiple epitopes. Accordingly, it may have an amino acid sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0032] An "antigen" is a molecule or combination of molecules that is intended to elicit an immune response, allowing the immune system of a living animal to recognize it. Antigens can be self-antigens, proteins expressed by host cells, such as tumor antigens. Antigens can be complete proteins or any portion of a protein, such as an immunogenic peptide.

[0033] In the present invention, the antigen may also be composed of a synthetic protein or molecule containing multiple immunogenic peptides (fusion polypeptide) in which several immunogenic peptides are linked.

[0034] In this specification, the terms “immunogenic peptide”, “epitope (antigenic determinant)”, and “antigenic determinant peptide” are used interchangeably.

[0035] The KRAS immunogenic peptide according to the present invention can induce an immune response, preferably T cell-mediated immunity, against KRAS. In particular, it selectively stimulates type 1 helper T cells (Th1 cells) that can recognize KRAS as an antigen among T cells capable of being immunogenic, thereby inducing antigen-specific immunogenicity, while simultaneously having the effect of not stimulating immune cells that suppress the immune response, thereby directly or indirectly killing cancer cells.

[0036] Specifically, the peptide of the present invention can act in a direction that increases the secretion of interferon gamma (IFN-γ) from type 1 helper T cells activated by stimulation, and does not stimulate the secretion of interleukin 10 (IL-10), a substance that suppresses cytotoxicity against cancer cells. Cell presentation for Th1 cell stimulation occurs through MHC class II. Therefore, the peptide according to the present invention can exhibit excellent efficacy in that it acts by selectively and preferentially binding to MHC class II.

[0037] In addition, in order to derive the peptide of the present invention during the development process, a pan-HLA DR binding epitope (PADRE) that has strong binding affinity to MHC class II but can exhibit effectiveness regardless of polymorphism was searched.

[0038] In addition, unlike MHC class I, MHC class II has the characteristic of causing an MHC class-T cell receptor reaction even if only a portion of the entire epitope matches. In other words, the immunogenic peptide according to the present invention does not exhibit HLA type restriction and thus has universality (off-the-shelf).

[0039] In addition, since the immunogenic peptide according to the present invention additionally includes a non-mutated region as an antigen, it has the versatility to be used for treating various subjects without the need to separately select the subjects.

[0040] Therefore, the peptide according to the present invention can exhibit an effect as an immunogenic peptide, and can be utilized for the purpose of a cancer vaccine or for the purpose of preventing or treating cancer.

[0041] The peptide according to the present invention may exhibit one or more of the following functional effects, but is not limited thereto:

[0042] CD4 by selectively binding to MHC class II and forming the immunopeptidome + Induces T cell responses;

[0043] Does not stimulate immune inhibitory cells;

[0044] Selectively stimulates type 1 helper T cells (Th1 cells) to induce antigen-specific immunogenicity;

[0045] Stimulates type 1 helper T cells (Th1 cells) to stimulate the secretion of interferon gamma (IFN-γ), which can directly or indirectly kill cancer cells.

[0046] Does not stimulate secretion of interleukin 10 (IL-10);

[0047] Does not stimulate immune inhibitory cells;

[0048] CD8 + It also increases the immunogenicity of T cells;

[0049] In addition to short-term immunogenicity, it activates memory T cells;

[0050] Shows the effect of epitope spreading of different cancer-related epitopes in cancer tissues;

[0051] Inhibits tumor recurrence or progression by increasing the immunogenicity of type 1 helper T cells (Th1 cells);

[0052] In addition to T cells, it shows an increased efficacy of NK cells.

[0053] It may be one or more peptides selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18 according to the present invention.

[0054] Sequence number Numbering based on SEQ ID NO. 1 (full sequence) Peptide sequence 21-15MTEYKLVVVGAGGVG33-17EYKLVVVGAGGVGKS415-29GKSALTIQLIQNHFV518-32ALTIQLIQNHFVDEY675-89GEGFLCVFAINNTKS776-90EGFLCVFAINNTKSF8127-141TKQAQDLARSYGIPF9129-143QAQDLARSYGIPFIE10135-149RSYGIPFIETSAKTR111 37-151YGIPFIETSAKTRQR12150-164QRVEDAFYTLVREIR13157-171YTLVREIRQYRLKKI145-19KLVVVGAGGVGKSAL155-19KLVV VGACGVGKSAL165-19KLVVVGAVGVGKSAL175-19KLVVVGADGVGKSAL18127-164TKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIR

[0055] The numbering mentioned in Table 1 above is based on the numbering of the KRAS peptide sequence. The peptide according to the present invention having an amino acid sequence of 50 or less means that the number of consecutive amino acid sequences is at most 50 or less. Preferably, it means having an amino acid sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. More preferably, it may comprise or consist of a sequence of 10 to 50 consecutive amino acids, or 15 to 38 consecutive amino acids.

[0056] For example, when confirming SEQ ID NO: 2 (the 1st to 15th amino acid sequence based on the KRAS peptide sequence numbering), it may further include an amino acid sequence of the N-terminus and / or C-terminus of the 15 amino acid sequence. More specifically, it may be configured to have an amino acid sequence of up to 50 amino acids based on SEQ ID NO: 2, and thus may further include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminus and / or C-terminus of the Core sequence (the 1st to 15th amino acid sequence based on the KRAS peptide sequence). As above, for SEQ ID NO: 3 to SEQ ID NO: 18, an N-terminal and / or C-terminal amino acid sequence may be further included as long as the maximum number of amino acid sequences is 50 or less. The N-terminal and / or C-terminal amino acid sequence that may be further included is derived from the N-terminal and / or C-terminal sequence of the peptide of SEQ ID NO: 2 to SEQ ID NO: 18 in the full-length KRAS sequence of SEQ ID NO: 1.

[0057] Additionally, it may be a peptide having at least 90% homology with any one peptide selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18. More specifically, the homology refers to a peptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence homology.

[0058] More specifically, it may be a sequence having functional homology to a peptide having at least one amino acid deletion, insertion, and / or substitution in any one sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18 mentioned above. The functional homology may mean exhibiting the desired immunogenic efficacy in the present invention.

[0059] The above amino acid sequence may be modified by, for example, 1, 2, 3, 4, or 5 (i.e., up to 5) additions, deletions, or substitutions, provided that the polypeptide having the modified sequence exhibits the same or increased immunogenicity against KRAS as compared to a peptide having the unmodified sequence. “Same” should be understood to mean that the peptide of the modified sequence does not exhibit significantly reduced immunogenicity as compared to the peptide of the unmodified sequence.

[0060] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art. The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. Furthermore, peptides may be included that have increased structural stability against heat, pH, etc., or increased cell permeability due to mutations or modifications in the amino acid sequence.

[0061] If necessary, one or more peptides selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18 according to the present invention can be linked directly or via a linker at the N-terminus or C-terminus. The one or more peptides may be two or more peptides that are identical or different.

[0062] That is, the present invention also provides a fusion polypeptide comprising at least one peptide selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18; and a linker.

[0063] Alternatively, the present invention also provides a fusion polypeptide comprising at least one peptide selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13, and SEQ ID NO: 18; and a linker.

[0064] A fusion polypeptide is provided, comprising, as needed, at least one peptide selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18; at least one peptide selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 17; and a linker.

[0065] The term “linked” according to the present invention refers to the linking and / or joining of peptide sequences to the N-terminus or C-terminus. Here, the linking includes both direct linking and linking via a linker or spacer.

[0066] The term "linker" or "spacer" refers to a short amino acid sequence used to separate two peptides with different functions when forming a fusion polypeptide. The absence of a linker between two or more individual domains within a protein can result in reduced or inappropriate function of the protein domains due to steric hindrance, such as reduced catalytic activity or binding affinity for receptors / ligands. In the present invention, the use of an artificial linker to connect each peptide in a fusion protein can increase the space between the domains. Preferably, the linker or spacer allows one or more of the aforementioned peptides to be linked to form a fusion polypeptide. The linking mode via the linker or spacer is not particularly limited, as long as it exhibits the effect of enhancing its activity. While it will not have a specific biological activity other than linking the domains or preserving some minimal distance or other spatial relationship between them, the constituent amino acids may be selected to influence some property of the molecule, such as folding, net charge, or hydrophobicity.

[0067] These linkers include, but are not limited to, glycine, polyhistidine tags, (GGGGS)n, enzyme cleavage linkers, and the like, but it is also possible to use bis-maleimide linkers / spacers, disulfide linkers, or polyethylene glycol (PEG) linkers. Any linker known to be useful in the art of peptide chemistry is also useful as a linker according to the present invention.

[0068] The peptides according to the present invention can be prepared using available techniques known in the art. The peptides can be synthesized using any suitable procedure known to those skilled in the art, i.e., known peptide synthesis methods (e.g., genetic engineering methods, chemical synthesis).

[0069] A polynucleotide encoding a KRAS immunogenic peptide, a plasmid vector comprising the same, and / or a transformant modified to express the same

[0070] The present invention provides a polynucleotide encoding a peptide having an amino acid sequence of 50 or fewer amino acids and having at least 90% or more or 95% or more homology with any one sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 18, and / or a fusion polypeptide thereof.

[0071] The present invention provides a polynucleotide encoding a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13, and SEQ ID NO: 18.

[0072] The polynucleotide may be DNA, mRNA, or a commonly available modified nucleotide.

[0073] Additionally, if the form of the polynucleotide is mRNA, it can be chemically synthesized or synthesized in vitro using RNA polymerase.

[0074] The mRNA molecule may further comprise one or more selected from the group consisting of a 5'-cap, a 5'-untranslated region (UTR), a 3'-untranslated region, and a poly(A) tail. The poly-A tail may comprise about 100 nucleotides. Optionally, the mRNA each comprises a cap structure or a modified cap structure. The cap structure or modified cap structure is a 5' cap structure, a 5' cap-0 structure, a 5' cap-1 structure, or a 5' cap-2 structure.

[0075] Additionally, the mRNA molecule may be composed of a nucleic acid containing A (Adenine), T (Thymine), G (Guanine), C (Cytosine), U (Uridine), and / or modified forms thereof.

[0076] The above modified form means, for example, at least one chemical modification in mRNA. The modified form includes, but is not limited to, 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2' deoxyuridine (2'dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), It may include at least one selected from the group consisting of N2,7-dimethylguanosine (m-2,7G), N2,N2,7-trimethylguanosine (m-2,2,7G), and N1-methyl-pseudouridine.

[0077] The polynucleotide may be mutated by one or more base substitutions, deletions, insertions, or a combination thereof. When producing the nucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and may be synthesized using triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

[0078] In another aspect, the present invention provides a vector comprising the polynucleotide.

[0079] A "vector" refers to a nucleic acid molecule, preferably an artificial nucleic acid molecule. It is suitable for incorporating or carrying a desired nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Such a vector may be a storage vector, an expression vector, a cloning vector, a transfer vector, etc. A storage vector is a vector that allows for the convenient storage of a nucleic acid molecule, such as an mRNA molecule. A cloning vector is typically a vector that includes a cloning site that can be used to incorporate a nucleic acid sequence into the vector. A cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transfer vector may be a vector suitable for transporting a nucleic acid molecule into a cell or organism, such as a viral vector.

[0080] Preferably, a plasmid vector or viral vector comprising a polynucleotide is provided. Preferably, the vector is a plasmid vector, preferably a DNA plasmid vector.

[0081] The above plasmid vector can be produced by a recombination technique according to a genetic engineering method.

[0082] In addition, the present invention provides a transformant transformed with an expression vector comprising the polynucleotide.

[0083] More specifically, the present application provides immune effector cells transformed with the expression vector. Immune effector cells generally refer to immune cells that participate in the removal of foreign antigens in an immune response and also perform effector functions. Examples include plasma cells, dendritic cells, cytotoxic T cells, NK cells, APSC multifunctional cells, mast cells, and the like. Preferably, the immune effector cells include, but are not limited to, antigen-presenting cells represented by dendritic cells or CAR-T / CAR-NKT cells.

[0084] Cells (transformants) transformed with the expression vector of the present invention can have a therapeutic effect by being administered to a subject.

[0085] Composition comprising a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same

[0086] The present invention provides a composition comprising a peptide of the present invention, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0087] For example, the present invention provides a pharmaceutical composition comprising one or more peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same.

[0088] The present invention also provides an immunogenic composition comprising one or more peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same.

[0089] The present invention also provides a composition comprising one or more peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; and optionally one or more adjuvants, pharmaceutically acceptable carriers, preservatives, and / or excipients.

[0090] The present invention also provides a pharmaceutical composition comprising one or more peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; and optionally one or more adjuvants, pharmaceutically acceptable carriers, preservatives, and / or excipients.

[0091] The composition may comprise, for example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or more different peptides or fusion polypeptides of the present invention; and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0092] The composition may comprise at least one, two, three, four, five, six, seven, eight or more different polynucleotides of the present invention; and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative, and / or excipient.

[0093] Carriers, preservatives and excipients refer to ingredients that are compatible with other ingredients of the composition and are pharmaceutically usable.

[0094] The composition may also preferably include an adjuvant. The adjuvant is any substance whose mixing within the composition increases or otherwise modifies the immune response induced by the composition. Broadly defined, an adjuvant is a substance that promotes an immune response. Adjuvants are classified into three types according to their mechanism: i) for antigen delivery, ii) for enhancing an immune response, and iii) for a combination thereof. Representative adjuvants for antigen delivery include liposomes and Montanide emulsion (IFA; Incomplete Freund's adjuvant, Montanide emulsion), while representative adjuvants for enhancing an immune response include granulocyte-macrophage colony-stimulating factor (GM-CSF) and TLR agonists.

[0095] The above adjuvant may be selected from the group consisting of one or more of the following, regardless of its mechanism: AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also called nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), 2% squalene / Tween-80.RTM.RIBI (MPL+TDM+CWS) in emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g., poly IC and poly AU acids), wax D of Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21, various saponin extracts, GM-CSF, TLR agonists, etc. can be used as adjuvants.

[0096] Immunogenic carriers may be used as needed.

[0097] The carrier may exist independently of the adjuvant. The function of the carrier may be to increase the molecular weight of the peptide fragment, for example, to increase activity or immunogenicity, to impart stability, to increase biological activity, or to increase serum half-life. Furthermore, the carrier may aid in presenting the peptide or its fragment to T cells. Accordingly, in the composition, the peptide may be associated with a carrier, such as those described below. The carrier may be any suitable carrier known to those skilled in the art, such as a commonly used carrier protein or an antigen-presenting cell such as a dendritic cell (DC).

[0098] If a composition includes excipients, they must be "pharmaceutically acceptable" in the sense that they are compatible with the other ingredients of the composition and are not deleterious to the recipient. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliaries can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0099] The formulation of suitable compositions can be accomplished using standard pharmaceutical formulation chemistries and methodologies, all of which are readily available to a reasonably skilled practitioner. These compositions can be manufactured, packaged, or sold in forms suitable for bolus or continuous administration. Injectable compositions can be manufactured, packaged, or sold in unit-dose form, such as ampoules or multi-dose containers containing a preservative.

[0100] Additionally, if the polynucleotide encoding the KRAS immunogenic peptide is mRNA, it may be formulated as, for example, but not limited to, lipid nanoparticles.

[0101] The composition of the present invention may be provided in the form of a kit including instructions and the like.

[0102] For example, the kit of the present invention may include a cancer vaccine composition and instructions, etc. In addition, one or more adjuvants may be included in the kit to increase the immunogenicity of the composition.

[0103] Methods of using and pharmaceutical uses of KRAS immunogenic peptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof

[0104] The present invention provides a method for treating or preventing a disease or condition comprising administering to a subject in need thereof a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof.

[0105] The present invention provides the use of KRAS immunogenic peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof, in the manufacture of medicaments for treating or preventing diseases or conditions.

[0106] The present invention provides KRAS immunogenic peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof, for treating or preventing a disease or condition.

[0107] The present invention provides a pharmaceutical composition for treating or preventing a disease or condition comprising a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0108] The method comprises administering to a subject a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof.

[0109] Administration may be a therapeutically or prophylactically effective amount of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof, to a subject in need thereof.

[0110] The disease or condition is caused by selective binding to MHC class II to form an immunopeptidome and CD4 through a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof. + It may refer to a disease or condition that can be prevented or treated by stimulating T cells, particularly type 1 helper T cells, stimulating immune cells with immunosuppressive function, and not stimulating T-reg cells.

[0111] More specifically, it may refer to a disease or condition that can be prevented or treated by stimulating the secretion of interferon gamma (IFN-gamma) and not stimulating the secretion of interleukin 10 (IL-10) through a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof.

[0112] More specifically, the disease or condition is cancer.

[0113] The cancer may be any one selected from the group consisting of breast cancer, brain cancer, neurological cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0114] Specifically, the cancer may be a cancer that expresses KRAS.

[0115] Additionally, the cancer may be colon cancer, esophageal cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, breast cancer, non-small cell lung cancer, lung cancer, endometrial cancer, or skin cancer.

[0116] Accordingly, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need thereof a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof.

[0117] The present invention provides the use of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof, in the manufacture of a medicament for treating or preventing cancer.

[0118] The present invention provides KRAS immunogenic peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof, for treating or preventing cancer.

[0119] The present invention provides a pharmaceutical composition for treating or preventing cancer, comprising a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0120] The present invention provides the use of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof, in the manufacture of a cancer vaccine.

[0121] The present invention provides KRAS immunogenic peptides, fusion polypeptides, polynucleotides encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof, for use as cancer vaccines.

[0122] The present invention provides a cancer vaccine composition comprising a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0123] The cancer vaccine composition according to the present invention is preferably a cancer vaccine composition comprising a KRAS immunogenic peptide.

[0124] However, in one feasible embodiment of the above vaccine, the vaccine may also be utilized in the form of a DNA vaccine, an mRNA vaccine, an adenovirus vaccine, or other viral vector vaccine.

[0125] Additionally, the vaccine can be utilized in the form of immune effector cells transformed with a vector containing a polynucleotide encoding the immunogenic peptide.

[0126] The vaccine composition or pharmaceutical composition according to the present invention can be used in mammals, preferably humans, and can deliver the desired peptide to antigen presenting cells (APCs) in vivo by injection via routes such as intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation, oral, intralymphatic, and tumor tissue.

[0127] The above vaccine composition or pharmaceutical composition may be formulated and provided in an appropriate form. The above formulations may be formulated and used as parenteral formulations, such as transdermal preparations, suppositories, and sterile injectable solutions, according to conventional methods.

[0128] The dosage of the above composition may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist.

[0129] The above composition is administered to the subject at set intervals. If necessary, a booster shot is administered.

[0130] The target of administration of the above composition is preferably mammals such as humans.

[0131] Additionally, if necessary, the therapeutic effect can be enhanced through simultaneous or sequential administration with additional cancer treatment.

[0132] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer, further comprising a KRAS immunogenic peptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; and an additional cancer therapeutic agent.

[0133] Additional cancer treatments may include standard treatments for each cancer type, chemotherapy, targeted therapy, and immunotherapy. The KRAS immunogenic peptide, fusion polypeptide, polynucleotide encoding the same, vectors comprising the same, and / or transformants modified to express the same; or compositions thereof, when used in combination with other compositions, can reduce the therapeutic dose of the drug required to exhibit therapeutic efficacy and enhance the cancer treatment effect.

[0134] The composition according to the present invention can be used in combination with other drugs.

[0135] Combination use refers to the use of a combination of active ingredients, and these active ingredients may be at least two, three, or more. For example, combination use of a first active ingredient and a second active ingredient includes administration as a single formulation containing the first active ingredient and the second active ingredient, and administration of the first active ingredient and the second active ingredient as separate formulations, either simultaneously or separately.

[0136] This combination may be suitable for the treatment of cancers expressing KRAS. More specifically, it is suitable for the treatment of any cancer harboring a KRAS G12C mutation. Such cancer may be selected from any of the aforementioned cancers, and preferably may be colorectal cancer, esophageal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, ovarian cancer, head and neck cancer, breast cancer, non-small cell lung cancer, lung cancer, endometrial cancer, or skin cancer.

[0137] Here, administration for combination therapy may be coordinated, referred to as "co-administration" or "co-administration." For example, co-administration may be simultaneous, sequential, overlapping, spaced, or continuous, or a combination thereof.

[0138] Specifically, the present invention provides a combined use of a composition comprising the composition and an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same.

[0139] The immunogenic peptide containing the above KRAS mutation site may be a peptide containing an amino acid sequence of SEQ ID NO: 15 to 17, and preferably a peptide containing an amino acid sequence of SEQ ID NO: 15. SEQ ID NO: 15 to 17 are amino acid sequences numbered from the 5th to the 19th based on the full-length KRAS peptide sequence (SEQ ID NO: 1), and refer to sequences in which the 12th amino acid, glycine (G), is substituted with cysteine ​​(C), valine (V), and aspartic acid (D), respectively.

[0140] The present invention also provides a combination use of the composition and an additional cancer therapeutic agent. The additional cancer therapeutic agent may be a KRAS inhibitor, such as sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963 BBP-454, and preferably sotorasib (LUMAKRAS).

[0141] In addition, the present invention provides a pharmaceutical composition comprising the composition; an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; and a use in combination with an additional cancer therapeutic agent. Here, the additional cancer therapeutic agent may be at least one selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454 as a KRAS inhibitor, and preferably sotorasib (LUMAKRAS).

[0142] Accordingly, the present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; and

[0143] A method for treating or preventing cancer is provided, comprising administering to a subject in need thereof an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof.

[0144] The present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a composition thereof; and

[0145] A method for treating or preventing cancer is provided, comprising administering to a subject in need thereof at least one selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454.

[0146] The present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof;

[0147] An immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof; and

[0148] A method for treating or preventing cancer is provided, comprising administering to a subject in need thereof at least one third composition selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454.

[0149] The present invention provides the use of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; and an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof, in the manufacture of a medicament for treating or preventing cancer.

[0150] The present invention provides the use of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; and at least one compound selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454, in the manufacture of a medicament for treating or preventing cancer.

[0151] The present invention provides the use of a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof; and at least one third composition selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454; in the manufacture of a medicament for treating or preventing cancer.

[0152] The present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; and

[0153] A combination of an immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof is provided.

[0154] The present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; and

[0155] A combination of any one or more selected from the group consisting of sotorasib (LUMAKRAS), adagraasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454 is provided.

[0156] The present invention relates to a KRAS immunogenic peptide, a fusion polypeptide, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a first composition thereof; for treating or preventing cancer.

[0157] An immunogenic peptide comprising a KRAS mutation site, a polynucleotide encoding the same, a vector comprising the same, and / or a transformant modified to express the same; or a second composition thereof; and

[0158] A combination of at least one third composition selected from the group consisting of sotorasib (LUMAKRAS), adagraasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454 is provided.

[0159] When the mutant sequence immunogenic peptide of the present invention is used, not only is the vaccine effect not reduced even if an acquired mutation occurs, but it also has the advantage of being able to treat patients with non-mutations, and for an additive effect, a novel vaccine can be developed by mixing non-mutant and mutant immunogenic peptides.

[0160] The KRAS immunogenic peptide according to the present invention is an excellent cancer vaccine that selectively binds to MHC class II and selectively enhances the immunogenicity of only specific immune cells capable of killing cancer cells, thereby minimizing the immune escape mechanism of cancer cells, and can be usefully used for the prevention and / or treatment of cancer.

[0161] In particular, the KRAS immunogenic peptide according to the present invention induces T cell-mediated immunity, selectively stimulates type 1 helper T cells (Th1 cells) that can recognize KRAS as an antigen among T cells that can have immunogenicity, and does not stimulate immune inhibitory cells and regulatory T cells with an immune suppression function, thereby inducing antigen-specific immunogenicity. Through this, it can exhibit excellent cancer vaccine efficacy by promoting the secretion of interferon gamma (IFN-gamma) that can directly or indirectly kill cancer cells and acting in a direction that does not stimulate the secretion of interleukin 10 (IL-10), a substance that suppresses the function of immune cells around cancer cells.

[0162] Moreover, CD4 + Not only T cells but also CD8 + It has the advantage of increasing the immunogenicity of T cells, increasing short-term immunogenicity, and stimulating memory immune cells (memory T cells) that react to KRAS antigens, thereby demonstrating long-term preventive and therapeutic efficacy.

[0163] In addition, since it utilizes a KRAS immunogenic peptide specific for type 1 helper T cells (Th1 cells), it is expected that various cancer-related epitopes will spread in cancer tissues through immunization, and the increase in immunogenicity through this can suppress tumor recurrence or progression, and it has high utility as a single vaccine or in combination with other anticancer drugs.

[0164] Figure 1 is a graph (A) showing positive interferon-gamma (IFN-γ) response PBMCs stimulated with a KRAS epitope peptide according to the present invention through ELISPOT analysis, and a SPOT image (B) thereof.

[0165] Figure 2 shows a graph (A) and a spot image (B) showing PBMCs positive for interleukin-10 (IL-10) stimulated with a KRAS epitope peptide according to the present invention through ELISPOT analysis.

[0166] Figure 3 is a graph showing the interferon-gamma (IFN-γ) response in spleen cells confirmed through ELISPOT analysis after administering the KRAS antigenic determinant peptide according to the present invention to immunized mice.

[0167] Figure 4 is a graph confirming the anti-tumor effect when the KRAS antigenic determinant peptide according to the present invention is used in combination with an antigenic determinant peptide including a KRAS mutation site, or when the KRAS antigenic determinant peptide including a KRAS mutation site is used in combination with a KRAS inhibitor.

[0168] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0169] Example 1. Prediction of immunogenic peptides forming the immunopeptidome with MHC class II.

[0170] Using the ASEP (Aston epitope prediction in-silico system) program, immunogenic peptides with a high probability of binding to MHC class II and forming an immunopeptidome among the full-length KRAS peptide sequence were predicted.

[0171] Specifically, the ASEP program utilized a variety of predictive models, cross-validated them, and calculated the ASEP score using a proprietary calculation algorithm. High-scoring amino acid sequences were selected as immunogenic peptide candidates, and the core sequence locations and ranges of the candidates' immunogenic peptides were cross-validated with commercially available programs to secure a group of MHC class II-selective immunogenic peptide candidates.

[0172] Through this, immunogenic peptides having a sequence of 15 to 38 amino acids were obtained from the full-length sequence of KRAS (SEQ ID NO: 1) and the main mutant full-length sequence, such as the mutation at codon 12, and these are shown in Table 2 below.

[0173] Sequence number (peptide number) Numbering based on SEQ ID NO. 1 (based on full-length sequence) Peptide sequence 2 (p1) 1-15MTEYKLVVVGAGGVG3 (p2) 3-17EYKLVVVGAGGVGKS4 (p3) 15-29GKSALTIQLIQNHFV5 (p4) 18-32ALTIQLIQNHFVDEY6 (p5) 75-89GEGFLCVFAINNTKS7 (p6) 76-90EGFLCVFAINNTKSF8 (p7) 127-141TKQAQDLARSYGIPF9 (p8) 129-143QAQDLARSYGIPFIE10 (p9) 135-149RSYGIPFIETSAKTR11 (p1 0)137-151YGIPFIETSAKTRQR12(p11)150-164QRVEDAFYTLVREIR13(p12)157-171YTLVREIRQYRLKKI14(p13)5-19KLVVVGAGGVGKSAL15(p14)5-19 KLVVVGACGVGKSAL16(p15)5-19KLVVVGAVGVGKSAL17(p16)5-19KLVVVGADGVGKSAL18(#18)127-164TKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIR

[0174] The peptide sequences identified through the above ASEP program were synthesized and used in the examples below.

[0175] Example 2. Identification of immunogenic peptides specific for type 1 helper T cells using ELISPOT analysis.

[0176] Type 1 helper T cell-specific immunogenic peptides were identified through ELISPOT analysis.

[0177] (1) Experimental method using human peripheral blood mononuclear cells (PBMC)

[0178] The frequency of interferon-gamma (IFN-γ, 3420-4HPW-10, MabTech, Stockholm, Sweden) and interleukin-10 (IL-10, 3430-4HPW-10, MabTech)-producing T cells was assessed using ELISpot assay.

[0179] Specifically, the pre-coated plates were washed four times by adding 200 μL / well of PBS. 2 x 10 of healthy human PBMC cells purchased from ATCC 6 cells / mL, 1 x 10 cells mixed with multiple healthy PBMCs purchased from Zenbio 6cells / mL and dispensed onto coated plates, and KRAS peptides were dispensed at 10 μg / mL each and reacted with PBMC cells. The plates reacted in a total volume of 200 uL / well and incubated at 5% CO2, 37°C for 70-72 hours. Then, the plates were washed five times with 200 μL of PBS, and 200 μL of diluted detection antibodies (IFN-γ:7-B6-1-biotin; IL-10:12G8-biotin) were added to the wells of the plates and reacted for 2 hours at room temperature. After the reaction, the plates were washed five times with 200 μL of PBS, streptavidin-HRP was diluted 1:1000, added at 100 μL / well, and reacted for 1 hour at room temperature. Then, after washing five times with 200 μL of PBS, 100 μL / well of TMB substrate solution was added and reacted at RT for up to 5–10 min. Color development was stopped by washing with tap water, and the plate was dried overnight in the dark at room temperature. Spots were counted using the AID iSpot reader system (AID, Strassberg, Germany).

[0180] (2) Experimental results using human peripheral blood mononuclear cells (PBMCs)

[0181] PBMCs with positive responses to interferon-gamma (IFN-γ) and interleukin-10 (IL-10) stimulated with KRAS immunogenic peptide candidates were counted and compared with the control group, and the results of each analysis are shown in Figures 1 and 2.

[0182] Figure 1 shows the immunogenic peptide results of SEQ ID NOs: 2 to 17, respectively, and shows the results of positive PBMC responses to interferon-gamma (IFN-γ) stimulated with the immunogenic peptide candidates. P1 to P16 mentioned in the figure correspond to peptide numbers p1 to p16 mentioned in Table 2 above.

[0183] Figure 1A shows the numerical results of ELISPOT analysis, and Figure 1B shows the results of ELISPOT analysis.

[0184] As can be seen in Fig. 1, it was confirmed that the antigenic determinant peptides of SEQ ID NOs: 8, 9, 10, 11, 12, and 13 dramatically increased the number of interferon-gamma (IFN-γ) positive PBMC cells compared to the control group.

[0185] Figure 2 shows the results of PBMCs responding to interleukin 10 (IL-10) stimulated with a group of immunogenic peptide candidates. Figure 2A shows the numerical results of ELISPOT analysis, and Figure 2B shows the results of ELISPOT analysis. p1 to p16 mentioned in the figure correspond to p1 to p16 mentioned in Table 2 above.

[0186] As can be seen in Figure 2, the KRAS immunogenic peptide candidate group was found to hardly stimulate interleukin-10 (IL-10).

[0187] As a result, it was confirmed that the KRAS immunogenic peptides of the present invention exhibit excellent effects in stimulating interferon-gamma (IFN-γ) secretion and not stimulating interleukin-10 (IL-10) secretion. These results demonstrate that the immunogenic peptides of the present invention exhibit excellent efficacy in that they act by selectively and preferentially binding to MHC class II.

[0188] (3) Experimental method using mouse animal-derived cells (spleen cells)

[0189] ICR mice were purchased from 6-week-old mice weighing 25-28 g and acclimatized for at least 3-7 days, and each group was divided as follows:

[0190] Group 1 (G1, CFA-IFA in Figure 3): PBS + CFA / IFA,

[0191] Group 2 (G2, GM-CSF in Figure 3): CFA / IFA + mGM-CSF,

[0192] Group 3 (G3, KRAS4a in Figure 3): SEQ ID NO: 18 epitope peptide + CFA / IFA + mGM-CSF,

[0193] Group 4 (G4, KRASG12C in Figure 3): KRAS G12C mutant peptide + CFA / IFA + mGM-CSF,

[0194] Group 5 (G5, KRAS P4 in Figure 3): SEQ ID NO: 5 epitope peptide + CFA / IFA+mGM-CSF.

[0195] Each group was administered intradermally 100 μg of the antigenic determinant peptide according to the present invention once a week for a total of three times. On the final day of the test, mice were anesthetized by intramuscular injection of an anesthetic (mixture of Zoletil50 50 mg / kg and Rompun 10 mg / kg) and bled to induce euthanasia. The spleens were removed and spleen cells were isolated.

[0196] The frequency of interferon-gamma (IFN-γ)-producing T cells in isolated mouse splenocytes was assessed using ELISpot assay.

[0197] Specifically, 30 μL / well of 35% ethanol was added to an IP Sterile Clear 96-well plate (MAIPS4510, Merck Millipore, Darmstadt, Germany) for 1 min, and then washed with 200 μL PBS. Then, the IP Sterile Clear 96-well plate was coated with 50 μL / well of anti-mouse IFN-γ antibody (AN81, MebTech) at a concentration of 10 μg / mL in PBS. After overnight incubation at 4°C, the plate was blocked with 200 μL mouse T cell medium for 2 h at 37°C. Mouse spleen cells were cultured at 2.5 × 10 6 / mL, 3.5 x 10 with 10 μg / mL of K-RAS peptide 6 / mL, 3.5 x 10 with 10 μg / mL of tetanus toxoid (TT) peptide 6 / mL, 3.5 x 10 with 2.5 μg / mL concanavalin A 6 / mL concentration, or in the medium alone, in a total volume of 200 μL / well at 37°C in 5% CO2 for 70–72 h. The plates were then washed with 200 μL of 0.05% Tween-20 (PBS-T), and 50 μL of diluted biotinylated anti-mouse IFN-γ antibody (R46A2, MabTech) was added to the wells for 16–24 h at 4°C. After washing three times with PBS, streptavidin-HRP (3310-9, MabTech) was diluted 1:250 and added at 50 μL / well for 1 h at room temperature. After washing three times with PBS, 50 μL / well of AEC substrate set (551951, BD Biosciences, CA, USA) developing solution was added for up to 5–10 min at RT. After stopping the color development and drying overnight in a dark room at room temperature, spots were counted using the AID iSpot reader system (AID, Strassberg, Germany).

[0198] (4) Results of experiments using mouse animal-derived cells (spleen cells)

[0199] As can be seen in Fig. 3, compared to the control group (G1) and the negative control group (G4), the epitope peptides according to the present invention were found to increase the number of cells secreting interferon-gamma (IFN-γ). In particular, the epitope peptide of SEQ ID NO: 8 (KRAS-P7), the epitope peptide of SEQ ID NO: 9 (KRAS-P8), and the epitope peptide of SEQ ID NO: 18 (#18) dramatically increased the secretion of interferon-gamma (IFN-γ).

[0200] Example 3. Confirmation of cancer vaccine efficacy in a xenograft cancer mouse model.

[0201] Athymic-nude mice (athymic-NCr-nu strain) were purchased from Coretec at 5 weeks of age and weighing 16-18 g, and were acclimated for at least 1 week.

[0202] Cell lines expressing human KRAS wild-type or KRAS mutants were cultured at 5X10 per individual. 6 Cells were suspended in 200 μl of PBS and injected subcutaneously into the axillary region between the right shoulder and chest wall of the mouse.

[0203] In order to analyze the cancer vaccine effect of the immunogenic peptide according to the present invention, among the mice transplanted with the cells, the tumor size was 150 mm on the 11th day after cell transplantation. 3 30 mice reached (mean tumor size 150.5±16.4 mm) 3 ) were selected and assigned to each group of 6 animals.

[0204] Then, the immunogenic peptide of sequence number 18 according to the present invention was administered intradermally three times in total on days 0, 7, and 14 in an amount of 10 to 500 ug per mouse.

[0205] Then, all mice were observed for general symptoms once a day.

[0206] During the test period, body weight was measured a total of 14 times (days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31), and changes in body weight up to day 31 were observed based on the body weight on the day of administration (100%).

[0207] Tumor size and tumor growth inhibition rate were calculated by measuring three directions for each individual using a vernier caliper eight times (8, 11, 15, 18, 22, 25, 30, 32) from the 8th day to the 32nd day after cancer cell transplantation, and the overall average tumor size on the 11th day after cancer cell transplantation was 150 mm. 3 From the time point (day 0) to day 31, the tumor size was calculated for each individual 14 times (days 0, 3, 5, 7, 10 12, 14, 17, 19, 21, 24, 26, 28, 31), and the tumor growth change rate was calculated by comparing it with the control group of each test group.

[0208] For autopsy and examination, on the last day of the test, mice were euthanized by intramuscular injection of an anesthetic (mixture of Zoletil50 50 mg / kg and Rompun 10 mg / kg) under anesthesia, followed by exsanguination, and the tumors and spleens were removed, weighed, and photographed.

[0209] Example 4. Confirmation of the antitumor effect of a cancer vaccine alone in a syngeneic cancer mouse model.

[0210] Syngeneic mice were purchased at 5 weeks of age, weighing 16–18 g, and acclimatized for at least 3–7 days.

[0211] LL / 2 (LLC1) cell line, a mouse-derived lung cancer cell line, was cultured at 5X10 per individual. 6 The cells were mixed 1:1 with 100 μl of PBS and Matrigel, suspended, and injected subcutaneously into the axillary region between the scapula and chest wall of the right mouse. On the 9th day after transplantation of LL / 2 cells, the tumor size was approximately 150 mm. 3 24 mice reached (mean tumor size 149.2 ± 3.6 mm) 3 ) were selected and assigned to each group of 8 animals, and each group was divided as follows:

[0212] Group 1 (G1): PBS + CFA / IFA,

[0213] Group 2 (G2): SEQ ID NO: 18 epitope peptide (#18) + CFA / IFA + mGM-CSF.

[0214] Each group was administered subcutaneously with the antigenic determinant peptide (#18) of sequence number 18 according to the present invention at a dose of 100 μg per mouse on days 0, 7, 14, and 21 (the first administration day, which is day 10 after cell transplantation, was defined as day 0) for a total of four times. The tumor size of each individual was measured eight times (days 0, 3, 6, 10, 13, 18, 20, and 24) up to day 24 based on the first administration day of the test drug (day 0), and the tumor growth inhibition rate (TGI rate) was calculated by comparing it with the control group.

[0215] The results of this analysis are shown in Table 3 and Figure 4.

[0216] Group Treatment Tumor Growth Inhibition Rate (TGI rate) Best Tumor Growth Inhibition Rate (Best TGI rate) Final Tumor Growth Inhibition Rate (TGI rate at the end date) G1 PBS + CFA / IFA (Control) - G2 Sequence number 18 epitope peptide (#18) + CFA / IFA + mGM-CSF 4 3.7 % 35.1 %

[0217] As shown in Table 3 and Figure 4, the non-mutant peptide alone according to the present invention exhibited superior antitumor effects (tumor growth inhibition rate) compared to the control group (G1). Notably, the peak tumor growth inhibition effect was observed 13 days after vaccine drug administration, providing information on the rapid onset of drug efficacy.

[0218] Example 5. Confirmation of the antitumor effect of the KRAS epitope peptide and the epitope peptide containing the KRAS mutation site according to the present invention in a syngeneic cancer mouse model.

[0219] In order to confirm whether the antitumor effect can be enhanced by combining the antigenic determinant peptide (#18) of sequence number 18 with other peptides or compositions, an experiment was conducted on a syngeneic cancer mouse model under the same conditions as in Example 4 to confirm the effect.

[0220] Each group was divided as follows:

[0221] Group 1 (G1): PBS + CFA / IFA,

[0222] Group 2 (G2): SEQ ID NO: 18 epitope peptide (#18) + CFA / IFA + mGM-CSF,

[0223] Group 3 (G3): SEQ ID NO: 18 epitope peptide (#18) + epitope peptide containing KRAS mutation site (mt KRAS peptide) + CFA / IFA + mGM-CSF,

[0224] Group 4 (G4): SEQ ID NO: 18 epitope peptide (#18) + epitope peptide containing the KRAS mutation site (mt KRAS peptide) + CFA / IFA + mGM-CSF + KRAS inhibitor (Sotorasib)

[0225] Each group was administered subcutaneously four times with the same schedule as Example 4, including the peptide containing the SEQ ID NO: 18 epitope peptide (#18) and the KRAS mutation site.

[0226] The results of the analysis are shown in Table 4 and Figure 4.

[0227] Group Treatment Tumor growth inhibition rate (TGI rate) Best tumor growth inhibition rate (TGI rate at the end date) G1 PBS + CFA / IFA (control) - G2 SEQ ID NO: 18 epitope peptide (#18) + CFA / IFA + mGM-CSF 4 3.7 % 35.1 % G3 SEQ ID NO: 18 epitope peptide (#18) + KRAS mutation site-containing epitope peptide (mt KRAS peptide) + CFA / IFA + mGM-CSF 5 7.1 % 41.9 % G4 SEQ ID NO: 18 epitope peptide (#18) + KRAS mutation site-containing epitope peptide (mt KRAS peptide) + CFA / IFA + mGM-CSF + KRAS inhibitor (Sotorasib) 81.1 % 74.2 %

[0228] As can be seen in Table 4 and Figure 4 above, the SEQ ID NO: 18 epitope peptide (#18) has an antitumor effect even when used alone, but exhibited a superior antitumor effect when used in combination with an epitope peptide containing a KRAS mutation site. In addition, it was confirmed that the best synergistic effect was exhibited when a KRAS inhibitor (Sotorasib) was additionally used in addition to the combination therapy of the two epitopes above. Consequently, the non-mutated MHC class 2 KRAS immunogenic epitope of the present invention not only had an antitumor effect as a drug in the form of an individual peptide, but could also induce a superior antitumor effect when used in combination with an epitope peptide containing a KRAS mutation site, or in combination with an epitope peptide containing a KRAS mutation site and a KRAS inhibitor. Therefore, it can be expected that the antigenic determinant peptide according to the present invention will have a therapeutic effect of preventing or inhibiting cancer cell growth in various clinically KRAS mutant cancers.

[0229] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

A peptide having an amino acid sequence of 1.50 or less and comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13, and SEQ ID NO:

18.

2. A peptide having a continuous amino acid sequence of 15 to 38 amino acids based on the peptide of sequence number 1 in the first paragraph.

3. A peptide having at least 90% homology with any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18 in the first paragraph.

4. A peptide comprising the sequence of SEQ ID NO: 18 in the first paragraph.

5. A polynucleotide encoding a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13, and SEQ ID NO:

18.

6. A polynucleotide encoding a peptide comprising the amino acid sequence of SEQ ID NO: 18 in the fifth paragraph.

7. An immunogenic composition comprising at least one peptide according to any one of claims 1 to 4, or at least one polynucleotide according to any one of claims 5 to 6; and at least one pharmaceutically acceptable carrier.

8. An immunogenic composition according to claim 7, wherein the composition further comprises a preservative or excipient.

9. An immunogenic composition according to claim 7, wherein the composition further comprises an adjuvant.

10. A pharmaceutical composition for preventing or treating cancer, comprising at least one peptide according to any one of claims 1 to 4, or at least one polynucleotide according to any one of claims 5 to 6.

11. A pharmaceutical composition for preventing or treating cancer, according to claim 10, further comprising a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17, or a polynucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17.

12. A pharmaceutical composition for preventing or treating cancer, wherein the composition comprises an additional cancer treatment agent in claim 10.

13. A pharmaceutical composition for preventing or treating cancer, wherein the additional anticancer agent in claim 12 is at least one selected from the group consisting of sotorasib (LUMAKRAS), adagrasib (Krazati), ARS-3248, GDC-6036, BI 1701963, and BBP-454.

14. A pharmaceutical composition for preventing or treating cancer, wherein the additional cancer treatment agent in claim 12 is sotorasib (LUMAKRAS).

15. In the 10th paragraph, the cancer is any one selected from the group consisting of breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. Pharmaceutical composition.

16. An anticancer vaccine composition comprising at least one peptide according to any one of claims 1 to 4, or at least one polynucleotide according to any one of claims 5 to 6.

17. An anticancer vaccine composition according to claim 16, wherein the composition further comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17, or a polynucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17. A method for preventing or treating cancer, comprising administering to a subject in need thereof a peptide having an amino acid sequence of 18.50 or less and comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18, or a polynucleotide encoding the peptide.

19. A method for preventing or treating cancer, further comprising administering a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17, or a polynucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 17.

20. In the 18th paragraph, the cancer is any one selected from the group consisting of breast cancer, brain cancer, nerve cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. method.

21. Use of a peptide having an amino acid sequence of 50 or fewer amino acids for manufacturing a medicine for preventing or treating cancer, and comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18, or a polynucleotide encoding the peptide.

22. The use according to claim 21, wherein the cancer is any one selected from the group consisting of breast cancer, brain cancer, neurological cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

23. A pharmaceutical composition comprising a peptide having an amino acid sequence of 50 or fewer amino acids for use in the prevention or treatment of cancer, and comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 13 and SEQ ID NO: 18, or a polynucleotide encoding the peptide.

24. A pharmaceutical composition according to claim 23, wherein the composition further comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 17, or a polynucleotide encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO:

17.

25. A pharmaceutical composition according to claim 23, wherein the cancer is any one selected from the group consisting of breast cancer, brain cancer, neurological cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

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