Novel CRS fragment peptides with immunoenhancing activity and their use

Abstract

The present invention relates to a novel CRS fragment peptide having immune enhancing activity and its use, more specifically, to a novel peptide consisting of the amino acid sequence of SEQ ID NO: 2 and its use as a vaccine adjuvant and a cancer therapeutic agent. The peptide disclosed in the present invention exhibits anti-cancer activity and immune function enhancing activity as the CRS fragment disclosed for the first time in the present specification.

Description

Technical Field

[0001] This application claims priority based on Korean Patent Application No. 2020-0172565, filed on December 10, 2020, and the entire specification of the said application is incorporated herein by reference.

[0002] The present invention relates to a novel CRS fragment peptide having immune-enhancing activity and its use, and more specifically, to a novel peptide consisting of the amino acid sequence of SEQ ID NO: 2, its vaccine adjuvant, and its use as a cancer therapeutic agent.

Background Art

[0003] Aminoacyl-tRNA synthetase (ARS, or AARS), which catalyzes the aminoacylation of tRNA molecules, is essential for decoding genetic information during the translation process. Each of the eukaryotic tRNA synthetases consists of a core enzyme (closely related to the prokaryotic counterpart of the tRNA synthetase) and an additional domain (added to the amino terminus or carboxy terminus of the said core enzyme). Therefore, there are significant differences in the enzyme composition between eukaryotes and prokaryotes. For example, human tyrosyl-tRNA synthetase (TyrRS) has a carboxy-terminal domain that is not present in the TyrRS molecules of prokaryotes and lower eukaryotes.

[0004] In recent years, several aminoacyl-tRNA synthetases have been shown to possess non-canonical functions separate from their involvement in the translation process. Specifically, it has been discovered that some fragments of ARS proteins exhibit extracellular signaling activity that modulates different types of pathways beyond protein translation, thus retaining unexpected activities unrelated to aminoacylation. While such unexpected activities may be therapeutically applicable to certain diseases, they may also be highly likely to induce disease states in humans. For example, lysyl-tRNA synthetase (KRS) has been shown to have activity that promotes cancer metastasis (Patent Document 1). Furthermore, mini-tyrosyl-tRNA synthase (mini-TRS, corresponding to amino acid residues 1-364), the N-terminal domain of TRS that is cleaved by polymorphonuclear cell elastase and plasmin, exhibits non-canonical biological activities not seen in the full-length protein. In vitro studies have shown that mini-TRS stimulates endothelial cell proliferation and migration, and exhibits pro-angiogenic activity in mouse Matrigel assays. This function of promoting neoangiogenesis is generally closely associated with cancer metastasis.

[0005] Thus, the unexpected activities described above are not observed in the natural full-length protein sequence (or do not show significant effects at the natural full-length protein level), but certain activities may be prominently displayed when certain regions are isolated, and these effects may possess properties that make them unsuitable for therapeutic use. To overcome the difficulties associated with such unpredictability and to utilize the therapeutic potential of this ARS family protein, various efforts are needed to elucidate the biologically relevant morphologies of other aminoacyl-tRNA synthetase proteins.

[0006] Meanwhile, the pharmaceutical industry is shifting from traditional natural product and chemically synthesized drugs to the development of protein or peptide drugs. The global market for protein or peptide drugs expanded from $43.7 billion in 2006 to $88.5 billion in 2011, and the share of the South Korean domestic protein drug market in the global market is projected to expand from 3% in 2006 to 7% in 2021. Protein or peptide drugs are valued as an innovative area of ​​pharmaceuticals because they have fewer side effects and faster efficacy compared to synthetic drugs.

[0007] Currently, the importance of biopharmaceuticals is gradually increasing in the pipelines of major pharmaceutical companies, but significant technical challenges remain before certain biopharmaceuticals, such as peptides, can be launched. For example, the low delivery rate of peptide drugs to target sites and the difficulty of synthesizing long-chain peptides act as obstacles to commercialization. It is known that the key to success with peptide drugs is to discover short sequences that exhibit activity, that is, to select the smallest unit (motif) with excellent physiological activity from full-length proteins. It is known that long peptides are expensive to synthesize, difficult to manufacture, and have problems with human absorption.

[0008] In this regard, Patent Document 2 discloses a CRS protein fragment (106-228 aa) and its use. However, the CRS fragment peptide disclosed in the above document not only forms a multimer, but also degrades when the affinity tags attached to the N-terminus and C-terminus are removed, which limits its potential for development as a pharmaceutical product. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Korean Registered Patent Publication No. 10-1453141 [Patent Document 2] Korean Patent Application No. 10-2019-0026490 [Overview of the project] [Problems that the invention aims to solve]

[0010] In previous research, the inventors found that fragment peptides of cysteinyl-tRNA synthetase (hereinafter referred to as "CRS") possess anticancer and immune-enhancing activity. However, they confirmed that development as a pharmaceutical product was difficult due to the aforementioned limitations. Therefore, the inventors diligently conducted research to develop a novel CRS fragment peptide that could overcome the aforementioned limitations. As a result, they found that a novel CRS fragment peptide consisting of the amino acid sequence of SEQ ID NO: 2 exhibits comparable levels of anticancer and immune-enhancing activity to the conventional CRS fragment peptides mentioned above. Furthermore, it not only maintains its shape as a monomer but also does not degrade even without an affinity tag, thus completing the present invention.

[0011] Therefore, the object of the present invention is to provide a peptide comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence exhibiting 95% or more sequence homology thereto.

[0012] Another object of the present invention is to provide a polynucleotide comprising the base sequence encoding the peptide.

[0013] Another object of the present invention is to provide a vector containing the polynucleotide.

[0014] Another object of the present invention is to provide host cells transformed with the vector.

[0015] Another object of the present invention is to provide a vaccine adjuvant comprising at least one selected from the group consisting of (i) to (iv) below. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) A vector including (ii) above, and (iv) Host cells transformed in (iii) above.

[0016] Another object of the present invention is to provide a vaccine composition containing the vaccine adjuvant and an antigen.

[0017] Another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment containing at least one selected from the group consisting of the following (i) to (iv).

[0018] Furthermore, another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment consisting of at least one selected from the group consisting of the following (i) to (iv).

[0019] Furthermore, another object of the present invention is to provide a pharmaceutical composition for cancer prevention or treatment consisting essentially of at least one selected from the group consisting of the following (i) to (iv). (i) The peptide, (ii) A polynucleotide encoding the (i), (iii) A vector containing the (ii), and (iv) A host cell transformed with the (iii).

[0020] Another object of the present invention is to provide the use of one or more selected from the group consisting of the following (i) to (iv) for producing a preparation for cancer treatment. (i) The peptide, (ii) A polynucleotide encoding the (i), (iii) A vector containing the (ii), and (iv) A host cell transformed with the (iii).

[0021] Another object of the present invention is to provide a cancer treatment method including administering an effective amount of a composition containing at least one selected from the group consisting of the following (i) to (iv) to an individual who needs it. (i) The peptide, (ii) A polynucleotide encoding the (i), (iii) A vector including (ii) above, and (iv) Host cells transformed in (iii) above. [Means for solving the problem]

[0022] To achieve the above-mentioned objectives of the present invention, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence exhibiting 95% or more sequence homology thereto.

[0023] To achieve another objective of the present invention, the present invention provides a polynucleotide comprising a base sequence encoding the peptide.

[0024] To achieve another objective of the present invention, the present invention provides a vector comprising the polynucleotide.

[0025] To achieve another objective of the present invention, the present invention provides host cells transformed with the vector.

[0026] To achieve another objective of the present invention, the present invention provides a vaccine adjuvant comprising at least one selected from the group consisting of (i) to (iv) below. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0027] To achieve another objective of the present invention, the present invention provides a vaccine composition comprising the vaccine adjuvant and the antigen.

[0028] To achieve another objective of the present invention, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising at least one selected from the group consisting of (i) to (iv) below. Furthermore, the present invention provides a pharmaceutical composition for cancer prevention or treatment comprising at least one selected from the group consisting of (i) to (iv) below. Furthermore, the present invention provides a pharmaceutical composition for cancer prevention or treatment that essentially comprises at least one selected from the group consisting of (i) to (iv) below. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0029] To achieve another objective of the present invention, the present invention provides at least one use selected from the group consisting of (i) to (iv) below for manufacturing a cancer treatment formulation. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0030] To achieve another objective of the present invention, the present invention provides a method for treating cancer, comprising administering an effective amount of a composition comprising at least one selected from the group consisting of (i) to (iv) below to an individual in need. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0031] The present invention will be described in detail below.

[0032] The examples of the present invention, unless otherwise indicated, utilize conventional methods of molecular biology and recombinant DNA technology within the art to which the present invention pertains, and much of the explanation is described below (references). Such techniques are described in detail in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I&II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B. Perbal, ed., 1984).

[0033] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

[0034] Throughout the disclosures herein, various aspects or conditions relating to the present invention can be proposed in range form. Unless otherwise specified, range values ​​described herein include the corresponding boundary values, meaning all values ​​from the lower limit to the upper limit. It should be understood that range descriptions are merely for convenience and simplicity and should not be interpreted as inflexible limitations on the scope of the present invention. Therefore, a range description should be considered to specifically disclose all possible subranges as well as the individual numerical values ​​within that range. For example, a range description such as 7 to 170 should be considered to specifically disclose the individual numerical values ​​within that range, such as 9, 27, 35, 101, and 155, as well as subranges such as 10 to 127, 23 to 35, 80 to 100, 50 to 169, etc. This applies regardless of the width of the range.

[0035] In this invention, the term "contains" is used interchangeably with "contains" or "characterizes" and does not exclude any additional components or method steps not mentioned in the composition or method. The term "consists of" means excluding any additional elements, processes, or components not separately described. The term "essentially consists of" means that, within the scope of the composition or method, it may include materials or processes that do not substantially affect its fundamental properties in addition to the described materials or processes.

[0036] As used herein, the terms “peptide” and “protein” are used according to their ordinary (conventional) meanings, i.e., meaning an arrangement of amino acids. A peptide is not limited to a specific length, but in the context of the present invention, generally represents a fragment of a full-length protein and may include post-translational modifications, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art (naturally occurring and unnaturally occurring modifications), and may be expressed as a “polypeptide.” The peptides and proteins of the present invention can be prepared using any of the various known recombinant and / or synthetic techniques, exemplary embodiments thereof are further described below.

[0037] This invention stems from the discovery that CRS and CRS-derived peptides retain therapeutically relevant non-standard biological activity.

[0038] In this specification, “non-standard activity” generally refers to the activity possessed by the CRS peptide of the present invention in addition to the addition of cysteine ​​to the tRNA molecule. As described in detail herein, in certain embodiments, the non-standard biological activity exhibited by the CRS fragment of the present invention may be selected from the group consisting of, but is not limited to, anti-cancer activity, activation of innate immunity, and activation of adaptive immunity.

[0039] It should be understood that the scope of the present invention includes not only CRS fragment peptides having at least one non-standard biological activity, but also mutants that substantially maintain the said non-standard activity.

[0040] Specifically, the inventors have demonstrated that the novel CRS fragment peptide exhibits activity that suppresses tumor growth by activating innate and adaptive immunity, and is also structurally very stable. The region of the above CRS fragment peptide is disclosed for the first time in this invention.

[0041] Therefore, the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence exhibiting 95% or more sequence homology thereto.

[0042] In the present invention, the peptide of Sequence ID No. 2 is a cleaved form of the CRS protein, and the peptide consists of amino acids from the 99th to the 200th position of the full-length CRS protein sequence consisting of the amino acid sequence of Sequence ID No. 1, with the cysteine ​​at position 182 being replaced by serine.

[0043] The peptides of the present invention can be prepared using available techniques known in the art. For example, they can be prepared using any of various proteolytic enzymes. Examples of proteases (protein-degrading enzymes) include, for example, achromopeptidase, aminopeptidase, ancrod, angiotensin-converting enzyme, bromelain, calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase G, carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2, caspase 3, caspase 4. 4) cathepsin 5, cathepsin 6, cathepsin 7, cathepsin 8, cathepsin 9, cathepsin 10, cathepsin 11, cathepsin 12, cathepsin 13, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin H, cathepsin L L), chymopapain, chymase, chymotrypsin, clostripain, collagenase, complement C1rC1r), complement C1s, complement Factor D, complement Factor I, cucumisin, dipeptidyl peptidase IV, leukocyte elastase, pancreatic elastase, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIV protease (HIV Protease), Igase, kallikrein tissue, general leucine aminopeptidase, cytosol leucine aminopeptidase, microsomal leucine aminopeptidase, matrix metalloprotease, methionine aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E, prostate-specific antigen, alkaliphilic protease from Streptomyces griseus, protease from AspergillusProtease from Aspergillus saitoi, protease from Aspergillus sojae, protease from B. licheniformis (alkaline or alcalase), protease from Bacillus polymyxa, protease from Bacillus sp., protease from Rhizopus sp., protease S, proteasomes, proteinase from Aspergillus oryzae, proteinase 3, proteinase A A) Examples include proteinase K, protein C, pyroglutamate aminopeptidase, rennin, streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase, and urokinase. Those skilled in the art can easily determine which proteolytic enzyme is appropriate by considering the chemical specificity of the fragment to be prepared.

[0044] The polypeptides described herein can be prepared by any suitable procedure known to those skilled in the art, such as recombinant techniques. In addition to recombinant manufacturing methods, the polypeptides of the present invention can be prepared by direct peptide synthesis using solid-phase techniques.

[0045] Solid-phase peptide synthesis (SPPS) can be initiated by attaching functional units called linkers to small porous beads to guide the linking of peptide chains. Unlike liquid-phase methods, the peptides covalently bond to the beads and are prevented from being detached by a filtration process until they are cleaved by specific reactants such as trifluoroacetyldic acid (TFA). Synthesis proceeds through a cycle (deprotection-wash-coupling-wash) in which the N-terminal amine of the peptide attached to the solid phase bonds with the N-protected amino acid unit, followed by a deprotection step, and then a coupling step where the re-exposed amine group bonds with a new amino acid. SPPS can be performed in conjunction with microwave technology, which can shorten the time required for coupling and deprotection in each cycle by applying heat during the peptide synthesis process. Thermal energy can prevent folding or aggregation of the elongating peptide chain and promote chemical bonding.

[0046] Furthermore, the peptides of the present invention can be produced by liquid-phase peptide synthesis, and for specific methods, please refer to the following document: U.S. Patent No. 5,516,891. In addition, the peptides of the present invention can be synthesized by various methods, such as a combination of the solid-phase synthesis method and the liquid-phase synthesis method, and the methods of production are not limited to those described herein.

[0047] Protein synthesis can be carried out using manual techniques or by automation. Automated synthesis can be achieved, for example, using the Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and then combined using chemical methods to produce the target molecule.

[0048] The peptides provided in the present invention include mutants having a sequence homology of 95% or more to the peptide of Sequence ID No. 2. A mutant means an active mutant of the peptide, and such an active mutant means that it retains at least one desired non-standard activity (e.g., anti-cancer activity and immunoenhancing activity) from the peptide from which it is derived. An example of such a mutant may be a splice mutant that, whether naturally occurring or not, retains at least one non-standard activity as described herein. Alternatively, the mutant may comprise one or more point mutations in the peptide sequence, whether naturally occurring or not, and the mutant peptide retains at least one non-standard activity as described herein. That is, the mutants (or the term "active mutant") in the present invention are understood as functional equivalents of the peptide of Sequence ID No. 2.

[0049] More specifically, the variant is characterized by being a functional equivalent having at least 95%, 96%, 97%, 98%, or 99% sequence homology to the peptide sequence along its length.

[0050] In one embodiment of the present invention, the peptide may be characterized by comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0051] A mutant is a change in the amino acid sequence of a "peptide," which may include one or more substitutions, deletions, additions, and / or insertions. Such mutants may be naturally occurring or may be synthetically produced by modifying or altering one or more of the peptide sequences of the present invention using any of the many techniques known in the art, and evaluating their biological activity as described herein.

[0052] In one embodiment of the present invention, the mutant includes a conservative substitution. A "conservative substitution" is a substitution in which one amino acid is replaced by another amino acid having similar properties, and a person skilled in the art can predict that the secondary structure and hydropathic nature (hydrophobic or hydrophilic properties) of the peptide will remain substantially unchanged. Generally, the following amino acid groups exhibit a conservative change: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; (5) phe, tyr, trp, his.

[0053] Modification can be carried out within the structure of the peptide of the present invention to obtain functional molecules encoding peptide variants or derivatives having desired (preferred) characteristics. If it is desired to change the amino acid sequence of the peptide in order to produce equivalent or improved variants of the peptide of the present invention, those skilled in the art can change one or more codons based on protein codon information known in the art.

[0054] For example, a particular amino acid can be substituted with another amino acid in a protein or peptide structure without significant loss of interactive binding ability, such as in receptors, antigen-binding sites on antibodies, or binding sites on substrate molecules. This is because, as is generally defined, protein interactive ability and properties contribute to the biological functional activity of proteins, and specific amino acid sequence substitutions may occur within the protein or peptide sequence, or of course, within the underlying DNA coding sequence, and nevertheless, proteins with the same or similar properties can be obtained.

[0055] Therefore, various modifications are intended to be made to the peptide sequences disclosed above or the DNA sequences encoding those peptides without significant loss of desired utility or activity. Hydropathic (hydrophobic or hydrophilic) indices of amino acids may also be considered in such modifications. The importance of hydrophobic amino acid indices in conferring biological functions that interact with proteins is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). For example, the relative hydrophobicity of amino acids has been found to contribute to the secondary structure of the resulting protein, which ultimately determines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Each amino acid is assigned a hydropathic index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982). The values ​​are as follows: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / Cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); Arginine (-4.5).

[0056] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophobicity indices or scores to obtain proteins with similar biological activity (i.e., still obtain proteins that are biologically functionally equivalent). In such modifications, amino acids with hydrophobicity indices of ±2 are preferred for substitution, amino acids with hydrophobicity indices of ±1 are particularly preferred, and amino acids with hydrophobicity indices of ±0.5 are even more particularly preferred.

[0057] It is also understood in the art that the same amino acid substitutions can be effectively carried out based on hydrophilicity. As described in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilicity values, thereby obtaining biologically equivalent proteins. In such modifications, substitutions of amino acids with a hydrophilicity value of ±2 are preferred, substitutions of amino acids with a hydrophilicity value of ±1 are particularly preferred, and substitutions of amino acids with a hydrophilicity value of ±0.5 are even more particularly preferred.

[0058] As outlined above, amino acid substitutions may be based on the relative similarity of amino acid side chain substituents, for example, on their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various characteristics described above are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0059] Amino acid substitutions can also be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid. Positively charged amino acids include lysine and arginine. Amino acids with uncharged polar head groups that have similar hydrophilic values ​​include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine, threonine, phenylalanine, and tyrosine.

[0060] Furthermore, the variants may include non-conservative modifications. In preferred embodiments, the variant peptide may differ from the native sequence by substitution, deletion, or addition of five or fewer amino acids. The variant may also be modified, for example, by the deletion or addition of amino acids that have minimal effect on the peptide's secondary structure and hydrophilicity.

[0061] Peptides may contain a signal (or leader) sequence at the N-terminus of a protein, which directs the transport of the protein either concurrently or post-translation. Peptides can also be conjugated to linker sequences or other sequences to facilitate their synthesis, purification, or identification (e.g., polyHis), or to enhance their binding to solid supports. For example, peptides can be conjugated to immunoglobulin Fc regions.

[0062] When peptide sequences are compared, as will be described later, two sequences are said to be "identical" if, when aligned to the greatest degree of matching, the amino acid sequences in the two sequences are identical. Comparison between two sequences is typically performed by identifying and comparing local regions of sequence similarity by comparing the sequences over a comparison window. As used herein, a "comparison window" refers to a region of at least about 20 consecutive positions, usually 30–75 or 40–50 consecutive positions, where the sequences are two sequences. After this optimal alignment, the sequences can be compared to a reference sequence at the same number of consecutive positions.

[0063] Optimal alignment of sequences for comparison can be performed using basic parameters, for example, with the Megaalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.). This program includes several alignment schemes described in the following references. Dayhoff, MO (1978) In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425. Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-The Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, WJ and Lipman, DJ (1983) Proc. Nat'l Acad., Sci. USA 80: 726-730.

[0064] Alternatively, optimal alignment of sequences for comparison may be performed by the partial identity algorithm of Smith and Waterman (1981) Add. APL. Math 2: 482, or by the identity sorting algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, or by the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, or by computerized execution of these algorithms (GAP, BESTFIT, BLAST, FASTA), and by TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., or by inspection.

[0065] Examples of suitable algorithms for determining sequence identity and sequence similarity percentages may be the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST2.0 can be used to determine the percentage of sequence homology to the polynucleotides and polypeptides of the present invention and can be used, for example, with the parameters described herein. Software for performing BLAST analysis is available through the National Center for Biotechnology Information. For amino acid sequences, a cumulative score can be calculated using a scoring matrix.

[0066] The progression of word matching in each direction stops when the number of matching residues becomes zero or less due to the accumulation of aligned residues, or when the end of a sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0067] In one exemplary approach, the "sequence homology percentage" is determined by comparing two optimal alignment sequences across at least 20 comparison windows, where the polypeptide sequence portion within the comparison window may contain 20% or less, typically 5–15%, or 10–12% additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions). The percentage is calculated by determining the number of positions where the same amino acid residues exist in the positive sequence, obtaining the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size). This result is then multiplied by 100 to obtain the sequence homology percentage.

[0068] The peptides provided in the present invention may be linear or cyclic, which can be understood by referring to embodiments of the present invention.

[0069] In the present invention, the preparation of the cyclic peptide as a mutant is not particularly limited in terms of the specific cyclization method and the resulting cyclization form, as long as it is carried out by a known peptide cyclization method known in the art. Preferably, the preparation of the cyclic peptide of the present invention may be carried out by preparing a linear peptide by cleaving or substituting such that cysteine ​​is located at both ends (N-terminus and C-terminus), and by causing a monosulfide bond to occur between the cysteine ​​residues present at both ends.

[0070] The peptides provided herein, in one embodiment, consider the use of modified polypeptides, such modified polypeptides include modifications that improve the desired properties of the isolated polypeptide as described herein. Exemplary modifications of the polypeptides of the present invention may include, but are not limited to, chemical and / or enzymatic derivatization of one or more constituent amino acids, and the derivatization includes side chain modifications, skeletal modifications, and N-terminal and C-terminal modifications, including acetylation, hydroxylation, methylation, amidation, and the addition of carbohydrate or lipid components, cofactors, etc. Exemplary modifications include PEGylation of polypeptides.

[0071] In certain embodiments, chemoselective ligation techniques can be used to modify the peptides of the present invention, for example, by adhering polymers in a site-specific and controlled manner. Such techniques typically rely on the attachment of chemoselective anchors to the protein backbone by one of chemical or recombinant means, followed by modification to polymers supporting complementary linkers. As a result, the assembly process and the covalent structure of the resulting protein-polymer conjugates are controlled, thereby enabling rational optimization of pharmaceutical properties such as efficacy and pharmacokinetic properties. For example, their pharmacokinetic properties can be improved by enabling selective attachment of PEGs.

[0072] The peptide of the present invention may include pharmaceutically acceptable salt forms. Examples of pharmaceutically acceptable salts include, but are not limited to, hydrochloride, sulfate, phosphate, acetate, citrate, stannate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, or p-toluenesulfonate.

[0073] The present invention also provides a polynucleotide encoding the peptide.

[0074] As used herein, the terms “DNA,” “polynucleotide,” and “nucleic acid” refer to DNA molecules isolated from the whole genomic DNA of a particular species. Therefore, a DNA fragment (part, segment) encoding a polypeptide is one or more coding sequences substantially isolated or purified from the whole genomic DNA of the species from which that DNA fragment can be obtained. The terms “DNA fragment” and “polynucleotide” include DNA fragments and smaller fragments thereof, and also refer to recombinant vectors (e.g., plasmids, cosmids, phagemids, bactericidal viruses, viruses, etc.).

[0075] As will be understood by those skilled in the art, the polynucleotide sequences of the present invention express or are modified to express proteins, peptides, etc., and include genomic sequences, extragenomic sequences, plasmid-encoded sequences, and smaller, manipulated gene fragments. Such fragments can be isolated from nature or modified synthetically by humans.

[0076] As will be recognized by those skilled in the art, polynucleotides may be single-stranded (coding sequence or antisense sequence) or double-stranded, and may be DNA molecules (genomic, cDNA, or synthetic) or RNA molecules. Further coding or non-coding sequences may be present in the polynucleotides of the present invention. Furthermore, polynucleotides can be linked to other molecules and / or supporting materials.

[0077] Polynucleotides may include native sequences, variants, or biologically functional equivalents thereof. Polypeptide variants may include one or more substitutions, additions, deletions, and / or insertions as further described below, preferably such modifications are made in a line in which the desired activity of the encoded polypeptide is not substantially reduced compared to the unmodified polypeptide. The effect on the activity of the encoded polypeptide can generally be evaluated as described herein.

[0078] The polynucleotides provided in the present invention are not particularly limited in their specific sequence, as long as they encode the peptide or a variant peptide thereof, and any combination of base sequence (nucleic acid sequence) configurations is permitted. For example, the peptide consisting of the amino acid sequence of SEQ ID NO: 2 can be expressed by a polynucleotide containing the base sequence represented by SEQ ID NO: 4, but is not limited to these examples.

[0079] The polynucleotides of the present invention, regardless of the length of their coding sequence itself, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multicloning sites, and other coding fragments (parts, segments). As a result, their total length can vary significantly. Therefore, it is considered that polynucleotide fragments of almost any length can be applied, and preferably, their total length can be limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0080] Furthermore, as a result of the degeneracy of the genetic code, it will be obvious to those skilled in the art that many nucleotide sequences exist that encode the peptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. Nevertheless, due to differences in codon usage, other polynucleotides (e.g., polynucleotides optimized for human and / or primate codon selection) are specifically considered in the present invention.

[0081] Furthermore, alleles of genes comprising polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes modified as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions. The resulting mRNA and proteins may (but may not necessarily) have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0082] Polynucleotides and their fusions can be manufactured, manipulated, and / or expressed using any of the well-established techniques known and available in the art. For example, a polynucleotide sequence encoding the peptide of the present invention or its functional equivalent can be utilized in a recombinant DNA molecule directed toward the expression of the polypeptide in a suitable host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences can be generated, and these sequences can be used for cloning and expression of a given polypeptide.

[0083] As will be understood by those skilled in the art, in some cases it may be advantageous to produce nucleotide sequences (nucleotide sequences encoding polypeptides) that hold codons not found in nature. For example, preferred codons in a particular prokaryotic or eukaryotic host can be selected to produce recombinant RNA transcripts with increased protein expression ratios or desired properties (e.g., a longer half-life than transcripts produced from naturally occurring sequences).

[0084] Furthermore, the polynucleotide sequences of the present invention can be manipulated using methods commonly known in the art to modify peptide coding sequences for a variety of reasons, including, but not limited to, cloning, processing, expression, and / or modification of the gene product to alter its activity.

[0085] Furthermore, the present invention provides a vector containing the polynucleotide and a host cell transformed with the vector.

[0086] To express a desired polypeptide, a nucleotide sequence encoding the polypeptide or its functional equivalent can be inserted into a suitable expression vector (i.e., a vector containing the elements necessary for the transcription and translation of the inserted coding sequence). An expression vector containing the sequence encoding the target polypeptide and appropriate transcriptional and translational regulatory elements can be constructed by methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. These methods are described in the following literature: Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0087] Various expression vectors / host systems are known and can be used to contain and express polynucleotide sequences. Expression vectors / host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophages, plasmids, or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with viral expression vectors (e.g., baculovirus), plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

[0088] The "regulatory elements" or "regulatory sequences" present within an expression vector are untranslated regions (enhancers, promoters, 5' and 3' untranslated regions) that interact with host cell proteins to perform transcription and translation. Such elements can have altered strength and specificity. Depending on the vector system and host used, any suitable transcription and translation elements (including constitutive and inducible promoters) can be used.

[0089] For example, when cloning into bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemide (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) can be used. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. When polypeptide encoding requires the generation of cell lines containing multiple copies of the sequence, SV40 or EBV-based vectors can be usefully utilized with appropriate selection markers.

[0090] In bacterial systems, multiple expression vectors can be selected depending on the intended use for peptide expression. For example, when large quantities are required, vectors designed to express easily purified fusion proteins at high levels can be used. Such vectors include, but are not limited to, pIN vectors (Van Heeke and Schuster, J. Biol. Chem. 264: 5503-5509 (1989)) in which the amino-terminal Met and the following seven residues of the drug are ligated in-frame within the vector, resulting in the production of a hybrid protein. pGEX vectors (Promega, Madison, Wis.) can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads and subsequent elution in the presence of free glutathione. Proteins produced using such a system can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that replicated polypeptides are released from the GST moiety.

[0091] In yeast (Saccharomyces cerevisiae), numerous vectors containing antigenic or inducible promoters (e.g., factor α, alcohol oxidase, and PGH) can be used. This can be understood by referring to Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153: 516-544 (1987), among others.

[0092] When using plant expression vectors, the expression of the polypeptide-encoding sequence can be driven by any number of promoters. For example, viral promoters (e.g., the 35S and 19S promoters of CaMV) can be used alone or in combination with omega (ω) leader sequences derived from TMV (Takamatsu, EMBO J. 6: 307-311 (1987)). Alternatively, plant promoters (e.g., the small subunit of RUBISCO or the heat shock promoter) can be utilized. These constructs are introduced into plant cells by direct DNA transformation or pathogen-mediated transfection, techniques known in the art.

[0093] Insect-based organisms can also be used to express desired polypeptides. For example, in one system, AcNPV (Autographa californica nuclear polyhedrosis virus) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The polypeptide-encoding sequence can be cloned into a non-essential region of the virus and placed under the control of a polyhedrin promoter, such as a polyhedrin gene. Successful insertion of the polypeptide-encoding sequence produces a recombinant virus in which the polyhedrin gene is inactivated and the coat protein is deficient. This recombinant virus can then be used to infect cells, such as S. frugiperda cells or Trichoplusia larvae, to express the desired polypeptide.

[0094] Many virus-based expression systems are commonly available in mammalian host cells. For example, when adenovirus is used as an expression vector, the sequence encoding the desired polypeptide can be ligated into an adenovirus transcription / translation complex consisting of a late promoter and a tripartite leader sequence. By inserting into non-essential E1 or E3 regions of the viral genome, viable viruses capable of expressing polypeptides in infected host cells can be obtained. Furthermore, transcriptional enhancers (e.g., Rous Sarcoma Virus (RSV) enhancers) can be used to increase expression in mammalian host cells.

[0095] Furthermore, specific start signals can be used for more efficient translation of the sequence encoding the desired polypeptide. Such signals include the ATG start codon and adjacent sequences. If the polypeptide encoding sequence, its start codon, and upstream sequence are inserted into a suitable expression vector, further transcriptional or translational regulatory signals may not be necessary. However, if only the coding sequence, or only a portion thereof, is inserted, an exogenous translational regulatory signal, including the ATG start codon, must be provided. Furthermore, the start codon must be within the correct reading frame to ensure translation of the entire insertion. Exogenous translational elements and start codons can originate from various sources (both natural and synthetic). Expression efficiency can be enhanced by introducing appropriate enhancers for the specific cell line being used.

[0096] Furthermore, host cell lines can be selected based on their ability to regulate the expression of the inserted sequence or to process the expressed protein in the desired manner. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can be used to facilitate accurate insertion, folding, and / or function. Other host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138, which have specific cellular and characteristic mechanisms for such post-translational activity) can be selected to ensure accurate modification and processing of foreign proteins.

[0097] For long-term, stable expression is generally preferred for high-yield production of recombinant proteins. For example, cell lines that stably express a desired polynucleotide can be transformed using an expression vector that may contain a viral replication origin and / or endogenous expression elements, and a selection marker gene on the same vector or a different vector.

[0098] After vector introduction, cells are grown in growth medium for 1-2 days, and then converted to selective medium. The purpose of the selection marker is to confer resistance to selection; its presence allows for the growth and harvesting of cells that successfully express the introduced sequence. Stablely transformed, resistant clones of cells can be grown using tissue culture techniques appropriate to their cell type.

[0099] Transformed cell lines can be recovered using a variety of selection systems. Examples of such selection systems, though not limited to these, include the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, which can be used for TK cells or APRT cells, respectively.

[0100] Furthermore, antimetabolite resistance, antibiotic resistance, or herbicide resistance can be used as a basis for selection. For example, dhfr confers resistance to methotrexate; npt confers resistance to aminoglycosides, neomycin, and G-418; als or pat confers resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (see Murry, reference above); additional selection genes are known, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine. Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin have gained popularity and are widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression resulting from specific vector systems.

[0101] Various protocols are known in the art for detecting and measuring the expression of polynucleotide-encoded products using either polyclonal or monoclonal antibodies specific to the polynucleotide-encoded product. Examples include ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), and FACS (fluorescence-activated cell sorting). For assay methods and further other assay methods, refer to methods known in the art. Various labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid assays and amino acid assays. Means for producing labeled hybridization probes or labeled PCR probes to detect sequences related to polynucleotides include oligo-labeling, nick translation, terminal labeling, or PCR amplification using labeled nucleotides. Alternatively, a sequence or any portion thereof can be cloned into a vector for the production of mRNA probes. Such vectors are known and commercially available in the art and can be used to synthesize RNA probes in vivo by adding a suitable RNA polymerase (e.g., T7, T3, or SP6) and labeled nucleotides. These procedures can be carried out using a variety of commercially available kits. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescent or chromogenic agents, substrates, carriers, inhibitors, and magnetic particles.

[0102] Host cells transformed with a desired polynucleotide sequence can be cultured under conditions suitable for protein expression and recovery from cell culture. Proteins produced by recombinant cells may be secreted or contained within the cell according to their sequence and / or vector.

[0103] As will be understood by those skilled in the art, the polynucleotide-containing expression vectors of the present invention can be designed to include a signal sequence directed toward the secretion of polypeptides across the prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to link the sequence encoding the desired polypeptide with a sequence encoding a polypeptide domain that facilitates the purification of water-soluble proteins.

[0104] In addition to recombinant production methods, the polypeptides and their fragments of the present invention can be produced by direct peptide synthesis using solid-phase technology. Protein synthesis may be carried out using manual techniques or by automation. Automated synthesis can be achieved, for example, using the Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and then combined using chemical methods to produce full-length molecules.

[0105] According to another aspect of the present invention, the polynucleotides encoding the polypeptide of the present invention can be delivered to a subject in vivo, for example, using gene therapy technology. Gene therapy generally refers to the transfer of heterologous nucleic acids into specific cells, or target cells, of mammals, particularly humans, that have the disorder or condition requiring such treatment. The nucleic acid is introduced into the selected target cells, heterologous DNA is expressed, and a correspondingly encoded therapeutic product is produced.

[0106] Various viral vectors available for gene therapy as disclosed herein include RNA viruses such as adenoviruses, herpesviruses, vaccinia, adeno-associated viruses (AAVs), or preferably retroviruses. Preferably, the retroviral vector may be a rodent or algae retroviral derivative or a lentiviral vector. A preferred retroviral vector may be a lentiviral vector. Examples of retroviral vectors into which a single exogenous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous Sarcoma Virus (RSV). Multiple additional retroviral vectors may contain multiple genes. All of these vectors may contain a selection marker gene so that transduced cells are identified and produced. For example, a vector may be made target-specific by inserting a desired zinc finger-derived DNA-binding polypeptide sequence into a viral vector along with other genes encoding ligands for receptors on specific target cells.

[0107] Retroviral vectors may be target-specific, for example, by inserting polynucleotides encoding polypeptides. Exemplarily, targeting can be achieved by utilizing antibodies that target retroviral vectors. Those skilled in the art are familiar with specific polynucleotide sequences that can be inserted into retroviral genomes to enable target-specific delivery of retroviral vectors containing zinc finger-nucleotide-binding protein polynucleotides. This can be easily confirmed without excessive experimentation.

[0108] Recombinant retroviruses have defects and therefore require assistance to produce infectious vector particles. This assistance can be provided, for example, by utilizing a helper cell line containing plasmids encoding all of the retrovirus's structural genes under the control of regulatory sequences within the LTR. These plasmids lack nucleotide sequences that enable a packaging mechanism that recognizes the RNA transcript for encapsulation. Helper cell lines lacking the packaging signal include, but are not limited to, PSI.2, PA317, and PA12. These cell lines produce empty virions because their genomes are not packaged. When a retroviral vector is introduced into cells in which the packaging signal is intact and the structural genes are replaced with other desired genes, the vector can be packaged and produce vector virions. These vector virions can then be used to infect tissue cell lines (e.g., NIH3T3 cells), thereby producing a large number of chimeric retroviral virions.

[0109] Furthermore, "non-viral" delivery techniques for gene therapy, such as DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct DNA injection, CaPO4 precipitation, gene gun technology, electroporation, liposome methods, and lipofection, can be used. Any of these methods is widely available to those skilled in the art and is suitable for use in the present invention. Other suitable methods are also available to those skilled in the art, and it is clearly understood that the present invention can be achieved using any available transduction method. Lipofection can be achieved by encapsulating isolated DNA molecules within liposome particles and bringing the liposome particles into contact with the cell membrane of a target cell. Liposomes are self-assembling colloidal molecules in which a lipid bilayer consisting of amphiphilic molecules such as phosphatidylserine or phosphatidylcholine encapsulates a portion of the surrounding medium, resulting in the lipid bilayer surrounding a hydrophilic interior. Monolayer or multilayer liposomes can be constructed, resulting in the inclusion of a desired chemical substance, pharmaceutical, or isolated DNA molecule as in the present invention.

[0110] The present invention also provides a vaccine adjuvant comprising at least one selected from the group consisting of (i) to (iv) below. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0111] In the present invention, a vaccine adjuvant can be defined as a substance that can present an antigen to the immune system in such a way that an immune response to the antigen, or an increase in the immune response, is induced when the antigen is administered together with the vaccine adjuvant. In other words, a vaccine adjuvant is an immunostimulant, a substance that promotes an immune response to an antigen, and is not an immunogen to the host, but rather a substance that strengthens immunity by increasing the activity of immune system cells.

[0112] To analyze antigen-specific immune responses induced by vaccine adjuvants, the immune response can be compared to the immune response induced in the presence of the antigen without the vaccine adjuvant. This induction can be evaluated in subjects or target cells.

[0113] In the present invention, the immune response may be one or more selected from the group consisting of macrophages, dendritic cells, mononuclear cells, B cells, and T cell responses. In particular, the immune response may be a B cell response, meaning that it can specifically produce antibodies against an antigen. The antibodies are preferably IgG antibodies, more preferably IgG2a and / or IgG1 antibodies. The immune response may be a T cell response, preferably a Th1 response, a Th2 response, or a balanced Th2 / Th1 response. Those skilled in the art will recognize that disease-dependent B cell and / or T cell responses may need to be induced to modulate them. In a modified example, the immune response can be detected by measuring the production of cytokines such as IFNgamma, IL-6, TNFalpha, or IL-10. The production of such cytokines can be evaluated by ELISA, preferably as performed in the examples.

[0114] According to one embodiment of the present invention, the peptide of Sequence ID No. 2 was found to have excellent effects in activating innate and acquired immunity, and was found to be able to significantly improve the increase in the immune response of the target during treatment together with the antigen.

[0115] Therefore, it is obvious to any ordinary engineer that the peptide of Sequence ID No. 2, or a peptide containing an amino acid sequence showing 95% or more homology to this peptide, the polynucleotide encoding it, the vector containing the polynucleotide, and host cells transformed with the vector will also exhibit the same kind of physiological activity.

[0116] In one embodiment of the present invention, when a vaccine adjuvant is administered together with the antigen, it can induce improved innate and adaptive immune responses compared to those obtained with the antigen alone. More specifically, it can induce improved cellular and humoral immune responses.

[0117] In one embodiment of the present invention, detection of an antigen-specific induced immune response means that the detection occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more after administration of the vaccine adjuvant of the present invention, or at least 1 day, at least 2 days, at least 3 days, at least 4 days or more. The detection can be evaluated in subjects or target cells, preferably as performed in the examples.

[0118] In one embodiment of the present invention, antigen-specific induced immune response preferably means a detectable immune response to the antigen. A detectable increase may mean an increase of at least 5%, or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or more in the amount of immune cells, antibodies, and / or cytokines at least 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 11, 12 hours, or more, or at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days, or more, in the amount of immune cells, antibodies, and / or cytokines. Detection can be evaluated in the subject or target cells, preferably as performed in the examples.

[0119] The present invention also provides a vaccine composition comprising the above-mentioned vaccine adjuvant and antigen.

[0120] In the present invention, the vaccine refers to a formulation containing an antigen consisting of all disease-inducing organisms (hereinafter referred to as structured or attenuated) or components of these organisms, such as proteins, peptides, or polysaccharides, and used to confer immunity against diseases induced by the organism. As described above, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, or a peptide containing an amino acid sequence showing 95% or more homology to this peptide, can be used as a vaccine composition because it activates the innate and adaptive immune systems and, when administered together with the antigen, significantly enhances the target immune response.

[0121] In the present invention, the antigens contained in the vaccine composition are not particularly limited in type and may include peptides, proteins, glycoproteins, glycolipids, lipids, carbohydrates, nucleic acids, polysaccharides, and viruses, bacteria, allergens, tissues, cells, etc., that contain these. These non-exclusive examples include: pollen-derived antigens, hepatitis A virus-derived antigens, hepatitis B virus-derived antigens, hepatitis C virus-derived antigens, hepatitis D virus-derived antigens, hepatitis E virus-derived antigens, hepatitis F virus-derived antigens, HIV virus-derived antigens, influenza virus-derived antigens, herpesvirus (HSV-1, HSV-2)-derived antigens, anthrax-derived antigens, chlamydia-derived antigens, pneumococcal-derived antigens, Japanese encephalitis virus-derived antigens, measles virus-derived antigens, rubella virus-derived antigens, tetanus-derived antigens, varicella-derived antigens, SARS virus-derived antigens, EB virus-derived antigens, papillomavirus-derived antigens, Helicobacter pylori-derived antigens, rabies virus-derived antigens, West Nile virus-derived antigens, hantavirus-derived antigens, streptococcal-derived antigens, staphylococcal-derived antigens, Bordetella pertussis-derived antigens, and Mycobacterium tuberculosis-derived antigens. Examples include antigens derived from tuberculosis, Plasmodium (malaria parasite), poliovirus, zoonotic diseases, cancer, and various food allergens.

[0122] The antigens contained in the vaccine composition of the present invention do not need to be single. This is because, considering the applications of the present invention, it may trigger an immune response against cancer cells, bacteria, viruses, or other substances composed of multiple components that are not single proteins or peptides. In this case, it may include multiple types of proteins capable of triggering an immune response, or a mixture of unspecified types. Furthermore, one possible use of the vaccine composition of the present invention is to include multiple types of antigens with the aim of actively triggering an immune response against multiple types of antigens.

[0123]

[0124] The vaccine composition of the present invention may preferably be an anti-cancer vaccine. Furthermore, the anti-cancer vaccine may be a vaccine for cancer prevention or a vaccine for cancer treatment.

[0125] According to an aspect of the present invention, a vaccine composition comprising a peptide consisting of the amino acid sequence of Sequence ID No. 2 of the present invention (or a peptide consisting of an amino acid sequence showing 95% or more sequence homology thereto) and a specific tumor antigen can exhibit a preventive effect that inhibits cancer growth by activating the immune response of the subject when administered in advance before cancer formation. Furthermore, the vaccine composition of the present invention has been shown to inhibit cancer growth or kill cancer cells when administered to a subject after cancer formation, confirming its efficacy as a therapeutic vaccine.

[0126] In the present invention, the type of cancer is not particularly limited, and can preferably be selected from the group consisting of breast cancer, colorectal cancer, prostate cancer, cervical cancer, stomach cancer, skin cancer, oral cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, pharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, esophageal cancer, rectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, bladder cancer, non-Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, childhood cancer, bronchial cancer, colon cancer, and ovarian cancer.

[0127] The vaccine composition of the present invention may further comprise at least one selected from the group consisting of any vaccine adjuvant and an immune checkpoint inhibitor.

[0128] According to one embodiment of the present invention, the peptide of the present invention was found to exhibit a more pronounced effect in activating the target immune function when treated in combination with any additional vaccine adjuvants and / or immune checkpoint inhibitors. This effect is particularly desirable in that it can minimize side effects that may occur from administering high doses of the substance, and achieve maximum effect with minimum dosage.

[0129] The vaccine adjuvants that may further be included in the vaccine composition of the present invention are not particularly limited in type and should be understood to include vaccine adjuvants currently widely used in the industry or newly developed vaccine adjuvants. Non-limiting examples of any of the above vaccine adjuvants include 1018 ISS, aluminum salt, Amplibax, AS15, BCG, CP-870, 893, CpG ODN, CpG7909, CIA-A, dSLIM, GM-CSF, IC30, IC31, imiquimod, Imfact IMP321, IS patch, IScomatrix, Jubuimmun, Lipovax, MF59, monophosphoryl lipid A, montanaid IMS 1312, montanaid ISA 206, and montanaid ISA This includes 50V, Montanaid, OK-432, OM-174, OM-197-MP-EC, Ontac, Peptel Vector System, PLG microparticles, Reshimode, SRL172, viralosomes and other viral particles, YF-17DBCG, Accuras QS21 Stimulon, Livis Detoxkill, Superforce, Proindos, GM-CSF, cholera toxin, immunological adjuvants, MF59, and cytokines, most preferably CpG ODN.

[0130] In the present invention, the checkpoint inhibitor is a substance that blocks immune checkpoints. Immune checkpoints can be stimulant or suppressive. Blocking suppressive immune checkpoints activates immune system function and can be used in cancer immunotherapy [see Pardoll, Nature Reviews. Cancer 12:252-64 (2012)]. When tumor cells attach to specific T cell receptors, they eliminate activated T cells. Immune checkpoint inhibitors prevent tumor cells from attaching to T cells, thereby leaving the T cells activated. In fact, the synergistic action of cells and soluble components counteracts damage caused by pathogens and cancer. Modulation of immune system pathways involves altering the expression or functional activity of at least one component of the pathway, thereby regulating the immune system's response. Immune checkpoint inhibitors include PD-1 (programmed cell death-1) antagonists, PD-L1 (programmed cell death-ligand 1) antagonists, PD-L2 (programmed cell death-ligand 2) antagonists, CD27 (cluster of differentiation 27) antagonists, CD28 (cluster of differentiation 28) antagonists, CD70 (cluster of differentiation 70) antagonists, CD80 (cluster of differentiation 80, also known as B7-1) antagonists, CD86 (cluster of differentiation 86, also known as B7-2) antagonists, CD137 (cluster of differentiation 137) antagonists, CD276 (cluster of differentiation 276) antagonists, and KIRs (killer-cell immunoglobulin-like) inhibitors. receptors) antagonist, LAG3 (lymphocyte-activation gene 3) antagonist, TNFRSF4 (tumor necrosis factor receptor superfamily,Antagonist of member 4 (also known as CD134), GITR (glucocorticoid-induced TNFR-related protein) antagonist, GITRL (glucocorticoid-induced TNFR-related protein ligand) antagonist, 4-1BBL (4-1BB ligand) antagonist, CTLA-4 (cytolytic T lymphocyte associated antigen-4) antagonist, A2AR (Adenosine A2A receptor) antagonist, VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, BTLA (B- and T-lymphocy) antagonist, IDO (Indoleamine 2,3-dioxygenase) antagonist, TIM-3 (T-cell immunoglobulin domain and Mucindomain 3) antagonist, VISTA (V-domain Ig suppressor of T cell It can be an activation antagonist and a KLRA (killer cell lectin-like receptor subfamily A) antagonist.

[0131] In one embodiment of the present invention, an immune checkpoint inhibitor may be a PD-1 antagonist. PD-1 is a T-cell co-suppressive receptor that plays a central role in the ability of tumor cells to evade the host immune system. Blocking the interaction between PD-1 and PD-L1, and the ligands of PD-1 antagonists, enhances immune function and mediates antitumor activity. Examples of PD-1 antagonists include antibodies that specifically bind to PD-1. Specific anti-PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, STI-1014, and pizillizumab.

[0132] In another embodiment of the present invention, the immune checkpoint inhibitor may be a PD-L1 antagonist. Examples of PD-L1 antagonists include antibodies that specifically bind to PD-L1. Specific anti-PD-L1 antibodies include, but are not limited to, avelumab, atezolizumab, durvalumab, and BMS-936559.

[0133] In another embodiment, the immune checkpoint inhibitor may be a LAG3 antagonist. LAG3 (lymphocyte activation gene 3) is a negative costimulatory receptor that regulates T cell homeostasis, proliferation, and activation. Furthermore, LAG3 has been reported to be involved in regulatory T cell (Treg) inhibitory function. The majority of LAG3 molecules are retained in cells near the microtubule organizing center and are induced only after activation of antigen-specific T cells (see US 2014 / 0286935). Examples of LAG3 antagonists include antibodies that specifically bind to LAG3. Certain anti-LAG3 antibodies include, but are not limited to, GSK2831781.

[0134] In the present invention, antibodies mean that, insofar as they exhibit the desired biological activity, they include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments. In another embodiment, antibodies mean that they include soluble receptors that do not retain the Fc portion of the antibody. In one embodiment, antibodies may be humanized monoclonal antibodies and their fragments prepared by recombinant gene manipulation.

[0135] Another class of immune checkpoint inhibitors comprises polypeptides that bind to and block the PD-1 receptor on T cells without inducing inhibitor signal transduction. Such peptides include B7-DC polypeptide, B7-H1 polypeptide, B7-1 polypeptide, and B7-2 polypeptide, as disclosed in U.S. Patent No. 8,114,845, as well as their soluble fragments.

[0136] Another class of immune checkpoint inhibitors includes compounds having a peptide moiety that inhibits PD-1 signaling. Examples of such compounds are disclosed in U.S. Patent No. 8,907,053.

[0137] Another class of immune checkpoint inhibitors includes inhibitors of specific metabolic enzymes, such as indoleamine-2,3-deoxygenase (IDO), which are expressed by infiltrating bone marrow cells and tumor cells. IDO enzymes inhibit the immune response by depleting amino acids necessary for anabolism in T cells, or by synthesizing specific native ligands for cytoplasmic receptors that can alter lymphocyte function.

[0138] The compositions of the present invention (e.g., polypeptides, polynucleotides, etc.) can generally be formulated in pharmaceutically acceptable or physiologically acceptable solutions, either alone or in combination with one or more other therapeutic methods, for administration to cells, tissues, or animals. If desired (as needed), the compositions of the present invention can be administered in combination with other agents (e.g., other proteins, polypeptides, or various pharmaceutically active agents). There are virtually no limitations on other components that may be included in the compositions of the present invention, as long as the additional agents do not adversely affect the properties of the peptides, etc. of the present invention.

[0139] The formulations of pharmaceutically acceptable excipients and carriers in the compositions of the present invention are well known to those skilled in the art. Furthermore, appropriate dosages and therapeutic methods in the use of the specific compositions described herein can be those well known to those skilled in the art, and may include, for example, oral, parenteral, intravenous, intranasal, intracerebral, and intramuscular administration and formulations (combinations) for such administration.

[0140] For specific applications, the pharmaceutical compositions disclosed herein may be delivered by oral administration to a subject. As such, the compositions may be formulated (compounded) with an inert diluent or with an absorbable food carrier, or the compositions may be encapsulated in hard-shell or soft-shell gelatin capsules, or the compositions may be compressed into tablets, or the compositions may be directly incorporated into food.

[0141] In certain circumstances, the pharmaceutical compositions disclosed herein may be preferably delivered parenterally, intravenously, intramuscularly, or intraperitoneally, and such routes of administration may be referenced from, for example, those described in U.S. Patents 5,543,158, 5,641,515, 5,399,363, etc. Solutions of the active compound (as a free base or as a pharmaceutically acceptable salt) can be prepared by appropriately mixing with a surfactant such as hydroxypropylcellulose in water. Furthermore, dispersions can be prepared in glycerol, liquid polyethylene glycol, or a mixture thereof, or in oil. To prevent microbial growth under general storage and use conditions, the preparations may contain preservatives.

[0142] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions, and sterile powders that enable the immediate preparation of sterile injectable solutions or dispersions (see U.S. Patent No. 5,466,468). In all cases, the pharmaceutical form must be sterile and fluid enough to be easily injected. It must be stable under production and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating agent such as lecithin to maintain the particle size required in dispersions, and by using a surfactant. Prevention of microbial action may be possible with various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include an isotonic agent (e.g., sugar or sodium chloride). Extended absorption of injectable compositions can be achieved by incorporating absorption-delaying agents (e.g., aluminum monostylate and gelatin) into the composition.

[0143] For parenteral administration of aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonicized with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media that can be used in this regard are known to those skilled in the art. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the proposed injection site. Depending on the symptoms of the subject to which the pharmaceutical composition is treated, some variation in dose is inherently necessary. Those skilled in the art can determine the appropriate dose for individual subjects by conventional knowledge of the art. Furthermore, for administration to humans, the preparation must meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA Office of Biologics standards.

[0144] Sterile injectable solutions can be prepared by including the required amount of the active compound in a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions can be prepared by including various sterilized active components in a sterile vehicle containing a basic dispersion medium and other necessary components listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred manufacturing methods may be vacuum drying and freeze-drying techniques, and the sterile powder is obtained from a pre-sterilized filtered solution. Powders of any additional desired components in addition to the active component can be manufactured.

[0145] The compositions disclosed herein can be formulated (compounded) in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, stinic acid, mandelic acid). Free carboxyl groups and the resulting salts can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine). Depending on the formulation, the solution is administered in a manner appropriate to the formulation and in a therapeutically effective amount. The formulations can be readily administered in various formulation forms, such as injection solutions and drug-release capsules.

[0146] As used herein, the term “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antimicrobial and antifungal agent, isotonic and absorption retardant, buffer, carrier solution, suspension, colloid, etc. The use of media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is used in therapeutic compositions unless it is incompatible with the active ingredient. Auxiliary active ingredients may also be included in the compositions of the present invention.

[0147] The term "pharmaceutically acceptable" refers to molecular materials and compositions that do not cause allergic reactions or similar inappropriate reactions when administered to humans. Methods for producing aqueous compositions containing proteins as active ingredients are well known in the art. Typically, such compositions are prepared as injectable liquid solutions or suspensions. Solid forms suitable for dissolving or suspending in liquid before injection can also be produced. Furthermore, the product may be an emulsion.

[0148] In certain embodiments, pharmaceutical compositions can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles. Methods for direct delivery of gene, polynucleotide, and peptide compositions to the lungs via nasal aerosol spray can be found, for example, in U.S. Patents 5,756,353 and 5,804,212. Similarly, the delivery of pharmaceuticals using intranasal particulate resins (Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U.S. Patent 5,725,871) are also well known in the pharmaceutical field. Likewise, mucosal drug delivery in the form of a polytetrafluoroetheylene support matrix can be found in U.S. Patent 5,780,045.

[0149] In certain embodiments, delivery can be carried out by the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., to introduce the composition of the present invention into suitable cells. In particular, the composition of the present invention can be encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc., and formulated for delivery. The formulation and the use of such delivery vehicles can be carried out using known prior art.

[0150] Furthermore, the pharmaceutical compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. The pharmaceutical compositions formulated as described above can be administered in an effective amount through several routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, as described above. In the above, “effective amount” means the amount of substance that, when administered to a patient, allows for the tracking of diagnostic or therapeutic effects. The pharmaceutical compositions containing the polypeptide of the present invention may vary in the content of the active ingredient depending on the severity of the disease, but can usually be administered several times a day in an effective dose of 0.1 μg to 10,000 mg, preferably 1 mg to 5,000 mg, based on an adult. However, the dosage of the pharmaceutical compositions according to the present invention can be appropriately selected depending on the route of administration, the target, the target disease and its severity, age, sex, weight, individual differences, and disease state, and such techniques are known to those skilled in the art.

[0151] In another aspect, the present invention provides a method for using the compositions of the present invention (e.g., polynucleotides, polypeptides, etc.) on cells, tissues, or subjects to achieve a desired cellular effect and / or therapeutic effect. The cells or tissues that can be controlled by the present invention may preferably be mammalian cells or tissues, and more preferably human cells or tissues. Such cells or tissues may be in a healthy state or in a diseased state.

[0152] The present invention also provides a pharmaceutical composition for cancer prevention or treatment comprising at least one selected from the group consisting of (i) to (iv) below. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0153] According to one aspect of the present invention, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, or a peptide containing an amino acid sequence showing 95% or more homology to that peptide, can both exert an effect of suppressing tumor growth and killing tumors, thereby demonstrating cancer prevention or therapeutic effects.

[0154] The present invention also provides at least one use selected from the group consisting of (i) to (iv) below for manufacturing cancer treatment formulations. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0155] The present invention also provides a method for treating cancer, comprising administering an effective amount of a composition comprising at least one selected from the group consisting of (i) to (iv) below to an individual in need. (i) The peptide, (ii) Polynucleotide encoding the above (i), (iii) vectors including (ii) above, and (iv) Host cells transformed in (iii) above.

[0156] In this invention, the "effective amount" refers to the amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing, or suppressing or reducing cancer. The "individual" may be an animal, preferably a mammal, and may include humans, or it may be animal-derived cells, tissues, organs, etc. The individual may be a patient who requires the effect.

[0157] In this invention, "treatment" broadly refers to improving cancer or the symptoms of cancer, which may include curing, substantially preventing, or improving the condition of cancer. It includes, but is not limited to, reducing, curing, or preventing symptoms or most of the symptoms. [Effects of the Invention]

[0158] The peptide disclosed in this invention, as the CRS fragment first disclosed herein, exhibits anticancer activity and immune function-enhancing activity.

[0159] Furthermore, the above-mentioned peptide, the polynucleotide encoding it, the vector containing the polynucleotide, the host cells transformed with the vector, or the full-length CRS protein exhibit excellent anti-cancer activity and immune-enhancing activity, and can be very usefully utilized in the development of vaccine adjuvants, vaccine compositions, and cancer treatment compositions. [Brief explanation of the drawing]

[0160] [Figure 1] Figure 1 is a schematic diagram illustrating the development method of C-VAX in a simplified manner. [Figure 2] Figure 2 shows the results of confirming protein stability after purifying the intermediates derived during the C-VAX development process, performing SDS-PAGE using an acrylamide gel, and then staining the protein with Coomassie blue stain. [Figure 3] Figure 3 shows the results of determining whether UNE-C1-4H and C-VAX are multimers or not, using size exclusion chromatography (FPLC). [Figure 4] Figure 4 shows the results of treating the developed C-VAX with his-UNE-C1-his in PMA (50 ng / ml) for 48 hours, then treating the differentiated THP-1 cells with TNF-α in culture medium for 4 hours, and confirming the levels via ELISA. [Figure 5] Figures 5 and 6 show the results of ELISA testing for IL-6(e) and IL-12p70(f) present in BMDC after treating it with his-UNE-C1-his and C-VAX at different concentrations for 24 hours, respectively. [Figure 6]Figures 5 and 6 show the results of ELISA testing for IL-6(e) and IL-12p70(f) present in BMDC after treating it with his-UNE-C1-his and C-VAX at different concentrations for 24 hours, respectively. [Figure 7] Figure 7 shows the experimental results after treating each HEK-Blue cell line with his-UNE-C-his and C-VAX at different concentrations for 24 hours, collecting the supernatant, and reacting it with QUANTI-Blue solution to confirm whether each cell line exhibited immunoactivity. [Figure 8] Figure 8 shows the results of transplanting Eg7-OVA cells (5x10⁵) subcutaneously into the right isocutaneous tissue of c57bl / 6 mice, and treating them with OVA (10ug / mouse), OVA (10ug / mouse) + his-UNE-C1-his (100ug / mouse), or OVA (10ug / mouse) + C-VAX (100ug / mouse) on day 3 and day 10, followed by observation of tumor size until day 17. [Modes for carrying out the invention]

[0161] [Specific details for carrying out the invention] The present invention will be described in detail below. However, the following embodiments are illustrative of the present invention, and the content of the present invention is not limited to the following embodiments.

[0162] Experimental method 1. Mouse C57BL / 6 and BALB / c mice were obtained from DooYeol Biotech. OVA-specific T cell receptor gene-modified OT-1 mice were provided courtesy of Professor Kang Chang-yul of Seoul National University. All mouse experiments were conducted in accordance with guidelines approved by Seoul National University. All mice were maintained in Friendship BSC using institutionally approved protocols.

[0163] 2. Cell lines CT26 and B16f10 CRS overexpressing cells were generated using the G418 selection method. Briefly, lipofectamine 2000 was used to transform each cell with pEXPR-IBA105 EV and CRS. To select overexpressing cells, 1 mg / ml of G418 was used in the growth medium, and resistant monoclonals were selected. The expression level of each clone was tested by immunoblotting, and clones with similar in vitro growth were selected for experimentation.

[0164] 3. Allogeneic mouse tumor model and tumor measurement CT26 and B16F10 were maintained in DMEM containing 10% FBS and 1% streptomycin. A total of 5 × 10⁶ CT26 and B16F10 were maintained. 5 The cells were subcutaneously injected into the right side of BALB / c and C57BL / 6 mice approximately 6-8 weeks old. On days 7, 8, and 9, 200 μg of each protein was injected intraperitoneally. The tumor was measured using a digital caliper and calculated using the following formula (0.52 × length × width). 2 The calculation was performed according to the formula. The tumor was 1500 mm. 3 At that point, the mice were euthanized.

[0165] 4. Cell binding assay After isolating the spleen of C57Bl / 6 mice, physical force was applied to separate the cells to the single-cell level, and they were then aliquoted. These aliquot cells, stained with Alexa647 dye and containing BSA and CRS proteins, were cultured at 4°C for 1 hour. The cultured cells were stained with CD11b, CD11c, CD3, CD19, Ly6C, Ly6G, and F4 / 80 FACS antibodies, and then analyzed by FACS. CD3: T cell, CD11b+, F4 / 80+: macrophage, CD11b, Ly6C: monocyte, CD11b+, Ly6G: neutrophil, CD19: B cell, CD11b+, CD11c+: dendritic cell

[0166] 5. Analysis of tumor-infiltrating immune cells and spleen immune cells CT26 tumor-carrying mice were sacrificed on days 10 and 12. Tumor cells were dissociated using a tumor dissociation kit, mouse (130-096-730, Miltenyi Biotec) according to the preparation protocol. Splenocytes were dissociated in a medium containing RPMI medium with 2% FBS and 1% streptomycin. After lysing red blood cells (Lysing Buffer (555899, BD biosciences)), tumor and spleen cells were stained with CD8, CD4, CD3, CD45, Foxp3, and CD69 antibodies. Monoclonal antibodies were stained at 4°C for 30 minutes. For intracellular staining, a fixation / permeabilization solution kit with BD GolgiPlug (trademark) (555028, BD biosciences) was used.

[0167] 6. Protein purification CRS and CRS(106-228) proteins were purified using a pET28a plasmid vector containing an N-terminal 6×his tag. His-UNE-C1-4H and C-VAX proteins were purified using a pHIS. Parallell plasmid vector containing an N-terminal 6×his tag and a region cleavable by rTEV. BL21-codon plus cells were transformed, and colonies were inoculated into culture medium and grown. Large cells were grown in LB until OD 600 reached 0.5, and protein expression was induced using 0.5 mM IPTG at 4°C for 16 hours. Cell pellets were obtained by centrifugation and sonicated with 50 mM Tris buffer pH 7.5 containing 300 mM NaCl. The supernatant was then obtained by centrifugation at 20,000 g for 30 minutes. This supernatant was poured onto a column containing Ni-NTA resin. The washing step was performed with 50 mM Tris, pH 7.5, containing 300 mM NaCl, 5% glycerol, and 15 mM imidazole. Proteins were separated from the column with 10 ml of elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 300 mM imidazole). For His-UNE-C1-4H and C-VAX, rTEV protease was mixed with the total amount of target protein at a ratio of 1:20 (g), followed by diagnosis, and endotoxin was removed using TX-114 (REF: Removal of endotoxin from protein solutions by phase separation using Triton X-114). After packing the polyprep column with SM-2 beads, the protein was passed through according to the manufacturer's recommended protocol, and any remaining Triton X-114 was removed. Based on LAL analysis, titration proteins of 0.04 EU / mg or less were used in all experiments.

[0168] 7. ELISA The tests were conducted to confirm cytokine secretion from THP1-PMA and BMDC (bone marrow-derived DC) cells. 5 × 10⁶ cells were used in a 24-well plate. 5Cells were processed at a rate of cells / ml, and each well was changed to serum-free medium 2 hours before reagent treatment. For THP1-PMA cells, 100 nM protein was treated for 4 hours. For BMDC cells, 100 nM protein was treated for 24 hours. The supernatant was centrifuged at 500 g for 10 minutes, and ELISA was performed using the IL-6, TNF-α, and IL-12 ELISA set (BD).

[0169] 8. Enabling BMDC and BMM BMDCs (bone marrow-derived dendritic cells) were prepared using a standard method. In short, bone marrow cells were obtained from female C57BL / 6 mice and cultured in RPMI medium containing 10% FBS, 1% streptomycin, and 10 ng / ml GM-CSF (R&D, BMDC) or 10 ng / ml M-CSF (R&D, BMM). On day 3, GM-CSF or M-CSF and fresh RPMI medium were added, and on day 6, BMDCs were collected from non-adherent and loosely adhered cells, and BMMs from adhered cells. Each protein was treated with 100 nM and 1 μg / ml LPS for 18 hours, and FACS analysis was performed using antibodies against FACS CD11c, CD40, CD80, and CD86.

[0170] 9. Mouse model for therapeutic anti-cancer vaccines In the therapeutic cancer vaccine model using C-VAX, E.G7-OVA cells were injected via sc into the right dorsal side. On days 3 and 10, OVA (10 ug / mouse), OVA (10 ug / mouse) + his-UNE-C1-his (100 ug / mouse), and OVA (10 ug / mouse) + C-VAX (100 ug / mouse) were injected into the left dorsal side, and tumor volume was measured as described above.

[0171] 10. Size Exclusion Chromatography (FPLC: Fast Protein Liquid Chromatography) Protein samples were concentrated to the required concentration using a buffer consisting of 50 mM Tris, pH 7.5, and 300 mM NaCl, and an Amicon Ultra-15 centrifugal filter unit. After connecting the column (GE Healthcare, Chicago, Illinois, USA) to AKTA pure (GE Healthcare), it was washed with three times the column volume of buffer to equilibrate with the protein buffer. After equilibration, the protein samples were injected into AKTA pure, and the experiment was performed according to the protocol under the conditions described by the manufacturer.

[0172] 11. HEK-blue SEAP assay HEK cells were cultured in a 5% CO2, 37°C incubator in DMEM containing 1% antibiotic, 10% FBS, and 100 μg / ml Normocin (Invivogen, San Diego, CA, USA). After processing the proteins to be treated in 24-well plates, hTLR2, hTLR1 / TLR2, and TLR2KO-hTLR1 / TLR2-HEK-Blue cells were plated in 5 × 10⁶ wells. 5 Each cell was added to a well. The plate was incubated for 24 hours, and the supernatant was collected. The collected supernatant was mixed with QUANTI-Blue solution (InvivoGen), incubated at 37°C for 15 minutes to 6 hours, and the results were obtained by measuring at OD 620 nm.

[0173] Experimental results 1. Development of C-VAX, a form of drug development, and verification of its immune activity and anti-cancer efficacy. After confirming the efficacy of CRS(106-228) in existing studies, the inventors proceeded with research to make CRS(106-228) into a form suitable for drug development. A form suitable for drug development must satisfy three conditions: (1) tagless, (2) stable without proteolysis, and (3) exhibiting efficacy equivalent to or higher than the initial form.

[0174] The CRS(106-228) used in previous studies is a form in which his-tags are bound to both ends of the protein, and in this invention, this CRS(106-228) fragment has been named his-UNE-C1-his.

[0175] In this invention, we have conducted research to remove the his-tags at both ends of the his-UNE-C1-his molecule and convert it from a multiform morphology due to cysteine ​​to a single-form morphology (Figure 1).

[0176] (1) Removal of His-tag and regulation of protein sequence (UNE-C1-4H production) After removing the C-terminal his-tag from the His-UNE-C1-his protein (his-UNE-C1), protein degradation was observed. To stabilize this, the protein sequence was adjusted from 106-228 aa to 99-200 aa in CRS (SEQ ID NO: 1), and the sequenced CRS(99-200 aa) peptide (hereinafter referred to as "UNE-C1-4H") was confirmed to be a stable form that does not undergo protein degradation (Figure 2).

[0177] (2) Production of single-form peptides without multimers The previously prepared UNE-C1-4H was confirmed to exist in two forms, trimer and monomer, via FPLC (Fast protein liquid chromatography). To resolve this, the cysteine ​​residue in UNE-C1-4H was modified to a serine residue, and when examined via FPLC, only the monomer was confirmed to exist (Figure 3). The modified UNE-C1-4H was named C-VAX.

[0178] (3) Immune activity of C-VAX The efficacy of his-UNE-C1-his and C-VAX in macrophage immunoactivation was confirmed using a PMA-differentiated THP-1 cell line. The experimental results confirmed that both C-VAX and his-UNE-C1-his exhibited sufficient immunoactivating efficacy (Figure 4).

[0179] Furthermore, the immunoactive efficacy of his-UNE-C1-his and C-VAX was confirmed using BMDC (bone marrow-derived dendrictic cell). The experimental results confirmed that both C-VAX and his-UNE-C1-his increased IL-6 and IL-12p70 cytokine secretion in BMDC as well (Figures 5 and 6).

[0180] (4) C-VAX immunoactivation mechanism by TLR2 / 6 Previous studies have confirmed that His-UNE-C1-his induces immune activity via TLR2 / 6. Therefore, in this invention, we investigated whether C-VAX, which is based on his-UNE-C1-his, also possesses immune activity via TLR2 / 6.

[0181] Experiments using the hTLR2-HEK-blue cell line, hTLR2 KO-TLR1 / 6-HEK-Blue cell line, and hTLR1 / 2-HEK-blue cell line showed that his-UNE-C1-his and C-VAX exhibited immunoactive activity in the same pattern, confirming that TLR2 is extremely important and that neither TLR1 / 6 showed any efficacy. Therefore, it was found that C-VAX also utilizes TLR2 / 6 as a receptor, and that C-VAX exhibits immunoactive activity through the same mechanism as his-UNE-C1-his (Figure 7).

[0182] (5) Anticancer efficacy of C-VAX Previous studies have confirmed the efficacy of his-UNE-C1-his as an anti-cancer vaccine, so we attempted to confirm the efficacy of C-VAX as an anti-cancer vaccine as well.

[0183] E.G7-OVA cell line 5x10 5 Cell was injected subcutaneously on the right side (right subcutaneous injection), and his-UNE-C1-his, C-VAX 5mpk, and OVA protein were injected subcutaneously on the left side (left subcutaneous injection) on day 3 and day 7 (left subcutaneous injection). The size of the tumor and the body weight of the mice were then measured.

[0184] When OVA was treated with C-VAX, it was confirmed to exhibit a potent anticancer effect, similar to when OVA was treated with his-UNE-C1-his (Figure 8). [Industrial applicability]

[0185] The peptides disclosed in this invention exhibit anticancer activity and immune function-enhancing activity as CRS fragments first disclosed herein.

[0186] Furthermore, the peptide, the polynucleotide encoding it, the vector containing the polynucleotide, the host cells transformed with the vector, or the full-length CRS protein exhibit excellent anti-cancer activity and immune-enhancing activity, making them highly useful in the development of vaccine adjuvants, vaccine compositions, and cancer treatment compositions, thus possessing excellent industrial applicability.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

2. The peptide according to claim 1, wherein the peptide activates innate immunity and acquired immunity.

3. A polynucleotide comprising a base sequence encoding the peptide described in claim 1.

4. The polynucleotide according to claim 3, characterized in that the polynucleotide consists of the base sequence of SEQ ID NO:

4.

5. A vector comprising the polynucleotide described in claim 4.

6. A host cell transformed with the vector described in claim 5.

7. Vaccine adjuvants comprising at least one selected from the following groups (i) to (iv): (i) The peptide according to claim 1, (ii) Polynucleotides representing (i) above, (iii) A vector including the above (ii), and (iv) Host cells transformed in (iii) above.

8. A vaccine composition comprising the vaccine adjuvant and antigen according to claim 7.

9. The aforementioned antigens include alpha-fetal protein, carcinoembryonic antigen, cdk4, β-catenin, CA125, caspase-8, epithelial tumor antigen, HPV antigen, HPV16 antigen, CTL epitope derived from HPV16 E7 antigen, melanoma-associated antigen (MAGE)-1, MAGE-3, tyrosinase, surface Ig idiotype, Her-2 / neu, MUC-1, prostate-specific antigen (PSA), sialyl Tn (STn), heat shock protein, gp96, ganglioside molecules GM2, GD2, GD3, and carcinoembryonic antigen (CEA). (antigen), PRAME, WT1, Survivin, Cyclin D, Cyclin E, HER2, MAGE, NY-ESO, EGF, GP100, Cathepsin G, Human papillomavirus (HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, Her / 2-neu antigen, Chimeric Her2 antigen, Prostate-specific antigen (PSA), Bivalent PSA, ERG, Androgen receptor (AR), PAK6, Prostate stem cell antigen (PSCA), NY-ESO-1, Stratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1 (WT)-1, HIV-1 gag, human telomerase reverse transcriptase (hTERT), proteinase 3, tyrosinase-related protein 2 (TRP2), high molecular weight melanoma-associated antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, female embryo antigen (CEA), melanoma-associated antigen E (MAGE-A, MAGE1, MAGE2, MAGE3, MAGE4), interleukin-13 receptor α (IL13-Rα), carbonate dehydratase IX (CAIX), survivorbin, GP100, angiogenesis antigen, rastan Protein, p53 protein, p97 melanoma antigen, KLH antigen, cancer embryo antigen (CEA), gp100, MART1 antigen, TRP-2, HSP-70, β-HCG, testisin, 1A01_HLA-A / m; 1A02; 5T4; ACRBP; AFP; AKAP4; α-actinin-_4 / m; α-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1 / m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1;BASI; BCL-2; bcr / abl; β-catenin / m; BING-4; BIRC7; BRCA1 / m; BY55; calreticulin; CAMEL; CASPA; caspase_8; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_homolog_1; CD44_homolog_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27 / m; CDE 30; CDK4 / m; CDKN2A / m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1 / m; coactosin-like protein; collagen_XXIII; COX-2; CP1B1; CSAG2; CT-9 / BRD6; CT45A1; CT55; CTAG2 homolog_LAGE-1A; CTAG2 homology_LAGE-1B; CTCFL; Cten; cyclin_B1; cyclin_D1; cyp-B; DAM-10; DEP1A; E7; EF1A2 ;EFTUD2 / m;EGFR;EGLN3;ELF2 / m;EMMPRIN;EpCam;EphA2;EphA3;ErbB3;ERBB4;ERG;ETV6;EWS;EZH2;FABP7;FCGR3A_version_1;FCGR3A_version_2;FGF5;FGFR2;FibronectinFOS;FOXP3;FUT1;G250;GAGE-1;GAGE-2;GAGE-3;GAGE-4;GAGE-5;GAGE-6;GAGE7b;GAGE-8_(GAGE-2D);GASR;GnT-V;GPC3;GPNMB / m; GRM3; HAGE; hepsin; Her2 / neu; HLA-A2 / m; Homeobox_NKX3.1; HOM-TES-85; HPG1; HS71A; HS71B; HST-2; hTERT; iCE; IF2B3; IL-10; IL-13Ra2; IL2-RA; IL 2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2; kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K; MAGA5;MAGA8,MAGAB,MAGE-_B1,MAGE-_E1,MAGE-A1,MAGE-A10,MAGE-A12,MAGE-A 2MAGE-A3,MAGE-A4,MAGE-A6,MAGE-A9,MAGE-B10,MAGE-B16,MAGE-B17;M AGE-B2,MAGE-B3,MAGE-B4,MAGE-B5,MAGE-B6,MAGE-C1,MAGE-C2,MAGE-C3 STOMACH-D1,STOMACH-D2,STOMACH-D4,STOMACH-E1_(STOMACH1)STOMACH-E2,STOMACH-F1,STOMACH-H 1MAGEL2:MART_1 / メラン(melan)-A:MART -2:MC1_RM-CSF(mesothelin)MITF:MMP1_1:MMP7:MUC-1:MUM-1 / m MUM-2 / mMYO1A,MYO1B,MYO1C,MYO1D,MYO1E,MYO1F,MYO1G,MYO1H,NA1 NA88-ANeo-PAP:NFYC / mNGEP:N-myc:NPM:NRCAM:NSE:NUF2:NY-ESO-1OA 1OGT:OS-9CarlCarp53PAGE-4PAI-1PAI-2PAP:PATEPA X3,PAX5,PD1L1,PDCD1,PDEF,PECA1,PGCB,PGFRB,Pim-1_-キナゼPin-1 / PL AC1,PMEL, PML, POTE, POTEF, PRAME, PRDX5 / m, PRM2, Pr PSA-3):PSA,PSB9,PSCA,PSGR,PSM,PTPRC,RAB8A,RAGE-1,RARA,RASH,RAS KRASN:RGS5:RHAMM / CD168:RHOCRSSA:RU1:RU2:RUNX1:S-100:SAGE:SART -1:SART-2;SART-3:SEPR:SERPINB5:SIA7F)SIA8A:SIAT9:SIRT2 / m:SOX10; SP17,SPNXA, SPXN3, SSX-1, SSX-2, SSX3, SSX-4, ST1A1, STAG2, STAMP-1, ST EAP-1, glucocorticoid-2B, SYCP1, SYT-SSX-1, SYT-SSX-2, TARP, TCRg, TF2AA.TGFbeta1; TGFR2; TGM-4; TIE2; TKTL1; TPI / m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101; Tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; XAGE1; α-Actinin-4; ARTC1; BCR-ABL fusion protein (b3a2); B-RAF; CASP-5; CASP-8; β-catenin; Cdc27; CDK4; CDKN2A; COA-1; dek-can fusion protein; EFTUD2; Elongation factor 2 2) ETV6-AML1 fusion protein; FN1; GPNMB; LDLR-fucosyltransferase AS fusion protein; HLA-A2d; HLA-A11d; hsp70-2; KIAAO205; MART2; ME1; MUM-If; MUM-2; MUM-3; neo-PAP; myosin class I; NFYC; OGT; OS-9; pml-RAR alpha fusion protein; PRDX5; PTPRK; K-ras; N-ras; RBAF600; SIRT2; SNRPD1; SYT-SSX1 or SSX2 fusion protein; triosephosphate isomerase Isomerase); BAGE-1; GAGE-1,2,8; GAGE-3,4,5,6,7; GnTVf; HERV-K-MEL; KK-LC-1; KM-HN-1; LAGE -1;MAGE-A1;MAGE-A2;MAGE-A3;MAGE-A4;MAGE-A6;MAGE-A9;MAGE-A10;MAGE-A12;MAGE-C2;mucin k;NA-88;NY-ESO-1 / LAGE-2;SAGE;Sp17;SSX-2;SSX-4;TRAG-3;TRP2-INT2g;CEA;gp100 / Pmel17;Kallikrein 4 4) ;mammaglobin-A;Melan-A / MART-1;NY-BR-1;OA1;PSA;RAB38 / NY-MEL-1;TRP-1 / gp75;TRP-2;tyrosinase;adipophilin;AIM-2;BING-4;CPSF;cyclin D1;Ep-CAM;EphA3;FGF5;G250 / MN / CAIX;HER-2 / neu;IL13R alpha-2; Intestinal carboxyl esterase; alpha-fetoprotein; M-CSF; mdm-2; MMP-2; MUC1; p53; PBF; PRAME; PSMA; RAGE-1; RNF43; RU2AS; secernin 1; SOX10; STEAP1; Survivin; Telomerase; WT1; FLT3-ITD; BCLX(L); DKK1; ENAH(hMena); MCSP; RGS5; Gastrin-17; Human Chorionic Gonadotropin, EGFRvIII, HER2, HER2 / neu, P501, Guanylyl Cyclase C The vaccine composition according to claim 8, characterized in that it is at least one selected from the group consisting of C), PAP, OVA (ovalbumin), and MART-1.

10. The vaccine composition according to claim 8 or 9, wherein the vaccine is an anti-cancer vaccine.

11. The vaccine composition according to claim 10, wherein the anti-cancer vaccine is a vaccine for cancer prevention or a vaccine for cancer treatment.

12. The vaccine composition according to claim 10, characterized in that the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, prostate cancer, cervical cancer, stomach cancer, skin cancer, head and neck cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, nasopharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, esophageal cancer, rectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, childhood cancer, bronchial cancer, colon cancer, and ovarian cancer.

13. The vaccine composition according to claim 10, further comprising at least one selected from the group consisting of vaccine adjuvants, immune checkpoint inhibitors, and combinations thereof.

14. The aforementioned vaccine adjuvants include 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG ODN, CpG7909, CIA-A, dSLIM, GM-CSF, IC30, IC31, imiquimod, Imfact IMP321, IS patch, IScomatrix, Jubuimmun, Lipovax, MF59, monophosphoryl lipid A, montanaid IMS 1312, montanaid ISA 206, and montanaid ISA The vaccine composition according to claim 13, characterized in that it is at least one selected from the group consisting of 50V, Montanaid, OK-432, OM-174, OM-197-MP-EC, Ontac, Peptel Vector System, PLG microparticles, Reshikimode, SRL172, virosoms and other viral particles, YF-17DBCG, Accuras QS21 Stimulon, Livis Detoxkill, Superforce, Proindos, GM-CSF, cholera toxin, immunological adjuvant, MF59, and cytokines.

15. The aforementioned immune checkpoint inhibitors include PD-1 (programmed cell death-1) antagonists, PD-L1 (programmed cell death-ligand 1) antagonists, PD-L2 (programmed cell death-ligand 2) antagonists, CD27 (cluster of differentiation 27) antagonists, CD28 (cluster of differentiation 28) antagonists, CD70 (cluster of differentiation 70) antagonists, CD80 (cluster of differentiation 80, also known as B7-1) antagonists, CD86 (cluster of differentiation 86, also known as B7-2) antagonists, CD137 (cluster of differentiation 137) antagonists, CD276 (cluster of differentiation 276) antagonists, and KIRs (killer-cell immunoglobulin-like) inhibitors. receptors) antagonist, LAG3 (lymphocyte-activation gene 3) antagonist, TNFRSF4 (tumor necrosis factor receptor superfamily, member 4,Also known as CD134 antagonist, GITR (glucocorticoid-induced TNFR-related protein) antagonist, GITRL (glucocorticoid-induced TNFR-related protein ligand) antagonist, 4-1BBL (4-1BB ligand) antagonist, CTLA-4 (cytolytic T lymphocyte associated antigen-4) antagonist, A2AR (Adenosine A2A receptor) antagonist, VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, BTLA (B- and T-lymphocy) antagonist, IDO (Indoleamine 2,3-dioxygenase) antagonist, TIM-3 (T-cell Immunoglobulin domain and Mucindomain 3) antagonist, VISTA (V-domain Ig suppressor of T cell activation) antagonist, and KLRA (killer The vaccine composition according to claim 13, characterized by being at least one selected from the group consisting of cell lectin-like receptor subfamily A) antagonists.

16. A pharmaceutical composition for cancer prevention or treatment comprising at least one selected from the group consisting of (i) to (iv) below: (i) The peptide according to claim 1, (ii) Polynucleotides representing (i) above, (iii) A vector including the above (ii), and (iv) Host cells transformed in (iii) above.

17. Use of one or more of the following groups (i) to (iv) for the manufacture of cancer treatment agents: (i) The peptide according to claim 1, (ii) Polynucleotides representing (i) above, (iii) A vector including the above (ii), and (iv) Host cells transformed in (iii) above.

18. A method for treating cancer, comprising administering an effective amount of a composition containing at least one selected from the group consisting of (i) to (iv) below to an individual (excluding humans) in need of it: (i) The peptide according to claim 1, (ii) Polynucleotides representing (i) above, (iii) A vector including the above (ii), and (iv) Host cells transformed in (iii) above.

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