KR20200107402A - Fragmented cysteinyl-tRNA synthetase peptide and use thereof - Google Patents

Abstract

The present invention relates to a fragmented CRS peptide and uses thereof and, more particularly, to a peptide containing an amino acid sequence of SEQ ID NO: 2; a peptide containing an amino acid sequence showing at least 80% homology with the peptide; a polynucleotide encoding the peptide; a vector comprising the polynucleotide; a host cell transformed with the vector; and uses thereof as vaccine adjuvants. The peptide disclosed in the present invention is a CRS fragment first disclosed in the present specification and exhibits anticancer activity and immune function enhancing activity. In addition, the peptide, the polynucleotide encoding the same, the vector containing the polynucleotide, the host cell transformed with the vector, or the CRS full-length protein are excellent in anticancer activity and immune function enhancing activity, and thus can be very useful in development of vaccine adjuvants, vaccine compositions and compositions for cancer treatment.

Description

Fragmented CRS Peptide and Use thereof {Fragmented Cysteinyl-tRNA Synthetase Peptide and Use thereof}

The present invention relates to a fragmented CRS peptide and a use thereof, and more particularly, to a peptide comprising the amino acid sequence of SEQ ID NO: 2; A peptide comprising an amino acid sequence showing at least 80% homology with the peptide; A polynucleotide encoding the peptide; A vector containing the polynucleotide; Host cells transformed with the vector; And to their use as vaccine adjuvants.

Aminoacyl-tRNA synthetase (ARS or AARS), which catalyzes the aminoacylation of tRNA molecules, is essential to deciphering genetic information during the translation process. Each of the eukaryotic tRNA synthase is composed of a core enzyme (which is closely related to the prokaryotic counterpart of the tRNA synthetase) and an additional domain (which is added to the amino or carboxy terminus of the core enzyme). Therefore, there are significant differences in the composition of the enzyme between eukaryotes and prokaryotes. For example, human tyrosyl-tRNA synthetase (TyrRS) has a carboxy terminal domain that is absent from prokaryotic and lower eukaryotic TyrRS molecules.

Recently, some aminoacyl-tRNA synthase has been demonstrated to have a non-canonical function independent of their involvement in the translation process. That is, in some fragments of the ARS protein, it has been identified that exhibit extracellular signaling (signaling) activity that regulates other types of pathways beyond protein translation, and possesses unexpected activities that are not related to aminoacylation. . Such an unexpected activity may sometimes be an activity that can be used therapeutically for a specific disease or the like, but there are cases where there is a high concern that it may function to induce a disease state in humans. As an example, it has been found that lysyl-t-RNA synthetase (KRS) has an activity to promote cancer metastasis (Korean Patent Registration No. 10-1453141). In addition, the mini-tyrosyl tRNA synthetase (mini-TRS, corresponding to amino acid residues 1 to 364), which is the N-terminal domain of TRS, cleaved by polymorphonuclear cell elastase and plasmin It exhibits irregular biological activity that is not found in full-length proteins.In vitro mini-TRS has been shown to stimulate endothelial cell proliferation and migration, and in mouse matrigel assay, it promotes angiogenesis (pro- It has angiogenic) activity. The ability to promote neovascularization is usually closely associated with cancer metastasis.

Such unexpected activity is not observed in the natural full-length protein sequence (or exhibits an insignificant effect at the level of the natural full-length protein), but certain activities are remarkably displayed when some regions are isolated. Sometimes the effect has properties that are inappropriate for therapeutic use. In order to overcome the difficulties caused by this unpredictability and utilize the therapeutic potential for this ARS family protein, various efforts to identify biologically related forms of other aminoacyl-tRNA synthase proteins are required. .

Meanwhile, the pharmaceutical industry is changing from the past natural or chemically synthesized drugs to the development of protein or peptide drugs, and the market of protein or peptide drugs in the global pharmaceutical market expanded from $43.7 billion in 2006 to 88.5 billion in 2011. The proportion of the domestic protein drug market in the global market is expected to expand from 3% in 2006 to 7% in 2021. Protein or peptide drugs are evaluated as an innovative field of pharmaceuticals because they have fewer side effects and faster efficacy than synthetic drugs.

Currently, the importance of biopharmaceuticals is gradually increasing in the pipeline of major pharmaceutical companies, but there are major technical problems until specific biopharmaceuticals such as peptides are released. For example, the low delivery rate of peptide drugs to the target site and the synthesis of long-chain peptides are acting as obstacles to commercialization. In order to succeed as a peptide drug, it is known that the key to discovering a short sequence and an activity exhibiting activity, that is, selecting the smallest unit (motif) with excellent physiological activity from the full length protein is known. If the length of the peptide is long, the synthesis cost is high, it is not easy to manufacture, and it is known that there is a problem in human absorption.

Accordingly, the present inventors confirmed that cysteinyl-tRNA synthetase (hereinafter referred to as “CRS”) has anticancer activity and immune function enhancing activity, and then the key motif of CRS showing such an effect (motif ) Peptide consisting of consecutive amino acids selected from 180 to 228 amino acids from any one selected from 101 to 119 of the amino acid sequence of SEQ ID NO: 1 as a result of exerting great efforts to identify); Alternatively, the present invention was completed by confirming that the peptide consisting of an amino acid sequence exhibiting 80% or more homology with the peptide has an anticancer activity and immune function enhancing activity equal to or higher than the CRS full-length sequence.

Accordingly, an object of the present invention is a peptide comprising consecutive amino acids from any one selected from the 101 to 119 amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228 amino acid sequence; Or it is to provide a peptide comprising an amino acid sequence that exhibits 80% or more homology with the peptide.

Another object of the present invention is to provide a polynucleotide comprising a nucleotide sequence encoding the peptide.

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

Another object of the present invention is to provide a host cell transformed with the vector.

Another object of the present invention is to provide a vaccine adjuvant comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

Another object of the present invention is to provide a vaccine composition comprising the vaccine adjuvant and antigen.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

In order to achieve the object of the present invention as described above, the present invention is a peptide comprising consecutive amino acids from any one selected from the 101 to 119th amino acid in the amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228th amino acid; Alternatively, it provides a peptide comprising an amino acid sequence exhibiting 80% or more homology with the peptide.

In order to achieve another object of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the peptide.

In order to achieve another object of the present invention, the present invention provides a vector comprising the polynucleotide.

In order to achieve another object of the present invention, the present invention provides a host cell transformed with the vector.

In order to achieve another object of the present invention, the present invention provides a vaccine adjuvant comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

In order to achieve another object of the present invention, the present invention provides a vaccine composition comprising the vaccine adjuvant and an antigen.

In order to achieve another object of the present invention, the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

Hereinafter, the present invention will be described in detail.

The practice of the present invention uses conventional methods of molecular biology and recombinant DNA technology within the technical field to which the present invention pertains, unless otherwise indicated, and most of them are described in the following (literature) for the purpose of explanation. This technique is described in detail in the literature: 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).

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Throughout the content disclosed herein, various aspects or conditions related to the present invention may be proposed in a range format. In the present specification, the description of a range value is meant to include a corresponding threshold value unless otherwise stated, that is, to include all values above the lower limit and below the upper limit. It should be understood that the description of the range form is merely for convenience and simplicity and should not be construed as an inflexible limitation to the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges, as well as individual numerical values within the range. For example, a description of a range such as 7 to 170 may refer to individual values within the range, e.g. 9, 27, 35, 101, and 155, as well as 10 to 127, 23 to 35, 80 to 100, 50. It should be regarded as specifically disclosed subranges such as to 169 and the like. This applies regardless of the width of the range.

The term'comprising' in the present invention is used in the same way as'containing' or'as a feature', and does not exclude additional component elements or method steps that are not mentioned in the composition or method. .

The terms "peptide" and "protein" as used herein are used according to their usual (conventional) meaning, that is, they mean an amino acid sequence. Peptides are not limited to a specific length, but in the context of the present invention generally refer to a fragment of a full length protein, and post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other known in the art. It can contain a formula (a formula of naturally occurring and a formula of non-naturally occurring) and can also be expressed as a "polypeptide". The peptides and proteins of the present invention can be prepared using any of a variety of known recombinant and/or synthetic techniques, and exemplary embodiments thereof are further described below.

The present invention stems from the discovery that CRS and CRS-derived peptides possess therapeutically relevant non-canonical biological activity.

In the present specification, the term'irregular 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 atypical biological activity exhibited by the CRS or fragment thereof of the present invention is, but is not limited to, anticancer activity, activation of innate immune and adaptive immune) can be selected from the group consisting of activation.

It is to be understood that the present invention encompasses not only the CRS fragment peptide having at least one irregular biological activity, but also a variant substantially maintaining the irregular activity.

Specifically, the present inventors have identified that the CRS protein exhibits an activity of inhibiting tumor growth by activating innate immunity and acquired immunity, and as a fragment domain exhibiting such activity, in amino acids 101 to 119 of the amino acid sequence of SEQ ID NO: 1 A peptide region consisting of consecutive amino acids from any one selected to any one selected from 180 to 228 amino acids was identified. The irregular biological activity of the CRS and the region of the fragment peptide thereof are disclosed for the first time in the present invention.

Accordingly, the present invention provides a peptide comprising consecutive amino acids from any one selected from the 101 to 119 amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228 amino acid sequence; Alternatively, it provides a peptide comprising an amino acid sequence exhibiting 80% or more homology with the peptide.

In the present invention, the peptide is a cleavage type of CRS protein, and the peptide is selected from any one selected from the 101 to 119 amino acid sequence of the CRS whole protein sequence consisting of the amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228 amino acid It may be of any length including a contiguous amino acid sequence of, and may also be one that maintains (retains) at least one desired non-canonical biological activity. More specifically, in the present invention, the peptide is a peptide consisting of a total of 62 amino acids, which are consecutive amino acids from the 119th amino acid to the 180th amino acid in the amino acid sequence of SEQ ID NO: 1, and the SEQ ID NO: 1 consisting of 748 amino acids. It can be understood to include a CRS protein of, and even a peptide comprising an amino acid sequence exhibiting 80% or more homology with them.

Preferably, in the present invention, the peptide is a peptide consisting of consecutive amino acids selected from 180 to 228 amino acids from any one selected from 101 to 119 of the amino acid sequence of SEQ ID NO: 1; Alternatively, as a peptide consisting of an amino acid sequence exhibiting 80% or more homology with the peptide, it may be of any length, and may also maintain (retain) at least one desired irregular biological activity. More specifically, in the present invention, the peptide is from a peptide consisting of a total of 62 amino acids, which are consecutive amino acids from the 119th amino acid to the 180th amino acid in the amino acid sequence of SEQ ID NO: 1, and the longest It can be understood that a peptide consisting of a total of 128 amino acids, which is a continuous amino acid from the 101st amino acid to the 28th amino acid of the amino acid sequence, and even a peptide including an amino acid sequence exhibiting 80% or more homology with them.

Even more preferably, the peptide may be composed of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7 or an amino acid sequence that exhibits 80% or more homology thereto.

Most preferably, the peptide may be composed of SEQ ID NO: 2 or an amino acid sequence showing 80% or more homology thereto.

The peptide of the present invention can be prepared using available techniques known in the art. For example, it can be prepared using any of a variety of proteolytic enzymes. Exemplary proteases (proteinases) include, for example, achromopeptidase, aminopeptidase, ancrod, angiotensin converting enzyme, and bromelain. , Calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase B, carboxypeptidase G G), carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2, caspa Caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9 (caspase 9), caspase 10 (caspase 10), caspase 11 (caspase 11), caspase 12 (caspase 12), caspase 13 (caspase 13), cathepsin B, cathepsin C (cathepsin C), cathepsin D, cathepsin E, cathepsin G, cathepsin H, cathepsin L, chymopapain, key Chymase, chymotrypsin, cross-tripain, collagenase, complement C1r, complement C1s, complement D factor D, complement I person Complement factor I, cucumisin, dipeptidyl peptidase IV (dipeptidyl peptidase IV), leukocyte elastase (leukocyte), pancreatic elastase (pancreatic), endoproteinase Arg-C (endoproteinase Arg-C), endoproteinase Asp-N (endoproteinase Asp-N), endoproteinase Glu-C (endoproteinase Glu-C), endoproteinase Lys-C (endoproteinase) Lys-C), enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIV protease , IGase, kallikrein tissue, general leucine aminopeptidase (general), cell substrate leucine aminopeptidase (cytosol), microsomal leucine aminopeptidase (microsomal) ), matrix metalloprotease, methionine aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, Pronase E, prostate specific antigen, protease alkalophilic from Streptomyces griseus, protease from Aspergillus, protease from Aspergillus saitoi f rom Aspergillus saitoi), Aspergillus sojae-derived protease (protease from Aspergillus sojae), B. licheniformis protease (protease B. licheniformis, alkaline or alcalase), Bacillus polymyxa-derived protease (protease from Bacillus polymyxa), Bacillus sp-derived protease from Bacillus sp), Rhizopus sp.-derived protease (protease from Rhizopus sp.), protease S (protease S), proteasomes, Aspergillus oryzae-derived proteinase (proteinase from Aspergillus oryzae), pro Proteinase 3, proteinase A, proteinase K, protein C, pyroglutamate aminopeptidase, renin ), streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase, and Urokinase and the like. Those skilled in the art can easily determine which proteolytic enzyme is appropriate in consideration of the chemical specificity of the fragment to be produced.

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 production methods, the polypeptides of the present invention can be prepared by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)).

The solid-phase peptide synthesis (SPPS) method can initiate synthesis by attaching functional units called linkers to small porous beads to induce the peptide chain to continue. Unlike the liquid phase method, the peptide covalently binds to the bead and prevents it from being separated by filtration until it is cleaved by a specific reactant such as trifluoroacetic acid (TFA). The protection process, deprotection process, in which the N-terminal amine of the peptide attached to the solid phase and the N-protected amino acid unit are combined, the re-exposed amine group and the new Synthesis is performed by repeating the cycle (deprotection-wash-coupling-wash) of the coupling process in which amino acids are bound. The SPPS method may be performed using a microwave technology together, and the microwave technology may shorten the time required for coupling and deprotection of each cycle by applying heat during the peptide synthesis process. The thermal energy may prevent folding or aggregation of the expanded peptide chain and promote chemical bonding.

In addition, the peptide of the present invention can be prepared by the liquid peptide synthesis method, and the specific method thereof is referred to the following documents: US Patent No. 5,516,891. In addition, the peptide of the present invention can be synthesized by various methods such as a method of mixing the solid-phase synthesis method and the liquid-phase synthesis method, and the preparation method is not limited to the method described herein.

Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished using, for example, Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, the various fragments can be chemically synthesized separately and combined using chemical methods to produce the molecule of interest.

The peptide provided in the present invention has 80 sequence homology with a peptide containing consecutive amino acids selected from 180 to 228 amino acids from any one selected from 101 to 119 of the amino acid sequence of SEQ ID NO: 1 Contains more than% variants. The variant refers to an active variant of the peptide, and such active variant means maintaining at least one or more desired irregular activities (e.g., anticancer activity and immunostimulating activity) from the peptide from which it is derived. As an example of the variant, whether naturally or non-naturally occurring (generated), it may be a splice variant, the splice variant is, for example, at least one non-canonical activity described herein Holds. As another example, the variant, whether naturally or non-naturally occurring (generated), comprises one or more point mutations to the peptide sequence, wherein the variant peptide is, for example, herein It retains at least one non-canonical activity described. That is, in the present invention, the variant (or the term'active variant') includes consecutive amino acids from any one selected from the 101 to 119th amino acid of the amino acid sequence of SEQ ID NO: 1 to any one selected from the 180th to 228th amino acid It is understood as a functional equivalent of a peptide that does.

More specifically, the variant is at least 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% along its length with respect to the aforementioned peptide sequence. More than, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more It is characterized by being a functional equivalent having sequence homology.

The variant is that any change has occurred in the amino acid sequence of the'peptide', and may include one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring, or using any of a number of techniques well known in the art, for example, modifying or modifying one or more of the above peptide sequences of the present invention and It can be produced synthetically by evaluating biological activity.

In one embodiment of the present invention, the variant includes conservative substitutions. 'Conservative substitution' is a substitution in which an amino acid is substituted with another amino acid having similar properties, so that a person skilled in the art can predict that the secondary structure and sensitivity (hydropathic nature, hydrophobicity or hydrophilic nature) of the peptide are substantially unchanged. . In general, the following amino acid groups exhibit conservative changes: (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; And (5) phe, tyr, trp, his.

Modifications may be carried out within the structure of the peptides of the present invention, and functional molecules encoding peptide variants or derivatives having desired (desired) characteristics may be obtained. When it is desired to change the amino acid sequence of the peptide to produce an equivalent or improved variant 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. have.

Certain amino acids can be substituted for other amino acids within the protein or peptide structure, without significant loss of interactive binding capacity, which has the same structure as, for example, the receptor, antigen-binding site of an antibody, or binding site on a substrate molecule. Because this is due to the interactive ability and nature of the protein as a generally defined biological and functional activity of the protein, specific amino acid sequence substitutions can be made in or in the protein or peptide sequence, and of course, in the underlying DNA coding sequence. And, nevertheless, it is possible to obtain proteins having the same or similar properties.

Thus, it is contemplated that various changes are made in the disclosed peptide sequence or the DNA sequence encoding the peptide, without significant loss of desired utility or activity. In this modification, the hydropathic (hydrophobic or hydrophilic nature) index of the amino acid may also be considered. The importance of the hydropathic amino acid index conferring an interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). For example, the relative hydropathic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn regulates the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It became known. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). These 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); And arginine (-4.5).

Certain amino acids can be substituted by other amino acids with similar susceptibility indexes or scores, and make it possible to obtain proteins with similar biological activity (i.e. still obtain a biologically functionally equivalent protein). Is well known in the art. In such a change, amino acids having a sensitivity index within ±2 are preferable for substitution, an amino acid substitution with a sensitivity index within ±1 is particularly preferable, and a substitution of an amino acid having a sensitivity index within ±0.5 is more particularly preferable.

It is also understood in the art that substitutions of the same amino acids can be effectively carried out based on hydrophilicity. As described in US Pat. 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, and that biologically equivalent proteins can be obtained. In such a change, substitution of amino acids whose hydrophilicity value is within ±2 is preferable, substitution of amino acids whose hydrophilicity value is within ±1 is particularly preferable, and substitution of amino acids whose hydrophilicity value is within ±0.5 is more It is particularly preferred.

As outlined above, amino acid substitutions can be based on the relative similarity of amino acid side-chain substituents, e.g. based on the hydrophobicity, hydrophilicity, charge, size, etc. of these (relative similarity of side-chain substituents). can do. Exemplary substitutions taking into account the various features of the foregoing are well known to those of skill in the art, and include: arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and asparagine; Valine, leucine and isoleucine.

Amino acid substitutions may also be those performed based on the similarity of 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; And as amino acids having a non-charged polar head group having a similar hydrophilicity value, leucine, isoleucine and valine; Glycine and alanine; Asparagine and glutamine; Serine, threonine, phenylalanine, and tyrosine.

In addition, the variant may contain non-conservative modifications. In a preferred embodiment, the variant peptide may differ from its native sequence by substitution, deletion or addition of 5 amino acids or fewer amino acids. Variants can also be altered, for example, by deletion or addition of amino acids with minimal effect on the secondary structure and hydropathic properties of the peptide.

The peptide may contain a signal (or leader) sequence at the N-terminus of the protein, which sequence directs the transfer of the protein either simultaneously with or after translation. The peptide may also be conjugated (conjugated) with a linker sequence or other sequence to facilitate synthesis, purification or identification of the peptide (e.g. polyHis), or to enhance the binding of the peptide to a solid support. I can. For example, the peptide may be bound (conjugated) to an immunoglobulin Fc region.

When the peptide sequences are compared, when the two sequences are aligned with maximum correspondence, as described below, if the sequence of amino acids in the two sequences is the same, the two sequences are said to be "identical". The comparison between the two sequences is typically performed by comparing the arrangement on a comparison window to identify and compare local regions of sequence similarity. As used herein, "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to 75, 40 to 50 consecutive positions, wherein the sequence is two sequences After this optimal alignment, it can be compared to a reference sequence of the same number of consecutive positions.

Optimal alignment of sequences for comparison may be performed using, for example, a Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. The program includes several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. 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, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'l Acad., Sci. USA 80:726-730.

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

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

The elongation of word hits in each direction stops in the following cases: if the cumulative alignment score is reduced by quantity X from its maximum reached, the cumulative score is at least one negative scoring. ) When it becomes 0 (zero) or less due to the accumulation of alignment of the residues; Or when it reaches the end of any sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment.

In one exemplary approach, the'percentage of sequence homology' is determined by comparing two optimal alignment sequences on a comparison window of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window is the reference sequence (this Additions or deletions (i.e., gaps) of 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent, compared to (not including additions or deletions) may be included. The percentage is determined by determining the number of positions in which the same amino acid residue exists in both sequences to obtain the number of matching positions, and the number of matching positions is divided by the total number of positions in the reference sequence (i.e., window size). , And this result is multiplied by 100 to obtain the percent sequence homology.

The peptides provided by the present invention may be in a linear form or a cyclic form, which can be understood with reference to examples of the present invention.

In the present invention, the production of the cyclic peptide as the variant is not particularly limited, as long as it is by a known peptide cyclization method known in the art, a specific cyclization method and a cyclization form accordingly. Preferably, the preparation of the cyclic peptide of the present invention is prepared by cutting or replacing a linear peptide such that cysteine is located at both ends (N-terminal and C-terminal), and both ends It may be performed by allowing a monosulfide bond to occur between cysteine residues present in the.

The peptides provided herein contemplate the use of a modified polypeptide as an aspect, and 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, but are not limited thereto, may include chemical derivatization and/or enzymatic derivatization at one or more constituent amino acids, and the derivatization is acetylation, hydroxylation, methylation. , Amidation, and side chain alteration, backbone alteration, and alteration of the N-terminus and C-terminus, including the addition of carbohydrate or lipid components, cofactors, and the like. An exemplary modification includes pegylation of the polypeptide (see, eg, Veronese and Harris, Advanced Drug Delivery Reviews 54:453-456, 2002).

In certain embodiments, chemoselective ligation techniques may be used to modify the peptides of the present invention, for example, by attaching a polymer in a site-specific and controlled manner. . These techniques rely on the binding of chemoselective anchors into the protein backbone, typically by either chemical or recombinant means, and the subsequent modification to a polymer carrying a complementary linker. As a result, the assembly process and the covalent bond structure of the resulting protein-polymer conjugate are controlled, thereby enabling rational optimization of drug properties such as efficacy and pharmacokinetic properties (e.g., Kochendoerfer , Current Opinion in Chemical Biology 9:555-560, 2005). They improve their pharmacokinetic properties, such as by allowing the selective attachment of PEGs.

The peptide of the present invention may include a pharmaceutically acceptable salt form. The pharmaceutically acceptable salts include, for example, hydrochloride, sulfate, phosphate, acetate, citrate, stannate, succinate, lactate, maleate, fumarate, oxalate, methanesulfonate, or paratoluenesulfonate. However, it is not limited thereto.

The present invention also provides a polynucleotide encoding the peptide.

As used herein, the terms “DNA”, “polynucleotide” and “nucleic acid” refer to a DNA molecule isolated from the total genomic DNA of a specific species. Therefore, a DNA fragment (segment) encoding a polypeptide is substantially isolated or purified from the total genomic DNA of the species from which the DNA fragment can be obtained, as one or more coding sequences. It refers to a piece of DNA that is formed. The terms'DNA fragment' and'polynucleotide' include DNA fragments and smaller fragments of the fragment, and also include recombinant vectors (e.g., plasmids, cosmids, phagemids, sterile viruses, viruses, etc.). Include.

As understood by those skilled in the art, the polynucleotide sequence of the present invention expresses or is modified to express a protein, a peptide, etc., a genomic sequence, an extra-genomic sequence, a sequence encoded in a plasmid, and And smaller engineered gene fragments. These pieces can be isolated naturally, or they can be transformed synthetically by human hands.

As recognized by one of skill in the art, polynucleotides can be single-stranded (code sequence or antisense sequence), or can be double-stranded, and can be DNA molecules (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention. In addition, polynucleotides can be linked to other molecules and/or support materials.

Polynucleotides may include native sequences, or may include variants, or biologically functional equivalents of such sequences. Polypeptide variants may comprise one or more substitutions, additions, deletions, and/or insertions, such as those described further below, preferably such modifications have substantially the desired activity of the encoded polypeptide compared to the unmodified polypeptide. It is performed in a way that is not reduced to The effect on the activity of the encoded polypeptide can generally be assessed as described herein.

The polynucleotide provided in the present invention is not particularly limited in its specific sequence as long as it encodes the peptide of the present invention or a variant peptide thereof, and any combination of base sequence (nucleic acid sequence) construction is allowed. For example, a peptide consisting of the amino acid sequence of SEQ ID NO: 2 is a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 8, and a peptide consisting of the amino acid sequence of SEQ ID NO: 6 includes a nucleotide sequence represented by SEQ ID NO: 9 The polynucleotide and the peptide consisting of the amino acid sequence of SEQ ID NO: 7 may be expressed by a polynucleotide including the nucleotide sequence of SEQ ID NO: 10, but are not limited thereto.

The polynucleotide of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences such as promoters, polyadenylation signals, additional restriction sites, multiple cloning sites, and other coding fragments (parts). You can, and as a result your overall length can vary considerably. Therefore, it is considered that polynucleotide fragments of almost any length can be applied, and the total length may be limited by ease of manufacture and use, preferably in the intended recombinant DNA protocol.

Moreover, it will be apparent to those skilled in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the peptides described herein. Some of these polynucleotides retain minimal homology to the nucleotide sequence of any native gene. Nevertheless, other polynucleotides (eg polynucleotides optimized for codon selection in humans and/or primates) due to differences in codon usage are specifically contemplated by the present invention.

In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are modified as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may have an altered structure or function (although not necessarily required). Alleles can be identified using standard techniques (eg, hybridization, amplification and/or database sequence comparison).

Polynucleotides and fusions thereof can be prepared, manipulated and/or expressed using any of the well-established techniques known and available in the art. For example, a polynucleotide sequence encoding a peptide of the present invention, or a functional equivalent thereof, can be used in a recombinant DNA molecule directed to expression of the polypeptide in an appropriate host cell. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially identical or functionally equivalent amino acid sequences can be generated, which sequences can be cloned and selected for a given polypeptide. It can be used to express.

As will be appreciated by those skilled in the art, in some cases it may be advantageous to produce a nucleotide sequence (a nucleotide sequence encoding a polypeptide) with a codon that does not exist in nature. For example, in a particular prokaryotic host or eukaryotic host, preferred codons increase the rate of protein expression or have the desired properties (e.g., a half-life longer than the half-life of a transcript produced from a naturally occurring sequence). Can be chosen to produce

In addition, the polynucleotide sequence of the present invention is a method generally known in the art to alter the peptide coding sequence for various reasons (including, but not limited to, alterations that alter the cloning, processing, expression and/or activity of the gene product). Can be manipulated using

In addition, the present invention provides a vector comprising the polynucleotide and a host cell transformed with the vector.

In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide or functional equivalent can be inserted into an appropriate expression vector (i.e., a vector including elements (elements) necessary for transcription and translation of the inserted coding sequence). An expression vector including a sequence encoding a polypeptide of interest and an appropriate transcription control element (element) and a translation control element (element) can be constructed by a method well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene 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).

A variety of expression vector/host systems are known and can be used to contain and express polynucleotide sequences. The expression vector/host system may include, but is not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vector; Yeast transformed with a yeast expression vector; Insect cell lines infected with a viral expression vector (eg baculovirus); Plant cell lines transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmid); Or microorganisms such as animal cell systems.

“Control elements (elements)” or “regulatory sequences” present in expression vectors are untranslated regions (enhancers, promoters, 5'and 3'untranslated regions) that interact with host cell proteins to effect transcription and translation. . These elements (elements) may have different strength and specificity. Depending on the vector system and host used, any number of suitable transcription elements and translation elements (including constitutive promoters and inducible promoters) can be used.

For example, when cloning into a bacterial system, an inducible promoter such as a hybrid lacZ promoter of PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) may be used. For mammalian cell lines, promoters derived from mammalian genes or mammalian viruses are generally preferred. When it is necessary to generate a cell line containing multiple copies of a sequence encoding a polypeptide, SV40 or EBV-based vectors can be usefully used with appropriate selection markers.

In the bacterial system, a number of expression vectors can be selected according to the intended use for peptide expression. For example, if necessary in large quantities, vectors directed to high level expression of easily purified fusion proteins can be used. Such vectors include, but are not limited to, the following: multifunctional E.coli cloning vectors and expression vectors, such as BLUESCRIPT ((Stratagene), the sequence encoding the polypeptide of interest is β-galactosidase. The sequence for the first amino-terminal Met and the 7 residues following it is ligated into the vector in frame, resulting in a hybrid protein); pIN vector (Van Heeke and Schuster, J. Biol. Chem. 264:5503 5509(1989)); etc. The pGEX vector (Promega, Madison, Wis.) can also be used to express foreign polypeptides as a fusion protein with glutathione S transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorbing to glutathione-agarose beads and then eluting in the presence of free glutathione. Proteins made in this system can be designed to contain heparin, thrombin, or Factor Xa protease cleavage sites so that the cloned polypeptide can be released from the GST moiety.

For yeast (Saccharomyces cerevisiae), a number of vectors can be used, including constitutive or inducible promoters (eg alpha factor, alcohol oxidase and PGH). This is Ausubel et al (supra). And Grant et al., Methods Enzymol. 153:516-544 (1987).

When using a plant expression vector, the expression of the sequence encoding the polypeptide can be driven by any number of promoters. For example, viral promoters (e.g., the 35S promoter and 19S promoter of CaMV) may be used alone, or may be used in combination with an ω (omega) leader sequence derived from TMV (Takamatsu, EMBO J .6:307-311 (1987)). Alternatively, a plant promoter (eg, a small subunit of RUBISCO or a heat shock promoter) may be used (Coruzzi, G. et al., EMBO J. 3:1671-1680 (1984); Broglie et al. al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)).These constructs are either direct DNA transformation or pathogen-mediated transfection. Such technology is known in the art, and the following documents may be referred to: Hobbs in McGraw Hill, Yearbook of Science and Technology, pp.191-196 (1992), etc.

The insect family can also be used to express the desired polypeptide. For example, in one system, AcNPV (Autographa californica nuclear polyhedrosis virus) is used as a vector for expressing foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The sequence encoding the polypeptide may be cloned into a non-essential region of the virus, and may be located under the control of a polyhedrin promoter, such as a polyhedrin gene. Successful insertion of the sequence encoding the polypeptide results in the inactivation of the polyhedrin gene and produces a recombinant virus that lacks the coat protein. The recombinant virus can then be used, for example, to infect S.frugiperda cells or Trichoplusia larvae, etc., where the desired polypeptide can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)).

For mammalian host cells, a number of virus-based expression systems are generally available. For example, when adenovirus is used as an expression vector, a sequence encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of a late promoter and a tripartite leader. have. Insertion of the viral genome into the non-essential E1 or E3 regions can be used to obtain a viable virus capable of expressing the polypeptide in an infected host cell. (Logan and Shenk, Proc. Natl. Acad. Sci. U.S.A. 81: 3655-3659 (1984)). In addition, a transcription enhancer (for example, Rous Sarcoma Virus (RSV) enhancer) may be used to increase expression of mammalian host cells.

In addition, for more efficient translation of the sequence encoding the desired polypeptide, a specific initiation signal may be used. Such signals include the ATG start codon and adjacent sequences. If the sequence encoding the polypeptide, its start codon, and its upstream sequence are inserted into an appropriate expression vector, no additional transcriptional control signals or translation control signals may be required. However, when only the coding sequence, or only a part thereof, is inserted, an exogenous translational control signal including the ATG start codon should be provided. Also, the start codon must be within the correct reading frame, to ensure translation of the entire insert. Exogenous translation elements (elements) and start codons can come from a variety of origins (both natural and synthetic). The efficiency of expression can be enhanced by adding appropriate enhancers (e.g., enhancers described in Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)) to the specific cell line used. .

In addition, the host cell line may be selected according to its ability to regulate the expression of the inserted sequence or to process (process) the expressed protein in a desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves the'prepro' form of the protein, can be used to facilitate correct insertion, folding and/or function. Other host cells (e.g. CHO, HeLa, MDCK, HEK293 and W138, which have specific cellular machinery and characteristic mechanisms for such post-translational activity) can be used for precise modification of foreign proteins and It can be chosen to ensure processing.

In the long term, for high yield of recombinant protein production, stable expression is generally preferred. For example, a cell line stably expressing a desired polynucleotide may be transformed using an expression vector, and the expression vector is an origin of viral replication and/or an endogenous expression element (element), and the same vector or a separate vector It may contain a selection marker gene on the phase.

After the introduction of the vector, cells can be proliferated for 1 to 2 days in enriched media, and then converted into selective media. The purpose of the selection marker is to confer resistance to selection, and its presence allows the growth and recovery of cells in which the introduced sequence is being successfully expressed. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

Transformed cell lines can be recovered using multiple selection systems. As such a selection system, but not limited thereto, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy), which can be used for tk-cells or aprt-cells, respectively. et al., Cell 22:817~823 (1990)).

Also, antimetabolite resistance, antibiotic resistance, or herbicide resistance can be used as a basis for selection. For example, dhfr conferring resistance to methotrexate (Wigler et al., Proc.Natl.Acad.Sci.U.S.A.77:3567-70 (1980)); Npt conferring resistance to aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150: 1-14 (1981)); And als or pat conferring resistance to chlorsulfone and phosphinotricin acetyltransferase, respectively (see Murry, above). Additional selection genes are known, such as trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histidine instead of histidine (Hartman and Mulligan ,Proc.Natl.Acad.Sci.USA 85:8047-51 (1988)). Visible markers such as anthocyanin, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin, are gaining popularity, and not only to identify transformants, but also to specific vectors. It is also widely used to quantify the amount of transient protein expression or stable protein expression due to the system (Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

Various protocols are known in the art for detecting and measuring the expression of a product encoded by the polynucleotide using either a polyclonal antibody or a monoclonal antibody specific for the product encoded by the polynucleotide. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), and the like. This assay method and further other assay methods may be referred to in the following literature: Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158: 1211-1216 (1983). A variety of labeling and conjugation techniques are known to those of skill in the art, and can be used for a variety of nucleic acid and amino acid assays. As a means for constructing a labeled hybridization probe or a labeled PCR probe to detect a sequence related to a polynucleotide, oligolabeling, nick translation, end-labeled or labeled PCR amplification using nucleotides is mentioned. Alternatively, for production of an mRNA probe, the sequence or any portion thereof can be cloned into a vector. Such vectors are known and commercially available in the art, and can be used to synthesize RNA probes in vito by adding appropriate RNA polymerases (eg T7, T3, or SP6) and labeled nucleotides. These procedures can be performed using a variety of commercially available kits. Examples of suitable reporter molecules or labels include radionuclides, enzymes, fluorescent agents, chemiluminescent or chromogenic agents, substrates, carriers, inhibitors, magnetic particles, and the like.

Host cells transformed with the desired polynucleotide sequence can be cultured under conditions suitable for expression of the protein and recovery from cell culture. The protein produced by the recombinant cell may be secreted or contained within the cell depending on its sequence and/or vector.

As will be appreciated by those skilled in the art, the expression vector comprising the polynucleotide of the present invention can be designed to contain a signal sequence directed to secretion of the polypeptide through a prokaryotic membrane or a eukaryotic membrane. Other recombinant constructs can be used to link the sequence encoding the polypeptide of interest with the sequence encoding the polypeptide domain that facilitates purification of the water-soluble protein.

In addition to the recombinant production method, the polypeptides and fragments thereof of the present invention can be produced by direct peptide synthesis using a solid phase technique (Merrifield, J.Am.Chem.Soc.85:2149-2154 ( 1963)). Protein synthesis can be performed using a manual technique or can be performed by automation. Automated synthesis can be achieved using, for example, Applied Biosystems 431 A Peptide Syntthesizer (Perkin Elmer). Alternatively, various fragments can be individually chemically synthesized and then combined using chemical methods to produce full length molecules.

According to another aspect of the present invention, the polynucleotide encoding the polypeptide of the present invention can be delivered to a subject in vivo using, for example, gene therapy technology. Gene therapy generally refers to heterologous nucleic acid transduction into specific cells, target cells of mammals, especially humans, having a disorder or condition for which such treatment is required. The nucleic acid is introduced into the selected target cell, and the heterologous DNA is expressed, thereby producing the encoded therapeutic product.

As various viral vectors usable for gene therapy as disclosed herein, there may be mentioned adenovirus, herpes virus, vaccinia, adeno-associated virus (AAV), or preferably an RNA virus such as a retrovirus. Preferably, the retroviral vector may be a retroviral derivative of a rodent or avian, 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). A number of additional retroviral vectors may contain multiple genes. All of these vectors may contain selectable marker genes so that the transduced cells can be identified and produced. For example, by inserting a desired zinc finger-derived DNA binding polypeptide sequence into a viral vector along with another gene encoding a ligand for a receptor on a specific target cell, the vector will become target specific. I can.

Retroviral vectors can be targeted specific, for example, by inserting a polynucleotide encoding a polypeptide. Illustratively, targeting can be achieved by using antibodies that target retroviral vectors. Those skilled in the art are familiar with specific polynucleotide sequences that can be inserted into the retroviral genome to enable target-specific delivery of retroviral vectors containing zinc finger-nucleotide binding protein polynucleotides. It can be easily checked without undue experimentation.

Because recombinant retroviruses are defective, assistance is needed to produce infectious vector particles. Such assistance can be provided, for example, by using a helper cell line comprising a plasmid encoding all structural genes of a retrovirus under the control of regulatory sequences in the LTR. These plasmids are missing a nucleotide sequence that enables a packaging mechanism to recognize RNA transcripts for encapsulation. The helper cell line in which the packaging signal is deficient is not limited thereto, but includes, for example, PSI.2, PA317, PA12, and the like. These cell lines produce empty virions because the genome is not packaged. If the packaging signal is intact and the retroviral vector is introduced into a cell in which the structural gene has been replaced with another gene of interest, the vector can be packaged and produced vector virion. The vector virions produced by the method can then be used to infect tissue cell lines (e.g., NIH3T3 cells), thereby producing large amounts of chimeric retroviral virions.

In addition,'non viral' delivery technology for gene therapy, such as DNA-ligand complex, adenovirus-ligand-DNA complex, direct injection of DNA, CaPO4 precipitation, gene gun technology, electroporation, liposome Methods, lipofection, and the like can be used. Any of these methods are widely available to those skilled in the art and are suitable for use in the present invention. Other suitable methods are also available to those of skill in the art, and it is clearly understood that the present invention may be achieved using any available transfection method. Lipofection can be achieved by encapsulating the isolated DNA molecule within the liposome particle and by contacting the liposome particle with the cell membrane of the target cell. Liposomes are self-assembled colloidal molecules, and a lipid bilayer composed of amphiphilic molecules such as phosphatidylserine or phosphatidylcholine encapsulates part of the surrounding media, resulting in a lipid bilayer surrounding the hydrophilic interior. . Unilamellar or multilamellar liposomes can be constructed, and as a result, desired chemicals, drugs, or DNA molecules isolated as in the present invention are contained therein.

The present invention also provides a vaccine adjuvant comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

In the present invention, the vaccine adjuvant may be defined as a substance capable of presenting an antigen to the immune system in a manner in which 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. . That is, the vaccine adjuvant is an immune enhancing agent, which is a substance that promotes an immune response to an antigen, and is not an immunogen to a host, but refers to a substance that enhances immunity by increasing the activity of cells of the immune system.

To analyze the antigen-specific induced immune response by the vaccine adjuvant, the immune response can be compared with the immune response induced in the presence of the antigen without the vaccine adjuvant. The induction can be assessed in the subject or in the subject's cells.

In the present invention, the immune response may be one or more selected from the group consisting of macrophages, dendritic cells, monocytes, B cells, and T cell responses. In particular, the immune response may be a B cell response, that is, it means that an antibody can be produced specifically for the antigen. The antibody is preferably an IgG antibody, more preferably an IgG2a and/or IgG1 antibody. The immune response may be a T cell response, preferably a Th1 response, a Th2 response or a balanced Th2/Th1 response. The skilled artisan recognizes that disease-dependent B and/or T cell responses may need to be induced to regulate them. Alternatively, the immune response can be detected, for example, by measuring the production of cytokines such as IFNgamma, IL-6, TNFalpha or IL-10. The production of these cytokines can be evaluated by ELISA, preferably as performed in the Examples.

According to an embodiment of the present invention, it was confirmed that the CRS protein or a fragment peptide thereof has a very excellent effect of activating innate immunity and acquired immunity, so that when treated with an antigen, an increase in the subject's immune response can be remarkably improved. Has been done.

Accordingly, a peptide comprising consecutive amino acids from any one selected from the 101 to 119th amino acid to any one selected from the 180 to 228th amino acid in the amino acid sequence of SEQ ID NO: 1; Or a peptide containing an amino acid sequence that exhibits 80% or more homology with the peptide, a polynucleotide encoding the same, a vector containing the polynucleotide, and a host cell transformed with the vector will also exhibit the same physiological activity. Will be apparent to those skilled in the art.

In one embodiment of the present invention, when the vaccine adjuvant is administered with the antigen and the vaccine adjuvant compared to that obtained by the antigen itself, an improved innate immune response and activation of the acquired immune response may be induced, and more specifically An improved cellular immune response and activation of a humoral immune response can be induced.

As an embodiment of the present invention, the detection of the antigen-specific induced immune response, wherein the detection is at least administered with the vaccine adjuvant of the present invention, at least 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12 hours or more, or after at least 1 day, or at least 2 days or at least 3 days or at least 4 days, or more time. The detection can be evaluated in a subject or in a cell of a subject, preferably as performed in the examples.

As an embodiment of the present invention, the antigen-specific induced immune response preferably refers to a detectable immune response against the antigen. A detectable increase is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more hours after administration of the vaccine adjuvant and antigen to a subject, or at least 1 At least 5%, or 10%, 15%, 20% of the amount of immune cells, antibodies and/or cytokines after days, or after at least 2 days or at least 3 days or after at least 4 days, or more time , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150 It may mean an increase of %, 200% or more. The detection can be evaluated in a subject or in a cell of a subject, preferably as performed in the examples.

In the present invention, the cysteinyl-TARNA synthase (CRS) refers to a full-length protein of CRS, and the specific amino acid sequence constituting it can be easily identified through a known sequence search. In the present invention, the CRS is not specifically limited in its amino acid sequence, but preferably may be a human CRS isoform b, and more preferably, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence showing 80% or more homology thereto. It may have been done.

The present invention also provides a vaccine composition comprising the vaccine adjuvant and antigen.

In the present invention, the vaccine contains antigens composed of whole disease-causing organisms (hereinafter, structured or weakened) or components of these organisms, such as proteins, peptides, or polysaccharides, and to impart immunity to diseases caused by the organism. It refers to the formulation used. As described above, a peptide comprising consecutive amino acids up to any one selected from 180 to 228 amino acids from any one selected from 101 to 119 amino acids in the amino acid sequence of SEQ ID NO: 1; Alternatively, the peptide and CRS full-length protein comprising an amino acid sequence that exhibits 80% or more homology with the peptide exerts the effect of remarkably enhancing the immune response of the subject when administered with an antigen by activating the innate and acquired immune systems. Can be used as

Antigens included in the vaccine composition in the present invention are not particularly limited in type, and peptides, proteins, glycoproteins, glycolipids, lipids, carbohydrates, nucleic acids, polysaccharides, and viruses, cells, allergens, tissues, cells containing them Etc. As a non-limiting example, pollen-derived antigen, hepatitis A virus-derived antigen, hepatitis B virus-derived antigen, hepatitis C virus-derived antigen, hepatitis D virus-derived antigen, hepatitis E virus-derived antigen, hepatitis F virus Derived antigen, HIV virus-derived antigen, influenza virus-derived antigen, herpes virus (HSV-1, HSV-2)-derived antigen, anthrax-derived antigen, Chrymia-derived antigen, pneumococcal-derived antigen, Japanese encephalitis virus-derived antigen, measles virus Derived antigen, rubella virus-derived antigen, tetanus-derived antigen, chickenpox virus-derived antigen, SARS virus-derived antigen, EB virus-derived antigen, papyroma virus-derived antigen, Helicobacter pylori-derived antigen, rabies virus-derived antigen, West Nile virus-derived antigen, Hanta virus-derived antigen, streptococcus-derived antigen, staphylococcus-derived antigen, pertussis (Bordetellapertussis)-derived antigen, Mycobacterium tuberculosis-derived antigen, malaria protozoa (Plasmodium)-derived antigen, poliovirus-derived antigen, various human animals And antigens derived from common infectious diseases, cancer antigens, and antigens derived from various food allergies.

The antigen contained in the vaccine composition of the present invention need not be single. When considering the application of the present invention, there may be cases in which an immune response is caused for cancer cells, bacteria, viruses, etc. that are not a single protein or peptide, and in this case, multiple types that can cause an immune response It may be a mixture of proteins or the like of which the type cannot be specified. In addition, the purpose of actively generating an immune response to a plurality of antigens, and containing a plurality of types of antigens may be one of the uses of the vaccine composition of the present invention.

The antigen included in the vaccine composition of the present invention may preferably be a tumor antigen. The tumor antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA). Several tumor antigens and their expression patterns are known in the art and can be selected depending on the type of tumor being treated. Non-limiting examples of tumor antigens include alpha fetal protein, oncogenic embryonic antigen, cdk4, beta-catenin, CA125, caspase-8, epithelial tumor antigen, HPV antigen, HPV16 antigen, CTL epitope derived from HPV16 E7 antigen, melanoma. 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 molecule GM2, GD2, GD3, carcinoembryonic antigen (CEA), 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 Enemy 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-related antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, cancer embryonic antigen (CEA), melanoma-related antigen E (MAGE-A, MAGE1, MAGE2, MAGE3, MAGE4), interleukin-13 receptor alpha (IL13-R alpha), carbonic anhydrase IX (CAIX) ), survivin, GP100, angiogenic antigen, ras protein, p53 protein, p97 melanoma antigen, KLH antigen, cancer embryonic antigen (CEA), gp100, MART1 antigen, TRP-2, HSP-70, beta-HCG, Testicin, 1A01 -HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4; Alpha-actinine-_4/m; Alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl; Beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; Calreticulin; CAMEL; CASPA; Caspase_8; Cathepsin_B; Cadepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_Isomorph_1; CD44_Isomorph_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30; 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_Isomorph_LAGE-1A; CTAG2_Isomorph_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; Fibronectin; FOS; 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; IL2-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-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16; MAGE-B17; MAGE-B2; MAGE-B3; MAGE-B4; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; MAGE-C3; MAGE-D1; MAGE-D2; MAGE-D4; MAGE-E1_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H1; MAGEL2; Mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF; Mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; N-myc; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9; Osteocalcin; Osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3; PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-kinase; Pin-1; PLAC1; PMEL; PML; POTE; POTEF; PRAME; PRDX5/m; PRM2; Prostein; Proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA; RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; 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; STEAP-1; Survivin; Survivin-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, alpha-actinin-4; ARTC1; BCR-ABL fusion protein (b3a2); B-RAF; CASP-5; CASP-8; Beta-catenin; Cdc27; CDK4; CDKN2A; COA-1; dek-can fusion protein; EFTUD2; Elongation factor 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-RARalpha fusion protein; PRDX5; PTPRK; K-ras; N-ras; RBAF600; SIRT2; SNRPD1; SYT-SSX1 or SSX2 fusion protein; Triosephosphate 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; 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; CyClean D1; Ep-CAM; EphA3; FGF5; G250 / MN / CAIX; HER-2/neu; IL13Ralpha2; Intestinal carboxyl esterase; Alpha-foetoprotein; 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, PAP. OVA (ovalbumin) and MART-1.

The vaccine composition of the present invention may preferably be an anticancer vaccine. In addition, the anticancer vaccine may be a vaccine for preventing cancer or a vaccine for treating cancer.

According to an embodiment of the present invention, the vaccine composition containing the peptide or CRS full-length protein and a specific tumor antigen of the present invention is pre-administered prior to cancer formation to activate the subject's immune response, thereby inhibiting the growth of cancer. It was confirmed to represent. In addition, the vaccine composition of the present invention is administered to a subject after formation of cancer, thereby inhibiting the growth of cancer or killing cancer, and thus the efficacy as a therapeutic vaccine has been confirmed.

In the present invention, the type of cancer is not particularly limited, and preferably breast cancer, colon cancer, prostate cancer, cervical cancer, gastric cancer, skin cancer, mouth cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, or 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 cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, It may be selected from the group consisting of leukemia, childhood cancer, bronchial cancer, colon cancer and ovarian cancer.

Any vaccine adjuvant of the vaccine composition of the present invention and at least one selected from the group consisting of an immune checkpoint inhibitor may be further included.

According to an embodiment of the present invention, it was confirmed that the effect of activating the immune function of the subject is more remarkable when the peptide of the present invention and the CRS full-length protein are treated in combination with any additional vaccine adjuvant and/or immune checkpoint inhibitor. have. In particular, such an effect may be particularly desirable in that side effects and the like that may occur by administering a high-dose substance can be minimized and maximum effects can be achieved with minimal administration.

The type of the vaccine adjuvant which may be further included in the vaccine composition of the present invention is not particularly limited, and it should be understood that vaccine adjuvants currently widely used in the art or vaccine adjuvants to be newly developed in the future are included therein. As a non-limiting example of any of the above vaccine adjuvants, 1018 ISS, aluminum salt, amplivax, AS15, BCG, CP-870,893, CpG ODN, CpG7909, CIAA, dSLIM, GM-CSF, IC30, IC31, imiqui Mod, Imupact IMP321, IS Patch, Iskomatrix, Juv Immun, Lipoback, MF59, Monophosphoryl Lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK -432, OM-174, OM-197-MP-EC, Ontag, Peptel vector system, PLG microparticles, reciquimod, SRL172, virosomes and other virus-like particles, YF-17DBCG, Aquilas QS21 stymulon , Libis detox quill, Superforce, Freunds, GM-CSF, cholera toxin, immunological adjuvant, MF59 and cytokines may be included, and most preferably CpG ODN.

In the present invention, the immune checkpoint inhibitor is a substance that blocks the immune system inhibitor checkpoint. Immune checkpoints can be stimulating or inhibitory. Blocking of inhibitory immune checkpoints activates immune system function and can be used in cancer immunotherapy [Pardoll, Nature Reviews. Cancer 12:252-64 (2012)]. Tumor cells lose activated T cells when they attach to specific T-cell receptors. Immune checkpoint inhibitors prevent tumor cells from attaching to T cells, which keeps the T cells active. In fact, the cooperative action by cells and soluble components combats damage caused by pathogens and cancer. Modulation of the immune system pathway involves modulating the response by the immune system by altering the expression or functional activity of at least one component of the pathway. The immune checkpoint inhibitor is a PD-1 (programmed cell death-1) antagonist, a PD-L1 (programmed cell death-ligand 1) antagonist, a PD-L2 (programmed cell death-ligand 2) antagonist, a CD27 (cluster of differentiation 27) Antagonist, CD28 (cluster of differentiation 28) antagonist, CD70 (cluster of differentiation 70) antagonist, CD80 (cluster of differentiation 80, also known as B7-1) antagonist, CD86 (cluster of differentiation 86, also known as B7-2) Antagonist, CD137 (cluster of differentiation 137) antagonist, CD276 (cluster of differentiation 276) antagonist, killer-cell immunoglobulin-like receptors (KIRs) antagonist, lymphocyte-activation gene 3 (LAG3) 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 antign-4) antagonist, A2AR (Adenosine A2A receptor) antagonist, VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, BTLA (B- and T-lymphocyte attenuato) r) Antagonist, IDO (Indoleamine 2,3-dioxygenase) antagonist, TIM-3 (T-cell Immunoglobulin domain and Mucindomain 3) antagonist, VISTA (V-domain Ig suppressorof T cell activation) antagonist, and killer cell lectin-like (KLRA) receptor subfamilyA) antagonists, preferably a PD-1 antagonist, a PD-L1 antagonist, or a LAG3 antagonist, and most preferably a PD-L1 antagonist.

In one embodiment of the present invention, the immune checkpoint inhibitor may be a PD-1 antagonist. PD-1 is a T-cell co-inhibitory receptor that plays a central role in the ability of tumor cells to evade the host's immune system. Blocking of the interaction between PD-1 and PD-L1, a ligand of the PD-1 antagonist enhances immune function and mediates anti-tumor activity. Examples of the PD-1 antagonist include antibodies that specifically bind to PD-1. Certain anti-PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, STI-1014 and pidilzumab.

In another embodiment of the present invention, the immune checkpoint inhibitor may be a PD-L1 antagonist. Examples of the PD-L1 antagonist include an antibody that specifically binds to PD-L1. Certain anti-PD-L1 antibodies include, but are not limited to, abelumab, atezolizumab, durvalumab and BMS-936559.

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

In the present invention, the antibodies are meant to include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments formed from at least two intact antibodies as long as they exhibit the desired biological activity. In another embodiment, an antibody is meant to comprise a soluble receptor that does not retain the Fc portion of the antibody. In one embodiment, the antibody may be a humanized monoclonal antibody and fragments thereof produced by recombinant genetic engineering.

Another class of immune checkpoint inhibitors includes polypeptides that bind to and block the PD-1 receptor on T-cells without causing inhibitor signal transduction. Such peptides include B7-DC polypeptides, B7-H1 polypeptides, B7-1 polypeptides and B7-2 polypeptides and soluble fragments thereof, as disclosed in US Pat. No. 8,114,845.

Another class of immune checkpoint inhibitors includes compounds with peptide moieties that inhibit PD-1 signaling. Examples of such compounds are disclosed in US Pat. No. 8,907,053.

Another class of immune checkpoint inhibitors includes inhibitors of certain metabolic enzymes, such as indoleamine 2,3-dioxygenase (IDO), which are expressed by infiltrating bone marrow cells and tumor cells. IDO enzymes suppress immune responses by depleting amino acids required for anabolic action in T cells, or through the synthesis of specific natural ligands for cytoplasmic receptors that can alter lymphocyte function.

The compositions of the present invention (e.g., polypeptides, polynucleotides, etc.), alone or in combination with one or more other therapeutic modalities, are pharmaceutically-acceptable or physiologically- for administration to cells, tissues or animals. It can be formulated (formulated) generally in an acceptable solution. If desired (if necessary), the composition of the present invention can be administered in combination with other agents (eg, other proteins, polypeptides, or various pharmaceutically active agents). In fact, there are no restrictions on other ingredients that may be included in the composition of the present invention as long as the additional agent does not negatively affect the properties of the peptide or the like of the present invention.

In the composition of the present invention, the combination of a pharmaceutically-acceptable excipient and a carrier is well known to those skilled in the art. In addition, in the use of the specific composition described herein, the appropriate dosage and treatment regimen may be performed in a manner well known to those skilled in the art, for example, oral, parenteral, intravenous, intranasal, intracranial and intramuscular, etc. It may include the administration and preparations (combinations) therefor.

In certain applications, the pharmaceutical compositions disclosed herein can be delivered to a subject by oral administration. As such, the composition may be formulated (blended) with an inert diluent or with an absorbable edible carrier, or the composition may be in a hard-shell or soft-shell gelatin capsule. It can be enclosed, or the compositions can be compressed into a tablet, or the composition can be included directly in the food.

In certain circumstances, it may be desirable for the pharmaceutical compositions disclosed herein to be delivered parenterally, intravenously, intramuscularly or intraperitoneally, such routes of administration, for example in US Pat. Nos. 5,543,158; US Patent No. 5,641,515; Reference may be made to those described in U.S. Patent No. 5,399,363. Solutions of the active compound (as a free base or as a pharmaceutically acceptable salt) can be prepared by mixing appropriately with a surfactant such as hydroxypropylcellulose in water. In addition, the dispersion may be prepared in glycerol, liquid polyethylene glycol, or a mixture thereof, or may be prepared in oil. The preparations may contain preservatives to prevent the growth of microorganisms under normal conditions of storage and use.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders that enable extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468 may be referred to). In all cases, the pharmaceutical form must be sterile and must be fluid enough to allow easy injection. It must be stable under the conditions of production and storage and must be conservative against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof and/or vegetable oil, etc. ) Can be. Appropriate fluidity can be maintained, for example by using a coating agent such as lecithin, by maintaining the required particle size in the case of dispersion, and by using a surfactant. Prevention of the action of microorganisms may be possible with various antimicrobial and antifungal agents (e.g., paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to include an isotonic agent (eg sugar or sodium chloride, etc.). Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption (e.g., aluminum monostearate and gelatin, etc.) in the composition.

For parenteral administration of an aqueous solution, for example, the solution must be adequately buffered as necessary, and the liquid diluent must first be isotonic with sufficient physiological saline or glucose. These particular aqueous solutions are particularly suitable for intravenous administration, intramuscular administration, subcutaneous administration and intraperitoneal administration. Sterile aqueous media that can be used in this connection are known to those skilled in the art. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution, added to 1000 ml of hypodermoclysis fluid, or injected at the proposed injection site (e.g. Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1571-1580). Depending on the symptoms of the subject to which the pharmaceutical composition is treated, some change in dosage necessarily occurs. Those skilled in the art can determine the appropriate dosage for an individual subject through common knowledge in the art. In addition, for administration to humans, these preparations must conform to the sterility, pyrogenic, and general safety and purity standards required by the FDA Office of Biologics standards.

Sterile injection solutions can be prepared by including the active compound in the required amount in an appropriate solvent, along with various other ingredients listed above, as needed, followed by filtration sterilization. In general, dispersions can be prepared by incorporating a variety of sterilized active ingredients into a sterilized vehicle comprising a basic dispersion medium and other necessary components listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, a preferred method of preparation may be a vacuum-drying and freeze-drying technique, which technique is from the previously sterilized-filtered solution. In addition to the active ingredient, a powder of any additional desired ingredients is produced.

The composition disclosed herein may be formulated (blended) in the form of neutral or salt. As pharmaceutically-acceptable salts, acid addition salts (formed with free amino groups of proteins) may be included, and the acid addition salts are inorganic acids (e.g., hydrochloric acid or phosphoric acid, etc.) or organic acids (acetic acid, Oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, etc.) and organic bases (isopropylamine, trimethylamine, histidine, procaine, etc.). . Depending on the formulation, the solution is administered in a manner suitable for the dosage form and in a therapeutically effective amount. The formulations can be easily administered in various dosage forms, such as for example injection solutions, drug-release capsules, and the like.

The term ``carrier'' as used herein refers to any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids. And the like. The use of such media and medicaments for pharmaceutically active substances is well known in the art. They are used in therapeutic compositions, except where any conventional medium or medicament is unsuitable for the active ingredient. Supplementary active ingredients may also be included in the compositions of the present invention.

The expression "pharmaceutically acceptable" refers to a molecular entity and composition that does not cause allergies or similar inappropriate reactions when administered to humans. Methods for preparing aqueous compositions comprising 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 dissolution or suspension in a liquid prior to injection can also be prepared. In addition, the product can be emulsified (emulsified).

In certain embodiments, the pharmaceutical composition may be delivered by intranasal spray, inhalation and/or other aerosol delivery vehicles. Methods for direct delivery of genes, polynucleotides and peptide compositions to the lungs via a nasal aerosol spray may be referred to, for example, those described in U.S. Patent Nos. 5,756,353 and U.S. Patent Nos. 5,804,212. Similarly, delivery of drugs using intranasal microparticle resins (Takenaga et al, 1998) and rizophosphatidyl-glycerol compounds (US Pat. No. 5,725,871) is also well known in the pharmaceutical field. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix may be referred to as described in US Patent No. 5,780,045.

In a specific embodiment, the delivery is a liposome, nanocapsule, microparticle, microsphere, lipid particle, for introduction of the composition of the present invention into an appropriate cell. It can be carried out by the use of vesicles or the like. In particular, the composition of the present invention may be encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, and formulated for delivery. The formulation and use of such delivery vehicles can be carried out using known prior art techniques.

In addition, the pharmaceutical compositions of the present invention may 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 composition formulated in the above manner may be administered in an effective amount, as described above, through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular. In the above, "effective amount" refers to an amount of substance that enables diagnosis or tracking of therapeutic effects when administered to a patient. The pharmaceutical composition containing the polypeptide of the present invention can vary the content of the active ingredient depending on the degree of disease, but is usually from 0.1 μg to 10,000 mg, preferably from 1 mg to 10,000 mg per administration, based on an adult. It can be administered repeatedly several times a day in an effective dose of 5,000 mg. However, the dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, the subject of administration, the target disease and its severity, age, sex and weight, individual differences, and disease states, and such techniques are known to those skilled in the art. .

In another aspect (aspect), the present invention relates to a method of using the composition (e.g., polynucleotide, polypeptide, etc.) of the present invention in a cell, tissue or subject to achieve a desired cellular effect and/or therapeutic effect. to provide. Cells or tissues that can be regulated by the present invention may be preferably mammalian cells or tissues, more preferably human cells or tissues. Such cells or tissues may be in a healthy state or may be in a disease state.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of the following (i) to (v):

(i) the peptide,

(ii) a polynucleotide encoding the (i),

(iii) a vector comprising (ii) above,

(iv) a host cell transformed with (iii) above, and

(v) cysteinyl-tRNA synthetase (CRS).

According to an embodiment of the present invention, a peptide comprising consecutive amino acids from any one selected from the 101 to 119 amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228 amino acid sequence; Alternatively, it was confirmed that the peptide and the CRS full-length protein comprising an amino acid sequence showing 80% or more homology with the peptide exhibited an effect of inhibiting and killing tumor growth by themselves, thereby exhibiting a cancer prevention or treatment effect.

The peptide disclosed in the present invention is a CRS fragment first disclosed in the present specification and exhibits anticancer activity and immune function enhancing activity.

In addition, the peptide, the polynucleotide encoding the same, the vector containing the polynucleotide, the host cell transformed with the vector, or the CRS full-length protein are excellent in anticancer activity and immune function enhancement activity, so that vaccine adjuvants, vaccine compositions and cancer It can be very usefully used in the development of therapeutic compositions.

1A to 1H show the results of confirming the anticancer effect of the CRS protein.
1A to 1C show that CT26 or B16F10 cells (5x10 ⁵ ) were implanted by subcutaneous injection on the right side of a Balb/c or c57bl/6 mouse, and then CRS protein was 200 ug/mouse (10 mg) on Days 7, 8, and 9, respectively. /kg) intraperitoneally, the change in cancer size over time (a, b), and the result of measuring the B16F10 arm weight (c).
Figure 1d is a result of observing changes in cancer size over time after CT26 cells were transplanted into balb/c mice, and CRS (7mg/kg) was administered intraperitoneally on Days 8, 9 and 10, respectively.
Figures 1e and 1f show changes in cancer size over time (e) and weight of cancer on Day 20 after implanting CT26 stable cell (1x10 ⁶ ) overexpressing CRS under the right back of a balb/c mouse. Is the result of measuring.
Figures 1g and 1h show the B16F10 stable cell (1x10 ⁶ ) overexpressing CRS was implanted subcutaneously on the right back of a c57bl/6 mouse, and then the mouse was sacrificed on Day 20 to measure the weight (g) of the cancer and observed with the naked eye. One result (h).
2a to 2f are results confirming that the CRS protein activates the in vivo acquired immune system.
2A to 2C show CT26 cells (5x10 ⁵ ) implanted subcutaneously on the right back of a balb/c mouse, and CRS 200 ug/mouse (10 mg/kg) intraperitoneally administered on Days 8, 9 and 10, respectively, and on Day 12 This is the result of analyzing the change in the number of immune cells in the spleen and tumor using FACS. Total T cell: CD45+, CD3+, CD8: CD45+, CD3+, CD8+, CD4: CD45+ CD3+, CD4+, Tregs: CD45+, CD3+, CD4+, Foxp3+.
2D and 2E are the results of observing changes in CD69, which are the active marks of CD4 and CD8 T cells in the spleen and tumor, using FACS.
2F is a CT26 cell (5x10 ⁵ ) implanted subcutaneously on the right back of a nude mouse, and CRS was administered intraperitoneally at 200 ug/mouse (10 mg/kg) on Days 7, 8, and 9, respectively, and then cancer according to time. This is the result of observing the change in size.
3A and 3B are results of analyzing the interaction between CRS and antigen-presenting cells.
Figures 3a and 3b are obtained by obtaining immune cells from the spleen of c57bl/6 mice, CRS by concentration (3a: BSA 200nM, CRS 100nM, CRS 200nM, 3b: BSA 100nM CRS 100nM) after 1 hour treatment, monocytes (CD11b + , Ly6c+), dendritic cells (CD11b+, CD11c+), neutrophils (CD11b+, LY6g+), B cells (CD19+), T cells (CD3+), and macrophages (CD11b+, F4/80+) were analyzed by FACS, respectively.
4A to 4C show the results of confirming the active fragment peptide of CRS.
4A is a schematic diagram briefly listing the sequence of the CRS full-length protein and its fragments (F2 and F4).
4B is a diagram showing a THP-1 cell treated with PMA (50 ng/ml) for 48 hours to differentiate, and then polymycin B (10 ug/ml), Protease K (20 ug/ml) and CRS protein or a fragment thereof. This is the result of treatment for 4 hours and confirming the level of TNF-α in the medium through ELISA.
Figure 4c is after differentiation by treating THP-1 cells with PMA (50 ng/ml) for 48 hours, and then treating various CRS fragments shown in the figure for 4 hours, and confirming the level of TNF-α in the medium through ELISA. It is the result.
5A to 5D are results confirming that the F2 fragment of CRS (CRS (106-228)) exhibits immune cell recruitment activity.
5A to 5D show OVA (10 ug/mouse), CRS (106-228) (100 ug/mouse), CpG ODN 1826 (40 ug/mouse) or CRS (100 ug/mouse) on the right back of the C57bl/6 mouse by time This is the result of cell analysis through FACS after injecting, cutting off skin such as mice. (A: monocytes (CD11b+, Ly6g-, Ly6c+, F4/80mid), B: neutrophils (CD11b+, ly6g+ ly6c+, F4/80-), C: macrophages (CD11b+, F4/80+), D: dendritic cells ( CD11b+, CD11c+, MHC-II+))
6A to 6E are results of confirming in vitro that CRS and CRS (106-228), a fragment peptide of CRS, induces activation of bone marrow derived macrophages (BMM) .
6A to 6E show the amounts of CD40, CD80 and CD86 cells through FACS analysis after treatment with CRS(106-228) (100nM), CRS (100nM) or LPS (100ng/ml) on BMM for 16 hours, respectively. Confirmation (ac), and the results of confirming TNF-α (d) and IL-6 (e) present in the medium through ELISA.
7A to 7F are results of confirming in vitro that CRS and CRS (106-228), a fragment peptide of CRS, induces activation of bone marrow derived dendritic cells (BMDC) .
7A to 7F show the amounts of CD40, CD80 and CD86 cells through FACS analysis after treating BMDC with CRS (106-228) (100nM), CRS (100nM) or LPS (100ng/ml) for 16 hours, respectively. Confirmation (ac), and treatment with CRS (106-228) (100nM), CRS (100nM) or LPS (100ng/ml) to BMDC for 24 hours, respectively, and then TNF-α(d) and IL- present in the medium 6(e), IL-12 p70 (f) was confirmed by ELISA.
8 is a result of confirming that CRS and CRS (106-228), a fragment peptide of CRS, improves antigen presentation of dendritic cells.
Figure 8 shows BMDC after treatment with OVA (SIINFEKL) (10ug/ml), CRS (100nM), CRS (106-228) (100nM), and CpG ODN 1826 (100nM) for 48 hours, FITC-CD11c, PE- This is the result of FACS analysis of the OVA peptide presented by BMDC using H-2kb antibody.
9A and 9B are results confirming that CRS and CRS (106-228), a fragment peptide of CRS, induces the activation of dendritic cells in lymph nodes.
9A and 9B show OVA (10 ug/mouse), CRS (106-228) (100 ug/mouse), CpG ODN 1826 (40 ug/mouse), or CRS (100 ug/mouse) by time, respectively, on the right back of C57bl/6 mice. After subcutaneous administration and separating the lymph nodes in the mouse inguinal region, the amount of CD40(a) or CD86(b) positive cells was analyzed by FACS through CD86-FITC, CD11c-APC, MHC II-percp, and CD40-PE. to be.
10A to 10I show the results of confirming in vivo that CRS and CRS (106-228), which is a fragment peptide of CRS, activates the acquired immune system .
Figures 10a to 10c are after implantation of Eg7-OVA cells (1x10 ⁶ ) subcutaneously on the right back of c57bl/6 mice, OVA (10ug/mouse), CRS (100ug/mouse) or CRS (106-228) (100ug/ mouse) was administered to the left side of the mouse on Day 3 and Day 10, and blood was collected from the mouse on Day 17, and the total amount of IgG(a), IgG1a(b), and IgG2c(c) in the blood was analyzed through ELISA. to be.
Figures 10d to 10f are after implantation of Eg7-OVA cells (1x10 ⁶ ) subcutaneously on the right back of c57bl/6 mice, OVA (10ug/mouse), CRS (100ug/mouse) or CRS (106-228) (100ug/ mouse) was administered to the left side of the mouse on Day 3 and Day 10, and the ratio of CD8 T cells and OVA-tetramer (f) in the blood (d) and tumor (e) of the mouse on Day 17 were analyzed through FACS. .
10g to 10h show that OVA (10ug/mouse), CRS (100ug/mouse), or CRS (106-228) (100ug/mouse) was administered on Day 7 and Day 14 to the subcutaneous right back of c57bl/6 mouse, and on Day 21 This is the result of FACS analysis of OVA-tetramer in mouse inguinal lymph nodes (g) and spleen (h).
Figure 10i shows that OVA (10ug/mouse), CRS (100ug/mouse), or CRS (106-228) (100ug/mouse) was administered on Day 7 and Day 14 and OVA peptide on Day 20 to the subcutaneous right back of c57bl/6 mouse. (SIINFEKL) pulsed or unpulseed naive mouse spleen cells were stained with each high and low concentration of CFSE, and then intravenously administered to the immunized mice. After 20 hours, specific apoptosis was confirmed through the proportion of cells in the pulsed and unpulsed groups in the splenocytes of the immunized mice.
11a and 11b are results confirming the effect of CRS as a vaccine for preventing cancer.
11A and 11B show a process of subcutaneous administration of OVA (10 ug/mouse) and CRS (20 ug/mouse or 100 ug/mouse) to the left back once a week to c57bl/6 mice for 3 weeks, and then Eg7-OVA cell (1x10 ⁶ ) was subcutaneously eaten on the right side of the mouse, and the change in tumor size (a) and the survival rate (b) of the mouse over time were observed.
12A and 12B are results of evaluating the effect of a vaccine for treating cancer according to the combination of a known vaccine adjuvant and CRS (106-228).
12A is a schematic diagram schematically showing an experiment schedule.
Figure 12b is a c57bl/6 mouse after avoiding Eg7-OVA cell (1x10 ⁶ ) on the right back, OVA (10ug/mouse), CRS (106-228) (100ug/mouse) or CpG on Day 3 and Day 7 ODN 1826 (40ug/mouse) was administered subcutaneously to the left side of the mouse according to the combination shown in the figure, and the change in the size of the tumor was observed until Day 18.
13A and 13B show the in vivo effect of a vaccine for cancer treatment according to the combination of CRS (106-228), an immune checkpoint inhibitor (PD-L1 mAb), and an antigen (OVA), which is a fragment peptide of CRS. These are the results of evaluation in transplanted mice.
13A is a schematic diagram briefly showing an experiment schedule.
Figure 13b is an Eg7-OVA cell (1x10 ⁶ ) implanted subcutaneously on the right side of a c57bl/6 mouse, OVA (10ug/mouse), CRS (100ug/mouse) or CRS (106-228) (100ug/mouse) Treatment on Days 3 and 10, PD-L1, PD-L1 isotype mAb (200 ug/mouse) was intraperitoneally administered on Days 3, 6 and 9, and the tumor size was observed until Day 18.

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Experiment method

(1) mouse

C57BL/6 and BALB/c mice were obtained from Dooyeol Biotech. And OVA-specific T cell receptor transgenic OT-1 mice were kindly provided by Professor Chang-Yul Kang of Seoul National University. All mouse experiments were performed according to guidelines approved by Seoul National University. All mice were maintained in friendship bsc using an institutionally approved protocol.

(2) cell line

CT26 and B16f10 CRS overexpressing cells were generated by the G418 selection method. Briefly, lipofectamine 2000 was used to transform pEXPR-IBA105 EV and CRS for each cell. For selection of overexpressed cells, 1 mg/ml of G418 was used in the growth medium and resistant single clones were selected. The expression level of each clone was tested through immunoblotting, and clones having similar in vitro growth were selected and used in the experiment.

(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 x 10 5 cells of CT26 and B16F10 were injected subcutaneously into the right side of BALB/c and C57BL/6 mice for 6-8 weeks, respectively. On days 7, 8, and 9, 200 μg of each protein was injected intraperitoneally into each protein. In the case of the immune checkpoint inhibitor, tumor bearing mice were intraperitoneally injected with 200 μg of each reagent (Isotype, PD1, PDL1, LAG3 antibody) on days 8, 11, and 14. Tumors were measured with a digital caliper and calculated according to the following equation (0.52 X length X width 2). When the tumor was larger than 1500 mm ³ , the mouse was euthanized.

(4) Cell binding Assay

After separating the spleen of C57Bl/6 mice, they were separated and dispensed at the level of single cells by applying physical force. Incubate for 1 hour at 4 degrees with the splenocytes dispensed using BSA and CRS proteins stained with Alexa647 dye. The cultured cells were stained with CD11b, CD11c, CD3, CD19, Ly6C, Ly6G, F4/80 FACS antibody, and then analyzed through FACS analysis. CD3: T cell, CD11b+, F4/80+: macrophage, CD11b, Ly6C: monocyte, CD11b+, Ly6G: neutrophil, CD19: B cell, CD11b+, CD11c+: Dendritic cell

(5) Analysis of tumor infiltrating immune cells and spleen immune cells

CT26 tumor bearing mice were sacrificed on days 10 and 12. Tumor cells were dissociated using a tumor dissociation kit, Tumor dissociation kit, and mouse (130-096-730, Miltenyi Biotec) according to the manufacturing protocol. Splenocytes were dissociated in a medium containing RPMI medium containing 2% FBS and 1% streptomycin. After lysing red blood cells (Lysing Buffer (555899, BD biosciences)), tumor and spleen cells were stained with antibodies CD8, CD4, CD3, CD45, Foxp3 and CD69. Monoclonal antibodies were stained at 4° C. for 30 minutes. For intracellular staining, a Fixation/Permeabilization Solution Kit with BD GolgiPlug?? (555028, BD biosciences) was used.

(6) protein purification

CRS and CRS (106-228) proteins were purified using a pET28a plasmid vector containing an N, C-terminal 6X his tag. BL21-codon plus cells were transformed and colonies were grown by inoculating the medium. Large-scale 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 from centrifugation, and crushed by sonication in 50mM Tris buffer pH7.5 containing 300 mM NaCl. Thereafter, centrifugation was performed at 20,000 g for 30 minutes to obtain a supernatant. It was poured onto a column containing Ni-NTA resin. A washing step was performed with 50 mM Tris, pH 7.5 containing 300 mM NaCl, 5% glycerol and 15 mM imidazole. After separating the protein from the column with 10 ml of elution buffer (50mM Tris pH7.5, 300mM NaCl, glycerol 5%, 300mM imidazole), endotoxin was removed using TX-114 (REF: Removal of endotoxin from protein solutions by phase separation using Triton X-114). From the LAL analysis, an appropriate protein of 0.04 EU/mg or less was used for the entire experiment.

(7) ELISA

RAW264.7, THP1-PMA, bone marrow derived macrophage (BMM) and bone marrow derived DC (BMDC) cells were tested to confirm cytokine secretion by CRS. Each cell type was treated with 5 X 10 ⁵ cells / ml in a 24 well plate overnight, and each well was changed to serum free medium for 2 hours before drug treatment. For RAW264,7 and THP1-PMA, 100 nM of protein was treated for 4 hours. In the case of BMM and BMDC cells, a protein at a concentration of 100 nM was treated for 24 hours. The supernatant was centrifuged at 500 g for 10 minutes to perform ELISA using IL-6, TNF-α, IL-12 p70, and IL-10 ELISA set (BD).

(8) BMDC, BMM activation

BMDC (bone marrow derived dendritic cell), BMM (bone marrow derived macrophage) were prepared using standard methods. Briefly, bone marrow cells were obtained from female C57BL/6 mice and contained 10% FBS, 1% streptomycin and 10 ng/ml GM-CSF (R&D, BMDC) or 10 ng/ml M-CSF (R&D, BMM). Cultured in RPMI medium. On the third day, GM-CSF or M-CSF and fresh RPMI medium were added and BMDC was harvested from non-adherent and loosely attached cells on day 6, and BMM was harvested from adherent cells. Each protein 100 nM and LPS 1 μg/ml were treated for 18 hours and FACS analysis was performed using antibodies against FACS CD11c, CD40, CD80 and CD86.

(9) Prophylactic and therapeutic anticancer vaccine mouse model

In the case of a prophylactic anticancer vaccine tumor model, OVA protein (10 ug) alone or OVA protein + 5 mg/kg CRS, CRS (106-228) was injected into the back flank of C57BL/6 females. One week after the final immunization injection, 1×10 ⁶ E.G7-OVA cells were injected into the opposite side of the mouse vaccination site. In the therapeutic cancer vaccine model, E.G7-OVA cells were injected with sc on the right side of the dorsal side. On days 3 and 10, OVA, CRS (5 mg/kg), and CRS (106-228) (5 mg/kg) were injected on the left dorsal side, respectively, and PD-L1 isotype antibody (10 mg/kg) was injected on days 3, 6 and 9 kg), PD-L1 antibody (10 mg/kg) was injected intraperitoneally. Tumor volume was measured as mentioned above

(10) In vivo CD8 priming

6-8 week old female C57BL/6 mice were injected subcutaneously with 10 ug OVA alone or OVA + 5 mg/kg CRS (106-228) twice a week. Spleen, blood and inguinal lymph nodes, and cancer cells were harvested 3 days after the last injection. The amount of OVA-specific tetramer in CD8+ T cell was analyzed by FACS.

(11) In vivo CTL killing assay

C57BL/6 mice were injected once a week with CRS, CRS (106-228), and CpG ODN 1826 with 10 ug of OVA on the back. Four days after the second injection, splenocytes of unimmunized normal C57BL/6 were harvested. Half of the splenocytes were cultured with SIINFEKL1 peptide (pulse group) and the other half without peptide (non-pulse group). After 2 hours, the pulsed and non-pulse groups were labeled with 2.5 uM or 0.25 uM of CFSE for 15 minutes, then the remaining CFSE dye was inactivated with serum medium. The same number of pulsed and non-pulse group cells were evenly mixed and injected intravenously into immunized mice for 2 weeks before. After 24 hours, splenocytes were collected, high and low CFSE cells were measured, and specific target cell death was calculated according to the following formula. [1-(High CFSE in immunized sample% / Low CFSE in immunized sample %)/ (High CFSE in unimmunized sample %/ Low CFSE in unimmunized sample %)] x 100.

(12) Anti-ova IgG detection ELISA

After 2 weeks of immunization, serum from the immunized mice was obtained and ELISA was performed. 10 μg/ml of OVA protein was coated on a Maxisorp plate with sodium carbonate buffer at 4° C. for 12 hours. After washing 3 times with PBS-T, the plate was blocked with 5% BSA for 1 hour. Then, the plate was washed 3 times with PBS-T, and the diluted sample was incubated for 2 hours. Anti-mouse IgG, IgG1 and IgG2c conjugated with HRP were added for 1 hour after 5 washing steps. After washing 7 times, TMB substrate was added and developed, and absorbance was measured at 450 nm.

(13) Immune cell recruitment

C57BL/6 mice were treated with OVA, CRS, CRS (106-228) (100 ug/mouse) CPG ODN 1826 (40 ug/mouse). At the indicated time, the injection site near the skin is cut out and minced very small with a razor. The minced skin samples were cultured in RPMI medium containing 5% FBS, 300µg/ml collagenase, and 50U/ml DNase1. After incubation in the digestion medium at 37° C. for 2 hours, the cell suspension was then passed through a 70 um filter, stained with antibodies against Ly6C, CD11b, CD11c, Ly6G, and F4/80, followed by FACS analysis.

(14) In vitro Antigen presentation

BMDC was generated with GM-CSF as described above. On day 6, non-adherent and loosely attached DCs were collected. BMDC was cultured with 100 nM of CRS, CRS (106-228), CpG ODN 1826 and OVA peptide (10 ug/ml) in growth RPMI medium. Treated BMDCs were stained with CD11c, CD11b and SIINFEKL.H-2Kb antibodies and analyzed by FACS.

Experiment result

1. Confirmation of anticancer effect of CRS (cysteinyl-tRNA synthetase)

An experiment was designed to confirm the anticancer effect of the CRS protein consisting of the amino acid sequence of SEQ ID NO: 1. It was confirmed that the anticancer effect appeared when CRS was injected intraperitoneally (intraperitoneal (IP)) or intratumorally (intratumoral (IT)) in an in vivo syngeneic mouse model using CT26 and B16F10 cell lines (FIGS. 1A to 1D).

Thereafter, the anticancer effect of CRS was again confirmed by confirming that the size of the cancer was reduced in the CRS overexpression cell line in the in vivo syngeneic mouse model using the CT26 and B16F10 CRS stable cell lines (FIGS. 1e to 1h ).

2. Confirmation of the mechanism of CRS anticancer effect

Next, in order to confirm the mechanism of the in vivo anticancer effect of CRS, after intraperitoneal administration of CRS, an adaptive immune system was observed in the tumor and spleen of mice. Briefly, CRS was administered intraperitoneally to mice implanted with CT26 cells, and immune cells were obtained 12 days later and observed.

As a result, the proportion of T cells among the total immune cells in the spleen was not significantly changed in the CRS-treated group compared to the normal control group (Con), but the proportion of T cells among the total immune cells in the tumor, the proportion of CD8 T cells, and The amount increased in the CRS-administered group, and it was confirmed that the CD8 T:Treg ratio also increased (FIGS. 2a to 2c). That is, the increase in the proportion and amount of CD8 T cells and the CD8 T:Treg ratio in the tumor means that the activity of Cytotoxic T lymphocyte (CTL) is increased. These results can be said to be an indicator of the ability to remove tumors by T cells.

In addition, it was confirmed that CD69, a marker of T cell activation, was increased in the CRS-administered group in the tumor and spleen (FIGS. 2D and 2E ). This means that CTL is activated during CRS processing.

Next, in vivo experiments using nude mice were conducted to confirm the relationship between CRS and the immune system. As a result of administering CRS intraperitoneally to nude mice implanted with CT26 cells, it was confirmed that there was no anticancer effect (FIG. 2F ). These results imply that an acquired immune system is required for CRS to exhibit anticancer effects.

Next, an experiment was conducted to determine what the main target immune cells of CRS were. Ex vivo cell binding experiments were performed using immune cells extracted from the spleen of mice. Through this, CRS is relatively more effective in binding to antigen presenting cells such as monocytes, macrophages, dendritic cells, and neutrophils than T cells and B cells. It could be confirmed, and among them, it could be seen that it binds most strongly to monocytes (FIGS. 3A and 3B ).

3. Confirmation of immune cell activity of CRS fragment peptide

Based on the above experimental results, a fragment peptide of the CRS protein consisting of the amino acid sequence of SEQ ID NO: 1 was prepared to confirm the activity against immune cells. Specifically, a fragment peptide of SEQ ID NO: 2 (F2) consisting of 106 to 228 amino acids of the amino acid sequence of SEQ ID NO: 1 and a fragment peptide (F4) of SEQ ID NO: 3 consisting of amino acids 571 to 748 were prepared, and human monocytes After THP-1 was differentiated using PMA, CRS of SEQ ID NO: 1 and the fragment peptides were treated, respectively.

As a result, it was confirmed that the F2 peptide activates monocytes with superior ability when compared to the CRS protein, and it was confirmed that the F4 peptide does not activate monocytes (Fig. 4b). For reference, in the case of boiled and polymyxin B treatment group in FIG. 4B, it was treated to confirm that the activity of the protein was not caused by LPS contamination. In the case of Protease K, it was treated to confirm that activity was not caused by substances other than proteins.

Thereafter, based on the experimental results of the F2 peptide (hereinafter referred to as “CRS (106-228)”), the following CRS fragment peptides were additionally produced, and the activity of each fragment peptide with monocytes was confirmed (in parentheses The number means the sequence number of the amino acids constituting the CRS protein of SEQ ID NO: 1. For example, CRS (101-179) refers to a peptide consisting of amino acids from 101 to 179 of the N-terminal CRS protein of SEQ ID NO: 1. .).

SEQ ID NO: 4: CRS (101-179)

SEQ ID NO: 5: CRS (119-179)

SEQ ID NO: 6: CRS (101-200)

SEQ ID NO: 7: CRS (119-200)

As a result, it was confirmed that CRS (101-200) and CRS (119-200) were also very excellent in activity with monocytes, as can be seen in FIG. 4C.

4. Evaluation of vaccine adjuvant activity of CRS and its fragment peptide

In general, in order for a specific substance to be used as a vaccine adjuvant, cellular and humoral immunity must be activated through activation of the immune system obtained after activation of innate immunity such as immune cell recruitment, activation, and differentiation during processing of the substance. do. Through this, the immune system in vivo can have immune activity against specific antigens. Accordingly, the present inventors confirmed the activity of CRS and its F2 fragment (CRS (106-228)) as a vaccine adjuvant through subsequent experiments.

(1) Innate immune activation effect

First, recruitment of antigen-presenting cells such as monocytes, neutrophils, macrophages and dendritic cells by CRS or CRS (106-228) treatment was confirmed. Specifically, after administering CRS or CRS (106-228) to the mouse skin, the amount of immune cells rushing to the skin for each time period was confirmed.

As a result of the experiment, monocytes were recruited the most due to CRS or CRS (106-228) treatment, which was consistent with previous experimental results. In addition, it was confirmed that the recruitment of other antigen-presenting cells (neutrophils, macrophages, dendritic cells) was also increased (FIGS. 5A to 5D ). The recruitment efficiency of antigen-presenting cells was generally highest in the group treated with CRS (106-228), an F2 fragment of CRS.

Next, in order to confirm the degree of activation of the clustered antigen-presenting cells, the ratio of CD40, CD80, and CD86, which are activation markers of macrophages and dendritic cells, and the amount of secretion of cytokines (TNF-alhpa, IL-6 and IL-12) Was confirmed in vitro .

In the case of macrophages, the ratio of CD40 and CD86 increased by treatment with CRS or CRS (106-228), and the amount of TNF-α secretion did not increase, but the amount of IL-6 secretion increased in these treatment groups (Fig. 6A to 6d).

In the case of dendritic cells, the ratio of CD40, CD80 and CD86 were all increased by treatment with CRS or CRS (106-228), and the secretion of IL-6, TNF-α, and IL-12 p70 were all increased (FIGS. 7A to 7F ). ).

Next, for the vaccine to be effective, the presentation of the antigen's cell membrane is important. To this end, the effects of CRS and CRS (106-228) on the uptake and presentation of OVA antigens treated together were confirmed.

As a result, it was confirmed that the OVA antigen presentation was increased in the CRS (106-228) and CRS-treated groups, and this effect was confirmed to be superior when compared to the known vaccine adjuvant CpG ODN 1826 (FIG. 8).

Next, the degree of dendritic cell activation in lymph nodes according to CRS or CRS (106-228) treatment was confirmed through CD40 and CD86. When 24 hours elapsed after each test substance was treated with dendritic cells, it was confirmed that CD40 and CD86, which are dendritic cell activation markers, increased in lymph nodes. In particular, an increase in CD40 and CD86 was observed in the CRS (106-228) treatment group than in the CpG ODN 1826 treatment group (FIGS. 9A and 9B ).

Through the above four experiments, the innate immune activation effect of CRS and its fragment, CRS (106-228) as a vaccine adjuvant, was confirmed.

(2) adaptive immune activation effect

The above-described innate immunity activation indicates immune activity against a specific antigen through acquired immunity activation. This activation of acquired immunity is essential for the effectiveness of the vaccine. Acquired immune activation of vaccine adjuvants can be confirmed through two ways. First, the cellular immune response is to look at the activity of cytotoxic T lymphocytes, through the presence of specific antigen-related peptides on TCR and induction of apoptosis of cells with specific antigens. Can be checked. Second, the humoral immune response confirms the activity of B cells and confirms an increase in the production capacity of specific antigen-related IgG. Specifically, there are total IgG, IgG1a, IgG2c, and the like.

First, in order to confirm the activation effect of the humoral immune response, the ability of OVA-related IgG production in blood was measured. Cancer was planted in mice and administered with OVA and CRS or CRS (106-228) once a week for a total of 2 weeks on the left side of the mouse, and then the amount of IgG in the blood was confirmed through ELISA.

As a result of the experiment, it was confirmed that the secretion of total IgG, IgG1a indicating Th2 activity, and IgG2c indicating Th1 activity were increased in the CRS and CRS (106-228) treatment groups (FIGS. 10A to 10C ). These results mean that CRS and CRS (106-228) can enhance antigen-specific antibody production upon vaccination with specific antigens.

Next, in order to confirm the activation effect of the cellular immune response, it was confirmed whether the antigen-specific peptide was expressed on the T cell surface. Specifically, after implanting cancer in a mouse and administering it to the left side of the mouse once a week with OVA and CRS or CRS (106-228) for a total of 2 weeks, the level of surface expression of antigen-specific peptides on blood and tumor-penetrating CD8 T cells Was confirmed. As a result of the experiment, it was confirmed that CRS (106-228) increases the level of surface expression of antigen-specific peptides in all parts (Figs. 10D to 10E). In addition, it was confirmed that the amount of tumor-penetrating CD8 T cells was significantly increased in the CRS (106-228) treatment group (FIG. 10F). Even when OVA, CRS, and CRS (106-228) were treated on the right side of the mouse for 2 weeks without tumor, it was confirmed that the level of surface expression of antigen-specific peptides in CD8 T cells of lymph nodes and spleens increased. (Fig. 10G to 10H)

In order to confirm the effect of activating the cellular immune response, antigen-specific cell death was confirmed. Specifically, when a cell expressing an antigen on the cell surface was administered from the outside, it was confirmed whether the immune cells of the mouse could recognize it and kill it. As a result of the experiment, in the case of mice immunized with CRS and CRS (106-228), it was confirmed that cells that label the antigen from outside can be recognized and killed (Fig. 10i).

5. Validation of effectiveness as vaccine for cancer prevention

After confirming the effect of increasing the immune activity of CRS through a previous experiment, it was confirmed whether or not it can actually show a preventive effect against a specific cancer when an animal is immunized using an antigen and CRS. Specifically, before transplanting Eg7-OVA cells into mice, CRS and OVA antigens were administered to mice once a week for a total of 3 weeks to sufficiently activate the immune system specific to OVA antigens. Thereafter, Eg7-OVA cells were implanted subcutaneously in mice, and tumor size changes were observed over time.

As a result, in the CRS + OVA treatment group, it was confirmed that the tumor grows significantly more slowly than in the untreated control group and the OVA treatment group, and it was also confirmed that the survival rate of the animal was increased (FIGS. 11A and 11B). Therefore, it was confirmed that CRS can be administered together with a specific tumor antigen to be used as an active ingredient in a tumor vaccine adjuvant or a tumor vaccine composition that can prevent the occurrence of the corresponding cancer.

6. Validation of effectiveness as a vaccine for cancer treatment

(1) Evaluation of combination with other vaccine adjuvants

In order to establish a more effective tumor vaccine method, the present inventor proceeded to screening for combination with other adjuvants. In the case of adjuvants, there is generally a high probability of toxicity when the dosage is increased. If a synergistic effect appears when two or more types of adjuvants are used in combination in a small amount, it can be used as the most effective and stable vaccine adjuvant. In the present invention, an experiment was conducted to select a vaccine adjuvant that can be used in combination with CRS or CRS (106-228). First, CpG ODN 1826, a known vaccine adjuvant, was selected as an adjuvant. CpG ODN 1826 is a TLR9 ligand and is currently being studied as an adjuvant for tumor vaccines.

The experiment was performed in combination with OVA, each adjuvant, and a combination of these adjuvants to mice subcutaneously implanted with EG-7 OVA, and specific experimental schedules and administration groups are shown in FIGS. 12A and 12B.

As a result of the experiment, the group co-administered with CRS (106-228) and CpG ODN showed the best tumor growth inhibitory effect (FIG. 12B ).

(2) Evaluation of combination with an immune checkpoint inhibitor (Immune checkpoint inhibitor)

Immune checkpoint inhibitors are a group of drugs currently in clinical trials for use with anticancer vaccines. In order to increase the effectiveness of an anticancer vaccine containing CRS or CRS (106-228), a combination experiment with an immune checkpoint inhibitor was conducted (FIG. 13A).

As a result of the experiment, (OVA antigen + CRS (106-228) + anti-PD-L1 antibody) co-administration group (OVA antigen + anti-PD-L1 antibody) co-administration group, and (OVA antigen + CRS (106-228) It was confirmed that tumor growth was significantly suppressed compared to the combination administration group) (Fig. 13B).

The peptide disclosed in the present invention is a CRS fragment first disclosed in the present specification and exhibits anticancer activity and immune function enhancing activity.

In addition, the peptide, the polynucleotide encoding the same, the vector containing the polynucleotide, the host cell transformed with the vector, or the CRS full-length protein are excellent in anticancer activity and immune function enhancement activity, so that vaccine adjuvants, vaccine compositions and cancer It can be used very usefully in the development of therapeutic compositions, so it has excellent industrial applicability.

<110> Medicinal Bioconvergence Research Center <120> Fragmented cysteinyl-tRNA synthetase peptide and use thereof <130> NP19-0013 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 748 <212> PRT <213> Artificial Sequence <220> <223> Cysteinyl tRNA synthetase <400> 1 Met Ala Asp Ser Ser Gly Gln Gln Gly Lys Gly Arg Arg Val Gln Pro 1 5 10 15 Gln Trp Ser Pro Pro Ala Gly Thr Gln Pro Cys Arg Leu His Leu Tyr 20 25 30 Asn Ser Leu Thr Arg Asn Lys Glu Val Phe Ile Pro Gln Asp Gly Lys 35 40 45 Lys Val Thr Trp Tyr Cys Cys Gly Pro Thr Val Tyr Asp Ala Ser His 50 55 60 Met Gly His Ala Arg Ser Tyr Ile Ser Phe Asp Ile Leu Arg Arg Val 65 70 75 80 Leu Lys Asp Tyr Phe Lys Phe Asp Val Phe Tyr Cys Met Asn Ile Thr 85 90 95 Asp Ile Asp Asp Lys Ile Ile Lys Arg Ala Arg Gln Asn His Leu Phe 100 105 110 Glu Gln Tyr Arg Glu Lys Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp 115 120 125 Val Gln Ala Ala Leu Lys Pro Phe Ser Val Lys Leu Asn Glu Thr Thr 130 135 140 Asp Pro Asp Lys Lys Gln Met Leu Glu Arg Ile Gln His Ala Val Gln 145 150 155 160 Leu Ala Thr Glu Pro Leu Glu Lys Ala Val Gln Ser Arg Leu Thr Gly 165 170 175 Glu Glu Val Asn Ser Cys Val Glu Val Leu Leu Glu Glu Ala Lys Asp 180 185 190 Leu Leu Ser Asp Trp Leu Asp Ser Thr Leu Gly Cys Asp Val Thr Asp 195 200 205 Asn Ser Ile Phe Ser Lys Leu Pro Lys Phe Trp Glu Gly Asp Phe His 210 215 220 Arg Asp Met Glu Ala Leu Asn Val Leu Pro Pro Asp Val Leu Thr Arg 225 230 235 240 Val Ser Glu Tyr Val Pro Glu Ile Val Asn Phe Val Gln Lys Ile Val 245 250 255 Asp Asn Gly Tyr Gly Tyr Val Ser Asn Gly Ser Val Tyr Phe Asp Thr 260 265 270 Ala Lys Phe Ala Ser Ser Glu Lys His Ser Tyr Gly Lys Leu Val Pro 275 280 285 Glu Ala Val Gly Asp Gln Lys Ala Leu Gln Glu Gly Glu Gly Asp Leu 290 295 300 Ser Ile Ser Ala Asp Arg Leu Ser Glu Lys Arg Ser Pro Asn Asp Phe 305 310 315 320 Ala Leu Trp Lys Ala Ser Lys Pro Gly Glu Pro Ser Trp Pro Cys Pro 325 330 335 Trp Gly Lys Gly Arg Pro Gly Trp His Ile Glu Cys Ser Ala Met Ala 340 345 350 Gly Thr Leu Leu Gly Ala Ser Met Asp Ile His Gly Gly Gly Phe Asp 355 360 365 Leu Arg Phe Pro His His Asp Asn Glu Leu Ala Gln Ser Glu Ala Tyr 370 375 380 Phe Glu Asn Asp Cys Trp Val Arg Tyr Phe Leu His Thr Gly His Leu 385 390 395 400 Thr Ile Ala Gly Cys Lys Met Ser Lys Ser Leu Lys Asn Phe Ile Thr 405 410 415 Ile Lys Asp Ala Leu Lys Lys His Ser Ala Arg Gln Leu Arg Leu Ala 420 425 430 Phe Leu Met His Ser Trp Lys Asp Thr Leu Asp Tyr Ser Ser Asn Thr 435 440 445 Met Glu Ser Ala Leu Gln Tyr Glu Lys Phe Leu Asn Glu Phe Phe Leu 450 455 460 Asn Val Lys Asp Ile Leu Arg Ala Pro Val Asp Ile Thr Gly Gln Phe 465 470 475 480 Glu Lys Trp Gly Glu Glu Glu Ala Glu Leu Asn Lys Asn Phe Tyr Asp 485 490 495 Lys Lys Thr Ala Ile His Lys Ala Leu Cys Asp Asn Val Asp Thr Arg 500 505 510 Thr Val Met Glu Glu Met Arg Ala Leu Val Ser Gln Cys Asn Leu Tyr 515 520 525 Met Ala Ala Arg Lys Ala Val Arg Lys Arg Pro Asn Gln Ala Leu Leu 530 535 540 Glu Asn Ile Ala Leu Tyr Leu Thr His Met Leu Lys Ile Phe Gly Ala 545 550 555 560 Val Glu Glu Asp Ser Ser Leu Gly Phe Pro Val Gly Gly Pro Gly Thr 565 570 575 Ser Leu Ser Leu Glu Ala Thr Val Met Pro Tyr Leu Gln Val Leu Ser 580 585 590 Glu Phe Arg Glu Gly Val Arg Lys Ile Ala Arg Glu Gln Lys Val Pro 595 600 605 Glu Ile Leu Gln Leu Ser Asp Ala Leu Arg Asp Asn Ile Leu Pro Glu 610 615 620 Leu Gly Val Arg Phe Glu Asp His Glu Gly Leu Pro Thr Val Val Lys 625 630 635 640 Leu Val Asp Arg Asn Thr Leu Leu Lys Glu Arg Glu Glu Lys Arg Arg 645 650 655 Val Glu Glu Glu Lys Arg Lys Lys Lys Glu Glu Ala Ala Arg Arg Lys 660 665 670 Gln Glu Gln Glu Ala Ala Lys Leu Ala Lys Met Lys Ile Pro Pro Ser 675 680 685 Glu Met Phe Leu Ser Glu Thr Asp Lys Tyr Ser Lys Phe Asp Glu Asn 690 695 700 Gly Leu Pro Thr His Asp Met Glu Gly Lys Glu Leu Ser Lys Gly Gln 705 710 715 720 Ala Lys Lys Leu Lys Lys Leu Phe Glu Ala Gln Glu Lys Leu Tyr Lys 725 730 735 Glu Tyr Leu Gln Met Ala Gln Asn Gly Ser Phe Gln 740 745 <210> 2 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> CRS (106-228) <400> 2 Ala Arg Gln Asn His Leu Phe Glu Gln Tyr Arg Glu Lys Arg Pro Glu 1 5 10 15 Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala Leu Lys Pro Phe Ser 20 25 30 Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys Lys Gln Met Leu Glu 35 40 45 Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu Pro Leu Glu Lys Ala 50 55 60 Val Gln Ser Arg Leu Thr Gly Glu Glu Val Asn Ser Cys Val Glu Val 65 70 75 80 Leu Leu Glu Glu Ala Lys Asp Leu Leu Ser Asp Trp Leu Asp Ser Thr 85 90 95 Leu Gly Cys Asp Val Thr Asp Asn Ser Ile Phe Ser Lys Leu Pro Lys 100 105 110 Phe Trp Glu Gly Asp Phe His Arg Asp Met Glu 115 120 <210> 3 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> CRS (571-748) <400> 3 Val Gly Gly Pro Gly Thr Ser Leu Ser Leu Glu Ala Thr Val Met Pro 1 5 10 15 Tyr Leu Gln Val Leu Ser Glu Phe Arg Glu Gly Val Arg Lys Ile Ala 20 25 30 Arg Glu Gln Lys Val Pro Glu Ile Leu Gln Leu Ser Asp Ala Leu Arg 35 40 45 Asp Asn Ile Leu Pro Glu Leu Gly Val Arg Phe Glu Asp His Glu Gly 50 55 60 Leu Pro Thr Val Val Lys Leu Val Asp Arg Asn Thr Leu Leu Lys Glu 65 70 75 80 Arg Glu Glu Lys Arg Arg Val Glu Glu Glu Lys Arg Lys Lys Lys Glu 85 90 95 Glu Ala Ala Arg Arg Lys Gln Glu Gln Glu Ala Ala Lys Leu Ala Lys 100 105 110 Met Lys Ile Pro Pro Ser Glu Met Phe Leu Ser Glu Thr Asp Lys Tyr 115 120 125 Ser Lys Phe Asp Glu Asn Gly Leu Pro Thr His Asp Met Glu Gly Lys 130 135 140 Glu Leu Ser Lys Gly Gln Ala Lys Lys Leu Lys Lys Leu Phe Glu Ala 145 150 155 160 Gln Glu Lys Leu Tyr Lys Glu Tyr Leu Gln Met Ala Gln Asn Gly Ser 165 170 175 Phe Gln <210> 4 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> CRS (101-179) <400> 4 Lys Ile Ile Lys Arg Ala Arg Gln Asn His Leu Phe Glu Gln Tyr Arg 1 5 10 15 Glu Lys Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala 20 25 30 Leu Lys Pro Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys 35 40 45 Lys Gln Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu 50 55 60 Pro Leu Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val 65 70 75 <210> 5 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> CRS (119-179) <400> 5 Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala Leu Lys 1 5 10 15 Pro Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys Lys Gln 20 25 30 Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu Pro Leu 35 40 45 Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val 50 55 60 <210> 6 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> CRS (101-200) <400> 6 Lys Ile Ile Lys Arg Ala Arg Gln Asn His Leu Phe Glu Gln Tyr Arg 1 5 10 15 Glu Lys Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala 20 25 30 Leu Lys Pro Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys 35 40 45 Lys Gln Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu 50 55 60 Pro Leu Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val Asn 65 70 75 80 Ser Cys Val Glu Val Leu Leu Glu Glu Ala Lys Asp Leu Leu Ser Asp 85 90 95 Trp Leu Asp Ser 100 <210> 7 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> CRS (119-200) <400> 7 Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala Leu Lys 1 5 10 15 Pro Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys Lys Gln 20 25 30 Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu Pro Leu 35 40 45 Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val Asn Ser Cys 50 55 60 Val Glu Val Leu Leu Glu Glu Ala Lys Asp Leu Leu Ser Asp Trp Leu 65 70 75 80 Asp Ser <210> 8 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> CRS (106-228) polynucleotide <400> 8 gcccggcaga accacctgtt cgagcagtat cgggagaaga ggcctgaagc ggcacagctc 60 ttggaggatg ttcaggccgc cctgaagcca ttttcagtaa aattaaatga gaccacggat 120 cccgataaaa agcagatgct cgaacggatt cagcacgcag tgcagcttgc cacagagcca 180 cttgagaaag ctgtgcagtc cagactcacg ggagaggaag tcaacagctg tgtggaggtg 240 ttgctggaag aagccaagga tttgctctct gactggctgg attctacact tggctgtgat 300 gtcactgaca attccatctt ctccaagctg cccaagttct gggaggggga cttccacaga 360 gacatggaa 369 <210> 9 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> CRS (101-200) polynucleotide <400> 9 aagatcatca agagggcccg gcagaaccac ctgttcgagc agtatcggga gaagaggcct 60 gaagcggcac agctcttgga ggatgttcag gccgccctga agccattttc agtaaaatta 120 aatgagacca cggatcccga taaaaagcag atgctcgaac ggattcagca cgcagtgcag 180 cttgccacag agccacttga gaaagctgtg cagtccagac tcacgggaga ggaagtcaac 240 agctgtgtgg aggtgttgct ggaagaagcc aaggatttgc tctctgactg gctggattct 300 300 <210> 10 <211> 246 <212> DNA <213> Artificial Sequence <220> <223> CRS (119-200) polynucleotide <400> 10 aggcctgaag cggcacagct cttggaggat gttcaggccg ccctgaagcc attttcagta 60 aaattaaatg agaccacgga tcccgataaa aagcagatgc tcgaacggat tcagcacgca 120 gtgcagcttg ccacagagcc acttgagaaa gctgtgcagt ccagactcac gggagaggaa 180 gtcaacagct gtgtggaggt gttgctggaa gaagccaagg atttgctctc tgactggctg 240 gattct 246

Claims (19)

A peptide comprising consecutive amino acids from any one selected from the 101 to 119 amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228 amino acid sequence; Or a peptide comprising an amino acid sequence exhibiting 80% or more homology with the peptide.
A peptide consisting of consecutive amino acids from any one selected from the 101 to 119th amino acid in the amino acid sequence of SEQ ID NO: 1 to any one selected from the 180 to 228th amino acid; Or a peptide consisting of an amino acid sequence showing 80% or more homology to the peptide.
The peptide according to claim 1, wherein the peptide comprises SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 7 or an amino acid sequence having 80% or more homology thereto.
The peptide according to claim 1, wherein the peptide activates innate immunity and adaptive immunity.
A polynucleotide comprising a nucleotide sequence encoding the peptide of claim 1.
The polynucleotide according to claim 5, wherein the polynucleotide is selected from the group consisting of SEQ ID NOs: 8 to 10.
A vector comprising the polynucleotide of claim 5.
A host cell transformed with the vector of claim 7.
A vaccine adjuvant comprising at least one selected from the group consisting of the following (i) to (v).
(i) the peptide of claim 1,
(ii) a polynucleotide encoding the (i),
(iii) a vector comprising (ii) above,
(iv) a host cell transformed with (iii) above, and
(v) cysteinyl-tRNA synthetase (CRS).
A vaccine composition comprising the vaccine adjuvant and antigen of claim 9.
The method of claim 10, wherein the antigen is alpha fetal protein, oncogenic embryonic antigen, cdk4, beta-catenin, CA125, caspase-8, epithelial tumor antigen, HPV antigen, HPV16 antigen, CTL epitope derived from HPV16 E7 antigen, black 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, carcinoembryonic antigen (CEA), 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-related antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, cancer embryonic antigen (CEA), melanoma-related antigen E (MAGE-A, MAGE1, MAGE2, MAGE3, MAGE4), interleukin-13 receptor alpha (IL13-R alpha), carbonic anhydrase IX ( CAIX), survivin, GP100, angiogenic antigen, ras protein, p53 protein, p97 melanoma antigen, KLH antigen, cancer embryonic antigen (CEA), gp100, MART1 antigen, TRP-2, HSP-70, beta-HCG , Testicin, 1A01 -HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4; Alpha-actinine-_4/m; Alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl; Beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; Calreticulin; CAMEL; CASPA; Caspase_8; Cathepsin_B; Cadepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD4; CD44_Isomorph_1; CD44_Isomorph_6; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30; 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_Isomorph_LAGE-1A; CTAG2_Isomorph_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; Fibronectin; FOS; 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; IL2-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-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16; MAGE-B17; MAGE-B2; MAGE-B3; MAGE-B4; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; MAGE-C3; MAGE-D1; MAGE-D2; MAGE-D4; MAGE-E1_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H1; MAGEL2; Mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF; Mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; N-myc; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9; Osteocalcin; Osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3; PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-kinase; Pin-1; PLAC1; PMEL; PML; POTE; POTEF; PRAME; PRDX5/m; PRM2; Prostein; Proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA; RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; 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; STEAP-1; Survivin; Survivin-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, alpha-actinin-4; ARTC1; BCR-ABL fusion protein (b3a2); B-RAF; CASP-5; CASP-8; Beta-catenin; Cdc27; CDK4; CDKN2A; COA-1; dek-can fusion protein; EFTUD2; Elongation factor 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-RARalpha fusion protein; PRDX5; PTPRK; K-ras; N-ras; RBAF600; SIRT2; SNRPD1; SYT-SSX1 or SSX2 fusion protein; Triosephosphate 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; 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; CyClean D1; Ep-CAM; EphA3; FGF5; G250 / MN / CAIX; HER-2/neu; IL13Ralpha2; Intestinal carboxyl esterase; Alpha-foetoprotein; 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, PAP. Vaccine composition, characterized in that at least one selected from the group consisting of OVA (ovalbumin) and MART-1.
The vaccine composition according to claim 10 or 11, wherein the vaccine is an anti-cancer vaccine.
The vaccine composition of claim 12, wherein the anticancer vaccine is a vaccine for preventing cancer or a vaccine for treating cancer.
The method of claim 12, wherein the cancer is breast cancer, colon cancer, prostate cancer, cervical cancer, gastric cancer, skin cancer, head and neck cancer, lung cancer, glioblastoma, oral cancer, pituitary adenoma, glioma, brain tumor, upper pharyngeal cancer, laryngeal cancer, thymoma, mesothelioma , Esophageal cancer, rectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureter cancer, renal cell cancer, bladder cancer, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, childhood cancer, bronchial cancer , A vaccine composition, characterized in that at least one selected from the group consisting of colon cancer and ovarian cancer.
The vaccine composition of claim 12, further comprising at least one selected from the group consisting of a vaccine adjuvant, an immune checkpoint inhibitor, and a combination thereof.
The method of claim 15, wherein the vaccine adjuvant is 1018 ISS, aluminum salt, amplivax, AS15, BCG, CP-870,893, CpG ODN, CpG7909, SaiA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, Imupact IMP321, IS Patch, Iscomatrices, Juv Immun, Lipobag, MF59, Monophosphoryl Lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51 , OK-432, OM-174, OM-197-MP-EC, Ontag, Peptel Vector System, PLG microparticles, Reciquimod, SRL172, Virosome and other virus-like particles, YF-17DBCG, Aquilas QS21 Stimulon, Libis detox quill, Superforce, Freunds, GM-CSF, cholera toxin, immunological adjuvant, MF59, and a vaccine composition, characterized in that at least one selected from the group consisting of cytokines.
The method of claim 15, wherein the immune checkpoint inhibitor is a PD-1 (programmed cell death-1) antagonist, a PD-L1 (programmed cell death-ligand 1) antagonist, a PD-L2 (programmed cell death-ligand 2) antagonist, CD27. (cluster of differentiation 27) antagonist, CD28 (cluster of differentiation 28) antagonist, CD70 (cluster of differentiation 70) antagonist, CD80 (cluster of differentiation 80, also known as B7-1) antagonist, CD86 (cluster of differentiation 86, also Known as B7-2) antagonist, CD137 (cluster of differentiation 137) antagonist, CD276 (cluster of differentiation 276) antagonist, KIRs (killer-cell immunoglobulin-like 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 antign-4) antagonist, A2AR (Adenosine A2A receptor) antagonist, VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, BTLA (B- and T-lymphocyt) e attenuator) antagonist, IDO (Indoleamine 2,3-dioxygenase) antagonist, TIM-3 (T-cell Immunoglobulin domain and Mucindomain 3) antagonist, VISTA (V-domain Ig suppressorof T cell activation) antagonist and KLRA (killer cell lectin- like receptor subfamilyA) vaccine composition, characterized in that at least one selected from the group consisting of antagonists.
11. The vaccine composition according to claim 10, wherein the cysteinyl TIR synthase is composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more homology thereto.
A pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of the following (i) to (v):
(i) the peptide of claim 1,
(ii) a polynucleotide encoding the (i),
(iii) a vector comprising (ii) above,
(iv) a host cell transformed with (iii) above, and
(v) cysteinyl-tRNA synthetase (CRS).

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