WO2023003380A1 - Novel cell-penetrating peptide and use thereof - Google Patents

Novel cell-penetrating peptide and use thereof Download PDF

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WO2023003380A1
WO2023003380A1 PCT/KR2022/010681 KR2022010681W WO2023003380A1 WO 2023003380 A1 WO2023003380 A1 WO 2023003380A1 KR 2022010681 W KR2022010681 W KR 2022010681W WO 2023003380 A1 WO2023003380 A1 WO 2023003380A1
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peptide
endoplasmic reticulum
seq
delivery
linked
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PCT/KR2022/010681
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French (fr)
Korean (ko)
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조인호
박정현
신재민
박혜진
맹재열
김민석
장지연
장재근
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주식회사 에이조스바이오
이화여자대학교 산학협력단
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Publication of WO2023003380A1 publication Critical patent/WO2023003380A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to novel cell penetrating peptides and uses thereof.
  • CPPs cell penetrating peptides
  • CPP cell penetrating peptides
  • a delivery system for intracellular drug delivery is cell penetrating peptides composed of about 10 to 30 short peptides, and are mostly derived from protein-transduction domains or membrane translocating sequences.
  • CPP is an oligopeptide capable of undergoing endocytosis or directly penetrating the cell membrane in response to the cell membrane, and has electrochemical and physicochemical properties capable of penetrating the cell membrane.
  • Such CPP is well known in the field of pharmacology as a study related to application methods to drug delivery systems.
  • a lot of research has been conducted and used as a tool for regulating gene expression by being applied in a specific biological system.
  • endoplasmic reticulum (ER) target sequences are largely unknown.
  • ER endoplasmic reticulum
  • the endoplasmic reticulum is one of the organelles found in all eukaryotic cells, and consists of a net shape of tubules, vesicles, and cisterna.
  • the endoplasmic reticulum makes proteins and transports them throughout the cell.
  • the basic structure and composition of the endoplasmic reticulum is similar to that of a general cell membrane, although it is an extension of the nuclear membrane (Endomembrane System).
  • the endoplasmic reticulum is where the synthesis of proteins to be secreted out of the cell, the formation of secondary structures, and transport of proteins, including proteins to be part of the cell membrane, take place.
  • the lumen of the endoplasmic reticulum is a specialized cellular environment for post-translational modification and protein folding.
  • About 1/3 of intracellular proteins undergo post-translational modification from mRNA to protein in the rough endoplasmic reticulum, that is, folding and assembly, glycosylation, disulfide bonds, etc. This process results in a protein structure that assumes an active form.
  • the smooth endoplasmic reticulum is a synthesis site of lipids and steroid hormones and plays an important role in regulating intracellular calcium concentration as a calcium store.
  • the endoplasmic reticulum performs many roles, including facilitating protein processing and transporting the storage sacs of produced proteins called cisternae.
  • the formation of secondary and tertiary structures of newly created primary proteins is carried out by endoplasmic reticulum proteins such as protein disulfide isomerase (PDI), Hsc70 family, calnexin, calreticulin or peptidylpropyl isomerase family. Then, only correctly formed proteins can be transported from the rough endoplasmic reticulum to the Golgi apparatus.
  • PDI protein disulfide isomerase
  • This endoplasmic reticulum stress response is particularly well observed in plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts, which are active in synthesizing and secreting proteins. It has been shown to act as the etiology of various diseases such as hyperhomocysteinemia and mutation.
  • the present inventors have made diligent efforts to develop novel endoplasmic reticulum target cell penetrating peptides.
  • the present invention was completed by developing novel fusion polypeptides and confirming that these sequences can enter the endoplasmic reticulum and induce various biological function changes to exhibit therapeutic effects.
  • the present invention provides cell penetrating peptides and uses thereof.
  • the present invention provides a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • the present invention provides a composition for intracellular endoplasmic reticulum-targeting peptide delivery comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum delivery peptide.
  • the present invention provides a pharmaceutical use comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • One aspect of the present invention for achieving the above object provides a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • peptide refers to a molecule formed by binding amino acid residues to each other via an amide bond (or a peptide bond).
  • the peptide may be synthesized using genetic recombination and a protein expression system, preferably synthesized in vitro through a peptide synthesizer or the like.
  • the fusion polypeptide of the present invention includes a derivative thereof, in which an amino acid fragment or part of the peptide is substituted or deleted, or a part of the amino acid sequence is modified into a structure capable of increasing stability in vivo, or to increase hydrophilicity It may be fragments in which part of the amino acid sequence is modified, part or all of the amino acids are substituted with L- or D-amino acids, or part of the amino acids is modified.
  • the fusion polypeptide or a derivative thereof has cell permeability and intracellular permeability, and in particular, has delivery capability to the endoplasmic reticulum.
  • cell permeability refers to the ability or property of a peptide to permeate a cell membrane and penetrate into a cell.
  • targeting the endoplasmic reticulum' of the present invention means that a fusion polypeptide is delivered to the endoplasmic reticulum within a cell.
  • the endoplasmic reticulum target cell penetrating peptide according to the present invention preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the cell penetrating peptide may be composed of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. At this time, at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, 96%, 97%, 98%, 99% of the amino acid sequence represented by SEQ ID NO: 1 or 2 It may include an amino acid sequence having a sequence homology of % or more.
  • a polypeptide having a sequence different from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 according to the present invention by at least one amino acid residue may be included.
  • Amino acid exchanges in proteins and polypeptides that do not entirely alter the activity of the molecule are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • peptides with increased structural stability against heat, pH, etc. or increased cell permeability may be included by mutation or modification in the amino acid sequence.
  • the peptide sequence according to the present invention is a polypeptide sequence whose cell permeability is confirmed through cell permeability analysis and has the ability to target the endoplasmic reticulum.
  • a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide is specifically, an endoplasmic reticulum target cell penetrating peptide; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • the connection of the peptide for endoplasmic reticulum delivery can be performed at either the N-terminus or the C-terminus of the endoplasmic reticulum target cell-penetrating peptide.
  • the expression "linked thereto" means linkage and/or binding of the peptide sequence to the N-terminus or C-terminus.
  • the connection includes both direct bonding and connection through a linker or spacer.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is an endoplasmic reticulum target cell penetrating peptide; and a endoplasmic reticulum delivery peptide linked to its N-terminus.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide may include an endoplasmic reticulum target cell penetrating peptide; and a endoplasmic reticulum delivery peptide linked to its C-terminus.
  • peptide for endoplasmic reticulum delivery refers to any peptide intended for delivery to the endoplasmic reticulum and exhibiting a desired activity after being delivered to the endoplasmic reticulum.
  • Any peptide that can exhibit pharmacological activity by regulating its activity in the endoplasmic reticulum after delivery into cells may be included in the scope of the present invention.
  • it may include, but is not limited to, immunogenic peptides, antigens, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors.
  • Peptides for endoplasmic reticulum delivery according to the present invention are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 It may have any number of amino acids within 1, 40, or 50.
  • peptides having 1 to 30, 2 to 25, and 3 to 20 amino acids are preferred.
  • an "immunogenic or immunostimulatory peptide” is a composition capable of generating an immune response in an animal when administered to said animal.
  • An immunogenic peptide is a composition that contains an antigen and is capable of generating an immune response.
  • the immune response generated may be a cellular (T-cell mediated) or humoral (B-cell mediated, antibody producing) immune response.
  • Immunogenic peptides are also capable of inducing both cellular and humoral immune responses.
  • the cellular immune response can be a CD8+ T lymphocyte mediated response (ie a cytotoxic response), or a CD4+ T lymphocyte mediated response (helper response). It is also possible to combine cytotoxic and helper cell immune responses.
  • the helper response may include Th1, Th2 or Th17 lymphocytes (such lymphocytes are capable of eliciting different cytokine responses as is known in the art). Immunogenic peptides may allow for better presentation of antigens present therein via the MHC1 or MHC2 pathway.
  • an “antigen” is a molecule or combination of molecules that seeks to elicit an immune response in order to allow the immune system of a living animal to recognize it. These antigens may be heterologous in the body of the host to which the immune response is added. In this case, the antigen may be a protein expressed by bacteria or viruses. Antigens can also be self antigens, ie proteins expressed by the host's cells, such as tumor antigens.
  • An antigen can be a complete protein or any part of a protein, such as an epitope of a protein.
  • Antigens in the context of the present invention may also consist of synthetic proteins or molecules containing multiple epitopes linked together.
  • the antigen may be a protein containing multiple epitopes of the same antigen, these epitopes being specific for the MHC haplotype.
  • a unique immune stimulatory molecule as described herein can be used to obtain an immune response in a different genetic (MHC) context.
  • Antigenic peptides used as immunogenic peptides contain one or several MHC epitopes.
  • An antigen as used in an immunogenic peptide, is flanked at its N and/or C terminus by several amino acids (1 to 10, preferably 1 to 6 amino acids at one or both C and N termini). It consists of MHC epitopes located on
  • MHC epitopes are presented on the surface of antigen-presenting cells bound to MHC molecules.
  • T cell epitopes presented by MHC class I molecules are peptides typically 8-11 amino acids in length, while MHC class II molecules present longer peptides 13-17 amino acids in length.
  • MHC epitopes can be synthesized in vitro (with or without added amino acids at their C and/or N termini). MHC binding peptides can be extracted from viable cells, particularly tumor cells, by any method known in the art, such as acid treatment, particularly with hydrochloric acid.
  • the antigen comprises one or several B cell epitopes, ie the portion of a protein recognized by an antibody, preferably a linear epitope, formed by a contiguous sequence of amino acids from the antigen.
  • the immunogenic peptide may include at least one epitope selected from the group consisting of an MHC epitope and a B-cell epitope as an antigen.
  • MHC epitopes and B-cell epitopes can be sufficiently derived by those skilled in the art based on known sequences, and the positions and sequences of these epitopes are included within the scope of the present invention.
  • antigens as used in immunogenic peptides consist of B cell epitopes.
  • the antigen, as used in the immunogenic peptide is modified by several amino acids (from 1 to 10, preferably 1 to 6 amino acids at the C and N termini of one or both of them) to their N and / or C-terminally flanked by B cell epitopes.
  • Other methods in the literature on epitope mapping make it possible to identify T cell or B cell epitopes from a given antigen.
  • an MHC class I antigenic peptide (SEQ ID NO: 3: SIINFEKL) derived from Ovalbumin (OVA) was used as an example of the immunogenic peptide (immunogenic epitope).
  • the peptide for endoplasmic reticulum delivery may be directly connected to the cell-penetrating peptide, or may be connected through a linker or spacer peptide.
  • linker refers to a short amino acid sequence used to separate two peptides with different functions in constructing a fusion protein.
  • the absence of a linker between two or more individual domains in a protein can result in reduced or inappropriate function of the protein domains due to steric hindrance, such as reduced catalytic activity or binding affinity for a receptor/ligand.
  • the spacing between the domains can be increased by linking the protein domains in the chimeric protein using artificial linkers.
  • the linker or spacer is not particularly limited as long as it exhibits an effect of enhancing the activity of the conjugate of the cell-permeable peptide and the endoplasmic reticulum delivery peptide.
  • the constituent amino acids affect some property of the molecule, such as folding, net charge, or hydrophobicity. can be selected to
  • a sequence cleavable with a protease may be included as a linker.
  • the protease cleavage site can be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site.
  • the present invention it is preferable to cut through the sequence including the sequence recognized by the signal peptidase enzyme. That is, a sequence that can be recognized by the signal peptidase enzyme can be added between the cell-permeable peptide and the drug target peptide sequence.
  • signal peptidase cleavage peptide refers to a linker sequence that is recognized by signal peptidase and can provide cleavage between the endoplasmic reticulum delivery peptide and the endoplasmic reticulum target cell-permeable peptide sequence.
  • the "signal peptidase cleavage peptide” can recognize the C-terminus of the cell-permeable peptide sequence and the stomach cleavage peptide sequence to provide peptide cleavage between them.
  • the cell-permeable peptide sequence according to the present invention is derived from a signal sequence
  • "AXA (SEQ ID NO: 5)” or “VXA (SEQ ID NO: 6)” (where X is any amino acid sequence) is added at the C-terminus.
  • the signal peptidase enzyme recognizes the cleavage position between the above "AXA” or “VXA” and “EA” sequences and cleaves them.
  • the "EA” sequence may be preferably used as a linker sequence.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide according to the present invention is specifically, the endoplasmic reticulum target cell penetrating peptide; Linker or spacer peptides linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is specifically, the endoplasmic reticulum target cell penetrating peptide; a signal peptidase cleavage peptide linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; and an immunogenic peptide linked thereto That is, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or 2; Following the signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto, any peptide capable of exhibiting immunogenicity may be conjugated.
  • An example of an endoplasmic reticulum target penetrating peptide sequence to which an immunogenic peptide can be linked and a sequence in which a cleavage peptide is linked are as follows:
  • sequences used in experiments in which immunogenic peptides are bound are listed as follows. Immunogenic peptides are easy to vary depending on the purpose and are very diverse, so the examples of such immunogenic peptide sequences are not intended to limit the scope of the present invention.
  • immunogenic peptide sequence SIINFEKL is only one experimental example for confirming the action of the ER-CPP sequence according to the present invention, and the immunogenic peptide is not limited thereto, and any known immunogenic peptide may be used.
  • Polypeptides according to the present invention can be prepared using available techniques known in the art. Polypeptides can be synthesized using any suitable procedure known to those skilled in the art, ie known polypeptide synthesis methods (eg genetic engineering methods, chemical synthesis).
  • the polypeptide according to the present invention can be produced by recombinant techniques according to genetic engineering methods.
  • nucleic acids polynucleotides
  • the nucleic acid can be prepared by PCR amplification using appropriate primers.
  • DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer.
  • the constructed nucleic acid is operably linked thereto and inserted into a vector containing one or more expression control sequences (eg, promoter, enhancer, etc.) that regulates the expression of the nucleic acid to construct a recombinant expression vector,
  • expression control sequences eg, promoter, enhancer, etc.
  • the cell is cultured in a medium and under conditions suitable for expression of the desired polypeptide, and substantially pure polypeptide expressed from the nucleic acid is recovered from the culture.
  • the recovery may be performed using a method known in the art.
  • extraction, recrystallization, various chromatographies (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc. are separated by methods known in the art. and can be purified.
  • 'substantially pure polypeptide' means that the polypeptide according to the present invention does not substantially contain any other proteins derived from host cells.
  • polypeptides according to the present invention can be prepared by chemical synthetic methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
  • the polypeptide of the present invention can be prepared by direct peptide synthesis using solid phase techniques.
  • SPPS solid-phase peptide synthesis
  • synthesis can be initiated by attaching functional units, called linkers, to small porous beads to guide the peptide chain.
  • linkers functional units
  • the peptide is covalently bound to the beads and prevents them from being separated by filtration until they are cleaved by a specific reactant, such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • a protection process in which the N-terminal amine of a peptide attached to a solid phase is combined with an N-protected amino acid unit, a deprotection process, a re-exposed amine group and a new Synthesis is performed by repeating a cycle (deprotection-wash-coupling-wash) of a coupling process in which amino acids are combined.
  • the SPPS method can be performed using microwave technology together, and microwave technology can shorten the time required for coupling and deprotection of each cycle by applying heat in the peptide synthesis process.
  • the thermal energy may prevent folding or aggregation of the extended peptide chain and promote chemical bonding.
  • the present invention provides a polynucleotide encoding the fusion polypeptide.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is specifically, the endoplasmic reticulum target cell penetrating peptide; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is specifically, the endoplasmic reticulum target cell penetrating peptide; a signal peptidase cleavage peptide linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • the polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases.
  • a synthesis method well known in the art for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other auto-primer methods, and oligonucleotide synthesis methods on solid supports.
  • the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the fusion polypeptide using the transformant.
  • expression vector of the present invention is a recombinant vector capable of expressing a desired peptide in a desired host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert.
  • the expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator.
  • the start codon and stop codon are generally regarded as part of a nucleotide sequence encoding a polypeptide, and when the gene construct is administered, the subject must be functional and must be in frame with the coding sequence.
  • the vector's promoter may be constitutive or inducible.
  • operably linked means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence.
  • Operational linkage with the expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
  • the expression vector may include a signal sequence for excretion of the polypeptide in order to promote protein separation from the cell culture medium.
  • a specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences.
  • an exogenous translation control signal which may include an ATG initiation codon, must be provided. These exogenous translation control signals and initiation codons can be from a variety of natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcriptional or translational enhancers.
  • the expression vector may further include a protein tag that can be optionally removed using endopeptiase to facilitate detection of the fusion polypeptide according to the present invention.
  • the term "tag” refers to a molecule exhibiting a quantifiable activity or characteristic, and is a chemical fluorophore such as fluorescein, a polypeptide fluorophore such as a fluorescent protein (GFP) or a related protein. It may be a fluorescent molecule including; It may also be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a streptavidin tag. In particular, when an epitope tag is used, a peptide tag preferably composed of 6 or more amino acid residues, and more preferably composed of 8 to 50 amino acid residues may be used.
  • the expression vector may include a nucleotide sequence encoding the fusion polypeptide of the present invention described above, but the vector used in this case is not particularly limited thereto as long as it can produce the fusion polypeptide of the present invention , preferably plasmid DNA, phage DNA, etc., more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E.
  • coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), animal virus vector (retrovirus (retrovirus), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the expression vector shows different protein expression levels and modifications depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.
  • the transformant provided in the present invention can be prepared by introducing the expression vector provided in the present invention into a host and transforming it, and expressing the polynucleotide contained in the expression vector to produce the fusion polypeptide of the present invention.
  • the transformation may be performed by various methods, but is not particularly limited thereto as long as the fusion polypeptide of the present invention can be produced, but a CaCl 2 precipitation method and a reducing material called DMSO (dimethyl sulfoxide) are used in the CaCl 2 precipitation method.
  • Hanahan method with increased efficiency by using electroporation, calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fibers, agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate , lipofectamine and desiccation/inhibition mediated transformation methods and the like can be used.
  • the host used for the production of the transformant can also produce the cell-permeable peptide of the present invention, it is not particularly limited thereto, but E.
  • coli Streptomyces, bacterial cells such as Salmonella typhimurium ; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or plant cells.
  • compositions for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide.
  • a composition for delivering an endoplasmic reticulum-targeting peptide comprising the endoplasmic reticulum-targeting cell-penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2 is provided.
  • endoplasmic reticulum target cell penetrating peptides; and a linker or spacer peptide linked thereto.
  • endoplasmic reticulum target cell penetrating peptides More specifically, endoplasmic reticulum target cell penetrating peptides; and a signal peptidase cleavage peptide linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and a signal peptidase cleavage peptide linked thereto.
  • composition for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum-targeting peptide linked thereto.
  • compositions for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum-targeting peptide linked to its N-terminus.
  • composition for delivery of an endoplasmic reticulum targeting peptide comprising an endoplasmic reticulum target cell penetrating peptide and an endoplasmic reticulum delivery peptide linked to its C-terminus.
  • endoplasmic reticulum target means that a substance can be delivered targeting the endoplasmic reticulum, the endoplasmic reticulum lumen, and/or the inside of the endoplasmic reticulum present in a cell.
  • composition for intracellular endoplasmic reticulum-targeting peptide delivery can directly deliver the endoplasmic reticulum-targeting peptide linked to the endoplasmic reticulum target cell-penetrating peptide or via a linker or spacer into the endoplasmic reticulum.
  • the endoplasmic reticulum target cell penetrating peptide comprising an endoplasmic reticulum delivery peptide linked thereto.
  • endoplasmic reticulum target cell penetrating peptides a signal peptidase cleavage peptide linked thereto; and an endoplasmic reticulum-targeted peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
  • endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; and an endoplasmic reticulum-targeted peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
  • composition for delivery of an endoplasmic reticulum-targeting peptide can deliver various functional effects by regulating protein expression in the endoplasmic reticulum by delivering the "ER-targeting peptide" intended for delivery to the endoplasmic reticulum.
  • ER-targeting peptide intended for delivery to the endoplasmic reticulum.
  • membrane proteins secreted from cells or distributed in the cell inner membrane must move to the lumen of the endoplasmic reticulum (ER) and undergo proper folding and processing.
  • chaperone proteins such as protein disulfide isomerase (PDI), heavy chain binding protein (BiP), calnexin (CNX), and calreticulin (CRT) exist to help proteins biosynthesized in the endoplasmic reticulum to be properly folded and processed.
  • PDI protein disulfide isomerase
  • BiP heavy chain binding protein
  • CNX calnexin
  • CRT calreticulin
  • endoplasmic reticulum target cell penetrating peptides and the signal peptidase cleavage peptide linked thereto can show excellent effects by linking various peptides for endoplasmic reticulum delivery to deliver only the target peptide into the endoplasmic reticulum.
  • an endoplasmic reticulum target cell penetrating peptide is linked to an endoplasmic reticulum delivery peptide via a signal peptidase cleavage peptide, so that the endoplasmic reticulum target cell penetrating peptide transfers the endoplasmic reticulum delivery peptide to the endoplasmic reticulum, and then the endoplasmic reticulum cleavage sequence results in the transfer of the endoplasmic reticulum to the endoplasmic reticulum.
  • the peptide is cleaved, allowing the desired endoplasmic reticulum delivery peptide to be delivered into the endoplasmic reticulum.
  • Peptides according to the present invention are preferably used in mammals. Such mammals are preferably humans, but other mammals when the composition is used in the veterinary field, in particular for inducing immunity in livestock such as cattle (dairy cows), sheep, goats or horses, as well as pets such as dogs or cats. It could be.
  • the present invention provides a vaccine composition
  • a vaccine composition comprising an endoplasmic reticulum target cell penetrating peptide and an endoplasmic reticulum delivery peptide.
  • a vaccine is a composition administered to create or artificially increase immunity against a specific antigen.
  • a vaccine composition comprising an endoplasmic reticulum target cell penetrating peptide and an immunogenic peptide is provided. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and an immunogenic peptide linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And the immunogenic peptide linked thereto may be connected by a linker or spacer peptide. More specifically, this linker or spacer peptide may be a signal peptidase cleavage peptide of SEQ ID NO: 4.
  • the salpin fusion polypeptide and the embodiment of the salpin specific peptide configuration in the composition for delivering the endoplasmic reticulum-targeting peptide can also be applied to the present vaccine composition.
  • a composition according to the present invention is a composition capable of generating an immune response in an animal.
  • the immune response generated may be a cellular (T-cell mediated) or humoral (B-cell mediated, antibody producing) immune response.
  • Immunogenic compositions are also capable of inducing both cellular and humoral immune responses.
  • the cellular immune response can be a CD8+ T lymphocyte mediated response (ie a cytotoxic response), or a CD4+ T lymphocyte mediated response (helper response). It is also possible to combine cytotoxic and helper cell immune responses. Helper responses can include Th1, Th2 or Th17 lymphocytes (such lymphocytes can elicit different cytokine responses as is known in the art). The composition may allow for better presentation of antigens present therein via the MHC1 or MHC2 pathway.
  • the vaccine composition of the present invention requires the presentation of appropriate antigens as immunogenic peptides, and these antigens can be considered as follows.
  • antigens that enter the body from outside, eg by inhalation, ingestion or injection; these antigens are usually presented by MHC II molecules.
  • antigens produced within normal cells as a result of normal cellular metabolism or due to viral or intracellular bacterial infection; these antigens are usually presented by MHC I molecules.
  • tumor antigens such as epitopes derived from viral open reading frames in virus-associated tumors, or other tumor antigens presented by MHC I or MHC II molecules on the surface of tumor cells.
  • Allergens (antigens that can stimulate type 1 hypersensitivity reactions in atopic individuals via an immunoglobulin E (IgE) response).
  • IgE immunoglobulin E
  • tumor antigens examples include alphafetoprotein (AFP) found in germ cell tumors and hepatocellular carcinoma, carcino embryonic antigen (CEA) found in intestinal cancer, CA-125 found in ovarian cancer, and breast cancer.
  • MUC-1 found in breast cancer
  • epithelial tumor antigen (ETA) found in breast cancer
  • tyrosinase found in malignant melanoma
  • MAGE melanoma-associated antigen
  • p53 found in various tumors
  • gp100 melanocyte protein PMEL, melanosome-rich type I transmembrane glycoprotein
  • TRP2 tyrosinase-related protein 2: Tyrosinase-Related Protein 2
  • EPHA2 receptor tyrosine kinase, Frequently overexpressed in a wide range of advanced cancers such as glioma
  • survivin a baculovirus inhibitor of apoptosis repeat-containing 5 or
  • pathogens whose antigens can be used in the immunogenic composition, mention may be made of any pathogen associated with infectious diseases (viruses, bacteria, parasites, mycoses).
  • preferred pathogens include human immune deficiency virus (HIV), hepatitis A and B viruses, hepatitis C virus (HCV), Rous sarcoma virus (RSV). ), Ebola virus, cytomegalovirus, herpes virus, varicella zoster virus, Epstein Barr virus (EBV), influenza virus, adenovirus, rotavirus, measles and rubella virus, smallpox virus, staphylococcus ( Staphylococcus ), Chlamydiae ( Chlamydiae ), Mycobacterium tuberculosis ( Mycobacterium tuberculosis ), Streptococcus pneumoniae ( Streptococcus pneumoniae ), Bacillus anthracis ( Bacillus anthracis ), Vibrio cholerae ( Vibrio cholerae ), Helicobacter pylorii ( Helicobacter Pilorii ), Salmonella ( Salmonella ), Plasmodium sp
  • Plasmodium sp . P. falciparum ( P. falciparum ), P. Bibox ( P. vivax ), etc.
  • Pneumocystis carinii Pneumocystis carinii )
  • Giardia duodenalis duodenalis Giardiose
  • Schistosoma Schistosoma
  • Bilharziose Aspergillus ( Aspergillus ), Cryptococcus ( Cryptococcus ), Candida albicans ( Candida albicans ), Listeria mono Cytogenes ( Listeria monocytogenes ), or Toxoplasma gondii .
  • cancer benign or malignant tumors
  • Chronic diseases such as blood cancer, allergies, autoimmune diseases, atherosclerosis or Alzheimer's disease.
  • Antigens are thus preferably bacterial or viral antigens (or peptides containing one or more epitopes isolated from bacterial or viral antigens). Antigens are self antigens (endogenous or neoantigens), especially tumor specific antigens (or isolated from such antigens). An antigen is a peptide containing one or more epitopes isolated from such an antigen.
  • the immunogenic peptide may contain at least one epitope selected from the group consisting of an MHC epitope and a B-cell epitope as an antigen.
  • any of the antigens mentioned above can be used as immunogenic peptides according to the present invention.
  • the SARS-CoV-2 MHC I epitope sequence is KLWAQCVQL (SEQ ID NO: 21), SPRWYFYYL (SEQ ID NO: 22), TTDPSFLGRY (SEQ ID NO: 23), SPRWYFYYL (SEQ ID NO: 24), etc. can be considered, but is not limited thereto.
  • SFQDILLRM (SEQ ID NO: 25), VTVTHSVNL (SEQ ID NO: 26), YQNIHPVTI (SEQ ID NO: 27), etc. may be considered as an influenza virus MHC I epitope sequence as an example of an immunogenic peptide, but is not limited thereto.
  • the KRAS mutant cancer MHC I epitope sequence may be VVVGAVGVGK (SEQ ID NO: 28), but is not limited thereto.
  • an MHC class I antigenic peptide (SEQ ID NO: 3: SIINFEKL) derived from Ovalbumin (OVA) was used as an example of an immunogenic peptide (immunogenic epitope). Through this, it was confirmed that it exhibited an excellent MHC1 presentation action.
  • Vaccines may be prophylactic (ie, intended to protect the recipient against the development of a disease) or therapeutic (ie, intended to help the recipient fight a pre-existing disease) vaccine.
  • a disease thus involves or relates to a cell that expresses a target antigen.
  • Such expression may include secretion of an antigen (eg, the antigen may be a bacterial toxin) or surface expression of an antigen or epitope thereof (an antigen may be a surface protein of a virus or a tumor-specific antigen or epitope thereof expressed on the surface of a tumor cell). may), or presentation of an antigen or epitope thereof on a cell surface (eg, MHC presentation of an antigen or epitope thereof by a target cell).
  • composition of the present invention may further include an adjuvant.
  • an “adjuvant” is a substance having the ability to modify or enhance an immune response to an antigen.
  • the immune response to the antigen may be higher or different in the presence of adjuvant than in the absence of adjuvant (when the response is modified, e.g., subtypes of T cells activated in the presence of adjuvant) (including when the set differs from the subset activated in the absence of adjuvant).
  • adjuvants are known in the art and have been widely used in the field of vaccines.
  • Alum emulsions (oil-in-water or water-in-oil, such as Freund's incomplete adjuvant (IFA) and MF59®), PRR (pattern recognition receptor) ligands, TLR3 (tall-like receptor 3) and RLR (RIG- I-like receptor) ligands such as double-stranded RNA (dsRNA), or synthetic analogs of dsRNA, such as poly(I:C), TLR4 ligands such as bacterial lipopolysaccharide (LPS), MPLA (monophosphoryl lipid A), especially alum Formulated with, TLR5 ligands such as bacterial flagellins, TLR7/8 ligands such as imidazoquinolines (i.e.
  • TLR9 ligands such as oligodeoxynucleotides containing specific CpG motifs (CpG ODN ) or NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) ligand.
  • CpG ODN CpG ODN
  • NOD2 Nucleotide-binding oligomerization domain-containing protein 2
  • ligand preferably describes an agonist of a receptor, i.e. a substance that binds to and activates the receptor, especially for the TLR3 and TLR9 receptors.
  • endoplasmic reticulum stress occurs due to changes in various cellular environments, cells try to relieve it through various pathways, and when the stress is prolonged, cells suffer fatal damage and cause apoptosis.
  • This endoplasmic reticulum stress response is particularly well observed in plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts, which are active in synthesizing and secreting proteins. It has been shown to act as the etiology of various diseases such as hyperhomocysteinemia and mutation.
  • the present invention is an endoplasmic reticulum target cell penetrating peptide; And it provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a peptide for endoplasmic reticulum delivery linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And it provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a peptide for endoplasmic reticulum delivery linked thereto.
  • the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And the peptide for endoplasmic reticulum delivery linked thereto may be linked by a linker or spacer peptide. More specifically, this linker or spacer peptide may be a signal peptidase cleavage peptide of SEQ ID NO: 4.
  • the pharmaceutical composition comprising the endoplasmic reticulum target cell penetrating peptide and the peptide for endoplasmic reticulum delivery linked thereto according to the present invention can treat endoplasmic reticulum stress-related diseases by delivering an appropriate peptide for expression in the endoplasmic reticulum.
  • the endoplasmic reticulum stress-related diseases include type 1 diabetes, type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, ALS), haemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease ( Kreutzfeldt-Jakob disease), familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, Coron's disease, ulcerative colitis, breast and prostate cancer, and the like.
  • composition of the present invention can be used in various fields of medicine by specifically and efficiently delivering a desired peptide into the endoplasmic reticulum.
  • the desired peptide into the endoplasmic reticulum for treatment may be a peptide capable of exhibiting antioxidant action.
  • active oxygen is known to have various effects on cells, such as proliferation, growth, or apoptosis.
  • the half-life of active oxygen in vivo is very short, and therapeutic efficacy can be exhibited through its local adjustment.
  • targeted antioxidants it is possible to consider the development of targeted antioxidants to remove harmful reactive oxygen species generated at specific locations within cells, which can be used as an important therapeutic agent for various diseases caused by excessive reactive oxygen species generated over a long period of time.
  • Peptides having such an antioxidant effect are not limited thereto, but, for example, acetylcysteine (N-Acetylcysteine), glutathione (glutathione), SOD-like (SODmimicking) peptides and Zeto-Schiller-peptides (Szeto-Schiller-peptides) It may be any one selected from the group consisting of
  • DMTU N,N'-dimethylthiourea
  • PTIO 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide
  • ⁇ -tocopherol ⁇ -tocopherol
  • manganese(III)-tetrakis 4-benzoic Compound-based antioxidants such as acid)porphyrin (MnTBAP) can also be used together with endoplasmic reticulum-targeting cell-penetrating peptides.
  • the vaccine composition or pharmaceutical composition according to the present invention can be used in mammals, preferably humans, such as intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal , It is possible to deliver the desired peptide to the endoplasmic reticulum in vivo by injecting it through routes such as nasal, mucosal, inhalation, and oral.
  • treatment means suppression or alleviation of a disease or disorder.
  • therapeutically effective amount in the present invention means an amount sufficient to achieve the pharmacological effect.
  • the vaccine composition or pharmaceutical composition may be formulated and provided in an appropriate form.
  • the above formulations may be formulated into oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, etc., or parenteral formulations in the form of transdermal preparations, suppositories and sterile injection solutions, etc. It can be formulated and used.
  • the formulation may further contain adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients and diluents.
  • Carriers, excipients and diluents that may be included in the vaccine composition or pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used.
  • the formulation may include a carrier for formulation in addition to the vaccine composition or pharmaceutical composition (active ingredient).
  • the carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersing agent, a suspending agent, a colorant, a flavoring agent, and the like.
  • the composition may be administered alone, but in general, it may be administered in combination with a pharmaceutical carrier selected in consideration of the method of administration and standard pharmaceutical practice. there is.
  • topical administration such as solutions, gels, cleaning compositions, tablets for insertion, suppositories, creams, ointments, dressing solutions, sprays, and other coating agents.
  • It may be a liquid formulation such as a solution type, a suspension type, an emulsion type, and a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, a cream, an ointment, a jelly, a foam, a detergent or an insert, preferably External skin preparations such as liquid preparations, gel preparations, cleansing compositions, and tablets for insertion may be included.
  • the formulation may be prepared by adding, for example, a solubilizing agent, an emulsifying agent, a buffering agent for pH control, etc. to sterilized water.
  • a solubilizing agent for example, a solubilizing agent, an emulsifying agent, a buffering agent for pH control, etc.
  • a buffering agent for pH control etc.
  • non-aqueous solvent or suspension propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • the preparation when the preparation is provided for oral use, for example, in the form of a tablet containing starch or lactose, or in the form of a capsule containing alone or an excipient, or an elixir containing a flavoring or coloring chemical. Or it can be administered orally, buccally or sublingually in the form of a suspension.
  • the dosage of the formulation may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be divided into once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. there is.
  • the daily dosage is 0.001 to 10000 mg/kg, 0.01 to 10000 mg/kg, 0.1 to 10000 mg/kg, 0.5 to 10000 mg/kg, 0.001 to 1000 mg/kg, 0.01 to 1000 0.1 to 1000 mg/kg, 0.5 to 1000 mg/kg, 0.001 to 500 mg/kg, 0.01 to 500 mg/kg, 0.1 to 500 mg/kg, 0.5 to 500 mg/kg, 0.001 to 300 mg /kg, 0.01 to 300 mg/kg, 0.1 to 300 mg/kg, or 0.5 to 300 mg/kg.
  • the above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.
  • the daily dose of the formulation is less than the dose, a significant effect cannot be obtained, and if it exceeds the dose, it is not only uneconomical but also out of the range of the usual dose, so undesirable side effects may occur. It's good to have a range.
  • the administration target of the agent may be mammals such as humans, cells, tissues, body fluids, or cultures thereof isolated from mammals.
  • the present invention provides a method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the vaccine composition to a subject in need thereof.
  • the present invention provides a method for treating or preventing ER stress-related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof.
  • the present invention provides a method for delivering a peptide for endoplasmic reticulum delivery to intracellular endoplasmic reticulum, comprising the step of treating a cell with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • the present invention provides a method for delivering peptides to the endoplasmic reticulum, comprising the step of treating a subject with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • the present invention provides a method for selective delivery of peptides for endoplasmic reticulum delivery to intracellular endoplasmic reticulum, comprising the step of treating cells with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • the present invention provides a method for selectively delivering a peptide for endoplasmic reticulum delivery to the endoplasmic reticulum, comprising the step of treating a subject with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
  • the present invention provides a vaccine composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for use in the treatment or prevention of the above diseases or disorders.
  • the present invention provides a pharmaceutical composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for use in the treatment or prevention of ER stress-related disorders.
  • the present invention provides a composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for delivering an endoplasmic reticulum delivery peptide to an intracellular endoplasmic reticulum.
  • the present invention provides a composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for delivery of an endoplasmic reticulum delivery peptide to the endoplasmic reticulum.
  • the present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a vaccine for the treatment or prevention of the above diseases or disorders.
  • the present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a medicament for use in the treatment or prevention of ER stress-related disorders.
  • the present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the preparation of a formulation for delivering an endoplasmic reticulum delivery peptide to an intracellular endoplasmic reticulum.
  • the present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a formulation for delivery of an endoplasmic reticulum delivery peptide to the endoplasmic reticulum.
  • the present invention also provides uses and methods of utilizing the aforementioned compositions.
  • the fusion polypeptide of the present invention can efficiently deliver a desired peptide to the intracellular endoplasmic reticulum. Accordingly, it has an excellent advantage in that it can be used as a vaccine, a drug delivery system, and a therapeutic agent, and can be used for preventive and therapeutic purposes by controlling various physiological phenomena in vivo.
  • Figure 1 shows the results of confirming the endoplasmic reticulum target of the endoplasmic reticulum target cell penetrating peptide sequence according to the present invention using confocal microscopy.
  • Figure 2 shows an example of a schematic schematic diagram of a fusion peptide according to the present invention.
  • Figure 3 shows the results of analyzing the MHC class I loading efficiency by treating the fusion peptide according to the present invention.
  • Figure 4 shows the results of analyzing the CD8+ T cell activation efficiency by treating the fusion peptide according to the present invention.
  • FIG. 5 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in vascular endothelial cells.
  • ER endoplasmic reticulum
  • FIG. 6 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in cervical cancer-derived epithelial cells.
  • ER endoplasmic reticulum
  • FIG. 7 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in macrophages.
  • ER endoplasmic reticulum
  • FIG. 8 shows the result of analyzing the intracellular location of peptides according to the treatment with Penetratin in epithelial cells of cervical cancer origin.
  • Figure 14 shows the results confirming that the antioxidant-loaded ER-CPP inhibits ER-stress-induced apoptosis.
  • Figure 15 shows the results confirming that ER-CPP loaded with antioxidants inhibits apoptosis caused by ER-stress.
  • sequences capable of targeting the endoplasmic reticulum with cell permeability were identified through deep learning-based cell permeability analysis.
  • Sequences classified as high ranks according to deep learning-based cell permeability analysis are as follows:
  • SEQ ID NO: 1 MLPGLALLLLAAWTARA (signal sequence derived from Amyloid-beta precursor protein, Human)
  • SEQ ID NO: 2 MLP S LALLLLAAWT V RA (signal sequence derived from Amyloid-beta precursor protein, Mouse)
  • polypeptide sequences used in the present invention were synthesized by Peptron Co., Ltd., and the following experiments were performed using them.
  • Example 2 it was confirmed that the endoplasmic reticulum target cell penetrating peptide according to the present invention was located in the endoplasmic reticulum.
  • a fusion polypeptide was prepared to confirm whether the desired peptide could be delivered to the endoplasmic reticulum using the above peptide (sequence).
  • the peptide sequence for delivery to the endoplasmic reticulum is directly linked, or a signal peptidase enzyme recognition sequence (signal peptidase cleavage peptide sequence) is linked to the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence, then delivery The desired sequence (peptide for endoplasmic reticulum delivery) was linked.
  • Figure 2 shows a schematic sequence structure of a fusion polypeptide in which a signal peptidase enzyme sequence is linked to the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence and then a peptide sequence for endoplasmic reticulum delivery is linked.
  • the peptidase cleavage recognition sequence for the signal sequence (ER-CPP) at the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence (ER-CPP) (signal peptidase cleavage peptide sequence, for example "EA" ), the signal peptidase enzyme recognizes and cuts it, and the ER-CPP sequence and the peptide sequence for delivery to the endoplasmic reticulum for delivery can be separated. Accordingly, it may be constituted by delivering a peptide sequence (eg, an epitope, etc.) for delivery to the endoplasmic reticulum for delivery into the endoplasmic reticulum.
  • a peptide sequence eg, an epitope, etc.
  • siinfekl SEQ ID NO: 3
  • Ovalbumin Ovalbumin
  • SEQ ID NO: 10 is the endoplasmic reticulum target cell-permeable sequence of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 3 according to the present invention; signal peptidase cleavage peptide sequence; and a sequence linking the immunogenic peptide sequence.
  • SEQ ID NO: 9 represents the sequence according to the present invention excluding the signal peptidase cleavage peptide sequence;
  • SEQ ID NO: 13 is a sequence for confirming whether the MHC1 presentation effect occurs by changing the OVA sequence.
  • Example 3 Using the fusion polypeptide sequences synthesized in Example 3, it was confirmed whether the endoplasmic reticulum target cell penetrating peptide could exhibit a desired effect after delivering the desired peptide to the endoplasmic reticulum.
  • MHC class I antigenic peptide derived from Ovalbumin which is the exemplary delivery peptide
  • a fusion polypeptide in which an antigen and an endoplasmic reticulum target cell-penetrating sequence are bonded directly or via a linker is processed in dendritic cells to determine whether the antigen peptide is presented (cross-presented) as MHC class I by adding 4 ⁇ M peptide to dendritic cells. was exposed for 3 hours. Then, the peptide remaining in the medium was removed by replacing the culture medium after 3 hours.
  • SINFEKL-MHC class I complex MHC I-antigen peptide complex
  • an antibody recognizing the SINFEKL-MHC class I complex was attached, and cells with the antibody among all dendritic cells The ratio of was analyzed by flow cytometry.
  • SEQ ID NO: 10 which added the signal peptidase cleavage peptide sequence recognized by the signal peptidase enzyme, showed the best effect, and it was further confirmed that the signal peptidase enzyme can play an important role in the delivery of the desired peptide sequence, especially the epitope. .
  • Example 4 and Example 5 show that the fusion polypeptide according to the present invention can exhibit the desired effect through the MHC class I epitope, which is the target peptide for delivery into the endoplasmic reticulum.
  • Fluorescein-5-isothiocyanate was bound to the N' terminus of ER-CPP (SEQ ID NO: 1: N'-acetyl-MLPGLALLLLAAWTARA-NH 2 -C'), and intracellular localization was performed using this.
  • the above fusion polypeptide is treated with human umbilical vein endothelial cells (HUVEC), cervical cancer-originated epithelial cells (HeLa cells), or macrophages (Raw 264.7 cells). and treated with ER tracker red to stain the endoplasmic reticulum and Hoechst 33342 to stain the nucleus. Then, the intracellular localization of ER-CPP was analyzed using confocal microscopy (Zeiss).
  • HUVEC human umbilical vein endothelial cells
  • HeLa cells cervical cancer-originated epithelial cells
  • macrophages Raw 264.7 cells
  • FIG. 5 shows the analysis results in human umbilical vein endothelial cells (HUVEC)
  • FIG. 6 shows the analysis results in cervical cancer-originated epithelial cells (HeLa cells)
  • FIG. Analysis results are shown in macrophages (Raw 264.7 cells).
  • ER-CPP targets intracellular endoplasmic reticulum in various cells. That is, it was confirmed that the ER-CPP sequence according to the present invention was transmitted to the endoplasmic reticulum after cell penetration regardless of cell type.
  • Penetratin (SEQ ID NO: 14: N'-acetyl-RQIKIWFQNRRMKWKK-NH 2 -C') was identified as a control. After binding FITC to the N' terminus of penetratin and treating HeLa cells, the endoplasmic reticulum and nucleus were stained to analyze the intracellular location, and the results are shown in FIG. 8 .
  • FITC-ER-CPP (SEQ ID NO: 1: N'-FITC-MLPGLALLLLAAWTARA-NH 2 -C') was administered at various times (10 minutes, 20 minutes, 40 minutes, 2 hours or 4 hours) and concentrations (1, 2, or After treatment with macrophages (Raw 264.7 cells) with 4 ⁇ M), cell permeation efficiency was analyzed by comparing the intensity of FITC using a flow cytometer (Fluorescence-activated cell sorting, FACS, Novocyte).
  • FIG. 9 The results are shown in FIG. 9 . As can be seen in FIG. 9, it was confirmed that the cell penetration efficiency was high at about 70% even 10 minutes after the cells were treated with the ER-CPP sequence, and when treated for 20 minutes or more, most of the ER-CPP was 90% or more. It was confirmed that the sequence penetrated the cells.
  • Antigen presentation efficiency was analyzed using a sequence in which the SIINFEKL peptide derived from ovalbumin (OVA) was linked.
  • sequence name order Peptide 1 (SEQ ID NO: 10) N'-Ac-MLPGLALLLLAAWTARAEASIINFEKL-NH 2 -C' Peptide 2 (SEQ ID NO: 9) N'-Ac-MLPGLLALLLLAAWTARASIINFEKL-NH 2 -C' Peptide 3 (Negative) (SEQ ID NO: 13) N'-Ac-MLPGLALLLLAAWTARAEASINYEKL-NH 2 -C' Peptide 4 (Current CPP) (SEQ ID NO: 15) N'-Ac-RQIKIWFQNRRMKWKKSIINFEKL-NH 2 -C'
  • DC 2.4 cells were treated for each concentration and cytotoxicity was analyzed. As a result, cytotoxicity was not confirmed at all. In addition, cell penetration efficiency was analyzed by comparing the intensity of FITC using a flow cytometer (Fluorescence-activated cell sorting, FACS, Novocyte), and the antigen presenting ability of the endoplasmic reticulum targeting peptide was analyzed.
  • DC 2.4 cells were treated with OVA 323-339 (MHC II epitope sequence (SEQ ID NO: 16: ISQAVHAAHAEINEAGR), OVA 257-264 (SIINFEKL sequence), Peptide 1, Peptide 2, Peptide 3, and Peptide 4 at different concentrations and time). After that, the cell membrane surface was analyzed by FACS using an antibody binding to MHC I-loaded SIINFEKL.
  • CPRG chlorophenol red- ⁇ -galactoside
  • treatment with the sequence including ER-CPP according to the present invention showed excellent effects on CD8+ T cell activity.
  • ER-CPP increased the activity of CD8+ T cells more than twice as compared to the use of OVA 257-264 or general CPP (SEQ ID NO: 15).
  • MHC I epitope loading complex must go through ER quality control before stable presentation on cell surface plasma membrane. Appropriate time is required for T cells to find antigen-expressing cells even in vivo , but the results of the present invention show the results of stable cell surface antigen presentation. That is, the results according to the present invention provide specific results for specifically targeting an antigen to the endoplasmic reticulum.
  • Example 10 Immune action boosting effect in a mouse model using ER-CPP
  • CD8+ T cell activity of the peptides was analyzed. After administering 5 ⁇ g of the peptide (SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 15) to the footpad of an 8-week-old C57BL/6 mouse, 20 hours later, CFSE-labeled OTI-CD8+ T cells were counted in the tail vein (5x10 6 cells). Intravenous, IV) injection. Then, after 72 hours, mouse blood was collected to analyze changes in CFSE staining amount and at the same time, changes in cells secreting interferon- ⁇ (INF- ⁇ ) among CD8+ T cells in the blood were analyzed.
  • the peptide SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 15
  • the use of the ER-CPP sequence according to the present invention increased the proliferation of CD8+ T cells more than 4 times compared to penetratin, a known CPP.
  • cells secreting interferon gamma were increased more than twice.
  • CD8+ T cells in the blood recognized the antigen and increased their activity in a mouse animal model because the MHC I epitope was loaded in the intracellular endoplasmic reticulum and antigen presentation on the cell surface was maintained for a long time.
  • cell-penetrating peptides targeting the endoplasmic reticulum, an organelle in cells can be used for various immune-related diseases as an important antigen transporter for immune action.
  • Example 11 Analysis of the intracellular localization of the endoplasmic reticulum (ER) target cell-penetrating peptide of an antioxidant
  • a peptide in which an antioxidant was fused to ER-CPP of SEQ ID NO: 1 was synthesized.
  • sequence name order Peptide 5 (SEQ ID NO: 17) N'FITC-MLPGLLALLLAAWTARA- ⁇ E-CG-amidation C Peptide 6 (SEQ ID NO: 18) N'FITC-CMLPGLALLLLAAWTARA-amidation C' Peptide 7 (SEQ ID NO: 19) N'MLPGLALLLLAAWTARA- ⁇ E-CG-amidation C' Peptide 8 (SEQ ID NO: 20) N'acetyl-CMLPGLALLLLAAWTARA-amidation C'
  • peptide 5 binds FITC to the N' end of ER-CPP and glutathione ( ⁇ E-CG, GSH), an antioxidant, to the C' end.
  • Peptide 6 was synthesized by fusing the N' terminus of ER-CPP with FITC and cysteine. These sequences were used as fluorescent peptides for intracellular localization.
  • Peptide 7 or Peptide 8 is a peptide fused with an antioxidant to which FITC is not added.
  • Peptide 7 has glutathione ( ⁇ E-CG, GSH), an antioxidant, linked to the C' terminus, and peptide 8 has acetylated cysteine linked to the N-terminus.
  • Example 12 Analysis of the inhibitory effect of endoplasmic reticulum-targeted antioxidants on apoptosis induced by endoplasmic reticulum stress
  • Peptide 7 or Peptide 8 and well-known antioxidants such as glutathione (GSH) or N-acetyl-cysteine (NAC) were administered to breast cancer cells. After pre-treatment for 1 hour, changes in apoptosis were analyzed by treatment with thapsigargin at a concentration of 10 ⁇ M for 24 hours.
  • This action is an effect that occurs through the delivery of a target substance to the endoplasmic reticulum, indicating that the endoplasmic reticulum stress can be resolved through the delivery of a target substance to the endoplasmic reticulum.
  • Example 13 Analysis of the inhibitory effect of endoplasmic reticulum-targeted antioxidants on apoptosis induced by endoplasmic reticulum stress
  • thapsigargin was treated for 24 hours at each concentration in neuroblastoma cells (neuroblastoma, SH-sy5y), and apoptotic changes were analyzed using 2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent. .
  • MTT 2,5-diphenyl-2H-tetrazolium bromide
  • peptide 7 or peptide 8 and well-known antioxidants glutathione (GSH) or N-acetyl-cysteine (NAC) were applied to neurotumor cells. After pre-treatment for 1 hour, thapsigargin was treated at a concentration of 20 ⁇ M for 24 hours, and changes in apoptosis were analyzed.
  • the endoplasmic reticulum target cell penetrating peptide sequence of the present invention transfers the target sequence to the endoplasmic reticulum.
  • peptides for treatment or improvement of ER stress or endoplasmic reticulum-related diseases could be well delivered into the ER.
  • suppressing endoplasmic reticulum stress known as a major cause of diabetes, degenerative brain diseases, cancer, and chronic inflammatory diseases
  • using an endoplasmic reticulum-targeting antioxidant makes it possible to suppress and treat disease progression.

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Abstract

The present invention relates to a fusion polypeptide including an endoplasmic reticulum-targeted cell-penetrating peptide and a use thereof. The fusion polypeptide of the present invention can effectively deliver a peptide of interest to the endoplasmic reticulum within cells. Accordingly, the fusion polypeptide has the excellent advantage in that it can be available as a vaccine, a drug carrier, and a therapeutic agent and can be applied to prophylactic and therapeutic purposes by controlling various physiological phenomena in vivo.

Description

신규 세포 투과성 펩타이드 및 이의 용도Novel cell penetrating peptides and uses thereof
본 발명은 신규 세포 투과성 펩타이드 및 이의 용도에 관한 것이다.The present invention relates to novel cell penetrating peptides and uses thereof.
세포 내 약물을 전달하기 위한 전달체로서 최근 세포 투과 펩타이드 (Cell Penetrating Peptides: CPP)가 주목받고 있다. 세포 투과 펩타이드는 약 10~30개 정도의 짧은 펩타이드로 이루어진 세포막 투과성 펩타이드로 대부분 단백질-투과 도메인 (protein-transduction domain)이나 막-이동 시퀀스 (membrane translocating sequence)로부터 유도되었다. 또한, CPP는 세포막에 반응하여 엔도사이토시스(endocytosis)나 직접 세포막을 투과할 수 있는 성질을 갖고 있는 올리고펩타이드로 세포막을 투과할 수 있는 전기화학 및 물리화학적 특성을 갖는다. 이러한 CPP는 약물 전달 시스템으로의 응용방법과 연관된 연구로 약학 분야에서 잘 알려져 있다. 또한, 특이적인 생물학적 시스템에서 응용되어 유전자 발현을 조절하는 하나의 도구로서 연구가 많이 진행되어 사용되고 있다.Recently, cell penetrating peptides (CPPs) are attracting attention as a delivery system for intracellular drug delivery. Cell penetrating peptides are cell membrane penetrating peptides composed of about 10 to 30 short peptides, and are mostly derived from protein-transduction domains or membrane translocating sequences. In addition, CPP is an oligopeptide capable of undergoing endocytosis or directly penetrating the cell membrane in response to the cell membrane, and has electrochemical and physicochemical properties capable of penetrating the cell membrane. Such CPP is well known in the field of pharmacology as a study related to application methods to drug delivery systems. In addition, a lot of research has been conducted and used as a tool for regulating gene expression by being applied in a specific biological system.
세포 투과성 펩타이드 서열과 관련하여 약물이 작용하는 세포소기관을 표적하는 많은 시도가 있어 왔는데, 핵 혹은 미토콘드리아로의 전달은 표적 시퀀스 targeting sequence)를 이용하는 방법들이 알려져 있다.Many attempts have been made to target organelles on which drugs act in relation to cell-penetrating peptide sequences, and methods using a targeting sequence are known for delivery to the nucleus or mitochondria.
그러나 소포체 (endoplasmic reticulum, ER) 표적 시퀀스는 거의 알려져 있지 않은 실정이다. 일부 연구 결과로, KDEL의 아미노산 서열을 이용하는 것이 알려져 있었으나, 실제 KDEL을 인지하는 receptor는 소포체 lumen에 존재하므로 CPP가 세포막을 통과하여 ER lumen 안으로 들어갈 수 없는 문제가 존재한다.However, endoplasmic reticulum (ER) target sequences are largely unknown. As a result of some studies, it has been known to use the amino acid sequence of KDEL, but since the receptor that actually recognizes KDEL exists in the endoplasmic reticulum lumen, there is a problem that CPP cannot pass through the cell membrane and enter the ER lumen.
한편, 소포체(Endoplasmic Reticulum, ER)는 모든 진핵세포에서 발견되는 세포소기관의 하나로 소포세관(tubule), 소포(vesicle), 시스터나(cisterna)가 그물모양으로 이루어져 있다. 소포체는 단백질을 만들어서 세포 곳곳에 전달한다. 소포체의 기본구조 및 구성은 비록 핵막의 연장(Endomembrane System)이지만 일반적인 세포막과 비슷하다. 소포체는 세포막의 일부가 될 단백질을 비롯하여 세포 밖으로 분비될 단백질의 합성, 단백질 2차 구조 형성 및 수송이 이루어지는 곳이다. 소포체(ER, endoplasmic reticulum)의 내강은 번역 후 변형과 단백질의 폴딩을 위해 특수화된 세포적 환경이다. 핵막으로부터 뻗어 나와 가지를 친 것 같은 막성분의 구조로서 리보좀이 붙어있는 조면소포체와 리보좀이 없는 활면소포체의 두 종류가 있다. 세포 내 단백질의 약 1/3이 조면소포체에서 mRNA에서 단백질로 번역 후 수정(post-translational modification), 즉 폴딩 (folding)과 조립 (assembly), 당화(glycosylation), 이황화결합(disulfide bond) 등의 과정을 통해 활성 형태를 띠는 단백질 구조가 된다. 또한, 활면소포체는 지질과 스테로이드 호르몬의 합성장소이며 칼슘 저장소로 세포 내 칼슘 농도를 조절하는데 중요한 역할을 한다. 소포체는 단백질 가공촉진 및 시스터나라고 불리는 생성된 단백질의 저장낭의 수송을 포함한 많은 역할을 수행한다. 새로 만들어진 1차 구조의 단백질의 2 및 3차 구조의 형성은 단백질 이황화 이성질화효소(PDI,Proteindisulfide isomerase), Hsc70 family, 칼넥신, 칼레티큘린 또는 peptidylpropyl isomerase family와 같은 소포체 단백질에 의해 행해진다. 그리고 나서, 올바르게 형성된 단백질만이 조면소포체에서 골지체로 운반될 수 있다.On the other hand, the endoplasmic reticulum (ER) is one of the organelles found in all eukaryotic cells, and consists of a net shape of tubules, vesicles, and cisterna. The endoplasmic reticulum makes proteins and transports them throughout the cell. The basic structure and composition of the endoplasmic reticulum is similar to that of a general cell membrane, although it is an extension of the nuclear membrane (Endomembrane System). The endoplasmic reticulum is where the synthesis of proteins to be secreted out of the cell, the formation of secondary structures, and transport of proteins, including proteins to be part of the cell membrane, take place. The lumen of the endoplasmic reticulum (ER) is a specialized cellular environment for post-translational modification and protein folding. There are two types of membranous components that seem to branch out from the nuclear membrane, the rough endoplasmic reticulum with ribosomes and the smooth endoplasmic reticulum without ribosomes. About 1/3 of intracellular proteins undergo post-translational modification from mRNA to protein in the rough endoplasmic reticulum, that is, folding and assembly, glycosylation, disulfide bonds, etc. This process results in a protein structure that assumes an active form. In addition, the smooth endoplasmic reticulum is a synthesis site of lipids and steroid hormones and plays an important role in regulating intracellular calcium concentration as a calcium store. The endoplasmic reticulum performs many roles, including facilitating protein processing and transporting the storage sacs of produced proteins called cisternae. The formation of secondary and tertiary structures of newly created primary proteins is carried out by endoplasmic reticulum proteins such as protein disulfide isomerase (PDI), Hsc70 family, calnexin, calreticulin or peptidylpropyl isomerase family. Then, only correctly formed proteins can be transported from the rough endoplasmic reticulum to the Golgi apparatus.
바이러스나 미생물에 의한 감염이나 특정한 세포 분화, 혹은 각종 비생물적인 스트레스(abiotic stress)나 외부에서 도입된 단백질의 과발현 등으로 소포체의 단백질 폴딩과 가공 과정이 방해를 받게 되면 소포체 내부에 적절하게 폴딩이 되지 않거나 당쇄 첨가 등의 적절한 가공이 이루어지지 않은 단백질이 축적되는데 이를 소포체 스트레스(ER stress)라고 한다. 다양한 세포 환경의 변화로 소포체 스트레스가 발생하면 세포는 다양한 경로를 통해 이를 해소하기 위해 노력하고, 스트레스가 장기화되면 세포는 치명적인 손상을 입고 아폽토시스(apoptosis)를 일으키게 된다. 이와 같은 소포체 스트레스 반응은 특히 단백질을 합성하여 분비하는 기능이 활발한 형질세포, 췌장의 베타세포, 간세포, 조골세포와 같은 곳에서 잘 관찰되고 있으며 최근 많은 연구들은 소포체 스트레스가 허혈, 당뇨병, 바이러스 감염, 고호모시스테인증, 돌연변이와 같은 여러 가지 질환의 병인으로 작용함을 보여 주고 있다.When the protein folding and processing process of the endoplasmic reticulum is disturbed due to infection by viruses or microorganisms, specific cell differentiation, various abiotic stresses, or overexpression of proteins introduced from the outside, proper folding inside the endoplasmic reticulum is not possible. Proteins that have not been processed or have not been properly processed, such as sugar chain addition, accumulate, which is called ER stress. When endoplasmic reticulum stress occurs due to changes in various cellular environments, cells try to relieve it through various pathways, and when the stress is prolonged, cells suffer fatal damage and cause apoptosis. This endoplasmic reticulum stress response is particularly well observed in plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts, which are active in synthesizing and secreting proteins. It has been shown to act as the etiology of various diseases such as hyperhomocysteinemia and mutation.
그러므로 이러한 소포체 내의 다양한 반응에 관여하는 분자적 메커니즘에 대한 규명은 항상 요구되어 왔고 끊임없이 연구되고 있는 실정이다.Therefore, identification of molecular mechanisms involved in various reactions within the endoplasmic reticulum has always been required and is being continuously studied.
이러한 기술적 배경하에서, 본 발명자들은 신규의 소포체 표적 세포 투과성 펩타이드를 개발하고자 예의 노력하였다. 그 결과, 신규 융합 폴리펩타이드를 개발하고 이러한 서열이 소포체 내로 들어가 다양한 생물학적 기능 변화를 유도함으로써 치료 효과를 나타낼 수 있음을 확인하여 본 발명을 완성하였다.Under these technical backgrounds, the present inventors have made diligent efforts to develop novel endoplasmic reticulum target cell penetrating peptides. As a result, the present invention was completed by developing novel fusion polypeptides and confirming that these sequences can enter the endoplasmic reticulum and induce various biological function changes to exhibit therapeutic effects.
본 발명은 세포 투과성 펩타이드 및 이의 용도를 제공하는 것이다.The present invention provides cell penetrating peptides and uses thereof.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 제공하는 것이다.The present invention provides a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명은 소포체 표적 세포 투과성 펩타이드 및 소포체 전달용 펩타이드를 포함하는 세포 내 소포체 표적 펩타이드 전달용 조성물을 제공하는 것이다.The present invention provides a composition for intracellular endoplasmic reticulum-targeting peptide delivery comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum delivery peptide.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 포함하는 의약 용도를 제공하는 것이다.The present invention provides a pharmaceutical use comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.
상기의 목적을 달성하기 위한 본 발명의 하나의 양태는, 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 제공한다.One aspect of the present invention for achieving the above object provides a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명에서 용어, "펩타이드"란 아미드 결합 (또는 펩타이드 결합)에 의해 아미노산 잔기들이 서로 결합되어 형성된 분자를 의미한다. 상기 펩타이드는 유전자 재조합과 단백질 발현 시스템을 이용하여 합성된 것일 수 있고, 바람직하게는 펩타이드 합성기 등을 통하여 시험관 내에서 합성된 것일 수 있다.As used herein, the term "peptide" refers to a molecule formed by binding amino acid residues to each other via an amide bond (or a peptide bond). The peptide may be synthesized using genetic recombination and a protein expression system, preferably synthesized in vitro through a peptide synthesizer or the like.
본 발명의 융합 폴리펩타이드는 이의 유도체를 포함하는 의미로 상기 펩타이드의 아미노산 단편 또는 일부가 치환 또는 결실되거나, 아미노산 서열의 일부가 생체 내에서 안정성을 증가시킬 수 있는 구조로 변형되거나, 친수성을 높이기 위해 아미노산 서열의 일부가 변형되거나, 아미노산의 일부 또는 전부가 L-또는 D-아미노산으로 치환되거나 또는 아미노산의 일부가 변형된 단편들일 수 있다.The fusion polypeptide of the present invention includes a derivative thereof, in which an amino acid fragment or part of the peptide is substituted or deleted, or a part of the amino acid sequence is modified into a structure capable of increasing stability in vivo, or to increase hydrophilicity It may be fragments in which part of the amino acid sequence is modified, part or all of the amino acids are substituted with L- or D-amino acids, or part of the amino acids is modified.
상기 융합 폴리펩타이드 또는 이의 유도체는 세포투과성을 가지면서 세포 내 투과성을 가지며, 특히 소포체로의 전달능을 가지는 것이다.The fusion polypeptide or a derivative thereof has cell permeability and intracellular permeability, and in particular, has delivery capability to the endoplasmic reticulum.
본 발명의 용어, "세포 투과성"이란, 펩타이드가 세포막을 투과하여 세포 내부로 침투할 수 있는 능력 또는 성질을 의미한다.As used herein, the term "cell permeability" refers to the ability or property of a peptide to permeate a cell membrane and penetrate into a cell.
본 발명의 용어 '소포체 표적"이란, 세포 내에서 소포체로 융합 폴리펩타이드가 전달되는 것을 의미한다.The term 'targeting the endoplasmic reticulum' of the present invention means that a fusion polypeptide is delivered to the endoplasmic reticulum within a cell.
본 발명에 따른 소포체 표적 세포 투과성 펩타이드는 바람직하게 서열번호 1 또는 서열번호 2의 아미노산 서열을 포함하거나, 이로 필수적으로 구성되거나, 이로 구성된다.The endoplasmic reticulum target cell penetrating peptide according to the present invention preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
세포 투과성 펩타이드는 서열번호 1 또는 서열번호 2로 표시되는 아미노산 서열로 이루어진 것일 수 있다. 이때, 상기 서열번호 1 또는 2로 표시되는 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95%, 96%, 97%, 98%, 99% 이상의 서열 상동성을 가지는 아미노산 서열을 포함할 수 있다.The cell penetrating peptide may be composed of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. At this time, at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, 96%, 97%, 98%, 99% of the amino acid sequence represented by SEQ ID NO: 1 or 2 It may include an amino acid sequence having a sequence homology of % or more.
상기 서열 상동성 측면에서, 본 발명에 따른 서열번호 1 또는 서열번호 2의 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 폴리펩타이드를 포함할 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 폴리펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 또한, 아미노산 서열상의 변이 또는 수식에 의해서 펩타이드의 열, pH 등에 대한 구조적 안정성이 증가하거나 세포투과성이 증가한 펩타이드를 포함할 수 있다.In terms of sequence homology, a polypeptide having a sequence different from the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 according to the present invention by at least one amino acid residue may be included. Amino acid exchanges in proteins and polypeptides that do not entirely alter the activity of the molecule are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ Exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In addition, peptides with increased structural stability against heat, pH, etc. or increased cell permeability may be included by mutation or modification in the amino acid sequence.
본 발명에 따른 펩타이드 서열은 세포 투과성 분석을 통해 세포 투과성이 확인되고 소포체를 표적하는 능력을 가진 폴리펩타이드 서열이다.The peptide sequence according to the present invention is a polypeptide sequence whose cell permeability is confirmed through cell permeability analysis and has the ability to target the endoplasmic reticulum.
소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 구체적으로, 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다. 여기서 소포체 전달용 펩타이드의 연결은 소포체 표적 세포 투과성 펩타이드의 N-말단 또는 C-말단에서 모두 가능하다.A fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide is specifically, an endoplasmic reticulum target cell penetrating peptide; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto. Here, the connection of the peptide for endoplasmic reticulum delivery can be performed at either the N-terminus or the C-terminus of the endoplasmic reticulum target cell-penetrating peptide.
즉, 본 발명에 따른 "이에 연결된"의 표현은 N-말단 또는 C-말단으로의 펩타이드 서열의 연결 및/또는 결합을 의미한다. 여기서 연결은 직접 결합되어 연결된 것이나 링커 또는 스페이서를 통해 연결되는 것을 모두 포함한다.That is, the expression "linked thereto" according to the present invention means linkage and/or binding of the peptide sequence to the N-terminus or C-terminus. Here, the connection includes both direct bonding and connection through a linker or spacer.
즉, 상기 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 소포체 표적 세포 투과성 펩타이드; 및 이의 N-말단에 연결된 소포체 전달용 펩타이드를 포함하는 융합폴리펩타이드일 수 있다. 또한, 상기 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 소포체 표적 세포 투과성 펩타이드; 및 이의 C-말단에 연결된 소포체 전달용 펩타이드를 포함하는 융합폴리펩타이드일 수 있다.That is, the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is an endoplasmic reticulum target cell penetrating peptide; and a endoplasmic reticulum delivery peptide linked to its N-terminus. In addition, the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide may include an endoplasmic reticulum target cell penetrating peptide; and a endoplasmic reticulum delivery peptide linked to its C-terminus.
본 발명의 용어 "소포체 전달용 펩타이드"란 소포체로 전달을 목적하여 소포체로 전달된 후 목적하는 활성을 나타내기 위한 임의의 펩타이드를 의미한다. 세포 내로 전달된 후 소포체에서 이의 활성을 조절하는 약리학적 활성을 나타낼 수 있는 펩타이드라면 어떠한 것이라도 본 발명의 범주에 포함될 수 있다. 예를 들어, 면역원성 펩타이드, 항원, 사이토카인, 케모카인, 림포카인, 리간드, 수용체, 호르몬, 효소, 항체 및 항체 단편, 및 성장 인자 등을 포함할 수 있으나, 이에 제한되지는 않는다.The term "peptide for endoplasmic reticulum delivery" as used herein refers to any peptide intended for delivery to the endoplasmic reticulum and exhibiting a desired activity after being delivered to the endoplasmic reticulum. Any peptide that can exhibit pharmacological activity by regulating its activity in the endoplasmic reticulum after delivery into cells may be included in the scope of the present invention. For example, it may include, but is not limited to, immunogenic peptides, antigens, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors.
본 발명에 따른 소포체 전달용 펩타이드는 예를 들어, 1개, 2개, 3개, 4개, 5개, 6개, 7개, 8개, 9개, 10개, 15개, 20개, 30개, 40개, 50개 내의 임의의 아미노산 개수를 가질 수 있다. 바람직하게 1개 내지 30개, 2개 내지 25개, 3개 내지 20개의 아미노산을 가지는 펩타이드가 바람직하다.Peptides for endoplasmic reticulum delivery according to the present invention are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 It may have any number of amino acids within 1, 40, or 50. Preferably, peptides having 1 to 30, 2 to 25, and 3 to 20 amino acids are preferred.
바람직하게 면역원성 또는 면역 자극을 일으킬 수 있는 펩타이드를 사용하는 할 수 있다. "면역원성 또는 면역자극 펩타이드"는 동물에게 투여될 때 상기 동물에서 면역 반응을 생성할 수 있는 조성물이다.Preferably, it is possible to use peptides capable of causing immunogenicity or immune stimulation. An "immunogenic or immunostimulatory peptide" is a composition capable of generating an immune response in an animal when administered to said animal.
면역원성 펩타이드는 항원을 함유하고 면역 반응을 생성할 수 있는 조성물이다. 생성된 면역 반응은 세포(T-세포 매개) 또는 체액(B-세포 매개, 항체 생성) 면역 반응일 수 있다. 면역원성 펩타이드는 또한 세포 및 체액 면역 반응 모두를 유도할 수 있다.An immunogenic peptide is a composition that contains an antigen and is capable of generating an immune response. The immune response generated may be a cellular (T-cell mediated) or humoral (B-cell mediated, antibody producing) immune response. Immunogenic peptides are also capable of inducing both cellular and humoral immune responses.
세포 면역 반응은 CD8+ T 림프구 매개 반응(즉 세포독성 반응), 또는 CD4+ T 림프구 매개 반응(헬퍼 반응)일 수 있다. 또한 세포독성 및 헬퍼 세포 면역 반응을 조합할 수도 있다. 헬퍼 반응은 Th1, Th2 또는 Th17 림프구(이러한 림프구는 당업계에 공지된 바와 같이 상이한 사이토카인 반응을 유도할 수 있음)를 포함할 수 있다. 면역원성 펩타이드는 MHC1 또는 MHC2 경로를 통해 그 안에 존재하는 항원을 보다 잘 제시하도록 허용할 수 있다.The cellular immune response can be a CD8+ T lymphocyte mediated response (ie a cytotoxic response), or a CD4+ T lymphocyte mediated response (helper response). It is also possible to combine cytotoxic and helper cell immune responses. The helper response may include Th1, Th2 or Th17 lymphocytes (such lymphocytes are capable of eliciting different cytokine responses as is known in the art). Immunogenic peptides may allow for better presentation of antigens present therein via the MHC1 or MHC2 pathway.
"항원"은 살아있는 동물의 면역계가 그것을 인식하도록 하기 위해 면역 반응을 유도하고자 하는 분자 또는 분자의 조합이다. 이러한 항원은 면역 반응이 추가하는 숙주의 본체에서 이종일 수 있다. 이 경우, 항원은 세균 또는 바이러스에 의해 발현되는 단백질일 수 있다. 항원은 또한 자가 항원, 즉 종양 항원과 같은 숙주의 세포에 의해 발현되는 단백질일 수 있다.An “antigen” is a molecule or combination of molecules that seeks to elicit an immune response in order to allow the immune system of a living animal to recognize it. These antigens may be heterologous in the body of the host to which the immune response is added. In this case, the antigen may be a protein expressed by bacteria or viruses. Antigens can also be self antigens, ie proteins expressed by the host's cells, such as tumor antigens.
항원은 완전한 단백질 또는 단백질의 에피토프와 같은 단백질의 임의의 부분 일 수 있다. 본 발명의 문맥에서의 항원은 또한 함께 연결된 다중 에피토프를 함유하는 합성 단백질 또는 분자로 구성될 수 있다.An antigen can be a complete protein or any part of a protein, such as an epitope of a protein. Antigens in the context of the present invention may also consist of synthetic proteins or molecules containing multiple epitopes linked together.
특히, 항원은 동일한 항원의 다중 에피토프를 함유하는 단백질일 수 있으며, 이들 에피토프는 MHC 일배체형에 특이적이다. 이 경우, 본원에 기재된 바와 같은 특유의 면역 자극 분자를 사용하여 상이한 유전적(MHC) 정황의 면역 반응을 얻을 수 있다.In particular, the antigen may be a protein containing multiple epitopes of the same antigen, these epitopes being specific for the MHC haplotype. In this case, a unique immune stimulatory molecule as described herein can be used to obtain an immune response in a different genetic (MHC) context.
면역원성 펩타이드로 사용되는 항원 펩타이드는 하나 또는 수개의 MHC 에피토프를 포함한다.Antigenic peptides used as immunogenic peptides contain one or several MHC epitopes.
면역원성 펩타이드에서 사용된 바의 항원은 몇 개의 아미노산(하나, 또는 둘 모두의 C 및 N 말단에서 1 내지 10개, 바람직하게는 1 내지 6개의 아미노산)에 의해 그의 N 및/또는 C 말단에서 측면에 위치한 MHC 에피토프로 구성된다.An antigen, as used in an immunogenic peptide, is flanked at its N and/or C terminus by several amino acids (1 to 10, preferably 1 to 6 amino acids at one or both C and N termini). It consists of MHC epitopes located on
MHC 에피토프(또는 T 세포 에피토프)는 MHC 분자에 결합되어 있는 항원-제시 세포의 표면상에 제시된다. MHC 클래스 I 분자에 의해 제시되는 T 세포 에피토프는 전형적으로 8 내지 11개의 아미노산 길이의 펩타이드이며, 한편 MHC 클래스 II 분자는 13 내지 17개의 아미노산 길이인 더 긴 펩타이드를 제시한다.MHC epitopes (or T cell epitopes) are presented on the surface of antigen-presenting cells bound to MHC molecules. T cell epitopes presented by MHC class I molecules are peptides typically 8-11 amino acids in length, while MHC class II molecules present longer peptides 13-17 amino acids in length.
MHC 에피토프는 시험 관내에서 합성될 수 있다(그의 C 및/또는 N 말단부에서 아미노산이 첨가되거나 또는 첨가되지 않음). MHC 결합 펩타이드는 특히 염산을 사용한 산 처리와 같은 당업계에 공지된 임의의 방법에 의해 살이있는 세포, 특히 종양 세포로부터 추출될 수 있다.MHC epitopes can be synthesized in vitro (with or without added amino acids at their C and/or N termini). MHC binding peptides can be extracted from viable cells, particularly tumor cells, by any method known in the art, such as acid treatment, particularly with hydrochloric acid.
또 다른 실시양태에서, 항원은 하나 또는 수 개의 B 세포 에피토프, 즉 항원으로부터 아미노산의 연속 서열에 의해 형성된 항체, 바람직하게는 선형 에피토프에 의해 인식되는 단백질의 일부를 포함한다.In another embodiment, the antigen comprises one or several B cell epitopes, ie the portion of a protein recognized by an antibody, preferably a linear epitope, formed by a contiguous sequence of amino acids from the antigen.
즉, 상기 면역원성 펩타이드는 항원으로서, MHC 에피토프 및 B-세포 에피토프로 구성된 군에서 선택된 적어도 하나의 에피토프를 포함하는 것일 수 있다. 이러한 MHC 에피토프 및 B-세포 에피토프는 통상의 기술자가 알려진 서열을 기반으로 해당 에피토프의 위치 및 서열을 충분히 도출할 수 있으며, 이러한 서열은 본 발명의 범주 내에 포함된다.That is, the immunogenic peptide may include at least one epitope selected from the group consisting of an MHC epitope and a B-cell epitope as an antigen. Such MHC epitopes and B-cell epitopes can be sufficiently derived by those skilled in the art based on known sequences, and the positions and sequences of these epitopes are included within the scope of the present invention.
특정 실시양태에서, 면역원성 펩타이드에서 사용된 바의 항원은 B 세포 에피토프로 구성된다. 또 다른 실시양태에서, 면역원성 펩타이드에서 사용된 바의 항원은 몇 개의 아미노산(하나, 또는 둘 모두의 C 및 N 말단에서 1 내지 10개, 바람직하게는 1 내지 6개의 아미노산)에 의해 그의 N 및/또는 C 말단에서 측면에 위치한 B 세포 에피토프로 구성된다. 에피토프 맵핑에 관한 문헌의 다른 방법은 제공된 항원으로부터 T 세포 또는 B 세포 에피토프를 식별하는 것을 가능하게 한다.In certain embodiments, antigens as used in immunogenic peptides consist of B cell epitopes. In another embodiment, the antigen, as used in the immunogenic peptide, is modified by several amino acids (from 1 to 10, preferably 1 to 6 amino acids at the C and N termini of one or both of them) to their N and / or C-terminally flanked by B cell epitopes. Other methods in the literature on epitope mapping make it possible to identify T cell or B cell epitopes from a given antigen.
본 발명의 일예시에 따르면, 상기 면역원성 펩타이드(면역원성 에피토프)의 일 예시로 Ovalbumin (OVA) 유래 MHC class I antigenic peptide (서열번호 3: SIINFEKL)을 사용하였다.According to an example of the present invention, an MHC class I antigenic peptide (SEQ ID NO: 3: SIINFEKL) derived from Ovalbumin (OVA) was used as an example of the immunogenic peptide (immunogenic epitope).
상기 세포투과성 펩타이드에 소포체 전달용 펩타이드의 연결은 직접적으로 연결될 수도 있고, 링커 또는 스페이서 펩타이드를 두고 연결될 수도 있다.The peptide for endoplasmic reticulum delivery may be directly connected to the cell-penetrating peptide, or may be connected through a linker or spacer peptide.
용어 "링커" 또는 "스페이서"는 융합 단백질 구성시 기능이 다른 두 펩타이드를 분리하는데 사용되는 짧은 아미노산 서열을 지칭하는 것이다. 단백질 내의 두 개 또는 그 이상의 개별 도메인 사이에 링커가 부재하면 입체 장애로 인하여 단백질 도메인의 감소된 또는 부적절한 기능, 예를 들어 수용체/리간드에 대한 촉매 활성 또는 결합 친화력의 감소를 초래할 수 있다. 인공적인 링커를 사용하여 키메라 단백질에서 단백질 도메인을 연결하면 도메인 사이의 공간을 증가시킬 수 있다. 바람직하게는, 상기 링커 또는 스페이서는 세포투과성 펩타이드와 소포체 전달용 펩타이드의 결합체의 활성을 향상시키는 효과를 나타내게 하는 한 특별히 이에 제한되지 않는다. 상기 영역들을 결합시키거나 또는 이들 간의 일부 최소 거리 또는 다른 공간적 관계를 보존하는 것 이외에 특정한 생물 활성을 갖지 않을 것이나, 구성 아미노산들은 상기 분자의 일부 성질, 예를 들어 폴딩, 순 전하, 또는 소수성에 영향을 미치도록 선택될 수 있다. The term "linker" or "spacer" refers to a short amino acid sequence used to separate two peptides with different functions in constructing a fusion protein. The absence of a linker between two or more individual domains in a protein can result in reduced or inappropriate function of the protein domains due to steric hindrance, such as reduced catalytic activity or binding affinity for a receptor/ligand. The spacing between the domains can be increased by linking the protein domains in the chimeric protein using artificial linkers. Preferably, the linker or spacer is not particularly limited as long as it exhibits an effect of enhancing the activity of the conjugate of the cell-permeable peptide and the endoplasmic reticulum delivery peptide. Other than joining the regions or preserving some minimal distance or other spatial relationship between them, they will not have any particular biological activity, but the constituent amino acids affect some property of the molecule, such as folding, net charge, or hydrophobicity. can be selected to
예를 들어, 상기 소포체 전달용 펩타이드가 소포체 내에서 활성을 향상시키는 효과를 나타내게 하기 위하여, 프로테아제로 절단 가능한 서열을 링커로 포함할 수 있다. 예를 들어, 프로테아제 절단 부위는 자연적으로 발생하는 프로테아제 절단 부위 또는 인공적으로 조작된 프로테아제 절단 부위일 수 있다.For example, in order for the peptide for endoplasmic reticulum delivery to exhibit an effect of enhancing activity in the endoplasmic reticulum, a sequence cleavable with a protease may be included as a linker. For example, the protease cleavage site can be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site.
이에 따라 본 발명에서는 signal peptidase 효소에 의해 인지되는 서열을 포함하여 이를 통해 절단되는 것이 바람직하다. 즉, signal peptidase 효소가 인지할 수 있는 서열을 세포투과성 펩타이드와 약물 대상 펩타이드 서열 간에 추가할 수 있다.Accordingly, in the present invention, it is preferable to cut through the sequence including the sequence recognized by the signal peptidase enzyme. That is, a sequence that can be recognized by the signal peptidase enzyme can be added between the cell-permeable peptide and the drug target peptide sequence.
본 발명에서 용어 "시그널 펩티다아제(signal peptidase) 절단 펩타이드"란 시그널 펩티다아제에 의해 인식되어 소포체 전달용 펩타이드와 소포체 표적 세포투과성 펩타이드 서열 간의 절단을 제공할 수 있는 링커 서열을 의미한다. "시그널 펩티다아제(signal peptidase) 절단 펩타이드"는 세포투과성 펩타이드 서열의 C-말단과 위 절단 펩타이드 서열을 인지하여 이들 사이에 펩타이드 절단을 제공할 수 있다.In the present invention, the term "signal peptidase cleavage peptide" refers to a linker sequence that is recognized by signal peptidase and can provide cleavage between the endoplasmic reticulum delivery peptide and the endoplasmic reticulum target cell-permeable peptide sequence. The "signal peptidase cleavage peptide" can recognize the C-terminus of the cell-permeable peptide sequence and the stomach cleavage peptide sequence to provide peptide cleavage between them.
본 발명에 따른 세포투과성 펩타이드 서열은 신호 서열로부터 유래되기 때문에, C-말단에 "AXA(서열번호: 5)" 또는 "VXA(서열번호: 6)"(여기서 X는 임의의 아미노산 서열임)을 가질 수 있다. 이에 연결되는 "EA(서열번호: 4)" 서열을 링커 서열로 추가하는 경우, signal peptidase 효소는 위 "AXA" 또는 "VXA"와 "EA"서열 사이를 절단 위치로 인지하여 이를 절단할 수 있다. 이에 따라, 본 발명에서는 바람직하게, 링커 서열로 "EA" 서열을 이용할 수 있다.Since the cell-permeable peptide sequence according to the present invention is derived from a signal sequence, "AXA (SEQ ID NO: 5)" or "VXA (SEQ ID NO: 6)" (where X is any amino acid sequence) is added at the C-terminus. can have When the "EA (SEQ ID NO: 4)" sequence linked thereto is added as a linker sequence, the signal peptidase enzyme recognizes the cleavage position between the above "AXA" or "VXA" and "EA" sequences and cleaves them. . Accordingly, in the present invention, the "EA" sequence may be preferably used as a linker sequence.
이에 본 발명에 따른 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 구체적으로, 소포체 표적 세포 투과성 펩타이드; 이에 연결된 링커 또는 스페이서 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다.Accordingly, the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide according to the present invention is specifically, the endoplasmic reticulum target cell penetrating peptide; Linker or spacer peptides linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
이에 본 발명에 따른 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 구체적으로, 소포체 표적 세포 투과성 펩타이드; 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다.Accordingly, the fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide according to the present invention is specifically, the endoplasmic reticulum target cell penetrating peptide; a signal peptidase cleavage peptide linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
보다 구체적으로, 서열 번호 1 또는 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다.More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
보다 구체적으로, 서열 번호 1 또는 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 면역원성 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다. 즉, 서열 번호 1 또는 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드에 이어서 면역원성을 나타낼 수 있는 임의의 펩타이드가 결합될 수 있다.More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; and an immunogenic peptide linked thereto. That is, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or 2; Following the signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto, any peptide capable of exhibiting immunogenicity may be conjugated.
면역원성 펩타이드가 연결될 수 있는 소포체 표적 투과성 펩타이드 서열과 절단 펩타이드가 연결된 서열의 예시는 아래와 같다:An example of an endoplasmic reticulum target penetrating peptide sequence to which an immunogenic peptide can be linked and a sequence in which a cleavage peptide is linked are as follows:
서열번호 1의 ER-CPP+서열번호 4의 EA 서열(서열번호 7):MLPGLALLLLAAWTARA EAER-CPP of SEQ ID NO: 1 + EA sequence of SEQ ID NO: 4 (SEQ ID NO: 7): MLPGLALLLLAAWTARA EA
서열번호 2의 ER-CPP+서열번호 4의 EA 서열(서열번호 8):MLPSLALLLLAAWTVRA EAER-CPP of SEQ ID NO: 2 + EA sequence of SEQ ID NO: 4 (SEQ ID NO: 8): MLPSLALLLLAAWTVRA EA
본 발명의 일실시양태에 따라, 면역원성 펩타이드가 결합된 실험에 사용된 서열을 하기와 같이 나열하였다. 면역원성 펩타이드는 목적에 따라 그 종류가 달라지기 쉽고, 매우 다양하기 때문에, 이러한 면역원성 펩타이드 서열의 예시는 본 발명의 내용을 한정하는 것은 아니다. According to one embodiment of the present invention, sequences used in experiments in which immunogenic peptides are bound are listed as follows. Immunogenic peptides are easy to vary depending on the purpose and are very diverse, so the examples of such immunogenic peptide sequences are not intended to limit the scope of the present invention.
서열번호 1의 ER-CPP+서열번호 3의 면역원성 펩타이드(서열번호 9):MLPGLALLLLAAWTARA SIINFEKLER-CPP of SEQ ID NO: 1 + immunogenic peptide of SEQ ID NO: 3 (SEQ ID NO: 9): MLPGLALLLLAAWTARA SIINFEKL
서열번호 1의 ER-CPP+서열번호 4의 EA 서열+서열번호 3의 면역원성 펩타이드(서열번호 10):MLPGLALLLLAAWTARA EA SIINFEKLER-CPP of SEQ ID NO: 1 + EA sequence of SEQ ID NO: 4 + immunogenic peptide of SEQ ID NO: 3 (SEQ ID NO: 10): MLPGLALLLLAAWTARA EA SIINFEKL
서열번호 2의 ER-CPP+서열번호 3의 면역원성 펩타이드(서열번호 11):MLPSLALLLLAAWTVRA SIINFEKLER-CPP of SEQ ID NO: 2 + immunogenic peptide of SEQ ID NO: 3 (SEQ ID NO: 11): MLPSLALLLLAAWTVRA SIINFEKL
서열번호 2의 ER-CPP+서열번호 4의 EA 서열+서열번호 3의 면역원성 펩타이드(서열번호 12):MLPSLALLLLAAWTVRA EA SIINFEKLER-CPP of SEQ ID NO: 2 + EA sequence of SEQ ID NO: 4 + immunogenic peptide of SEQ ID NO: 3 (SEQ ID NO: 12): MLPSLALLLLAAWTVRA EA SIINFEKL
위 언급된 면역원성 펩타이드 서열 SIINFEKL은 본 발명에 따른 ER-CPP 서열의 작용을 확인하기 위한 하나의 실험예일뿐, 면역원성 펩타이드는 이에 국한되지 않고 임의의 알려진 면역원성 펩타이드를 모두 사용할 수 있다.The above-mentioned immunogenic peptide sequence SIINFEKL is only one experimental example for confirming the action of the ER-CPP sequence according to the present invention, and the immunogenic peptide is not limited thereto, and any known immunogenic peptide may be used.
본 발명에 따른 폴리펩타이드는 당업계에 공지된 이용 가능한 기술을 이용하여 제조될 수 있다. 폴리펩타이드는 당분야의 숙련자에게 공지된 임의의 적합한 절차, 즉 공지의 폴리펩타이드 합성 방법(ex. 유전공학적 방법, 화학적 합성)을 이용하여 합성될 수 있다. Polypeptides according to the present invention can be prepared using available techniques known in the art. Polypeptides can be synthesized using any suitable procedure known to those skilled in the art, ie known polypeptide synthesis methods (eg genetic engineering methods, chemical synthesis).
예컨대 본 발명에 따른 폴리펩타이드는 유전공학적 방법에 따른 재조합 기법에 의해 제조될 수 있다. 유전공학적 방법에 의한 펩타이드의 제작은, 예를 들어, 우선 통상적인 방법에 따라 상기 본 발명의 폴리펩타이드 또는 이의 기능적 동등물을 암호화하는 핵산(폴리뉴클레오타이드)을 작제한다. 상기 핵산은 적절한 프라이머를 사용하여 PCR로 증폭하여 준비할 수 있다. 또는 당업계에 공지된 표준 방법에 의해, 예컨대, 자동 DNA 합성기를 사용하여 DNA 서열을 합성할 수도 있다. 작제된 핵산은 이에 작동가능하게 연결되어(operatively linked) 핵산의 발현을 조절하는 하나 이상의 발현 조절 서열(expression control sequence; 예: 프로모터, 인핸서 등)을 포함하는 벡터에 삽입시켜 재조합 발현 벡터를 제작하고 숙주세포에 형질전환시킨 후, 상기 세포를 목적하는 폴리펩타이드가 발현되기에 적절한 배지 및 조건 하에서 배양하여, 배양물로부터 상기 핵산으로부터 발현된, 실질적으로 순수한 폴리펩타이드를 회수하게 된다. 상기 회수는 당업계에 공지된 방법을 이용하여 수행할 수 있다. 이에 제한되지 않으나, 예를 들어 예를 들면 추출법, 재결정법, 다양한 크로마토크래피(겔 여과법, 이온 교환, 침전, 흡착, 역전상), 전기영동, 역류 분배법 등 당업계에 공지된 방법으로 분리 및 정제할 수 있다. 상기에서 '실질적으로 순수한 폴리펩타이드(substally pure polypeptide)'라 함은 본 발명에 따른 폴리펩타이드가 숙주세포로부터 유래된 어떠한 다른 단백질도 실질적으로 포함하지 않는 것을 의미한다.For example, the polypeptide according to the present invention can be produced by recombinant techniques according to genetic engineering methods. In the production of peptides by genetic engineering methods, for example, first, nucleic acids (polynucleotides) encoding the polypeptides of the present invention or functional equivalents thereof are constructed according to conventional methods. The nucleic acid can be prepared by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer. The constructed nucleic acid is operably linked thereto and inserted into a vector containing one or more expression control sequences (eg, promoter, enhancer, etc.) that regulates the expression of the nucleic acid to construct a recombinant expression vector, After transformation of the host cell, the cell is cultured in a medium and under conditions suitable for expression of the desired polypeptide, and substantially pure polypeptide expressed from the nucleic acid is recovered from the culture. The recovery may be performed using a method known in the art. Although not limited thereto, for example, extraction, recrystallization, various chromatographies (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc., are separated by methods known in the art. and can be purified. As used herein, 'substantially pure polypeptide' means that the polypeptide according to the present invention does not substantially contain any other proteins derived from host cells.
또한, 예컨대 본 발명에 따른 폴리펩타이드는 당업계에 공지된 화학적 합성 방법에 의해 제조될 수 있다. 대표적인 방법으로서, 이들로 한정되는 것은 아니지만, 액체 또는 고체상 합성, 단편 응축, F-MOC 또는 T-BOC 화학법이 포함된다.In addition, for example, polypeptides according to the present invention can be prepared by chemical synthetic methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
구체적 일례로, 본 발명의 폴리펩타이드는 고상 기법을 이용한 직접적 펩타이드 합성에 의해 제조될 수 있다. 고체상 펩타이드 합성(SPPS) 방법은 작은 다공성의 비드(beads)에 링커(linkers)라 불리는 기능성 유닛(functional units)을 부착하여 펩타이드 사슬을 이어 나갈 수 있도록 유도함으로써 합성을 개시할 수 있다. 액체상 방법과 달리 펩타이드는 비드와 공유 결합하여 TFA(trifluoroacetic acid)와 같은 특정 반응물에 의해 절단되기 전까지 여과(filtration) 과정에 의해 떨어져 나가는 것을 방지한다. 고체상에 부착된 펩타이드의 N-말단 아민과 N-보호 아미노산 유닛(N-protected amino acid unit)이 결합하는 보호(protection) 과정, 탈보호(deprotection) 과정, 다시 드러난 아민 그룹(amine group)과 새로운 아미노산이 결합하는 커플링(coupling) 과정의 사이클(cycle, deprotection-wash-coupling-wash)이 반복되면서 합성이 이루어지게 된다. 상기 SPPS 방법은 마이크로파(microwave) 기술을 함께 이용하여 수행할 수 있으며, 마이크로파 기술은 펩타이드 합성 과정에서 열을 가해줌으로써 각 사이클의 커플링과 탈보호에 요구되는 시간을 단축시킬 수 있다. 상기 열 에너지는 확장되는 펩타이드 사슬이 접히거나(folding) 집합체를 형성하는 것(aggregation)을 방지하고 화학적 결합을 촉진시킬 수 있다.In a specific example, the polypeptide of the present invention can be prepared by direct peptide synthesis using solid phase techniques. In the solid-phase peptide synthesis (SPPS) method, synthesis can be initiated by attaching functional units, called linkers, to small porous beads to guide the peptide chain. Unlike the liquid-phase method, the peptide is covalently bound to the beads and prevents them from being separated by filtration until they are cleaved by a specific reactant, such as trifluoroacetic acid (TFA). A protection process in which the N-terminal amine of a peptide attached to a solid phase is combined with an N-protected amino acid unit, a deprotection process, a re-exposed amine group and a new Synthesis is performed by repeating a cycle (deprotection-wash-coupling-wash) of a coupling process in which amino acids are combined. The SPPS method can be performed using microwave technology together, and microwave technology can shorten the time required for coupling and deprotection of each cycle by applying heat in the peptide synthesis process. The thermal energy may prevent folding or aggregation of the extended peptide chain and promote chemical bonding.
본 발명은 상기 융합 폴리펩타이드를 코딩하는 폴리뉴클레오티드를 제공한다.The present invention provides a polynucleotide encoding the fusion polypeptide.
상기 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 구체적으로, 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다.The fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is specifically, the endoplasmic reticulum target cell penetrating peptide; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
상기 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드는 구체적으로, 소포체 표적 세포 투과성 펩타이드; 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드일 수 있다.The fusion polypeptide comprising the endoplasmic reticulum target cell penetrating peptide is specifically, the endoplasmic reticulum target cell penetrating peptide; a signal peptidase cleavage peptide linked thereto; And it may be a fusion polypeptide comprising an endoplasmic reticulum delivery peptide linked thereto.
상기 폴리뉴클레오티드는 하나 이상의 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있다. 뉴클레오티드 서열을 화학적으로 합성하여 제조하는 경우, 당업계에 널리 공지된 합성법, 예를 들어 문헌(Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988)에 기술된 방법을 이용할 수 있으며, 트리에스테르, 포스파이트, 포스포르아미다이트 및 H-포스페이트 방법, PCR 및 기타 오토프라이머 방법, 고체 지지체상의 올리고뉴클레오타이드 합성법 등을 이용하여 합성할 수 있다.The polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When preparing a nucleotide sequence by chemical synthesis, a synthesis method well known in the art, for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used , triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other auto-primer methods, and oligonucleotide synthesis methods on solid supports.
또 다른 하나의 양태로서, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현벡터, 상기 발현벡터를 포함하는 형질전환체 및 상기 형질전환체를 이용하여 상기 융합 폴리펩타이드를 제조하는 방법을 제공한다.As another aspect, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the fusion polypeptide using the transformant.
본 발명의 용어 "발현 벡터"란, 목적하는 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 의미한다. 상기 발현벡터는 개시코돈, 종결코돈, 프로모터, 오퍼레이터 등의 발현조절 요소들을 포함하는데, 상기 개시코돈 및 종결코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되고, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내어야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다.The term "expression vector" of the present invention is a recombinant vector capable of expressing a desired peptide in a desired host cell, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator. The start codon and stop codon are generally regarded as part of a nucleotide sequence encoding a polypeptide, and when the gene construct is administered, the subject must be functional and must be in frame with the coding sequence. The vector's promoter may be constitutive or inducible.
본 발명의 용어 "작동가능하게 연결(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질 또는 RNA를 코딩하는 핵산 서열이 기능적으로 연결(functional linkage)되어 있는 상태를 의미한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 발현 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다.The term "operably linked" of the present invention means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operational linkage with the expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.
또한, 상기 발현벡터는 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널 및 개시코돈은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.In addition, the expression vector may include a signal sequence for excretion of the polypeptide in order to promote protein separation from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translation control signal, which may include an ATG initiation codon, must be provided. These exogenous translation control signals and initiation codons can be from a variety of natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcriptional or translational enhancers.
아울러, 상기 발현벡터는 본 발명에 따른 융합 폴리펩타이드의 검출이 용이하도록 하기 위하여, 임의로 엔도펩티아이제를 사용하여 제거할 수 있는 단백질 태그를 추가로 포함할 수 있다.In addition, the expression vector may further include a protein tag that can be optionally removed using endopeptiase to facilitate detection of the fusion polypeptide according to the present invention.
본 발명의 용어 "태그(tag)"란, 정량가능한 활성 또는 특성을 나타내는 분자를 의미하며, 플로오레세인과 같은 화학적 형광물질(fluoracer), 형광 단백질(GFP) 또는 관련 단백질과 같은 폴리펩타이드 형광물질을 포함한 형광 분자일 수도 있고; Myc 태그, 플래그(Flag) 태그, 히스티딘 태그, 루신 태그, IgG 태그, 스트랩타비딘 태그 등의 에피토프 태그일 수도 있다. 특히, 에피토프 태그를 사용할 경우, 바람직하게는 6개 이상의 아미노산 잔기로 구성되고, 보다 바람직하게는 8개 내지 50개의 아미노산 잔기로 구성된 펩타이드 태그를 사용할 수 있다.As used herein, the term "tag" refers to a molecule exhibiting a quantifiable activity or characteristic, and is a chemical fluorophore such as fluorescein, a polypeptide fluorophore such as a fluorescent protein (GFP) or a related protein. It may be a fluorescent molecule including; It may also be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a streptavidin tag. In particular, when an epitope tag is used, a peptide tag preferably composed of 6 or more amino acid residues, and more preferably composed of 8 to 50 amino acid residues may be used.
본 발명에 있어서, 상기 발현벡터는 상술한 본 발명의 융합 폴리펩타이드를 코딩하는 뉴클레오티드 서열을 포함할 수 있는데, 이때 사용되는 벡터는 본 발명의 융합 폴리펩타이드를 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 바람직하게는 플라스미드 DNA, 파아지 DNA 등이 될 수 있고, 보다 바람직하게는 상업적으로 개발된 플라스미드(pUC18, pBAD, pIDTSAMRT-AMP 등), 대장균 유래 플라스미드(pYG601BR322, pBR325, pUC118, pUC119 등), 바실러스 서브틸리스 유래 플라스미드(pUB110, pTP5 등), 효모-유래 플라스미드(YEp13, YEp24, YCp50 등), 파아지 DNA(Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP 등), 동물바이러스 벡터 (레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 백시니아 바이러스(vaccinia virus) 등), 곤충 바이러스 벡터(배큘로바이러스(baculovirus) 등)가 될 수 있다. 상기 발현벡터는 숙주세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용함이 바람직하다.In the present invention, the expression vector may include a nucleotide sequence encoding the fusion polypeptide of the present invention described above, but the vector used in this case is not particularly limited thereto as long as it can produce the fusion polypeptide of the present invention , preferably plasmid DNA, phage DNA, etc., more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vector (retrovirus (retrovirus), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the expression vector shows different protein expression levels and modifications depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.
본 발명에서 제공하는 형질전환체는 상기 본 발명에서 제공하는 발현벡터를 숙주에 도입하여 형질전환시킴으로써 제작될 수 있고, 상기 발현벡터에 포함된 폴리뉴클레오티드를 발현시켜서, 본 발명의 융합 폴리펩타이드를 생산하는데 사용될 수 있다. 상기 형질전환은 다양한 방법에 의하여 수행될 수 있는데, 본 발명의 융합 폴리펩타이드를 생산할 수 있는 한, 특별히 이에 제한되지 않으나, CaCl2 침전법, CaCl2 침전법에 DMSO(dimethyl sulfoxide)라는 환원물질을 사용함으로써 효율을 높인 Hanahan 방법, 전기천공법(electroporation), 인산칼슘 침전법, 원형질 융합법, 실리콘 카바이드 섬유를 이용한 교반법, 아그로박테리아 매개된 형질전환법, PEG를 이용한 형질전환법, 덱스트란 설페이트, 리포펙타민 및 건조/억제 매개된 형질전환 방법 등이 사용될 수 있다. 또한, 상기 형질전환제의 제작에 사용되는 숙주 역시 본 발명의 세포투과성 펩타이드를 생산할 수 있는 한, 특별히 이에 제한되지 않으나, 대장균(E. coli), 스트렙토마이세스, 살모넬라 티피뮤리움 등의 박테리아 세포; 사카로마이세스 세레비지애, 스키조사카로마이세스 폼베 등의 효모 세포; 피치아 파스토리스 등의 균류세포; 드로소필라, 스포도프테라 Sf9 세포 등의 곤충 세포; CHO, COS, NSO, 293, 보우 멜라노마 세포 등의 동물 세포; 또는 식물 세포가 될 수 있다.The transformant provided in the present invention can be prepared by introducing the expression vector provided in the present invention into a host and transforming it, and expressing the polynucleotide contained in the expression vector to produce the fusion polypeptide of the present invention. can be used to The transformation may be performed by various methods, but is not particularly limited thereto as long as the fusion polypeptide of the present invention can be produced, but a CaCl 2 precipitation method and a reducing material called DMSO (dimethyl sulfoxide) are used in the CaCl 2 precipitation method. Hanahan method with increased efficiency by using electroporation, calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fibers, agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate , lipofectamine and desiccation/inhibition mediated transformation methods and the like can be used. In addition, as long as the host used for the production of the transformant can also produce the cell-permeable peptide of the present invention, it is not particularly limited thereto, but E. coli , Streptomyces, bacterial cells such as Salmonella typhimurium ; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or plant cells.
상기의 목적을 달성하기 위한 본 발명의 다른 하나의 양태는, 소포체 표적 세포 투과성 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다. 구체적으로, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.Another aspect of the present invention for achieving the above object provides a composition for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide. Specifically, a composition for delivering an endoplasmic reticulum-targeting peptide comprising the endoplasmic reticulum-targeting cell-penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2 is provided.
보다 구체적으로, 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 링커 또는 스페이서 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.More specifically, endoplasmic reticulum target cell penetrating peptides; and a linker or spacer peptide linked thereto.
보다 구체적으로, 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다. 보다 더 구체적으로, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.More specifically, endoplasmic reticulum target cell penetrating peptides; and a signal peptidase cleavage peptide linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and a signal peptidase cleavage peptide linked thereto.
보다 구체적으로, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다. 보다 더 구체적으로 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드는 서열번호 7 또는 8의 펩타이드일 수 있다. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and a signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And the signal peptidase cleavage peptide linked thereto may be the peptide of SEQ ID NO: 7 or 8.
보다 구체적으로, 소포체 표적 세포 투과성 펩타이드 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공하는 것이다.More specifically, it is to provide a composition for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum-targeting peptide linked thereto.
보다 구체적으로, 소포체 표적 세포 투과성 펩타이드 및 이의 N-말단에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공하는 것이다. 또는, 소포체 표적 세포 투과성 펩타이드 및 이의 C-말단에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공하는 것이다.More specifically, it is to provide a composition for delivering an endoplasmic reticulum-targeting peptide comprising an endoplasmic reticulum-targeting cell-penetrating peptide and an endoplasmic reticulum-targeting peptide linked to its N-terminus. Alternatively, it is to provide a composition for delivery of an endoplasmic reticulum targeting peptide comprising an endoplasmic reticulum target cell penetrating peptide and an endoplasmic reticulum delivery peptide linked to its C-terminus.
본 발명의 용어 "소포체 표적"이란, 세포 내 존재하는 소포체, 소포체 Lumen 및/또는 소포체 내부를 대상으로 하여 물질이 전달될 수 있음을 의미한다.The term "endoplasmic reticulum target" of the present invention means that a substance can be delivered targeting the endoplasmic reticulum, the endoplasmic reticulum lumen, and/or the inside of the endoplasmic reticulum present in a cell.
본 발명에 따른 세포 내 소포체 표적 펩타이드 전달용 조성물은 소포체 표적 세포 투과성 펩타이드를 이용하여 이에 직접 또는 링커나 스페이서를 통해 연결된 소포체 전달용 펩타이드를 소포체 내로 직접 전달할 수 있다.The composition for intracellular endoplasmic reticulum-targeting peptide delivery according to the present invention can directly deliver the endoplasmic reticulum-targeting peptide linked to the endoplasmic reticulum target cell-penetrating peptide or via a linker or spacer into the endoplasmic reticulum.
이에 따라, 구체적으로, 소포체 표적 세포 투과성 펩타이드; 이에 연결된 링커 또는 스페이서 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.Accordingly, specifically, the endoplasmic reticulum target cell penetrating peptide; Linker or spacer peptides linked thereto; and an endoplasmic reticulum-targeted peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
보다 구체적으로, 소포체 표적 세포 투과성 펩타이드; 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.More specifically, endoplasmic reticulum target cell penetrating peptides; a signal peptidase cleavage peptide linked thereto; and an endoplasmic reticulum-targeted peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
보다 구체적으로, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물을 제공한다.More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; and an endoplasmic reticulum-targeted peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
본 발명에 따른 소포체 표적 펩타이드 전달용 조성물은 소포체로의 전달을 목적하는 "소포체 표적 펩타이드"를 소포체로 전달하여 소포체에서의 단백질 발현 조절을 통하여 다양한 작용 효과를 나타낼 수 있다. 세포에서 생합성되는 단백질 중 세포에서 분비되거나 세포 내막에 분포하는 막단백질은 반드시 소포체(Endoplasmic Reticulum, ER)의 내부(lumen)로 이동하여 적절한 폴딩(folding)과 가공 과정을 거치게 된다. 소포체 내부에는 PDI(protein disulfide isomerase), BiP(heavy chain binding protein), calnexin(CNX), calreticulin(CRT) 등과 같은 샤페론(chaperone) 단백질들이 존재하여 소포체에서 생합성되는 단백질들이 적절한 폴딩을 이루고 가공되도록 도와주는데, 이러한 작용이 원활하게 이루어지지 않는 경우 다양한 질환의 문제를 발생시킬 수 있다.The composition for delivery of an endoplasmic reticulum-targeting peptide according to the present invention can deliver various functional effects by regulating protein expression in the endoplasmic reticulum by delivering the "ER-targeting peptide" intended for delivery to the endoplasmic reticulum. Among the proteins biosynthesized in cells, membrane proteins secreted from cells or distributed in the cell inner membrane must move to the lumen of the endoplasmic reticulum (ER) and undergo proper folding and processing. Inside the endoplasmic reticulum, chaperone proteins such as protein disulfide isomerase (PDI), heavy chain binding protein (BiP), calnexin (CNX), and calreticulin (CRT) exist to help proteins biosynthesized in the endoplasmic reticulum to be properly folded and processed. However, if these actions are not performed smoothly, problems of various diseases may occur.
본 발명에서는 위와 같은 소포체로의 펩타이드 전달 측면에서 목적하는 특정의 소포체 전달용 펩타이드를 소포체 내로 적절히 전달함으로써 목적하는 다양한 작용을 달성할 수 있다는 점에 장점을 가진다.In the present invention, in terms of peptide delivery to the endoplasmic reticulum, it is advantageous in that various desired actions can be achieved by appropriately delivering a specific target peptide for endoplasmic reticulum delivery into the endoplasmic reticulum.
특히, 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 시그널 펩티다아제(signal peptidase) 절단 펩타이드는 이에 다양한 소포체 전달용 펩타이드를 연결하여 소포체 내로 위 목적 펩타이드만을 전달함으로써 우수한 효과를 나타낼 수 있다. 본 발명에 따르면, 시그널 펩티다아제(signal peptidase) 절단 펩타이드를 통해 소포체 표적 세포 투과성 펩타이드를 소포체 전달용 펩타이드와 연결시켜서 소포체 표적 세포 투과성 펩타이드가 소포체 전달용 펩타이드를 소포체로 전달한 후에 위 절단 서열로 인해 위 두 펩타이드가 절단되고, 목적하는 소포체 전달용 펩타이드가 소포체 내로 전달될 수 있도록 한다.In particular, endoplasmic reticulum target cell penetrating peptides; And the signal peptidase cleavage peptide linked thereto can show excellent effects by linking various peptides for endoplasmic reticulum delivery to deliver only the target peptide into the endoplasmic reticulum. According to the present invention, an endoplasmic reticulum target cell penetrating peptide is linked to an endoplasmic reticulum delivery peptide via a signal peptidase cleavage peptide, so that the endoplasmic reticulum target cell penetrating peptide transfers the endoplasmic reticulum delivery peptide to the endoplasmic reticulum, and then the endoplasmic reticulum cleavage sequence results in the transfer of the endoplasmic reticulum to the endoplasmic reticulum. The peptide is cleaved, allowing the desired endoplasmic reticulum delivery peptide to be delivered into the endoplasmic reticulum.
본 발명에 따른 펩타이드는 포유동물에서 사용되는 것이 바람직하다. 이러한 포유동물은 바람직하게는 인간이지만, 조성물이 수의학 분야에서, 특히 가축 예컨대 소(젖소), 양, 염소 또는 말, 뿐만 아니라 개 또는 고양이와 같은 애완동물에서 면역을 유도하기 위해 사용될 때 다른 포유동물일 수도 있다.Peptides according to the present invention are preferably used in mammals. Such mammals are preferably humans, but other mammals when the composition is used in the veterinary field, in particular for inducing immunity in livestock such as cattle (dairy cows), sheep, goats or horses, as well as pets such as dogs or cats. It could be.
의약 용도medicinal use
백신 용도 vaccine use
본 발명은 소포체 표적 세포 투과성 펩타이드 및 소포체 전달용 펩타이드를 포함하는 백신 조성물을 제공한다. 백신은 특정 항원에 대하여 면역을 생성하거나 또는 인위적으로 증가시키기 위해 투여되는 조성물이다.The present invention provides a vaccine composition comprising an endoplasmic reticulum target cell penetrating peptide and an endoplasmic reticulum delivery peptide. A vaccine is a composition administered to create or artificially increase immunity against a specific antigen.
구체적으로, 소포체 표적 세포 투과성 펩타이드 및 면역원성 펩타이드를 포함하는 백신 조성물을 제공한다. 보다 더 구체적으로 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 면역원성 펩타이드를 포함하는 백신 조성물을 제공한다. 보다 더 구체적으로 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 면역원성 펩타이드는 링커 또는 스페이서 펩타이드로 연결될 수 있다. 보다 더 구체적으로, 이러한 링커 또는 스페이서 펩타이드는 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드일 수 있다.Specifically, a vaccine composition comprising an endoplasmic reticulum target cell penetrating peptide and an immunogenic peptide is provided. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and an immunogenic peptide linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And the immunogenic peptide linked thereto may be connected by a linker or spacer peptide. More specifically, this linker or spacer peptide may be a signal peptidase cleavage peptide of SEQ ID NO: 4.
바람직한 실시 양태는 앞서 살핀 융합 폴리펩타이드 및 소포체 표적 펩타이드 전달용 조성물에서 살핀 구체적 펩타이드 구성에 대한 실시 양태가 본 백신 조성물에서도 적용될 수 있다.In a preferred embodiment, the salpin fusion polypeptide and the embodiment of the salpin specific peptide configuration in the composition for delivering the endoplasmic reticulum-targeting peptide can also be applied to the present vaccine composition.
본 발명에 따른 조성물은 동물에서 면역 반응을 생성할 수 있는 조성물이다. 항원을 함유하고 그러한 항원에 대하여 면역 반응을 생성할 수 있는 조성물이다. 생성된 면역 반응은 세포(T-세포 매개) 또는 체액(B-세포 매개, 항체 생성) 면역 반응일 수 있다. 면역원성 조성물은 또한 세포 및 체액 면역 반응 모두를 유도할 수 있다.A composition according to the present invention is a composition capable of generating an immune response in an animal. A composition that contains an antigen and is capable of generating an immune response against such antigen. The immune response generated may be a cellular (T-cell mediated) or humoral (B-cell mediated, antibody producing) immune response. Immunogenic compositions are also capable of inducing both cellular and humoral immune responses.
세포 면역 반응은 CD8+ T 림프구 매개 반응(즉 세포독성 반응), 또는 CD4+ T 림프구 매개 반응(헬퍼 반응)일 수 있다. 또한 세포독성 및 헬퍼 세포 면역 반응을 조합할 수도 있다. 헬퍼 반응은 Th1, Th2 또는 Th17 림프구(이 러한 림프구는 당업계에 공지된 바와 같이 상이한 사이토카인 반응을 유도할 수 있음)를 포함할 수 있다. 조성물은 MHC1 또는 MHC2 경로를 통해 그 안에 존재하는 항원을 보다 잘 제시하도록 허용할 수 있다.The cellular immune response can be a CD8+ T lymphocyte mediated response (ie a cytotoxic response), or a CD4+ T lymphocyte mediated response (helper response). It is also possible to combine cytotoxic and helper cell immune responses. Helper responses can include Th1, Th2 or Th17 lymphocytes (such lymphocytes can elicit different cytokine responses as is known in the art). The composition may allow for better presentation of antigens present therein via the MHC1 or MHC2 pathway.
이에 따라, 본 발명의 백신 조성물은 면역원성 펩타이드로 적절 항원의 제시가 필요하며, 이러한 항원은 아래와 같이 고려될 수 있다. Accordingly, the vaccine composition of the present invention requires the presentation of appropriate antigens as immunogenic peptides, and these antigens can be considered as follows.
- 외인성 항원(예를 들어 흡입, 섭취 또는 주사에 의해 외부에서 몸으로 들어온 항원; 이들 항원은 일반적으로 MHC II 분자에 의해 제시된다).- exogenous antigens (antigens that enter the body from outside, eg by inhalation, ingestion or injection; these antigens are usually presented by MHC II molecules).
- 내인성 항원(정상 세포 대사의 결과로서 또는 바이러스 또는 세포 내 세균 감염으로 인해 정상 세포 내에서 생성된 항원; 이들 항원은 일반적으로 MHC I 분자에 의해 제시된다).- endogenous antigens (antigens produced within normal cells as a result of normal cellular metabolism or due to viral or intracellular bacterial infection; these antigens are usually presented by MHC I molecules).
- 네오항원(예컨대 종양 항원, 예컨대 바이러스 관련 종양에서의 바이러스 개방 판독 프레임으로부터 유래된 에피토프, 또는 종양 세포의 표면상에서 MHC I 또는 MHC II 분자에 의해 제시되는 다른 종양 항원).- neoantigens (eg tumor antigens, such as epitopes derived from viral open reading frames in virus-associated tumors, or other tumor antigens presented by MHC I or MHC II molecules on the surface of tumor cells).
- 알레르겐(면역글로불린E(IgE) 반응을 통해 아토피성 개인에서 유형-1 과민성 반응을 자극할 수 있는 항원).- Allergens (antigens that can stimulate type 1 hypersensitivity reactions in atopic individuals via an immunoglobulin E (IgE) response).
종양 항원의 예로서, 생식 세포 종양 및 간세포 암종에서 발견되는 알파태아단백질(AFP: alphafetoprotein), 장암에서 발견되는 암종배아 항원(CEA: carcino embryonic antigen), 난소암에서 발견되는 CA-125, 유방암에서 발견되는 MUC-1, 유방암에서 발견되는 상피성 종양 항원(ETA: epithelial tumor antigen), 라스(ras)의 비정상적인 생성물인 악성 흑색종에서 발견되는 티로시나아제 또는 흑색종 관련 항원(MAGE: melanoma-associated antigen), 다양한 종양에서 발견되는 p53, gp100(멜라닌세포 단백질 PMEL, 멜라노솜이 풍부한 I 형 막관통 당단백질), TRP2(티로시나아제 관련 단백질 2: Tyrosinase-Related Protein 2), EPHA2(수용체 티로신 키나아제, 신경아교종과 같은 넓은 범위의 진행성 암에서 빈번하게 과발현됨), 서바이빈(세포자멸 반복 함유 5의 바큘로바이러스 억제제 또는 BIRC5, 특히 유방암 및 폐암에서 발현됨), EGFRvIII(표피 성장 인자 수용체 돌연변이체, 특히 아교모세포종에서 발현됨)을 언급할 수 있다.Examples of tumor antigens include alphafetoprotein (AFP) found in germ cell tumors and hepatocellular carcinoma, carcino embryonic antigen (CEA) found in intestinal cancer, CA-125 found in ovarian cancer, and breast cancer. MUC-1 found in breast cancer, epithelial tumor antigen (ETA) found in breast cancer, tyrosinase found in malignant melanoma, an abnormal product of ras, or melanoma-associated antigen (MAGE) antigen), p53 found in various tumors, gp100 (melanocyte protein PMEL, melanosome-rich type I transmembrane glycoprotein), TRP2 (tyrosinase-related protein 2: Tyrosinase-Related Protein 2), EPHA2 (receptor tyrosine kinase, Frequently overexpressed in a wide range of advanced cancers such as glioma), survivin (a baculovirus inhibitor of apoptosis repeat-containing 5 or BIRC5, especially expressed in breast and lung cancer), EGFRvIII (an epidermal growth factor receptor mutant) , especially expressed in glioblastoma).
면역원성 조성물에 항원을 사용할 수 있는 병원체의 예로서, 감염성 질환(바이러스, 세균, 기생충, 진균증)과 관련된 임의의 병원체를 언급할 수 있다.As examples of pathogens whose antigens can be used in the immunogenic composition, mention may be made of any pathogen associated with infectious diseases (viruses, bacteria, parasites, mycoses).
감염성 질환에 대하여, 바람직한 병원체는 인간 면역 결핍 바이러스(HIV: human immune deficiency virus), A형 및 B형 간염 바이러스, C형 간염 바이러스(HCV: hepatitis C virus), 라우스 육종 바이러스(RSV: Rous sarcoma virus), 에볼라 바이러스, 세포거대바이러스, 헤르페스 바이러스, 수두대상포진 바이러스, 엡스타인 바바이러스(EBV: Epstein Barr virus), 인플루엔자 바이러스, 아데노바이러스, 로타바이러스, 홍역 및 풍진 바이러스, 천연두 바이러스, 스타필로코쿠스(Staphylococcus), 클라미디아에(Chlamydiae), 미코박테리움 투베르쿨로시스(Mycobacterium tuberculosis), 스트렙토코쿠스 뉴모니아에(Streptococcus pneumoniae), 바실루스 안트라시스(Bacillus anthracis), 비브리오 콜레라에(Vibrio cholerae), 헬리코박터 필로리이(Helicobacter Pilorii), 살모넬라(Salmonella), 플라스모디움 에스피.(Plasmodium sp .)(피. 팔시파룸(P. falciparum), 피.비박스(P. vivax), 등), 뉴모시스티스 카리니이(Pneumocystis carinii), 기아르디아 듀오데날리스(Giardia duodenalis)(람블편모충증(Giardiose)), 스키스토소마(Schistosoma)(주혈흡충증(Bilharziose)), 아스페르길루스(Aspergillus), 크립토코쿠스(Cryptococcus), 칸디다 알비칸스(Candida albicans), 리스테리아 모노사이토게네스(Listeria monocytogenes), 또는 톡소플라스마 곤디이(Toxoplasma gondii)로부터 선택된다.For infectious diseases, preferred pathogens include human immune deficiency virus (HIV), hepatitis A and B viruses, hepatitis C virus (HCV), Rous sarcoma virus (RSV). ), Ebola virus, cytomegalovirus, herpes virus, varicella zoster virus, Epstein Barr virus (EBV), influenza virus, adenovirus, rotavirus, measles and rubella virus, smallpox virus, staphylococcus ( Staphylococcus ), Chlamydiae ( Chlamydiae ), Mycobacterium tuberculosis ( Mycobacterium tuberculosis ), Streptococcus pneumoniae ( Streptococcus pneumoniae ), Bacillus anthracis ( Bacillus anthracis ), Vibrio cholerae ( Vibrio cholerae ), Helicobacter pylorii ( Helicobacter Pilorii ), Salmonella ( Salmonella ), Plasmodium sp. ( Plasmodium sp . ) (P. falciparum ( P. falciparum ), P. Bibox ( P. vivax ), etc.), Pneumocystis carinii ( Pneumocystis carinii ) , Giardia duodenalis duodenalis ) ( Giardiose ), Schistosoma ( Schistosoma ) ( Bilharziose ), Aspergillus ( Aspergillus ), Cryptococcus ( Cryptococcus ), Candida albicans ( Candida albicans ), Listeria mono Cytogenes ( Listeria monocytogenes ), or Toxoplasma gondii .
적절한 항원을 사용한 면역화의 혜택을 받을 수 있는 질환의 예로는 하기를 언급할 수 있다: 암(양성 또는 악성 종양); 혈액암, 알레르기, 자가 면역 질환, 죽상 동맥 경화증과 같은 만성 질환 또는 알츠하이머병.As examples of diseases that may benefit from immunization with appropriate antigens, mention may be made of: cancer (benign or malignant tumors); Chronic diseases such as blood cancer, allergies, autoimmune diseases, atherosclerosis or Alzheimer's disease.
따라서 항원은 바람직하게는 세균 또는 바이러스 항원(또는 세균 또는 바이러스 항원으로부터 단리된 하나 이상의 에피토프를 함유하는 펩타이드이다. 항원은 자가 항원(내인성 또는 네오항원), 특히 종양 특이적 항원(또는 이러한 항원으로부터 단리된 하나 이상의 에피토프를 함유하는 펩타이드)이다. 항원은 이러한 항원으로부터 단리된 하나 이상의 에피토프를 함유하는 펩타이드이다.Antigens are thus preferably bacterial or viral antigens (or peptides containing one or more epitopes isolated from bacterial or viral antigens). Antigens are self antigens (endogenous or neoantigens), especially tumor specific antigens (or isolated from such antigens). An antigen is a peptide containing one or more epitopes isolated from such an antigen.
바람직하게, 상기 면역원성 펩타이드는 항원으로서, MHC 에피토프 및 B-세포 에피토프로 구성된 군에서 선택된 적어도 하나의 에피토프를 포함하는 것일 수 있다.Preferably, the immunogenic peptide may contain at least one epitope selected from the group consisting of an MHC epitope and a B-cell epitope as an antigen.
상기 언급된 임의의 항원이 본 발명에 따른 면역원성 펩타이드로 이용될 수 있다.Any of the antigens mentioned above can be used as immunogenic peptides according to the present invention.
예를 들어, 면역원성 펩타이드의 임의의 예시로 SARS-CoV-2 MHC I epitope 서열은 KLWAQCVQL(서열번호 21), SPRWYFYYL(서열번호 22), TTDPSFLGRY (서열번호 23), SPRWYFYYL (서열번호 24) 등을 고려할 수 있으며, 이에 한정되지 않는다.For example, as an example of an immunogenic peptide, the SARS-CoV-2 MHC I epitope sequence is KLWAQCVQL (SEQ ID NO: 21), SPRWYFYYL (SEQ ID NO: 22), TTDPSFLGRY (SEQ ID NO: 23), SPRWYFYYL (SEQ ID NO: 24), etc. can be considered, but is not limited thereto.
또한, 면역원성 펩타이드의 임의의 예시로 Influenza virus MHC I epitope 서열은 SFQDILLRM(서열번호 25), VTVTHSVNL(서열번호 26), YQNIHPVTI(서열번호 27) 등을 고려할 수 있으며, 이에 한정되지 않는다.In addition, SFQDILLRM (SEQ ID NO: 25), VTVTHSVNL (SEQ ID NO: 26), YQNIHPVTI (SEQ ID NO: 27), etc. may be considered as an influenza virus MHC I epitope sequence as an example of an immunogenic peptide, but is not limited thereto.
예를 들어, 면역원성 펩타이드의 임의의 예시로 KRAS mutant cancer MHC I epitope 서열은 VVVGAVGVGK(서열번호 28) 등을 고려할 수 있으며, 이에 한정되지 않는다.For example, as an example of an immunogenic peptide, the KRAS mutant cancer MHC I epitope sequence may be VVVGAVGVGK (SEQ ID NO: 28), but is not limited thereto.
본 발명에서는 면역원성 펩타이드(면역원성 에피토프)의 일 예시로 Ovalbumin (OVA) 유래 MHC class I antigenic peptide (서열번호 3: SIINFEKL)을 사용하였다. 이를 통해 우수한 MHC1 presentation 작용을 나타냄을 확인하였다.In the present invention, an MHC class I antigenic peptide (SEQ ID NO: 3: SIINFEKL) derived from Ovalbumin (OVA) was used as an example of an immunogenic peptide (immunogenic epitope). Through this, it was confirmed that it exhibited an excellent MHC1 presentation action.
백신은 예방(즉, 질환의 발생에 대하여 수혜자를 보호하고자 하는 것) 또는 치료(즉, 수혜자가 이미 존재하는 질환과 싸우는 것을 돕고자 하는 것) 백신일 수 있다.Vaccines may be prophylactic (ie, intended to protect the recipient against the development of a disease) or therapeutic (ie, intended to help the recipient fight a pre-existing disease) vaccine.
그 질환은 백신 조성물에 사용된 표적 항원과 연결되어 있다. 결과적으로, 항원 또는 그의 에피토프는 질환의 과정 동안 동물의 세포(또는 병원체)에 의해 발현되거나 또는 제시된다. 질환은 따라서 표적 항원을 발현하는 세포를 포함하거나 세포에 관한 것이다. 이러한 발현은 항원(예시로서, 항원은 세균 독소일 수 있다)의 분비 또는 항원 또는 이의 에피토프의 표면 발현(항원은 바이러스의 표면 단백질 또는 종양 세포의 표면에서 발현되는 종양-특이적 항원 또는 이의 에피토프일 수 있다), 또는 세포 표면에서의 항원 또는 이의 에피토프의 제시(예컨대, 표적 세포에 의한 항원 또는 이의 에피토프의 MHC 제시)일 수 있다.The disease is linked to the target antigen used in the vaccine composition. Consequently, the antigen or its epitope is expressed or presented by the animal's cells (or pathogen) during the course of disease. A disease thus involves or relates to a cell that expresses a target antigen. Such expression may include secretion of an antigen (eg, the antigen may be a bacterial toxin) or surface expression of an antigen or epitope thereof (an antigen may be a surface protein of a virus or a tumor-specific antigen or epitope thereof expressed on the surface of a tumor cell). may), or presentation of an antigen or epitope thereof on a cell surface (eg, MHC presentation of an antigen or epitope thereof by a target cell).
필요에 따라, 본 발명의 조성물은 애쥬번트를 더 포함할 수 있다.If necessary, the composition of the present invention may further include an adjuvant.
"애쥬번트"는 항원에 대한 면역 반응을 변형 또는 증강시키는 능력을 갖는 물질이다. 다시 말해, 항원에 대한 면역 반응은 애쥬번트가 존재하지 않을 때보다 애쥬번트의 존재하에 더 높거나 상이할 수 있다(반응이 변형될 때, 예를 들어 애쥬번트의 존재하에 활성화된 T 세포의 서브세트가 애쥬번트의 부재하에 활성화된 서브세트와 상이할 때를 포함한다). 애쥬번트는 당업계에 공지되어 있으며 백신 분야에서 널리 사용되어왔다.An "adjuvant" is a substance having the ability to modify or enhance an immune response to an antigen. In other words, the immune response to the antigen may be higher or different in the presence of adjuvant than in the absence of adjuvant (when the response is modified, e.g., subtypes of T cells activated in the presence of adjuvant) (including when the set differs from the subset activated in the absence of adjuvant). Adjuvants are known in the art and have been widely used in the field of vaccines.
알럼(alum), 에멀션(수중유 또는 유중수, 예컨대 프로인트 불완전 애쥬번트(IFA) 및 [0195] MF59®), PRR(패턴 인식 수용체) 리간드, TLR3(톨유사 수용체 3) 및 RLR(RIG-I 유사 수용체) 리간드 예컨대 이중 가닥 RNA(dsRNA), 또는 dsRNA의 합성 유사체, 예컨대 폴리(I:C), TLR4 리간드 예컨대 세균 리포폴리사카라이드(LPS), MPLA(모노포스포릴 리피드 A), 특히 알럼과 함께 제형화됨, TLR5 리간드 예컨대 세균 플라젤린, TLR7/8 리간드 예컨대 이미다조퀴놀린(즉 이미퀴모드, 가르디퀴모드 및 R848), TLR9 리간드 예컨대 특이적 CpG 모티프를 함유하는 올리고데옥시뉴클레오티드(CpG ODN) 또는 NOD2(Nucleotide-binding oligomerization domain-containing protein 2: 뉴클레오티드 결합 올리고머화 도메인 함유 단백질 2) 리간드를 언급할 수 있다. 상기 용어 리간드는 바람직하게는 수용체의 작용제, 즉 수용체에 결합하고 특히 TLR3 및 TLR9 수용체에 대해 수용체를 활성화시키는 물질을 기술한다.Alum, emulsions (oil-in-water or water-in-oil, such as Freund's incomplete adjuvant (IFA) and MF59®), PRR (pattern recognition receptor) ligands, TLR3 (tall-like receptor 3) and RLR (RIG- I-like receptor) ligands such as double-stranded RNA (dsRNA), or synthetic analogs of dsRNA, such as poly(I:C), TLR4 ligands such as bacterial lipopolysaccharide (LPS), MPLA (monophosphoryl lipid A), especially alum Formulated with, TLR5 ligands such as bacterial flagellins, TLR7/8 ligands such as imidazoquinolines (i.e. imiquimod, gardiquimod and R848), TLR9 ligands such as oligodeoxynucleotides containing specific CpG motifs (CpG ODN ) or NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) ligand. The term ligand preferably describes an agonist of a receptor, i.e. a substance that binds to and activates the receptor, especially for the TLR3 and TLR9 receptors.
ER 스트레스 관련 질환ER stress-related disorders
바이러스나 미생물에 의한 감염이나 특정한 세포 분화, 혹은 각종 비생물적인 스트레스(abiotic stress) 등으로 소포체의 단백질 폴딩과 가공 과정이 방해를 받게 되면 소포체 내부에 적절하게 폴딩이 되지 않거나 당쇄 첨가 등의 적절한 가공이 이루어지지 않은 단백질이 축적되는데 이를 소포체 스트레스(ER stress)라고 한다. 다양한 세포 환경의 변화로 소포체 스트레스가 발생하면 세포는 다양한 경로를 통해 이를 해소하기 위해 노력하고, 스트레스가 장기화되면 세포는 치명적인 손상을 입고 아폽토시스(apoptosis)를 일으키게 된다. 이와 같은 소포체 스트레스 반응은 특히 단백질을 합성하여 분비하는 기능이 활발한 형질세포, 췌장의 베타세포, 간세포, 조골세포와 같은 곳에서 잘 관찰되고 있으며 최근 많은 연구에서 소포체 스트레스가 허혈, 당뇨병, 바이러스 감염, 고호모시스테인증, 돌연변이와 같은 여러 가지 질환의 병인으로 작용함을 보여 주고 있다.If the protein folding and processing process of the endoplasmic reticulum is hindered due to infection by viruses or microorganisms, specific cell differentiation, or various abiotic stresses, the endoplasmic reticulum may not be properly folded or appropriate processing such as addition of sugar chains may occur. The accumulation of unfinished proteins is called ER stress. When endoplasmic reticulum stress occurs due to changes in various cellular environments, cells try to relieve it through various pathways, and when the stress is prolonged, cells suffer fatal damage and cause apoptosis. This endoplasmic reticulum stress response is particularly well observed in plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts, which are active in synthesizing and secreting proteins. It has been shown to act as the etiology of various diseases such as hyperhomocysteinemia and mutation.
본 발명은 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물을 제공한다. 보다 구체적으로, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물을 제공한다. 보다 더 구체적으로 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드는 링커 또는 스페이서 펩타이드로 연결될 수 있다. 보다 더 구체적으로, 이러한 링커 또는 스페이서 펩타이드는 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드일 수 있다.The present invention is an endoplasmic reticulum target cell penetrating peptide; And it provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a peptide for endoplasmic reticulum delivery linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And it provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising a peptide for endoplasmic reticulum delivery linked thereto. More specifically, the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And the peptide for endoplasmic reticulum delivery linked thereto may be linked by a linker or spacer peptide. More specifically, this linker or spacer peptide may be a signal peptidase cleavage peptide of SEQ ID NO: 4.
본 발명에 따른 소포체 표적 세포 투과성 펩타이드 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 약제학적 조성물은 소포체 내에 발현을 목적하는 적절 펩타이드를 전달함으로써 소포체 스트레스 관련 질환의 치료가 가능하다.The pharmaceutical composition comprising the endoplasmic reticulum target cell penetrating peptide and the peptide for endoplasmic reticulum delivery linked thereto according to the present invention can treat endoplasmic reticulum stress-related diseases by delivering an appropriate peptide for expression in the endoplasmic reticulum.
상기 소포체 스트레스 관련 질환은 제1형 당뇨, 제2형 당뇨, 알츠하이머 질환 (Alzheimer's disease), 면역글로블린 경쇄 아밀로이드증 (immumoglobulin light chain amyloidosis), 파킨슨병 (Parkinson's disease), 근위축성 측삭경화증 (amyotrophic lateral sclerosis, ALS), 혈액투석 연관성 아밀로이드증 (haemodialysis-related amyloidosis), 활동성 아밀로이드증 (reactive amyloidosis), 낭포성 섬유증 (cystic fibrosis), 겸상적혈구빈혈증 (sickle cell anemia), 헌팅턴병 (Huntington's disease), 크로이츠펠트-야콥 질환 (Kreutzfeldt-Jakob disease), 가족성 고콜레스테롤혈증 (familial hypercholesterolaemia), 알파1-항트립신 결핍증 (Alpha1-antitrypsin deficiency), 경변 (cirrhosis), 전신성 폐기종 (emphysema systemic), 뇌성 유전성 아밀로이드증(cerebral hereditary amyloidoses), 울콧-랠리스 증후군 (Wolcott-Rallison syndrome), 울프람 증후군(Wolfram syndrome), 염증성 장질환, 코론씨 병, 궤양성 대장염, 유방암 및 전립선암 등을 포함한다.The endoplasmic reticulum stress-related diseases include type 1 diabetes, type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, ALS), haemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease ( Kreutzfeldt-Jakob disease), familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, Coron's disease, ulcerative colitis, breast and prostate cancer, and the like.
즉, 본 발명의 조성물은 소포체 내로 목적하는 펩타이드를 특이적 및 효율적으로 전달함으로써 다양한 의약 분야에 이용될 수 있다.That is, the composition of the present invention can be used in various fields of medicine by specifically and efficiently delivering a desired peptide into the endoplasmic reticulum.
본 발명의 일실시양태에 따르면, 치료를 목적하는 소포체 내로 목적하는 펩타이드는 항산화제 작용을 나타낼 수 있는 펩타이드일 수 있다. 예를 들어, 활성산소는 세포에 증식과 성장 혹은 세포사멸 등 다양한 작용을 한다고 알려져 있는데, 활성산소의 생체내 반감기는 매우 짧아 국소적으로 이의 조정을 통해 치료 효능을 나타낼 수 있다. 예를 들어, 세포 내 특정 위치에서 발생되는 유해한 활성산소를 제거하기 위해 표적화한 항산화제 개발을 고려할 수 있고, 이는 과량의 장기간 발생하는 활성산소로 인한 다양한 질환에 중요한 치료제로 이용 가능하다. 이러한 항산화 효능을 가지는 펩타이드는 이에 국한되지 않으나, 예를 들어, 아세틸시스테인 (N-Acetylcysteine), 글루타치온 (glutathione), SOD-유사(SODmimicking) 펩타이드 및 제토-실러-펩타이드(Szeto-Schiller-peptides)로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.According to one embodiment of the present invention, the desired peptide into the endoplasmic reticulum for treatment may be a peptide capable of exhibiting antioxidant action. For example, active oxygen is known to have various effects on cells, such as proliferation, growth, or apoptosis. However, the half-life of active oxygen in vivo is very short, and therapeutic efficacy can be exhibited through its local adjustment. For example, it is possible to consider the development of targeted antioxidants to remove harmful reactive oxygen species generated at specific locations within cells, which can be used as an important therapeutic agent for various diseases caused by excessive reactive oxygen species generated over a long period of time. Peptides having such an antioxidant effect are not limited thereto, but, for example, acetylcysteine (N-Acetylcysteine), glutathione (glutathione), SOD-like (SODmimicking) peptides and Zeto-Schiller-peptides (Szeto-Schiller-peptides) It may be any one selected from the group consisting of
또한, N,N'-dimethylthiourea(DMTU), 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide(PTIO), α-tocopherol, manganese(III)-tetrakis(4-benzoic acid)porphyrin(MnTBAP)와 같은 화합물 기반 항산화제들도 소포체 표적 세포투과성 펩타이드와 함께 사용 가능하다.In addition, N,N'-dimethylthiourea (DMTU), 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), α-tocopherol, manganese(III)-tetrakis (4-benzoic Compound-based antioxidants such as acid)porphyrin (MnTBAP) can also be used together with endoplasmic reticulum-targeting cell-penetrating peptides.
본 발명에 따른 백신 조성물 또는 약제학적 조성물은 포유류, 바람직하게는 인간에게 사용될 수 있으며 예컨대 정맥내 (intravein), 복막내 (intraperitoneal), 근육내 (intramuscular), 피하내 (subcutaneous), 피내 (intradermal), 비내 (nasal), 점막내 (mucosal), 흡입 (inhalation) 및 경구 (oral) 등의 경로로 주입함으로써 생체 내 소포체에 목적하는 펩타이드를 전달할 수 있다.The vaccine composition or pharmaceutical composition according to the present invention can be used in mammals, preferably humans, such as intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal , It is possible to deliver the desired peptide to the endoplasmic reticulum in vivo by injecting it through routes such as nasal, mucosal, inhalation, and oral.
본 발명에서 용어, "치료"는 질병 또는 질환의 억제, 경감을 의미한다. 따라서, 본 발명에서 용어 "치료학적 유효량"은 상기 약리학적 효과를 달성하는데 충분한 양을 의미한다.As used herein, the term "treatment" means suppression or alleviation of a disease or disorder. Accordingly, the term "therapeutically effective amount" in the present invention means an amount sufficient to achieve the pharmacological effect.
상기 백신 조성물 또는 약제학적 조성물은 적절한 형태로 제제화되어 제공될 수 있다. 상기 제제는 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 연고제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 또는 경피제, 좌제 및 멸균 주사용액의 형태의 비경구 제형 등으로 제형화하여 사용될 수 있다.The vaccine composition or pharmaceutical composition may be formulated and provided in an appropriate form. According to conventional methods, the above formulations may be formulated into oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, etc., or parenteral formulations in the form of transdermal preparations, suppositories and sterile injection solutions, etc. It can be formulated and used.
또한, 상기 제제는 약제학적으로 적합하고 생리학적으로 허용되는 담체, 부형제 및 희석제 등의 보조제를 추가로 함유하는 것일 수 있다. 본 발명의 백신 조성물 또는 약제학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다.In addition, the formulation may further contain adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients and diluents. Carriers, excipients and diluents that may be included in the vaccine composition or pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used.
보다 구체적으로, 상기 제제는 상기 백신 조성물 또는 약제학적 조성물 (유효성분)에 추가하여 이를 제형화하기 위한 담체를 포함할 수 있다. 상기 담체는 결합제, 활탁제, 현탁용제, 가용화제, 완충제, 보존제, 윤활제, 등장제, 부형제, 안정화제, 분산제, 현탁화제, 색소, 향료 등을 사용할 수 있다.More specifically, the formulation may include a carrier for formulation in addition to the vaccine composition or pharmaceutical composition (active ingredient). The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersing agent, a suspending agent, a colorant, a flavoring agent, and the like.
상기 백신 조성물 또는 약제학적 조성물의 구체예에 있어서, 상기 조성물은 단독으로 투여될 수 있으나, 일반적으로 투여방식과 표준 약제학적 관행(standard phamaceutical practice)을 고려하여 선택된 약제학적 담체와 혼합되어 투여될 수 있다.In the specific embodiment of the vaccine composition or pharmaceutical composition, the composition may be administered alone, but in general, it may be administered in combination with a pharmaceutical carrier selected in consideration of the method of administration and standard pharmaceutical practice. there is.
예를 들면, 상기 제제를 비경구용으로 제공하는 경우, 일 예로 액제, 겔(gel)제, 세정 조성물, 삽입용 정제, 좌제 형태, 크림, 연고, 드레싱 용액, 분무제, 기타 도포제등의 국소 투여제, 용액형, 현탁형, 유제형 등의 액상제형일 수 있으며, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제, 크림, 연고, 젤리, 거품, 세척제 또는 삽입물, 바람직하게는 액제, 겔 (gel)제, 세정 조성물, 삽입용 정제 등의 피부 외용제가 포함될 수 있다. 상기 제형은 일 예로 멸균수에 용해보조제, 유화제, pH 조절을 위한 완충제 등을 첨가하여 제조할 수 있다. 상기 비수성용제 또는 현탁용제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌글리콜 (polyethylene glycol), 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.For example, when the preparation is provided for parenteral use, for example, topical administration such as solutions, gels, cleaning compositions, tablets for insertion, suppositories, creams, ointments, dressing solutions, sprays, and other coating agents. , It may be a liquid formulation such as a solution type, a suspension type, an emulsion type, and a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, a cream, an ointment, a jelly, a foam, a detergent or an insert, preferably External skin preparations such as liquid preparations, gel preparations, cleansing compositions, and tablets for insertion may be included. The formulation may be prepared by adding, for example, a solubilizing agent, an emulsifying agent, a buffering agent for pH control, etc. to sterilized water. As the non-aqueous solvent or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
또한, 상기 제제를 경구용으로 제공하는 경우, 일 예로 전분 또는 락토오즈를 함유하는 정제 형태로, 또는 단독 또는 부형제를 함유하는 캡슐 형태로, 또는 맛을 내거나 색을 띄게 하는 화학 약품을 함유하는 엘릭시르 또는 현탁제 형태로 경구, 구강 내 또는 혀 밑 투여될 수 있다.In addition, when the preparation is provided for oral use, for example, in the form of a tablet containing starch or lactose, or in the form of a capsule containing alone or an excipient, or an elixir containing a flavoring or coloring chemical. Or it can be administered orally, buccally or sublingually in the form of a suspension.
상기 제제의 투여 용량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 의사 또는 약사의 판단에 따라 일정 시간간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다. 예컨대, 유효성분 함량을 기준으로 1일 투여량이 0.001 내지 10000 ㎎/kg, 0.01 내지 10000 ㎎/kg, 0.1 내지 10000 ㎎/kg, 0.5 내지 10000 ㎎/kg, 0.001 내지 1000 ㎎/kg, 0.01 내지 1000 ㎎/kg, 0.1 내지 1000 ㎎/kg, 0.5 내지 1000 ㎎/kg, 0.001 내지 500 ㎎/kg, 0.01 내지 500 ㎎/kg, 0.1 내지 500 ㎎/kg, 0.5 내지 500 ㎎/kg, 0.001 내지 300 ㎎/kg, 0.01 내지 300 ㎎/kg, 0.1 내지 300 ㎎/kg, 또는 0.5 내지 300 ㎎/kg일 수 있다. 상기한 투여량은 평균적인 경우를 예시한 것으로서 개인적인 차이에 따라 그 투여량이 높거나 낮을 수 있다.The dosage of the formulation may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be divided into once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. there is. For example, based on the active ingredient content, the daily dosage is 0.001 to 10000 mg/kg, 0.01 to 10000 mg/kg, 0.1 to 10000 mg/kg, 0.5 to 10000 mg/kg, 0.001 to 1000 mg/kg, 0.01 to 1000 0.1 to 1000 mg/kg, 0.5 to 1000 mg/kg, 0.001 to 500 mg/kg, 0.01 to 500 mg/kg, 0.1 to 500 mg/kg, 0.5 to 500 mg/kg, 0.001 to 300 mg /kg, 0.01 to 300 mg/kg, 0.1 to 300 mg/kg, or 0.5 to 300 mg/kg. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.
상기 제제의 1일 투여량이 상기 투여 용량 미만이면 유의성 있는 효과를 얻을 수 없으며, 그 이상을 초과하는 경우 비경제적일 뿐만 아니라 상용량의 범위를 벗어나므로 바람직하지 않은 부작용이 나타날 우려가 발생할 수 있으므로, 상기 범위로 하는 것이 좋다.If the daily dose of the formulation is less than the dose, a significant effect cannot be obtained, and if it exceeds the dose, it is not only uneconomical but also out of the range of the usual dose, so undesirable side effects may occur. It's good to have a range.
상기 제제의 투여 대상은 인간 등의 포유류, 포유류로부터 분리된 세포, 조직, 체액, 또는 이들의 배양물일 수 있다.The administration target of the agent may be mammals such as humans, cells, tissues, body fluids, or cultures thereof isolated from mammals.
본 발명은 상기 백신 조성물의 치료 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 질병 또는 장애의 치료 또는 예방 방법을 제공한다.The present invention provides a method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount of the vaccine composition to a subject in need thereof.
본 발명은 상기 약제학적 조성물의 치료 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, ER 스트레스 관련 질환의 치료 또는 예방 방법을 제공한다.The present invention provides a method for treating or preventing ER stress-related diseases, comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 세포에 처리하는 단계를 포함하는 세포 내 소포체로의 소포체 전달용 펩타이드 전달 방법을 제공한다.The present invention provides a method for delivering a peptide for endoplasmic reticulum delivery to intracellular endoplasmic reticulum, comprising the step of treating a cell with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 대상체에 처리하는 단계를 포함하는 소포체로 소포체 전달용 펩타이드 전달 방법을 제공한다.The present invention provides a method for delivering peptides to the endoplasmic reticulum, comprising the step of treating a subject with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 세포에 처리하는 단계를 포함하는 세포 내 소포체로의 소포체 전달용 펩타이드의 선택적 전달 방법을 제공한다.The present invention provides a method for selective delivery of peptides for endoplasmic reticulum delivery to intracellular endoplasmic reticulum, comprising the step of treating cells with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명은 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 대상체에 처리하는 단계를 포함하는 소포체로 소포체 전달용 펩타이드의 선택적 전달 방법을 제공한다.The present invention provides a method for selectively delivering a peptide for endoplasmic reticulum delivery to the endoplasmic reticulum, comprising the step of treating a subject with a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide.
본 발명은 상기 질병 또는 장애의 치료 또는 예방에 사용하기 위한 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 포함하는 백신 조성물을 제공한다.The present invention provides a vaccine composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for use in the treatment or prevention of the above diseases or disorders.
본 발명은 ER 스트레스 관련 질환의 치료 또는 예방에 사용하기 위한 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 포함하는 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for use in the treatment or prevention of ER stress-related disorders.
본 발명은 세포 내 소포체로의 소포체 전달용 펩타이드를 전달하기 위한 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 포함하는 조성물을 제공한다.The present invention provides a composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for delivering an endoplasmic reticulum delivery peptide to an intracellular endoplasmic reticulum.
본 발명은 소포체로의 소포체 전달용 펩타이드를 전달하기 위한 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드를 포함하는 조성물을 제공한다.The present invention provides a composition comprising a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide for delivery of an endoplasmic reticulum delivery peptide to the endoplasmic reticulum.
본 발명은 상기 질병 또는 장애의 치료 또는 예방을 위한 백신의 제조에 있어 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드의 용도를 제공한다.The present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a vaccine for the treatment or prevention of the above diseases or disorders.
본 발명은 ER 스트레스 관련 질환의 치료 또는 예방에 사용하기 위한 약제의 제조에 있어 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드의 용도를 제공한다.The present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a medicament for use in the treatment or prevention of ER stress-related disorders.
본 발명은 세포 내 소포체로의 소포체 전달용 펩타이드를 전달하기 위한 제제의 제조에 있어 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드의 용도를 제공한다.The present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the preparation of a formulation for delivering an endoplasmic reticulum delivery peptide to an intracellular endoplasmic reticulum.
본 발명은 소포체로의 소포체 전달용 펩타이드를 전달하기 위한 제제의 제조에 있어 소포체 표적 세포 투과성 펩타이드를 포함하는 융합 폴리펩타이드의 용도를 제공한다.The present invention provides the use of a fusion polypeptide comprising an endoplasmic reticulum target cell penetrating peptide in the manufacture of a formulation for delivery of an endoplasmic reticulum delivery peptide to the endoplasmic reticulum.
본 발명은 또한, 상기 언급된 조성물들의 용도 및 활용 방법을 제공한다.The present invention also provides uses and methods of utilizing the aforementioned compositions.
본 발명의 융합 폴리펩타이드는 세포 내 소포체로 목적하는 펩타이드 전달을 효율적으로 할 수 있다. 이에 따라, 백신, 의약품 전달체 및 치료제로 이용 가능하며 생체 내 다양한 생리현상을 조절하여 예방 및 치료적 목적으로 이용 가능하다는 점에 우수한 장점을 가진다.The fusion polypeptide of the present invention can efficiently deliver a desired peptide to the intracellular endoplasmic reticulum. Accordingly, it has an excellent advantage in that it can be used as a vaccine, a drug delivery system, and a therapeutic agent, and can be used for preventive and therapeutic purposes by controlling various physiological phenomena in vivo.
도 1은 confocal microscopy를 이용하여 본 발명에 따른 소포체 표적 세포 투과성 펩타이드 서열의 소포체 표적을 확인한 결과를 나타낸다.Figure 1 shows the results of confirming the endoplasmic reticulum target of the endoplasmic reticulum target cell penetrating peptide sequence according to the present invention using confocal microscopy.
도 2는 본 발명에 따른 융합펩타이드의 개략적 모식도의 일예시를 나타낸다.Figure 2 shows an example of a schematic schematic diagram of a fusion peptide according to the present invention.
도 3은 본 발명에 따른 융합펩타이드를 처리하여 MHC class I loading 효율을 분석한 결과를 나타낸다.Figure 3 shows the results of analyzing the MHC class I loading efficiency by treating the fusion peptide according to the present invention.
도 4는 본 발명에 따른 융합펩타이드를 처리하여 CD8+ T cell 활성 효율을 분석한 결과를 나타낸다.Figure 4 shows the results of analyzing the CD8+ T cell activation efficiency by treating the fusion peptide according to the present invention.
도 5는 혈관내피세포에서 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 펩타이드(cell-penetrating peptide)의 세포 내 위치를 분석한 결과를 나타낸다.5 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in vascular endothelial cells.
도 6은 자궁경부암 기원 상피세포에서 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 펩타이드(cell-penetrating peptide)의 세포 내 위치를 분석한 결과를 나타낸다.6 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in cervical cancer-derived epithelial cells.
도 7은 대식세포에서 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 펩타이드(cell-penetrating peptide)의 세포 내 위치를 분석한 결과를 나타낸다.7 shows the results of analyzing the intracellular location of endoplasmic reticulum (ER) target cell-penetrating peptides in macrophages.
도 8은 자궁경부암 기원 상피세포에서 Penetratin의 처리에 따른 펩타이드의 세포 내 위치를 분석한 결과를 나타낸다.8 shows the result of analyzing the intracellular location of peptides according to the treatment with Penetratin in epithelial cells of cervical cancer origin.
도 9는 세포에 ER-CPP 서열을 처리한 후 시간 및 농도에 따른 세포 투과 효율을 확인한 결과를 나타낸다.9 shows the result of confirming cell penetration efficiency according to time and concentration after treating cells with ER-CPP sequences.
도 10은 DC 2.4 세포에서 ER-CPP의 항원 제시 효율을 세포표면의 MHC I-loaded epitope의 발현 수준을 통해 확인한 결과를 나타낸다.10 shows the result of confirming the antigen presentation efficiency of ER-CPP in DC 2.4 cells through the expression level of MHC I-loaded epitope on the cell surface.
도 11는 ER-CPP의 CD8+ T 세포 활성 효과를 확인한 결과를 나타낸다.11 shows the results confirming the CD8+ T cell activation effect of ER-CPP.
도 12는 마우스 모델에 ER-CPP의 처리를 통하여 혈액 내의 CD8+ T세포가 그 항원을 인식하여 활성이 증가하는 것을 확인한 결과를 나타낸다.12 shows the result of confirming that CD8+ T cells in the blood recognize the antigen and increase their activity through treatment of the mouse model with ER-CPP.
도 13은 항산화제를 탑재한 ER-CPP가 세포 내 소포체로 표적함을 확인한 결과를 나타낸다.13 shows the results confirming that ER-CPP loaded with antioxidants target the intracellular endoplasmic reticulum.
도 14는 항산화제를 탑재한 ER-CPP가 ER-스트레스에 의한 세포사멸을 억제하는 것을 확인한 결과를 나타낸다.Figure 14 shows the results confirming that the antioxidant-loaded ER-CPP inhibits ER-stress-induced apoptosis.
도 15는 항산화제를 탑재한 ER-CPP가 ER-스트레스에 의한 세포사멸을 억제하는 것을 확인한 결과를 나타낸다.Figure 15 shows the results confirming that ER-CPP loaded with antioxidants inhibits apoptosis caused by ER-stress.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예 1. 소포체 표적 세포 투과성 서열의 확인 및 합성Example 1. Identification and synthesis of endoplasmic reticulum target cell penetrating sequences
viral, bacterial 및 mammalian signal peptide 서열들을 기초로 Deep learning 기반 세포 투과성 분석을 통해 세포 투과성을 가지면서 소포체를 표적할 수 있는 서열들을 확인하였다.Based on viral, bacterial and mammalian signal peptide sequences, sequences capable of targeting the endoplasmic reticulum with cell permeability were identified through deep learning-based cell permeability analysis.
Deep learning 기반 세포 투과성 분석에 따라 상위 랭크로 분류된 서열은 아래와 같다:Sequences classified as high ranks according to deep learning-based cell permeability analysis are as follows:
서열번호 1: MLPGLALLLLAAWTARA (Amyloid-beta precursor protein 유래 signal sequence, Human)SEQ ID NO: 1: MLPGLALLLLAAWTARA (signal sequence derived from Amyloid-beta precursor protein, Human)
서열번호 2: MLP S LALLLLAAWT V RA (Amyloid-beta precursor protein 유래 signal sequence, Mouse)SEQ ID NO: 2: MLP S LALLLLAAWT V RA (signal sequence derived from Amyloid-beta precursor protein, Mouse)
상기 서열을 비롯하여 본 발명에서 사용된 폴리펩타이드 서열은 ㈜펩트론에 합성 의뢰하였으며, 이를 이용하여 이하 실험들을 수행하였다.The polypeptide sequences used in the present invention, including the above sequences, were synthesized by Peptron Co., Ltd., and the following experiments were performed using them.
실시예 2. 소포체 표적 세포 투과성 펩타이드 서열의 소포체 표적화 확인Example 2. Confirmation of endoplasmic reticulum targeting of endoplasmic reticulum target cell penetrating peptide sequences
상기 실시예 1에서 합성한 서열번호 1의 아미노산 서열의 N-terminus에 TITC를 결합시킨 후, 세포 투과성 펩타이드 서열이 소포체로 전달되는지 여부를 확인하였다.After binding TITC to the N-terminus of the amino acid sequence of SEQ ID NO: 1 synthesized in Example 1, it was confirmed whether the cell-permeable peptide sequence was delivered to the endoplasmic reticulum.
구체적으로, 세포 내 투과 및 소포체 내 도달을 confocal microscopy를 이용하여 확인하였다.Specifically, intracellular penetration and arrival in the endoplasmic reticulum were confirmed using confocal microscopy.
상기 실험 결과를 도 1에 나타내었다.The experimental results are shown in FIG. 1 .
도 1에서 확인할 수 있는 바와 같이, 본 발명에 따른 소포체 표적 세포 투과성 펩타이드가 소포체에 위치하는 것을 확인하였다. 상기 결과를 통해 본 발명에 따른 소포체 표적 세포 투과성 서열을 포함하는 경우 세포 내 및/또는 세포 내 세포 소기관 중 소포체로 펩타이드의 전달이 이루어지는 것을 확인하였다.As can be seen in Figure 1, it was confirmed that the endoplasmic reticulum target cell penetrating peptide according to the present invention is located in the endoplasmic reticulum. Through the above results, it was confirmed that when the endoplasmic reticulum target cell-penetrating sequence according to the present invention was included, the peptide was delivered to the endoplasmic reticulum of intracellular and/or intracellular organelles.
실시예Example 3. 소포체 표적 세포 투과성 3. Endoplasmic Reticulum Target Cell Permeability 펩타이드peptide 서열을 포함하는 융합 Fusions involving sequences 폴리펩타이드의of the polypeptide 제조 Produce
위 실시예 2에서 본 발명에 따른 소포체 표적 세포 투과성 펩타이드가 소포체에 위치하는 것을 확인하였다.In Example 2 above, it was confirmed that the endoplasmic reticulum target cell penetrating peptide according to the present invention was located in the endoplasmic reticulum.
이에 따라, 위 펩타이드(서열)를 이용하여 목적하는 펩타이드를 소포체로 전달할 수 있는지 확인하기 위하여 융합 폴리펩타이드를 제조하였다.Accordingly, a fusion polypeptide was prepared to confirm whether the desired peptide could be delivered to the endoplasmic reticulum using the above peptide (sequence).
서열번호 1을 기초로 하여 소포체에 전달을 목적으로 하는 펩타이드 서열을 직접 연결시키거나, 소포체 표적 세포 투과성 펩타이드 서열의 C terminus에 signal peptidase 효소 인지 서열(시그널 펩티다아제 절단 펩타이드 서열)을 연결한 후, 전달을 목적하는 서열(소포체 전달용 펩타이드)을 연결하였다.Based on SEQ ID NO: 1, the peptide sequence for delivery to the endoplasmic reticulum is directly linked, or a signal peptidase enzyme recognition sequence (signal peptidase cleavage peptide sequence) is linked to the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence, then delivery The desired sequence (peptide for endoplasmic reticulum delivery) was linked.
소포체 표적 세포 투과성 펩타이드 서열의 C terminus에 signal peptidase 효소 서열을 연결한 후 전달을 목적하는 소포체 전달용 펩타이드 서열을 연결한, 융합 폴리펩타이드의 개략적인 서열 구조를 도 2에 나타내었다. 도 2에서 확인할 수 있는 바와 같이, 소포체 표적 세포 투과성 펩타이드 서열(ER-CPP)의 C terminus에 신호 서열(ER-CPP)에 대한 펩티다아제 절단 인지 서열(시그널 펩티다아제 절단 펩타이드 서열, 일예시로 "EA")을 연결시켜 signal peptidase 효소가 이를 인지하여 절단하고, ER-CPP 서열과 전달을 목적하는 소포체 전달용 펩타이드 서열을 분리시킬 수 있다. 이에 따라 전달을 목적하는 소포체 전달용 펩타이드 서열(예를 들어, 에피토프 등)을 소포체 내에 전달시키는 것으로 구성될 수 있다.Figure 2 shows a schematic sequence structure of a fusion polypeptide in which a signal peptidase enzyme sequence is linked to the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence and then a peptide sequence for endoplasmic reticulum delivery is linked. As can be seen in Figure 2, the peptidase cleavage recognition sequence for the signal sequence (ER-CPP) at the C terminus of the endoplasmic reticulum target cell penetrating peptide sequence (ER-CPP) (signal peptidase cleavage peptide sequence, for example "EA" ), the signal peptidase enzyme recognizes and cuts it, and the ER-CPP sequence and the peptide sequence for delivery to the endoplasmic reticulum for delivery can be separated. Accordingly, it may be constituted by delivering a peptide sequence (eg, an epitope, etc.) for delivery to the endoplasmic reticulum for delivery into the endoplasmic reticulum.
본 발명에서는 소포체 내로 전달을 목적하는 소포체 전달용 펩타이드의 일 예시로 Ovalbumin (OVA) 유래 MHC class I antigenic peptide인 siinfekl(서열번호 3)을 사용하였다.In the present invention, siinfekl (SEQ ID NO: 3), an MHC class I antigenic peptide derived from Ovalbumin (OVA), was used as an example of an endoplasmic reticulum delivery peptide intended for delivery into the endoplasmic reticulum.
실험 진행을 위한 구체적 합성 진행은 하기 표 1와 같이 진행하였다.Specific synthetic progress for the experiment proceeded as shown in Table 1 below.
서열명sequence name 서열order 서열번호sequence number
ER-CPP+EA+OVAER-CPP+EA+OVA MLPGLALLLLAAWTARA EA SIINFEKL MLPGLALLLLAAWTARA EA SIINFEKL 1010
ER-CPP+OVAER-CPP+OVA MLPGLALLLLAAWTARA SIINFEKLMLPGLALLLLAAWTARA SIINFEKL 99
ER-CPP+EA+OVA(Negative)ER-CPP+EA+OVA(Negative) MLPGLALLLLAAWTARA EA SIIN Y EKLMLPGLALLLLAAWTARA EA SIIN Y EKL 1313
상기 서열번호 10은 서열번호 1, 서열번호 4 및 서열번호 3의 본 발명에 따른 소포체 표적 세포 투과성 서열; 시그널 펩티다아제 절단 펩타이드 서열; 및 면역원성 펩타이드 서열을 연결한 서열을 나타낸다.서열번호 9는 시그널 펩티다아제 절단 펩타이드 서열을 제외한 본 발명에 따른 서열을 나타내고,SEQ ID NO: 10 is the endoplasmic reticulum target cell-permeable sequence of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 3 according to the present invention; signal peptidase cleavage peptide sequence; and a sequence linking the immunogenic peptide sequence. SEQ ID NO: 9 represents the sequence according to the present invention excluding the signal peptidase cleavage peptide sequence;
서열번호 13은 OVA의 서열을 달리하여 MHC1 presentation 효과가 나타나는지 여부를 확인하기 위한 서열이다.SEQ ID NO: 13 is a sequence for confirming whether the MHC1 presentation effect occurs by changing the OVA sequence.
실시예Example 4. 소포체 표적 세포 투과성 4. Endoplasmic Reticulum Target Cell Permeability 펩타이드peptide 서열을 이용한 using sequence MHCMHC I I epitopeepitope presentation 효과 확인 Check presentation effect
상기 실시예 3에서 합성한 융합 폴리펩타이드 서열들을 이용하여, 소포체 표적 세포 투과성 펩타이드가 목적하는 펩타이드를 소포체로 전달한 후 원하는 작용을 나타낼 수 있는지 여부를 확인하였다.Using the fusion polypeptide sequences synthesized in Example 3, it was confirmed whether the endoplasmic reticulum target cell penetrating peptide could exhibit a desired effect after delivering the desired peptide to the endoplasmic reticulum.
구체적으로, 상기 예시적인 전달 펩타이드인 Ovalbumin (OVA) 유래 MHC class I antigenic peptide를 이용하여 MHC 1 presentation이 이루어지는지 여부를 확인하였다.Specifically, using the MHC class I antigenic peptide derived from Ovalbumin (OVA), which is the exemplary delivery peptide, it was confirmed whether MHC 1 presentation was made.
구체적으로, 항원과 소포체 표적 세포 투과성 서열이 직접 또는 링커를 통해 결합된 융합 폴리펩타이드가 수지상 세포 내에서 process되어 항원 peptide가 MHC class I으로 presentation (교차제시)되었는지를 측정하기 위해 수지상세포에 4μM peptide를 3시간 동안 노출시켰다. 그리고 나서, 3시간 뒤 배양액을 교체해줌으로써 배지에 남아있는 peptide를 제거하였다. 24시간 뒤 수지상 세포 세포막에 SINFEKL-MHC class I complex (MHC I-항원 펩타이드 complex)가 발현하고 있는지를 알아보기 위해 SINFEKL-MHC class I complex를 인식하는 항체를 붙인 뒤 전체 수지상세포 중 항체를 지닌 세포의 비율을 flow cytometry로 분석하였다.Specifically, a fusion polypeptide in which an antigen and an endoplasmic reticulum target cell-penetrating sequence are bonded directly or via a linker is processed in dendritic cells to determine whether the antigen peptide is presented (cross-presented) as MHC class I by adding 4 μM peptide to dendritic cells. was exposed for 3 hours. Then, the peptide remaining in the medium was removed by replacing the culture medium after 3 hours. After 24 hours, in order to determine whether the SINFEKL-MHC class I complex (MHC I-antigen peptide complex) is expressed on the cell membrane of dendritic cells, an antibody recognizing the SINFEKL-MHC class I complex was attached, and cells with the antibody among all dendritic cells The ratio of was analyzed by flow cytometry.
상기 실험 결과를 도 3에 나타내었다. The experimental results are shown in FIG. 3 .
도 3에서 확인할 수 있는 바와 같이, 본 발명에 따른 소포체 표적 세포 투과성 펩타이드 서열을 사용하는 경우 우수한 MHC1 presentation 작용을 나타냄을 확인하였다.As can be seen in Figure 3, it was confirmed that the endoplasmic reticulum target cell penetrating peptide sequence according to the present invention exhibits excellent MHC1 presentation.
특히, signal peptidase 효소가 인지하는 시그널 펩티다아제 절단 펩타이드 서열을 더한 서열번호 10의 경우 가장 우수한 효과를 나타내어, signal peptidase 효소가 전달을 목적하는 펩타이드 서열, 특히 에피토프 전달에 중요하게 작용할 수 있음을 추가적으로 확인하였다.In particular, SEQ ID NO: 10, which added the signal peptidase cleavage peptide sequence recognized by the signal peptidase enzyme, showed the best effect, and it was further confirmed that the signal peptidase enzyme can play an important role in the delivery of the desired peptide sequence, especially the epitope. .
실시예Example 5. 소포체 표적 세포 투과성 5. Endoplasmic Reticulum Target Cell Permeability 펩타이드peptide 서열을 이용한 using sequence SIINFEKLSIINFEKL 특이적 CD8+ T cell 활성 분석 Specific CD8+ T cell activity assay
상기 실시예 2의 융합 폴리펩타이드를 수지상 세포에 프라이밍 시킨 후에 전달을 목적하는 서열인 "SIINFEKL" 특이적인 T cell receptor transgenic mice (OT-I mice)에서 분리한 CD8+ T cell과 반응시키고 CD8+ T 세포와의 활성을 비교하였다.After priming dendritic cells with the fusion polypeptide of Example 2, it was reacted with CD8+ T cells isolated from T cell receptor transgenic mice (OT-I mice) specific for "SIINFEKL", the sequence to be delivered, and CD8+ T cells and activity was compared.
구체적으로, 수지상세포에 앞서 합성한 융합 폴리펩타이드를 3시간 동안 노출한 뒤 배양액을 교체하고 24시간 동안 배양하였다. 이후 Ovalbumin 유래 SINFEKL peptide를 인식할 수 있는 T-cell receptor (TCR)과 E. Coli lacZ reporter gene을 지닌 T세포 (B3Z cell)을 peptide에 의해 pulse 된 수지상세포와 공동 배양하였다 (DC:T cell = 2:1). 24시간 동안 공동배양한 후, 세포를 lysis하고, chlorophenol red-b-galactoside (CPRG)를 넣어 4시간 동안 배양하고 560㎚ 파장의 흡광도를 측정함으로 lacZ의 활성을 분석하였다.Specifically, after exposing the previously synthesized fusion polypeptide to dendritic cells for 3 hours, the culture medium was replaced and cultured for 24 hours. Then, the T-cell receptor (TCR) and E. coli that can recognize Ovalbumin-derived SINFEKL peptide T cells (B3Z cells) carrying the lacZ reporter gene were co-cultured with peptide-pulsed dendritic cells (DC:T cell = 2:1). After co-cultivation for 24 hours, the cells were lysed, chlorophenol red-b-galactoside (CPRG) was added, cultured for 4 hours, and the activity of lacZ was analyzed by measuring absorbance at a wavelength of 560 nm.
상기 실험 결과를 도 4에 나타내었다. The experimental results are shown in FIG. 4 .
도 4에서 확인할 수 있는 바와 같이, 본 발명에 따른 소포체 표적 세포 투과성 펩타이드 서열을 사용하는 경우 우수한 CD8+ T cell 활성 효과를 나타냄을 확인하였다.As can be seen in FIG. 4 , it was confirmed that an excellent CD8+ T cell activation effect was exhibited when the endoplasmic reticulum target cell penetrating peptide sequence according to the present invention was used.
상기 실시예 4 및 실시예 5의 결과는 본 발명에 따른 융합 폴리펩타이드가 소포체 내로 전달을 목적하는 목적 펩타이드인 MHC class I epitope를 통해 목적하는 효과를 나타낼 수 있음을 보여주고 있다.The results of Example 4 and Example 5 show that the fusion polypeptide according to the present invention can exhibit the desired effect through the MHC class I epitope, which is the target peptide for delivery into the endoplasmic reticulum.
실시예Example 6. 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 6. Endoplasmic reticulum (ER) target cell permeability 펩타이드peptide (cell-penetrating peptide)의 세포 내 위치 분석(cell-penetrating peptide) intracellular localization analysis
ER-CPP(서열번호 1: N'-acetyl-MLPGLALLLLAAWTARA-NH2-C')의 N' terminus에 Fluorescein-5-isothiocyanate(FITC)를 결합시키고, 이를 이용하여 세포 내 위치 분석을 수행하였다.Fluorescein-5-isothiocyanate (FITC) was bound to the N' terminus of ER-CPP (SEQ ID NO: 1: N'-acetyl-MLPGLALLLLAAWTARA-NH 2 -C'), and intracellular localization was performed using this.
구체적으로, 혈관내피세포(human umbilical vein endothelial cells, HUVEC), 자궁경부암 기원 상피세포(Cervical cancer-originated epithelial cells, HeLa cells), 혹은 대식세포(macrophage, Raw 264.7 cells)에 위 융합 폴리펩타이드를 처리하고 소포체를 염색하는 ER tracker red와 핵을 염색하는 Hoechst 33342를 처리하였다. 그리고 나서, 공초점 현미경(Confocal Microscopy, Zeiss)을 이용하여 ER-CPP의 세포 내 위치를 분석하였다.Specifically, the above fusion polypeptide is treated with human umbilical vein endothelial cells (HUVEC), cervical cancer-originated epithelial cells (HeLa cells), or macrophages (Raw 264.7 cells). and treated with ER tracker red to stain the endoplasmic reticulum and Hoechst 33342 to stain the nucleus. Then, the intracellular localization of ER-CPP was analyzed using confocal microscopy (Zeiss).
그 결과를 도 5 내지 7에 나타내었다.The results are shown in Figures 5 to 7.
도 5는 혈관내피세포(human umbilical vein endothelial cells, HUVEC)에서 분석 결과를 나타내며, 도 6은 자궁경부암 기원 상피세포(Cervical cancer-originated epithelial cells, HeLa cells)에서 분석 결과를 나타내며, 도 7은 대식세포(macrophage, Raw 264.7 cells)에서 분석 결과를 나타낸다.5 shows the analysis results in human umbilical vein endothelial cells (HUVEC), FIG. 6 shows the analysis results in cervical cancer-originated epithelial cells (HeLa cells), and FIG. Analysis results are shown in macrophages (Raw 264.7 cells).
도 5 내지 7을 통해 확인할 수 있는 바와 같이, ER-CPP는 다양한 세포에서 세포 내 소포체로 표적(targeting)함을 확인하였다. 즉, 세포 종류에 관계없이 본 발명에 따른 ER-CPP 서열이 세포 투과 후 소포체로 서열이 전달되는 점을 확인하였다.As can be seen through FIGS. 5 to 7 , it was confirmed that ER-CPP targets intracellular endoplasmic reticulum in various cells. That is, it was confirmed that the ER-CPP sequence according to the present invention was transmitted to the endoplasmic reticulum after cell penetration regardless of cell type.
위와 같은 본 발명의 ER-CPP 서열의 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 펩타이드(cell-penetrating peptide)의 소포체 국재화 효과를 명확히 확인하고자 일반적으로 사용되는 세포투과성 펩타이드인 penetratin(서열번호 14: N'-acetyl-RQIKIWFQNRRMKWKK-NH2-C')를 대조군으로 확인하였다. Penetratin의 N' terminus에 FITC를 결합시키고 HeLa 세포에 처리한 후 소포체와 핵을 염색하여 세포 내 위치를 분석하였고, 그 결과를 도 8에 나타내었다.Penetratin (SEQ ID NO: 14: N'-acetyl-RQIKIWFQNRRMKWKK-NH 2 -C') was identified as a control. After binding FITC to the N' terminus of penetratin and treating HeLa cells, the endoplasmic reticulum and nucleus were stained to analyze the intracellular location, and the results are shown in FIG. 8 .
도 8에서 확인할 수 있는 바와 같이, penetratin은 비 특이적으로 세포막을 포함하여 세포 전체에 분포되어 있음이 확인되었다(FITC 결과). 반면, 앞선 도 6의 결과에서 확인할 수 있는 바와 같이 ER-CPP의 처리는 특이적으로 세포 내 소기관인 소포체에 표적하는 효과를 나타내어 핵 주위의 소포체에 염색을 나타내었다.As can be seen in Figure 8, it was confirmed that penetratin was non-specifically distributed throughout the cell including the cell membrane (FITC result). On the other hand, as can be seen from the results of FIG. 6 above, treatment with ER-CPP showed an effect specifically targeting the endoplasmic reticulum, which is an organelle within the cell, and showed staining of the endoplasmic reticulum around the nucleus.
위 대조군에 대한 결과를 고려할 때, 본 발명에 따른 ER-CPP 서열의 우수한 소포체 전달능이 확인되었다.Considering the results for the above control group, the excellent endoplasmic reticulum delivery capability of the ER-CPP sequence according to the present invention was confirmed.
실시예 7. ER-CPP의 세포 투과 효율 분석Example 7. Analysis of cell penetration efficiency of ER-CPP
FITC-ER-CPP(서열번호 1: N'-FITC-MLPGLALLLLAAWTARA-NH2-C')를 다양한 시간(10분, 20분, 40분, 2시간 혹은 4시간)과 농도(1, 2, 혹은 4μM)로 대식세포(Raw 264.7 세포)에 처리한 후 유세포 분석기(Fluorescence-activated cell sorting, FACS, Novocyte)를 이용하여 FITC의 intensity를 비교함으로써 세포 투과 효율을 분석하였다.FITC-ER-CPP (SEQ ID NO: 1: N'-FITC-MLPGLALLLLAAWTARA-NH 2 -C') was administered at various times (10 minutes, 20 minutes, 40 minutes, 2 hours or 4 hours) and concentrations (1, 2, or After treatment with macrophages (Raw 264.7 cells) with 4 μM), cell permeation efficiency was analyzed by comparing the intensity of FITC using a flow cytometer (Fluorescence-activated cell sorting, FACS, Novocyte).
그 결과를 도 9에 나타내었다. 도 9를 통해 확인할 수 있는 바와 같이, 세포에 ER-CPP 서열을 처리한 후 10분 뒤에도 약 70% 가까이 세포 투과 효율이 높은 것을 확인하였으며, 20분 이상 처리시에는 90% 이상 대부분의 ER-CPP 서열이 세포를 투과함을 확인하였다.The results are shown in FIG. 9 . As can be seen in FIG. 9, it was confirmed that the cell penetration efficiency was high at about 70% even 10 minutes after the cells were treated with the ER-CPP sequence, and when treated for 20 minutes or more, most of the ER-CPP was 90% or more. It was confirmed that the sequence penetrated the cells.
또한, 4시간 처리시 매우 낮은 농도인 1μM 농도에서도 70% 가까이 세포 투과 효율이 높은 것을 확인하였으며, 2μM 농도부터는 90% 이상 대부분의 ER-CPP 서열이 세포를 투과함을 확인하였다.In addition, it was confirmed that cell penetration efficiency was high by nearly 70% even at a very low concentration of 1 μM when treated for 4 hours, and most of the ER-CPP sequences penetrated cells by 90% or more from a concentration of 2 μM.
위 결과를 통해 본 발명에 따른 서열의 우수한 세포 투과도를 확인하였다.Through the above results, excellent cell permeability of the sequence according to the present invention was confirmed.
실시예 8. ER-CPP의 항원 제시 효율 분석Example 8. Analysis of antigen presentation efficiency of ER-CPP
ovalbumin(OVA)에서 유래한 SIINFEKL 펩타이드가 연결된 서열을 이용하여 항원 제시 효율을 분석하였다.Antigen presentation efficiency was analyzed using a sequence in which the SIINFEKL peptide derived from ovalbumin (OVA) was linked.
서열명sequence name 서열order
Peptide 1 (서열번호 10)Peptide 1 (SEQ ID NO: 10) N'-Ac-MLPGLALLLLAAWTARAEASIINFEKL-NH2-C'N'-Ac-MLPGLALLLLAAWTARAEASIINFEKL-NH 2 -C'
Peptide 2 (서열번호 9)Peptide 2 (SEQ ID NO: 9) N'-Ac-MLPGLALLLLAAWTARASIINFEKL-NH2-C'N'-Ac-MLPGLLALLLLAAWTARASIINFEKL-NH 2 -C'
Peptide 3 (Negative)(서열번호 13)Peptide 3 (Negative) (SEQ ID NO: 13) N'-Ac-MLPGLALLLLAAWTARAEASIINYEKL-NH2-C'N'-Ac-MLPGLALLLLAAWTARAEASINYEKL-NH 2 -C'
Peptide 4 (Current CPP)(서열번호 15)Peptide 4 (Current CPP) (SEQ ID NO: 15) N'-Ac-RQIKIWFQNRRMKWKKSIINFEKL-NH2-C'N'-Ac-RQIKIWFQNRRMKWKKSIINFEKL-NH 2 -C'
DC 2.4 세포에 농도별로 처리하여 세포 독성을 분석하였다. 그 결과 세포 독성은 전혀 확인되지 않았다.또한, 유세포 분석기(Fluorescence-activated cell sorting, FACS, Novocyte)를 이용하여 FITC의 intensity를 비교함으로써 세포 투과 효율을 분석하고, 소포체 표적 펩타이드의 항원 제시 능력을 분석하기 위해 OVA 323-339(MHC II epitope 시퀀스 (서열번호 16: ISQAVHAAHAEINEAGR), OVA 257-264(SIINFEKL 시퀀스), Peptide 1, Peptide 2, Peptide 3, Peptide 4를 농도와 시간에 따라 DC 2.4 세포에 처리한 후 세포막 표면에 MHC I-loaded SIINFEKL과 결합하는 항체를 이용하여 FACS로 분석하였다.DC 2.4 cells were treated for each concentration and cytotoxicity was analyzed. As a result, cytotoxicity was not confirmed at all. In addition, cell penetration efficiency was analyzed by comparing the intensity of FITC using a flow cytometer (Fluorescence-activated cell sorting, FACS, Novocyte), and the antigen presenting ability of the endoplasmic reticulum targeting peptide was analyzed. DC 2.4 cells were treated with OVA 323-339 (MHC II epitope sequence (SEQ ID NO: 16: ISQAVHAAHAEINEAGR), OVA 257-264 (SIINFEKL sequence), Peptide 1, Peptide 2, Peptide 3, and Peptide 4 at different concentrations and time). After that, the cell membrane surface was analyzed by FACS using an antibody binding to MHC I-loaded SIINFEKL.
그 결과를 도 10에 나타내었다. 24시간이 지난 후에 항원 제시 능력이 ER-CPP 서열을 포함하는 peptide 1 및 peptide 2를 처리한 군에서 월등하게 증가됨을 확인하였다.The results are shown in FIG. 10 . After 24 hours, it was confirmed that the antigen presenting ability was remarkably increased in the group treated with peptide 1 and peptide 2 containing the ER-CPP sequence.
실시예 9. ER-CPP의 CD8+ T 세포 활성 효과 분석Example 9. Analysis of CD8+ T cell activity effect of ER-CPP
소포체 표적 펩타이드의 CD8+ T 세포의 활성 증가 효과를 확인하기 위해 수지상세포에 peptide(서열번호 10, 서열번호 13 또는 서열번호 15)를 4시간 노출한 뒤 SIINFEKL peptide를 인식할 수 있는 T-cell receptor과 lacZ reporter gene을 지닌 T세포(B3Z cell)을 peptide에 의해 pulse되어진 수지상세포와 공동 배양하였다(DC:T cell = 1:5). 12시간 혹은 24시간 동안 공동배양한 후, 세포를 lysis하고, chlorophenol red-β-galactoside (CPRG)를 넣어 4시간 동안 배양한 후 560㎚ 파장의 흡광도를 측정함으로 lacZ의 활성을 분석하였다.In order to confirm the effect of the endoplasmic reticulum-targeting peptide on increasing the activity of CD8+ T cells, dendritic cells were exposed to the peptide (SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 15) for 4 hours, and then the T-cell receptor and T-cell receptor that can recognize the SIINFEKL peptide T cells (B3Z cells) carrying the lacZ reporter gene were co-cultured with peptide-pulsed dendritic cells (DC:T cell = 1:5). After co-culture for 12 or 24 hours, the cells were lysed, chlorophenol red-β-galactoside (CPRG) was added and incubated for 4 hours, and the activity of lacZ was analyzed by measuring absorbance at a wavelength of 560 nm.
그 결과를 도 11에 나타내었다.The results are shown in Figure 11.
도 11을 통해 확인할 수 있는 바와 같이, 본 발명에 따른 ER-CPP를 포함하는 서열의 처리는 CD8+ T 세포 활성에 우수한 효과를 나타내었다.As can be seen from FIG. 11 , treatment with the sequence including ER-CPP according to the present invention showed excellent effects on CD8+ T cell activity.
특히, 본 발명에 따른 ER-CPP를 사용하는 경우 OVA 257-264 혹은 일반적인 CPP(서열번호 15)를 사용하는 것에 비해 2배 이상 CD8+ T 세포의 활성을 증가시켰다.In particular, the use of ER-CPP according to the present invention increased the activity of CD8+ T cells more than twice as compared to the use of OVA 257-264 or general CPP (SEQ ID NO: 15).
MHC I epitope loading complex는 ER quality control 과정을 거쳐야만 cell surface plasma membrane에 stable presentation이 이루어진다. in vivo에서도 T cell이 항원을 발현하는 세포를 찾기 위해선 적절한 시간이 필요한데, 본 발명의 결과들은 stable cell surface antigen presentation이 이루어진 결과들을 보여주고 있다. 즉, 본 발명에 따른 결과들은 소포체에 항원을 특이적으로 표적시킨 것에 대한 구체적 결과를 제공한다.MHC I epitope loading complex must go through ER quality control before stable presentation on cell surface plasma membrane. Appropriate time is required for T cells to find antigen-expressing cells even in vivo , but the results of the present invention show the results of stable cell surface antigen presentation. That is, the results according to the present invention provide specific results for specifically targeting an antigen to the endoplasmic reticulum.
실시예 10. ER-CPP를 활용한 마우스 모델에서의 면역 작용 부스팅 효과 Example 10. Immune action boosting effect in a mouse model using ER-CPP
In vivo 마우스 모델을 활용하여, 펩타이드들의 CD8+ T 세포의 활성을 분석하였다. 8주령 C57BL/6 mouse의 footpad에 peptide(서열번호 10, 서열번호 13 또는 서열번호 15)를 5㎍ 투여한 후 20시간 후에 CFSE-labeled OTI-CD8+ T cell를 5x106 개의 세포수로 tail vein(intravenous, I.V.)으로 injection 하였다. 그리고 나서, 72시간 후에 마우스 혈액을 채취하여 CFSE의 염색량의 변화를 분석함과 동시에 혈액 내의 CD8+ T 세포 중 interferon-γ(INF- γ)를 분비하는 세포의 변화를 분석하였다.Using an in vivo mouse model, CD8+ T cell activity of the peptides was analyzed. After administering 5 μg of the peptide (SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 15) to the footpad of an 8-week-old C57BL/6 mouse, 20 hours later, CFSE-labeled OTI-CD8+ T cells were counted in the tail vein (5x10 6 cells). Intravenous, IV) injection. Then, after 72 hours, mouse blood was collected to analyze changes in CFSE staining amount and at the same time, changes in cells secreting interferon-γ (INF-γ) among CD8+ T cells in the blood were analyzed.
위 결과를 도 12에 나타내었다. The above results are shown in FIG. 12 .
도 12를 통해 확인할 수 있는 바와 같이, 본 발명에 따른 ER-CPP 서열의 사용은 기존의 알려진 CPP인 penetratin 보다 CD8+ T 세포의 증식이 4배 이상 증가시켰다. 또한, 인터페론 감마를 분비하는 세포를 2배 이상 증가시켰다.As can be seen from FIG. 12, the use of the ER-CPP sequence according to the present invention increased the proliferation of CD8+ T cells more than 4 times compared to penetratin, a known CPP. In addition, cells secreting interferon gamma were increased more than twice.
즉, 세포 내 소포체에서 MHC I epitope이 탑재되어 세포 표면에서의 항원 제시가 오랜 시간 동안 유지되어 마우스 동물 모델에서 혈액 내의 CD8+ T 세포가 그 항원을 인식하여 활성이 증가함을 확인하였다.That is, it was confirmed that CD8+ T cells in the blood recognized the antigen and increased their activity in a mouse animal model because the MHC I epitope was loaded in the intracellular endoplasmic reticulum and antigen presentation on the cell surface was maintained for a long time.
또한, 세포 내 소기관인 소포체를 표적하는 세포 투과성 펩타이드가 면역 작용의 중요한 항원 전달체로서 다양한 면역 관련 질환들에 활용 가능함을 확인하였다.In addition, it was confirmed that cell-penetrating peptides targeting the endoplasmic reticulum, an organelle in cells, can be used for various immune-related diseases as an important antigen transporter for immune action.
실시예Example 11. 항산화제(antioxidant) 융합 소포체(endoplasmic reticulum, ER) 표적 세포 투과성 펩타이드(cell-penetrating peptide)의 세포 내 위치 분석 11. Analysis of the intracellular localization of the endoplasmic reticulum (ER) target cell-penetrating peptide of an antioxidant
서열 번호 1의 ER-CPP에 항산화제를 융합시킨 펩타이드를 합성하였다. A peptide in which an antioxidant was fused to ER-CPP of SEQ ID NO: 1 was synthesized.
구체적으로, 하기 표 3과 같이 펩타이드 서열을 합성하였다. Specifically, peptide sequences were synthesized as shown in Table 3 below.
서열명sequence name 서열 order
Peptide 5 (서열번호 17)Peptide 5 (SEQ ID NO: 17) N' FITC-MLPGLALLLLAAWTARA-γE-CG-amidation CN'FITC-MLPGLLALLLAAWTARA-γE-CG-amidation C
Peptide 6 (서열번호 18)Peptide 6 (SEQ ID NO: 18) N' FITC-CMLPGLALLLLAAWTARA-amidation C'N'FITC-CMLPGLALLLLAAWTARA-amidation C'
Peptide 7 (서열번호 19)Peptide 7 (SEQ ID NO: 19) N' MLPGLALLLLAAWTARA-γE-CG-amidation C'N'MLPGLALLLLAAWTARA-γE-CG-amidation C'
Peptide 8 (서열번호 20) Peptide 8 (SEQ ID NO: 20) N' acetyl-CMLPGLALLLLAAWTARA-amidation C'N'acetyl-CMLPGLALLLLAAWTARA-amidation C'
위 표를 통해 확인할 수 있는 바와 같이, Peptide 5는 ER-CPP의 N' 말단을 FITC로 C' 말단을 항산화제인 glutathione(γE-CG, GSH)로 결합시켰다. peptide 6은 ER-CPP의 N' 말단을 FITC와 cysteine로 융합하여 합성하였다. 이들 서열은 세포 내 위치 분석을 위한 형광 펩타이드로 사용되었다.또한, Peptide 7 혹은 Peptide 8는 FITC를 첨가하지 않은 항산화제와 융합시킨 형태의 펩타이드이다. Peptide 7은 C' 말단을 항산화제인 glutathione(γE-CG, GSH)을 결합시킨 것이고, Peptide 8은 N-말단에 대하여 아세틸화된 시스테인을 결합시킨 것이다.As can be seen from the table above, peptide 5 binds FITC to the N' end of ER-CPP and glutathione (γE-CG, GSH), an antioxidant, to the C' end. Peptide 6 was synthesized by fusing the N' terminus of ER-CPP with FITC and cysteine. These sequences were used as fluorescent peptides for intracellular localization. In addition, Peptide 7 or Peptide 8 is a peptide fused with an antioxidant to which FITC is not added. Peptide 7 has glutathione (γE-CG, GSH), an antioxidant, linked to the C' terminus, and peptide 8 has acetylated cysteine linked to the N-terminus.
상기 peptide 5 및 6을 유방암 유래 상피세포(breast cancer-originated epithelial cell, MCF-7)에 10μM의 농도로 4시간 동안 처리한 후 ER tracker red와 Hoechst로 염색하여 세포 내 위치를 형광현미경 (Fluorescence microscopy, Leica)으로 확인하였다. 그 결과를 도 13에 나타내었다.After treating the peptides 5 and 6 at a concentration of 10 μM for 4 hours in breast cancer-originated epithelial cells (MCF-7), they were stained with ER tracker red and Hoechst, and the intracellular location was examined by fluorescence microscopy (Fluorescence microscopy). , Leica). The results are shown in Figure 13.
도 13을 통해 확인할 수 있는 바와 같이, 항산화제를 탑재한 펩타이드는 세포 내 소포체를 표적함을 확인하였다.As can be seen in Figure 13, it was confirmed that the antioxidant-loaded peptides target intracellular endoplasmic reticulum.
실시예Example 12. 소포체 표적 항산화제의 소포체 스트레스로 유도된 세포사멸 억제 효과 분석 12. Analysis of the inhibitory effect of endoplasmic reticulum-targeted antioxidants on apoptosis induced by endoplasmic reticulum stress
소포체 스트레스 유도체인 thapsigargin을 유방암 세포에 농도별로 24시간 동안 처리한 후 2,5-diphenyl-2H-tetrazolium bromide(MTT) 시약을 사용하여 세포사멸 변화를 분석하였다. 그 결과, thapsigargin 10μM 농도에서 50%의 세포사멸이 일어남을 확인하고, 해당 농도를 소포체 스트레스 유발을 위한 농도로 사용하였다.After treating breast cancer cells with thapsigargin, an endoplasmic reticulum stress inducer, at different concentrations for 24 hours, apoptotic changes were analyzed using 2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent. As a result, it was confirmed that 50% of apoptosis occurred at a concentration of 10 μM thapsigargin, and the corresponding concentration was used as a concentration for inducing endoplasmic reticulum stress.
구체적으로, 소포체를 표적하는 항산화제의 소포체 스트레스 유도 세포사멸 억제 효과를 분석하기 위해 Peptide 7 혹은 Peptide 8 그리고 기존의 잘 알려진 항산화제인 glutathione(GSH) 혹은 N-acetyl-cysteine(NAC)을 유방암 세포에 1시간 동안 전 처리한 후 thapsigargin을 10μM의 농도로 24시간 동안 처리하여 세포사멸의 변화를 분석하였다.Specifically, to analyze the inhibitory effect of endoplasmic reticulum-targeting antioxidants on endoplasmic reticulum stress-induced apoptosis, Peptide 7 or Peptide 8 and well-known antioxidants such as glutathione (GSH) or N-acetyl-cysteine (NAC) were administered to breast cancer cells. After pre-treatment for 1 hour, changes in apoptosis were analyzed by treatment with thapsigargin at a concentration of 10 μM for 24 hours.
그 결과를 도 14에 나타내었다. The results are shown in FIG. 14 .
도 14를 통해 확인할 수 있는 바와 같이, Peptide 7 혹은 Peptide 8이 처리된 세포에서 세포사멸이 현저하게 저해됨을 확인하였다. 이러한 작용은 단독의 glutathione(GSH) 혹은 N-acetyl-cysteine(NAC)와 대비하였을 때도 현저한 차이를 나타내는 것이었다.As can be seen in FIG. 14 , it was confirmed that apoptosis was significantly inhibited in cells treated with Peptide 7 or Peptide 8. This action showed a remarkable difference even when compared with glutathione (GSH) or N-acetyl-cysteine (NAC) alone.
이러한 작용은 소포체에 대한 목적 물질의 전달을 통하여 발생하는 효과로 소포체 스트레스를 소포체를 표적하는 물질 전달을 통해 해소할 수 있음을 나타낸다. This action is an effect that occurs through the delivery of a target substance to the endoplasmic reticulum, indicating that the endoplasmic reticulum stress can be resolved through the delivery of a target substance to the endoplasmic reticulum.
실시예Example 13. 소포체 표적 항산화제의 소포체 스트레스로 유도된 세포사멸 억제 효과 분석 13. Analysis of the inhibitory effect of endoplasmic reticulum-targeted antioxidants on apoptosis induced by endoplasmic reticulum stress
실시예 12과 유사하게 Thapsigargin을 신경종양세포 (neuroblastoma, SH-sy5y)에 농도별로 24시간 동안 처리한 후 2,5-diphenyl-2H-tetrazolium bromide(MTT) 시약을 사용하여 세포사멸 변화를 분석하였다. 그 결과, thapsigargin 20μM 농도에서 50%의 세포사멸이 일어남을 확인하고, 해당 농도를 소포체 스트레스 유발을 위한 농도로 사용하였다.Similarly to Example 12, thapsigargin was treated for 24 hours at each concentration in neuroblastoma cells (neuroblastoma, SH-sy5y), and apoptotic changes were analyzed using 2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent. . As a result, it was confirmed that 50% of apoptosis occurred at a concentration of 20 μM thapsigargin, and the corresponding concentration was used as a concentration for inducing endoplasmic reticulum stress.
구체적으로, 소포체를 표적하는 항산화제의 소포체 스트레스 유도 세포사멸 억제 효과를 분석하기 위해 Peptide 7 혹은 Peptide 8 그리고 기존의 잘 알려진 항산화제인 glutathione(GSH) 혹은 N-acetyl-cysteine(NAC)을 신경종양 세포에 1시간 동안 전 처리한 후 thapsigargin을 20μM의 농도로 24시간 동안 처리하여 세포사멸의 변화를 분석하였다.Specifically, in order to analyze the inhibitory effect of endoplasmic reticulum-targeting antioxidants on endoplasmic reticulum stress-induced apoptosis, peptide 7 or peptide 8 and well-known antioxidants glutathione (GSH) or N-acetyl-cysteine (NAC) were applied to neurotumor cells. After pre-treatment for 1 hour, thapsigargin was treated at a concentration of 20 μM for 24 hours, and changes in apoptosis were analyzed.
그 결과를 도 15에 나타내었다.The results are shown in FIG. 15 .
도 15를 통해 확인할 수 있는 바와 같이, Peptide 7 혹은 Peptide 8이 처리된 세포에서 세포사멸이 현저하게 저해됨을 확인하였다. 이러한 작용은 단독의 glutathione(GSH) 혹은 N-acetyl-cysteine(NAC)와 대비하였을 때도 현저한 차이를 나타내는 것이었다.As can be confirmed through FIG. 15, it was confirmed that apoptosis was significantly inhibited in cells treated with Peptide 7 or Peptide 8. This action showed a remarkable difference even when compared with glutathione (GSH) or N-acetyl-cysteine (NAC) alone.
상기 결과들을 통해 세포 내 소포체 스트레스로 유도되는 세포사멸은 기존의 항산화제인 GSH 혹은 NAC를 처리를 하여도 억제 효과가 전혀 없는데 비하여 소포체를 표적하는 항산화제는 현저하게 세포사멸을 억제함을 확인하였다.Through the above results, apoptosis induced by intracellular endoplasmic reticulum stress was confirmed to have no inhibitory effect even when treated with GSH or NAC, which are conventional antioxidants, whereas antioxidants targeting endoplasmic reticulum significantly inhibit apoptosis.
상기와 같은 결과들로부터 본 발명의 소포체 표적 세포 투과성 펩타이드 서열이 소포체로 목적 서열을 전달하는 것을 보여준다. From the above results, it is shown that the endoplasmic reticulum target cell penetrating peptide sequence of the present invention transfers the target sequence to the endoplasmic reticulum.
이를 통해 ER 스트레스, 또는 소포체 관련 매개 질환의 치료 또는 개선을 위한 펩타이드를 ER 내로 잘 전달할 수 있는 것을 확인하였다. 이외에도 ER 내로의 펩타이드 전달을 통해 생체 내 다양한 생리현상 등에 적용하여, 다양한 용도로 이용될 수 있음을 확인하였다. 또한, 당뇨병, 퇴행성 뇌질환, 암, 만성 염증 질환의 주요한 원인으로 알려진 소포체 스트레스를 소포체 표적 항산화제를 이용하여 억제하게 되면 질환의 진행 억제 및 치료를 가능하게 하는 것을 보여준다.Through this, it was confirmed that peptides for treatment or improvement of ER stress or endoplasmic reticulum-related diseases could be well delivered into the ER. In addition, it was confirmed that it can be used for various purposes by applying it to various physiological phenomena in vivo through the delivery of peptides into the ER. In addition, it is shown that suppressing endoplasmic reticulum stress, known as a major cause of diabetes, degenerative brain diseases, cancer, and chronic inflammatory diseases, using an endoplasmic reticulum-targeting antioxidant makes it possible to suppress and treat disease progression.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (26)

  1. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물.A composition for delivering an endoplasmic reticulum-targeting peptide comprising the endoplasmic reticulum-targeting cell-penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2.
  2. 제1항에 있어서, 소포체 표적 펩타이드 전달용 조성물은 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 링커 또는 스페이서 펩타이드를 포함하는 소포체 표적 펩타이드 전달용 조성물.The method of claim 1, wherein the composition for delivering the endoplasmic reticulum targeting peptide comprises the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and a linker or spacer peptide linked thereto.
  3. 제2항에 있어서, 링커 또는 스페이서는 시그널 펩티다아제(signal peptidase) 절단 펩타이드인 소포체 표적 펩타이드 전달용 조성물.The composition for delivering an endoplasmic reticulum-targeted peptide according to claim 2, wherein the linker or spacer is a signal peptidase cleaved peptide.
  4. 제3항에 있어서, 시그널 펩티다아제(signal peptidase) 절단 펩타이드는 서열번호 4의 펩타이드인 소포체 표적 펩타이드 전달용 조성물.The composition for delivering the endoplasmic reticulum-targeting peptide according to claim 3, wherein the signal peptidase cleavage peptide is a peptide of SEQ ID NO: 4.
  5. 제2항에 있어서, 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 링커 또는 스페이서 펩타이드는 서열번호 7 또는 8의 펩타이드인 소포체 표적 펩타이드 전달용 조성물.The method of claim 2, wherein the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and a linker or spacer peptide linked thereto is a peptide of SEQ ID NO: 7 or 8.
  6. 제1항에 있어서, 소포체 표적 펩타이드 전달용 조성물은 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 것인 소포체 표적 펩타이드 전달용 조성물.The method of claim 1, wherein the composition for delivering the endoplasmic reticulum targeting peptide comprises the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; And an endoplasmic reticulum targeting peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
  7. 제1항에 있어서, 소포체 표적 펩타이드 전달용 조성물은 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 것인 소포체 표적 펩타이드 전달용 조성물.The method of claim 1, wherein the composition for delivering the endoplasmic reticulum targeting peptide comprises the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And an endoplasmic reticulum targeting peptide delivery composition comprising an endoplasmic reticulum delivery peptide linked thereto.
  8. 제6항에 있어서, 소포체 전달용 펩타이드는 면역원성 펩타이드, 항원, 사이토카인, 케모카인, 림포카인, 리간드, 수용체, 호르몬, 효소, 항체 및 항체 단편, 및 성장 인자로 이루어진 군으로부터 선택되는 어느 하나인 소포체 표적 펩타이드 전달용 조성물.The method of claim 6, wherein the peptide for endoplasmic reticulum delivery is any one selected from the group consisting of immunogenic peptides, antigens, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors A composition for delivering a phosphorus endoplasmic reticulum-targeted peptide.
  9. 제7항에 있어서, 소포체 전달용 펩타이드는 면역원성 펩타이드, 항원, 사이토카인, 케모카인, 림포카인, 리간드, 수용체, 호르몬, 효소, 항체 및 항체 단편, 및 성장 인자로 이루어진 군으로부터 선택되는 어느 하나인 소포체 표적 펩타이드 전달용 조성물.The method of claim 7, wherein the peptide for endoplasmic reticulum delivery is any one selected from the group consisting of immunogenic peptides, antigens, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors A composition for delivering a phosphorus endoplasmic reticulum-targeted peptide.
  10. 제8항에 있어서, 상기 소포체 전달용 펩타이드는 면역원성 펩타이드 또는 항원인 소포체 표적 펩타이드 전달용 조성물.The composition for delivery of an endoplasmic reticulum-targeted peptide according to claim 8, wherein the peptide for endoplasmic reticulum delivery is an immunogenic peptide or an antigen.
  11. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 면역원성 펩타이드를 포함하는 백신 조성물.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and an immunogenic peptide linked thereto.
  12. 제11항에 있어서, 상기 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 면역원성 펩타이드는 링커 또는 스페이서 펩타이드로 연결된 것인 백신 조성물.The method of claim 11, wherein the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and the immunogenic peptide is linked by a linker or spacer peptide.
  13. 제12항에 있어서, 상기 링커 또는 스페이서 펩타이드는 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드인 백신 조성물.The vaccine composition according to claim 12, wherein the linker or spacer peptide is a signal peptidase cleavage peptide of SEQ ID NO: 4.
  14. 제11항에 있어서, 면역원성 펩타이드는 MHC 에피토프 또는 B-세포 에피토프인 백신 조성물.12. The vaccine composition of claim 11, wherein the immunogenic peptide is an MHC epitope or a B-cell epitope.
  15. 제11항에 있어서, 상기 백신 조성물은 애쥬번트를 더 포함하는 것인 백신 조성물.The vaccine composition according to claim 11, further comprising an adjuvant.
  16. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases comprising an endoplasmic reticulum delivery peptide linked thereto.
  17. 제16항에 있어서, 상기 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드는 링커 또는 스페이서 펩타이드로 연결된 것인 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물.The method of claim 16, wherein the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases in which the endoplasmic reticulum delivery peptide linked thereto is linked by a linker or spacer peptide.
  18. 제17항에 있어서, 링커 또는 스페이서 펩타이드는 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드인 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물.The pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases according to claim 17, wherein the linker or spacer peptide is a signal peptidase cleavage peptide of SEQ ID NO: 4.
  19. 제16항에 있어서, 상기 소포체 스트레스 관련 질환은 제1형 당뇨, 제2형 당뇨, 알츠하이머 질환 (Alzheimer's disease), 면역글로블린 경쇄 아밀로이드증 (immumoglobulin light chain amyloidosis), 파킨슨병 (Parkinson's disease), 근위축성 측삭경화증 (amyotrophic lateral sclerosis, ALS), 혈액투석 연관성 아밀로이드증 (haemodialysis-related amyloidosis), 활동성 아밀로이드증 (reactive amyloidosis), 낭포성 섬유증 (cystic fibrosis), 겸상적혈구빈혈증 (sickle cell anemia), 헌팅턴병 (Huntington's disease), 크로이츠펠트-야콥 질환 (Kreutzfeldt-Jakob disease), 가족성 고콜레스테롤혈증 (familial hypercholesterolaemia), 알파1-항트립신 결핍증 (Alpha1-antitrypsin deficiency), 경변 (cirrhosis), 전신성 폐기종 (emphysema systemic), 뇌성 유전성 아밀로이드증(cerebral hereditary amyloidoses), 울콧-랠리스 증후군 (Wolcott-Rallison syndrome), 울프람 증후군(Wolfram syndrome), 염증성 장질환, 코론씨 병, 궤양성 대장염, 유방암 및 전립선암으로 이루어진 군으로부터 선택되는 어느 하나인 소포체 스트레스 관련 질환 예방 또는 치료용 약제학적 조성물.The method of claim 16, wherein the endoplasmic reticulum stress-related disease is type 1 diabetes, type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral striatum Sclerosis (amyotrophic lateral sclerosis (ALS), haemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Kreutzfeldt-Jakob disease, familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, hereditary cerebral amyloidosis Any one selected from the group consisting of (cerebral hereditary amyloidoses), Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, Coron's disease, ulcerative colitis, breast cancer and prostate cancer A pharmaceutical composition for preventing or treating phosphorus endoplasmic reticulum stress-related diseases.
  20. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드를 세포에 처리하는 단계를 포함하는 세포 내 소포체로의 소포체 전달용 펩타이드 전달 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And a method for delivering peptides for endoplasmic reticulum delivery to intracellular endoplasmic reticulum comprising the step of processing a cell with a fusion polypeptide comprising a peptide for endoplasmic reticulum delivery linked thereto.
  21. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드를 세포에 처리하는 단계를 포함하는 세포 내 소포체로의 소포체 전달용 펩타이드 전달 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; And a method for delivering peptides for endoplasmic reticulum delivery to intracellular endoplasmic reticulum comprising the step of processing a cell with a fusion polypeptide comprising a peptide for endoplasmic reticulum delivery linked thereto.
  22. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드를 대상체에 처리하는 단계를 포함하는 소포체로의 소포체 전달용 펩타이드 전달 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; And a method for delivering a peptide for endoplasmic reticulum delivery to the endoplasmic reticulum comprising the step of processing a subject with a fusion polypeptide comprising the peptide for endoplasmic reticulum delivery linked thereto.
  23. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드를 대상체에 처리하는 단계를 포함하는 소포체로의 소포체 전달용 펩타이드 전달 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; And a method for delivering a peptide for endoplasmic reticulum delivery to the endoplasmic reticulum comprising the step of processing a subject with a fusion polypeptide comprising a peptide for endoplasmic reticulum delivery linked thereto.
  24. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 면역원성 펩타이드를 포함하는 백신 조성물의 치료 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는 면역 증진 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and administering to a subject in need thereof a therapeutically effective amount of a vaccine composition comprising an immunogenic peptide linked thereto.
  25. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드의 치료 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, ER 스트레스 관련 질환의 치료 또는 예방 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; and administering to a subject in need thereof a therapeutically effective amount of a fusion polypeptide comprising a peptide for endoplasmic reticulum delivery linked thereto.
  26. 서열번호 1 또는 서열번호 2의 소포체 표적 세포 투과성 펩타이드; 이에 연결된 서열번호 4의 시그널 펩티다아제(signal peptidase) 절단 펩타이드; 및 이에 연결된 소포체 전달용 펩타이드를 포함하는 융합 폴리펩타이드의 치료 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, ER 스트레스 관련 질환의 치료 또는 예방 방법.the endoplasmic reticulum target cell penetrating peptide of SEQ ID NO: 1 or SEQ ID NO: 2; Signal peptidase cleavage peptide of SEQ ID NO: 4 linked thereto; and administering to a subject in need thereof a therapeutically effective amount of a fusion polypeptide comprising a peptide for endoplasmic reticulum delivery linked thereto.
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