WO2023003380A1 - Nouveau peptide à pénétration cellulaire et son utilisation - Google Patents

Nouveau peptide à pénétration cellulaire et son utilisation 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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English (en)
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/fr

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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

La présente invention concerne un polypeptide de fusion comprenant un peptide de pénétration cellulaire ciblant le réticulum endoplasmique et son utilisation. Le polypeptide de fusion de la présente invention peut efficacement administrer un peptide d'intérêt au réticulum endoplasmique à l'intérieur des cellules. Par conséquent, le polypeptide de fusion présente l'excellent avantage qu'il peut être disponible comme vaccin, support médicamenteux, et agent thérapeutique et peut être appliqué à des fins prophylactiques et thérapeutiques en régulant divers phénomènes physiologiques in vivo.
PCT/KR2022/010681 2021-07-22 2022-07-21 Nouveau peptide à pénétration cellulaire et son utilisation WO2023003380A1 (fr)

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Citations (4)

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WO2012159164A1 (fr) * 2011-05-23 2012-11-29 Phylogica Limited Procédé de détermination, d'identification ou d'isolement de peptides pénétrant dans des cellules
EP2662389A1 (fr) * 2011-01-07 2013-11-13 Toagosei Co., Ltd Procédé de préparation d'un antigène en vue d'obtenir un anticorps anti-peptide hydrophobe
WO2015138435A1 (fr) * 2014-03-11 2015-09-17 Molecular Templates, Inc. Protéines comportant des régions de liaison, des régions effectrices à sous-motifs a de shiga-toxine, et des motifs signal de localisation du réticulum endoplasmique carboxy-terminaux
US20210061856A1 (en) * 2018-02-07 2021-03-04 Dana-Farber Cancer Institute, Inc. Cell-permeable stapled peptide modules for cellular delivery

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EP2662389A1 (fr) * 2011-01-07 2013-11-13 Toagosei Co., Ltd Procédé de préparation d'un antigène en vue d'obtenir un anticorps anti-peptide hydrophobe
WO2012159164A1 (fr) * 2011-05-23 2012-11-29 Phylogica Limited Procédé de détermination, d'identification ou d'isolement de peptides pénétrant dans des cellules
WO2015138435A1 (fr) * 2014-03-11 2015-09-17 Molecular Templates, Inc. Protéines comportant des régions de liaison, des régions effectrices à sous-motifs a de shiga-toxine, et des motifs signal de localisation du réticulum endoplasmique carboxy-terminaux
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