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WO2018212540A2 - Composite repebody-anticancéreux protéique à capacité de pénétration cellulaire améliorée, son procédé de préparation et utilisation - Google Patents

Composite repebody-anticancéreux protéique à capacité de pénétration cellulaire améliorée, son procédé de préparation et utilisation Download PDF

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WO2018212540A2
WO2018212540A2 PCT/KR2018/005526 KR2018005526W WO2018212540A2 WO 2018212540 A2 WO2018212540 A2 WO 2018212540A2 KR 2018005526 W KR2018005526 W KR 2018005526W WO 2018212540 A2 WO2018212540 A2 WO 2018212540A2
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cancer
protein drug
protein
repebody
anticancer
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Korean (ko)
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WO2018212540A3 (fr
Inventor
김학성
김희연
유정현
이중재
서효덕
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Korea Advanced Institute of Science and Technology KAIST
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Korea Advanced Institute of Science and Technology KAIST
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Priority claimed from KR1020170169409A external-priority patent/KR102098709B1/ko
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Publication of WO2018212540A2 publication Critical patent/WO2018212540A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a lipid-anticancer protein drug conjugate with improved cell permeability. More specifically, the present invention relates to a leucine-rich repeat (LRR) family protein having an alpha helical capping motif. Protein drug having anti-cancer activity through cell permeation domain to protein derivatives having lipid-based or ribibodies as basic skeletons by linking LRR structures derived from VLR (variable lymphocyte receptor) to N-terminus The present invention relates to a lipid body-anti-cancer protein drug complex having improved bound cell permeability, a method for preparing the same, and a use thereof.
  • LRR leucine-rich repeat
  • the lipidi-drug complex has a disadvantage of low efficiency in delivering drugs into cells.
  • the substance that binds to the lipid body is not a drug, but a protein, there is a disadvantage that can not be properly delivered into the cell is a situation in the development of pharmaceuticals using the lipid body.
  • Protein therapeutics are being actively developed for the effective treatment of diseases including cancer, but most of them are limited to disease targets outside the cell because proteins cannot cross the cell membrane. However, since there are more disease targets in cells, the importance of developing protein therapeutics for them is of great importance. Therefore, in order to develop a protein therapeutic agent having a high therapeutic effect against a wide range of diseases, the development of a technology capable of safely delivering a therapeutic protein targeting specific intracellular disease targets into specific cells is required.
  • TDP Pseudomonas exotoxin
  • the present inventors have combined the translocation domain of Pseudomonas exotoxin (TDP) and the anticancer protein drug, which are lipibody and bacterial toxin, to develop a more effective anticancer protein drug delivery system. It delivers anticancer protein drugs into cells based on the cell permeation domains of toxin proteins produced by the body and bacteria, and the advantages of Lipibody are expressed in soluble form in Escherichia coli and can be easily mass-produced at low cost. While maintaining the point, and the Tm of the Lipibody is thermodynamically very stable above 85 °C, it was confirmed that the cell permeability is improved can significantly increase the therapeutic efficacy as an anti-cancer protein drug conjugate, and the present invention Completed.
  • TDP Pseudomonas exotoxin
  • An object of the present invention is a lipid body anti-cancer protein drug complex with improved cell permeability, a polynucleotide encoding the complex, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced and the recombinant microorganism using the recombinant microorganism. It provides a method for producing a body-anticancer protein drug complex.
  • the present invention provides a lipid body-anticancer protein drug conjugate (repebody-anticancer protein drug conjugate) is coupled to the protein body having a cell permeation domain and anti-cancer activity to the lipid body (repebody) do.
  • the present invention also provides a polynucleotide encoding a lipid-anti-cancer protein drug complex having improved cell permeability, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced, and an improved cell permeability using the recombinant microorganism.
  • a method for preparing a lipidi-anticancer protein drug complex is provided.
  • the present invention also provides a composition for treating cancer, which contains the lipid-anti-cancer protein drug complex having improved cell permeability as an active ingredient.
  • Figure 1 confirms the performance of the lipid body-permeable domain-EGFP conjugate according to an embodiment of the present invention
  • (a) is the EGFR specific Lipibody-Pseudomonas aeruginosa cell permeation domain (Translocation Domain of Pseudomonas aeruginosa exotoxin A) -EGFP Schematic diagram showing the structure of the conjugate
  • (b) is a Western blot result that the EGFP increases in proportion to the injection concentration when the complex is injected into the cells
  • (c) is a cancer cell line with a different degree of EGFR expression Confocal microscopy fluorescence confirming receptor-based endocytosis
  • (d) is a confocal microscopy fluorescence confirming the movement of EGFP after 6 hours when the complex is administered to A431 cells
  • (e) Six hours after injecting the complex into A431 cells, a confocal microscopic fluorescence image was taken with a z
  • Figure 2 shows the intragrade retrograde transport pathway of the EGFP injected into the Lipibody-cell permeation domain-EGFP conjugate
  • (a) is a schematic diagram showing the intracellular transport pathway of EGFP
  • (b) Is a confocal microscopy fluorescence image of the time course of EGFP transported into cells.
  • FIG. 3 is a result confirming the EGFP transport capacity of the control group, (a) is a confocal microscopy fluorescence image confirming the intracellular transport of off-target lipid body (EGFR non-specific) -TDP-EGFP complex, (b) is lipi Confocal microscopy fluorescence image confirming the intracellular transport progress of the body (EGFR specific) -EGFP complex.
  • Figure 4 confirms the effect of the Lipibody-TDP-toxin protein (Gelonin, Glo) complex
  • (a) is a schematic diagram showing the structure of the Lipibody-TDP-Glo complex
  • (b) is a different amount of EGFR expression
  • (c) are the cell lines with high EGFR expression (MDA- MB-468) and the low cell line (MCF-7) after injection of the Lipibody-TDP-Glo complex, and TUNEL analysis confirmed the cell killing effect.
  • Figure 5 confirms the intracellular anticancer activity of the Lipibody-TDP-Glo complex
  • (a) is a schematic diagram showing the schedule of injecting cancer cells and Lipibody-TDP-Glo complex into the mouse
  • (b) is a xenograft Tumor reduction was observed in the mice
  • (c) is a picture of the tumor obtained in the mouse at 30 days after the injection of PBS, free lipid and Lipibody-TDP-Glo complex
  • (d) is a mouse
  • E) is the result of measuring hepatotoxicity
  • (f) is the result of measuring kidney toxicity.
  • the present invention provides a new lipid permeability-enhanced lipid-anti-cancer protein drug complex in which cell-permeable domains and protein drugs are directly bound to 'lipibody' through genetic binding.
  • lipibody refers to a water-soluble fusion polypeptide in which the N-terminus of an internalin protein and LUC (Leucine rich repeat) family protein are fused, and a polypeptide partially modified with the basic structure thereof.
  • Repebody Korean Patent Publication No. 10-2011-0099600 A1, Korean Registered Patent Publication No. 1356075, International Publication No. WO2013-129852 A1 and Lipibody
  • the lipid body is microorganism-derived N-terminus of the Leucine rich repeat (LRR) family protein having an alpha helical capping motif, a modified repeat module of a variable lymphocyte receptor (VLR) protein and a VLR protein.
  • LRR Leucine rich repeat
  • VLR variable lymphocyte receptor
  • the lipid body has a property of specifically binding to a specific target protein, such as a receptor specifically expressed or overexpressed only in cancer cells, and repeat module It can include all proteins belonging to the LRR family having, and all fusion LRR family proteins with improved biophysical properties.
  • the N-terminus of the internalin protein which is a constituent of the lipida body, may be selected from the N-terminus having a high structural similarity according to the type of LRR family protein that can be fused, and the most stable through calculation of binding energy.
  • an amino acid it is possible to change the amino acid of the module.
  • the present invention relates to a Lipibody-anti-cancer protein drug conjugate (Repebody-protein toxin conjugate) in which a protein drug having anti-cancer activity is coupled to a lipid body (repebody) through a cell permeation domain, thereby improving cell permeability.
  • Repebody-protein toxin conjugate a Lipibody-anti-cancer protein drug conjugate in which a protein drug having anti-cancer activity is coupled to a lipid body (repebody) through a cell permeation domain, thereby improving cell permeability.
  • the lipbodies constituting the lipbodi-anti-cancer protein drug complex having improved cell permeability are the known lipbodi monomers or lipbody multimers composed of two or more lipbodi monomers (hereinafter referred to as “lipiva body multimers”).
  • a protein such as Fc protein or an organic / inorganic compound such as polyethylene glycol (PEG) is introduced into these lipid body monomers or lipid body multimers.
  • PEG polyethylene glycol
  • various kinds of proteins, specific amino acid sequences, organic / inorganic compounds, etc. may be obtained by methods well known in the art. It is possible to introduce and the resulting lipid bodies can also be used without limitation so long as they do not depart from the object of the present invention.
  • anticancer protein drug of the present invention may be used as long as it is a protein capable of performing apoptosis as a protein, and preferably, protein toxin, interferon, interleukin, hematopoietic growth factor, tumor necrosis factor, and self-assembled anticancer protein. It may be characterized in that selected from the group consisting of, but is not limited thereto.
  • the self-assembled protein may be used as long as it is a protein that is self-assembled when expressed in cells, preferably ferritin, ferritin-like protein, magnetosome constituent protein and virus. It may be selected from the group consisting of constitutive proteins.
  • the anticancer agent can be used as long as it is a small compound (small-molecule) capable of binding or injecting the self-assembled protein nanoparticles, preferably a group consisting of a nucleic acid alkylating agent, metabolic antagonist, natural-derived alkaloid agent and hormone agent. It may be characterized in that selected from, but is not limited thereto.
  • the nucleic acid alkylating agent is bischloroethylamines, aziridines, nitrosoureas, tetrazine, tetrazine, alkyl alkane sulphonates, non-classical agents ( non-classical agent) and platinum compounds may be selected from the group consisting of, but is not limited to these.
  • the bischloroethylamine may be cyclophosphamide, melphalan, chlorambucil, or ifosphamide, but is not limited thereto. It may be thiotepa or mitomycin c, but is not limited thereto, and the nitrosourea may be carmustine, lomustine, fotemustine or streptoto.
  • the tetrazine may be, but not limited thereto, but may be dacarbazine, temozolomide, or mitozolomide, but is not limited thereto.
  • Alkane sulfonates may be busulphan, but are not limited thereto, and the non-classical agent may be altretamine or Procarbazine may be, but is not limited to, the platinum compound may be cisplatin, cicarlatin, carboplatin, or oxalooplatin, but is not limited thereto.
  • the metabolic antagonist may be selected from the group consisting of pyrimidine derivatives, folate derivatives and purine derivatives, but is not limited thereto.
  • the pyrimidine derivative may be fluorouracil (5-FU), cytarabine, or gemcitabine, but is not limited thereto.
  • the folate derivative may be mesoprexate, but is not limited thereto.
  • the purine derivative may be mercaptopurine (6-MP), but is not limited thereto.
  • the natural-derived alkaloid agent may be selected from the group consisting of vinca alkalroid, taxane, and antibiotics, but is not limited thereto.
  • the vinca alkaloid may be vinblastine, vincristine or vinorelbine, but is not limited thereto
  • the taxane may be paclitaxel or docetaxel, but is not limited thereto
  • the antibiotic may be doxorubicin, It may be daunorubicin or mitomycin, but is not limited thereto.
  • the hormonal agent may be selected from the group consisting of corticosteroids, sex hormones, and antiestrogens, but is not limited thereto.
  • the adrenal cortex steroid may be prednisone or prednisolone, but is not limited thereto, and the sex hormone may be progestin, estrogen or androgen, but is not limited thereto, and the antiestogen may be tamoxifen, It is not limited to this.
  • a complex having a form of self-assembled protein nanoparticles by fusion of a lipid body and a cell-permeable domain in turn to the N-terminus of the self-assembled protein, and a protein having anticancer activity at the C-terminus are also within the scope of the present invention. It is obvious to those skilled in the art to belong.
  • the protein toxin may be Pseudomonas aeruginosa exotoxin A, diphtheria sulfur toxin, botulinum toxin, tetanus antitoxin, heterogeneous toxin, cholera toxin, siguatoxin, gelonin (Gelonin) or lysine, but is not limited thereto.
  • the interferon may be interferon-alpha, -beta or -gamma, but is not limited thereto.
  • the interleukin may be interleukin 2, but is not limited thereto.
  • the hematopoietic factor may be G-CSF or GM-CSF. However, the present invention is not limited thereto, and the tumor necrosis factor may be TNF- ⁇ or TNF- ⁇ , but is not limited thereto.
  • cell permeation domain refers to a part that allows a biomolecule to enter a cell through a cell membrane.
  • the domain is Pseudomonas exotoxin A (Pseudomonas exotoxin A), Shiga toxin (shiga like toxin), Tat, antp (antennapedia protein), R9 (RRRRRRRRR), Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, It may be characterized in that it is selected from the group consisting of 2IL-1a and dNP2, more preferably a part of Pseudomonas Exotoxin A, Pseudomonas Exotoxin A, may be part of Shiga like toxin of E. coli hemorrhagic, It is not limited.
  • the cell permeation domain was used as part of the cell permeation domain of Pseudomonas aeruginosa exotoxin A, which was referred to as TDP (Translocation Domain of Pseudomona aeruginosa exotoxin A).
  • TDP Translocation Domain of Pseudomona aeruginosa exotoxin A
  • the cell permeation domain may be linked to the C-terminus of the Lipibody through a linker for the purpose of preserving the protein's original structure and maximizing the activity, and the anticancer protein drug is originally
  • the linker may be linked to the C-terminus of the cell permeation domain for the purpose of preserving the structure in its entirety and maximizing activity.
  • the linker is generally composed of amino acids, but the type and length thereof are not particularly limited, and as in one embodiment of the present invention, a (G3S) n spacer having flexable properties, that is, three glycines and one serine may be used.
  • EAAAK alpha-helix likner with rigid properties or GSAGSAAGSG linker, ie (EAAAK) n
  • EAAAK alpha-helix likner with rigid properties or GSAGSAAGSG linker
  • n is an integer greater than or equal to 1, but in the case of G3S, various variants of glycine and serine may be possible, and DRDD (where D is aspartate and R may be used to introduce more hydrophilic properties in consideration of physical properties).
  • Various linkers containing charged amino acids such as arginine can also be used.
  • the lipid body of the lipid body-enhanced lipid body anti-cancer protein drug complex is CD19, CD20, CD21, CD22, CD37, CD70, CD72, CD79a / b, CD180, CD30, CD33, CD43, CD56, CD74, CD138, endothelin B receptor, EGFR, VEGF, CRIPTO, FAP, mesothelin, G2D, 5T4, alpha v beta6, CD174, CD227 (MUC-1), CD326 (Epcam), ED-B, CD227 (MUC-1), nectin-4 (ASG-22ME), HER2, GPNMB, LIV1A, MUC16 (CA125), TIM-1 (CDX-014), GD2, GPNMB, PMEL 17, SMA, STEAP-1, TENB2 and CAIX It may be characterized by binding specifically.
  • the target protein is a protein that is specific or overexpressed in cancer cells, and is a non-Hodgkin.
  • lymphoma may be CD19, CD20, CD21, CD22, CD37, CD70, CD72, CD79a / b, and CD180, and CD30 if non-Hodgkin lymphoma is the main indication.
  • the main indications are CD33 and CD43 for acute myeloid leukemia, CD43 for multiple myeloma leukemia, and multiple myeloma for multiple myeloma.
  • CD56, CD74, CD138, and EGFR for endothelin B receptor, head and neck cancer
  • EGFR, CD56, C for lung cancer
  • the main indications for D326, CRIPTO, FAP, mesothelin, G2D, 5T4, and alpha v beta6, glioblastoma are EGFR, CDEG, CD174, CD227 (MUC) for EGFR, EGFRvIII, and colorectal cancer.
  • MUC16 CA125
  • TIM-1 CDX-014
  • mesothelin for ovarian cancer GD2, GPNMB, ED-B, PMEL 17, and endothelin B receptor, melanoma as the main indications, PSMA, STEAP-1, and TENB2, kidney cancer as the main indications CAIX, TIM-1 (CDX-014), and mesothelin may be the main indications for mesothelioma.
  • the indications listed above and the target proteins do not necessarily have to match, and in particular, EGFR and HER2 are often overexpressed in various cancers and need not be limited to the indications.
  • the anti-EGFR lipido-anti-cancer protein drug complex having improved cell permeability was prepared using a lipidi that binds to EGFR (endothelial growth factor receptor), and confirmed its efficacy.
  • the lipida-anti-cancer protein drug complex may further comprise an ER retention sequence at the carboxy terminal end of the anti-cancer protein drug.
  • the vesicle-conserved sequence may be used without limitation as long as it is an amino acid sequence capable of promoting intracellular retrograde transport pathway of anticancer protein drug transported into cells by the complex of the present invention.
  • K -aspartic acid
  • E -glutamic acid
  • L leucine
  • the lipido-anti-cancer protein drug complex may be represented by an amino acid sequence of any one of SEQ ID NOs: 1 to 7.
  • the present invention also relates to a polynucleotide encoding a lipid body anti-cancer protein drug complex with improved cell permeability.
  • the present invention also relates to a vector comprising the polynucleotide.
  • the term "vector” refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence to express the target protein in a suitable host cell.
  • the regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation, It can be produced in various ways.
  • the promoter of the vector may be constitutive or inducible. After the vector has been transformed into a suitable host, it can replicate or function independently of the host genome and integrate into the genome itself.
  • the vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophages.
  • phage vector or cosmid vector can be used as a phage vector or cosmid vector, and pBR, pUC, and pBluescriptII based , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
  • the vector usable in the present invention is not particularly limited and known expression vectors can be used.
  • pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used.
  • pACYC177, pCL, pCC1BAC vectors can be used.
  • the present invention also relates to a recombinant microorganism into which the polynucleotide or the vector comprising the polynucleotide is introduced.
  • the term “recombinant microorganism” means that a vector having a polynucleotide encoding at least one target protein is introduced into a host cell, or a polynucleotide encoding at least one target protein is introduced at a microorganism so that the polynucleotide is incorporated into a chromosome. It means a cell infected with a trait to express the target protein, and can be any cell such as eukaryotic cells, prokaryotic cells, etc., but is not particularly limited to these, bacterial cells such as E.
  • coli coli, Streptomyces, Salmonella typhimurium
  • Yeast cells Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells
  • Animal cells such as CHO, COS, NSO, 293, Bow Melanoma cells, and the like.
  • the term “transformation” refers to introducing a vector comprising a polynucleotide encoding a target protein into a host cell or integrating a polynucleotide encoding a target protein into a chromosome of the host cell to complete the polynucleotide in the host cell. Means that the protein that is encoded by the gene can be expressed. Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, whether they are inserted into or located outside the chromosome of the host cell. The polynucleotide also includes DNA and RNA encoding the target protein.
  • the polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression.
  • the expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide.
  • the expression cassette may be in the form of an expression vector capable of self replication.
  • the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell.
  • the present invention comprises the steps of: (i) culturing the recombinant microorganism to express the Lipibody-anticancer protein drug complex with improved cell permeability; And (ii) recovering the expressed lipid permeability-enhanced lipido-anti-cancer protein drug complex.
  • the step of culturing the transformant is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto.
  • a basic compound e.g. sodium hydroxide, potassium hydroxide or ammonia
  • an acidic compound e.g. phosphoric acid or sulfuric acid
  • the incubation temperature can be maintained at 20 to 45 °C, preferably 25 to 40 °C, about 10 to 160 hours Incubating is preferred.
  • the polypeptide produced by the culture may be secreted into the medium or remain in the cell.
  • the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources.
  • sugars and carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
  • fats and fats e.g. soybean oil, sunflower seeds
  • fatty acids e.g. palmitic acid, stearic acid and linoleic acid
  • alcohols e.g. glycerol and ethanol
  • organic acids e.g. acetic acid
  • Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination;
  • As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination;
  • Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
  • the method for recovering the lipid permeability-enhanced lipid body anti-cancer protein drug complex produced in the present invention is lipi using a suitable method known in the art according to a culture method, for example, batch, continuous or fed-batch culture method. Body-toxin protein complexes can be collected.
  • the present invention relates to a composition for preventing or treating a disease, wherein the composition contains a lipida anti-cancer protein drug complex having improved cell permeability as an active ingredient.
  • the present invention relates to the use of the lipid-anti-cancer protein drug complex with improved cell permeability for disease prevention or treatment.
  • the disease is not limited but may include all diseases commonly known, for example, digestive diseases, respiratory diseases, cardiovascular diseases, kidney diseases, endocrine diseases, immune diseases, blood diseases, genetic diseases, congenital Metabolic disorders, sexually transmitted diseases, cancers, psychiatric diseases, addictions or diseases corresponding to surgical diseases, and the like.
  • cancers such as brain cancer, leukemia, stomach cancer, colon cancer, rectal cancer, liver cancer, lung cancer, esophageal cancer, prostate cancer, breast cancer, skin cancer, uterine cancer, cold, pneumonia, tuberculosis, AIDS, and plague , Infectious diseases such as prion disease, hepatitis B, arteriosclerosis, hernias, smallpox, anemia, polio, allergies, asthma, diabetes, arteriosclerosis, kidney disease, stroke, Alzheimer's disease, and other diseases such as obesity.
  • cancers such as brain cancer, leukemia, stomach cancer, colon cancer, rectal cancer, liver cancer, lung cancer, esophageal cancer, prostate cancer, breast cancer, skin cancer, uterine cancer, cold, pneumonia, tuberculosis, AIDS, and plague , Infectious diseases such as prion disease, hepatitis B, arteriosclerosis, hernias, smallpox, anemia, polio, allergies, asthma, diabetes, arterio
  • the disease may preferably be cancer, more preferably non-Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, acute lymphocytic Leukemia (acute-lymphoid leukemia), multiple myeloma, head and neck cancer, lung cancer, glioblastoma, colon / rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma, prostate cancer , Kidney cancer and mesothelioma may be selected from the group consisting of.
  • treatment means not only inhibiting or alleviating the disease or one or more symptoms caused by the administration of the composition, but also preventing the progression of sepsis or the treatment of a disease that reverses the symptoms of the disease.
  • prevention in the present invention means any action that inhibits or delays the onset of the disease by administration of the composition.
  • the prevention or treatment of disease is achieved by combining the lipid permeability-enhanced lipid-anti-cancer protein drug complex developed in the present invention with the substrate of the lipid body, for example, if the substrate of the lipid body is EGFR, The body and EGFR bind, and anticancer protein drugs act on cells overexpressing EGFR to prevent or treat disease.
  • composition for preventing or treating a disease containing the lipid permeability-enhanced lipidi-anticancer protein drug complex of the present invention as an active ingredient may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • the amount of the Lipibodi-anti-cancer protein drug complex having improved composition cell permeability is not limited but may be added at 0.01 to 95% by weight of the total composition weight.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Cancer preventive or therapeutic composition comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier is applicable to any formulation comprising it as an active ingredient, it can be prepared in oral or parenteral formulations.
  • Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.
  • Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do.
  • lactose For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax
  • a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as.
  • the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials.
  • solutions or suspensions which may be formulated for unit administration of ampoules or vials.
  • a rectal composition such as suppositories or enemas, including conventional suppository bases such as cocoa butter or other glycerides.
  • a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
  • the present invention relates to a method for preventing or treating cancer comprising administering a composition for preventing or treating cancer, including the lipid peri-anti-cancer protein drug complex having improved cell permeability.
  • the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner.
  • the route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhaled, intraocular or intradermal routes.
  • the treatment method of the present invention includes administering a composition for preventing or treating cancer of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily usage may be determined by treatment within the correct medical judgment.
  • the specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, the duration of treatment, and the various factors and similar factors well known in the medical arts, including drugs used with or concurrent with the specific composition. Therefore, the effective amount of the composition for preventing or treating cancer suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.
  • the resulting lipobody construct was transformed into Escherichia coli (BL21 (DE3) or OrigamiB (DE3)), respectively, and one colony containing each construct was inoculated into 10 ml LB medium containing 100 ⁇ g of ampicillin. Then, it was incubated overnight at 37 °C. For free lipida (rEgH9) it was incubated in a medium additionally containing kanamycin (50 ⁇ g / ml) and tetracycline (10 ⁇ g / ml).
  • the culture was diluted 1: 100 and incubated at 37 ° C. up to OD 0.5, then IPTG was added to (0.5 mM) and then grown overnight at 18 ° C. to induce protein expression.
  • the culture solution was centrifuged at 6,000 rpm for 15 minutes to separate the cells, and then the pellets were dissolved in Lysis buffer (20 mM Tris, 150 mM NaCl, 10 mM Imidazole pH 8.0), and the cells were pulverized by ultra sonication and then 13,000. Centrifugation at rpm for 60 minutes yielded a supernatant, which was filtered through a 0.22 ⁇ m filter.
  • the filtered supernatant was loaded into a Ni-NTA chromatography column (Qiagen, USA0) and washed three times with washing buffer (20 mM Tris, 500 mM NaCl, Rb-Apo, 150 mM NaCl, 20 mM Imidazole, pH 8.0), and then Protein was obtained (20 mM Tris, 150 mM NaCl, 250 mM Imidazole, pH 8.0) and the buffer was replaced with Tris or PBS buffer at pH 7.4.
  • Obtained lipid body protein was stored at 4 °C, each protein concentration was calculated by measuring the absorbance at 280nm using a UV-Visible spectrophotometer (GE healthcare, USA).
  • the concentration of the fusion protein used in the present invention is expressed by the molar concentration of the monomeric fusion protein.
  • the cells to be tested were placed on an 8-well slide glass (SPL, Korea), and then 2 ⁇ M of lipido-TDP-EGFP (Rb-TDP-EGFP) protein was mixed with DMEM medium and treated for different times. Incubated for 6 hours in a 37 °C incubator containing 5% carbon dioxide. Cells were washed three times with DPBS and then fixed for 20 minutes with 4% paraformaldehyde.
  • the cross sections of the cells were measured at 0.7 ⁇ m intervals to determine how the green fluorescence protein (EGFP) was distributed in the cells by Z-stacking technique (confocal microscopy technique where the cross section of cells could be seen using light).
  • EGFP green fluorescence protein
  • Human cancer cell lines (A431 and MCF7) were incubated in a 37 ° C. 5% carbon dioxide incubator in 100 mm culture dish cell culture plates.
  • the A431 cell line used DMEM medium and the MCF7 cell line used PRMI medium. 10% fetal bovine serum was added to all culture media. After washing the cells with DPBS, trypsin-EDTA was treated for 5-10 minutes at 37 °C separated to the surface, and then released to each culture medium again.
  • Experimental cell lines were dispensed 5 ⁇ 10 3 cells per well on an 8-well chamber slide (SPL., Korea), and then incubated for 24 hours in a 37 °C, 5% carbon dioxide incubator. The following day, different concentrations of Rb-TDP-EGFP were treated in each well for 6 hours.
  • the cell lines of the subjects were washed with DPBS, and then lysed with Lysis buffer (Tris-HCl 50 mM, pH 8, NaCl 150 mM and 1% (v / v) Triton X-100). Supernatants were electrophoresed on 12% SDS PAGE and then transferred to nitrocellulose membrane (Bio-Rad) for 100 V for 2 hours. The membrane was blocked with PBS solution (TPBSA) containing 1% BSA and 0.05% Tween 20 at 25 ° C. for 2 hours. The membrane was then subjected to western blotting using goat anti-EGFP polyclonal antibody (Abcam) and rabbit anti-chlorine IgG-HRP conjugated antibody (Bio-Rad).
  • Lysis buffer Tris-HCl 50 mM, pH 8, NaCl 150 mM and 1% (v / v) Triton X-100.
  • Supernatants were electrophoresed on 12% SDS PAGE and then transferred to
  • the resulting bands were analyzed by LAS-3000 imaging system (Fuji-Film) using enhanced chemiluminescence solution (Millipore).
  • the cell lines of the experiment were dispensed at an density of 1 ⁇ 10 4 cells in an 8-well chamber slide (SPL) and then treated with Rb-TDP-Gelonin for 72 hours.
  • SPL 8-well chamber slide
  • Rb-TDP-Gelonin Fixed with 4% (w / v) formaldehyde solution for 15 minutes, washed with DPBS, treated with permeabilization buffer for 20 minutes, washed with 3% (v / v) hydrogen peroxide and DPBS, followed by TDT. (erminal deoxynucleotidyl Transferase) enzyme was labeled for 1 hour, and the label was stopped by treatment with DPBS in a stop buffer.
  • mice Six male BALB / c nu / nu mice were organized into a total of three groups and injected with PBS, lipido or complex corresponding to the tail vein of the rat. Mice were raised for 4 weeks and mortality was measured.
  • MDA-MB-468 cells (5 ⁇ 10 6 cells, 100 ⁇ l Martigel) were subcutaneously inoculated into the rat right flank. After 7 days, it was confirmed that the mouse tumor size reached about 100 mm 3 .
  • Six mice were prepared for each of the injected Lipibodies or Lipibody-complexes, and a total of three groups were prepared, and PBS, Lipibodi (4.2 mg / kg) and Lipibody-TDP-Glo complex (10 mg / kg) were injected intravenously at start, 3, 6, 9, and 12 days. That is, each mouse received the same concentration of drug (27 ⁇ M / dose).
  • the body weight and tumor size of the mice were measured twice a day for 30 days, and the tumor volume was measured by length and width with a caliper, and then volume was calculated by a formula of length ⁇ width ⁇ 0.5.
  • the tumors were euthanized when the tumor volume became 650 mm 3 or skin ulceration was observed.
  • Experimental data are expressed as ⁇ S.D mean.
  • statistical analysis was performed using ANOVA, and comparisons between corresponding groups were performed using Tukey's multiple comparison test.
  • TDP water-soluble Lipibody-Cell Permeable Domain
  • Fluorescently labeled Rb-TDP-EGFP was injected into A431 cell line overexpressing EGFR, MDA-MB-468 cell line, H1650 cell line expressing less EGFR and MCF-7 cell line expressing no EGFR, respectively. Incubated for 6 hours in the included 37 °C incubator. Subsequently, as a result of measuring the fluorescence, it was confirmed that the fluorescence of the green fluorescent protein (EGFP) appeared strongly in the A431 cells as a whole (FIG. 1C).
  • EGFP green fluorescent protein
  • Fluorescently labeled Rb-TDP-GEFP was injected into the A431 cell line overexpressing EGFR and analyzed for fluorescence of EGFP over time.
  • Lipidbody-TDP-EGFP complex and EGFR specific Lipibody-EGFP complex that do not specifically bind to EGFR were produced, injected into A431 cell line overexpressing EGFR, and analyzed by fluorescence over time. Confirmed that they did not properly deliver EGFP into the cytoplasm (Fig. 3).
  • a Lipibody-TDP-protein toxin (Gelonin, Glo) complex, a free Lipibody and an off-target Lipibody (Rboff) -TDP-Glo were prepared, which were each overexpressed EGFR.
  • Cell lines, MDA-MB-468 cell line, H1650 cell line expressing less EGFR, and MCF-7 cell line expressing no EGFR were injected and then cytotoxicity was measured.
  • apoptotic-TDP-Glo complex was analyzed by the TUNEL Assay analysis of the apoptosis rate of the cells treated with Lipibody-TDP-Glo complex and Glo alone in MDA-MB-468 cell line and MCF-7 cell line. Only cell suicide occurred was confirmed (Fig. 4C).
  • mice were transplanted with an MDA-MB-468 cell line overexpressing EGFR, and when the tumor size reached 100 mm 3, the complex, PBS, Tumor size and body weight were measured after each administration of the free EGFR specific Lipibody.
  • the lipid permeability-enhanced lipid body anti-cancer protein drug complex according to the present invention has a high substrate specificity provided by the lipid body and the cell-permeable domain, and a protein drug capable of selectively killing only cancer cells in combination with excellent tissue and tumor penetration ability.
  • a protein drug capable of selectively killing only cancer cells in combination with excellent tissue and tumor penetration ability As a result, it has superior efficacy compared to the use of Lipibody alone, and has the advantage of minimizing side effects by having specific selectivity for target cells compared to the use of protein drug alone, It is expressed in soluble form in E. coli, which can be easily mass-produced at low cost, is thermodynamically stable, and has excellent tissue penetrability.

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Abstract

La présente invention concerne un composite dans lequel un repebody, un domaine de pénétration cellulaire et un anticancéreux protéique sont conjugués, et son procédé de préparation et, plus précisément, un composite repebody-anticancéreux protéique ayant une capacité de pénétration cellulaire améliorée, où un domaine de pénétration cellulaire et un médicament protéique ayant une activité anticancéreuse sont conjugués au repebody ou à un dérivé de protéine comprenant le repebody à titre de squelette basique, son procédé de préparation et utilisation. Le composite repebody-anticancéreux protéique à capacité de pénétration cellulaire améliorée selon la présente invention est obtenu par conjugaison du médicament protéique, capable de détruire sélectivement uniquement les cellules cancéreuses, et doué en outre d'une spécificité de substrat élevée et d'un excellente excellente capacité d'infiltration des tissus et des tumeurs conférée par le repebody et le domaine de pénétration cellulaire. Par conséquent, le composite repebody-anticancéreux protéique est utile pour détruire des cellules cibles par liaison spécifique à des protéines cibles, tout en présentant les avantages d'une efficacité supérieure comparativement au cas où le repebody est utilisé seul, ainsi que les avantages d'une production de masse à bas coût par une expression sous forme soluble chez E. coli, qui est très stable sur le plan thermodynamique, et a une excellent pouvoir d'infiltration des tissus.
PCT/KR2018/005526 2017-05-18 2018-05-15 Composite repebody-anticancéreux protéique à capacité de pénétration cellulaire améliorée, son procédé de préparation et utilisation Ceased WO2018212540A2 (fr)

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CN112156189A (zh) * 2020-07-15 2021-01-01 华南师范大学 一种her2+乳腺癌靶向蛋白复合纳米粒及其制备方法和应用

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US10017570B2 (en) * 2012-02-27 2018-07-10 Korea Advanced Institute Of Science And Technology Repebody for novel interleukin-6 and use thereof
KR101572219B1 (ko) * 2013-11-13 2015-11-30 한국과학기술원 반복모듈을 포함하는 리피바디 단백질의 개량방법
KR20160015893A (ko) * 2014-08-01 2016-02-15 한국과학기술원 생체분자를 세포 내로 전달하는 폴리펩타이드 및 그 용도
KR101732380B1 (ko) * 2014-12-09 2017-05-24 전남대학교산학협력단 상피성장인자 수용체를 표적으로 하는 신규 항암제
KR20160080832A (ko) * 2014-12-29 2016-07-08 주식회사 레고켐 바이오사이언스 리피바디 유도체-약물 복합체, 그 제조방법 및 용도

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112156189A (zh) * 2020-07-15 2021-01-01 华南师范大学 一种her2+乳腺癌靶向蛋白复合纳米粒及其制备方法和应用
CN112156189B (zh) * 2020-07-15 2023-01-17 华南师范大学 一种her2+乳腺癌靶向蛋白复合纳米粒及其制备方法和应用

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