WO2022004707A1 - Sars-cov-2 adsorption carrier and column containing this - Google Patents

Sars-cov-2 adsorption carrier and column containing this Download PDF

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Publication number
WO2022004707A1
WO2022004707A1 PCT/JP2021/024503 JP2021024503W WO2022004707A1 WO 2022004707 A1 WO2022004707 A1 WO 2022004707A1 JP 2021024503 W JP2021024503 W JP 2021024503W WO 2022004707 A1 WO2022004707 A1 WO 2022004707A1
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Prior art keywords
ace2
carrier
partial peptide
peptide
immobilized
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PCT/JP2021/024503
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French (fr)
Japanese (ja)
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善隆 猪阪
善治 松浦
拓 吉矢
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国立大学法人大阪大学
株式会社ペプチド研究所
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Priority to JP2022534029A priority Critical patent/JPWO2022004707A1/ja
Publication of WO2022004707A1 publication Critical patent/WO2022004707A1/en

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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the present invention relates to a partial peptide of angiotensin converting enzyme 2 (ACE2), a carrier on which the peptide is immobilized, a column containing the carrier, and the like.
  • ACE2 angiotensin converting enzyme 2
  • SARS-CoV and SARS-CoV-2 cause a large-scale pandemic.
  • pneumonia caused by SARS-CoV-2 COVID-19
  • COVID-19 has become a pandemic worldwide and has become an extremely serious problem.
  • the coronavirus spike (S) protein contains two subunits, S1 and S2.
  • S1 contains a receptor binding domain (RBD) and is involved in the binding of host cell surfaces to receptors.
  • RBD receptor binding domain
  • the S2 subunit has a function necessary for membrane fusion, and membrane fusion proceeds when S2 is cleaved by TMPRSS2, which is a human serine protease.
  • TMPRSS2 a human serine protease.
  • HCoV-NL63, SARS-CoV and SARS-CoV-2 all utilize human ACE2 as a receptor.
  • the RBD of SARS-CoV-2 binds to the peptidase domain (PD) of ACE2 (Kd value of about 15 nM).
  • the RBD regions of SARS-CoV and SARS-CoV-2 that bind to ACE2 are conserved.
  • the PD region of ACE2 is considered to be useful for adsorbing SARS-CoV-2, and can be applied even when a variant of SARS-CoV appears in the future.
  • SARS-CoV which was prevalent in 2003, also caused acute respiratory distress syndrome (ARDS), and there is a report that blood adsorption therapy using an endotoxin adsorption column was useful as a treatment method (Non-Patent Document 1).
  • Non-Patent Documents 2 to 4 The decrease of IL-6 which is a cytokine and the decrease of inflammatory cells have been reported (Non-Patent Documents 2 to 4). There is also a report that plasmapheresis was effective against this SARS-CoV-2 infection (COVID-19) (Non-Patent Documents 5 and 6), but in the case of plasmapheresis, a waste liquid containing a virus. Processing becomes a problem.
  • a carrier and column for blood adsorption therapy that are effective in treating viral infections such as SARS-CoV and SARS-CoV-2 and that do not pose a problem in the treatment of waste liquid containing a virus are desired.
  • the present inventors have conducted extensive studies to solve the above problems, and have found that a carrier on which a specific partial peptide of ACE2 is immobilized efficiently adsorbs SARS-CoV-2, and completes the present invention. I arrived.
  • the present invention provides: (1) A carrier on which an angiotensin converting enzyme 2 (ACE2) partial peptide is immobilized. (2) The carrier according to (1), wherein the ACE2 partial peptide contains an amino acid sequence in the peptidase domain of the ACE2 protein. (3) The carrier according to (2), wherein the ACE2 partial peptide contains an amino acid sequence in the ⁇ -helix of the peptidase domain of the ACE2 protein. (4) The carrier according to (3), wherein the ACE2 partial peptide comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
  • ACE2 partial peptide comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
  • the carrier and column of the present invention efficiently adsorb viruses. Therefore, the carriers and columns of the present invention are effective for blood adsorption therapy for infectious diseases caused by viruses using ACE2 as a receptor.
  • the carriers and columns of the present invention on which the ACE2 partial peptide and polymyxin B are immobilized are particularly effective for blood adsorption therapy for severe infectious diseases caused by viruses that accept ACE2 as a receptor.
  • FIG. 1 is a scheme showing an outline of a carrier of the present invention obtained by immobilizing an ACE peptide on a commercially available polymyxin B-immobilized fiber and a column containing the same.
  • FIG. 2 is a scheme showing an example of combined use of a column containing the carrier of the present invention (denoted as a tremixin-improved column in the figure) and ECMO.
  • FIG. 3 shows the SARS-CoV-2 virus of MI on which an amine-type Purolite resin (N), a maleimide-bound Purolite resin (MI), and a peptide (1N, 1C, 2N, 2C, 3N, 3C, 4N, 4C) are immobilized.
  • FIG. 4 shows the results of examining the SARS-CoV-2 virus adsorption capacity of chloroacetylated Toray fibers (TF) on which 3N, 4N or 4C was immobilized, and TF on which 4N-Dibeg was immobilized. It is a graph which shows.
  • 4N-Dibeg-TF is a TF carrying a 4N peptide containing cysteine via two molecules of Diveg (described later) as a spacer.
  • FIG. 5 is a graph showing the results of examining the SARS-CoV-2 virus adsorption capacity of the sterilized peptide-immobilized TF. For each sterile instrument, the three bars show the results of TF immobilized with peptides 3N (left), 4N (middle) and 4C (right), respectively.
  • Peptides 3N and 4C contain 3 molecules of AEEA as spacers, and peptide 4N contains 2 molecules of Diveg.
  • the present invention provides, in one embodiment, a carrier on which the ACE2 partial peptide is immobilized.
  • ACE2 partial peptide refers to a peptide containing or consisting of a portion of the amino acid sequence of a human ACE2 protein.
  • the amino acid sequence of the human ACE2 protein is known.
  • ACE2 partial peptide includes not only human ACE2 partial peptide, but also a mutant peptide thereof and a modified peptide thereof.
  • a peptide containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 4 and having a spacer and a peptide having an attachment are also included in the "ACE2 partial peptide".
  • the mutant ACE2 partial peptide, the modified ACE2 partial peptide, and the ACE2 partial peptide having a spacer have a virus adsorption capacity equal to or higher than that of the original human ACE2 partial peptide.
  • Equivalent adsorption capacity means, for example, that the amount of virus adsorbed per molecule of mutant ACE2 partial peptide or modified ACE2 partial peptide is about 70% or more, preferably about 70% or more, as compared with the original human ACE2 partial peptide. Means that it is about 80% or more, more preferably about 90% or more, still more preferably about 100% or more.
  • the mutant ACE2 partial peptide may be a peptide containing or consisting of an amino acid sequence in which one or several amino acids are substituted, added, inserted or deleted in the amino acid sequence of the original human ACE2 partial peptide.
  • the number means 2, 3, 4, 5, 5, 6, 7, 8, or 9.
  • the mutant ACE2 partial peptide has an amino acid sequence identity of about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more with respect to the amino acid sequence of the original human ACE2 partial peptide. It may be a peptide containing or consisting of an amino acid sequence having.
  • the amino acid substitution is preferably a substitution between homologous amino acids. Homogeneous amino acids are known to those of skill in the art.
  • the identity of the amino acid sequence can be performed by using a known means such as BLAST search.
  • the mutant ACE2 partial peptide may contain a D-amino acid or an unnatural amino acid.
  • unnatural amino acids include, but are not limited to, ⁇ -alanine, 2-aminoisobutyric acid, cyclohexylalanine, tert-leucine, and various N-methyl amino acids.
  • One to several amino acid residues in the amino acid sequence of the ACE2 partial peptide may be replaced with unnatural amino acids to improve helix properties (see SEQ ID NO: 2 as an example).
  • One to all amino acid residues in the amino acid sequence of the ACE2 partial peptide may be used as the D-form to improve blood stability.
  • the reverse sequence of the amino acid sequence of the ACE2 partial peptide is also included in the mutant ACE2 partial peptide.
  • the modification mode of the modified ACE2 partial peptide may be any, for example, alkylation, esterification, amidation, halogenation and the like.
  • the preferred ACE2 partial peptide is a peptide comprising or consisting of the amino acid sequence in the peptidase domain of the ACE2 protein.
  • a more preferred ACE2 partial peptide is a peptide comprising or consisting of the amino acid sequence in the ⁇ -helix of the peptidase domain of the ACE2 protein.
  • ACE2 partial peptide examples include, but are not limited to, peptides containing or consisting of an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 1 to 4 shown below, variants thereof, and modified peptides. ..
  • SEQ ID NO: 1 IEEQAKTFLDKFNHEAEDLFYQS
  • SEQ ID NO: 2 IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS
  • SEQ ID NO: 3 sqyfldeaehnfkdlftkaqeei
  • SEQ ID NO: 4 sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei (Amino acid notation is a known one-letter notation. Uppercase letters represent L-amino acids, lowercase letters represent D-amino acids. Aib represents 2-aminoisobutyric acid.)
  • the ACE2 partial peptide containing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 is preferable, and the ACE2 partial peptide containing the amino acid sequence shown in SEQ ID NO: 4 is particularly preferable.
  • the ACE2 partial peptide may have a spacer (described later). An attachment (described later) may be attached to the spacer.
  • the ACE2 partial peptide can be produced by a chemical synthesis method (for example, Fmoc method, Boc method, etc.), a biological method (for example, a gene recombination method), and a combination thereof. These methods are known. Methods for producing the mutant ACE2 partial peptide and the modified ACE2 partial peptide are also known, and those skilled in the art can produce various mutant ACE2 partial peptides and modified ACE2 partial peptides.
  • a chemical synthesis method for example, Fmoc method, Boc method, etc.
  • a biological method for example, a gene recombination method
  • the ACE2 partial peptide immobilized on the carrier may be one kind or two or more kinds.
  • a water-insoluble material is used as the carrier material. Both polymer materials such as polymers and inorganic materials such as glass and ceramics are used.
  • As the shape of the carrier various shapes such as a particle shape, a fibrous shape, a film shape, and a hollow thread shape are used.
  • the functional group for peptide immobilization includes an acid derivative structure such as a maleic acid derivative, an activated halogen structure such as chloroacetamide, and a structure having a thiol group such as cysteine, and the carboxyl group, amino group or thiol of an amino acid. Any chemical structure capable of forming a covalent bond with the group can be used.
  • ACE2 partial peptide Methods of immobilizing the ACE2 partial peptide on the carrier are known to those skilled in the art. Covalent bonds, ionic bonds, physical adsorption and the like can be mentioned, but covalent bonds are preferable because they have strong bonds and the ACE2 partial peptide is difficult to drop off.
  • the ACE2 partial peptide is immobilized on the carrier by covalent bonding, the functional group in any amino acid residue of the ACE2 partial peptide can be reacted with the functional group in the carrier to form a covalent bond.
  • it reacts with the functional group of the carrier via the amino group of the N-terminal amino acid residue of the ACE2 partial peptide or the carboxyl group of the C-terminal amino acid residue to form a covalent bond.
  • immobilization on a carrier via the C-terminal means that the C-terminal amino acid residue of the peptide may be directly bound to the carrier, or may be bound to the carrier via a spacer or an attachment. It means that it is also good. The same applies to the case of "immobilization on the carrier via the N-terminal" of the peptide.
  • the ACE2 partial peptide may be covalently bonded to the resin via a spacer to be immobilized. It is preferable to use spacers of appropriate length in order to reduce steric hindrance due to the carrier.
  • a spacer can be covalently attached to a functional group in any amino acid residue of the ACE2 partial peptide. Preferably, the spacer is covalently attached to the amino group of the N-terminal amino acid residue of the ACE2 partial peptide or the carboxyl group of the C-terminal amino acid residue.
  • a plurality of spacers may be connected and used. An attachment portion may be provided at the tip of the spacer and covalently bonded to the carrier.
  • spacers are known and can be used as long as they do not interfere with the adsorption of the virus on the ACE2 partial peptide and are not harmful to the body.
  • spacers are "mini-PEG" based on short-chain polyethylene glycol, a repeating sequence consisting of glycine and serine (eg (GGGGS) n ), a repeating sequence of proline, glutamic acid and lysine or arginine and alanine.
  • n is a natural number of 2 or more
  • sequences consisting of their D-forms such as ⁇ -aminobutyric acid or ⁇ -aminocaproic acid or ⁇ -glutamic acid or ⁇ -lysine.
  • the length of the spacer is preferably about 20 to 50 atoms, more preferably about 30 to 40 atoms.
  • the spacers that can be used in the present invention are not limited to the above examples. Various attachments are known and can be used as long as they do not interfere with the adsorption of the virus on the ACE2 partial peptide and are not harmful to the body.
  • attachments include thiols adapted for reactions such as maleimide or chloroacetyl with thiols (eg, thiols in the cysteine residue side chain) or azides adapted for maleimide or chloroacetyl, azido-alkyne cyclization reactions, etc.
  • examples include, but are not limited to, aminooxys, hydrazides, aldehydes, etc. that are adapted to alkynes, oximes, hydrazone bond-forming reactions, and the like. Methods for adding spacers and attachments are known.
  • ACE2 partial peptide with a spacer include, but are not limited to, the following (1) to (8): (1) Spacer-IEEQAKTFLDKFNHEAEDLFYQS (2) IEEQAKTFLDKFNHEAEDLFYQS-Spacer (3) Spacer-IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS (4) IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-Spacer (5) sqyfldeaehnfkdlftkaqeei-Spacer (6) Spacer-sqyfldeaehnfkdlftkaqeei (7) sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-Spacer (8) Spacer-sqyflde-Aib-ehnfkd-Aib-ftk-Aib-
  • Specific examples of the preferred ACE2 partial peptide in the present invention are the peptide shown in (5) above [the peptide having a spacer at the C-terminal of the amino acid sequence shown in SEQ ID NO: 3] and the peptide shown in (7) [sequence].
  • the peptide shown in (7) is preferable.
  • the spacer may include an attachment.
  • the present invention provides a method for removing a virus from a liquid, which comprises contacting the above-mentioned carrier of the present invention with the liquid.
  • the liquid is as described below.
  • the liquid may be a liquid taken out of the body.
  • "contacting" means mixing a liquid containing a virus with a carrier so that the virus is adsorbed on the ACE2 partial peptide immobilized on the carrier.
  • the mixing includes mixing in various modes such as mixing in a batch system, mixing in a continuous system (eg, passing a liquid through a column). Those skilled in the art can appropriately select conditions such as the amount of liquid and carrier to be used, the flow rate of the liquid, and the adsorption temperature.
  • the carrier on which the above-mentioned ACE2 partial peptide is immobilized may be packed in a column and used. Columns of various sizes, shapes and materials are commercially available or can be manufactured.
  • the present invention provides a column comprising a carrier on which an ACE2 partial peptide is immobilized.
  • the column of the present invention may be used to adsorb and remove viruses in liquids.
  • a liquid containing a virus may be pumped to one end of the column of the present invention using a pump, and the liquid to which the virus has been adsorbed and removed may be taken out from the other end of the column.
  • the type of liquid is not particularly limited, and may be, for example, body fluids such as blood and urine, medical water, clean water, reclaimed water, sewage, groundwater, river water, seawater and the like.
  • the liquid is blood.
  • the column may be, for example, a cassette type.
  • the size, shape, and material of the column can be appropriately selected and changed according to the mode of use, and can be determined, for example, according to the symptoms of a patient with a virus infection, the required blood flow rate, the equipment used, and the like.
  • the column of the present invention can be connected to the patient in the same manner as in the case of conventional blood adsorption therapy.
  • the above column may be used in combination with another column for blood adsorption therapy.
  • Other columns for blood adsorption therapy may be those that adsorb activated immune cells, cytokines, endotoxins, and the like.
  • Polymyxin B is an antibiotic having a high affinity for endotoxin, and a column containing a carrier on which it is immobilized is used for removing endotoxin in blood. It has also been reported that columns using polymyxin B adsorb activated immune cells and cytokines.
  • the carrier on which the ACE2 partial peptide and polymyxin B are immobilized can adsorb activated immune cells and cytokines in addition to viruses such as SARS-CoV and SARS-COV-2. Therefore, a carrier on which the ACE2 partial peptide and polymyxin B are immobilized can be used to treat a patient with a severe viral infection and improve the survival rate.
  • the present invention provides a carrier on which an ACE2 partial peptide and polymyxin B are immobilized.
  • the present invention provides, in a further embodiment, a column comprising a carrier on which an ACE2 partial peptide and polymyxin B are immobilized.
  • a carrier capable of immobilizing polymyxin B and a method for immobilizing polymyxin B on the carrier are known. Therefore, one of ordinary skill in the art can produce a carrier on which the ACE2 partial peptide and polymyxin B are immobilized, and such a carrier can be packed in the column.
  • the column may be, for example, a cassette type. The size and shape of the column can be determined according to the patient's symptoms, required blood flow rate, equipment used, and the like.
  • the ACE2 partial peptide may be immobilized on a carrier (polymyxin B is immobilized) in the endotoxin adsorption column "Tremixin (registered trademark) (Toray Medical Co., Ltd.)" currently used (FIG. 1). reference).
  • Toremikishin (TM) is used to treat patients with severe condition by severe conditions or gram-negative bacteria infections associated with endotoxemia.
  • the carrier and column of the present invention may be used for blood adsorption therapy.
  • Blood adsorption therapy is a treatment method in which blood is taken out from a patient, the carrier that adsorbs a target substance or cell is brought into contact with the blood, and the blood is returned to the patient again.
  • the blood adsorption therapy may be a treatment for a viral infection.
  • the present invention provides a carrier for blood adsorption therapy on which an ACE2 partial peptide is immobilized, and a carrier for viral infection treatment on which an ACE2 partial peptide is immobilized.
  • the present invention provides, in a further aspect, the use of a carrier on which an ACE2 partial peptide is immobilized for blood adsorption therapy and the use of a carrier on which an ACE2 partial peptide is immobilized for the treatment of a viral infection.
  • the present invention provides a column for blood adsorption therapy, which comprises a carrier on which an ACE2 partial peptide is immobilized, and a column for treating a viral infection, which comprises a carrier on which the ACE2 partial peptide is immobilized.
  • the present invention in a further embodiment, is used for blood adsorption therapy of a column containing a carrier on which an ACE2 partial peptide is immobilized, and for treatment of a viral infection of a column containing a carrier on which the ACE2 partial peptide is immobilized.
  • the present invention uses the ACE2 partial peptide for the production of a carrier for blood adsorption therapy or a column containing the same, and the ACE2 partial peptide for the production of a carrier for the treatment of viral infections or a column containing the same. Provide use.
  • the invention comprises contacting a patient's blood with a carrier on which the ACE2 partial peptide is immobilized, a method of performing a blood adsorption therapy, and the patient's blood on a carrier on which the ACE2 partial peptide is immobilized.
  • the invention comprises a method of performing a blood adsorption therapy comprising contacting a patient's blood against a column comprising a carrier on which the ACE2 partial peptide is immobilized, and a carrier on which the ACE2 partial peptide is immobilized.
  • a method of performing a blood adsorption therapy comprising contacting a patient's blood against a column comprising a carrier on which the ACE2 partial peptide is immobilized, and a carrier on which the ACE2 partial peptide is immobilized.
  • the treatment of a viral infection is any of various symptoms such as pneumonia, bronchitis, fever, cough, joint pain, organ failure, acute renal injury, gastrointestinal symptom, thrombosis, and myocardial disease due to the viral infection. To reduce, suppress or eliminate one or more.
  • the virus adsorbed on the carrier of the present invention may be any virus as long as it has the ability to bind to ACE2.
  • viruses include, but are not limited to, coronavirus.
  • Coronaviruses include, but are not limited to, HCoV-NL63, SARS-CoV, SARS-CoV-2 and variants thereof.
  • a serine protease inhibitor such as nafamostat is used as an anticoagulant to suppress the cleavage of S2 subunit by TMPRSS2 and to fuse the virus. May be suppressed.
  • the carrier and column of the present invention may be used in combination with ECMO (see FIG. 2).
  • the virus-removing effect of the carrier and column of the present invention can enhance the effectiveness of ECMO.
  • ACE2 partial peptides (1) to (8) with spacers and attachments were chemically synthesized and named 1N, 1C, 2N, 2C, 3N, 3C, 4N, 4C, respectively. These peptides were immobilized on a beaded resin carrier.
  • IEEQAKTFLDKFNHEAEDLFYQS is the 21st to 42nd amino acid sequence from the N-terminus of human ACE2.
  • sqyfldeaehnfkdlftkaqeei is a retro-inverso peptide sequence of IEEQAKTFLDKFNHEAEDLFYQS.
  • Aib represents 2-aminoisobutyric acid.
  • the part of miniPEG is the one in which three molecules of AEEA shown below are bound (length of 27 atoms) or the one in which two molecules of Diveg are bound (length of 40 atoms). Cys represents a cysteine residue.
  • NH 2 indicates that the C-terminus of the peptide is amidated.
  • AEEA 2- (2- (2-Aminoethoxy) ethoxy) acetic acid
  • Dibeg Diethylene glycol bis (3-aminopropyl) ether glutarate
  • the immobilization procedure is as follows. 6-Maleimidehexanoic acid 1.05g, O- (6-chlorobenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphart 2.68g, 1-hydroxybenzotriazole 675 mg was dissolved in dimethylformamide, 1.7 mL of N, N'-diisopropylethylamine was added, and the mixture was stirred for 1 minute. The reaction solution was added to 1.3 g of beads (LS01391 manufactured by Purolite) and stirred at room temperature to obtain beads having a maleimide group.
  • 6-Maleimidehexanoic acid 1.05g, O- (6-chlorobenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphart 2.68g, 1-hydroxybenzotriazole 675 mg was dissolved in di
  • the obtained maleimide group-bearing beads was suspended in a phosphate buffer solution at pH 7 containing 20% acetonitrile and 6M guanidine hydrochloride, 37.5 mg of the peptide was added, and the mixture was stirred at room temperature for 20 hours. It was reacted. After the reaction, the beads were washed successively with 50% dimethylformamide water, dimethylformamide and water to obtain a bead-like virus adsorbent.
  • the SARS-CoV-2 virus adsorption capacity of the ACE2 partially peptide-immobilized beads obtained by the above procedure was examined by the following procedure. Peptide-unfixed beads and peptide-immobilized beads were incubated with a SARS-CoV-2 pseudotype VSV (SARS2pv) virus solution incorporating the luciferase gene for a period of time. After that, the virus solution was added to Vero E6-TMPRSS2 cells (1x10 4 cells / well, 96 well), and the luciferase activity after 24 hours was measured to quantify the virus adsorption ability. As the virus, SARS-CoV-2 pseudo-type VSV (SARS2pv) was used. The results are shown in FIG. Virus adsorption ability was observed in beads on which peptides 3N, 4N and 4C were immobilized. From these results, it was considered that peptides 3N, 4N and 4C have virus adsorption ability.
  • SARS2pv SARS-
  • Virus adsorption ability test (Part 2) Peptides (3N, 4N and 4C) confirmed to have high virus adsorption ability in the above test (1) were fixed on a fibrous carrier as a base material of a column.
  • the immobilization procedure is as follows. TF 12.56 cm 2 was co-washed with dimethyl sulfoxide. Separately, 30 mg of a peptide dissolved in dimethyl sulfoxide and 80 mg of sodium iodide were added to the fiber, 0.4 mL of N, N'-diisopropylethylamine was added, and the mixture was stirred at room temperature. After 30 minutes, 0.2 mL of tributylphosphine was added and the reaction was carried out at room temperature with stirring. After the reaction, the cells were washed successively with dimethyl sulfoxide, water, pH 7 phosphate buffer, and water.
  • SARS-CoV-2 pseudotype VSV (SARS2pv) virus solution incorporating the washed carrier and the luciferase gene (the virus is the same as in the above test (1)), and mix at 4 ° C. for 1 hour. Incubated to adsorb the virus on the carrier. 50 ⁇ l of the supernatant was added to VeroE6-TMPRSS2 cells (1x10 4 cells / well in a 96-well plate) and luciferase activity was measured 24 hours later.
  • TFs immobilized with peptides 3N, 4N and 4C were sterilized.
  • Gamma rays or high pressure were used for sterilization.
  • the sterilization procedure was as follows.
  • Gamma-ray sterilization-TF was suspended in physiological saline (raw food) and irradiated with ⁇ -rays (25 kGy).
  • High-pressure sterilization-TF was suspended in saline or phosphate buffered saline (PBS) and sterilized by high pressure steam (116.5 ° C., 90 minutes). Columns were filled with sterile or non-sterile carriers.
  • a SARS-CoV-2 pseudo-type VSV (SARS2pv) virus solution in which the luciferase gene was incorporated into a column (the virus is the same as in the above tests (1) and (2)) was filled, circulated for a certain period of time, and then released in the solution.
  • the virus was added to Vero E6-TMPRSS2 cells (1x10 4 cells / well, 96 well), and the luciferase activity after 24 hours was measured to quantify the virus adsorption capacity.
  • the virus adsorption capacity of the column was quantified from the amount of virus adsorbed on the column. The results are shown in FIG. It was found that the virus adsorption capacity of the carrier (that is, the peptide) was maintained even after sterilization.
  • the present invention can be used in the fields of medical materials and medical devices, research fields of viral infectious diseases, and the like.

Abstract

The present invention provides a partial peptide of angiotensin-converting enzyme (ACE2), a carrier where said peptide is fixed, and a column containing said carrier.

Description

ＳＡＲＳ－ＣｏＶ－２吸着担体およびそれを含むカラムSARS-CoV-2 adsorption carrier and column containing it
　本発明は、アンジオテンシン変換酵素２（ＡＣＥ２）の部分ペプチド、該ペプチドが固定化された担体、および該担体を含むカラム等に関する。 The present invention relates to a partial peptide of angiotensin converting enzyme 2 (ACE2), a carrier on which the peptide is immobilized, a column containing the carrier, and the like.
　いくつかのコロナウイルスはヒトに感染し、様々な重症度の呼吸器障害を引き起こす。なかでもＳＡＲＳ－ＣｏＶおよびＳＡＲＳ－ＣｏＶ－２は大規模なパンデミックを引き起こす。現在、ＳＡＲＳ－ＣｏＶ－２による肺炎（ＣＯＶＩＤ－１９）が世界的に大流行し、極めて深刻な問題となっている。 Some coronaviruses infect humans and cause various severe respiratory disorders. Among them, SARS-CoV and SARS-CoV-2 cause a large-scale pandemic. Currently, pneumonia caused by SARS-CoV-2 (COVID-19) has become a pandemic worldwide and has become an extremely serious problem.
　コロナウイルスのスパイク（Ｓ）タンパク質にはＳ１とＳ２の２つのサブユニットが含まれている。Ｓ１には受容体結合ドメイン（ＲＢＤ）が含まれており、宿主細胞表面の受容体への結合に関与する。Ｓ２サブユニットは膜融合に必要な機能を有しており、Ｓ２がヒトセリンプロテアーゼであるＴＭＰＲＳＳ２によって切断されることにより、膜融合が進行する。コロナウイルスのうち、ＨＣｏＶ－ＮＬ６３、ＳＡＲＳ－ＣｏＶおよびＳＡＲＳ－ＣｏＶ－２はいずれもヒトＡＣＥ２を受容体として利用している。 The coronavirus spike (S) protein contains two subunits, S1 and S2. S1 contains a receptor binding domain (RBD) and is involved in the binding of host cell surfaces to receptors. The S2 subunit has a function necessary for membrane fusion, and membrane fusion proceeds when S2 is cleaved by TMPRSS2, which is a human serine protease. Among the coronaviruses, HCoV-NL63, SARS-CoV and SARS-CoV-2 all utilize human ACE2 as a receptor.
　ＳＡＲＳ－ＣｏＶ－２のＲＢＤはＡＣＥ２のペプチダーゼドメイン（ＰＤ）に結合する（Ｋｄ値約１５ｎＭ）。ＡＣＥ２に結合するＳＡＲＳ－ＣｏＶとＳＡＲＳ－ＣｏＶ－２のＲＢＤ領域は保存されている。ＡＣＥ２のＰＤ領域はＳＡＲＳ－ＣｏＶ－２を吸着するために有用であると考えられ、将来的にＳＡＲＳ－ＣｏＶの変異型が出現した場合にも応用可能である。２００３年に流行したＳＡＲＳ－ＣｏＶもまた急性呼吸窮迫症候群（ＡＲＤＳ）を引き起こしたが、この治療法としてエンドトキシン吸着カラムを用いた血液吸着療法が有用であったとの報告があり（非特許文献１）、サイトカインであるＩＬ－６の減少や炎症性細胞の減少などが報告されている（非特許文献２～４）。今回のＳＡＲＳ－ＣｏＶ－２感染症（ＣＯＶＩＤ－１９）に対しても、血漿交換が有効であったという報告もあるが（非特許文献５、６）、血漿交換の場合、ウイルスを含んだ廃液の処理が問題となる。 The RBD of SARS-CoV-2 binds to the peptidase domain (PD) of ACE2 (Kd value of about 15 nM). The RBD regions of SARS-CoV and SARS-CoV-2 that bind to ACE2 are conserved. The PD region of ACE2 is considered to be useful for adsorbing SARS-CoV-2, and can be applied even when a variant of SARS-CoV appears in the future. SARS-CoV, which was prevalent in 2003, also caused acute respiratory distress syndrome (ARDS), and there is a report that blood adsorption therapy using an endotoxin adsorption column was useful as a treatment method (Non-Patent Document 1). , The decrease of IL-6 which is a cytokine and the decrease of inflammatory cells have been reported (Non-Patent Documents 2 to 4). There is also a report that plasmapheresis was effective against this SARS-CoV-2 infection (COVID-19) (Non-Patent Documents 5 and 6), but in the case of plasmapheresis, a waste liquid containing a virus. Processing becomes a problem.
　ＳＡＲＳ－ＣｏＶやＳＡＲＳ－ＣｏＶ－２などのウイルス感染症の治療に有効で、かつウイルスを含む廃液の処理が問題とならない血液吸着療法用の担体およびカラムが望まれている。 A carrier and column for blood adsorption therapy that are effective in treating viral infections such as SARS-CoV and SARS-CoV-2 and that do not pose a problem in the treatment of waste liquid containing a virus are desired.
　本発明者らは、上記課題を解決すべく鋭意研究を重ね、ＡＣＥ２の特定の部分ペプチドが固定化された担体が効率良くＳＡＲＳ－ＣｏＶ－２を吸着することを見いだし、本発明を完成させるに至った。 The present inventors have conducted extensive studies to solve the above problems, and have found that a carrier on which a specific partial peptide of ACE2 is immobilized efficiently adsorbs SARS-CoV-2, and completes the present invention. I arrived.
　すなわち、本発明は以下のものを提供する：
　（１）アンジオテンシン変換酵素２（ＡＣＥ２）部分ペプチドが固定化された担体。
　（２）ＡＣＥ２部分ペプチドが、ＡＣＥ２タンパク質のペプチダーゼドメイン中のアミノ酸配列を含むものである、（１）記載の担体。
　（３）ＡＣＥ２部分ペプチドが、ＡＣＥ２タンパク質のペプチダーゼドメインのαヘリックス中のアミノ酸配列を含むものである、（２）記載の担体。
　（４）ＡＣＥ２部分ペプチドが、配列番号：３または配列番号：４に示すアミノ酸配列から選択されるアミノ酸配列を含むものである、（３）記載の担体。
　（５）ＡＣＥ２部分ペプチドが、配列番号：４に示すアミノ酸配列を含むものである、（４）記載の担体。
　（６）ＡＣＥ２部分ペプチドが、そのＣ末端を介して担体に固定化されている、（５）記載の担体。
　（７）高圧滅菌されている、（６）記載の担体。
　（８）ＡＣＥ２部分ペプチドがスペーサーを介して固定化されている、（１）～（７）のいずれか記載の担体。
　（９）さらにポリミキシンＢが固定化された、（１）～（８）のいずれか記載の担体。
　（１０）（１）～（９）のいずれか記載の担体を含むカラム。
　（１１）液体中からウイルスを吸着除去するために用いられる（１０）記載のカラム。
　（１２）液体が血液である（１１）記載のカラム。
　（１３）配列番号：３または配列番号：４に示すアミノ酸配列を含むペプチド。
　（１４）スペーサーを有する（１３）記載のペプチド。
　（１５）スペーサーにアタッチメントが付いた（１４）記載のペプチド。 That is, the present invention provides:
(1) A carrier on which an angiotensin converting enzyme 2 (ACE2) partial peptide is immobilized.
(2) The carrier according to (1), wherein the ACE2 partial peptide contains an amino acid sequence in the peptidase domain of the ACE2 protein.
(3) The carrier according to (2), wherein the ACE2 partial peptide contains an amino acid sequence in the α-helix of the peptidase domain of the ACE2 protein.
(4) The carrier according to (3), wherein the ACE2 partial peptide comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
(5) The carrier according to (4), wherein the ACE2 partial peptide contains the amino acid sequence shown in SEQ ID NO: 4.
(6) The carrier according to (5), wherein the ACE2 partial peptide is immobilized on the carrier via its C-terminal.
(7) The carrier according to (6), which is sterilized under high pressure.
(8) The carrier according to any one of (1) to (7), wherein the ACE2 partial peptide is immobilized via a spacer.
(9) The carrier according to any one of (1) to (8), wherein polymyxin B is further immobilized.
(10) A column containing the carrier according to any one of (1) to (9).
(11) The column according to (10) used for adsorbing and removing a virus from a liquid.
(12) The column according to (11), wherein the liquid is blood.
(13) A peptide containing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.
(14) The peptide according to (13), which has a spacer.
(15) The peptide according to (14), which has an attachment attached to a spacer.
　本発明の担体およびカラムは効率良くウイルスを吸着する。したがって、本発明の担体およびカラムは、ＡＣＥ２を受容体とするウイルスによる感染症の血液吸着療法に有効である。ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された本発明の担体およびカラムは、特にＡＣＥ２を受容体とするウイルスによる重症の感染症の血液吸着療法に有効である。 The carrier and column of the present invention efficiently adsorb viruses. Therefore, the carriers and columns of the present invention are effective for blood adsorption therapy for infectious diseases caused by viruses using ACE2 as a receptor. The carriers and columns of the present invention on which the ACE2 partial peptide and polymyxin B are immobilized are particularly effective for blood adsorption therapy for severe infectious diseases caused by viruses that accept ACE2 as a receptor.
図１は、市販のポリミキシンＢ固定化繊維にＡＣＥペプチドを固定化して得られる本発明の担体およびそれを含むカラムの概要を示すスキームである。FIG. 1 is a scheme showing an outline of a carrier of the present invention obtained by immobilizing an ACE peptide on a commercially available polymyxin B-immobilized fiber and a column containing the same. 図２は、本発明の担体を含むカラム（図中トレミキシン改良カラムと表記）とＥＣＭＯの併用例を示すスキームである。FIG. 2 is a scheme showing an example of combined use of a column containing the carrier of the present invention (denoted as a tremixin-improved column in the figure) and ECMO. 図３は、アミン型Ｐｕｒｏｌｉｔｅ樹脂（Ｎ）、マレイミド結合Ｐｕｒｏｌｉｔｅ樹脂（ＭＩ）、ペプチド（１Ｎ、１Ｃ、２Ｎ、２Ｃ、３Ｎ、３Ｃ、４Ｎ、４Ｃ）を固定化したＭＩのＳＡＲＳ－ＣｏＶ－２ウイルス吸着能を調べた結果を示すグラフである。ペプチドはスペーサーとして３分子のＡＥＥＡ（後で説明）を含むものである。FIG. 3 shows the SARS-CoV-2 virus of MI on which an amine-type Purolite resin (N), a maleimide-bound Purolite resin (MI), and a peptide (1N, 1C, 2N, 2C, 3N, 3C, 4N, 4C) are immobilized. It is a graph which shows the result of having investigated the adsorption capacity. The peptide contains three molecules of AEEA (described later) as spacers. 図４は、３Ｎ、４Ｎまたは４Ｃを固定化したクロロアセチル化されている東レ製の繊維（ＴＦ）、および４Ｎ－Ｄｉｂｅｇを固定化したＴＦのＳＡＲＳ－ＣｏＶ－２ウイルス吸着能を調べた結果を示すグラフである。図中、４Ｎ－Ｄｉｂｅｇ－ＴＦは、スペーサーとして２分子のＤｉｂｅｇ（後で説明）を介してシステインを含む４ＮペプチドをＴＦに担持させたものである。それ以外は、スペーサーとして３分子のＡＥＥＡを介してペプチドをＴＦに担持させたものである。FIG. 4 shows the results of examining the SARS-CoV-2 virus adsorption capacity of chloroacetylated Toray fibers (TF) on which 3N, 4N or 4C was immobilized, and TF on which 4N-Dibeg was immobilized. It is a graph which shows. In the figure, 4N-Dibeg-TF is a TF carrying a 4N peptide containing cysteine via two molecules of Diveg (described later) as a spacer. Other than that, the peptide is supported on the TF via three molecules of AEEA as a spacer. 図５は、滅菌されたペプチド固定化ＴＦのＳＡＲＳ－ＣｏＶ－２ウイルス吸着能を調べた結果を示すグラフである。各滅菌手段について、３本のバーはそれぞれペプチド３Ｎ（左）、４Ｎ（中）および４Ｃ（右）を固定化したＴＦの結果を示す。ペプチド３Ｎと４Ｃはスペーサーとして３分子のＡＥＥＡを含むものであり、ペプチド４Ｎは２分子のＤｉｂｅｇを含むものである。FIG. 5 is a graph showing the results of examining the SARS-CoV-2 virus adsorption capacity of the sterilized peptide-immobilized TF. For each sterile instrument, the three bars show the results of TF immobilized with peptides 3N (left), 4N (middle) and 4C (right), respectively. Peptides 3N and 4C contain 3 molecules of AEEA as spacers, and peptide 4N contains 2 molecules of Diveg.
　本発明は１の態様において、ＡＣＥ２部分ペプチドが固定化された担体を提供する。 The present invention provides, in one embodiment, a carrier on which the ACE2 partial peptide is immobilized.
　本明細書において、「ＡＣＥ２部分ペプチド」は、ヒトＡＣＥ２タンパク質のアミノ酸配列の一部分を含むあるいはからなるペプチドをいう。ヒトＡＣＥ２タンパク質のアミノ酸配列は公知である。本明細書において、「ＡＣＥ２部分ペプチド」は、ヒトＡＣＥ２部分ペプチドのみならず、その変異型ペプチド、およびその修飾ペプチドを包含する。本明細書において、例えば、配列番号１～４のいずれかに示すアミノ酸配列を含むペプチドであって、スペーサーを有するペプチドおよびアタッチメントを有するペプチドも、「ＡＣＥ２部分ペプチド」に包含される。ただし、変異型ＡＣＥ２部分ペプチド、修飾ＡＣＥ２部分ペプチド、およびスペーサーを有するＡＣＥ２部分ペプチドは、元のヒトＡＣＥ２部分ペプチドと同等またはそれ以上のウイルス吸着能を有するものである。吸着能が同等とは、例えば、変異型ＡＣＥ２部分ペプチド１分子あたり、または修飾ＡＣＥ２部分ペプチド１分子あたり吸着されるウイルスの量が、元のヒトＡＣＥ２部分ペプチドと比べて、約７０％以上、好ましくは約８０％以上、より好ましくは約９０％以上、さらに好ましくは約１００％以上であることをいう。 As used herein, the term "ACE2 partial peptide" refers to a peptide containing or consisting of a portion of the amino acid sequence of a human ACE2 protein. The amino acid sequence of the human ACE2 protein is known. As used herein, "ACE2 partial peptide" includes not only human ACE2 partial peptide, but also a mutant peptide thereof and a modified peptide thereof. In the present specification, for example, a peptide containing the amino acid sequence shown in any of SEQ ID NOs: 1 to 4 and having a spacer and a peptide having an attachment are also included in the "ACE2 partial peptide". However, the mutant ACE2 partial peptide, the modified ACE2 partial peptide, and the ACE2 partial peptide having a spacer have a virus adsorption capacity equal to or higher than that of the original human ACE2 partial peptide. Equivalent adsorption capacity means, for example, that the amount of virus adsorbed per molecule of mutant ACE2 partial peptide or modified ACE2 partial peptide is about 70% or more, preferably about 70% or more, as compared with the original human ACE2 partial peptide. Means that it is about 80% or more, more preferably about 90% or more, still more preferably about 100% or more.
　変異型ＡＣＥ２部分ペプチドは、元のヒトＡＣＥ２部分ペプチドのアミノ酸配列において１個ないし数個のアミノ酸が置換、付加、挿入または欠失されたアミノ酸配列を含むあるいはからなるペプチドであってもよい。ここで数個とは、２個、３個、４個、５個、６個、７個、８個または９個を意味する。また変異型ＡＣＥ２部分ペプチドは、元のヒトＡＣＥ２部分ペプチドのアミノ酸配列に対して約６０％以上、約７０％以上、約８０％以上、約９０％以上、または約９５％以上のアミノ酸配列同一性を有するアミノ酸配列を含むあるいはからなるペプチドであってもよい。アミノ酸置換は同族アミノ酸間の置換が好ましい。同族アミノ酸は当業者に公知である。アミノ酸配列の同一性は、ＢＬＡＳＴ検索などの公知の手段を用いて行うことができる。 The mutant ACE2 partial peptide may be a peptide containing or consisting of an amino acid sequence in which one or several amino acids are substituted, added, inserted or deleted in the amino acid sequence of the original human ACE2 partial peptide. Here, the number means 2, 3, 4, 5, 5, 6, 7, 8, or 9. Further, the mutant ACE2 partial peptide has an amino acid sequence identity of about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more with respect to the amino acid sequence of the original human ACE2 partial peptide. It may be a peptide containing or consisting of an amino acid sequence having. The amino acid substitution is preferably a substitution between homologous amino acids. Homogeneous amino acids are known to those of skill in the art. The identity of the amino acid sequence can be performed by using a known means such as BLAST search.
　変異型ＡＣＥ２部分ペプチドは、Ｄ－アミノ酸や非天然アミノ酸を含むものであってもよい。非天然アミノ酸としては、β－アラニン、２－アミノイソ酪酸、シクロヘキシルアラニン、ｔｅｒｔ－ロイシン、種々のＮ－メチルアミノ酸などが挙げられるが、これらに限定されない。ＡＣＥ２部分ペプチドのアミノ酸配列中の１個～数個のアミノ酸残基を非天然アミノ酸に置換して、ヘリックス性を向上させてもよい（一例として配列番号２参照）。ＡＣＥ２部分ペプチドのアミノ酸配列中の１個～すべてのアミノ酸残基をＤ－体として血中安定性を向上させてもよい。本明細書において、ＡＣＥ２部分ペプチドのアミノ酸配列の逆配列も変異型ＡＣＥ２部分ペプチドに含まれる。 The mutant ACE2 partial peptide may contain a D-amino acid or an unnatural amino acid. Examples of unnatural amino acids include, but are not limited to, β-alanine, 2-aminoisobutyric acid, cyclohexylalanine, tert-leucine, and various N-methyl amino acids. One to several amino acid residues in the amino acid sequence of the ACE2 partial peptide may be replaced with unnatural amino acids to improve helix properties (see SEQ ID NO: 2 as an example). One to all amino acid residues in the amino acid sequence of the ACE2 partial peptide may be used as the D-form to improve blood stability. As used herein, the reverse sequence of the amino acid sequence of the ACE2 partial peptide is also included in the mutant ACE2 partial peptide.
　修飾ＡＣＥ２部分ペプチドの修飾様式はいかなるものであってもよく、例えばアルキル化、エステル化、アミド化、ハロゲン化等であってもよい。 The modification mode of the modified ACE2 partial peptide may be any, for example, alkylation, esterification, amidation, halogenation and the like.
　好ましいＡＣＥ２部分ペプチドは、ＡＣＥ２タンパク質のペプチダーゼドメイン中のアミノ酸配列を含むあるいはからなるペプチドである。 The preferred ACE2 partial peptide is a peptide comprising or consisting of the amino acid sequence in the peptidase domain of the ACE2 protein.
　より好ましいＡＣＥ２部分ペプチドは、ＡＣＥ２タンパク質のペプチダーゼドメインのαヘリックス中のアミノ酸配列を含むあるいはからなるペプチドである。 A more preferred ACE2 partial peptide is a peptide comprising or consisting of the amino acid sequence in the α-helix of the peptidase domain of the ACE2 protein.
　かかるＡＣＥ２部分ペプチドの具体例としては、以下に示す配列番号１～４に示すアミノ酸配列から選択されるアミノ酸配列を含むあるいはからなるペプチド、その変異体および修飾ペプチドが挙げられるが、これらに限定されない。
配列番号：１：IEEQAKTFLDKFNHEAEDLFYQS
配列番号：２：IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS
配列番号：３：sqyfldeaehnfkdlftkaqeei
配列番号：４：sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei
（アミノ酸の表記は公知の１文字表記である。大文字はＬ－アミノ酸、小文字はＤ－アミノ酸を表す。Aibは２－アミノイソ酪酸を表す。） Specific examples of such an ACE2 partial peptide include, but are not limited to, peptides containing or consisting of an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 1 to 4 shown below, variants thereof, and modified peptides. ..
SEQ ID NO: 1: IEEQAKTFLDKFNHEAEDLFYQS
SEQ ID NO: 2: IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS
SEQ ID NO: 3: sqyfldeaehnfkdlftkaqeei
SEQ ID NO: 4: sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei
(Amino acid notation is a known one-letter notation. Uppercase letters represent L-amino acids, lowercase letters represent D-amino acids. Aib represents 2-aminoisobutyric acid.)
　上記ＡＣＥ２部分ペプチドのなかでも、配列番号：３または配列番号：４に示すアミノ酸配列を含むＡＣＥ２部分ペプチドが好ましく、配列番号：４に示すアミノ酸配列を含むＡＣＥ２部分ペプチドが特に好ましい。ＡＣＥ２部分ペプチドはスペーサー（後で説明）を有していてもよい。スペーサーにアタッチメント（後で説明）が付されていてもよい。 Among the above ACE2 partial peptides, the ACE2 partial peptide containing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 is preferable, and the ACE2 partial peptide containing the amino acid sequence shown in SEQ ID NO: 4 is particularly preferable. The ACE2 partial peptide may have a spacer (described later). An attachment (described later) may be attached to the spacer.
　ＡＣＥ２部分ペプチドは、化学合成法（例えばＦｍｏｃ法、Ｂｏｃ法など）、生物学的方法（例えば遺伝子組換え法）、およびそれらの組み合わせによって製造することができる。これらの方法は公知である。変異型ＡＣＥ２部分ペプチドおよび修飾ＡＣＥ２部分ペプチドの製造方法も公知であり、当業者は様々な変異型ＡＣＥ２部分ペプチドおよび修飾ＡＣＥ２部分ペプチドを製造することができる。 The ACE2 partial peptide can be produced by a chemical synthesis method (for example, Fmoc method, Boc method, etc.), a biological method (for example, a gene recombination method), and a combination thereof. These methods are known. Methods for producing the mutant ACE2 partial peptide and the modified ACE2 partial peptide are also known, and those skilled in the art can produce various mutant ACE2 partial peptides and modified ACE2 partial peptides.
　担体に固定化されるＡＣＥ２部分ペプチドは１種類であってもよく、２種類以上であってもよい。 The ACE2 partial peptide immobilized on the carrier may be one kind or two or more kinds.
　担体の材料としては、水不溶性材料を用いる。ポリマー等の高分子材料、硝子やセラミックのような無機材料のいずれもが用いられる。担体の形状としては、粒子状、繊維状、フィルム状、中空糸状等の種々の形状が用いられる。また、ペプチド固定化のための官能基としては、マレイン酸誘導体等の酸誘導体構造、クロロアセトアミド等の活性化ハロゲン構造、システイン等のチオール基を有する構造等、アミノ酸のカルボキシル基、アミノ基あるいはチオール基と共有結合を形成することが可能な化学構造であれば、いずれも用いることが出来る。 A water-insoluble material is used as the carrier material. Both polymer materials such as polymers and inorganic materials such as glass and ceramics are used. As the shape of the carrier, various shapes such as a particle shape, a fibrous shape, a film shape, and a hollow thread shape are used. The functional group for peptide immobilization includes an acid derivative structure such as a maleic acid derivative, an activated halogen structure such as chloroacetamide, and a structure having a thiol group such as cysteine, and the carboxyl group, amino group or thiol of an amino acid. Any chemical structure capable of forming a covalent bond with the group can be used.
　担体へのＡＣＥ２部分ペプチドの固定化方法は当業者に公知である。共有結合、イオン結合、物理的吸着などが挙げられるが、結合が強力でＡＣＥ２部分ペプチドが脱落し難い共有結合が好ましい。共有結合によってＡＣＥ２部分ペプチドを担体に固定化する場合、ＡＣＥ２部分ペプチドのいずれかのアミノ酸残基中の官能基を担体中の官能基と反応させて共有結合を生じさせることができる。好ましくは、ＡＣＥ２部分ペプチドのＮ末端アミノ酸残基のアミノ基またはＣ末端アミノ酸残基のカルボキシル基を介して担体の官能基と反応させて共有結合を生じさせる。本明細書において、「Ｃ末端を介して担体に固定化」とは、ペプチドのＣ末端アミノ酸残基が直接担体と結合していてもよく、あるいはスペーサーやアタッチメントを介して担体に結合していてもよいことを意味する。ペプチドの「Ｎ末端を介して担体に固定化」という場合も同様である。 Methods of immobilizing the ACE2 partial peptide on the carrier are known to those skilled in the art. Covalent bonds, ionic bonds, physical adsorption and the like can be mentioned, but covalent bonds are preferable because they have strong bonds and the ACE2 partial peptide is difficult to drop off. When the ACE2 partial peptide is immobilized on the carrier by covalent bonding, the functional group in any amino acid residue of the ACE2 partial peptide can be reacted with the functional group in the carrier to form a covalent bond. Preferably, it reacts with the functional group of the carrier via the amino group of the N-terminal amino acid residue of the ACE2 partial peptide or the carboxyl group of the C-terminal amino acid residue to form a covalent bond. As used herein, "immobilization on a carrier via the C-terminal" means that the C-terminal amino acid residue of the peptide may be directly bound to the carrier, or may be bound to the carrier via a spacer or an attachment. It means that it is also good. The same applies to the case of "immobilization on the carrier via the N-terminal" of the peptide.
　スペーサーを介してＡＣＥ２部分ペプチドを樹脂に共有結合させて固定化してもよい。担体による立体障害を低減するために、適切な長さのスペーサーを用いることが好ましい。ＡＣＥ２部分ペプチドのいずれかのアミノ酸残基中の官能基にスペーサーを共有結合させることができる。好ましくは、ＡＣＥ２部分ペプチドのＮ末端アミノ酸残基のアミノ基またはＣ末端アミノ酸残基のカルボキシル基にスペーサーを共有結合させる。複数のスペーサーを連結して用いてもよい。スペーサーの先端にアタッチメント部分を設けて、これを担体に共有結合させてもよい。様々なスペーサーが公知であり、ＡＣＥ２部分ペプチドへのウイルスの吸着を妨げず、生体に有害でない限りそれらを使用できる。スペーサーの例としては、短鎖ポリエチレングリコールをベースにした「ミニペグ（ｍｉｎｉ－ＰＥＧ）」、グリシンとセリンからなる繰り返し配列（例えば（ＧＧＧＧＳ）ｎ）、プロリンの繰り返し配列、グルタミン酸とリシンまたはアルギニンとアラニンからなる繰り返し配列（例えば（ＥＡＡＡＫ）ｎや（ＥＡＡＡＲ）ｎ、ｎは２以上の自然数）およびそれらのＤ体からなる配列、γ－アミノ酪酸またはε－アミノカプロン酸またはγ－グルタミン酸またはε－リシンなどの繰り返しなどが挙げられる。スペーサーの長さは約２０～５０原子であることが好ましく、約３０～４０原子であることがより好ましい。本発明に使用できるスペーサーは上例に限定されない。様々なアタッチメントが公知であり、ＡＣＥ２部分ペプチドへのウイルスの吸着を妨げず、生体に有害でない限りそれらを使用できる。アタッチメントの例としては、マレイミドまたはクロロアセチルなどとチオールとの反応などに適応させられるチオール（例えばシステイン残基側鎖のチオール）またはマレイミドまたはクロロアセチル、アジド－アルキン環化反応などに適応させられるアジドまたはアルキン、オキシムまたはヒドラゾン結合形成反応などに適応させられるアミノオキシまたはヒドラジドまたはアルデヒドなどが挙げられるが、これらに限定されない。スペーサーやアタッチメントの付加方法は公知である。 The ACE2 partial peptide may be covalently bonded to the resin via a spacer to be immobilized. It is preferable to use spacers of appropriate length in order to reduce steric hindrance due to the carrier. A spacer can be covalently attached to a functional group in any amino acid residue of the ACE2 partial peptide. Preferably, the spacer is covalently attached to the amino group of the N-terminal amino acid residue of the ACE2 partial peptide or the carboxyl group of the C-terminal amino acid residue. A plurality of spacers may be connected and used. An attachment portion may be provided at the tip of the spacer and covalently bonded to the carrier. Various spacers are known and can be used as long as they do not interfere with the adsorption of the virus on the ACE2 partial peptide and are not harmful to the body. Examples of spacers are "mini-PEG" based on short-chain polyethylene glycol, a repeating sequence consisting of glycine and serine (eg (GGGGS) n ), a repeating sequence of proline, glutamic acid and lysine or arginine and alanine. Repeated sequences consisting of (eg, (EAAAK) n and (EAAAR) n , n is a natural number of 2 or more) and sequences consisting of their D-forms, such as γ-aminobutyric acid or ε-aminocaproic acid or γ-glutamic acid or ε-lysine. And so on. The length of the spacer is preferably about 20 to 50 atoms, more preferably about 30 to 40 atoms. The spacers that can be used in the present invention are not limited to the above examples. Various attachments are known and can be used as long as they do not interfere with the adsorption of the virus on the ACE2 partial peptide and are not harmful to the body. Examples of attachments include thiols adapted for reactions such as maleimide or chloroacetyl with thiols (eg, thiols in the cysteine residue side chain) or azides adapted for maleimide or chloroacetyl, azido-alkyne cyclization reactions, etc. Alternatively, examples include, but are not limited to, aminooxys, hydrazides, aldehydes, etc. that are adapted to alkynes, oximes, hydrazone bond-forming reactions, and the like. Methods for adding spacers and attachments are known.
　スペーサーを付したＡＣＥ２部分ペプチドの具体例としては以下の（１）～（８）が挙げられるが、これらに限定されない：
（１）スペーサー-IEEQAKTFLDKFNHEAEDLFYQS
（２）IEEQAKTFLDKFNHEAEDLFYQS-スペーサー
（３）スペーサー-IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS
（４）IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-スペーサー
（５）sqyfldeaehnfkdlftkaqeei-スペーサー
（６）スペーサー-sqyfldeaehnfkdlftkaqeei
（７）sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-スペーサー
（８）スペーサー-sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei
　上のペプチドにおいてスペーサーが１分子～数分子連結されていてもよい。ここで数分子は、２分子、３分子、４分子、５分子、６分子、７分子、８分子または９分子を意味する。スペーサーにアタッチメントが付加されていてもよい。 Specific examples of the ACE2 partial peptide with a spacer include, but are not limited to, the following (1) to (8):
(1) Spacer-IEEQAKTFLDKFNHEAEDLFYQS
(2) IEEQAKTFLDKFNHEAEDLFYQS-Spacer (3) Spacer-IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS
(4) IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-Spacer (5) sqyfldeaehnfkdlftkaqeei-Spacer (6) Spacer-sqyfldeaehnfkdlftkaqeei
(7) sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-Spacer (8) Spacer-sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei
In the above peptide, one to several spacers may be linked. Here, several molecules mean 2 molecules, 3 molecules, 4 molecules, 5 molecules, 6 molecules, 7 molecules, 8 molecules or 9 molecules. An attachment may be added to the spacer.
　本発明における好ましいＡＣＥ２部分ペプチドの特別な具体例は、上の（５）に示すペプチド［配列番号：３に示すアミノ酸配列のＣ末端にスペーサーを付したペプチド］、（７）に示すペプチド［配列番号：４に示すアミノ酸配列のＣ末端にスペーサーを付したペプチド］、および（８）に示すペプチド［配列番号：４に示すアミノ酸配列のＮ末端にスペーサーを付したペプチド］である。とりわけ（７）に示すペプチドが好ましい。スペーサーはアタッチメントを含んでいてもよい。 Specific examples of the preferred ACE2 partial peptide in the present invention are the peptide shown in (5) above [the peptide having a spacer at the C-terminal of the amino acid sequence shown in SEQ ID NO: 3] and the peptide shown in (7) [sequence]. No .: Peptide having a spacer at the C-terminal of the amino acid sequence shown in FIG. 4] and the peptide shown in (8) [Peptide having a spacer at the N-terminal of the amino acid sequence shown in SEQ ID NO: 4]. In particular, the peptide shown in (7) is preferable. The spacer may include an attachment.
　本発明のＡＣＥ２部分ペプチドが固定化された担体を用いて、液体中のウイルスを除去することができる。したがって、本発明は、さらなる態様において、液体からウイルスを除去する方法であって、上記した本発明の担体と該液体とを接触させることを含む方法を提供する。液体については以下に説明するとおりである。この方法において、液体は体外に取り出された液体であってもよい。ここで、接触させるとは、ウイルスを含む液体と担体とを混合し、担体に固定化されたＡＣＥ２部分ペプチドにウイルスが吸着されるようにすることをいう。該混合には、バッチ式の系での混合、連続式の系での混合（例えば、液体をカラムに通す）など様々な様式での混合が包含される。当業者は、使用する液体や担体の量、液体の流量、吸着温度などの条件を適宜選択することができる。 The virus in the liquid can be removed by using the carrier on which the ACE2 partial peptide of the present invention is immobilized. Therefore, in a further aspect, the present invention provides a method for removing a virus from a liquid, which comprises contacting the above-mentioned carrier of the present invention with the liquid. The liquid is as described below. In this method, the liquid may be a liquid taken out of the body. Here, "contacting" means mixing a liquid containing a virus with a carrier so that the virus is adsorbed on the ACE2 partial peptide immobilized on the carrier. The mixing includes mixing in various modes such as mixing in a batch system, mixing in a continuous system (eg, passing a liquid through a column). Those skilled in the art can appropriately select conditions such as the amount of liquid and carrier to be used, the flow rate of the liquid, and the adsorption temperature.
　上記のＡＣＥ２部分ペプチドが固定化された担体をカラムに充填して使用してもよい。様々なサイズ、形状および材料のカラムが市販されており、あるいは製作することができる。 The carrier on which the above-mentioned ACE2 partial peptide is immobilized may be packed in a column and used. Columns of various sizes, shapes and materials are commercially available or can be manufactured.
　したがって、本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体を含むカラムを提供する。本発明のカラムを用いて、液体中のウイルスを吸着し、除去してもよい。例えば、ポンプを用いてウイルスを含む液体を本発明のカラムの一端に送り込み、ウイルスが吸着、除去された液体をカラムの他端から取り出してもよい。液体の種類は特に限定されず、例えば血液、尿などの体液、医療用水、上水、中水、下水、地下水、河川水、海水などであってもよい。好ましくは、液体は血液である。カラムは例えばカセット式のものであってもよい。カラムのサイズや形状、材料は使用態様に応じて適宜選択、変更することができ、例えばウイスル感染症患者の症状、必要な血液流量、使用機器などに応じて決定されうる。 Therefore, in a further aspect, the present invention provides a column comprising a carrier on which an ACE2 partial peptide is immobilized. The column of the present invention may be used to adsorb and remove viruses in liquids. For example, a liquid containing a virus may be pumped to one end of the column of the present invention using a pump, and the liquid to which the virus has been adsorbed and removed may be taken out from the other end of the column. The type of liquid is not particularly limited, and may be, for example, body fluids such as blood and urine, medical water, clean water, reclaimed water, sewage, groundwater, river water, seawater and the like. Preferably, the liquid is blood. The column may be, for example, a cassette type. The size, shape, and material of the column can be appropriately selected and changed according to the mode of use, and can be determined, for example, according to the symptoms of a patient with a virus infection, the required blood flow rate, the equipment used, and the like.
　本発明のカラムを用いて患者の血液中のウイルスを効果的に吸着・除去できるので、ウイルス感染症の治療効果が大幅に改善される。本発明のカラムの患者への接続は、通常の血液吸着療法の場合と同様に行うことができる。 Since the virus in the blood of the patient can be effectively adsorbed and removed using the column of the present invention, the therapeutic effect on viral infections is greatly improved. The column of the present invention can be connected to the patient in the same manner as in the case of conventional blood adsorption therapy.
　上記カラムを他の血液吸着療法用カラムと組み合わせて使用してもよい。他の血液吸着療法用カラムは、活性化免疫細胞、サイトカイン、エンドトキシンなどを吸着するものであってもよい。 The above column may be used in combination with another column for blood adsorption therapy. Other columns for blood adsorption therapy may be those that adsorb activated immune cells, cytokines, endotoxins, and the like.
　ポリミキシンＢはエンドトキシンに対する高い親和性を有する抗生物質であり、これを固定化した担体を含むカラムが血中エンドトキシン除去に使用されている。ポリミキシンＢを利用したカラムは、活性化免疫細胞やサイトカインを吸着することも報告されている。ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された担体は、ＳＡＲＳ－ＣｏＶおよびＳＡＲＳ－ＣＯＶ－２などのウイルスに加え、活性化免疫細胞やサイトカインも吸着することができる。したがって、ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された担体を用いて、重症化したウイルス感染症患者を治療し、生存率を改善することができる。 Polymyxin B is an antibiotic having a high affinity for endotoxin, and a column containing a carrier on which it is immobilized is used for removing endotoxin in blood. It has also been reported that columns using polymyxin B adsorb activated immune cells and cytokines. The carrier on which the ACE2 partial peptide and polymyxin B are immobilized can adsorb activated immune cells and cytokines in addition to viruses such as SARS-CoV and SARS-COV-2. Therefore, a carrier on which the ACE2 partial peptide and polymyxin B are immobilized can be used to treat a patient with a severe viral infection and improve the survival rate.
　したがって、本発明はさらなる態様において、ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された担体を提供する。本発明は、さらなる態様において、ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された担体を含むカラムを提供する。ポリミキシンＢを固定化できる担体、ならびにポリミキシンＢの担体への固定化方法は公知である。したがって、当業者は、ＡＣＥ２部分ペプチドとポリミキシンＢが固定化された担体を製造することができ、このような担体をカラムに充填することができる。カラムは、例えばカセット式のものであってもよい。カラムのサイズや形状は患者の症状、必要な血液流量、使用機器などに応じて決定されうる。 Therefore, in a further embodiment, the present invention provides a carrier on which an ACE2 partial peptide and polymyxin B are immobilized. The present invention provides, in a further embodiment, a column comprising a carrier on which an ACE2 partial peptide and polymyxin B are immobilized. A carrier capable of immobilizing polymyxin B and a method for immobilizing polymyxin B on the carrier are known. Therefore, one of ordinary skill in the art can produce a carrier on which the ACE2 partial peptide and polymyxin B are immobilized, and such a carrier can be packed in the column. The column may be, for example, a cassette type. The size and shape of the column can be determined according to the patient's symptoms, required blood flow rate, equipment used, and the like.
　例えば、現在使用されているエンドトキシン吸着カラム「トレミキシン（登録商標）（東レ・メディカル株式会社）」中の担体（ポリミキシンＢが固定化されている）にＡＣＥ２部分ペプチドを固定化してもよい（図１参照）。トレミキシン（登録商標）は、エンドトキシン血症に伴う重症病態あるいはグラム陰性菌感染症による重症病態の患者の治療に使用されている。 For example, the ACE2 partial peptide may be immobilized on a carrier (polymyxin B is immobilized) in the endotoxin adsorption column "Tremixin (registered trademark) (Toray Medical Co., Ltd.)" currently used (FIG. 1). reference). Toremikishin (TM) is used to treat patients with severe condition by severe conditions or gram-negative bacteria infections associated with endotoxemia.
　本発明の担体およびカラムを血液吸着療法に使用してもよい。血液吸着療法は、患者から血液を取り出し、目的の物質や細胞を吸着する担体と血液を接触させて、再び血液を患者に返す治療法である。当該血液吸着療法はウイルス感染症の治療であってもよい。 The carrier and column of the present invention may be used for blood adsorption therapy. Blood adsorption therapy is a treatment method in which blood is taken out from a patient, the carrier that adsorbs a target substance or cell is brought into contact with the blood, and the blood is returned to the patient again. The blood adsorption therapy may be a treatment for a viral infection.
　したがって、本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された血液吸着療法用担体、およびＡＣＥ２部分ペプチドが固定化されたウイルス感染症治療用担体を提供する。 Therefore, in a further aspect, the present invention provides a carrier for blood adsorption therapy on which an ACE2 partial peptide is immobilized, and a carrier for viral infection treatment on which an ACE2 partial peptide is immobilized.
　本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体の血液吸着療法のための使用、およびＡＣＥ２部分ペプチドが固定化された担体のウイルス感染症治療のための使用を提供する。 The present invention provides, in a further aspect, the use of a carrier on which an ACE2 partial peptide is immobilized for blood adsorption therapy and the use of a carrier on which an ACE2 partial peptide is immobilized for the treatment of a viral infection.
　本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体を含む、血液吸着療法用カラム、およびＡＣＥ２部分ペプチドが固定化された担体を含む、ウイルス感染症治療用カラムを提供する。 In a further aspect, the present invention provides a column for blood adsorption therapy, which comprises a carrier on which an ACE2 partial peptide is immobilized, and a column for treating a viral infection, which comprises a carrier on which the ACE2 partial peptide is immobilized.
　本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体を含むカラムの血液吸着療法のための使用、およびＡＣＥ２部分ペプチドが固定化された担体を含むカラムのウイルス感染症治療のための使用を提供する。 The present invention, in a further embodiment, is used for blood adsorption therapy of a column containing a carrier on which an ACE2 partial peptide is immobilized, and for treatment of a viral infection of a column containing a carrier on which the ACE2 partial peptide is immobilized. Provide use.
　本発明は、さらなる態様において、血液吸着療法用担体またはそれを含むカラムの製造のためのＡＣＥ２部分ペプチドの使用、およびウイルス感染症治療用担体またはそれを含むカラムの製造のためのＡＣＥ２部分ペプチドの使用を提供する。 The present invention, in a further embodiment, uses the ACE2 partial peptide for the production of a carrier for blood adsorption therapy or a column containing the same, and the ACE2 partial peptide for the production of a carrier for the treatment of viral infections or a column containing the same. Provide use.
　本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体に患者の血液を接触させることを含む、血液吸着療法の実行方法、およびＡＣＥ２部分ペプチドが固定化された担体に患者の血液を接触させることを含む、ウイルス感染症の治療方法を提供する。 In a further embodiment, the invention comprises contacting a patient's blood with a carrier on which the ACE2 partial peptide is immobilized, a method of performing a blood adsorption therapy, and the patient's blood on a carrier on which the ACE2 partial peptide is immobilized. Provided are methods of treating viral infections, including contact.
　本発明は、さらなる態様において、ＡＣＥ２部分ペプチドが固定化された担体を含むカラムに患者の血液を接触させることを含む、血液吸着療法の実行方法、およびＡＣＥ２部分ペプチドが固定化された担体を含むカラムに患者の血液を接触させることを含む、ウイルス感染症の治療方法を提供する。 In a further aspect, the invention comprises a method of performing a blood adsorption therapy comprising contacting a patient's blood against a column comprising a carrier on which the ACE2 partial peptide is immobilized, and a carrier on which the ACE2 partial peptide is immobilized. Provided are methods of treating viral infections, including contacting the column with the patient's blood.
　本明細書において、ウイルス感染症の治療とは、ウイルス感染による肺炎、気管支炎、発熱、せき、関節痛、臓器不全、急性腎障害、消化器症状、血栓症、心筋症などの諸症状のいずれか１つまたはそれ以上を軽減、抑制または消失させることをいう。 In the present specification, the treatment of a viral infection is any of various symptoms such as pneumonia, bronchitis, fever, cough, joint pain, organ failure, acute renal injury, gastrointestinal symptom, thrombosis, and myocardial disease due to the viral infection. To reduce, suppress or eliminate one or more.
　本発明の担体に吸着されるウイルスは、ＡＣＥ２に対する結合能を有するものであればいかなるものであってもよい。かかるウイルスの例としてはコロナウイルスが挙げられるが、これに限定されない。コロナウイルスとしてはＨＣｏＶ－ＮＬ６３、ＳＡＲＳ－ＣｏＶ、ＳＡＲＳ－ＣｏＶ－２およびそれらの変異体が挙げられるが、これらに限定されない。 The virus adsorbed on the carrier of the present invention may be any virus as long as it has the ability to bind to ACE2. Examples of such viruses include, but are not limited to, coronavirus. Coronaviruses include, but are not limited to, HCoV-NL63, SARS-CoV, SARS-CoV-2 and variants thereof.
　本発明の担体およびカラムを用いて血液吸着療法を行う際に、抗凝固薬としてセリンプロテアーゼ阻害薬、例えばナファモスタットを使用することにより、ＴＭＰＲＳＳ２によるＳ２サブユニットの切断を抑制し、ウイルスの膜融合を抑制してもよい。 When performing blood adsorption therapy using the carrier and column of the present invention, a serine protease inhibitor such as nafamostat is used as an anticoagulant to suppress the cleavage of S2 subunit by TMPRSS2 and to fuse the virus. May be suppressed.
　本発明の担体およびカラムをＥＣＭＯと併用してもよい（図２参照）。本発明の担体およびカラムのウイルス除去効果により、ＥＣＭＯの有効性を高めることができる。 The carrier and column of the present invention may be used in combination with ECMO (see FIG. 2). The virus-removing effect of the carrier and column of the present invention can enhance the effectiveness of ECMO.
　本明細書の用語は、特に断らない限り、医学、生物学、生化学、化学、薬学等の分野において通常に理解されている意味に解される。 Unless otherwise specified, the terms used herein are understood to have commonly understood meanings in the fields of medicine, biology, biochemistry, chemistry, pharmacy, etc.
　以下に実施例を示して本発明をより詳細かつ具体的に説明するが、実施例は本発明の範囲を限定するものではない。 The present invention will be described in more detail and concretely by showing examples below, but the examples do not limit the scope of the present invention.
　（１）ウイルス吸着能試験（その１）
　スペーサーおよびアタッチメントを付したＡＣＥ２部分ペプチド（１）～（８）を化学合成し、それぞれ１Ｎ、１Ｃ、２Ｎ、２Ｃ、３Ｎ、３Ｃ、４Ｎ、４Ｃと命名した。これらのペプチドをビーズ状樹脂担体に固定化した。
（１）Cys-miniPEG-IEEQAKTFLDKFNHEAEDLFYQS-NH2　（１Ｎ）
（２）IEEQAKTFLDKFNHEAEDLFYQS-miniPEG-Cys-NH2　（１Ｃ）
（３）Cys-miniPEG-IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-NH2　（２Ｎ）
（４）IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-miniPEG-Cys-NH2　（２Ｃ）
（５）sqyfldeaehnfkdlftkaqeei-miniPEG-Cys-NH2　（３Ｎ）
（６）Cys-miniPEG-sqyfldeaehnfkdlftkaqeei-NH2　（３Ｃ）
（７）sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-miniPEG-Cys-NH2　（４Ｎ）
（８）Cys-miniPEG-sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-NH2　（４Ｃ）
　式中、アミノ酸は公知の１文字表記とし、大文字はＬ－アミノ酸、小文字はＤ－アミノ酸を表す。IEEQAKTFLDKFNHEAEDLFYQSはヒトＡＣＥ２のＮ末端から２１～４２番目のアミノ酸配列である。sqyfldeaehnfkdlftkaqeeiはIEEQAKTFLDKFNHEAEDLFYQSのretro-inversoペプチド配列である。Ａｉｂは２－アミノイソ酪酸を表す。ｍｉｎｉＰＥＧの部分は、以下に示すＡＥＥＡを３分子結合したもの（２７原子の長さ）またはＤｉｂｅｇを２分子結合したもの（４０原子の長さ）である。Ｃｙｓはシステイン残基を表す。ＮＨ２はペプチドのＣ末端がアミド化されていることを示す。

Figure JPOXMLDOC01-appb-C000001
AEEA: 2-(2-(2-Aminoethoxy)ethoxy)acetic acid
Dibeg: Diethylene glycol bis(3-aminopropyl) ether glutarate
(1) Virus adsorption ability test (1)
ACE2 partial peptides (1) to (8) with spacers and attachments were chemically synthesized and named 1N, 1C, 2N, 2C, 3N, 3C, 4N, 4C, respectively. These peptides were immobilized on a beaded resin carrier.
(1) Cys-miniPEG-IEEQAKTFLDKFNHEAEDLFYQS-NH 2 (1N)
(2) IEEQAKTFLDKFNHEAEDLFYQS-miniPEG-Cys-NH 2 (1C)
(3) Cys-miniPEG-IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-NH 2 (2N)
(4) IEEQ-Aib-KTF-Aib-DKFNHE-Aib-EDLFYQS-miniPEG-Cys-NH 2 (2C)
(5) sqyfldeaehnfkdlftkaqeei-miniPEG-Cys-NH 2 (3N)
(6) Cys-miniPEG-sqyfldeaehnfkdlftkaqeei-NH 2 (3C)
(7) sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-miniPEG-Cys-NH 2 (4N)
(8) Cys-miniPEG-sqyflde-Aib-ehnfkd-Aib-ftk-Aib-qeei-NH 2 (4C)
In the formula, amino acids are represented by a known one-letter notation, uppercase letters represent L-amino acids, and lowercase letters represent D-amino acids. IEEQAKTFLDKFNHEAEDLFYQS is the 21st to 42nd amino acid sequence from the N-terminus of human ACE2. sqyfldeaehnfkdlftkaqeei is a retro-inverso peptide sequence of IEEQAKTFLDKFNHEAEDLFYQS. Aib represents 2-aminoisobutyric acid. The part of miniPEG is the one in which three molecules of AEEA shown below are bound (length of 27 atoms) or the one in which two molecules of Diveg are bound (length of 40 atoms). Cys represents a cysteine residue. NH 2 indicates that the C-terminus of the peptide is amidated.

Figure JPOXMLDOC01-appb-C000001
AEEA: 2- (2- (2-Aminoethoxy) ethoxy) acetic acid
Dibeg: Diethylene glycol bis (3-aminopropyl) ether glutarate
　固定化手順は以下のとおり。６－マレイミドヘキサン酸１.０５ｇ、Ｏ－(６－クロロベンゾトリアゾール－１－イル)－Ｎ,Ｎ,Ｎ',Ｎ'－テトラメチルウロニウムヘキサフルオロホスファート２．６８ｇ、１－ヒドロキシベンゾトリアゾール６７５ｍｇをジメチルホルムアミドに溶解し、Ｎ,Ｎ'－ジイソプロピルエチルアミン１．７ｍＬを加えて、１分間攪拌した。その反応液を、ビーズ１．３ｇ（Purolite社製、ＬＳ０１３９１）に加えて、室温で攪拌させてマレイミド基を有するビーズを得た。得られたマレイミド基を有するビーズ７５ｍｇを２０％アセトニトリルと６Ｍグアニジン塩酸塩とを含有するｐＨ７のリン酸緩衝液中に懸濁させ、ペプチド３７．５ｍｇを添加し、２０時間、室温で撹拌しながら反応させた。反応後、ビーズを５０％ジメチルホルムアミド水、ジメチルホルムアミド、水で順次洗浄し、ビーズ状ウイルス吸着体とした。 The immobilization procedure is as follows. 6-Maleimidehexanoic acid 1.05g, O- (6-chlorobenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphart 2.68g, 1-hydroxybenzotriazole 675 mg was dissolved in dimethylformamide, 1.7 mL of N, N'-diisopropylethylamine was added, and the mixture was stirred for 1 minute. The reaction solution was added to 1.3 g of beads (LS01391 manufactured by Purolite) and stirred at room temperature to obtain beads having a maleimide group. 75 mg of the obtained maleimide group-bearing beads was suspended in a phosphate buffer solution at pH 7 containing 20% acetonitrile and 6M guanidine hydrochloride, 37.5 mg of the peptide was added, and the mixture was stirred at room temperature for 20 hours. It was reacted. After the reaction, the beads were washed successively with 50% dimethylformamide water, dimethylformamide and water to obtain a bead-like virus adsorbent.
　上記手順にて得られたＡＣＥ２部分ペプチド固定化ビーズのＳＡＲＳ－ＣｏＶ－２ウイルス吸着能を以下の手順にて調べた。
　ペプチド未固定化ビーズと、ペプチド固定化ビーズをルシフェラーゼ遺伝子を組み込んだSARS-CoV-2シュードタイプVSV（SARS2pv）ウイルス溶液とともに一定時間インキュベートした。その後ウイルス液をVeroE6-TMPRSS2細胞（1x104細胞/well, 96well）に加え、24時間後のルシフェラーゼ活性を測定し、ウイルス吸着能を定量化した。ウイルスとしてはＳＡＲＳ－ＣｏＶ－２シュードタイプＶＳＶ（ＳＡＲＳ２ｐｖ）を用いた。結果を図３に示す。ペプチド３Ｎ、４Ｎおよび４Ｃを固定化したビーズにウイルス吸着能が見られた。これらの結果より、ペプチド３Ｎ、４Ｎおよび４Ｃにウイルス吸着能があると考えられた。 The SARS-CoV-2 virus adsorption capacity of the ACE2 partially peptide-immobilized beads obtained by the above procedure was examined by the following procedure.
Peptide-unfixed beads and peptide-immobilized beads were incubated with a SARS-CoV-2 pseudotype VSV (SARS2pv) virus solution incorporating the luciferase gene for a period of time. After that, the virus solution was added to Vero E6-TMPRSS2 cells (1x10 4 cells / well, 96 well), and the luciferase activity after 24 hours was measured to quantify the virus adsorption ability. As the virus, SARS-CoV-2 pseudo-type VSV (SARS2pv) was used. The results are shown in FIG. Virus adsorption ability was observed in beads on which peptides 3N, 4N and 4C were immobilized. From these results, it was considered that peptides 3N, 4N and 4C have virus adsorption ability.
　（２）ウイルス吸着能試験（その２）
　上記試験（１）でウイルス吸着能が高いことを確認したペプチド（３Ｎ、４Ｎおよび４Ｃ）を、カラムの基材となる繊維状担体に固定した。 (2) Virus adsorption ability test (Part 2)
Peptides (3N, 4N and 4C) confirmed to have high virus adsorption ability in the above test (1) were fixed on a fibrous carrier as a base material of a column.
　固定化手順は以下のとおり。ＴＦ１２．５６ｃｍ２を、ジメチルスホキシドで共洗いした。別途、ジメチルスホキシドに溶解したペプチド３０ｍｇ、ヨウ化ナトリウム８０ｍｇを、繊維に添加し、Ｎ,Ｎ'－ジイソプロピルエチルアミン０．４ｍＬを加えて室温で攪拌した。３０分後、トリブチルホスフィン０．２ｍＬを加えて室温で撹拌しながら反応させた。反応後、ジメチルスホキシド、水、ｐＨ７のリン酸緩衝液、水で順次洗浄した。洗浄した担体とルシフェラーゼ遺伝子を組み込んだSARS-CoV-2シュードタイプVSV（SARS2pv）ウイルス溶液２５０μｌ（７．５ｘ１０３ＰＦＵ）（ウイルスは上記試験（１）と同じ）を混合し、４℃で１時間インキュベートして、ウイルスを担体に吸着させた。上清５０μｌをＶｅｒｏＥ６－ＴＭＰＲＳＳ２細胞（９６ウェルプレート中、１ｘ１０４個／ウェル）に添加し、２４時間後にルシフェラーゼ活性を測定した。 The immobilization procedure is as follows. TF 12.56 cm 2 was co-washed with dimethyl sulfoxide. Separately, 30 mg of a peptide dissolved in dimethyl sulfoxide and 80 mg of sodium iodide were added to the fiber, 0.4 mL of N, N'-diisopropylethylamine was added, and the mixture was stirred at room temperature. After 30 minutes, 0.2 mL of tributylphosphine was added and the reaction was carried out at room temperature with stirring. After the reaction, the cells were washed successively with dimethyl sulfoxide, water, pH 7 phosphate buffer, and water. Mix 250 μl (7.5x10 3 PFU) of the SARS-CoV-2 pseudotype VSV (SARS2pv) virus solution incorporating the washed carrier and the luciferase gene (the virus is the same as in the above test (1)), and mix at 4 ° C. for 1 hour. Incubated to adsorb the virus on the carrier. 50 μl of the supernatant was added to VeroE6-TMPRSS2 cells (1x10 4 cells / well in a 96-well plate) and luciferase activity was measured 24 hours later.
　結果を図４に示す。いずれのペプチド固定化担体もウイルス吸着能を示した。ペプチド４Ｎを固定化した担体がウイルスを最も多く吸着することが示された。 The results are shown in Fig. 4. Both peptide-immobilized carriers showed virus-adsorbing ability. It was shown that the carrier on which peptide 4N was immobilized most adsorbed the virus.
　（３）ウイルス吸着能試験（その３）
　ペプチド３Ｎ、４Ｎおよび４Ｃを固定化したＴＦを滅菌した。γ線または高圧を滅菌に使用した。滅菌手順は以下のとおりであった。γ線滅菌－ＴＦを生理食塩液（生食）に懸濁し、γ線（２５ｋＧｙ）を照射した。高圧滅菌－ＴＦを生理食塩液またはリン酸緩衝化生理食塩液（ＰＢＳ）に懸濁し、高圧蒸気滅菌した（１１６．５℃、９０分）。滅菌した担体または滅菌しなかった担体をカラムに充填した。カラムにルシフェラーゼ遺伝子を組み込んだSARS-CoV-2シュードタイプVSV（SARS2pv）ウイルス溶液（ウイルスは上記試験（１）、（２）と同じ）を充填し、一定時間循環させた後、溶液中の遊離ウイルスをVeroE6-TMPRSS2細胞（1x104細胞/well, 96well）に加え、２４時間後のルシフェラーゼ活性を測定し、ウイルス吸着能を定量化した。カラムに吸着したウイルス量から、当該カラムのウイルス吸着能を定量化した。結果を図５に示す。滅菌を行っても担体（すなわちペプチド）のウイルス吸着能が維持されることがわかった。 (3) Virus adsorption ability test (3)
TFs immobilized with peptides 3N, 4N and 4C were sterilized. Gamma rays or high pressure were used for sterilization. The sterilization procedure was as follows. Gamma-ray sterilization-TF was suspended in physiological saline (raw food) and irradiated with γ-rays (25 kGy). High-pressure sterilization-TF was suspended in saline or phosphate buffered saline (PBS) and sterilized by high pressure steam (116.5 ° C., 90 minutes). Columns were filled with sterile or non-sterile carriers. A SARS-CoV-2 pseudo-type VSV (SARS2pv) virus solution in which the luciferase gene was incorporated into a column (the virus is the same as in the above tests (1) and (2)) was filled, circulated for a certain period of time, and then released in the solution. The virus was added to Vero E6-TMPRSS2 cells (1x10 4 cells / well, 96 well), and the luciferase activity after 24 hours was measured to quantify the virus adsorption capacity. The virus adsorption capacity of the column was quantified from the amount of virus adsorbed on the column. The results are shown in FIG. It was found that the virus adsorption capacity of the carrier (that is, the peptide) was maintained even after sterilization.
　本発明は医療材料および医療機器の分野、ならびにウイルス感染症の研究分野等において利用可能である。 The present invention can be used in the fields of medical materials and medical devices, research fields of viral infectious diseases, and the like.

Claims (15)

  1. 　アンジオテンシン変換酵素２（ＡＣＥ２）部分ペプチドが固定化された担体。 A carrier on which angiotensin converting enzyme 2 (ACE2) partial peptide is immobilized.
  2. 　ＡＣＥ２部分ペプチドが、ＡＣＥ２タンパク質のペプチダーゼドメイン中のアミノ酸配列を含むものである、請求項１記載の担体。 The carrier according to claim 1, wherein the ACE2 partial peptide contains an amino acid sequence in the peptidase domain of the ACE2 protein.
  3. 　ＡＣＥ２部分ペプチドが、ＡＣＥ２タンパク質のペプチダーゼドメインのαヘリックス中のアミノ酸配列を含むものである、請求項２記載の担体。 The carrier according to claim 2, wherein the ACE2 partial peptide contains an amino acid sequence in the α-helix of the peptidase domain of the ACE2 protein.
  4. 　ＡＣＥ２部分ペプチドが、配列番号：３または配列番号：４に示すアミノ酸配列から選択されるアミノ酸配列を含むものである、請求項３記載の担体。 The carrier according to claim 3, wherein the ACE2 partial peptide contains an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.
  5. 　ＡＣＥ２部分ペプチドが、配列番号：４に示すアミノ酸配列を含むものである、請求項４記載の担体。 The carrier according to claim 4, wherein the ACE2 partial peptide contains the amino acid sequence shown in SEQ ID NO: 4.
  6. 　ＡＣＥ２部分ペプチドが、そのＣ末端を介して担体に固定化されている、請求項５記載の担体。 The carrier according to claim 5, wherein the ACE2 partial peptide is immobilized on the carrier via its C-terminal.
  7. 　高圧滅菌されている、請求項６記載の担体。 The carrier according to claim 6, which is sterilized under high pressure.
  8. 　ＡＣＥ２部分ペプチドがスペーサーを介して固定化されている、請求項１～７のいずれか１項記載の担体。 The carrier according to any one of claims 1 to 7, wherein the ACE2 partial peptide is immobilized via a spacer.
  9. 　さらにポリミキシンＢが固定化された、請求項１～８のいずれか１項記載の担体。 The carrier according to any one of claims 1 to 8, further immobilized with polymyxin B.
  10. 　請求項１～９のいずれか１項記載の担体を含むカラム。 A column containing the carrier according to any one of claims 1 to 9.
  11. 　液体中からウイルスを吸着除去するために用いられる請求項１０記載のカラム。 The column according to claim 10, which is used for adsorbing and removing a virus from a liquid.
  12. 　液体が血液である請求項１１記載のカラム。 The column according to claim 11, wherein the liquid is blood.
  13. 　配列番号：３または配列番号：４に示すアミノ酸配列を含むペプチド。 Peptide containing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.
  14. 　スペーサーを有する請求項１３記載のペプチド。 The peptide according to claim 13, which has a spacer.
  15. 　スペーサーにアタッチメントが付いた請求項１４記載のペプチド。 The peptide according to claim 14, which has an attachment attached to the spacer.
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