WO2022004707A1 - Support d'adsorption de sars-cov -2 et colonne contenant celui-ci - Google Patents

Support d'adsorption de sars-cov -2 et colonne contenant celui-ci Download PDF

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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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ace2
carrier
partial peptide
peptide
immobilized
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PCT/JP2021/024503
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English (en)
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/fr

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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.

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  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
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  • External Artificial Organs (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un peptide partiel de l'enzyme de conversion de l'angiotensine (ACE2), un support sur lequel ledit peptide est fixé, et une colonne contenant ledit support.
PCT/JP2021/024503 2020-06-30 2021-06-29 Support d'adsorption de sars-cov -2 et colonne contenant celui-ci WO2022004707A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004516896A (ja) * 2000-12-29 2004-06-10 アップフロント・クロマトグラフィー・アー／エス 細胞外体液からの特異的生体高分子物質の体外捕獲
JP2009524420A (ja) * 2006-01-25 2009-07-02 ヴィロ ファルマソイティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト ウイルス感染ペプチド（ｓｅｖｉ）のヒト精子増強剤およびその使用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004516896A (ja) * 2000-12-29 2004-06-10 アップフロント・クロマトグラフィー・アー／エス 細胞外体液からの特異的生体高分子物質の体外捕獲
JP2009524420A (ja) * 2006-01-25 2009-07-02 ヴィロ ファルマソイティカルズ ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト ウイルス感染ペプチド（ｓｅｖｉ）のヒト精子増強剤およびその使用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAN, YANXIAO ET AL.: "Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2", ACS NANO, vol. 14, no. 4, 14 April 2020 (2020-04-14), pages 5143 - 5147, XP055732485, DOI: 10.1021/acsnano.0c02857 *
SAURABH, SUMAN ET AL.: "A Modified ACE2 peptide mimic to block SARS-CoV2 entry", BIORXIV, 8 May 2020 (2020-05-08), XP055799138, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.05.07.082230v1.abstract> [retrieved on 20210810], DOI: https://doi.org/10.1101/ 2020.05.07.082230 *
TEMEL, SAHIN ET AL.: "Blood Purification Treatments in COVID-19", J. CRI. INT. CARE, vol. 11, no. 1, 22 June 2020 (2020-06-22), pages 32 - 35, XP055895932 *

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