WO2023090381A1 - Anti-hepatitis b virus agent targeting host factor dock11 - Google Patents

Anti-hepatitis b virus agent targeting host factor dock11 Download PDF

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WO2023090381A1
WO2023090381A1 PCT/JP2022/042642 JP2022042642W WO2023090381A1 WO 2023090381 A1 WO2023090381 A1 WO 2023090381A1 JP 2022042642 W JP2022042642 W JP 2022042642W WO 2023090381 A1 WO2023090381 A1 WO 2023090381A1
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polypeptide
sequence
amino acid
acid sequence
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弘志 柳川
典子 田畠
真由子 井手
裕子 米村
哲 伊藤
周一 金子
政夫 本多
和寿 村居
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ピューロテックバイオ株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to an anti-hepatitis B virus agent that targets the host factor DOCK11.
  • the present invention also relates to antibodies, antibody fragments, or single-chain antibodies that bind to asialoglycoprotein receptors that are excellent carriers for drug delivery into hepatocytes.
  • HBV hepatitis B virus
  • rcDNA incomplete double-stranded DNA
  • cccDNA covalently closed circular DNA
  • HBV utilizes the host's DNA repair mechanism, particularly the ATR signaling pathway, when synthesizing cccDNA from rcDNA (Non-Patent Document 3).
  • HBV can remain latent in cells as stably inactivated cccDNA.
  • HBV reactivation occurs when the immune system is compromised by chemotherapy or hematopoietic stem cell transplantation.
  • interactions between the host's immune system and the virus influence the development of advanced hepatocellular carcinoma (HCC). Treatments that successfully reduce viral replicas can delay the development of advanced liver disease and hepatocellular carcinoma.
  • targeting cccDNA is necessary to eliminate latent HBV.
  • HBV-infected hepatocytes to HCC cells express reduced amounts of HBV protein and mRNA intracellularly. Except for cells containing HBV DNA integrated into the host genome, cell lines established from HCC usually do not express HBV transcripts. Cell lines that continue to express HBV transcripts are very useful for identifying host genes required for HBV maintenance. Therefore, Hashimoto et al. established an HC1 cell line that is maintained at a very low rate of HBV infection (about 1 in 3000 cells), and performed single-cell transcriptome analysis (Nx1-Seq) to detect HBV infection. Four host genes, LIPG, DOCK11, DENND2A, and HECW2, which are highly expressed in cells, were identified (Non-Patent Document 4).
  • DOCK11 (dedicator of cytokinesis 11, NCBI Gene ID: 139818), also called Zizimin2
  • DOCK11 is a member of the DOCK-D subfamily and has a large molecular size ( ⁇ 240 kDa).
  • Cells use cytoskeleton such as actin filaments and microtubules to change and maintain their morphology.
  • the cytoskeleton is strictly controlled by intracellular signal transduction triggered by external stimuli, and Rho family low-molecular-weight GTP-binding proteins (G proteins) play an important role in the signal transduction.
  • Rho family G proteins act as molecular switches for intracellular signal transduction by being activated by GTP binding and inactivated by GDP binding.
  • Rho family G proteins are activated by the GDP-GTP exchange reaction caused by the action of guanine nucleotide exchange factor (GEF).
  • GEFs that activate Rho family G proteins are roughly classified into two groups: a group having a common Dbl homology domain (DH domain) and a group having a unique activation region called the DOCK family (Non-Patent Document 5. ). About 80 GEFs have been reported so far in mammals, including both groups. Since DOCK 180 was first reported in 1996, 11 types of DOCK family proteins have been confirmed in mammals. It is divided into C and DOCK-D (Non-Patent Document 6). DOCK11 belongs to the DOCK-D family.
  • DOCK family proteins have two regions, Dock homology region 1 (DHR1) and DHR2, whose amino acid sequences are well conserved among families, and each activates a specific Rho family G protein through DHR2. It is known to do (Non-Patent Document 6). DOCK11 associates with CDC42, a G protein, and is a molecule involved in cytoskeleton and transport.
  • DOCK11 has also been reported as one of the molecules that function to maintain the HBV gene in host cells (Patent Document 1).
  • Patent Document 1 discloses that suppression of DOCK11 expression by siRNA and lenti-shRNA reduces HBV replication.
  • a DOCK11 inhibitor applicable to HBV-infected patients has not yet been developed.
  • DDS drug delivery systems
  • the purpose of the present invention is to provide a novel anti-HBV drug that targets DOCK11.
  • DOCK11-binding peptides have shown effects such as inhibition of DOCK11-Ack1 binding to inhibit Ack1 activation, inhibition of DOCK11 guanine nucleotide exchange factor (GEF) activity, and inhibition of DOCK11-mediated activation of the ATR signaling pathway.
  • GEF DOCK11 guanine nucleotide exchange factor
  • the present invention provides an anti-hepatitis B virus agent (anti-HBV agent) containing, as an active ingredient, a substance that binds to DOCK11 and inhibits the function of DOCK11.
  • anti-HBV agent anti-hepatitis B virus agent
  • the substance used as an active ingredient binds to the region of residues 1516 to 2073 of DOCK11.
  • the inhibition of DOCK11 function is selected from inhibition of Ack1 activation by inhibition of binding of DOCK11 to Ack1, inhibition of guanine nucleotide exchange factor activity of DOCK11, and inhibition of activation of the ATR signaling pathway by DOCK11. is at least one
  • the substance used as an active ingredient is at least one polypeptide selected from the following polypeptides (1) to (16).
  • a polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1).
  • a polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
  • a polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
  • a polypeptide of the sequence TEKRRLMKPVLLTYNP SEQ ID NO:4
  • a polypeptide of the sequence IICPGAEVLNGDLVAS SEQ ID NO:5
  • a polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
  • a polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
  • a polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
  • a polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
  • a polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
  • a polypeptide of sequence HEEHRGMLREDSMMEYLK SEQ ID NO: 11
  • a polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
  • a partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
  • a partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
  • a partial polypeptide of ⁇ -centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
  • the polypeptide is in a form linked to a carrier molecule for delivery into hepatocytes.
  • the carrier molecule can be, for example, an antibody, antibody fragment or single-chain antibody that binds to the asialoglycoprotein receptor.
  • the polypeptide is in a form linked to a cell membrane permeabilization molecule.
  • a cell membrane permeabilization molecule can be, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO:38 or 39.
  • the polypeptide is in a form linked to a nuclear localization signal.
  • the present invention also provides the use of at least one polypeptide selected from the polypeptides (1) to (16) above as a DOCK11-binding peptide, and from the polypeptides (1) to (16) above.
  • DOCK11-binding peptides are provided that consist of at least one selected polypeptide.
  • a heavy chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence
  • a heavy chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence
  • a heavy chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence
  • a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence
  • a light chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35, or an amino acid sequence
  • the present invention provides a drug delivery carrier for delivering a drug into hepatocytes, containing the antibody, antibody fragment, or single-chain antibody of the present invention.
  • the present invention provides a pharmaceutical composition comprising a complex of the drug delivery carrier of the present invention and a drug to be delivered into hepatocytes.
  • the present invention provides for the first time an anti-HBV agent that binds to DOCK11 and has the action of inhibiting the function of DOCK11. Since the anti-HBV agent of the present invention can reduce intracellular HBV DNA and cccDNA and inhibit re-invasion of HBV particles into cells, it is expected to cure hepatitis B completely.
  • the anti-ASGR antibody, antibody fragment or single-chain antibody having a specific CDR sequence or modified sequence thereof provided by the present invention is very excellent as a carrier for delivering drugs into hepatocytes, and is an anti-HVB agent. In addition, it is useful as a delivery carrier for various drugs to act in the liver.
  • DNA library design for IVV screening Structure of the human DOCK180 superfamily. Explanatory diagram of the construction procedure of biotinylated full-length DOCK11. Explanatory drawing of selection experiments for peptides that bind to DOCK11 by the IVV method.
  • Day 18 Transfection with plasmid expressing peptide (1 ⁇ g with Lipofectamine 3000).
  • Day 20 Measure HBV-DNA, cccDNA.
  • Day 1 Seed cells in 12 well plates.
  • Day 2 Transfection with plasmid expressing peptide (1 ⁇ g with Lipofectamine 3000).
  • Day 3 HBV infection (medium change after 12 hours).
  • Day 5 Measure HBV-DNA and cccDNA. Anti-HBV activity of DOCK11-binding peptides in HepG2-NTCP-C4 cells (long-term administration).
  • A Schedule of HBV infection and sample administration.
  • B Early endosomes, peptides (GGP), actin, and nuclei were observed with a confocal microscope. Functional verification of membrane fusion-promoting peptides S28 and S39: Comparison of nuclear uptake of peptides by S28 and S39 Validation of the function of nuclear localization signals.
  • A Experimental procedure.
  • B Peptides (GGP), actin, and nuclei were observed with a confocal microscope.
  • DOCK1516-2073-Bio-His containing the DHR2 domain was added to a streptavidin-coated 96-well plate, fixed overnight at 4°C, reacted with 0-1.0 mol of DCS8-42TN at room temperature for 1 hour, and then subjected to GEF assay. gone.
  • A HepG2 cells were transfected with DOCK11 siRNA #1-3 using Lipofectamine 3000, and real-time RT-PCR was performed 72 hours later. GAPDH mRNA levels were used to normalize DOCK11 mRNA levels.
  • B HepG2 cells were transfected with DOCK11 or Ack1 siRNA #1-3 using Lipofectamine 3000.
  • HepG2 cells were transfected with DOCK11 or Ack1 siRNA #1-3 using Lipofectamine 3000. After 72 hours, the cells were fixed and permeabilized, and the actin filaments were stained with fluorescent phalloidin, followed by nuclear staining with DAPI.
  • A HepG2 cells were treated with 100 nM N-10M-D42TN for 0-48 hours, washed, and cultured for 24-72 hours. Then, the cells were fixed and permeabilized, and the actin filaments were stained with fluorescent phalloidin, followed by nuclear staining with DAPI.
  • C HepG2 cells were transfected with EYFP-NLS-Actin and treated 48 hours later with 100 nM N-10M-D42TN for 20 hours. After fixing and permeabilizing the cells and staining the nuclei with DAPI, the fluorescence of EYFP and GFP was observed.
  • Ack1 binds to and activates DOCK11-activated Cdc42, and binds to and phosphorylate EGFR.
  • both EGFR-Ack1 complexes are endocytosed and degraded, but by inhibiting DOCK11 function by N-10M-D42TN, phosphorylated EGFR is not degraded and Ack1 phosphorylation and Phosphorylation of WASP, which is phosphorylated by Ack1, is also inhibited.
  • Huh7 cells were treated with 100 nM N-10M-D42TN for 24 hours and then treated with 10 ng/ml EGF for 0-10 minutes, and Western blotting was performed using the cell lysate.
  • Ack1, pAck1, EGFR, pEGFR(Tyr845), pEGFR(Tyr1068), pEGFR(Tyr1045), WASP, pWASP, and GAPDH antibodies were used as primary antibodies.
  • HepG2 cells were treated with 100 nM N-10M-D42TN for 24 hours and then treated with 100 ng/ml EGF for 1 hour. Early endosomes were stained with CellLight Early Endosomes-RFP, immunostained with anti-Ack1 antibody, and nuclear stained with DAPI. White arrows indicate early endosomes where Ack1 is localized, and black arrows indicate early endosomes where Ack1 is not localized.
  • (B) shows the ratio of all pixels representing early endosomes to those colocalizing with Ack1 in the cells shown in Panel A (Colocalization coefficients).
  • A HepG2 cells were transfected with DOCK11 siRNA #1-3 using Lipofectamine 3000, and DNA damage was induced by UV irradiation 72 hours later. RNA was extracted from these cells and the amount of DOCK11 mRNA was measured by real-time RT-PCR. GAPDH mRNA was similarly measured and normalized.
  • This cell lysate was prepared and Western blotting was performed using anti-Chk1 and pChk1 antibodies as primary antibodies.
  • C In Fig. B, ImageLab was used to calculate the expression level ratio of pChk1 and Chk1, and the phosphorylation level of Chk1 in each cell was calculated. As a control, the amount of phosphorylation in cells that were not irradiated with UV after transfection with siEmpty was used.
  • D HepG2 cells were treated with 100 nM N-10M-D42TN for 24 hours, and DNA damage was induced by UV irradiation. This cell lysate was prepared and Western blotting was performed using anti-Chk1 and pChk1 antibodies as primary antibodies.
  • PXB cells were treated with 100 nM N-10M-D42TN for 24 hours and then UV-irradiated to induce DNA damage. Immunostaining was performed using an anti-DOCK11 antibody and nuclear staining was performed with DAPI.
  • B The fluorescence intensity of DOCK11 that showed a threshold value or higher inside the nucleus was calculated and normalized using the fluorescence intensity of DAPI.
  • DNA damage was induced by UV irradiation in PXB cells transfected with siRNA targeting DOCK11 and treated with 100 nM N-10M-D42TN for 24 h.
  • C, D Fluorescence intensity of DOCK11 (C) and ⁇ H2AX (D) in each cell in FIG. 37A. Both show the ratio of DAPI fluorescence intensity, and the results of cells transfected with siEmpty without UV exposure were used as a control. *p ⁇ 0.05, **p ⁇ 0.005.
  • N-10M-D42TN and control Measurement of blood HBV DNA in Example 16. Comparison of N-10M-D42TN and control (PBS). Measurement of h-Alb in Example 16. Comparison of N-10M-D42TN and control (PBS). Measurement of ALT in Example 16. Comparison of N-10M-D42TN and control (PBS). Measurement of HBV DNA in liver in Example 16. Comparison of N-10M-D42TN and control (PBS). Measurement of HBV DNA in liver in Example 16. Comparison of N-10M-D42TN and control (PBS).
  • anti-hepatitis B virus includes treatment of HBV infection, prevention of HBV infection, suppression of HBV proliferation, treatment of hepatitis B, and prevention of hepatitis B. be.
  • hepatitis B can be treated by suppressing the proliferation of HBV in the patient's body (inside the liver).
  • an anti-HBV agent to an HBV carrier before the onset of hepatitis B, the proliferation of HBV in the carrier can be prevented and the onset of hepatitis B can be prevented (prevention of hepatitis B).
  • the anti-HBV agent of the present invention contains as an active ingredient a substance that binds to DOCK11 and inhibits the function of DOCK11.
  • Human DOCK11 is a protein having the structure shown in FIG. 2, and has Dock homology region 1 (DHR1) and DHR2, which are domains whose amino acid sequences are well conserved among DOCK families.
  • DHR1 located at the N-terminus binds to phosphatidylinositol 3-phosphate.
  • DHR2 present on the C-terminal side is a domain that activates a specific Rho family G protein, and DHR2 of DOCK11 activates a small G protein Cdc42.
  • sequences shown in SEQ ID NOS: 41 and 42 in the Sequence Listing are the nucleotide sequence of the coding region of human DOCK11 mRNA and the amino acid sequence of DOCK11 encoded by this registered in GenBank of NCBI under NM_144658.4.
  • the region from 1st to 2036th residues is the DHR2 domain.
  • the substance used as the active ingredient of the anti-HBV agent of the present invention may be a substance that binds to the region of residues 1516 to 2073 (C-terminal region) of DOCK11, eg, the DHR2 domain.
  • Cdc42 is replaced from inactive (GDP-bound) to active (GTP-bound), and unlike other GEF proteins, active Cdc42 is known to bind to and further activate and induce positive feedback (Lin, Q. et al. J. Biol. Chem., 281, 35253-35262, 2006; Nishikimi, A. et al. Exp Cell Res 319, 2343-2349, 2013).
  • GEF guanine nucleotide exchange factor
  • Ack1 activated CDC42 kinase 1
  • GTP-bound Cdc42 Primary phosphate-maleic anhydride-semiconduct-binding protein
  • the present inventors demonstrated that DOCK11 and Ack1 bind intracellularly, and that Cdc42 and Ack1 competitively bind to DOCK11.
  • Inhibition of DOCK11 function is, for example, at least one selected from inhibition of Ack1 activation by inhibition of binding of DOCK11 and Ack1, inhibition of GEF activity of DOCK11, and inhibition of activation of ATR signaling pathway by DOCK11. It's okay.
  • the following examples demonstrate that a DOCK11-binding peptide with anti-HBV activity inhibits the binding of DOCK11 and Ack1 to inhibit Ack1 activation, inhibits the GEF activity of DOCK11, and activates the ATR signaling pathway by DOCK11.
  • Substances that bind to the region of residues 1516 to 2073 (C-terminal region) of DOCK11 or the DHR2 domain and inhibit the function of DOCK11 include, for example, recognizing and binding to the C-terminal region or DHR2 domain.
  • the term "antibody fragment” is synonymous with the term "antigen-binding fragment of antibody” and includes Fab, Fab', F(ab') 2 and the like. There is no particular limitation as long as it is an antibody fragment that maintains antigen-binding properties.
  • the anti-HBV agent of the present invention may contain, as an active ingredient, at least one polypeptide selected from the following polypeptides (1) to (16), for example.
  • polypeptides (1) to (16) are the names of each polypeptide used in the following examples. Hereinafter, these polypeptides may be referred to as "DOCK11-binding peptides".
  • DCS3-1] (2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
  • DCS5-4] (3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
  • [DCS5-5] (4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4).
  • [DCS5-15] (5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5).
  • [DCS8-6] (6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
  • [DCS8-29] (7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
  • [DCS8-59] (8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
  • [DCS8-72] (9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
  • [DCS8-42] (10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
  • [DCS8-42TN] (11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
  • [DCS8-59R] (12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
  • [DCS8-59C] (13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
  • a partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
  • a partial polypeptide of ⁇ -centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
  • Polypeptides (1) to (9) are polypeptides selected from an IVV library by a selection experiment for peptides that bind to DOCK11 using Biacore in the following examples, and confirmed to have anti-HBV activity by pull-down assay or the like.
  • (10) to (12) are polypeptides designed based on the homology search results of the sequences of (7) and (9) and confirmed to have anti-HBV activity by pull-down assays and the like.
  • the test substance prepared using DCS8-42TN in (10) was used in an in vivo administration experiment to confirm the anti-HBV activity. Agents other than 42TN are also useful as anti-HBV agents like DCS8-42TN.
  • the partial polypeptide of (13) is a partial region of Ack1 (NP_001374642.1, SEQ ID NO: 50), which is a region having high homology with DOCK11-binding peptide DCS8-42 PEQARPPPPLEDNLFL (SEQ ID NO: 10; SEQ ID NO: 50 674th to 689th amino acids in the amino acid sequence of Ack1 shown in )).
  • SEQ ID NO: 49 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 50 (nucleotide sequence of the coding region of NM_001387713.1).
  • the partial polypeptide of (14) is a partial region of radixin (NP_001247421.1, SEQ ID NO: 92) and has high homology with DOCK11-binding peptide DCS8-59 HEEHRGMLREDSMMEYLK (SEQ ID NO: 11; SEQ ID NO: 92 176th to 193rd amino acids in the radixin amino acid sequence shown in )).
  • SEQ ID NO:91 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:92 (nucleotide sequence of the coding region of NM_001260492.2).
  • the partial polypeptide of (15) is a partial region of ⁇ -centractin (NP_005726.1, SEQ ID NO: 94) and is a region having high homology with DOCK11-binding peptide DCS8-59 AEEHRGLLTIRYPMEH (SEQ ID NO: 12; It is a polypeptide composed of a partial region containing the 62nd to 77th amino acid region in the amino acid sequence of ⁇ -centractin shown in SEQ ID NO:94).
  • SEQ ID NO:93 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:94 (nucleotide sequence of the coding region of NM_005735.4).
  • DCS8-42TN a polypeptide of SEQ ID NO: 10, DCS8-59R, a polypeptide of SEQ ID NO: 11, and DCS8-59C, a polypeptide of SEQ ID NO: 12, have anti-HBV activity.
  • polypeptides composed of partial regions of each protein containing these regions also have anti-HBV activity and are useful as anti-HBV agents.
  • the chain length of the polypeptides (13) to (15) is not particularly limited. The size may be within 10 groups, within 50 residues, within 40 residues, within 30 residues, within 25 residues, or within 20 residues.
  • the polypeptide of (16) is a polypeptide having a sequence identity of 80% or more with the original polypeptide in which a part of the residues is modified in any of the polypeptides of (1) to (15). . It is well known in the art that a very small number of residues can be altered while maintaining equivalent or better activity of the polypeptide. Thus, the original sequence in which a few residues (eg, 1-4 residues, 1-3 residues, 1 or 2 residues, or 1 residue) of the polypeptides (1)-(15) are altered Polypeptides having 80% or more identity, such as 85% or more, or 90% or more identity with , can exhibit anti-HBV activity like the original polypeptide and are useful as anti-HBV agents. Modifications of residues herein are substitutions, deletions, insertions or additions, typically substitutions.
  • the identity of amino acid sequences means that two amino acid sequences to be compared are aligned so that as many amino acid residues as possible match each other, and the number of matched amino acid residues is divided by the total number of amino acid residues. It is expressed as a percentage.
  • gaps are appropriately inserted in one or both of the two sequences to be compared, if necessary.
  • sequence alignment can be performed using well-known programs such as BLAST, FASTA, CLUSTAL W, and the like.
  • the above total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences being compared, then the % identity is the total number of amino acid residues in the longer sequence and the number of matching amino acid residues. calculated by dividing
  • amino acids with similar side chains have similar chemical properties.
  • Grouping amino acids by side chain similarity includes, for example, the group of amino acids with aliphatic side chains (glycine, alanine, valine, leucine, isoleucine), the group of amino acids with aliphatic hydroxyl side chains (serine, threonine).
  • the group of amino acids with amide-containing side chains (asparagine, glutamine), the group of amino acids with aromatic side chains (phenylalanine, tyrosine, tryptophan), the group of amino acids with basic side chains (arginine, lysine, histidine), They can be classified into groups of amino acids with acidic side chains (aspartic acid, glutamic acid), groups of amino acids with sulfur-containing side chains (cysteine, methionine), and the like.
  • a substitution for another amino acid belonging to the same group is a conservative substitution.
  • polyethylene glycol (PEG) chains are added for the purpose of improving the stability of peptides in vivo and increasing the half-life in blood (Clin Nephrol. 2006 Mar;65(3): 180-90. and Proc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7.), mainly adding sugar chains to the N-terminus or C-terminus (J Am Chem Soc. 2004 Nov 3;126 (43): 14013-22 and Angew Chem Int Ed Engl. 2004 Mar 12;43(12): 1516-20), at least part of the amino acid residues are in the D form (J Pharmacol Exp Ther.
  • PEG polyethylene glycol
  • the Fc region of the antibody is appropriately modified and added (e.g., J.Immunol., 154( 10), 5590-5600 (1995), Nature, 332, 563-564 (1998), Nature, 332, 738-740 (1998), BioDrugs. 2008;22:11-26, etc.), C-terminal amidation , N-terminal acetylation, etc. are used.
  • the polypeptide used as an active ingredient in the anti-HBV agent of the present invention may be one to which such techniques are applied.
  • polypeptide having the sequence of SEQ ID NO: X refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: X and having a total length of N residues (such a polypeptide is referred to as polypeptide X for convenience), and the Fc region and It includes polypeptides having a structure in which other functional molecules are added or linked, such as polypeptides having the effect of enhancing delivery to the liver.
  • polypeptide having the sequence of SEQ ID NO: X means that the full length is N residues.
  • polypeptide having a structure in which another functional molecule is added to polypeptide X is included as an active ingredient. Any functional molecule may be linked as long as it does not impair the anti-HBV activity of the polypeptide.
  • polypeptides of (1) to (12) and the polypeptides of (16) that have 80% or more and less than 100% sequence identity with any of (1) to (12) among the polypeptides of (16) have a chain length Since it is short, it can be easily prepared by conventional chemical synthesis.
  • the polypeptides of (13) to (15) and the polypeptides of (16) that have 80% or more and less than 100% sequence identity with any of (13) to (15) among the polypeptides of (16) have a chain length Short ones can be easily prepared by chemical synthesis, and long chain ones that are difficult to prepare by chemical synthesis can be produced by genetic engineering techniques.
  • Polypeptides having a structure in which other functional molecules are linked are also produced by chemical synthesis when the functional molecule is a short-sized polypeptide, and by genetic engineering when the functional molecule is a long-sized polypeptide. can be manufactured by
  • chemical synthesis methods include the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Moreover, it can also synthesize
  • a cDNA encoding a full-length DOCK11-binding peptide with a long size or a full-length DOCK11-binding peptide linked to a functional molecule is prepared, the cDNA is incorporated into an appropriate expression vector, and the expression is transformed into an appropriate host cell.
  • the polypeptide of interest can be obtained by introducing it, producing the polypeptide in the host cell, and extracting and purifying it.
  • the DOCK11-binding peptide may have a tag sequence such as a Flag tag or His tag added for convenience of purification and detection.
  • a tag sequence can also be regarded as one example of a functional molecule.
  • SEQ ID NO: 90 for example, in the "cleaved sequence + DCS8-42TN peptide + S28 + nuclear localization signal" portion (SEQ ID NO: 90) of N-10M-D42TN prepared in the following example, Flag tag and His tag is added.
  • addition of such a tag sequence is optional, and it is also possible to prepare the anti-HBV agent of the present invention using a DOCK11-binding peptide without a tag sequence.
  • a functional molecule is a carrier molecule for delivery into hepatocytes. That is, the DOCK11-binding peptide may be in a form linked to a carrier molecule for delivery into hepatocytes.
  • ASGR asialoglycoprotein receptor
  • ApoE receptor which are abundant in hepatocytes
  • carrier molecules that deliver drugs into hepatocytes using ASGR or ApoE receptors can be used.
  • ASGR asialoglycoprotein receptor
  • ASGR is a type II single-pass transmembrane protein with the N-terminus directed intracellularly and the C-terminal carbohydrate recognition sites (CRDs) directed extracellularly, as shown in FIG.
  • CCDs carbohydrate recognition sites
  • ASGR-specific binding molecule used as a drug delivery carrier may bind to the extracellular domain of ASGR.
  • a specific binding molecule that can bind to both ASGR1 and ASGR2 can be particularly preferably used, but even a specific binding molecule that binds to only one of them can be used as a drug delivery carrier.
  • a specific binding molecule that binds only to either one it may be used alone as a drug delivery carrier, or a combination of a specific binding molecule that binds to one and a specific binding molecule that binds to the other may be used.
  • a DOCK11-binding peptide linked to an ASGR1-specific binding molecule and a DOCK11-binding peptide linked to an ASGR2-specific binding molecule may be prepared, mixed and used as an anti-HBV agent.
  • the base sequence of the coding region of human ASGR1 mRNA registered under NM_001671.5 in GenBank and the amino acid sequence of ASGR1 encoded by this are shown in SEQ ID NOs: 43 and 44, and the code of human ASGR2 mRNA registered under NM_001181.4.
  • the nucleotide sequence of the region and the amino acid sequence of ASGR2 encoded by this are shown in SEQ ID NOS: 45 and 46, respectively.
  • the 41st to 61st amino acids of SEQ ID NO: 44 (ASGR1) and the 59th to 79th amino acids of SEQ ID NO: 46 (ASGR2) are transmembrane domains, the N-terminal side of which is the intracellular domain, and the C-terminal side of which is the cell.
  • ASGR extracellular domain includes hetero-oligomerized extracellular domains.
  • hetero-oligomer is meant a hetero-oligomerized ASGR extracellular domain.
  • Anti-ASGR polyclonal antibodies can be obtained by immunizing a non-human animal using the ASGR extracellular domain as an immunogen, collecting blood, separating the serum, and collecting and purifying the antibody that binds to the ASGR extracellular domain from the serum. can be done.
  • Anti-ASGR monoclonal antibodies are produced by collecting antibody-producing cells such as splenocytes and lymphocytes from non-human animals immunized with the ASGR extracellular domain, fusing them with myeloma cells to prepare hybridomas, and binding to the ASGR extracellular domain.
  • Anti-ASGR monoclonal antibodies can be obtained from culture supernatants by selecting hybridomas that produce antibodies against the anti-ASGR and proliferating them.
  • An anti-ASGR antibody fragment can be obtained by treating an anti-ASGR antibody with a proteolytic enzyme such as papain or pepsin.
  • a proteolytic enzyme such as papain or pepsin.
  • the definition of the antibody fragment is as described above, and includes antibody fragments such as Fab, Fab', F(ab') 2 that maintain the binding ability to the antigen (ASGR extracellular domain).
  • cDNA is prepared by extracting mRNA from the hybridoma produced as described above, and PCR is performed using primers specific for immunoglobulin H chain and L chain to obtain immunoglobulin H chain gene and L chain.
  • PCR is performed using primers specific for immunoglobulin H chain and L chain to obtain immunoglobulin H chain gene and L chain.
  • anti-ASGR scFv can be obtained from scFv libraries by techniques such as the phage display method and IVV method.
  • a phage library with scFv displayed on the phage surface is prepared as an scFv library.
  • a naive scFv phage library may be prepared by the following procedure. mRNA is extracted from antibody-producing cells such as splenocytes and lymphocytes collected from healthy humans or non-human animals, cDNA is synthesized by reverse transcription reaction, and cDNA encoding the region containing the heavy chain variable region (VH) (VH cDNA) and cDNA encoding a region containing the light chain variable region (VL) (VL cDNA) are comprehensively amplified by PCR.
  • VH heavy chain variable region
  • VL light chain variable region
  • the amplified VH cDNA and VL cDNA are randomly ligated via a suitable linker (for example, a linker consisting of three repeated GGGGS units, etc.) by standard assembly PCR or Fusion PCR to obtain scFv-encoding cDNA.
  • a suitable linker for example, a linker consisting of three repeated GGGGS units, etc.
  • Fusion PCR Fusion PCR
  • Plasmid vectors for phage include all phage genes necessary for forming phage particles and capable of forming phage particles independently, and phage vectors containing the g3p gene but no other phage protein genes and containing phage Although there are two types of phagemid vectors that require helper phage for particle formation, phagemid vectors are preferably used.
  • a scFv cDNA library prepared using a phagemid vector is introduced into Escherichia coli, and the E. coli is superinfected with a helper phage to package each vector of the scFv cDNA library, and the scFv expressed by the vector is placed on the surface. Libraries of displaying phage can be generated.
  • a phage library of mutant scFv may be prepared by randomly introducing mutations into the prepared VH cDNA and VL cDNA or scFv cDNA.
  • phages displaying scFv that bind to the ASGR extracellular domain are selected (panning).
  • This panning step can be performed using a solid-phase carrier (chip, plate, magnetic bead, etc.) on which the extracellular domain of ASGR is immobilized, and a carrier on which a hetero-oligomer is immobilized is particularly preferably used.
  • the extracellular domain of ASGR1 (ASGR1ex) and the extracellular domain of ASGR2 (ASGR2ex) are biotinylated, and by contacting the biotinylated ASGR1ex and ASGR2ex with a carrier coated with avidins, ASGR1ex and ASGR2ex are hetero-oligomerized on the carrier surface.
  • the phage library is brought into contact with the ASGR extracellular domain-immobilized carrier, and after washing, the phages bound on the carrier are recovered.
  • the recovered phages are lysed, the packaged vector is recovered, introduced again into E. coli, and superinfected with helper phages to form phage particles again.
  • These phage particles are again brought into contact with the ASGR extracellular domain-immobilized carriers.
  • Phages enriched through multiple rounds of panning can be obtained as anti-ASGR scFv candidate clones, but clone selection may be performed to further narrow down the candidates.
  • the scFv expression vector is collected from the phage after concentration and introduced into an appropriate host cell such as E. coli to prepare host cell clones expressing scFv. and select clones with high specific binding to the ASGR extracellular domain. Reactivity can be confirmed by an immunoassay such as ELISA using the ASGR extracellular domain, preferably a hetero-oligomer, as an antigen.
  • clones may be grouped by confirming duplication of clones based on the base sequences of VH and VL. Through these clone selection activities, it is possible to select clones with high specific binding to the ASGR extracellular domain and further narrow down the candidates.
  • the scFv expression vector is recovered from the candidate clone, the scFv cDNA is amplified from the scFv expression vector, incorporated into an appropriate plasmid expression vector to express the scFv in an appropriate host cell, recovered and purified.
  • the reactivity with ASGR1ex and ASGR2ex or the reactivity with heterooligomers is finally confirmed for the scFv after purification, and scFv that specifically binds to the ASGR extracellular domain can be obtained.
  • IVV method (Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03/106675 A1) is a technique developed by Yanagawa, one of the inventors of the present application, and his collaborators.
  • puromycin a type of antibiotic
  • PEG polyethylene glycol
  • An mRNA-protein junction molecule (in vitro virus; IVV) is formed covalently linked to the mRNA molecule via puromycin. From the IVV library constructed in this way, IVVs containing proteins that bind to baits (proteins, peptides, antigens, etc.) are picked in vitro, and the genes (mRNA) linked to them are amplified by reverse transcription PCR. Then, by deciphering the base sequence with a DNA sequencer, interacting proteins, peptides and antibodies can be easily identified. Competitive elution with free bait is the common method for eluting and recovering IVV bound to bait.
  • IVV in vitro virus
  • the spacer can be cleaved by UV irradiation at 365 nm, and the mRNA portion can be eluted and recovered (Doi, N. et al., J. Biotechnol., 131:231-239, 2007).
  • mRNA is synthesized from the cDNA library by reverse transcription reaction, puromycin is bound to the 3' end of the mRNA via a PEG spacer, and scFv mRNA-puromycin library.
  • a cell-free translation reaction is carried out to construct a library of mRNA-scFv linking molecules (IVV) in which scFv molecules and mRNA molecules encoding them are covalently bound via puromycin.
  • a scFv that binds to the ASGR extracellular domain is selected from the scFv IVV library.
  • this selection step can also be performed using a solid-phase carrier (chip, plate, magnetic beads, etc.) on which ASGR extracellular domains, preferably hetero-oligomers, are immobilized as bait.
  • the scFv IVV library is brought into contact with the ASGR extracellular domain-immobilized carrier, and after washing, the IVV bound to the carrier is eluted and collected. IVV can be eluted and recovered by competitive elution using ASGR1ex or ASGR2ex.
  • Reverse transcription PCR is performed using the recovered IVV or mRNA part as a template to prepare a scFv cDNA library after the 1st round of selection.
  • An IVV library can be prepared again from this cDNA library and a second round of selection can be performed. It is preferable to perform multiple rounds of selection while sequentially increasing the selection pressure (contact time between IVV library and bait, amount of bait immobilized on carrier, etc.).
  • This vector is introduced into a suitable host cell such as E. coli to obtain a library of scFv clones after selection.
  • the resulting clone library is evaluated for reactivity to the ASGR extracellular domain, analyzed for the variable region sequence, and grouped.
  • Vectors are collected from the selected clones and scFv and final confirmation of reactivity with ASGR1ex and ASGR2ex or reactivity with heterooligomers, scFv with high specific binding to ASGR extracellular domain can be obtained.
  • anti-ASGR antibody, antibody fragment or scFv are a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31; a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32; a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33; a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35;
  • An antibody, antibody fragment or scFv having a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, 24, 30 or 36 can be mentioned.
  • anti-ASGR scFv having heavy chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 13-15 and light chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 16-18, amino acids shown in SEQ ID NOS: 19-21 an anti-ASGR scFv having the heavy chain CDRs 1-3 of the sequences shown in SEQ ID NOS: 22-24 and the light chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 22-24, the heavy chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 25-27 and SEQ ID NOS: 28-30 and anti-ASGR scFv with heavy chain CDRs 1-3 shown in SEQ ID NOS: 31-33 and light chain CDRs 1-3 shown in SEQ ID NOS: 34-36.
  • the CDR sequence is not limited to the specific amino acid sequence described above, and a CDR containing an amino acid sequence having 80% or more identity with the original amino acid sequence by substituting a part of the bases in the above amino acid sequence. It may be an antibody or the like having For example, heavy and light chain CDR1 allows substitution of 1 residue, heavy and light chain CDR2 allows substitution of 1-3, 1-2 or 1 residue, Heavy and light chain CDR3s tolerate substitutions of 1-2 or 1 residue.
  • An antibody, antibody fragment or scFv having such modified CDRs can also be used as an antibody, antibody fragment or scFv that specifically binds to ASGR.
  • Antibodies and the like having CDRs of a given sequence encode the amino acid sequences described above by, for example, introducing mutations into the CDR regions of the VH and VL genes of any cloned antibody or the scFv gene encoding any scFv. It can be easily prepared by modifying as follows. Any antibody and any scFv as a base for introducing the above CDR sequence may be an anti-ASGR antibody and anti-ASGR scFv having a CDR sequence different from the above, or an antibody and scFv against other antigens There may be.
  • the anti-ASGR antibody, antibody fragment or scFv having CDRs or modified CDRs containing the specific amino acid sequence described above can be used not only as a carrier for delivery of the anti-HVB agent of the present invention, but also as various drugs to be delivered into hepatocytes. It is an excellent delivery carrier for This anti-ASGR antibody, antibody fragment, or scFv can be used as a carrier for drug delivery into hepatocytes, and a drug to be delivered into hepatocytes can be complexed with the carrier by techniques known in the pharmaceutical field and used as a pharmaceutical composition. .
  • the anti-ASGR antibody may be a human antibody, a humanized antibody, a human-non-human animal chimeric antibody, or a non-human animal antibody.
  • anti-ASGR scFv antibodies may be derived from human antibodies, humanized antibodies, chimeric antibodies between human and non-human animals, or non-human animal antibodies.
  • those derived from human antibodies, humanized antibodies or chimeric antibodies, particularly those derived from human antibodies or humanized antibodies, particularly those derived from human antibodies are preferred.
  • DOCK11-binding peptide When linking a DOCK11-binding peptide to a carrier molecule for intrahepatocyte delivery such as an anti-ASGR antibody, it may be linked to either end of the DOCK11-binding peptide. If one or more other functional molecules are also used, they may be linked to the DOCK11-binding peptide so as to function properly in conjunction with those other functional molecules.
  • the carrier molecule for intrahepatic delivery is preferably linked to the DOCK11-binding peptide via a cleavage sequence that is cleaved by endogenous enzymes present in the cells of patients receiving the anti-HBV agent of the present invention.
  • An example of a cleavage sequence is the sequence RVRR (SEQ ID NO: 37) that Furin recognizes and cleaves, but the cleavage sequence is not limited to this.
  • a functional molecule is a cell membrane permeation promoting molecule. That is, the DOCK11-binding peptide may be in a form linked to a cell membrane permeabilization molecule.
  • a cell membrane permeation-enhancing molecule is a molecule that promotes release of a DOCK11-binding peptide that has been taken up by endocytosis into the cytoplasm from the endosome through the membrane.
  • S19 Sudo, K. et al., J. Control. Release, 255: 1-11, 2017, Sudo, K. et al., J. Control. Release, 255: 1-11, 2017, WO 2016/199674 A1).
  • 28-residue syntisin 1 partial peptide S28 (PFVIGAGVLGALGTGIGGITTSTQFYYK, SEQ ID NO: 38) and 39-residue syntisin 1 partial peptide S39 (PFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMER, SEQ ID NO: 39), which were developed as peptides that can function more efficiently, can be mentioned.
  • These peptides, particularly S28 and S39 are cell membrane permeation promoting molecules that can be preferably used in the present invention, but usable cell membrane permeation promoting molecules are not limited to these.
  • the cell membrane permeation promoting molecule may be linked to either end of the DOCK11-binding peptide. or to the end opposite to the carrier molecule.
  • a nuclear localization signal can be mentioned as a further example of a functional molecule. That is, the DOCK11-binding peptide may be in a form linked to a nuclear localization signal.
  • Various nuclear localization signals are known, and any of them may be used.
  • PAAKRVKLD SEQ ID NO: 40
  • the nuclear localization signal may be ligated to either end of the DOCK11-binding peptide. , or at the end opposite to the carrier molecule.
  • both ends may be linked individually, or both may be linked to one end.
  • the delivery carrier + cleavage sequence, cell membrane permeabilization molecule and nuclear localization signal are ligated to the DOCK11-binding peptide, the delivery carrier + cleavage sequence is at one end and the cell membrane permeabilization molecule and nuclear localization signal are at the other end. Link.
  • HBV-infected patients including hepatitis B patients and HBV-infected patients who have not developed hepatitis B (HBV carriers).
  • Patients are typically, but not limited to, mammals, particularly humans.
  • the dosage of the anti-HBV agent of the present invention may be any amount that provides an anti-HBV effect in the patient to whom it is administered.
  • An effective dose can be appropriately selected according to the patient's symptoms, viral load, age, body weight and the like.
  • the dosage of the anti-HBV agent of the present invention may be about 1 ⁇ g to 10000 mg, for example about 100 ⁇ g to 1000 mg per 1 kg body weight of the active ingredient per day for the subject.
  • the amount of active ingredient referred to here is the amount of the peptide portion only, and when using a DOCK11-binding peptide linked to one or more other functional molecules as an active ingredient.
  • the amount of active ingredients is the total amount.
  • the daily dose may be administered once or divided into several doses. Administration may be daily or every few days.
  • the administration route of the anti-HBV agent of the present invention may be either oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, and intraarterial administration is generally preferred.
  • the active ingredient of the anti-HBV agent of the present invention contains pharmaceutically acceptable carriers, diluents, excipients, binders, lubricants, disintegrants, sweeteners, suspending agents suitable for each administration route. , an emulsifier, a coloring agent, a corrigent, a stabilizer, and the like.
  • Formulations include oral agents such as tablets, capsules, granules, powders and syrups, and parenteral agents such as inhalants, injections, suppositories and liquid agents.
  • Formulation methods and usable excipients are well known in the field of pharmaceutical formulations, and any method and excipients can be used.
  • An anti-HBV agent containing two or more substances as active ingredients may be a combination drug containing all two or more active ingredients in the same formulation, or a single agent containing each active ingredient independently. may include a combination of In embodiments involving a combination of single agents, the single agents are usually administered simultaneously or sequentially, although each single agent may be administered at appropriate intervals.
  • FIG. 1 shows the design of a DNA library for IVV screening.
  • IVV in vitro virus
  • FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000 WO 03/106675 A1 is a technique proposed by Yanagawa, one of the inventors of the present application, and his co-researchers, who succeeded in constructing it for the first time in the world.
  • puromycin a type of antibiotic
  • a PEG polyethylene glycol
  • a cell-free translation reaction is performed using this as a template to bind puromycin to the protein and mRNA.
  • IVV is formed, a simple mRNA-protein linking molecule covalently linked via (Miyamoto-Sato, E. et al., Nucleic Acids Res., 31: e78, 2003). From the IVV library constructed in this way, IVV containing proteins that bind to baits (proteins, peptides, antigens, etc.) are picked in vitro, and the genes (mRNA) linked to them are analyzed by reverse transcription and PCR.
  • baits proteins, peptides, antigens, etc.
  • RNA portion can be eluted and recovered by cleaving the spacer by irradiation with UV at 365 nm (Doi, N. et al., J. Biotechnol., 131:231-239, 2007).
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • DNA was purified with Wizard SV Gel PCR Clean-Up System (Promega) and collected as 50 ⁇ l of GSP6-GFP-DNA solution.
  • Atail sequence 1 ⁇ l, 10 ⁇ KOD plus buffer (TOYOBO) 10 ⁇ l, 2 mM dNTPs (TOYOBO) 10 ⁇ l, 25 mM MgSO 4 4 ⁇ l
  • forward primer Flag-His-F (10 pmol/ ⁇ l) 3 ⁇ l
  • reverse primer Add RNase-free water to 3 ⁇ l of
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • DNA was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of Flag-His Atail-DNA solution.
  • Atail-DNA solution 1 ⁇ l, 10 ⁇ KOD plus buffer (TOYOBO) 10 ⁇ l, 2 mM dNTPs (TOYOBO) 10 ⁇ l, 25 mM MgSO 4 4 ⁇ l, forward primer: 16NNS-F or 9NNS-F (10 pmol/ ⁇ l) 3 ⁇ l , reverse primer: Atail (R) (10 pmol/ ⁇ l) 3 ⁇ l, and KOD plus polymerase (TOYOBO) 2 ⁇ l, add RNase-free water to make the total volume 100 ⁇ l, put this in one tube, total 300 ⁇ l (tube 3) were subjected to PCR reaction.
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of 16NNS Atail-DNA solution or 9NNS Atail-DNA solution.
  • cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of cDNA library 16NNSLib and 9NNSLib.
  • Example 2 Preparation of IVV library (2-1) Transcription of library 2 pmol each of cDNA libraries 16NNSLib and 9NNSLib, 8 ⁇ l of 5 ⁇ SP6 buffer, 2 ⁇ l of ATP (100 mM), 2 ⁇ l of CTP (100 mM), UTP (100 mM) 2 ⁇ l, GTP (10 mM) 4 ⁇ l, cap analog (m7G(5')PPP(5')G) (Thermo Fisher Scientific) (40 mM) 5 ⁇ l, Enzyme Mix SP6 RNA polymerase (Promega) 4 ⁇ l, RNase-Free water. After 3 hours of reaction at 37°C, 10 ⁇ l of RQ1 RNase-Free DNase (Promega) was added and further reacted at 37°C for 1 hour.
  • RNase-free water was added to the transcription reaction solution to bring the total volume to 100 ⁇ l, and 350 ⁇ l of RLT buffer (Qiagen), 3.5 ⁇ l of 2-mercaptoethanol, and 250 ⁇ l of (100%) ethanol were added to the RNeasy mini spin column.
  • Example 3 Preparation of biotinylated DOCK11
  • DOCK family proteins have two regions, Dock homology region 1 (DHR1) and DHR2, whose amino acid sequences are highly conserved among families.
  • DHR1 binds to phosphatidylinositol 3-phosphate.
  • DHR2 domains activate their own specific Rho family G proteins.
  • DOCK11 belongs to the DOCK-D group and has a PH domain in the N-terminal region. DOCK11 activates the small G protein Cdc42 and acts as a guanine nucleotide exchange factor (GEF).
  • GEF guanine nucleotide exchange factor
  • a biotinylation sequence, Flag-tag and His-tag were added to the C-terminus of the amino acid sequence of full-length DOCK11 (aa1-2073, SEQ ID NO: 42).
  • a biotinylation sequence, Flag-tag and His-tag were added to the C-terminus of the amino acid sequence of the DOCK11 C-terminal region peptide (aa1516-2073 in SEQ ID NO: 42) containing the DHR2 domain.
  • Bio-tag (Nucleic Acids Research, 2009 , Vol. 37, No. 8, page e64) was added with Flag-tag and His-tag.
  • Bio-tag 2.37 ⁇ l, 10 ⁇ KOD plus buffer solution (TOYOBO) 40 ⁇ l, 2 mM dNTPs (TOYOBO) 40 ⁇ l, 25 mM MgSO 4 16 ⁇ l, KstartBio-F (10 pmol/ ⁇ l) 12 ⁇ l, Bio-Flag-Histag A stop (10 pmol/ ⁇ l) and 8 ⁇ l of KOD plus polymerase (TOYOBO), RNase-free water was added to make the total volume 200 ⁇ l, and PCR reaction was performed.
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 20 or 25 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of DNA solution to obtain KstartBio-Flag-His.
  • KstartBio-Flag-His was introduced into the pcDNA 3.3 vector using the TOPO cloning kit (Invitrogen) according to the procedure. After confirming that the resulting clone had the correct sequence, a colony was inoculated and cultured at 37°C for 16 hours. Plasmid pcDNA3.3 TOPO KBioFlagHis was purified from the cell pellet with the PureYield TM Plasmid Maxiprep System (Promega).
  • DOCK11 vector pF1KE2360 (Kazusa DNA Res.Inst.) (1 ng/ ⁇ l) 0.5 ⁇ l, KAPA HiFi HS RM 12.5 ⁇ l, 10 ⁇ M TOPOKstartDOCK11-IF-F 0.75 ⁇ l, and 10 ⁇ M DOCK11Bio-IF-R 0.75 ⁇ l in RNase-Free water was added to make the total volume 25 ⁇ l, and the PCR reaction was performed. PCR was carried out at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 1 minute. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 30 ⁇ l of DNA solution to obtain Kstart-DOCK11-IF.
  • RNase-free water was added to 0.5 ⁇ l of pcDNA3.3 TOPO KBioFlagHis, 10 ⁇ l of KAPA HiFi HS RM, 0.6 ⁇ l of 10 ⁇ M F-Bio, and 0.6 ⁇ l of 10 ⁇ M TOPOKstartINV-R to make the total volume 20 ⁇ l, and a PCR reaction was performed. PCR was carried out by reacting at 95°C for 3 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 3 minutes, followed by reaction at 72°C for 1 minute. After confirming the PCR product by agarose gel electrophoresis, it was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 30 ⁇ l of DNA solution to obtain TOPO KBioFlagHisINV.
  • Kstart-DOCK11-IF 1.0 ⁇ l, TOPO KBioFlagHisINV 1.0 ⁇ l, 5x infusion HD Enzyme premix 1.0 ⁇ l (Takara) was added with RNase-free water to make the total volume 5 ⁇ l, and reacted at 50°C for 15 minutes. 2.5 ⁇ l was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain clones. Sequence analysis of the clone confirmed the plasmid pcDNA3.3 TOPO KDOCK11-BioFLAGHis.
  • RIPA buffer 25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6
  • 800 ⁇ l protease inhibitor
  • x50 16 ⁇ l of (4-(2-aminoethyl)fluoride, benzenesulfonyl hydrochloride (AEBSF), aprotinin, E-64, leupeptin hemisulfate monohydrate, bestatin, pepstatin A) and 8 ⁇ l of 0.1M PMSF were added to the cells.
  • Sample buffer LDS (4x) and 6.8 ⁇ l of 0.2 mM DTT were added to 15 ⁇ l of the eluted fraction E1, heated at 70° C. for 10 minutes, and subjected to SDS-PAGE.
  • SDS-PAGE was performed on a 4-12% Bis-Tris NuPAGE gel with MES electrophoresis buffer (Invitrogen) at 200V, 400mA, 35 minutes, followed by Mini Format, 0.2 ⁇ m PVDF, Single application (BIORAD) Trans-Blot Turbo. transcribed.
  • the membrane was blocked with Blocking One Buffer: TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer: TBST (1:9).
  • Detection was performed using ChemiDoc (BIORAD) using ECL (Enhanced ChemiLuminescence). Biotinylated full-length DOCK11 was identified as a band with a molecular weight of 246,762 Da, and biotinylated C-terminal region DOCK11 was identified as a band with a molecular weight of 73,404 Da.
  • Example 4 Selection of peptides that bind to DOCK11 A selection experiment for peptides that bind to DOCK11 was performed according to the procedure shown in Fig. 4 . (4-1) Immobilization of DOCK11 Biacore immobilized the biotinylated C-terminal region DOCK11 on the sensor chip SA using the Biacore 3000 system. Flow was performed at 10 ⁇ l/min in buffer HBS-P (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20). Pretreatment for immobilization was performed by injecting 10 ⁇ l of a solution containing 50 mM NaOH and 1 M NaCl into flow cells 1 to 4 three times.
  • Biotinylated C-terminal region DOCK11 (1 nM) was used to immobilize to flow cells 1-4.
  • Flow was in buffer HBS-P, 20 ⁇ l/min. Manual injection of 23 ⁇ l resulted in 1267.4 RU (average) binding to flow cells 1-4.
  • an extra wash was performed with a buffer solution HBS-P at 10 ⁇ l/min using 50% Isopropanol, 50 mM NaOH and 1M NaCl.
  • the sensor chip was extracted from the machine. 55 ⁇ l of RNase-free water was gently added to the gold film of the sensor chip, and UV light of 365 nm was applied for 20 minutes to cleave the spacer, and the mRNA part was eluted and collected.
  • Example 5 Cloning and nucleotide sequence determination (5-1) Cloning and nucleotide sequence Creation of an in-fusion cloning library insert from a peptide library that binds to DOCK11 Library subjected to 8 rounds of selection experiment 1 ⁇ l, 10 ⁇ KOD plus buffer (TOYOBO) 100 ⁇ l, 2 mM dNTPs (TOYOBO) 100 ⁇ l, 25 mM MgSO 4 40 ⁇ l, GFP-F in (10 pmol/ ⁇ l) 30 ⁇ l, His-S28-R in (10 pmol/ ⁇ l) 30 ⁇ l, RNase-free water was added to 20 ⁇ l of KOD plus polymerase (TOYOBO) to bring the total volume to 1000 ⁇ l, followed by PCR reaction.
  • TOYOBO 10 ⁇ KOD plus buffer
  • TOYOBO 2 mM dNTPs
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of DNA solution to obtain GFP-DC-S8in.
  • PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 ⁇ l of DNA solution to obtain KGFP-S28 3.3 vector.
  • a nuclear localization signal sequence gene CCTGCTGCCAAGAGGGGTCAAGTTGGAC (SEQ ID NO: 47) was introduced upstream of the GFP sequence, and 8 ⁇ l of pcDNA 3.3 vector containing a stop codon, 80 ⁇ l of 10 ⁇ KOD plus buffer (TOYOBO), 80 ⁇ l of 2 mM dNTPs (TOYOBO), 25 mM MgSO. 4 Add RNase-free water to 32 ⁇ l, S28iv-25F (10 pmol/ ⁇ l) 24 ⁇ l, GFPiv-71R (10 pmol/ ⁇ l) 24 ⁇ l, and KOD plus polymerase (TOYOBO) 16 ⁇ l to make the total volume 800 ⁇ l, and perform PCR reaction.
  • PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 ⁇ l of DNA solution to obtain NLS-GFP-S28 3.3 vector.
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of DNA solution to obtain NLS-GFP-DC-S8in.
  • NLS-GFP-DC-S8 in 0.08 ⁇ l, NLS-GFP-S28 3.3 vector 0.14 ⁇ l, 5x infusion HD Enzyme premix 1.0 ⁇ l (Takara), add RNase-Free water to bring the total volume to 5 ⁇ l, and heat at 50°C. React for 15 minutes. 2.5 ⁇ l of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain NLS-GFP-DCS8 clones. Sequence analysis of the obtained clones was performed by Eurofin DNA sequence contract service, ValueRead Premix.
  • Example 6 Evaluation of activity of DOCK11-binding peptide clones (6-1) Preparation of protein In-frame clones were inoculated from a master plate onto LB medium containing 20 ⁇ g/ml of carbenicillin and cultured at 37°C for 16 hours. bottom. Plasmids were purified from cell pellets with the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen).
  • the resin was transferred to a new tube, and 1000 ⁇ l of the culture supernatant expressing DCS3, DCS5 or DCS8 was mixed with 40 ⁇ l of biotinylated full-length DOCK11, biotinylated C-terminal region DOCK11, or bait-free treated resin, and mixed in a mini disc rotor. (Bio craft) and combined at 4° C. for 1 hour 30 minutes. After removing the solution, washing was performed three times with 500 ⁇ l of TBST (0.1% Tween 20), and the recovered resin was subjected to Western blotting.
  • a pull-down assay (Fig. 5) was performed against C-terminal DOCK11 containing the DHR2 region by DOCK11-binding peptides.
  • Three of the clones subjected to three rounds of selection experiments were evaluated and positive for DCS3-1 (SEQ ID NO: 1), DCS3-2 and DCS3-3.
  • DCS3-1 (SEQ ID NO: 1), DCS5-15 (SEQ ID NO: 4), DCS8-42 (SEQ ID NO: 9) and DCS8-42TN (SEQ ID NO: 10) among clones of 3 rounds, 5 rounds, and 8 rounds was subjected to a pull-down assay against the C-terminal DOCK11 containing the DHR2 region with a DOCK11-binding peptide (Fig. 6).
  • DCS3-1 SEQ ID NO: 1
  • DCS8-42 SEQ ID NO: 9
  • DCS8-42TN (SEQ ID NO: 10) obtained from 8 rounds strongly bind to DOCK11. I understand.
  • Example 7 Anti-HBV activity of screened DOCK11-binding peptide (7-1) Anti-HBV activity of DOCK11G-binding peptide in HepG2-NTCP-C4 cells Regarding clones that were positive in pull-down experiments against C-terminal DOCK11 containing DHR2 region , DOCK11-binding peptides in HepG2-NTCP-C4 cells (HepG2 cells overexpressing the HBV receptor NTCP, Iwamoto, M. et al. Biochem. Biophys. Res. Commun. 443:808-813, 2014) was assessed for anti-HBV activity (Fig. 7).
  • a peptide-containing plasmid (DCS3-1) for intracellular expression and a plasmid (N-DCS3-1) added with a nuclear localization signal PAAKRVKLD (SEQ ID NO: 40) were prepared.
  • the HBV used was HBV derived from HepG2.2.15 cells in which a helper plasmid lacking the packaging signal ( ⁇ ) present upstream of the HBV core was stably expressed in HepG2 cells. After HepG2-NTCP-C4 cells were infected with HepG2.2.15-derived HBV, plasmids encoding various peptides were transfected with Lipofectamine 3000, respectively. Samples were collected 3-5 days after transfection and evaluated for HBV-DNA and cccDNA.
  • DCS5-1, DCS5-7, DCS5 among DCS5-1, DCS5-4 (SEQ ID NO: 2), DCS5-5 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 4), DCS5-7, DCS5-33 -33 did not decrease HBV DNA copy number and cccDNA copy number compared to controls, but DCS5-4 (SEQ ID NO: 2), DCS5-5 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 3) number 4) decreased the copy number of HBV DNA compared to controls. In addition, the copy number of cccDNA was also decreased.
  • DCS8-42TN having a sequence homologous to DCS8-42 (peptide consisting of a partial region of TNK2 (Ack1) showing high homology with DCS8-42, SEQ ID NO: 10)
  • DCS8-59R having a sequence homologous to DCS8-59 (peptide consisting of a partial region of radixin showing high homology with DCS8-59, SEQ ID NO: 11)
  • DCS8-59C peptide consisting of a partial region of ⁇ -Centractin showing high homology with DCS8-59, SEQ ID NO: 12
  • HepG2-NTCP-C4 cells were infected with HBV for 20 days and evaluated. No. 8) and DCS8-42TN (SEQ ID NO: 10) also decreased the HBV DNA copy number and the cccDNA copy number (Fig. 9).
  • the liver has asialoglycoprotein receptors for specifically recognizing and taking up asialoglycoprotein in the blood.
  • Anti-asialoglycoprotein receptor antibodies are also thought to be taken up into liver cells after binding to the receptor.
  • selection experiments for single-chain antibodies that bind to asialoglycoprotein receptors were conducted using the IVV method. did.
  • Example 8 Preparation of biotinylated ASGR First, a biotinylated asialoglycoprotein receptor as an antigen was prepared.
  • Asialoglycoprotein receptor (ASGR) is a type II single-pass transmembrane protein with the N-terminus directed intracellularly and the carbohydrate recognition sites (CRDs) directed extracellularly, as shown in FIG.
  • CTDs carbohydrate recognition sites
  • a construct was prepared by removing the transmembrane domain and adding a biotinylated sequence and the like. Since human hepatocytes have receptors ASGR1 and ASGR2 and form hetero-oligomers, we created biotinylated ASGR1ex and biotinylated ASGR2ex by adding biotin to their extracellular domains.
  • the extracellular domains of ASGR1 and ASGR2 were cloned as shown in FIG. cDNA Library, Human Liver (1 ng/ ⁇ l) (Takara Bio) 1 ⁇ l, KAPA HiFi HS RM 10 ⁇ l, 10 ⁇ M ASGR1-ex-if-F1 0.6 ⁇ l or ASGR2-ex-if-F1 0.6 ⁇ l, and 10 ⁇ M ASGR1-if- RNase-free water was added to 0.6 ⁇ l of R2 or 0.6 ⁇ l of ASGR2-if-R2 to make the total volume 20 ⁇ l, and PCR reaction was performed.
  • PCR was carried out at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 1 minute. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and collected as a 30 ⁇ l DNA solution to obtain ASGR1-if or ASGR2-if.
  • TBS-washed M2 agarose beads (ANTI-FLAG (registered trademark) M2 Affinity Gel, Sigma, A2220) were added to the obtained cell extract and mixed at 4°C for 16 hours. After washing three times with TBST (10x TBS-t 1% Tween-20, Nakarai, 12749-21), competitive elution was performed with 150 ⁇ g/mL 3x FLAG peptide (Sigma, F4799) diluted with TBS.
  • EKMax buffer 500 mM Tris-HCl, pH 8.0, 10 mM CaCl 2 , 1% Tween-20.
  • EKMax TM Enterokinase (Thermo Fisher Scientific) was added, EKMax digestion was performed by rotating at 37°C O/N (16.5h), SA beads were adsorbed on a magnet stand, and the supernatant was recovered.
  • Pretreated EK Away resin was added to the recovered supernatant, and the mixture was rotated at room temperature for 15 minutes to remove EKMax. After centrifugation, the supernatant was recovered (5000 rcf 2 min x 2) and detected by Western blotting using SDS-PAGE and Flag antibody, and an ASGR1ex molecular weight 26394 Da band could be confirmed.
  • Example 9 Construction of single-chain antibody cDNA library (9-1) DNA library design ).
  • V H chain and V L chain are composed of three CDRs (Complementarity-determining regions) and four FRs (Framework regions) (Fig. 15 right).
  • CDRs are composed of a variety of amino acid sequences for each antibody, enabling them to bind to various antigens.
  • Artificial antibody fragments include single-chain antibodies (scFv), in which VH and VL chains are linked by a peptide linker (Fig. 16). A linker ( sequence 48) is widely used.
  • Example 10 Construction of mouse-derived single-chain antibody cDNA library As shown in Fig. 17, a mouse-derived single-chain antibody cDNA library was constructed using mouse spleen Poly A+ RNA as a starting material. Preparation of H chain DNA solution, preparation of L chain DNA solution, and unification PCR of H chain and L chain were carried out. (Nucleic Acids Research, 2009, Vol. 37, No. 8 e64).
  • H-chain DNA solution For preparation of a single-chain antibody cDNA library, first, an H-chain DNA solution was prepared. 11 ⁇ l of mouse spleen Poly A+ RNA (5 ⁇ g/ ⁇ l) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-free water, 22 ⁇ l of 5 ⁇ RT buffer (TOYOBO), (10 mM) Mix 11 ⁇ l of dNTPs (TOYOBO), 27.5 ⁇ l of forward primer MulgG1/2 (1 pmol/ ⁇ l), and 27.5 ⁇ l of forward primer MulgG3 (1 pmol/ ⁇ l), react at 65°C for 9 minutes, then immediately cool to 4°C and leave at 4°C for 2 minutes.
  • TOYOBO 5 ⁇ RT buffer
  • PCR reaction was carried out. PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes.
  • each DNA (19 types) was dissolved in 20 ⁇ l of RNase-free water.
  • 1 ⁇ l of each synthesized DNA solution (19 types) 2 ⁇ l of each corresponding HB primer (10 pmol/ ⁇ l) shown in HB primer, 10 ⁇ l of 10 ⁇ PCR buffer (TOYOBO), 10 ⁇ l of (2 mM) dNTPs (TOYOBO) , VH forward primer HF1:HF2:HF3:HF4 (1:1:1:1) mixture (10pmol/ ⁇ l) 2 ⁇ l, KOD DASH polymerase (TOYOBO) 0.5 ⁇ l, and RNase-free water were added to HF primer.
  • the total volume was 100 ⁇ l, and each was subjected to PCR reaction. PCR was performed at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes.
  • the amplified gene was subjected to 2% agarose gel electrophoresis to confirm bands of 500-900 bp, followed by phenol/chloroform treatment and ethanol precipitation. After centrifugation for about 15 minutes, each DNA (19 types) was dissolved in 10 ⁇ l of RNase-free water. The obtained 19 DNAs were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and respective bands were excised.
  • PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. Bands of 500-900 bp were confirmed for each amplified gene by 2% agarose gel electrophoresis, and treated with phenol/chloroform. Ethanol precipitation was performed on the resulting solution. After centrifugation for about 15 minutes, each DNA (18 types) was dissolved in 20 ⁇ l of RNase-free water.
  • PCR was performed at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes.
  • the amplified gene was subjected to 2% agarose gel electrophoresis to confirm bands of 500-900 bp, followed by phenol/chloroform treatment and ethanol precipitation. After centrifugation for about 15 minutes, each DNA (18 types) was dissolved in 10 ⁇ l of RNase-free water. The obtained 18 DNAs were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and respective bands were excised. Each DNA (18 species) was dissolved in 10 ⁇ l of RNase-free water.
  • PCR was performed at 96°C for 5 minutes, followed by 15 cycles of 96°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes.
  • the resulting DNA was electrophoresed on a 1% low melting point agarose gel (Sigma) and each band was excised.
  • the DNA was dissolved in 10 ⁇ l of RNase-free water to obtain mouse-derived single-chain antibody library DNA.
  • Example 11 Construction of human-derived single-chain antibody cDNA library
  • human bone-marrow-derived Poly A+ RNA was used as a starting material for construction of a single-chain antibody cDNA library from human bone marrow-derived lymphocytes. went to Using multiple specific primers as in the case of mice, H-chain DNA solution preparation, L-chain DNA solution preparation, and H- and L-chain unification PCR were performed to obtain a single human sample for IVV selection experiments.
  • a chain antibody cDNA library was constructed. (J. Mol. Biol. 1991, 222, 581-597, Nature Biotechnology, 2005, 23, 344-348, Antibody Engineering, Springer Lab. Manual (2001) 93-108).
  • mouse H-chain DNA and mouse L-chain DNA or human H-chain DNA and human L-chain DNA prepared as shown in Fig. 17 were each subjected to random mutagenesis by error-prone PCR using Mutazyme II.
  • a cDNA library of mouse mutated single-chain antibodies was prepared by PCR that unifies the H and L chains of mutated mouse H-chain DNA and mutated mouse L-chain DNA.
  • a cDNA library of human mutated single-chain antibodies was prepared by PCR for unifying H and L chains of human mutated H-chain DNA and human mutated L-chain DNA.
  • Example 12 Selection of single-chain antibodies that bind to ASGR A selection experiment for single-chain antibodies that bind to ASGR was performed according to the procedure shown in Fig. 19 .
  • RNA was purified by RNeasy Mini kit (Qiagen). That is, RNase-free water was added to the transcription reaction solution to bring the total volume to 100 ⁇ l, and 350 ⁇ l of RLT buffer (Qiagen), 5 ⁇ l of 2-mercaptoethanol, and 250 ⁇ l of (100%) ethanol were added and applied to an RNeasy mini spin column.
  • RPE buffer Qiagen
  • the biotinylated extracellular domain ASGR2ex was then used to immobilize to flow cells 3-4.
  • Flow was in buffer HBS-P, 20 ⁇ l/min.
  • the fixed amount of bait is shown in Table 7.
  • an extra wash was performed with a buffer solution HBS-P at 10 ⁇ l/min using 50% Isopropanol, 50 mM NaOH and 1M NaCl.
  • Biacore was used to increase the selection pressure in the selection experiment step by step as shown in Table 7.
  • Example 13 Cloning and base sequence determination (13-1) Cloning and base sequence An in-fusion cloning library was inserted from a single-chain antibody library that binds to ASGR. 1 ⁇ l of library subjected to three rounds of selection experiments, 100 ⁇ l of 10 ⁇ KOD plus buffer (TOYOBO), 100 ⁇ l of 2 mM dNTPs (TOYOBO), 40 ⁇ l of 25 mM MgSO 4 , 30 ⁇ l of T7-long-F in (10 pmol/ ⁇ l), RNase-free water was added to 30 ⁇ l of Flag (Histag) in R (10 pmol/ ⁇ l) and 20 ⁇ l of KOD plus polymerase (TOYOBO) to bring the total volume to 1000 ⁇ l, and a PCR reaction was performed.
  • TOYOBO 10 ⁇ KOD plus buffer
  • TOYOBO 2 mM dNTPs
  • 40 ⁇ l of 25 mM MgSO 4 40 ⁇ l of 25 mM M
  • PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 ⁇ l of DNA solution to obtain T7-ASGR3.
  • PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds.
  • the cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 ⁇ l of DNA solution to obtain KIgk-stop 3.3 vector.
  • T7-ASGR3 0.08 ⁇ l, KIgk-stop 3.3 vector 0.14 ⁇ l, 5x infusion HD Enzyme premix 1.0 ⁇ l (Takara) were mixed, RNase-Free water was added to make the total volume 5 ⁇ l, and the mixture was reacted at 50°C for 15 minutes.
  • 2.5 ⁇ l of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain a KIgk-ASGR3 clone. Sequence analysis of the obtained clones was performed by Eurofin DNA sequence contract service, ValueRead Premix.
  • Example 14 Activity evaluation of ASGR-binding single-chain antibody clone (14-1) Protein preparation Clones were inoculated from a master plate onto an LB medium containing 20 ⁇ g/ml of carbenicillin and cultured at 37°C for 16 hours. bottom. Plasmids were purified from cell pellets with the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen).
  • a pull-down assay (Figs. 20, 21) was performed using an ASGR-binding single-chain antibody.
  • ASGR3-10M recognized both antigens, and ASGR3-39D recognized the ASGR1 antigen particularly strongly.
  • Clones ASGR4-70D, ASGR5-24M subjected to 4 and 5 rounds of selection experiments recognized the antigen compared to controls.
  • ASGR4-70D and ASGR5-24M particularly strongly recognized the ASGR1 antigen.
  • Fig. 22 shows a schematic diagram of a method for selectively delivering DOCK11-binding peptides to hepatocytes using an anti-ASGR single-chain antibody as a carrier for intrahepatocyte delivery.
  • DOCK11-binding peptides conjugated with anti-ASGR antibodies are packaged into early endosomes by endocytosis after binding to receptors.
  • a cleavage sequence that is cleaved by an enzyme in the endosome upstream of the DOCK11-binding peptide, it is cleaved and the peptide is dissociated.
  • a membrane permeabilization peptide is added downstream of the DOCK11-binding peptide, thereby releasing the peptide into the cytoplasm. Since the target DOCK11 is a protein localized in cells or nuclear fractions, a nuclear localization signal is added when delivered to the nucleus.
  • the sequence recognized by the endogenous protease Furin was used as the cleavage sequence, the membrane fusion promoting peptide S28 or S39 as the membrane permeation promoting peptide, and PAAKRVKLD (SEQ ID NO: 40) as the nuclear localization signal.
  • the DOCK11-binding peptide DCS8-42TN causes actin morphology changes when the plasmid is introduced into cells by the transfection method and the protein is expressed. It was verified whether or not the fusion of the construct shown in FIG. 23A would also cause a similar morphological change in actin when it was directly extracellularly incorporated into HepG2 cells. At the same time, it was also verified whether the cleaved sequence was cleaved by Furin and functioned. Using the construct of FIG. 23B, it was verified whether the membrane fusion-promoting peptide was functioning. Using the construct in Figure 23C, it was verified whether the nuclear localization signal was functioning.
  • SEQ ID NO:90 shows the amino acid sequence of the cleavage sequence+DCS8-42TN peptide+S28+nuclear localization signal portion of N-10M-D42TN.
  • the 21st to 28th amino acids are the Flag tag
  • the 29th to 35th amino acids are the His tag.
  • a single-chain antibody-peptide fusion was added to HepG2 cells with or without the DCS8-42TN peptide.
  • actin fragmentation occurred in the cells with the peptide applied, whereas actin fragmentation was not observed in the cells without the peptide.
  • GFP signal indicating the localization of the peptide was observed in the cytoplasm, and it was found that the cleavage sequence was cleaved by Furin and functioned, and the fusion-promoting peptide functioned.
  • Example 15 Anti-HBV activity of DOCK11-binding peptide in PXB cells Liver cells, persistently infected with hepatitis B virus (HBV). Therefore, we evaluated the anti-HBV activity of DOCK11-binding peptides using PXB cells. Since the DOCK11-binding peptide was added from the outside of the cell, the anti-ASGR single-chain antibody-peptide fusion N-10M-D42TN was used and subjected to HBV assay in PXB cells.
  • HBV hepatitis B virus
  • Figures 27A and 27B show the anti-HBV activity of DOCK11-binding peptides in PXB cells.
  • N-10M-D42TN markedly decreased the copy numbers of HBV-DNA and cccDNA, and showed high anti-HBV activity in a dose-dependent manner. It was found to exhibit very high anti-HBV activity.
  • results with good reproducibility were obtained as shown in FIG. 27B.
  • the DOCK11-binding peptide DCS8-42TN is the sequence of residues 674-689 of non-receptor tyrosine kinase Ack1 (NM_001387713.1, SEQ ID NOS: 49, 50). Therefore, we examined whether DOCK11 and Ack1 interact in cells by co-immunoprecipitation experiments.
  • the peptide DCS8-42TN was fused with an anti-ASGR single-chain antibody so that it was taken up into cells.
  • the fusion of DCS8-42TN and a single chain antibody is hereinafter referred to as N-10M-D42TN.
  • DOCK11 is a member of the DOCK-D subfamily and is known to be a guanine nucleotide exchange factor (GEF) that converts Cdc42 from an inactive (GDP-bound) to an active (GTP-bound) form.
  • GEF guanine nucleotide exchange factor
  • Ack1 is an active Cdc42-binding protein and is activated by binding specifically to GTP-bound Cdc42 (Prieto-Echague, V., Miller, J., Signal Transduct, 1-9, 2011).
  • Ack1 is known to phosphorylate WASP and promote actin polymerization (Yokoyama, N., Lougheed, J., and Miller, W.T. (2005). Phosphorylation of WASP by the Cdc42-associated Kinase ACK1. Journal of Biological Chemistry 280, 42219-42226.). Transfection of HepG2 cells with siRNAs targeting DOCK11 and Ack1 and staining of actin filaments with fluorescent phalloidin showed fragmentation of actin filaments and increased microprojections in the cytoplasm (FIGS. 30A-C).
  • HBV binds to the receptor NTCP on the cell membrane and then enters the cell by endocytosis together with the epidermal growth factor receptor EGFR (Iwamoto, M. et al., Proc Natl Acad Sci USA, 116 , 8487-8492, 2019).
  • Ack1 activated by Cdc42 interacts with EGFR in response to EGF stimulation and contributes to EGFR endocytosis (Shen, F. et al., Mol. Biol. Cell, 18, 732-742, 2007; Lin, Q. et al., J. Biol. Chem.
  • FIG. 32A shows a schematic of this mechanism. That is, when EGFR is activated by EGF stimulation, it undergoes phosphorylation and ubiquitination. Ack1 binds to and activates DOCK11-activated Cdc42, and binds to and phosphorylate EGFR. As a result, both EGFR-Ack1 complexes are endocytosed and degraded. On the other hand, inhibition of DOCK11 function by N-10M-D42TN inhibits Ack1 activation, preventing Ack1 and phosphorylated EGFR from being endocytosed and degraded.
  • N-10M-D42TN does not affect EGFR phosphorylation and inhibits Ack1 activation, thereby inhibiting Ack1 and EGFR endocytosis.
  • HBV is known to utilize the host's DNA repair mechanism, especially the ATR signaling pathway, when synthesizing cccDNA from rcDNA (Luo, J. et al. mBio, 11, e03423-19, 2020). Since ATR is recruited according to actin accumulation at sites of DNA damage (Wang, Y-H. et al. Nat Commun, 8, 2118-2133, 2017), DOCK11 affects the ATR signaling pathway through actin polymerization. Possibility of contributing to cccDNA synthesis is conceivable. UV irradiation of HepG2 cells increased DOCK11 mRNA (FIG. 34A), suggesting that DOCK11 contributes to the DNA repair mechanism. In addition, knockdown of DOCK11 suppressed the activation of Chk1 during DNA repair (Fig. 34B,C), indicating that DOCK11 is required for the activation of the ATR signaling pathway.
  • N-10M-D42TN suppressed Chk1 phosphorylation in a time-dependent manner (Fig. 34D,E), indicating that inhibiting DOCK11 function inhibits the ATR signaling pathway. It has been suggested. Immunostaining of HepG2 cells with anti-pChk1 antibody showed that knockdown of DOCK11 suppressed the phosphorylation of Chk1 in the nucleus (FIG. 35A). In addition, phosphorylation of Chk1 in the nucleus was similarly suppressed by N-10M-D42TN treatment (Fig. 35B). These results also suggest that DOCK11 is essential for activation of the ATR signaling pathway in the nucleus and that N-10M-D42TN inhibits it.
  • ⁇ H2AX is a phosphorylated histone H2AX and a marker for sites of DNA damage, suggesting that DOCK11 accumulates at sites of DNA damage and activates the ATR signaling pathway.
  • N-10M-D42TN reduced the expression of ⁇ H2AX similarly to DOCK11 knockdown (Fig. 37D), but ⁇ H2AX did not co-localize with DOCK11 (Fig. 37F,G; Wang, Y-H. et al. Nat Commun, 8, 2118, 2017). This suggests that N-10M-D42TN inhibits DOCK11 activation of the ATR signaling pathway at sites of DNA damage.
  • HBV cannot utilize the DNA repair mechanism when synthesizing cccDNA from rcDNA, and it is thought that infection is suppressed.
  • the DOCK11-binding peptide N-10M-D42TN inhibited the binding of DOCK11 and Ack1 and inhibited the GEF activity of DOCK11.
  • actin filaments contribute to endocytosis (Toshima, JY. et al., eLife 5, e10276, 2016)
  • N-10M-D42TN inhibits actin polymerization to promote HBV activation. It may be blocking intrusions.
  • the DOCK11-binding peptide N-10M-D42TN may also inhibit the activation of the ATR signaling pathway by DOCK11 assembled and accumulated at sites of DNA damage, thus inhibiting the repair process from rcDNA to cccDNA. Conceivable.
  • the DOCK11-binding peptide N-10M-D42TN is thought to suppress HBV infection by inhibiting EGFR endocytosis and the repair process from rcDNA to cccDNA.
  • Example 16 Efficacy test using HBV-infected PXB mice PXB mice were infected with HBV, and the following experiment was performed for the purpose of confirming the efficacy of administration of the test substance (N-10M-D42TN).
  • Virus used The virus used was HBV provided by Phoenix Bio Co., Ltd.
  • Virus name Hepatitis B virus Strain name: PBB004 (Genotype C)
  • BSL 2 Viral titer: 1.1E+09 copies/mL (2 tubes of 10 ⁇ L/tube)
  • Storage Store in an ultra-low temperature freezer
  • Preparation method Thaw one bottle of frozen virus solution and adjust to 1.0E+06 copies/mL using physiological saline (Otsuka Pharmaceutical Factory Co., Ltd.).
  • Animal care conditions 16-4.1 Husbandry Conditions (SOP/Environment/504) Room temperature 24 ⁇ 3 °C, humidity 50 ⁇ 20%, ventilation (10 to 25 times/hour), lighting 12 hours (8:00 to 20:00)
  • mice 16-4.6 Grouping (SOP/Study/002) Six weeks after virus inoculation, blood is collected, and based on the amount of HBV DNA in the blood, mice are sorted into groups at eight weeks after virus inoculation (day of first administration of the test substance).
  • Virus Inoculation HBV is inoculated into the mouse tail vein at 1.0E+05 copies/100 ⁇ L/body.
  • Test Substance From the 8th week after virus inoculation (Day 0) to the day before sample collection (Day 27), 300 ⁇ L/body is intraperitoneally administered to mice once every 2 to 3 days (Mon, Wed, Fri).
  • livers are collected and weighed. Livers are partially cryopreserved and the rest formalin-fixed for histopathological examination for HBV DNA and cccDNA. Lungs, spleens and kidneys are also fixed in formalin.
  • HBsAg, HBeAg and HBcrAg Serum HBsAg concentration measurement was performed by SRL Co., Ltd. (Tokyo).
  • Lumipulse registered trademark
  • Presto II Flujirebio Co., Ltd., Tokyo
  • CLIA ChemiLuminescence Enzyme ImmunoAssay
  • the measurement range was 0.005 to 150 IU/mL.
  • the sample to be measured in this test was diluted 30 times, and the measurement range at the same dilution was 0.15 to 4500 IU/mL.
  • Serum HBsAg concentrations were measured by SRL Co., Ltd. (Tokyo).
  • Lumipulse registered trademark
  • Presto II Flujirebio Co., Ltd., Tokyo
  • ChemiLuminescence Enzyme ImmunoAssay (CLEIA)
  • the measurement range was from 0.1 to 1590 COI.
  • the sample to be measured in this test was diluted 30 times, and the measurement range at the same dilution was 3 to 47,700 COI.
  • Serum HBc-rAg concentrations were measured by SRL Co., Ltd. (Tokyo).
  • LUMIPULSE HBcrAg, LUMIPULSE F (Fujirebio Co., Ltd., Tokyo) using ChemiLuminescence Enzyme ImmunoAssay (CLEIA) was used.
  • the lower limit of measurement was 3.0 log U/mL.
  • the dilution ratio of the sample to be measured in this test was 300 times, and the lower limit of measurement at the same dilution ratio was 5.5 log U/mL.
  • h-Alb concentration was measured using a latex agglutination immunoturbidimetric assay (LZ test 'Eiken' U-ALB, Eiken Chemical Co., Ltd., Tokyo) using an automatic analyzer BioMajestyTM (JCA-BM6050, JEOL, Tokyo). measured in
  • ALT ALT was measured by PhoenixBio using serum at autopsy. Plasma ALT activity was measured using 10 ⁇ L of collected plasma.
  • the object to be measured is a diarylimidazole leuco dye (diarylimidazole leuco dye is colored blue by hydrogen peroxide generated by pyruvate oxidase and peroxidase), and DRYCHEM 7000/NX500sV was used for the measurement.
  • HBV DNA in Liver DNA was extracted from RNAlater-immersed liver samples using DNeasy (registered trademark) Blood & Tissue Kits (Qiagen Co., Ltd., Tokyo), and the DNA was dissolved in Nuclease-free water. After DNA concentration was measured with BioPhotometer (registered trademark) 6131 (Eppendorf Co., Ltd.), the final concentration was adjusted to 20 ng/ ⁇ L using Nuclease-free water.
  • a PCR reaction solution was prepared using 5 ⁇ L of dissolved DNA stock solution or diluted DNA and TaqMan (registered trademark) Fast Advanced Master Mix.
  • the CFX96 Touch TM Real-Time PCR Detection System was used for PCR reaction and analysis.
  • the PCR reaction was carried out as follows: 50°C 2 minutes ⁇ 95°C 20 seconds ⁇ (95°C 3 seconds ⁇ 60°C 32 seconds) ⁇ 53 cycles.
  • HBV DNA concentration in liver was calculated by averaging 2 wells.
  • the sequences of the primers and probes used are listed in Table 12 below.
  • the lower limit of detection by the quantitation method is 50 copies/100 ng DNA.
  • the HBV DNA standard used serum obtained from HBV-infected PXB mice. The HBV DNA concentration contained in this serum was quantified by digital PCR.
  • cccDNA When measuring HBV DNA in liver, 5 ⁇ L of purified DNA stock solution or diluted DNA was prepared using TaqMan® Fast Advanced Master Mix. Using the PCR Detection System, the PCR reaction was performed as follows: 50°C for 2 minutes ⁇ 95°C for 20 seconds ⁇ (95°C for 3 seconds ⁇ 60°C for 32 seconds) ⁇ 55 cycles. The HBV cccDNA concentration in the liver was the average of 2 wells. The sequences of the primers (Takara Bio Inc., Shiga) and the probes (Takara Bio Inc.) used are shown in Table 13. The lower limit of detection by the quantification method is 1.0 ⁇ 10 2 . copies/100 ng DNA, and the HBV cccDNA standard utilized a plasmid containing the entire HBV genome sequence.
  • HsAg immunostaining Liver tissue was fixed in 10% neutral buffered formalin solution and then replaced with 70% ethanol. These samples were requested to Nara Pathological Research Institute (Nara) to prepare paraffin-embedded blocks by standard methods, and then sliced. got After deparaffinization of paraffin sections, they were subjected to antigen retrieval by microwave. Primary antibody (HBsAg (anti-HBsAg antibody, Code: OBT0990, Bio-Rad AbD Serotec Limited, Oxford, UK) or HBcAg (anti-HBcAg antibody, Code: PAB14506, Abnova, Taipei City, Taiwa)) was incubated at 4°C.
  • HBsAg anti-HBsAg antibody, Code: OBT0990, Bio-Rad AbD Serotec Limited, Oxford, UK
  • HBcAg anti-HBcAg antibody, Code: PAB14506, Abnova, Taipei City, Taiwa
  • the primary antibody was reacted with a biotin-avidin-peroxidase complex and then developed with DAB. After staining cell nuclei with hematoxylin, these sections were dehydrated, cleared and mounted. After that, a microscopic examination was performed using an optical microscope at Hamley Co., Ltd.
  • N-10M-D42TN did not exacerbate hepatitis compared to control (PBS).
  • PBS control
  • N-10M-D42TN reduced the copy number of HBV DNA compared to the control administration group.
  • N-10M-D42TN decreased the copy number of HBV DNA compared to the control administration group.
  • DOCK11-binding peptide (N-10M-D42TN) reduced HBV-DNA and cccDNA in the liver in animal experiments using HBV-infected human hepatocyte chimeric mice, demonstrating a clear anti-HBV effect. has been proven to have Moreover, in the in vitro and in vivo assay systems, DOCK11-binding peptide (N-10M-D42TN) was administered after sufficient HBV infection. After being secreted outside the cell once, it is highly likely that it prevents the process of re-infection, in which it reenters the cell.
  • the DOCK11-binding peptide exhibited an anti-HBV effect by inhibiting the repair process from rcDNA to cccDNA and inhibiting re-entry of HBV particles into cells, such as by inhibiting EGFR endocytosis. It is suggested that

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Abstract

Disclosed is a new anti-HBV agent targeting host factor DOCK11. This anti-HBV agent contains, as an active ingredient, a substance which binds to DOCK11 and inhibits the function of DOCK11. In one embodiment, the anti-HBV according to the present invention contains, as an active ingredient, a DOCK11-binding peptide constituted by a specific amino acid sequence. The DOCK11-binding peptide may be a carrier molecule (for example, antibody, antibody fragment, or single-chain antibody binding to asialoglycoprotein receptor) for delivery into cells, a cell membrane permeation-promoting molecule, or in a form connected to a nuclear localization signal.

Description

宿主因子ＤＯＣＫ１１をターゲットとした抗Ｂ型肝炎ウイルス剤Anti-hepatitis B virus agent targeting host factor DOCK11
　本発明は、宿主因子であるDOCK11をターゲットとした抗B型肝炎ウイルス剤に関する。また、本発明は、肝細胞内への薬物の送達用担体として優れたアシアロ糖タンパク質受容体に結合する抗体、抗体断片又は一本鎖抗体に関する。 The present invention relates to an anti-hepatitis B virus agent that targets the host factor DOCK11. The present invention also relates to antibodies, antibody fragments, or single-chain antibodies that bind to asialoglycoprotein receptors that are excellent carriers for drug delivery into hepatocytes.
　慢性肝疾患の主な原因であるB型肝炎ウイルス（HBV）の慢性キャリアは、世界で3億5000万人と推定されている。毎年、78万人以上の人々が、肝硬変や肝がんなどのB型肝炎感染の合併症により亡くなっている(非特許文献１)。HBVキャリアは、非感染者に比べて肝臓がんを発症する可能性が非常に高いと言われている。HBVは、不完全二重鎖DNA（relaxed-circular DNA; rcDNA）として核内に存在している。HBVが肝細胞に感染すると、rcDNAは、核内で共有結合で閉じた環状DNA（cccDNA）に変換される(非特許文献２)。HBVがrcDNAからcccDNAを合成する際には宿主のDNA修復機構、特にATRシグナル伝達経路を利用することが知られている(非特許文献３)。HBVは、細胞内では、安定的に不活性化されたcccDNAとして潜伏状態を維持することができる。しかし、化学療法や造血幹細胞移植によって免疫系が障害されると、HBVの再活性化が引き起こされる。さらに、宿主の免疫系とウイルスの間の相互作用は、進行した肝細胞癌（HCC）の発症に影響を与える。ウイルスのレプリカを減らすことに成功した治療は、進行した肝疾患や肝細胞癌の発生を遅らせることができる。しかし、潜伏しているHBVを排除するためには、cccDNAを標的とすることが必要である。 It is estimated that 350 million people worldwide are chronic carriers of hepatitis B virus (HBV), the main cause of chronic liver disease. More than 780,000 people die each year from complications of hepatitis B infection such as cirrhosis and liver cancer (Non-Patent Document 1). HBV carriers are said to be much more likely to develop liver cancer than uninfected individuals. HBV exists in the nucleus as incomplete double-stranded DNA (relaxed-circular DNA; rcDNA). When HBV infects hepatocytes, rcDNA is converted into covalently closed circular DNA (cccDNA) in the nucleus (Non-Patent Document 2). It is known that HBV utilizes the host's DNA repair mechanism, particularly the ATR signaling pathway, when synthesizing cccDNA from rcDNA (Non-Patent Document 3). HBV can remain latent in cells as stably inactivated cccDNA. However, HBV reactivation occurs when the immune system is compromised by chemotherapy or hematopoietic stem cell transplantation. Furthermore, interactions between the host's immune system and the virus influence the development of advanced hepatocellular carcinoma (HCC). Treatments that successfully reduce viral replicas can delay the development of advanced liver disease and hepatocellular carcinoma. However, targeting cccDNA is necessary to eliminate latent HBV.
　HBVに感染した肝細胞からHCC細胞に形質転換された細胞は、細胞内に減少した量のHBVのタンパク質とmRNAを発現している。宿主ゲノムに組み込まれたHBV DNAを含む細胞を除いて、HCCから樹立された細胞株は通常、HBV転写産物を発現しない。HBVの転写産物を発現し続ける細胞株は、HBVの維持に必要な宿主遺伝子の同定に非常に有用である。そこで、橋本らは、HBV感染が非常に少ない割合（3000個に1個程度）で維持されるHC1細胞株を樹立し、シングルセルトランスクリプトーム解析（Nx1-Seq）を行うことで、HBV感染細胞で高発現するLIPG、DOCK11、DENND2A、HECW2の４つの宿主遺伝子を同定した(非特許文献４)。そのうちの一つであるDOCK11(dedicator of cytokinesis 11、NCBI Gene ID: 139818)は、Zizimin2とも呼ばれ、DOCK-Dサブファミリーのメンバーで、分子サイズは大きい（～240 kDa）。細胞はアクチンフィラメントや微小管などの細胞骨格を使ってその形態を変化させたり維持したりしている。細胞骨格は外界からの刺激によって引き起こされる細胞内のシグナル伝達によって厳密にコントロールされており、そのシグナル伝達に重要な役割を担っているのがRhoファミリー低分子量GTP結合タンパク質(Gタンハ゜ク質)て゛ある。RhoファミリーGタンパク質は、GTPが結合することにより活性状態に、GDPが結合することにより不活性状態になることで、細胞内シグナル伝達の分子スイッチとして働いている。また、RhoファミリーGタンパク質はグアニンヌクレオチド交換因子(guanine nucleotide exchange factor、GEF)の働きによって GDP-GTP交換反応が引き起こされ活性化される。RhoファミリーGタンパク質を活性化するGEFは、Dblホモロジードメイン(DHト゛メイン)を共通にもつグループと、DOCKファミリーと呼ばれる独自の活性化領域をもつグループの二つに大きく分類される(非特許文献５)。両グループを含めて哺乳類ではこれでに約80種類のGEFが報告されている。1996年にDOCK 180が最初に報告されて以来、現在では11種類のDOCKファミリータンパク質が哺乳類で確認されており、その構造の類似性から大きく４つのグループ、DOCK-A、DOCK-B、DOCK-C、DOCK-Dに分けられる(非特許文献６)。DOCK11はDOCK-Dファミリーに属する。DOCKファミリータンパク質にはファミリー間でアミノ酸配列か゛よく保存されている二つの領域、Dock homology region 1(DHR1)とDHR2が存在し、このうちDHR2を介してそれぞれ特異的なRhoファミリーGタンパク質を活性化することが知られている(非特許文献６)。DOCK11はG蛋白であるCDC42と会合し、細胞骨格及び物質輸送に係る分子である。 Cells transformed from HBV-infected hepatocytes to HCC cells express reduced amounts of HBV protein and mRNA intracellularly. Except for cells containing HBV DNA integrated into the host genome, cell lines established from HCC usually do not express HBV transcripts. Cell lines that continue to express HBV transcripts are very useful for identifying host genes required for HBV maintenance. Therefore, Hashimoto et al. established an HC1 cell line that is maintained at a very low rate of HBV infection (about 1 in 3000 cells), and performed single-cell transcriptome analysis (Nx1-Seq) to detect HBV infection. Four host genes, LIPG, DOCK11, DENND2A, and HECW2, which are highly expressed in cells, were identified (Non-Patent Document 4). One of them, DOCK11 (dedicator of cytokinesis 11, NCBI Gene ID: 139818), also called Zizimin2, is a member of the DOCK-D subfamily and has a large molecular size (~240 kDa). Cells use cytoskeleton such as actin filaments and microtubules to change and maintain their morphology. The cytoskeleton is strictly controlled by intracellular signal transduction triggered by external stimuli, and Rho family low-molecular-weight GTP-binding proteins (G proteins) play an important role in the signal transduction. Rho family G proteins act as molecular switches for intracellular signal transduction by being activated by GTP binding and inactivated by GDP binding. In addition, Rho family G proteins are activated by the GDP-GTP exchange reaction caused by the action of guanine nucleotide exchange factor (GEF). GEFs that activate Rho family G proteins are roughly classified into two groups: a group having a common Dbl homology domain (DH domain) and a group having a unique activation region called the DOCK family (Non-Patent Document 5. ). About 80 GEFs have been reported so far in mammals, including both groups. Since DOCK 180 was first reported in 1996, 11 types of DOCK family proteins have been confirmed in mammals. It is divided into C and DOCK-D (Non-Patent Document 6). DOCK11 belongs to the DOCK-D family. DOCK family proteins have two regions, Dock homology region 1 (DHR1) and DHR2, whose amino acid sequences are well conserved among families, and each activates a specific Rho family G protein through DHR2. It is known to do (Non-Patent Document 6). DOCK11 associates with CDC42, a G protein, and is a molecule involved in cytoskeleton and transport.
　また、DOCK11は、HBV遺伝子を宿主細胞内で維持するために機能している分子の１つとしても報告されている（特許文献１）。特許文献１には、siRNA、及びレンチshRNAによってDOCK11の発現を抑制することにより、HBVの複製が低下することが開示されている。しかしながら、HBV感染患者に適用し得るDOCK11阻害剤は未だ開発されていない。 DOCK11 has also been reported as one of the molecules that function to maintain the HBV gene in host cells (Patent Document 1). Patent Document 1 discloses that suppression of DOCK11 expression by siRNA and lenti-shRNA reduces HBV replication. However, a DOCK11 inhibitor applicable to HBV-infected patients has not yet been developed.
　医薬分野において、薬物を標的部位に効率よく送達するためのドラッグデリバリーシステム（DDS）が従来より種々検討、開発されており、肝細胞内に送達すべき薬物の場合には、肝実質細胞に豊富に存在するアシアロ糖タンパク質受容体やApoE受容体に着目したDDSが知られている（非特許文献７）。 In the pharmaceutical field, various drug delivery systems (DDS) have been studied and developed to efficiently deliver drugs to target sites. DDS focusing on asialoglycoprotein receptors and ApoE receptors that are present in E. coli is known (Non-Patent Document 7).
WO 2017/082202 A1WO 2017/082202 A1
　本願発明は、DOCK11をターゲットとした新規な抗HBV薬を提供することを目的とする。 The purpose of the present invention is to provide a novel anti-HBV drug that targets DOCK11.
　本願発明者らは、鋭意検討の結果、DOCK11への結合活性が強いペプチドを同定し、それらのペプチドが宿主細胞内のHBV DNA、cccDNAを低減する活性を有することを明らかにした。また、DOCK11結合ペプチドの肝細胞選択的送達法を確立するため、肝細胞表面に存在するアシアロ糖タンパク質受容体（ASGR）に結合する一本鎖抗体を鋭意作製し、ASGRを強く認識できる一本鎖抗体の確立に成功した。さらに、確立した抗ASGR結合一本鎖抗体を利用し、DOCK11結合ペプチドに細胞膜透過を促進するペプチドや核移行シグナル等を付加したコンストラクトを作製し、PXBマウス由来のPXB細胞及びPXBマウス体内で抗HBV活性を発揮できることを確認した。さらに、DOCK11結合ペプチドが、DOCK11とAck1の結合の阻害によるAck1活性化の阻害、DOCK11のグアニンヌクレオチド交換因子（GEF）活性の阻害、DOCK11によるATRシグナル伝達経路の活性化の阻害などの作用を有し、これらの作用により、rcDNAからcccDNAへの修復過程を阻害するとともに、EGFRのエンドサイトーシスの阻害等によるHBV粒子の細胞内への再侵入を阻害し、抗HBV効果を発揮することを見いだした。以上の鋭意研究の結果から、本願発明を完成した。 As a result of extensive studies, the inventors of the present application identified peptides with strong binding activity to DOCK11 and clarified that these peptides have the activity of reducing HBV DNA and cccDNA in host cells. In addition, in order to establish a method for selective delivery of DOCK11-binding peptides to hepatocytes, we diligently produced single-chain antibodies that bind to the asialoglycoprotein receptor (ASGR) present on the surface of hepatocytes, and produced a single-chain antibody that strongly recognizes ASGR. A chain antibody was successfully established. Furthermore, using the established anti-ASGR-binding single-chain antibody, we prepared a construct that added a peptide that promotes cell membrane penetration, a nuclear localization signal, etc. to the DOCK11-binding peptide, and produced an anti-antibody in PXB mouse-derived PXB cells and in the PXB mouse body. It was confirmed that HBV activity can be exhibited. Furthermore, DOCK11-binding peptides have shown effects such as inhibition of DOCK11-Ack1 binding to inhibit Ack1 activation, inhibition of DOCK11 guanine nucleotide exchange factor (GEF) activity, and inhibition of DOCK11-mediated activation of the ATR signaling pathway. However, it was found that these actions inhibit the repair process from rcDNA to cccDNA and inhibit re-invasion of HBV particles into cells due to inhibition of EGFR endocytosis, etc., and exert anti-HBV effects. rice field. As a result of the above earnest research, the present invention was completed.
　すなわち、本発明は、DOCK11に結合し、DOCK11の機能を阻害する物質を有効成分として含む、抗B型肝炎ウイルス剤（抗HBV剤）を提供する。 That is, the present invention provides an anti-hepatitis B virus agent (anti-HBV agent) containing, as an active ingredient, a substance that binds to DOCK11 and inhibits the function of DOCK11.
　１つの態様において、有効成分とする物質は、DOCK11の第1516番～第2073番残基の領域に結合する。 In one embodiment, the substance used as an active ingredient binds to the region of residues 1516 to 2073 of DOCK11.
　１つの態様において、DOCK11の機能の阻害は、DOCK11とAck1の結合の阻害によるAck1活性化の阻害、DOCK11のグアニンヌクレオチド交換因子活性の阻害、及びDOCK11によるATRシグナル伝達経路の活性化の阻害から選択される少なくとも１種である。 In one embodiment, the inhibition of DOCK11 function is selected from inhibition of Ack1 activation by inhibition of binding of DOCK11 to Ack1, inhibition of guanine nucleotide exchange factor activity of DOCK11, and inhibition of activation of the ATR signaling pathway by DOCK11. is at least one
　１つの態様において、有効成分とする物質は、下記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドである。
(1)　IITPGTEVLNSDLQAS（配列番号1）の配列のポリペプチド。
(2)　HNVLSVYNPAWGKYFH（配列番号2）の配列のポリペプチド。
(3)　NFPPNPMHNTDSCICA（配列番号3）の配列のポリペプチド。
(4)　TEKRRLMKPVLLTYNP（配列番号4）の配列のポリペプチド。
(5)　IICPGAEVLNGDLVAS（配列番号5）の配列のポリペプチド。
(6)　TEYRRCVTPVLLTYNN（配列番号6）の配列のポリペプチド。
(7)　TEEHRGLLPVLMTYNV（配列番号7）の配列のポリペプチド。
(8)　TEFCRWTWPVLCTYNA（配列番号8）の配列のポリペプチド。
(9)　TEQARPTPPPVLDTYNL（配列番号9）の配列のポリペプチド。
(10)　PEQARPPPPLEDNLFL（配列番号10）の配列のポリペプチド。
(11)　HEEHRGMLREDSMMEYLK（配列番号11）の配列のポリペプチド。
(12)　AEEHRGLLTIRYPMEH（配列番号12）の配列のポリペプチド。
(13)　PEQARPPPPLEDNLFL（配列番号10）の領域を含むAck1の部分ポリペプチド。
(14)　HEEHRGMLREDSMMEYLK（配列番号11）の領域を含むラディキシンの部分ポリペプチド。
(15)　AEEHRGLLTIRYPMEH（配列番号12）の領域を含むβ-セントラクチンの部分ポリペプチド。
(16)　(1)～(15)のいずれかと８０％以上１００％未満の配列同一性を有するポリペプチド。 In one embodiment, the substance used as an active ingredient is at least one polypeptide selected from the following polypeptides (1) to (16).
(1) A polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1).
(2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
(3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
(4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4).
(5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5).
(6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
(7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
(8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
(9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
(10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
(11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
(12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
(13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
(14) A partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
(15) A partial polypeptide of β-centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
(16) A polypeptide having 80% or more and less than 100% sequence identity with any of (1) to (15).
　１つの態様において、該ポリペプチドは、肝細胞内への送達のための担体分子と連結した形態にある。担体分子は、例えば、アシアロ糖タンパク質受容体に結合する抗体、抗体断片又は一本鎖抗体であり得る。 In one embodiment, the polypeptide is in a form linked to a carrier molecule for delivery into hepatocytes. The carrier molecule can be, for example, an antibody, antibody fragment or single-chain antibody that binds to the asialoglycoprotein receptor.
　１つの態様において、該ポリペプチドは、細胞膜透過促進分子と連結した形態にある。細胞膜透過促進分子は、例えば、配列番号３８又は３９に示すアミノ酸配列のポリペプチドであり得る。 In one embodiment, the polypeptide is in a form linked to a cell membrane permeabilization molecule. A cell membrane permeabilization molecule can be, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO:38 or 39.
　１つの態様において、該ポリペプチドは、核移行シグナルと連結した形態にある。 In one embodiment, the polypeptide is in a form linked to a nuclear localization signal.
　また、本発明は、上記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドの、DOCK11結合ペプチドとしての使用、及び、上記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドからなる、DOCK11結合ペプチドを提供する。 The present invention also provides the use of at least one polypeptide selected from the polypeptides (1) to (16) above as a DOCK11-binding peptide, and from the polypeptides (1) to (16) above. DOCK11-binding peptides are provided that consist of at least one selected polypeptide.
　また、本発明は、
　配列番号１３、１９、２５若しくは３１に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR1と、
　配列番号１４、２０、２６若しくは３２に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR2と、
　配列番号１５、２１、２７若しくは３３に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR3と、
　配列番号１６、２２、２８若しくは３４に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR1と、
　配列番号１７、２３、２９若しくは３５に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR2と、
　配列番号１８、２４、３０若しくは３６に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR3と
を有する、アシアロ糖タンパク質受容体に結合する抗体、抗体断片又は一本鎖抗体を提供する。 In addition, the present invention
A heavy chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
A heavy chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
A heavy chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
a light chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
light chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 18, 24, 30 or 36, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has 80% or more identity with the amino acid sequence , an antibody, antibody fragment or single chain antibody that binds to the asialoglycoprotein receptor.
　さらに、本発明は、上記本発明の抗体、抗体断片又は一本鎖抗体を含む、肝細胞内に薬物を送達するための薬物送達用担体を提供する。 Furthermore, the present invention provides a drug delivery carrier for delivering a drug into hepatocytes, containing the antibody, antibody fragment, or single-chain antibody of the present invention.
　さらに、本発明は、上記本発明の薬物送達用担体と肝細胞内に送達すべき薬物との複合体を含む、医薬組成物を提供する。 Furthermore, the present invention provides a pharmaceutical composition comprising a complex of the drug delivery carrier of the present invention and a drug to be delivered into hepatocytes.
　本発明により、DOCK11に結合し、DOCK11の機能を阻害する作用を有する抗HBV剤が初めて提供される。本発明の抗HBV剤によれば、細胞内のHBV DNA及びcccDNAを低減するとともに、HBV粒子の細胞内への再侵入も阻害できるので、B型肝炎の根治も期待される。また、本発明が提供する特定のCDR配列ないしはその改変配列を有する抗ASGR抗体、抗体断片又は一本鎖抗体は、肝細胞内への薬物の送達用担体として非常に優れており、抗HVB剤のみならず、肝臓内で作用させるべき様々な薬物の送達用担体として有用である。 The present invention provides for the first time an anti-HBV agent that binds to DOCK11 and has the action of inhibiting the function of DOCK11. Since the anti-HBV agent of the present invention can reduce intracellular HBV DNA and cccDNA and inhibit re-invasion of HBV particles into cells, it is expected to cure hepatitis B completely. In addition, the anti-ASGR antibody, antibody fragment or single-chain antibody having a specific CDR sequence or modified sequence thereof provided by the present invention is very excellent as a carrier for delivering drugs into hepatocytes, and is an anti-HVB agent. In addition, it is useful as a delivery carrier for various drugs to act in the liver.
IVVスクリーニングのためのDNAライブラリーのデザイン。DNA library design for IVV screening. ヒトDOCK180 superfamilyの構造。Structure of the human DOCK180 superfamily. ビオチン化全長DOCK11の構築手順の説明図。Explanatory diagram of the construction procedure of biotinylated full-length DOCK11. IVV法によるDOCK11と結合するペプチドの選択実験の説明図。Explanatory drawing of selection experiments for peptides that bind to DOCK11 by the IVV method. DOCK11結合ペプチドによるDHR2領域を含むC末端DOCK11に対するプルダウンアッセイ（１）Pull-down assay for C-terminal DOCK11 containing DHR2 region by DOCK11-binding peptide (1) DOCK11結合ペプチドによるDHR2領域を含むC末端DOCK11に対するプルダウンアッセイ（２）Pull-down assay for C-terminal DOCK11 containing DHR2 region by DOCK11-binding peptide (2) HepG2-NTCP-C4細胞におけるDOCK11結合ペプチドの抗HBV活性（１）Anti-HBV activity of DOCK11-binding peptide in HepG2-NTCP-C4 cells (1) HepG2-NTCP-C4細胞におけるDOCK11結合ペプチドの抗HBV活性（２）Anti-HBV activity of DOCK11-binding peptide in HepG2-NTCP-C4 cells (2) HepG2-NTCP-C4細胞におけるDOCK11結合ペプチドの抗HBV活性（長期投与）。Day 1:10 cm Dishに細胞を播種。Day 2:HBVを感染（12時間後にmedium change）。Day 17:12 well plateにHBV感染細胞を播種。Day 18:Peptideを発現するPlasmidをトランスフェクション（Lipofectamine 3000で1μg）。Day 20:HBV-DNA, cccDNAを測定。Anti-HBV activity of DOCK11-binding peptides in HepG2-NTCP-C4 cells (long-term administration). Day 1: Seed the cells on a 10 cm dish. Day 2: HBV infection (medium change after 12 hours). Day 17:12 Seed HBV-infected cells on a well plate. Day 18: Transfection with plasmid expressing peptide (1 μg with Lipofectamine 3000). Day 20:Measure HBV-DNA, cccDNA. HepG2-NTCP-C4細胞におけるDOCK11結合ペプチドの抗HBV活性（短期投与）。Day 1:12 well plateに細胞を播種。Day 2:Peptideを発現するPlasmidをトランスフェクション（Lipofectamine 3000で1μg）。Day 3:HBVを感染（12時間後にmedium change）。Day 5:HBV-DNAとcccDNAを測定。Anti-HBV activity of DOCK11-binding peptides in HepG2-NTCP-C4 cells (short-term administration). Day 1: Seed cells in 12 well plates. Day 2: Transfection with plasmid expressing peptide (1 μg with Lipofectamine 3000). Day 3: HBV infection (medium change after 12 hours). Day 5: Measure HBV-DNA and cccDNA. HepG2-NTCP-C4細胞におけるDOCK11結合ペプチドの抗HBV活性（長期投与）。A:HBVの感染とサンプル投与のスケジュール。B:（左図）HBV-DNAのコピー数の定量図、（右図）HBV-cccDNAのコピー数の定量図。Anti-HBV activity of DOCK11-binding peptides in HepG2-NTCP-C4 cells (long-term administration). A: Schedule of HBV infection and sample administration. B: (Left) Quantification of HBV-DNA copy number, (Right) Quantification of HBV-cccDNA copy number. ビオチン化アシアロ糖タンパク質受容体のデザイン。Design of the biotinylated asialoglycoprotein receptor. ASGR1とASGR2のそれぞれの細胞外ドメインのクローニング配列。Cloning sequences of the extracellular domains of ASGR1 and ASGR2, respectively. ビオチン化アシアロ糖タンパク質受容体のコンストラクト。Construct of biotinylated asialoglycoprotein receptor. 抗体の構造。(左) IgG抗体の基本構造。S-S結合により結合した2本のH鎖と2本のL鎖から構成され、抗原結合部位である可変領域 (VH、VL)と、それ以外の定常領域 (CH、CL)にわかれている。定常領域のFc領域は、Fcレセプター結合部位である。(右) 可変領域に存在する相補性決定領域 (Complementarity determining region : CDR)。それ以外の領域はフレームワーク領域である。Structure of an antibody. (Left) Basic structure of IgG antibody. It consists of two H chains and two L chains bound by S-S bonds, and is divided into a variable region (VH, VL), which is the antigen-binding site, and constant regions (CH, CL). The Fc region of the constant region is the Fc receptor binding site. (Right) Complementarity determining region (CDR) in the variable region. Other areas are framework areas. 一本鎖抗体 (scFv)の構造。VH鎖のC末端とVL鎖のN末端がペプチドリンカーを介して結合している。Structure of a single-chain antibody (scFv). The C-terminus of the VH chain and the N-terminus of the VL chain are linked via a peptide linker. マウス一本鎖抗体のcDNAライブラリーの作成のスキーム。A scheme for creating a mouse single-chain antibody cDNA library. ヒト一本鎖抗体のcDNAライブラリーの作成のスキーム。Scheme for construction of human single-chain antibody cDNA library. IVV法によるASGRと結合する一本鎖抗体の選択実験の説明図。Explanatory drawing of the selection experiment of the single-chain antibody which couple|bonds with ASGR by IVV method. ASGR結合一本鎖抗体によるプルダウンアッセイ（１）Pull-down assay with ASGR-binding single-chain antibody (1) ASGR結合一本鎖抗体によるプルダウンアッセイ（２）Pull-down assay with ASGR-binding single-chain antibody (2) DOCK11結合ペプチドとアシアロ糖タンパク質受容体との融合体の細胞内での機構。Intracellular mechanism of fusion between DOCK11-binding peptide and asialoglycoprotein receptor. DOCK11結合ペプチドとアシアロ糖タンパク質受容体との融合体のデザイン。A:DOCK11結合ペプチドの有無による機能の検証に使用した融合体。B:膜融合促進ペプチドの機能の検証に使用した融合体。C:核移行シグナルの機能の検証に使用した融合体。D:PXB細胞を使って抗HBV効果を調べる、あるいはキメラマウスを用いたin vivoでの評価に使用した融合体。Design of fusions between DOCK11-binding peptides and asialoglycoprotein receptors. A: Fusions used to validate function with and without DOCK11-binding peptide. B: Fusion used to verify the function of the membrane fusion-promoting peptide. C: Fusion used to verify the function of the nuclear localization signal. D: Fusions used to examine anti-HBV effects using PXB cells or to evaluate in vivo using chimeric mice. DOCK11結合ペプチドの有無による融合体の機能の検証。A:実験の手順。B:初期エンドソーム、ペプチド（GGP）、アクチン、核について共焦点顕微鏡で観察した。Validation of fusion function with and without DOCK11 binding peptide. A: Experimental procedure. B: Early endosomes, peptides (GGP), actin, and nuclei were observed with a confocal microscope. 膜融合促進ペプチドの機能の検証。A:実験の手順。B:初期エンドソーム、ペプチド（GGP）、アクチン、核について共焦点顕微鏡で観察した。Validation of the function of membrane fusion-promoting peptides. A: Experimental procedure. B: Early endosomes, peptides (GGP), actin, and nuclei were observed with a confocal microscope. 膜融合促進ペプチドS28とS39の機能検証:S28とS39によるペプチドの核内取り込み量の比較Functional verification of membrane fusion-promoting peptides S28 and S39: Comparison of nuclear uptake of peptides by S28 and S39 核移行シグナルの機能の検証。A:実験の手順。B:ペプチド（GGP）、アクチン、核について共焦点顕微鏡で観察した。Validation of the function of nuclear localization signals. A: Experimental procedure. B: Peptides (GGP), actin, and nuclei were observed with a confocal microscope. PXB細胞におけるDOCK11結合ペプチドの抗HBV活性(1)。（上):アッセイのタイムスケジュール。（下左）:DOCK11結合ペプチドのHBV-DNAのcopy数に対する影響。（下右）:DOCK11結合ペプチドのcccDNAのcopy数に対する影響。Anti-HBV activity of DOCK11-binding peptides in PXB cells (1). (Top): Assay time schedule. (Lower left): Effect of DOCK11-binding peptide on HBV-DNA copy number. (Lower right): Effect of DOCK11-binding peptide on cccDNA copy number. PXB細胞におけるDOCK11結合ペプチドの抗HBV活性(2)。（上)：アッセイのタイムスケジュール。（下左）:DOCK11結合ペプチドのHBV-DNAのcopy数に対する影響。（下右）:DOCK11結合ペプチドのcccDNAのcopy数に対する影響。Anti-HBV activity of DOCK11-binding peptides in PXB cells (2). (Top): Assay time schedule. (Lower left): Effect of DOCK11-binding peptide on HBV-DNA copy number. (Lower right): Effect of DOCK11-binding peptide on cccDNA copy number. （A）HEK293T細胞にHisBioFLAG-DOCK11あるいはHisBioFLAGとともにT7-Ack1-Hisを発現して24時間後、100 nM N-10M-D42TNで24時間処理し、IP Lysis bufferで可溶化した。この細胞可溶化液を用いて共免疫沈降実験を行い、検出には抗FLAGタグ抗体あるいは抗Ack1抗体を用いた。ImageLabを用いて、各条件において共免疫沈降されたAck1の量を比較した。（B）HEK293T細胞で発現させたHisBioFLAG-DOCK11およびT7-Ack1-HisをHisタグ精製し、HisBioFLAG-DOCK11をストレプトアビジンビーズに結合させた後、T7-Ack1-Hisを加え、0-100nM GST-Cdc42 WTあるいは100 nM DCS8-42TN存在下でプルダウンアッセイを行った。このビーズから、抗FLAGタグ抗体および抗T7タグ抗体を用いてWestern Blottingを行った。（C）HEK293T細胞で発現させたHisBioFLAG-DOCK11およびT7-Ack1-HisをHisタグ精製し、HisBioFLAG-DOCK11をストレプトアビジンビーズに結合させた後、T7-Ack1-Hisを加え、100nM GST-Cdc42 Q61Lあるいは100nM GST-Cdc42 T17N, 100 nM DCS8-42TN存在下でプルダウンアッセイを行った。このビーズから、抗FLAGタグ抗体および抗T7タグ抗体を用いてWestern Blottingを行った。(A) T7-Ack1-His was expressed in HEK293T cells together with HisBioFLAG-DOCK11 or HisBioFLAG 24 hours later, treated with 100 nM N-10M-D42TN for 24 hours, and solubilized with IP Lysis buffer. Co-immunoprecipitation experiments were performed using this cell lysate, and anti-FLAG tag antibody or anti-Ack1 antibody was used for detection. ImageLab was used to compare the amount of co-immunoprecipitated Ack1 in each condition. (B) HisBioFLAG-DOCK11 and T7-Ack1-His expressed in HEK293T cells were His-tag purified, HisBioFLAG-DOCK11 was bound to streptavidin beads, T7-Ack1-His was added, and 0-100 nM GST- A pull-down assay was performed in the presence of Cdc42 WT or 100 nM DCS8-42TN. Western blotting was performed from these beads using an anti-FLAG tag antibody and an anti-T7 tag antibody. (C) HisBioFLAG-DOCK11 and T7-Ack1-His expressed in HEK293T cells were His-tag purified, HisBioFLAG-DOCK11 was bound to streptavidin beads, T7-Ack1-His was added, and 100 nM GST-Cdc42 Q61L was added. Alternatively, a pull-down assay was performed in the presence of 100 nM GST-Cdc42 T17N and 100 nM DCS8-42TN. Western blotting was performed from these beads using an anti-FLAG tag antibody and an anti-T7 tag antibody. ストレプトアビジンコートされた96wellプレートに、DHR2ドメインを含むDOCK1516-2073-Bio-Hisを加えて4℃で一晩固定し、0-1.0 molのDCS8-42TNを室温で1時間反応後、GEFアッセイを行った。DOCK1516-2073-Bio-His containing the DHR2 domain was added to a streptavidin-coated 96-well plate, fixed overnight at 4°C, reacted with 0-1.0 mol of DCS8-42TN at room temperature for 1 hour, and then subjected to GEF assay. gone. （A）HepG2細胞にLipofectamine 3000を用いてDOCK11 siRNA #1～3をトランスフェクションし、72時間後にリアルタイムRT-PCRを行った。GAPDH mRNA量を用いてDOCK11 mRNA量を標準化した。（B）HepG2細胞にLipofectamine 3000を用いてDOCK11あるいはAck1 siRNA #1～3をトランスフェクションした。72時間後に細胞可溶化液を調製し、抗Ack1抗体を用いてWestern Blottingを行った。(C) HepG2細胞にLipofectamine 3000を用いてDOCK11あるいはAck1 siRNA #1～3をトランスフェクションした。72時間後に細胞を固定・透過し、蛍光ファロイジンでアクチンフィラメントを染色後、DAPIを用いて核染色した。(A) HepG2 cells were transfected with DOCK11 siRNA #1-3 using Lipofectamine 3000, and real-time RT-PCR was performed 72 hours later. GAPDH mRNA levels were used to normalize DOCK11 mRNA levels. (B) HepG2 cells were transfected with DOCK11 or Ack1 siRNA #1-3 using Lipofectamine 3000. After 72 hours, a cell lysate was prepared and Western blotting was performed using an anti-Ack1 antibody. (C) HepG2 cells were transfected with DOCK11 or Ack1 siRNA #1-3 using Lipofectamine 3000. After 72 hours, the cells were fixed and permeabilized, and the actin filaments were stained with fluorescent phalloidin, followed by nuclear staining with DAPI. （A）HepG2細胞を100 nM N-10M-D42TNで0-48時間処理後、洗浄し、さらに24-72時間培養した。その後、細胞を固定・透過し、蛍光ファロイジンでアクチンフィラメントを染色後、DAPIを用いて核染色した。（B）蛍光顕微鏡LSM800を用いて細胞質におけるアクチンフィラメントの長さを測定し、N-10M-D42TNで0時間処理した細胞における平均長をコントロールとして標準化した。（C）HepG2細胞にEYFP-NLS-Actinをトランスフェクションし、48時間後100 nMのN-10M-D42TNで20時間処理した。細胞を固定・透過し、DAPIで核染色後、EYFPおよびGFPの蛍光を観察した。(A) HepG2 cells were treated with 100 nM N-10M-D42TN for 0-48 hours, washed, and cultured for 24-72 hours. Then, the cells were fixed and permeabilized, and the actin filaments were stained with fluorescent phalloidin, followed by nuclear staining with DAPI. (B) Actin filament length in the cytoplasm was measured using a fluorescence microscope LSM800 and normalized to the average length in cells treated for 0 h with N-10M-D42TN as a control. (C) HepG2 cells were transfected with EYFP-NLS-Actin and treated 48 hours later with 100 nM N-10M-D42TN for 20 hours. After fixing and permeabilizing the cells and staining the nuclei with DAPI, the fluorescence of EYFP and GFP was observed. （A）EGFRのエンドサイトーシスに関するN-10M-D42TN作用機序の模式図。EGF刺激によって活性化されると、EGFRはリン酸化およびユビキチン化を受ける。Ack1はDOCK11によって活性化されたCdc42と結合して活性化し、EGFRと結合してリン酸化される。その結果、EGFR-Ack1複合体はともにエンドサイトーシスされて分解されるが、N-10M-D42TNがDOCK11の機能を阻害することで、リン酸化されたEGFRは分解されず、Ack1のリン酸化およびAck1によってリン酸化されるWASPのリン酸化も阻害される。（B）Huh7細胞を100 nM N-10M-D42TNで24時間処理後、10 ng/ml EGFで0-10分間処理し、この細胞可溶化液を用いてWestern Blottingを行った。１次抗体としてAck1, pAck1, EGFR, pEGFR(Tyr845), pEGFR(Tyr1068), pEGFR(Tyr1045), WASP, pWASP, GAPDH抗体を用いた。(A) Schematic representation of N-10M-D42TN mechanism of action for EGFR endocytosis. EGFR undergoes phosphorylation and ubiquitination when activated by EGF stimulation. Ack1 binds to and activates DOCK11-activated Cdc42, and binds to and phosphorylate EGFR. As a result, both EGFR-Ack1 complexes are endocytosed and degraded, but by inhibiting DOCK11 function by N-10M-D42TN, phosphorylated EGFR is not degraded and Ack1 phosphorylation and Phosphorylation of WASP, which is phosphorylated by Ack1, is also inhibited. (B) Huh7 cells were treated with 100 nM N-10M-D42TN for 24 hours and then treated with 10 ng/ml EGF for 0-10 minutes, and Western blotting was performed using the cell lysate. Ack1, pAck1, EGFR, pEGFR(Tyr845), pEGFR(Tyr1068), pEGFR(Tyr1045), WASP, pWASP, and GAPDH antibodies were used as primary antibodies. (A）HepG2細胞を100 nM N-10M-D42TNで24時間処理後、100 ng/ml EGFで1時間処理した。CellLight Early Endosomes-RFPを用いて初期エンドソームを染色し、抗Ack1抗体を用いて免疫染色を行い、DAPIで核染色した。白矢印でAck1が局在する初期エンドソームを、黒矢印でAck1が局在しない初期エンドソームを示す。（B）図Aで示した細胞における初期エンドソームをあらわす全ピクセルと、そのうちAck1と共局在しているピクセルの比（Colocalization coefficients）を示す。(A) HepG2 cells were treated with 100 nM N-10M-D42TN for 24 hours and then treated with 100 ng/ml EGF for 1 hour. Early endosomes were stained with CellLight Early Endosomes-RFP, immunostained with anti-Ack1 antibody, and nuclear stained with DAPI. White arrows indicate early endosomes where Ack1 is localized, and black arrows indicate early endosomes where Ack1 is not localized. (B) shows the ratio of all pixels representing early endosomes to those colocalizing with Ack1 in the cells shown in Panel A (Colocalization coefficients). （A）HepG2細胞にLipofectamine 3000を用いてDOCK11 siRNA #1～3をトランスフェクションし、72時間後にUV照射によってDNA損傷を誘導した。この細胞からRNAを抽出し、リアルタイムRT-PCRによってDOCK11 mRNA量を測定した。GAPDH mRNAも同様に測定し標準化した。（B）HepG2細胞にLipofectamine 3000を用いてDOCK11 siRNA #1～3をトランスフェクションし、72時間後にUV照射によってDNA損傷を誘導した。この細胞可溶化液を調製し、１次抗体として抗Chk1, pChk1抗体を用いてWestern Blottingを行った。（C）図BにおいてImageLabを用いてpChk1とChk1の発現量比を算出し、各細胞におけるChk1のリン酸化量を算出した。コントロールとして、siEmptyをトランスフェクション後UV照射しなかった細胞でのリン酸化量を用いた。（D）HepG2細胞を100 nM N-10M-D42TNで24時間処理後、UV照射によってDNA損傷を誘導した。この細胞可溶化液を調製し、１次抗体として抗Chk1, pChk1抗体を用いてWestern Blottingを行った。（E）図DにおいてImageLabを用いてpChk1とChk1の発現量比を算出し、各細胞におけるChk1のリン酸化量を算出した。コントロールとして、N-10M-D42TN処理およびUV照射処理しなかった細胞でのリン酸化量を用いた。(A) HepG2 cells were transfected with DOCK11 siRNA #1-3 using Lipofectamine 3000, and DNA damage was induced by UV irradiation 72 hours later. RNA was extracted from these cells and the amount of DOCK11 mRNA was measured by real-time RT-PCR. GAPDH mRNA was similarly measured and normalized. (B) HepG2 cells were transfected with DOCK11 siRNA #1-3 using Lipofectamine 3000, and DNA damage was induced by UV irradiation 72 hours later. This cell lysate was prepared and Western blotting was performed using anti-Chk1 and pChk1 antibodies as primary antibodies. (C) In Fig. B, ImageLab was used to calculate the expression level ratio of pChk1 and Chk1, and the phosphorylation level of Chk1 in each cell was calculated. As a control, the amount of phosphorylation in cells that were not irradiated with UV after transfection with siEmpty was used. (D) HepG2 cells were treated with 100 nM N-10M-D42TN for 24 hours, and DNA damage was induced by UV irradiation. This cell lysate was prepared and Western blotting was performed using anti-Chk1 and pChk1 antibodies as primary antibodies. (E) In Fig. D, ImageLab was used to calculate the expression level ratio of pChk1 and Chk1, and the phosphorylation level of Chk1 in each cell was calculated. As a control, the amount of phosphorylation in cells not treated with N-10M-D42TN and not treated with UV irradiation was used. UV照射によってPXB細胞にDNA損傷を誘導後、抗pChk1抗体を用いて免疫染色を行い、DAPIで核染色した。（A）UV照射前にEmptyあるいはDOCK11を標的としたsiRNAをトランスフェクションし、72時間培養した。（B）UV照射前に0 nMあるいは100 nM N-10M-D42TNで24時間処理した。After DNA damage was induced in PXB cells by UV irradiation, immunostaining was performed using an anti-pChk1 antibody, followed by nuclear staining with DAPI. (A) Empty or DOCK11-targeted siRNA was transfected before UV irradiation and cultured for 72 hours. (B) Treated with 0 nM or 100 nM N-10M-D42TN for 24 hours before UV irradiation. （A）PXB細胞を100 nM N-10M-D42TNで24時間処理後、UV照射によってDNA損傷を誘導した。抗DOCK11抗体を用いて免疫染色を行い、DAPIで核染色した。（B）核内部において閾値以上を示したDOCK11の蛍光強度を算出し、DAPIの蛍光強度を用いて標準化した。(A) PXB cells were treated with 100 nM N-10M-D42TN for 24 hours and then UV-irradiated to induce DNA damage. Immunostaining was performed using an anti-DOCK11 antibody and nuclear staining was performed with DAPI. (B) The fluorescence intensity of DOCK11 that showed a threshold value or higher inside the nucleus was calculated and normalized using the fluorescence intensity of DAPI. （A）DOCK11を標的としたsiRNAをトランスフェクションしたPXB細胞および100 nM N-10M-D42TNで24時間処理したPXB細胞に対して、UV照射によりDNA損傷を誘導し、抗DOCK11抗体および抗γH2AX抗体を用いて免疫染色した。白矢印はγH2AXと共局在しているDOCK11、黒矢印はγH2AXと共局在しないDOCK11を示す。（B, E-G）図37Aの矢印で示した点を含む5.0μmの直線上における、DOCK11およびγH2AXの蛍光強度の変化を示す。（B）Emptyを標的としたsiRNAをトランスフェクションし、UV照射した細胞。（E）DOCK11を標的としたsiRNAをトランスフェクションし、UV照射した細胞。（F）100 nM N-10M-D42TNで0時間処理し、UV照射した細胞。（G）100 nM N-10M-D42TNで24時間処理し、UV照射した細胞。（C, D）図37Aの各細胞におけるDOCK11(C)およびγH2AX（D）の蛍光強度を示す。いずれもDAPIの蛍光強度との比を示し、siEmptyをトランスフェクションしUVしなかった細胞での結果をコントロールとした。*p<0.05, **p<0.005.(A) DNA damage was induced by UV irradiation in PXB cells transfected with siRNA targeting DOCK11 and treated with 100 nM N-10M-D42TN for 24 h. was immunostained using White arrows indicate DOCK11 co-localized with γH2AX, black arrows indicate DOCK11 not co-localized with γH2AX. (B, E-G) show changes in fluorescence intensity of DOCK11 and γH2AX on a 5.0 μm straight line containing the points indicated by arrows in FIG. 37A. (B) Cells transfected with siRNA targeting Empty and UV-irradiated. (E) Cells transfected with siRNA targeting DOCK11 and irradiated with UV. (F) Cells treated with 100 nM N-10M-D42TN for 0 h and UV irradiated. (G) Cells treated with 100 nM N-10M-D42TN for 24 hours and UV irradiated. (C, D) Fluorescence intensity of DOCK11 (C) and γH2AX (D) in each cell in FIG. 37A. Both show the ratio of DAPI fluorescence intensity, and the results of cells transfected with siEmpty without UV exposure were used as a control. *p<0.05, **p<0.005. 実施例１６におけるサンプルの投与スケジュール。Dosing schedule for the samples in Example 16. 実施例１６におけるHBsAgの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of HBsAg in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６におけるHBeAgの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of HBeAg in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６におけるHBcrAgの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of HBcrAg in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６における血中HBV DNAの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of blood HBV DNA in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６におけるh-Albの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of h-Alb in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６におけるALTの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of ALT in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６における肝臓中HBV DNAの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of HBV DNA in liver in Example 16. Comparison of N-10M-D42TN and control (PBS). 実施例１６における肝臓中HBV DNAの測定。N-10M-D42TNとcontrol (PBS)の比較。Measurement of HBV DNA in liver in Example 16. Comparison of N-10M-D42TN and control (PBS).
　本発明において、「抗B型肝炎ウイルス（抗HBV）」という語には、HBV感染の治療、HBV感染の予防、HBVの増殖抑制、B型肝炎の治療、及びB型肝炎の予防が包含される。本発明の抗HBV剤は、B型肝炎患者に投与することで、該患者体内（肝臓内）でのHBVの増殖を抑制し、B型肝炎を治療することができる。また、抗HBV剤をB型肝炎発症前のHBVキャリアに投与することで、該キャリアにおけるHBVの増殖を防止し、B型肝炎の発症を防止することができる（B型肝炎の予防）。 In the present invention, the term "anti-hepatitis B virus (anti-HBV)" includes treatment of HBV infection, prevention of HBV infection, suppression of HBV proliferation, treatment of hepatitis B, and prevention of hepatitis B. be. By administering the anti-HBV agent of the present invention to a hepatitis B patient, hepatitis B can be treated by suppressing the proliferation of HBV in the patient's body (inside the liver). In addition, by administering an anti-HBV agent to an HBV carrier before the onset of hepatitis B, the proliferation of HBV in the carrier can be prevented and the onset of hepatitis B can be prevented (prevention of hepatitis B).
　本発明の抗HBV剤は、DOCK11に結合し、DOCK11の機能を阻害する物質を有効成分として含む。ヒトDOCK11は図２に示した構造を有するタンパク質であり、DOCKファミリー間でアミノ酸配列がよく保存されているドメインであるDock homology region 1(DHR1)及びDHR2を有する。N末側に存在するDHR1は、ホスファチジルイノシトール3-リン酸と結合する。C末側に存在するDHR2は、それぞれ特異的なRhoファミリーGタンパク質を活性化するドメインであり、DOCK11のDHR2は低分子量Gタンパク質Cdc42を活性化する。配列表の配列番号４１、４２に示した配列は、NCBIのGenBankにNM_144658.4で登録されているヒトDOCK11 mRNAのコード領域の塩基配列及びこれにコードされるDOCK11のアミノ酸配列であり、第1609番～第2036番残基の領域がDHR2ドメインである。本発明の抗HBV剤の有効成分として用いる物質は、DOCK11の第1516番～第2073番残基の領域（C末領域）、例えばDHR2ドメインに結合する物質であってよい。 The anti-HBV agent of the present invention contains as an active ingredient a substance that binds to DOCK11 and inhibits the function of DOCK11. Human DOCK11 is a protein having the structure shown in FIG. 2, and has Dock homology region 1 (DHR1) and DHR2, which are domains whose amino acid sequences are well conserved among DOCK families. DHR1 located at the N-terminus binds to phosphatidylinositol 3-phosphate. DHR2 present on the C-terminal side is a domain that activates a specific Rho family G protein, and DHR2 of DOCK11 activates a small G protein Cdc42. The sequences shown in SEQ ID NOS: 41 and 42 in the Sequence Listing are the nucleotide sequence of the coding region of human DOCK11 mRNA and the amino acid sequence of DOCK11 encoded by this registered in GenBank of NCBI under NM_144658.4. The region from 1st to 2036th residues is the DHR2 domain. The substance used as the active ingredient of the anti-HBV agent of the present invention may be a substance that binds to the region of residues 1516 to 2073 (C-terminal region) of DOCK11, eg, the DHR2 domain.
　DOCK11については、グアニンヌクレオチド交換因子（guanine nucleotide exchange factor, GEF）としてCdc42を非活性型（GDP結合型）から活性型（GTP結合型）へと置換すること、他のGEFタンパク質と異なり活性型Cdc42にも結合してさらに活性化し、ポジティブフィードバックをもたらすことが知られている（Lin, Q. et al. J. Biol. Chem., 281, 35253-35262, 2006; Nishikimi, A. et al. Exp Cell Res 319, 2343-2349, 2013）。また、活性型Cdc42結合タンパク質であるAck1（activated CDC42 kinase 1）が、GTP結合型Cdc42と特異的に結合して活性化されることが知られている（Prieto-Echague, V., Miller, J., Signal Transduct, 1-9, 2011）。下記実施例において、DOCK11とAck1が細胞内で結合すること、Cdc42とAck1は競合的にDOCK11に結合することが、本願発明者らによって示された。 For DOCK11, as a guanine nucleotide exchange factor (GEF), Cdc42 is replaced from inactive (GDP-bound) to active (GTP-bound), and unlike other GEF proteins, active Cdc42 is known to bind to and further activate and induce positive feedback (Lin, Q. et al. J. Biol. Chem., 281, 35253-35262, 2006; Nishikimi, A. et al. Exp Cell Res 319, 2343-2349, 2013). In addition, Ack1 (activated CDC42 kinase 1), an activated Cdc42-binding protein, is known to be activated by binding specifically to GTP-bound Cdc42 (Prieto-Echague, V., Miller, J. ., Signal Transduct, 1-9, 2011). In the following examples, the present inventors demonstrated that DOCK11 and Ack1 bind intracellularly, and that Cdc42 and Ack1 competitively bind to DOCK11.
　DOCK11の機能の阻害は、例えば、DOCK11とAck1の結合の阻害によるAck1活性化の阻害、DOCK11のGEF活性の阻害、及びDOCK11によるATRシグナル伝達経路の活性化の阻害から選択される少なくとも１種であってよい。下記実施例には、抗HBV活性を有するDOCK11結合ペプチドが、DOCK11とAck1の結合を阻害してAck1活性化を阻害すること、DOCK11のGEF活性を阻害すること、DOCK11によるATRシグナル伝達経路の活性化を阻害すること、これらの作用により、宿主のDNA修復機構（特にATR（ataxia telangiectasia and Rad-3 related）シグナル伝達経路）を利用したrcDNAからcccDNAの合成過程を阻害するとともに、EGFRのエンドサイトーシスの阻害等によるHBV粒子の細胞内への再侵入を阻害し、抗HBV効果を発揮することが示されている。 Inhibition of DOCK11 function is, for example, at least one selected from inhibition of Ack1 activation by inhibition of binding of DOCK11 and Ack1, inhibition of GEF activity of DOCK11, and inhibition of activation of ATR signaling pathway by DOCK11. It's okay. The following examples demonstrate that a DOCK11-binding peptide with anti-HBV activity inhibits the binding of DOCK11 and Ack1 to inhibit Ack1 activation, inhibits the GEF activity of DOCK11, and activates the ATR signaling pathway by DOCK11. These actions inhibit the process of synthesizing cccDNA from rcDNA using the host's DNA repair mechanism (especially the ATR (ataxia telangiectasia and Rad-3 related) signaling pathway), as well as inhibiting the EGFR end-cycle. It has been shown to exert an anti-HBV effect by inhibiting the re-entry of HBV particles into cells by inhibiting thosis.
　DOCK11の第1516番～第2073番残基の領域（C末領域）、又はDHR2ドメインに結合し、DOCK11の機能を阻害する物質としては、例えば、該C末領域又はDHR2ドメインを認識して結合する抗体、抗体断片若しくは一本鎖抗体又はアプタマー、該C末領域又はDHR2ドメインに選択的に結合するポリペプチド等を挙げることができる。本発明において、抗体断片という語は、抗体の抗原結合性断片という語と同義であり、Fab、Fab'、F(ab')2等が包含される。抗原との結合性を維持した抗体断片であれば特に限定されない。 Substances that bind to the region of residues 1516 to 2073 (C-terminal region) of DOCK11 or the DHR2 domain and inhibit the function of DOCK11 include, for example, recognizing and binding to the C-terminal region or DHR2 domain. antibodies, antibody fragments, single-chain antibodies, aptamers, polypeptides that selectively bind to the C-terminal region or DHR2 domain, and the like. In the present invention, the term "antibody fragment" is synonymous with the term "antigen-binding fragment of antibody" and includes Fab, Fab', F(ab') 2 and the like. There is no particular limitation as long as it is an antibody fragment that maintains antigen-binding properties.
　本発明の抗HBV剤は、例えば、下記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドを有効成分として含むものであってよい。[]内は、下記実施例において使用した各ポリペプチドの名称である。以下、これらのポリペプチドを「DOCK11結合ペプチド」ということがある。
(1)　IITPGTEVLNSDLQAS（配列番号1）の配列のポリペプチド。[DCS3-1]
(2)　HNVLSVYNPAWGKYFH（配列番号2）の配列のポリペプチド。[DCS5-4]
(3)　NFPPNPMHNTDSCICA（配列番号3）の配列のポリペプチド。[DCS5-5]
(4)　TEKRRLMKPVLLTYNP（配列番号4）の配列のポリペプチド。[DCS5-15]
(5)　IICPGAEVLNGDLVAS（配列番号5）の配列のポリペプチド。[DCS8-6]
(6)　TEYRRCVTPVLLTYNN（配列番号6）の配列のポリペプチド。[DCS8-29]
(7)　TEEHRGLLPVLMTYNV（配列番号7）の配列のポリペプチド。[DCS8-59]
(8)　TEFCRWTWPVLCTYNA（配列番号8）の配列のポリペプチド。[DCS8-72]
(9)　TEQARPTPPPVLDTYNL（配列番号9）の配列のポリペプチド。[DCS8-42]
(10)　PEQARPPPPLEDNLFL（配列番号10）の配列のポリペプチド。[DCS8-42TN]
(11)　HEEHRGMLREDSMMEYLK（配列番号11）の配列のポリペプチド。[DCS8-59R]
(12)　AEEHRGLLTIRYPMEH（配列番号12）の配列のポリペプチド。[DCS8-59C]
(13)　PEQARPPPPLEDNLFL（配列番号10）の領域を含むAck1の部分ポリペプチド。
(14)　HEEHRGMLREDSMMEYLK（配列番号11）の領域を含むラディキシンの部分ポリペプチド。
(15)　AEEHRGLLTIRYPMEH（配列番号12）の領域を含むβ-セントラクチンの部分ポリペプチド。
(16)　(1)～(15)のいずれかと８０％以上１００％未満の配列同一性を有するポリペプチド。 The anti-HBV agent of the present invention may contain, as an active ingredient, at least one polypeptide selected from the following polypeptides (1) to (16), for example. [] is the name of each polypeptide used in the following examples. Hereinafter, these polypeptides may be referred to as "DOCK11-binding peptides".
(1) A polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1). [DCS3-1]
(2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2). [DCS5-4]
(3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3). [DCS5-5]
(4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4). [DCS5-15]
(5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5). [DCS8-6]
(6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6). [DCS8-29]
(7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7). [DCS8-59]
(8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8). [DCS8-72]
(9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9). [DCS8-42]
(10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10). [DCS8-42TN]
(11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11). [DCS8-59R]
(12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12). [DCS8-59C]
(13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
(14) A partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
(15) A partial polypeptide of β-centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
(16) A polypeptide having 80% or more and less than 100% sequence identity with any of (1) to (15).
　(1)～(9)のポリペプチドは、下記実施例において、ビアコアを使ったDOCK11と結合するペプチドの選択実験によりIVVライブラリーから選択され、プルダウンアッセイ等により抗HBV活性を確認したポリペプチドである。(10)～(12)は、(7)及び(9)の配列のホモロジー検索結果に基づいて設計され、プルダウンアッセイ等により抗HBV活性を確認したポリペプチドである。下記実施例では、(10)のDCS8-42TNを用いて調製した被検物質をin vivo投与実験に使用して抗HBV活性を確認しているが、上記したポリペプチドの具体例は、DCS8-42TN以外のものもDCS8-42TNと同様に抗HBV剤として有用である。 Polypeptides (1) to (9) are polypeptides selected from an IVV library by a selection experiment for peptides that bind to DOCK11 using Biacore in the following examples, and confirmed to have anti-HBV activity by pull-down assay or the like. be. (10) to (12) are polypeptides designed based on the homology search results of the sequences of (7) and (9) and confirmed to have anti-HBV activity by pull-down assays and the like. In the examples below, the test substance prepared using DCS8-42TN in (10) was used in an in vivo administration experiment to confirm the anti-HBV activity. Agents other than 42TN are also useful as anti-HBV agents like DCS8-42TN.
　(13)の部分ポリペプチドとは、Ack1（NP_001374642.1、配列番号50）の部分領域であって、DOCK11結合ペプチドDCS8-42と高いホモロジーを有する領域であるPEQARPPPPLEDNLFL（配列番号10; 配列番号50に示すAck1のアミノ酸配列中の第674番～第689番アミノ酸の領域）を含む部分領域で構成されるポリペプチドである。配列番号50のアミノ酸配列をコードする塩基配列（NM_001387713.1のコード領域の塩基配列）を配列番号49に示す。(14)の部分ポリペプチドとは、ラディキシン（NP_001247421.1、配列番号92）の部分領域であって、DOCK11結合ペプチドDCS8-59と高いホモロジーを有する領域であるHEEHRGMLREDSMMEYLK（配列番号11; 配列番号92に示すラディキシンのアミノ酸配列中の第176番～第193番アミノ酸の領域）を含む部分領域で構成されるポリペプチドである。配列番号92のアミノ酸配列をコードする塩基配列（NM_001260492.2のコード領域の塩基配列）を配列番号91に示す。(15)の部分ポリペプチドとは、β-セントラクチン（NP_005726.1、配列番号94）の部分領域であって、DOCK11結合ペプチドDCS8-59と高いホモロジーを有する領域であるAEEHRGLLTIRYPMEH（配列番号12; 配列番号94に示すβ-セントラクチンのアミノ酸配列中の第62番～第77番アミノ酸の領域）を含む部分領域で構成されるポリペプチドである。配列番号94のアミノ酸配列をコードする塩基配列（NM_005735.4のコード領域の塩基配列）を配列番号93に示す。配列番号10の配列のポリペプチドであるDCS8-42TN、配列番号11の配列のポリペプチドであるDCS8-59R、及び配列番号12の配列のポリペプチドであるDCS8-59Cが抗HBV活性を有することは下記実施例において確認されているので、これらの領域を含む各タンパク質の部分領域で構成されるポリペプチドも同様に抗HBV活性を有し、抗HBV剤として有用である。(13)～(15)のポリペプチドの鎖長は特に限定されないが、合成や肝細胞内への送達などの便宜から、例えば100残基以内、80残基以内、70残基以内、60残基以内、50残基以内、40残基以内、30残基以内、25残基以内、又は20残基以内のサイズとしてもよい。 The partial polypeptide of (13) is a partial region of Ack1 (NP_001374642.1, SEQ ID NO: 50), which is a region having high homology with DOCK11-binding peptide DCS8-42 PEQARPPPPLEDNLFL (SEQ ID NO: 10; SEQ ID NO: 50 674th to 689th amino acids in the amino acid sequence of Ack1 shown in )). SEQ ID NO: 49 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 50 (nucleotide sequence of the coding region of NM_001387713.1). The partial polypeptide of (14) is a partial region of radixin (NP_001247421.1, SEQ ID NO: 92) and has high homology with DOCK11-binding peptide DCS8-59 HEEHRGMLREDSMMEYLK (SEQ ID NO: 11; SEQ ID NO: 92 176th to 193rd amino acids in the radixin amino acid sequence shown in )). SEQ ID NO:91 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:92 (nucleotide sequence of the coding region of NM_001260492.2). The partial polypeptide of (15) is a partial region of β-centractin (NP_005726.1, SEQ ID NO: 94) and is a region having high homology with DOCK11-binding peptide DCS8-59 AEEHRGLLTIRYPMEH (SEQ ID NO: 12; It is a polypeptide composed of a partial region containing the 62nd to 77th amino acid region in the amino acid sequence of β-centractin shown in SEQ ID NO:94). SEQ ID NO:93 shows the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:94 (nucleotide sequence of the coding region of NM_005735.4). DCS8-42TN, a polypeptide of SEQ ID NO: 10, DCS8-59R, a polypeptide of SEQ ID NO: 11, and DCS8-59C, a polypeptide of SEQ ID NO: 12, have anti-HBV activity. As confirmed in the examples below, polypeptides composed of partial regions of each protein containing these regions also have anti-HBV activity and are useful as anti-HBV agents. The chain length of the polypeptides (13) to (15) is not particularly limited. The size may be within 10 groups, within 50 residues, within 40 residues, within 30 residues, within 25 residues, or within 20 residues.
　(16)のポリペプチドは、(1)～(15)のいずれかのポリペプチドにおいて残基の一部を改変した、もとのポリペプチドと80％以上の配列同一性を有するポリペプチドである。ごく少数の残基を改変しても、ポリペプチドの活性が同等以上に維持されることは、この分野において周知である。従って、(1)～(15)のポリペプチドの少数の残基（例えば１～４残基、１～３残基、１若しくは２残基、又は１残基）を改変した、もとの配列との同一性が８０％以上、例えば８５％以上、又は９０％以上であるポリペプチドも、もとのポリペプチドと同様に抗HBV活性を発揮でき、抗HBV剤として有用である。ここでいう残基の改変は、置換、欠失、挿入又は付加であり、典型的には置換である。 The polypeptide of (16) is a polypeptide having a sequence identity of 80% or more with the original polypeptide in which a part of the residues is modified in any of the polypeptides of (1) to (15). . It is well known in the art that a very small number of residues can be altered while maintaining equivalent or better activity of the polypeptide. Thus, the original sequence in which a few residues (eg, 1-4 residues, 1-3 residues, 1 or 2 residues, or 1 residue) of the polypeptides (1)-(15) are altered Polypeptides having 80% or more identity, such as 85% or more, or 90% or more identity with , can exhibit anti-HBV activity like the original polypeptide and are useful as anti-HBV agents. Modifications of residues herein are substitutions, deletions, insertions or additions, typically substitutions.
　ここで、アミノ酸配列の同一性とは、比較すべき２つのアミノ酸配列のアミノ酸残基ができるだけ多く一致するように両アミノ酸配列を整列させ、一致したアミノ酸残基数を全アミノ酸残基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する２つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化は、例えばBLAST、FASTA、CLUSTAL W等の周知のプログラムを用いて行なうことができる。ギャップが挿入される場合、上記全アミノ酸残基数は、１つのギャップを１つのアミノ酸残基として数えた残基数となる。このようにして数えた全アミノ酸残基数が、比較する２つの配列間で異なる場合には、同一性（％）は、長い方の配列の全アミノ酸残基数で、一致したアミノ酸残基数を除して算出される。 Here, the identity of amino acid sequences means that two amino acid sequences to be compared are aligned so that as many amino acid residues as possible match each other, and the number of matched amino acid residues is divided by the total number of amino acid residues. It is expressed as a percentage. In the above alignment, gaps are appropriately inserted in one or both of the two sequences to be compared, if necessary. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, CLUSTAL W, and the like. When a gap is inserted, the above total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences being compared, then the % identity is the total number of amino acid residues in the longer sequence and the number of matching amino acid residues. calculated by dividing
　保存的置換、すなわち、化学的性質が類似するアミノ酸への置換であれば、タンパク質の性質・活性を損なわない蓋然性が高い。側鎖が類似するアミノ酸は、化学的性質が類似する。側鎖の類似性でアミノ酸をグループ分けすると、例えば、脂肪族側鎖を有するアミノ酸の群（グリシン、アラニン、バリン、ロイシン、イソロイシン）、脂肪族ヒドロキシル側鎖を有するアミノ酸の群（セリン、トレオニン）、アミド含有側鎖を有するアミノ酸の群（アスパラギン、グルタミン）、芳香族側鎖を有するアミノ酸の群（フェニルアラニン、チロシン、トリプトファン）、塩基性側鎖を有するアミノ酸の群（アルギニン、リジン、ヒスチジン）、酸性側鎖を有するアミノ酸の群（アスパラギン酸、グルタミン酸）、硫黄含有側鎖を有するアミノ酸の群（システイン、メチオニン）、などに分類することができる。同じ群に属する別のアミノ酸への置換が保存的置換である。 Conservative substitutions, that is, substitutions with amino acids that have similar chemical properties, are highly likely not to impair the properties and activities of the protein. Amino acids with similar side chains have similar chemical properties. Grouping amino acids by side chain similarity includes, for example, the group of amino acids with aliphatic side chains (glycine, alanine, valine, leucine, isoleucine), the group of amino acids with aliphatic hydroxyl side chains (serine, threonine). , the group of amino acids with amide-containing side chains (asparagine, glutamine), the group of amino acids with aromatic side chains (phenylalanine, tyrosine, tryptophan), the group of amino acids with basic side chains (arginine, lysine, histidine), They can be classified into groups of amino acids with acidic side chains (aspartic acid, glutamic acid), groups of amino acids with sulfur-containing side chains (cysteine, methionine), and the like. A substitution for another amino acid belonging to the same group is a conservative substitution.
　ペプチド製剤の分野では、ペプチドの生体内での安定性を向上させ、血中半減期を高めるなどの目的で、ポリエチレングリコール（PEG）鎖を付加する（Clin Nephrol. 2006 Mar;65(3):180-90.やProc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7.など）、主としてN末端又はC末端に糖鎖を付加する（J Am Chem Soc. 2004 Nov 3;126(43):14013-22やAngew Chem Int Ed Engl. 2004 Mar 12;43(12):1516-20など）、アミノ酸残基の少なくとも一部をD体とする（J Pharmacol Exp Ther. 2004 Jun;309(3):1190-7やJ Pharmacol Exp Ther. 2004 Jun;309(3):1183-9.など）、抗体のFc領域を適宜改変して付加する（例えば、J. Immunol., 154 (10), 5590-5600 (1995)、Nature, 332, 563-564 (1998)、Nature, 332, 738-740 (1998)、BioDrugs. 2008;22:11-26など）、C末端をアミド化する、N末端をアセチル化する等の技術が用いられている。本発明の抗HBV剤に有効成分として用いるポリペプチドは、そのような技術を適用したものであってもよい。 In the field of peptide formulations, polyethylene glycol (PEG) chains are added for the purpose of improving the stability of peptides in vivo and increasing the half-life in blood (Clin Nephrol. 2006 Mar;65(3): 180-90. and Proc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7.), mainly adding sugar chains to the N-terminus or C-terminus (J Am Chem Soc. 2004 Nov 3;126 (43): 14013-22 and Angew Chem Int Ed Engl. 2004 Mar 12;43(12): 1516-20), at least part of the amino acid residues are in the D form (J Pharmacol Exp Ther. 2004 Jun; 309(3):1190-7 and JPharmacolExpTher.2004Jun;309(3):1183-9., etc.), the Fc region of the antibody is appropriately modified and added (e.g., J.Immunol., 154( 10), 5590-5600 (1995), Nature, 332, 563-564 (1998), Nature, 332, 738-740 (1998), BioDrugs. 2008;22:11-26, etc.), C-terminal amidation , N-terminal acetylation, etc. are used. The polypeptide used as an active ingredient in the anti-HBV agent of the present invention may be one to which such techniques are applied.
　本発明において、「配列番号Xの配列のポリペプチド」、「配列番号Xで表されるポリペプチド」、「配列番号Xで構成されるポリペプチド」（配列番号Xはアミノ酸残基数Nのアミノ酸配列とする）という語は、配列番号Xに示されるアミノ酸配列を有し、全長がN残基であるポリペプチド（このようなポリペプチドを便宜的にポリペプチドXと呼ぶ）に、Fc領域や肝臓へのデリバリーを高める効果を有するポリペプチドのような他の機能性分子を付加ないし連結した構造を有するポリペプチドを包含する。「配列番号Xの配列のポリペプチド」、「配列番号Xで表されるポリペプチド」又は「配列番号Xで構成されるポリペプチド」を有効成分として含む」という語は、全長がN残基であるポリペプチドXを有効成分として含むという態様の他、ポリペプチドXに他の機能性分子を付加した構造を有するポリペプチドを有効成分として含む態様を包含する。ポリペプチドの抗HBV活性を損なわない限り、いかなる機能性分子が連結されていてもよい。 In the present invention, "polypeptide having the sequence of SEQ ID NO: X", "polypeptide represented by SEQ ID NO: X", "polypeptide composed of SEQ ID NO: X" (SEQ ID NO: X is an amino acid having N amino acid residues The term "sequence") refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: X and having a total length of N residues (such a polypeptide is referred to as polypeptide X for convenience), and the Fc region and It includes polypeptides having a structure in which other functional molecules are added or linked, such as polypeptides having the effect of enhancing delivery to the liver. The term "containing a polypeptide having the sequence of SEQ ID NO: X", "polypeptide represented by SEQ ID NO: X", or "polypeptide consisting of SEQ ID NO: X" as an active ingredient means that the full length is N residues. In addition to an embodiment in which a certain polypeptide X is included as an active ingredient, an embodiment in which a polypeptide having a structure in which another functional molecule is added to polypeptide X is included as an active ingredient. Any functional molecule may be linked as long as it does not impair the anti-HBV activity of the polypeptide.
　(1)～(12)のポリペプチド、及び(16)のポリペプチドのうちで(1)～(12)のいずれかと80％以上100％未満の配列同一性を有するポリペプチドは、鎖長が短いので、常法の化学合成により容易に調製できる。(13)～(15)のポリペプチド、及び(16)のポリペプチドのうちで(13)～(15)のいずれかと80％以上100％未満の配列同一性を有するポリペプチドは、鎖長が短いものは化学合成により容易に調製でき、化学合成による調製が難しい鎖長のものは遺伝子工学的手法により製造することができる。他の機能性分子が連結された構造を有するポリペプチドも、機能性分子がサイズの短いポリペプチドである場合は化学合成により、機能性分子がサイズの長いポリペプチドである場合は遺伝子工学的手法により製造することができる。 The polypeptides of (1) to (12) and the polypeptides of (16) that have 80% or more and less than 100% sequence identity with any of (1) to (12) among the polypeptides of (16) have a chain length Since it is short, it can be easily prepared by conventional chemical synthesis. The polypeptides of (13) to (15) and the polypeptides of (16) that have 80% or more and less than 100% sequence identity with any of (13) to (15) among the polypeptides of (16) have a chain length Short ones can be easily prepared by chemical synthesis, and long chain ones that are difficult to prepare by chemical synthesis can be produced by genetic engineering techniques. Polypeptides having a structure in which other functional molecules are linked are also produced by chemical synthesis when the functional molecule is a short-sized polypeptide, and by genetic engineering when the functional molecule is a long-sized polypeptide. can be manufactured by
　化学合成法の具体例としては、例えばFmoc法（フルオレニルメチルオキシカルボニル法）、tBoc法（t-ブチルオキシカルボニル法）等を挙げることができる。また、各種の市販のペプチド合成機を利用して常法により合成することもできる。 Specific examples of chemical synthesis methods include the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Moreover, it can also synthesize|combine by a conventional method using various commercially available peptide synthesizers.
　遺伝子工学的手法による製造方法も周知である。簡潔に記載すると、サイズの長いDOCK11結合ペプチドの全長、又は機能性分子に連結されたDOCK11結合ペプチドの全長をコードするcDNAを調製し、該cDNAを適当な発現ベクターに組み込んで適当な宿主細胞に導入し、該宿主細胞内でポリペプチドを生産させて抽出、精製することにより、目的とするポリペプチドを得ることができる。 Manufacturing methods using genetic engineering techniques are also well known. Briefly, a cDNA encoding a full-length DOCK11-binding peptide with a long size or a full-length DOCK11-binding peptide linked to a functional molecule is prepared, the cDNA is incorporated into an appropriate expression vector, and the expression is transformed into an appropriate host cell. The polypeptide of interest can be obtained by introducing it, producing the polypeptide in the host cell, and extracting and purifying it.
　また、DOCK11結合ペプチドは、精製や検出の便宜のためにFlagタグやHisタグ等のタグ配列を付加したものであってもよい。このようなタグ配列も、機能性分子の１つの例として捉えることができる。例えば、下記実施例で調製したN-10M-D42TNのうちの「切断配列＋DCS8-42TNペプチド＋S28＋核移行シグナル」部分（配列番号90）では、DOCK11結合ペプチドDCS8-42TNのC末にFlagタグとHisタグが付加されている。もっとも、このようなタグ配列の付加は任意であり、タグ配列を付加しないDOCK11結合ペプチドにて本発明の抗HBV剤を調製することも可能である。 In addition, the DOCK11-binding peptide may have a tag sequence such as a Flag tag or His tag added for convenience of purification and detection. Such a tag sequence can also be regarded as one example of a functional molecule. For example, in the "cleaved sequence + DCS8-42TN peptide + S28 + nuclear localization signal" portion (SEQ ID NO: 90) of N-10M-D42TN prepared in the following example, Flag tag and His tag is added. However, addition of such a tag sequence is optional, and it is also possible to prepare the anti-HBV agent of the present invention using a DOCK11-binding peptide without a tag sequence.
　機能性分子の1つの例として、肝細胞内への送達のための担体分子を挙げることができる。すなわち、DOCK11結合ペプチドは、肝細胞内への送達のための担体分子と連結した形態にあってよい。肝細胞内に送達すべき薬物のための担体分子としては、肝実質細胞に豊富に存在するアシアロ糖タンパク質受容体（ASGR）やApoE受容体を利用したDDSが知られている（非特許文献７）。本発明においても、ASGRやApoE受容体を利用して肝細胞内に薬物を送達する担体分子を用いることができる。 One example of a functional molecule is a carrier molecule for delivery into hepatocytes. That is, the DOCK11-binding peptide may be in a form linked to a carrier molecule for delivery into hepatocytes. DDS using asialoglycoprotein receptor (ASGR) and ApoE receptor, which are abundant in hepatocytes, is known as a carrier molecule for drugs to be delivered into hepatocytes (Non-Patent Document 7). ). Also in the present invention, carrier molecules that deliver drugs into hepatocytes using ASGR or ApoE receptors can be used.
　ASGRを利用した薬物送達用担体の例として、ASGRに結合する特異結合分子（抗体、抗体断片若しくは一本鎖抗体、又はアプタマーなど）、特にASGRに結合する抗体、抗体断片又は一本鎖抗体（scFv）を挙げることができる。アシアロ糖タンパク質受容体（ASGR）は、図12に示すように、N末端を細胞内、C末端の糖認識部位（CRDs）を細胞外に向けたII型の１回膜貫通タンパク質である。ヒトの肝細胞においては、ASGR1とASGR2のヘテロオリゴマーにより受容体が形成されている。薬物送達用担体として用いるASGR特異結合分子は、ASGRの細胞外ドメインと結合するものであればよい。ASGR1とASGR2の両方に結合できる特異結合分子を特に好ましく用いることができるが、いずれか一方にのみ結合する特異結合分子であっても薬物送達用担体として利用可能である。いずれか一方にのみ結合する特異結合分子の場合、単独で薬物送達用担体として利用してもよいし、一方に結合する特異結合分子と他方に結合する特異結合分子を組み合わせて利用してもよい。組み合わせる場合、ASGR1特異結合分子を連結したDOCK11結合ペプチドと、ASGR2特異結合分子を連結したDOCK11結合ペプチドを調製し、両者を混合して抗HBV剤として利用すればよい。 Examples of drug delivery carriers using ASGR include specific binding molecules that bind to ASGR (antibodies, antibody fragments or single-chain antibodies, or aptamers), particularly antibodies that bind to ASGR, antibody fragments or single-chain antibodies ( scFv). The asialoglycoprotein receptor (ASGR) is a type II single-pass transmembrane protein with the N-terminus directed intracellularly and the C-terminal carbohydrate recognition sites (CRDs) directed extracellularly, as shown in FIG. In human hepatocytes, receptors are formed by hetero-oligomers of ASGR1 and ASGR2. The ASGR-specific binding molecule used as a drug delivery carrier may bind to the extracellular domain of ASGR. A specific binding molecule that can bind to both ASGR1 and ASGR2 can be particularly preferably used, but even a specific binding molecule that binds to only one of them can be used as a drug delivery carrier. In the case of a specific binding molecule that binds only to either one, it may be used alone as a drug delivery carrier, or a combination of a specific binding molecule that binds to one and a specific binding molecule that binds to the other may be used. . In the case of combination, a DOCK11-binding peptide linked to an ASGR1-specific binding molecule and a DOCK11-binding peptide linked to an ASGR2-specific binding molecule may be prepared, mixed and used as an anti-HBV agent.
　GenBankにNM_001671.5で登録されているヒトASGR1 mRNAのコード領域の塩基配列及びこれにコードされるASGR1のアミノ酸配列を配列番号43、44に、NM_001181.4で登録されているヒトASGR2 mRNAのコード領域の塩基配列及びこれにコードされるASGR2のアミノ酸配列を配列番号45、46に、それぞれ示す。配列番号44（ASGR1）の第41番～第61番アミノ酸、配列番号46（ASGR2）の第59番～第79番アミノ酸が膜貫通ドメインであり、そのN末側が細胞内ドメイン、C末側が細胞外ドメインである。抗ASGR抗体、抗体断片又は一本鎖抗体の調製においては、ASGR1の第62番～第291番アミノ酸の領域と、ASGR2の第80番～第331番アミノ酸の領域を抗原ないし免疫原として利用すればよいが、これらの細胞外ドメインをヘテロオリゴマー化して用いることがより好ましい。以下、本明細書において、「ASGR細胞外ドメイン」という語には、ヘテロオリゴマー化した細胞外ドメインが包含される。「ヘテロオリゴマー」といった場合には、ヘテロオリゴマー化したASGR細胞外ドメインを意味する。 The base sequence of the coding region of human ASGR1 mRNA registered under NM_001671.5 in GenBank and the amino acid sequence of ASGR1 encoded by this are shown in SEQ ID NOs: 43 and 44, and the code of human ASGR2 mRNA registered under NM_001181.4. The nucleotide sequence of the region and the amino acid sequence of ASGR2 encoded by this are shown in SEQ ID NOS: 45 and 46, respectively. The 41st to 61st amino acids of SEQ ID NO: 44 (ASGR1) and the 59th to 79th amino acids of SEQ ID NO: 46 (ASGR2) are transmembrane domains, the N-terminal side of which is the intracellular domain, and the C-terminal side of which is the cell. Foreign domain. In the preparation of an anti-ASGR antibody, antibody fragment, or single-chain antibody, the 62nd to 291st amino acid region of ASGR1 and the 80th to 331st amino acid region of ASGR2 may be used as antigens or immunogens. However, it is more preferable to hetero-oligomerize these extracellular domains. Hereinafter, the term "ASGR extracellular domain" includes hetero-oligomerized extracellular domains. By "hetero-oligomer" is meant a hetero-oligomerized ASGR extracellular domain.
　抗ASGRポリクローナル抗体は、ASGR細胞外ドメインを免疫原として非ヒト動物を免役し、血液を採取して血清を分離し、血清からASGR細胞外ドメインに結合する抗体を回収・精製することで得ることができる。抗ASGRモノクローナル抗体は、ASGR細胞外ドメインで免役した非ヒト動物から脾細胞やリンパ球などの抗体産生細胞を採取し、これをミエローマ細胞と融合させてハイブリドーマを調製し、ASGR細胞外ドメインと結合する抗体を産生するハイブリドーマを選択し、これを増殖させて培養上清から抗ASGRモノクローナル抗体を得ることができる。 Anti-ASGR polyclonal antibodies can be obtained by immunizing a non-human animal using the ASGR extracellular domain as an immunogen, collecting blood, separating the serum, and collecting and purifying the antibody that binds to the ASGR extracellular domain from the serum. can be done. Anti-ASGR monoclonal antibodies are produced by collecting antibody-producing cells such as splenocytes and lymphocytes from non-human animals immunized with the ASGR extracellular domain, fusing them with myeloma cells to prepare hybridomas, and binding to the ASGR extracellular domain. Anti-ASGR monoclonal antibodies can be obtained from culture supernatants by selecting hybridomas that produce antibodies against the anti-ASGR and proliferating them.
　抗ASGR抗体断片は、抗ASGR抗体をパパインやペプシンのようなタンパク分解酵素で処理することにより得ることができる。抗体断片の定義は上記した通りであり、Fab、Fab'、F(ab')2等の、抗原（ASGR細胞外ドメイン）との結合性を維持した抗体断片が包含される。 An anti-ASGR antibody fragment can be obtained by treating an anti-ASGR antibody with a proteolytic enzyme such as papain or pepsin. The definition of the antibody fragment is as described above, and includes antibody fragments such as Fab, Fab', F(ab') 2 that maintain the binding ability to the antigen (ASGR extracellular domain).
　抗ASGR scFvは、例えば、上記の通りに作製したハイブリドーマのmRNAを抽出してcDNAを調製し、免疫グロブリンH鎖及びL鎖に特異的なプライマーを用いてPCRを行ない免疫グロブリンH鎖遺伝子及びL鎖遺伝子を増幅し、これらをリンカーで連結し、適切な制限酵素部位を付与してプラスミドベクターに導入し、該ベクターで大腸菌を形質転換してscFvを発現させ、これを大腸菌から回収することにより得ることができる。 For anti-ASGR scFv, for example, cDNA is prepared by extracting mRNA from the hybridoma produced as described above, and PCR is performed using primers specific for immunoglobulin H chain and L chain to obtain immunoglobulin H chain gene and L chain. By amplifying the chain genes, ligating them with a linker, adding an appropriate restriction enzyme site, introducing them into a plasmid vector, transforming E. coli with the vector to express scFv, and recovering it from E. coli. Obtainable.
　また、抗ASGR scFvは、ファージディスプレイ法やIVV法等の技術により、scFvライブラリーから取得することができる。 In addition, anti-ASGR scFv can be obtained from scFv libraries by techniques such as the phage display method and IVV method.
　ファージディスプレイ法では、scFvライブラリーとして、ファージ表面にscFvが提示されたファージのライブラリーを調製する。例えば次のような手順で、ナイーブscFvファージライブラリーを調製すればよい。健常なヒト又は非ヒト動物から採取された脾細胞やリンパ球などの抗体産生細胞からmRNAを抽出し、逆転写反応によりcDNAを合成し、重鎖可変領域（VH）を含む領域をコードするcDNA（VH cDNA）及び軽鎖可変領域（VL）を含む領域をコードするcDNA（VL cDNA）をPCRにより網羅的に増幅する。次いで、増幅したVH cDNA及びVL cDNAを、常法のアセンブリPCRないしはFusion PCRにより、適当なリンカー（例えば、GGGGSユニットを3回繰り返したリンカー等）を介してランダムに連結し、scFvをコードするcDNA（scFv cDNA）を調製する。scFv cDNAをファージ用プラスミドベクターに組み込んでベクターのライブラリーを調製した後、ファージディスプレイ法により各scFvを表面に提示するファージのライブラリーを作製する。ファージ用プラスミドベクターとしては、ファージ粒子を形成するために必要なファージ遺伝子が全て含まれ、単独でファージ粒子形成が可能なファージベクターと、g3p遺伝子を含むが他のファージタンパク質遺伝子を含まず、ファージ粒子形成のためにヘルパーファージを必要とするファージミドベクターの２種類があるが、ファージミドベクターを好ましく用いることができる。ファージミドベクターを用いて作製したscFv cDNAライブラリーを大腸菌に導入し、大腸菌にヘルパーファージを重感染させることにより、scFv cDNAライブラリーの個々のベクターがパッケージングされ、該ベクターが発現するscFvを表面に提示するファージのライブラリーを作製できる。調製したVH cDNA及びVL cDNAあるいはscFv cDNAにランダムに突然変異を導入し、変異scFvのファージライブラリーを作製してもよい。 In the phage display method, a phage library with scFv displayed on the phage surface is prepared as an scFv library. For example, a naive scFv phage library may be prepared by the following procedure. mRNA is extracted from antibody-producing cells such as splenocytes and lymphocytes collected from healthy humans or non-human animals, cDNA is synthesized by reverse transcription reaction, and cDNA encoding the region containing the heavy chain variable region (VH) (VH cDNA) and cDNA encoding a region containing the light chain variable region (VL) (VL cDNA) are comprehensively amplified by PCR. Next, the amplified VH cDNA and VL cDNA are randomly ligated via a suitable linker (for example, a linker consisting of three repeated GGGGS units, etc.) by standard assembly PCR or Fusion PCR to obtain scFv-encoding cDNA. (scFv cDNA) is prepared. After preparing a vector library by inserting the scFv cDNA into a phage plasmid vector, a phage library displaying each scFv on its surface is prepared by the phage display method. Plasmid vectors for phage include all phage genes necessary for forming phage particles and capable of forming phage particles independently, and phage vectors containing the g3p gene but no other phage protein genes and containing phage Although there are two types of phagemid vectors that require helper phage for particle formation, phagemid vectors are preferably used. A scFv cDNA library prepared using a phagemid vector is introduced into Escherichia coli, and the E. coli is superinfected with a helper phage to package each vector of the scFv cDNA library, and the scFv expressed by the vector is placed on the surface. Libraries of displaying phage can be generated. A phage library of mutant scFv may be prepared by randomly introducing mutations into the prepared VH cDNA and VL cDNA or scFv cDNA.
　作製したライブラリーより、ASGR細胞外ドメインに結合するscFvを提示したファージを選別する（パニング）。このパニング工程は、ASGR細胞外ドメインを固定化した固相担体（チップ、プレート、磁気ビーズ等）を使用して実施することができ、ヘテロオリゴマーを固定化した担体を特に好ましく使用できる。ASGR1の細胞外ドメイン（ASGR1ex）及びASGR2の細胞外ドメイン（ASGR2ex）をビオチン化し、アビジン類がコートされた担体とビオチン化ASGR1ex及びASGR2exを接触させることで、担体表面上でASGR1ex及びASGR2exをヘテロオリゴマー化させることができる。ファージライブラリーをASGR細胞外ドメイン固定化担体と接触させ、洗浄後、担体上に結合したファージを回収する。回収したファージを溶解し、パッケージングされていたベクターを回収して再度大腸菌に導入し、ヘルパーファージを重感染させてファージ粒子を再度形成させる。これらのファージ粒子を再度、ASGR細胞外ドメイン固定化担体と接触させる。この操作を複数回実施することで、ASGR細胞外ドメインに特異的なscFvを提示したファージクローンを濃縮できる。 From the prepared library, phages displaying scFv that bind to the ASGR extracellular domain are selected (panning). This panning step can be performed using a solid-phase carrier (chip, plate, magnetic bead, etc.) on which the extracellular domain of ASGR is immobilized, and a carrier on which a hetero-oligomer is immobilized is particularly preferably used. The extracellular domain of ASGR1 (ASGR1ex) and the extracellular domain of ASGR2 (ASGR2ex) are biotinylated, and by contacting the biotinylated ASGR1ex and ASGR2ex with a carrier coated with avidins, ASGR1ex and ASGR2ex are hetero-oligomerized on the carrier surface. can be made The phage library is brought into contact with the ASGR extracellular domain-immobilized carrier, and after washing, the phages bound on the carrier are recovered. The recovered phages are lysed, the packaged vector is recovered, introduced again into E. coli, and superinfected with helper phages to form phage particles again. These phage particles are again brought into contact with the ASGR extracellular domain-immobilized carriers. By performing this operation multiple times, phage clones displaying scFv specific to the ASGR extracellular domain can be enriched.
　複数ラウンドのパニングを経て濃縮したファージを抗ASGR scFv候補のクローンとして取得できるが、候補をさらに絞り込むためにクローンの選定を行なってもよい。濃縮後のファージよりscFv発現ベクターを回収し、大腸菌等の適当な宿主細胞に導入してscFvを発現する宿主細胞クローンを調製し、これらのクローンについて、ASGR細胞外ドメインへの反応性及び可変領域の配列を調べ、ASGR細胞外ドメインへの特異的結合性が高いクローンを選定する。反応性の確認は、ASGR細胞外ドメイン、好ましくはヘテロオリゴマーを抗原としたELISA等のイムノアッセイにより行なえばよい。可変領域の配列が同一ないし非常に類似したクローンが複数存在している可能性があるため、VH及びVLの塩基配列によりクローンの重複を確認してクローンのグルーピングを行なってもよい。これらのクローン選定作業により、ASGR細胞外ドメインへの特異的結合性が高いクローンを選定し、候補をさらに絞り込むことができる。 Phages enriched through multiple rounds of panning can be obtained as anti-ASGR scFv candidate clones, but clone selection may be performed to further narrow down the candidates. The scFv expression vector is collected from the phage after concentration and introduced into an appropriate host cell such as E. coli to prepare host cell clones expressing scFv. and select clones with high specific binding to the ASGR extracellular domain. Reactivity can be confirmed by an immunoassay such as ELISA using the ASGR extracellular domain, preferably a hetero-oligomer, as an antigen. Since there may be multiple clones with the same or very similar variable region sequences, clones may be grouped by confirming duplication of clones based on the base sequences of VH and VL. Through these clone selection activities, it is possible to select clones with high specific binding to the ASGR extracellular domain and further narrow down the candidates.
　候補クローンよりscFv発現ベクターを回収し、scFv発現ベクターからscFv cDNAを増幅し、適当なプラスミド発現ベクターに組み込んで適当な宿主細胞内でscFvを発現させ、これを回収、精製する。精製後のscFvについて、ASGR1ex及びASGR2exとの反応性又はヘテロオリゴマーとの反応性を最終確認し、ASGR細胞外ドメインに特異的に結合するscFvを得ることができる。 The scFv expression vector is recovered from the candidate clone, the scFv cDNA is amplified from the scFv expression vector, incorporated into an appropriate plasmid expression vector to express the scFv in an appropriate host cell, recovered and purified. The reactivity with ASGR1ex and ASGR2ex or the reactivity with heterooligomers is finally confirmed for the scFv after purification, and scFv that specifically binds to the ASGR extracellular domain can be obtained.
　IVV法（Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03/106675 A1）は、本願発明者の一人である柳川及びその共同研究者らが開発した技術である。IVV法では、mRNAの3'末端にPEG（ポリエチレングリコール）スペーサーを介して抗生物質の一種のピューロマイシンを結合させ、それを鋳型として無細胞翻訳反応を行うことにより、タンパク質分子とこれをコードするmRNA分子とがピューロマイシンを介して共有結合したmRNA－タンパク質連結分子（in vitro virus; IVV）が形成される。こうして構築されたIVVライブラリーの中からベイト（タンパク質、ペプチド、抗原など）と結合するタンパク質を含むIVVをin vitroで釣り上げた後、そこに連結している遺伝子（mRNA）を逆転写PCRで増幅し、DNAシーケンサーで塩基配列を解読することによって、相互作用するタンパク質やペプチドや抗体を容易に同定することができる。ベイトに結合したIVVを溶出・回収する方法は、フリーのベイトで競合溶出する方法が一般的であるが、競合溶出できない場合は、光開裂が可能な2-nitrobenzylリンカーを導入したPEGスペーサーを用い、365nmのUVを照射してスペーサーを切断してmRNA部を溶出・回収することができる(Doi,N. et al., J.Biotechnol.,131:231-239,2007)。 IVV method (Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03/106675 A1) is a technique developed by Yanagawa, one of the inventors of the present application, and his collaborators. In the IVV method, puromycin, a type of antibiotic, is attached to the 3' end of mRNA via a PEG (polyethylene glycol) spacer, and a cell-free translation reaction is performed using this as a template to encode a protein molecule. An mRNA-protein junction molecule (in vitro virus; IVV) is formed covalently linked to the mRNA molecule via puromycin. From the IVV library constructed in this way, IVVs containing proteins that bind to baits (proteins, peptides, antigens, etc.) are picked in vitro, and the genes (mRNA) linked to them are amplified by reverse transcription PCR. Then, by deciphering the base sequence with a DNA sequencer, interacting proteins, peptides and antibodies can be easily identified. Competitive elution with free bait is the common method for eluting and recovering IVV bound to bait. , the spacer can be cleaved by UV irradiation at 365 nm, and the mRNA portion can be eluted and recovered (Doi, N. et al., J. Biotechnol., 131:231-239, 2007).
　IVV法による抗ASGR scFvの作製方法の詳細は下記実施例に記載の通りである。以下、簡単に説明する。 The details of the method for producing anti-ASGR scFv by the IVV method are as described in the examples below. A brief description will be given below.
　上記のようにしてscFv cDNAライブラリーを調製した後、cDNAライブラリーから逆転写反応にてmRNAを合成し、PEGスペーサーを介してmRNAの3'末端にピューロマイシンを結合させ、scFv mRNA-ピューロマイシンのライブラリーとする。これを鋳型として無細胞翻訳反応を行ない、scFv分子とこれをコードするmRNA分子がピューロマイシンを介して共有結合したmRNA－scFv連結分子（IVV）のライブラリーを構築する。 After preparing the scFv cDNA library as described above, mRNA is synthesized from the cDNA library by reverse transcription reaction, puromycin is bound to the 3' end of the mRNA via a PEG spacer, and scFv mRNA-puromycin library. Using this as a template, a cell-free translation reaction is carried out to construct a library of mRNA-scFv linking molecules (IVV) in which scFv molecules and mRNA molecules encoding them are covalently bound via puromycin.
　scFv IVVライブラリーの中から、ASGR細胞外ドメインに結合するscFvを選択する。この選択工程も、ファージディスプレイ法と同様に、ASGR細胞外ドメイン、好ましくはヘテロオリゴマーをベイトとして固定化した固相担体（チップ、プレート、磁気ビーズ等）を使用して実施できる。scFv IVVライブラリーとASGR細胞外ドメイン固定化担体を接触させ、洗浄後、担体上に結合したIVVを溶出・回収する。ASGR1ex若しくはASGR2exを用いた競合溶出によりIVVを溶出・回収できる。PEGスペーサーとして2-nitrobenzylリンカーを導入したPEGスペーサーを使用した場合には、UV照射によりスペーサーを切断してIVVのmRNA部を溶出・回収することも可能である。 A scFv that binds to the ASGR extracellular domain is selected from the scFv IVV library. As in the phage display method, this selection step can also be performed using a solid-phase carrier (chip, plate, magnetic beads, etc.) on which ASGR extracellular domains, preferably hetero-oligomers, are immobilized as bait. The scFv IVV library is brought into contact with the ASGR extracellular domain-immobilized carrier, and after washing, the IVV bound to the carrier is eluted and collected. IVV can be eluted and recovered by competitive elution using ASGR1ex or ASGR2ex. When a PEG spacer into which a 2-nitrobenzyl linker is introduced is used as the PEG spacer, it is also possible to cleave the spacer by UV irradiation and to elute and recover the mRNA part of IVV.
　回収したIVV又はmRNA部を鋳型として逆転写PCRを行ない、1stラウンド選択後のscFv cDNAライブラリーを調製する。このcDNAライブラリーから再度IVVライブラリーを調製し、2ndラウンドの選択を実施することができる。選択圧（IVVライブラリーとベイトの接触時間、担体へのベイトの固定量等）を順次上げながら複数ラウンドの選択を実施することが好ましい。 Reverse transcription PCR is performed using the recovered IVV or mRNA part as a template to prepare a scFv cDNA library after the 1st round of selection. An IVV library can be prepared again from this cDNA library and a second round of selection can be performed. It is preferable to perform multiple rounds of selection while sequentially increasing the selection pressure (contact time between IVV library and bait, amount of bait immobilized on carrier, etc.).
　数回目以降のラウンドで得られた各scFv cDNAライブラリーからscFvコード領域を増幅し、適当なプラスミドベクターにクローニングして、選択後のscFv cDNAを組み込んだベクターのライブラリーを調製する。このベクターを大腸菌等の適当な宿主細胞に導入し、選択後のscFvのクローンのライブラリーを得る。得られたクローンライブラリーについて、ファージディスプレイ法のクローン選定と同様に、ASGR細胞外ドメインへの反応性の評価と可変領域配列の解析、グルーピングを行ない、選定されたクローンよりベクターを回収してscFvを調製し、ASGR1ex及びASGR2exとの反応性又はヘテロオリゴマーとの反応性を最終確認することで、ASGR細胞外ドメインへの特異的結合性が高いscFvを得ることができる。 Amplify the scFv coding region from each scFv cDNA library obtained in the subsequent rounds, clone it into an appropriate plasmid vector, and prepare a library of vectors incorporating the scFv cDNA after selection. This vector is introduced into a suitable host cell such as E. coli to obtain a library of scFv clones after selection. Similar to the clone selection by the phage display method, the resulting clone library is evaluated for reactivity to the ASGR extracellular domain, analyzed for the variable region sequence, and grouped. Vectors are collected from the selected clones and scFv and final confirmation of reactivity with ASGR1ex and ASGR2ex or reactivity with heterooligomers, scFv with high specific binding to ASGR extracellular domain can be obtained.
　抗ASGR抗体、抗体断片又はscFvの特に好ましい例として、
　配列番号１３、１９、２５若しくは３１に示すアミノ酸配列を含む重鎖CDR1と、
　配列番号１４、２０、２６若しくは３２に示すアミノ酸配列を含む重鎖CDR2と、
　配列番号１５、２１、２７若しくは３３に示すアミノ酸配列を含む重鎖CDR3と、
　配列番号１６、２２、２８若しくは３４に示すアミノ酸配列を含む軽鎖CDR1と、
　配列番号１７、２３、２９若しくは３５に示すアミノ酸配列を含む軽鎖CDR2と、
　配列番号１８、２４、３０若しくは３６に示すアミノ酸配列を含む軽鎖CDR3と
を有する抗体、抗体断片又はscFvを挙げることができる。これらのCDR配列は、下記実施例で取得された抗ASGR scFvのCDR配列に基づいている。下記実施例では、配列番号13～15に示すアミノ酸配列の重鎖CDR1～3及び配列番号16～18に示すアミノ酸配列の軽鎖CDR1～3を有する抗ASGR scFv、配列番号19～21に示すアミノ酸配列の重鎖CDR1～3及び配列番号22～24に示すアミノ酸配列の軽鎖CDR1～3を有する抗ASGR scFv、配列番号25～27に示すアミノ酸配列の重鎖CDR1～3及び配列番号28～30に示す軽鎖CDR1～3を有する抗ASGR scFv、並びに配列番号31～33に示す重鎖CDR1～3及び配列番号34～36に示す軽鎖CDR1～3を有する抗ASGR scFvが得られている。 Particularly preferred examples of the anti-ASGR antibody, antibody fragment or scFv are
a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31;
a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32;
a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33;
a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34;
a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35;
An antibody, antibody fragment or scFv having a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, 24, 30 or 36 can be mentioned. These CDR sequences are based on the CDR sequences of the anti-ASGR scFv obtained in the Examples below. In the following examples, anti-ASGR scFv having heavy chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 13-15 and light chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 16-18, amino acids shown in SEQ ID NOS: 19-21 an anti-ASGR scFv having the heavy chain CDRs 1-3 of the sequences shown in SEQ ID NOS: 22-24 and the light chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 22-24, the heavy chain CDRs 1-3 of the amino acid sequences shown in SEQ ID NOS: 25-27 and SEQ ID NOS: 28-30 and anti-ASGR scFv with heavy chain CDRs 1-3 shown in SEQ ID NOS: 31-33 and light chain CDRs 1-3 shown in SEQ ID NOS: 34-36.
　また、CDR配列は上記した特定のアミノ酸配列のものに限定されず、上記アミノ酸配列において一部の塩基が置換され、もとのアミノ酸配列と80％以上の同一性を有するアミノ酸配列を含むCDRを有した抗体等であってもよい。例えば、重鎖及び軽鎖CDR1は、1個の残基の置換が許容され、重鎖及び軽鎖CDR2は、1～3個、1～2個又は1個の残基の置換が許容され、重鎖及び軽鎖CDR3は、1～2個又は1個の残基の置換が許容される。このような改変型CDRを有する抗体、抗体断片又はscFvも、ASGRに特異的に結合する抗体、抗体断片又はscFvとして利用できる。 In addition, the CDR sequence is not limited to the specific amino acid sequence described above, and a CDR containing an amino acid sequence having 80% or more identity with the original amino acid sequence by substituting a part of the bases in the above amino acid sequence. It may be an antibody or the like having For example, heavy and light chain CDR1 allows substitution of 1 residue, heavy and light chain CDR2 allows substitution of 1-3, 1-2 or 1 residue, Heavy and light chain CDR3s tolerate substitutions of 1-2 or 1 residue. An antibody, antibody fragment or scFv having such modified CDRs can also be used as an antibody, antibody fragment or scFv that specifically binds to ASGR.
　所定の配列のCDRを有する抗体等は、例えば、クローニングされた任意の抗体のVH遺伝子及びVL遺伝子あるいは任意のscFvをコードするscFv遺伝子のCDR領域に変異を導入して上記のアミノ酸配列をコードするように改変することにより容易に調製できる。上記のCDR配列を導入するベースとなる任意の抗体及び任意のscFvは、上記とは異なる配列のCDRを有する抗ASGR抗体及び抗ASGR scFvであってもよいし、他の抗原に対する抗体及びscFvであってもよい。 Antibodies and the like having CDRs of a given sequence encode the amino acid sequences described above by, for example, introducing mutations into the CDR regions of the VH and VL genes of any cloned antibody or the scFv gene encoding any scFv. It can be easily prepared by modifying as follows. Any antibody and any scFv as a base for introducing the above CDR sequence may be an anti-ASGR antibody and anti-ASGR scFv having a CDR sequence different from the above, or an antibody and scFv against other antigens There may be.
　上記した特定のアミノ酸配列を含むCDR又は改変型CDRを有する抗ASGR抗体、抗体断片又はscFvは、本発明の抗HVB剤の送達用担体としてのみならず、肝細胞内に送達すべき様々な薬物のための送達用担体として非常に優れている。この抗ASGR抗体、抗体断片又はscFvを肝細胞内への薬物送達用担体とし、肝細胞内に送達すべき薬物を医薬分野で公知の技術により該担体と複合体化して医薬組成物として利用できる。当該抗ASGR抗体は、ヒト抗体、ヒト化抗体、ヒト－非ヒト動物間のキメラ抗体、非ヒト動物抗体のいずれであってもよいが、ヒト用医薬の送達用担体として利用する場合にはヒト抗体、ヒト化抗体又はキメラ抗体、特にヒト抗体又はヒト化抗体、とりわけヒト抗体が好ましい。抗ASGR scFv抗体も同様に、ヒト抗体、ヒト化抗体、ヒト－非ヒト動物間のキメラ抗体、非ヒト動物抗体のいずれに由来するものであってもよいが、ヒト用医薬の送達用担体として利用する場合にはヒト抗体、ヒト化抗体又はキメラ抗体に由来するもの、特にヒト抗体又はヒト化抗体に由来するもの、とりわけヒト抗体に由来するものが好ましい。 The anti-ASGR antibody, antibody fragment or scFv having CDRs or modified CDRs containing the specific amino acid sequence described above can be used not only as a carrier for delivery of the anti-HVB agent of the present invention, but also as various drugs to be delivered into hepatocytes. It is an excellent delivery carrier for This anti-ASGR antibody, antibody fragment, or scFv can be used as a carrier for drug delivery into hepatocytes, and a drug to be delivered into hepatocytes can be complexed with the carrier by techniques known in the pharmaceutical field and used as a pharmaceutical composition. . The anti-ASGR antibody may be a human antibody, a humanized antibody, a human-non-human animal chimeric antibody, or a non-human animal antibody. Antibodies, humanized antibodies or chimeric antibodies, especially human antibodies or humanized antibodies, especially human antibodies, are preferred. Similarly, anti-ASGR scFv antibodies may be derived from human antibodies, humanized antibodies, chimeric antibodies between human and non-human animals, or non-human animal antibodies. When used, those derived from human antibodies, humanized antibodies or chimeric antibodies, particularly those derived from human antibodies or humanized antibodies, particularly those derived from human antibodies are preferred.
　DOCK11結合ペプチドに抗ASGR抗体等の肝細胞内送達用担体分子を連結する場合、DOCK11結合ペプチドのどちらの末端に連結してもよい。1種以上の他の機能性分子も使用する場合には、それら他の機能性分子と併せて適切に機能するよう、DOCK11結合ペプチドに連結すればよい。肝細胞内送達用担体分子は、本発明の抗HBV剤の投与を受ける患者の細胞内に存在する内因性酵素によって切断される切断配列を介してDOCK11結合ペプチドと連結することが好ましい。切断配列の一例として、Furinが認識して切断する配列RVRR（配列番号37）を挙げることができるが、切断配列はこれに限定されない。 When linking a DOCK11-binding peptide to a carrier molecule for intrahepatocyte delivery such as an anti-ASGR antibody, it may be linked to either end of the DOCK11-binding peptide. If one or more other functional molecules are also used, they may be linked to the DOCK11-binding peptide so as to function properly in conjunction with those other functional molecules. The carrier molecule for intrahepatic delivery is preferably linked to the DOCK11-binding peptide via a cleavage sequence that is cleaved by endogenous enzymes present in the cells of patients receiving the anti-HBV agent of the present invention. An example of a cleavage sequence is the sequence RVRR (SEQ ID NO: 37) that Furin recognizes and cleaves, but the cleavage sequence is not limited to this.
　機能性分子の他の例として、細胞膜透過促進分子を挙げることができる。すなわち、DOCK11結合ペプチドは、細胞膜透過促進分子と連結した形態にあってよい。細胞膜透過促進分子とは、エンドサイトーシスにより細胞内に取り込まれたDOCK11結合ペプチドが、エンドソーム内から膜を透過して細胞質内に放出するのを促進する作用を有する分子である。細胞膜透過促進分子の具体例として、土居らが開発した、ヒト胎盤形成における細胞融合に関与するシンシチン１というタンパク質の部分ペプチドであるS19（Sudo, K. et al., J. Control. Release, 255:1-11, 2017、Sudo, K. et al., J. Control. Release, 255:1-11, 2017、WO 2016/199674 A1）や、その後の検討により、膜透過促進ペプチドとしてS19よりもさらに効率的に機能しうるペプチドとして開発された、28残基のシンチシン1部分ペプチドS28（PFVIGAGVLGALGTGIGGITTSTQFYYK、配列番号38）及び39残基のシンチシン1部分ペプチドS39（PFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMER、配列番号39）を挙げることができる。これらのペプチド、特にS28及びS39は、本発明において好ましく使用できる細胞膜透過促進分子であるが、使用可能な細胞膜透過促進分子はこれらに限定されない。 Another example of a functional molecule is a cell membrane permeation promoting molecule. That is, the DOCK11-binding peptide may be in a form linked to a cell membrane permeabilization molecule. A cell membrane permeation-enhancing molecule is a molecule that promotes release of a DOCK11-binding peptide that has been taken up by endocytosis into the cytoplasm from the endosome through the membrane. As a specific example of a cell membrane permeation promoting molecule, S19 (Sudo, K. et al., J. Control. Release, 255 : 1-11, 2017, Sudo, K. et al., J. Control. Release, 255: 1-11, 2017, WO 2016/199674 A1). 28-residue syntisin 1 partial peptide S28 (PFVIGAGVLGALGTGIGGITTSTQFYYK, SEQ ID NO: 38) and 39-residue syntisin 1 partial peptide S39 (PFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMER, SEQ ID NO: 39), which were developed as peptides that can function more efficiently, can be mentioned. can. These peptides, particularly S28 and S39, are cell membrane permeation promoting molecules that can be preferably used in the present invention, but usable cell membrane permeation promoting molecules are not limited to these.
　細胞膜透過促進分子は、DOCK11結合ペプチドのいずれの末端に連結してもよいが、肝細胞内送達用担体分子及び切断配列をDOCK11結合ペプチドに連結する場合には、切断配列よりもDOCK11結合ペプチド側に、あるいは担体分子とは逆の末端に連結する。 The cell membrane permeation promoting molecule may be linked to either end of the DOCK11-binding peptide. or to the end opposite to the carrier molecule.
　機能性分子のさらなる例として、核移行シグナルを挙げることができる。すなわち、DOCK11結合ペプチドは、核移行シグナルと連結した形態にあってよい。核移行シグナルも種々のものが公知であり、いずれを用いてもよい。下記実施例では核移行シグナルとしてPAAKRVKLD（配列番号40）を利用しているが、この具体例に限定されない。核移行シグナルは、DOCK11結合ペプチドのいずれの末端に連結してもよいが、肝細胞内送達用担体分子及び切断配列をDOCK11結合ペプチドに連結する場合には、切断配列よりもDOCK11結合ペプチド側に、あるいは担体分子とは逆の末端に連結する。細胞膜透過促進分子と核移行シグナルをDOCK11結合ペプチドに連結する場合には、両末端にそれぞれ連結してもよいし、一方の末端に両方を連結してもよい。後者の場合、どちらを最末端に配置してもよい。送達用担体＋切断配列、細胞膜透過促進分子及び核移行シグナルをDOCK11結合ペプチドに連結する場合は、送達用担体＋切断配列を一方の末端に、細胞膜透過促進分子及び核移行シグナルを他方の末端に連結する。 A nuclear localization signal can be mentioned as a further example of a functional molecule. That is, the DOCK11-binding peptide may be in a form linked to a nuclear localization signal. Various nuclear localization signals are known, and any of them may be used. Although PAAKRVKLD (SEQ ID NO: 40) is used as a nuclear localization signal in the following examples, it is not limited to this specific example. The nuclear localization signal may be ligated to either end of the DOCK11-binding peptide. , or at the end opposite to the carrier molecule. When a cell membrane permeabilization molecule and a nuclear localization signal are linked to a DOCK11-binding peptide, both ends may be linked individually, or both may be linked to one end. In the latter case, either may be placed at the extreme end. When the delivery carrier + cleavage sequence, cell membrane permeabilization molecule and nuclear localization signal are ligated to the DOCK11-binding peptide, the delivery carrier + cleavage sequence is at one end and the cell membrane permeabilization molecule and nuclear localization signal are at the other end. Link.
　本発明の抗HBV剤の投与する主たる対象はHBV感染患者であり、B型肝炎患者、及びB型肝炎を発症していないHBV感染患者（HBVキャリア）が包含される。患者は典型的には哺乳動物、特にヒトであるが、これに限定されない。 The main subjects to whom the anti-HBV agent of the present invention is administered are HBV-infected patients, including hepatitis B patients and HBV-infected patients who have not developed hepatitis B (HBV carriers). Patients are typically, but not limited to, mammals, particularly humans.
　本発明の抗HBV剤の投与量は、投与対象の患者において抗HBV効果が得られる量であればよい。有効量は、患者の症状、ウイルス量、年齢、体重等に応じて適宜選択されうる。特に限定されないが、本発明の抗HBV剤の投与量は、対象に対し1日当たりの有効成分量として、体重1kg当たり1μg～10000mg程度、例えば100μg～1000mg程度としてよい。なお、ここでいう有効成分量とは、DOCK11結合ペプチドの場合には該ペプチド部分のみの量であり、DOCK11結合ペプチドに1種以上の他の機能性分子を連結したものを有効成分として用いる場合には、当該1種以上の他の機能性分子の量は上記の有効成分量には含まれていない。また、複数種類の物質を有効成分として用いる場合、有効成分量はその合計量である。1日の投与は1回でもよいし、数回に分けて投与してもよい。投与は毎日でもよいし、数日おきでもよい。 The dosage of the anti-HBV agent of the present invention may be any amount that provides an anti-HBV effect in the patient to whom it is administered. An effective dose can be appropriately selected according to the patient's symptoms, viral load, age, body weight and the like. Although not particularly limited, the dosage of the anti-HBV agent of the present invention may be about 1 μg to 10000 mg, for example about 100 μg to 1000 mg per 1 kg body weight of the active ingredient per day for the subject. In the case of DOCK11-binding peptides, the amount of active ingredient referred to here is the amount of the peptide portion only, and when using a DOCK11-binding peptide linked to one or more other functional molecules as an active ingredient. does not include the amount of said one or more other functional molecules in the above amounts of active ingredients. Moreover, when using a multiple types of substance as an active ingredient, the amount of active ingredients is the total amount. The daily dose may be administered once or divided into several doses. Administration may be daily or every few days.
　本発明の抗HBV剤の投与経路は、経口投与でも非経口投与でもよいが、一般には筋肉内投与、皮下投与、静脈内投与、動脈内投与等の非経口投与が好ましい。 The administration route of the anti-HBV agent of the present invention may be either oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration, and intraarterial administration is generally preferred.
　本発明の抗HBV剤の有効成分は、各投与経路に適した、薬剤的に許容される担体、希釈剤、賦形剤、結合剤、滑沢剤、崩壊剤、甘味剤、懸濁化剤、乳化剤、着色剤、矯味剤、安定剤等の添加剤と適宜混合して製剤することができる。製剤形態としては、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤などの経口剤や、吸入剤、注射剤、座剤、液剤などの非経口剤などを挙げることができる。製剤方法及び使用可能な添加剤は、医薬製剤の分野において周知であり、いずれの方法及び添加剤をも用いることができる。 The active ingredient of the anti-HBV agent of the present invention contains pharmaceutically acceptable carriers, diluents, excipients, binders, lubricants, disintegrants, sweeteners, suspending agents suitable for each administration route. , an emulsifier, a coloring agent, a corrigent, a stabilizer, and the like. Formulations include oral agents such as tablets, capsules, granules, powders and syrups, and parenteral agents such as inhalants, injections, suppositories and liquid agents. Formulation methods and usable excipients are well known in the field of pharmaceutical formulations, and any method and excipients can be used.
　2種以上の物質を有効成分として含む抗HBV剤は、2種以上の有効成分の全てを同一の製剤中に含む配合剤であってもよいし、各有効成分をそれぞれ単独で含有する単剤の組み合わせを含むものであってもよい。単剤の組み合わせを含む態様では、通常、単剤を同時に又は順次に投与するが、適当な間隔を置いて各単剤を投与しても差し支えない。 An anti-HBV agent containing two or more substances as active ingredients may be a combination drug containing all two or more active ingredients in the same formulation, or a single agent containing each active ingredient independently. may include a combination of In embodiments involving a combination of single agents, the single agents are usually administered simultaneously or sequentially, although each single agent may be administered at appropriate intervals.
　以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[実施例1]: DNAライブラリーの作成
（1-1）DNAライブラリーデザイン
　IVVスクリーニングのためのDNAライブラリーのデザインを図1に示した。
　In vitro virus(IVV)法（Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03/106675 A1）は、本願発明者の一人である柳川及びその共同研究者らが提案し、その構築に世界に先がけて成功した手法である。IVV法では、mRNAの3'末端にPEG（ポリエチレングリコール）スペーサーを介して抗生物質の一種のピューロマイシンを結合させ、それを鋳型として無細胞翻訳反応を行うことにより、タンパク質とmRNAがピューロマイシンを介して共有結合した単純なmRNA-タンパク質連結分子であるIVVが形成される（Miyamoto-Sato,E. et al., Nucleic Acids Res.,31: e78,2003）。こうして構築されたIVVライブラリーの中からベイト（タンパク質、ペプチド、抗原など）と結合するタンパク質を含むIVVをin vitroで釣り上げた後、そこに連結している遺伝子（mRNA）を逆転写・PCRで増幅し、DNAシーケンサーで塩基配列を解読することによって、相互作用するタンパク質やペプチドや抗体を容易に同定することができる。ベイトに結合したIVVを溶出・回収する方法は、フリーのベイトで競合溶出する方法が一般的であるが、競合溶出できない場合は、光開裂が可能な2-nitrobenzylリンカーを導入したPEGスペーサーを用い、365nmのUVを照射してスペーサーを切断することでmRNA部を溶出・回収できる(Doi,N. et al., J.Biotechnol.,131:231-239,2007)。 [Example 1]: Preparation of DNA library (1-1) DNA library design Fig. 1 shows the design of a DNA library for IVV screening.
In vitro virus (IVV) method (Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000 WO 03/106675 A1) is a technique proposed by Yanagawa, one of the inventors of the present application, and his co-researchers, who succeeded in constructing it for the first time in the world. In the IVV method, puromycin, a type of antibiotic, is attached to the 3' end of mRNA via a PEG (polyethylene glycol) spacer, and a cell-free translation reaction is performed using this as a template to bind puromycin to the protein and mRNA. IVV is formed, a simple mRNA-protein linking molecule covalently linked via (Miyamoto-Sato, E. et al., Nucleic Acids Res., 31: e78, 2003). From the IVV library constructed in this way, IVV containing proteins that bind to baits (proteins, peptides, antigens, etc.) are picked in vitro, and the genes (mRNA) linked to them are analyzed by reverse transcription and PCR. By amplifying and decoding the base sequence with a DNA sequencer, interacting proteins, peptides and antibodies can be easily identified. Competitive elution with free bait is the common method for eluting and recovering IVV bound to bait. , the mRNA portion can be eluted and recovered by cleaving the spacer by irradiation with UV at 365 nm (Doi, N. et al., J. Biotechnol., 131:231-239, 2007).
（1-2）DNAライブラリーの合成
　緑色蛍光タンパク質GFP配列を有するDNA溶液 1μl、10×KOD plus緩衝液(TOYOBO) 10μl、2 mM dNTPs(TOYOBO) 10μl、25mM MgSO4 4μl、forwardプライマー：GSP6-GFP-F (10 pmol/μl) 3μl、reverseプライマー：GFP-R (10 pmol/μl) 3μl、及びKOD plus ポリメラーゼ(TOYOBO) 2μlにRNase-Free水を添加して全体量を100μlとし、これを1チューブに入れ、合計300μl（チューブ3本）をPCR反応した。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を16サイクル行った後、68℃5分間反応を行った。DNAはWizard SV Gel PCR Clean-Up System（Promega）で精製し、50μlのGSP6-GFP-DNA溶液として回収した。 (1-2) Synthesis of DNA Library 1 μl of DNA solution having green fluorescent protein GFP sequence, 10 μl of 10×KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO 4 , forward primer: GSP6- 3 μl of GFP-F (10 pmol/μl), 3 μl of reverse primer: GFP-R (10 pmol/μl), and 2 μl of KOD plus polymerase (TOYOBO) were added with RNase-free water to bring the total volume to 100 μl. A total of 300 μl (3 tubes) of the mixture was put into one tube and subjected to PCR reaction. PCR was carried out by reacting at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. DNA was purified with Wizard SV Gel PCR Clean-Up System (Promega) and collected as 50 μl of GSP6-GFP-DNA solution.
　Atail配列を有するDNA溶液 1μl、10×KOD plus緩衝液(TOYOBO) 10μl、2 mM dNTPs(TOYOBO) 10μl、25mM MgSO4 4μl、forwardプライマー：Flag-His-F (10 pmol/μl) 3μl、reverseプライマー：Atail (R) (10 pmol/μl) 3μl、及びKOD plus ポリメラーゼ(TOYOBO) 2μlにRNase-Free水を添加して全体量を100μlとし、これを1チューブに入れ、合計300μl（チューブ3本）をPCR反応した。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を16サイクル行った後、68℃5分間反応を行った。DNAはWizard SV Gel PCR Clean-Up System（Promega）で精製し50μlのFlag-His Atail-DNA溶液として回収した。 DNA solution with Atail sequence 1 μl, 10×KOD plus buffer (TOYOBO) 10 μl, 2 mM dNTPs (TOYOBO) 10 μl, 25 mM MgSO 4 4 μl, forward primer: Flag-His-F (10 pmol/μl) 3 μl, reverse primer : Add RNase-free water to 3 μl of Atail (10 pmol/μl) and 2 μl of KOD plus polymerase (TOYOBO) to make the total volume 100 μl, put this in one tube, total 300 μl (3 tubes) was PCR-reacted. PCR was carried out by reacting at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. DNA was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of Flag-His Atail-DNA solution.
　Flag-His Atail-DNA溶液 1μl、10×KOD plus緩衝液(TOYOBO) 10μl、2 mM dNTPs(TOYOBO) 10μl、25mM MgSO4 4μl、forwardプライマー：16NNS-Fあるいは9NNS-F (10 pmol/μl) 3μl、reverseプライマー：Atail (R) (10 pmol/μl) 3μl、及びKOD plus ポリメラーゼ(TOYOBO) 2μlにRNase-Free水を添加して全体量を100μlとし、これを1チューブに入れ、合計300μl（チューブ3本）をPCR反応した。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を12サイクル行った後、68℃5分間反応を行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し50μlの16NNS Atail-DNA溶液あるいは9NNS Atail-DNA溶液として回収した。 Flag-His Atail-DNA solution 1 μl, 10×KOD plus buffer (TOYOBO) 10 μl, 2 mM dNTPs (TOYOBO) 10 μl, 25 mM MgSO 4 4 μl, forward primer: 16NNS-F or 9NNS-F (10 pmol/μl) 3 μl , reverse primer: Atail (R) (10 pmol/μl) 3 μl, and KOD plus polymerase (TOYOBO) 2 μl, add RNase-free water to make the total volume 100 μl, put this in one tube, total 300 μl (tube 3) were subjected to PCR reaction. PCR was carried out by reacting at 94°C for 5 minutes, followed by 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of 16NNS Atail-DNA solution or 9NNS Atail-DNA solution.
　GSP6-GFP-DNA溶液1μl、16NNS Atail-DNA溶液あるいは9NNS Atail-DNA溶液 1μl、10×KOD plus緩衝液(TOYOBO) 10μl、2 mM dNTPs(TOYOBO) 10μl、25mM MgSO4 4μl、forwardプライマー：GSP6-GFP-F (10 pmol/μl) 3μl、reverseプライマー：Atail (R) (10 pmol/μl) 3μl、及びKOD plus ポリメラーゼ(TOYOBO) 2μlにRNase-Free水を添加して全体量を100μlとし、これを1チューブに入れ、合計300μl（チューブ3本）をオーバーラップPCR反応した。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を12サイクル行った後68℃5分間反応を行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し50μlのcDNAライブラリー16NNSLibならびに9NNSLibとして回収した。 1 μl of GSP6-GFP-DNA solution, 1 μl of 16NNS Atail-DNA solution or 9NNS Atail-DNA solution, 10 μl of 10×KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO 4 , forward primer: GSP6- 3 μl of GFP-F (10 pmol/μl), 3 μl of reverse primer: Atail (R) (10 pmol/μl), and 2 μl of KOD plus polymerase (TOYOBO) were added with RNase-free water to bring the total volume to 100 μl. was placed in one tube, and a total of 300 μl (3 tubes) was subjected to overlap PCR reaction. PCR was carried out at 94°C for 5 minutes, followed by 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of cDNA library 16NNSLib and 9NNSLib.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
[実施例2]: IVVライブラリーの調製
（2-1）ライブラリーの転写
　cDNAライブラリー16NNSLib ならびに9NNSLib をそれぞれ2pmol、5×SP6緩衝液 8μl、ATP (100mM) 2μl、CTP (100mM) 2μl、UTP (100mM) 2μl、GTP (10mM) 4μl、キャップアナログ(m7G(5')PPP(5')G) (Thermo Fisher Scientific) (40mM) 5μl、エンザイムミックスSP6RNA ポリメラーゼ（Promega） 4μl、RNase-Free水を添加し全体量を 40μlとし、37℃、3時間反応後、RQ1 RNase-Free DNase（Promega）10μlを添加し、さらに37℃、1時間反応させた。 [Example 2]: Preparation of IVV library (2-1) Transcription of library 2 pmol each of cDNA libraries 16NNSLib and 9NNSLib, 8 μl of 5×SP6 buffer, 2 μl of ATP (100 mM), 2 μl of CTP (100 mM), UTP (100 mM) 2 μl, GTP (10 mM) 4 μl, cap analog (m7G(5')PPP(5')G) (Thermo Fisher Scientific) (40 mM) 5 μl, Enzyme Mix SP6 RNA polymerase (Promega) 4 μl, RNase-Free water. After 3 hours of reaction at 37°C, 10 µl of RQ1 RNase-Free DNase (Promega) was added and further reacted at 37°C for 1 hour.
　RNAはRNeasy Mini kit (Qiagen)により精製した。すなわち転写反応液に、RNase-Free水を添加し全体量を100μlとし、RLT緩衝液(Qiagen) 350μl、2-メルカプトエタノール 3.5μl、(100%) エタノール 250μl、を加えRNeasy ミニスピンカラムに供し、25℃、12000 rpm、15秒間遠心後排出された溶液を除去し、RPE緩衝液(Qiagen)500μlを同カラムに加え、25℃、12000 rpm、15秒間遠心後排出された溶液を除去し、さらにRPE緩衝液(Qiagen)500μlを同カラムに加え、25℃、12000 rpm、2分間遠心後排出された溶液を除去し、同カラムを新しいチューブに差し替え、25℃、12000 rpm、1分間遠心分離し、再び同カラムを新しいチューブに差し替え同カラムに、RNase-Free水を33μl添加し、10分間室温で放置後25℃、13200 rpm、1分間遠心しRNA溶液として回収した。 　RNA was purified using the RNeasy Mini kit (Qiagen). That is, RNase-free water was added to the transcription reaction solution to bring the total volume to 100 μl, and 350 μl of RLT buffer (Qiagen), 3.5 μl of 2-mercaptoethanol, and 250 μl of (100%) ethanol were added to the RNeasy mini spin column. After centrifuging at 25°C, 12000 rpm for 15 seconds, remove the discharged solution, add 500 μl of RPE buffer (Qiagen) to the same column, remove the discharged solution after centrifuging at 25°C, 12000 rpm for 15 seconds, and Add 500 μl of RPE buffer (Qiagen) to the same column, centrifuge at 25°C, 12,000 rpm for 2 minutes, remove the discharged solution, replace the column with a new tube, and centrifuge at 25°C, 12,000 rpm for 1 minute. 33 μl of RNase-free water was added to the same column, which was again replaced with a new tube, allowed to stand at room temperature for 10 minutes, centrifuged at 25° C., 13200 rpm for 1 minute, and collected as an RNA solution.
（2-2）PEGスペーサーとのライゲーション
　RNA溶液 32μl、T4 ligation 10×緩衝液 5μl、0.1 M DTT 1μl、40 mM ATP 0.5μl、100% DMSO 5μl、0.1% BSA 1μl、RNase inhibitor(TOYOBO) 1μl、ピューロマイシン付きポリエチレングリコール光切断リンカー分子 (10 nmol) 0.5μl、ポリエチレングリコール（PEG） 2000 （日本油脂）(30 nmol) 1μl、T4 RNA リガーゼ (Takara)(40 U/μl) 3μlを混合し、遮光条件下15℃、15時間反応させた。得られたスペーサー分子が結合したPEG-RNAはRNeasy Mini kit (Qiagen)により精製した。 (2-2) Ligation RNA solution with PEG spacer 32 μl, T4 ligation 10× buffer 5 μl, 0.1 M DTT 1 μl, 40 mM ATP 0.5 μl, 100% DMSO 5 μl, 0.1% BSA 1 μl, RNase inhibitor (TOYOBO) 1 μl, 0.5 μl of puromycin-attached polyethylene glycol photocleavable linker molecule (10 nmol), 1 μl of polyethylene glycol (PEG) 2000 (NOF) (30 nmol), and 3 μl of T4 RNA ligase (Takara) (40 U/μl) were mixed and protected from light. The reaction was carried out at 15°C for 15 hours. The obtained PEG-RNA bound with a spacer molecule was purified using the RNeasy Mini kit (Qiagen).
（2-3）IVVライブラリーの調製
　PEG-RNA 10 pmol、小麦胚芽抽出液（ZoeGene）20 μl、クレアチンキナーゼ (40 μg/μl)（ZoeGene） 2μl、RNase inhibitor(TOYOBO) 3.2 μl、及び5×翻訳緩衝液(ZoeGene) 20μlにRNase-Free水を添加し全体量を100μlとし、遮光条件下26℃、1時間反応させ翻訳を行い、IVVライブラリーを調製した。 (2-3) IVV library preparation PEG-RNA 10 pmol, wheat germ extract (ZoeGene) 20 μl, creatine kinase (40 μg/μl) (ZoeGene) 2 μl, RNase inhibitor (TOYOBO) 3.2 μl, and 5× RNase-free water was added to 20 μl of translation buffer (ZoeGene) to make the total volume 100 μl, and reaction was performed for 1 hour at 26° C. under light-shielded conditions to carry out translation to prepare an IVV library.
[実施例3]: ビオチン化DOCK11の調製
　DOCK11のヒトDOCK180 superfamilyの構造を図2に示した。構造の類似性から大きく四つのグループに分けられる。DOCKファミリータンパク質にはファミリー間て゛アミノ酸配列か゛よく保存されている二つの領域，Dock homology region 1(DHR1)とDHR2か゛存在し、DHR1は、ホスファチジルイノシトール3-リン酸と結合する。DHR2ドメインは、それそ゛れ特異的なRhoファミリーGタンパク質を活性化する。DOCK11はDOCK-Dグループに属し、N末端領域にPHドメインを有している。DOCK11は低分子量Gタンパク質Cdc42を活性化し、グアニンヌクレオチド交換因子（GEF）としてはたらく。DOCK11結合ペプチドのIVVスクリーニングにおいて、全長のDOCK11(aa1-2073、配列番号42)のアミノ酸配列のC末端にビオチン化配列、Flag-tag及びHis-tagを付加した。またDHR2ドメインを含むDOCK11のC末側領域のペプチド(配列番号42のうちのaa1516-2073)のアミノ酸配列のC末端にビオチン化配列、Flag-tag及びHis-tagを付加した。 [Example 3]: Preparation of biotinylated DOCK11 The structure of the human DOCK180 superfamily of DOCK11 is shown in FIG. They are roughly divided into four groups based on structural similarities. DOCK family proteins have two regions, Dock homology region 1 (DHR1) and DHR2, whose amino acid sequences are highly conserved among families. DHR1 binds to phosphatidylinositol 3-phosphate. DHR2 domains activate their own specific Rho family G proteins. DOCK11 belongs to the DOCK-D group and has a PH domain in the N-terminal region. DOCK11 activates the small G protein Cdc42 and acts as a guanine nucleotide exchange factor (GEF). In IVV screening for DOCK11-binding peptides, a biotinylation sequence, Flag-tag and His-tag were added to the C-terminus of the amino acid sequence of full-length DOCK11 (aa1-2073, SEQ ID NO: 42). In addition, a biotinylation sequence, Flag-tag and His-tag were added to the C-terminus of the amino acid sequence of the DOCK11 C-terminal region peptide (aa1516-2073 in SEQ ID NO: 42) containing the DHR2 domain.
（3-1）ビオチン化全長DOCK11の発現ベクター構築
　図３に示したように、初めに全長のDOCK11遺伝子（配列番号41）にビオチン化配列、Flag-tag、His-tag配列を付加したpcDNA3.3 TOPO KDOCK11-BioFLAGHisベクターを作成した。 (3-1) Construction of an expression vector for biotinylated full-length DOCK11 As shown in FIG. 3, first, pcDNA3, in which a biotinylated sequence, a Flag-tag, and a His-tag sequence were added to the full-length DOCK11 gene (SEQ ID NO: 41). 3 TOPO KDOCK11-BioFLAGHis vector was generated.
　BioEaseTM plasmidよりF-Bioプライマー、R-Bioプライマーを用いてPCRにて調製したKlebsiella pneumoniaeのオキサロ酢酸脱炭酸酵素αサブユニットの遺伝子（72アミノ酸分）からなるBio-tag（Nucleic Acids Research, 2009, Vol. 37, No. 8, page e64）に、Flag-tagとHis-tagを付加させた。Bio-tag 2.37μl、10×KOD plus緩衝液(TOYOBO) 40μl、2 mM dNTPs(TOYOBO) 40μl、25mM MgSO4 16μl、KstartBio-F (10 pmol/μl) 12μl、Bio-Flag-Histag A stop (10 pmol/μl) 12μl、及びKOD plus ポリメラーゼ(TOYOBO) 8μlにRNase-Free水を添加して全体量を200μlとし、PCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を20あるいは25サイクル行った後68℃5分間反応を行った。PCR産物はアガロースゲル電気泳動でDNAのバンドを確認後、Wizard SV Gel PCR Clean-Up System(Promega)で精製し50μlのDNA溶液として回収しKstartBio-Flag-Hisを得た。 Bio-tag (Nucleic Acids Research, 2009 , Vol. 37, No. 8, page e64) was added with Flag-tag and His-tag. Bio-tag 2.37 μl, 10×KOD plus buffer solution (TOYOBO) 40 μl, 2 mM dNTPs (TOYOBO) 40 μl, 25 mM MgSO 4 16 μl, KstartBio-F (10 pmol/μl) 12 μl, Bio-Flag-Histag A stop (10 pmol/μl) and 8 μl of KOD plus polymerase (TOYOBO), RNase-free water was added to make the total volume 200 μl, and PCR reaction was performed. PCR was carried out by reacting at 94°C for 5 minutes, followed by 20 or 25 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of DNA solution to obtain KstartBio-Flag-His.
　KstartBio-Flag-HisのpcDNA 3.3ベクターへの導入はTOPO クローニングキット（Invitrogen）で手順に従って行った。得られたクローンが正しい配列であることを確認し、コロニーを植菌し37℃、16時間培養した。菌体ペレットからPureYieldTM Plasmid Maxiprep System (Promega)でプラスミドpcDNA3.3 TOPO KBioFlagHisを精製した。 KstartBio-Flag-His was introduced into the pcDNA 3.3 vector using the TOPO cloning kit (Invitrogen) according to the procedure. After confirming that the resulting clone had the correct sequence, a colony was inoculated and cultured at 37°C for 16 hours. Plasmid pcDNA3.3 TOPO KBioFlagHis was purified from the cell pellet with the PureYield ™ Plasmid Maxiprep System (Promega).
　DOCK11ベクター pF1KE2360 (Kazusa DNA Res.Inst.) (1 ng/μl) 0.5μl、KAPA HiFi HS RM 12.5μl、10μM TOPOKstartDOCK11-IF-F 0.75μl、及び10μM DOCK11Bio-IF-R 0.75μlにRNase-Free水を添加して全体量を25μlとし、PCR反応を行った。PCRは、95℃5分間反応後、98℃20秒間、60℃15秒間、72℃1分間を25サイクル行った後72℃1分間反応を行った。PCR産物はアガロースゲル電気泳動でDNAのバンドを確認後、Wizard SV Gel PCR Clean-Up System(Promega)で精製し30μlのDNA溶液として回収しKstart-DOCK11-IFを得た。 DOCK11 vector pF1KE2360 (Kazusa DNA Res.Inst.) (1 ng/μl) 0.5 μl, KAPA HiFi HS RM 12.5 μl, 10 μM TOPOKstartDOCK11-IF-F 0.75 μl, and 10 μM DOCK11Bio-IF-R 0.75 μl in RNase-Free water was added to make the total volume 25 μl, and the PCR reaction was performed. PCR was carried out at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 1 minute. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 30 μl of DNA solution to obtain Kstart-DOCK11-IF.
　pcDNA3.3 TOPO KBioFlagHis 0.5μl、KAPA HiFi HS RM　10μl、10μM F-Bio　0.6μl、10μM TOPOKstartINV-R 0.6μlにRNase-Free水を添加して全体量を20μlとし、PCR反応させた。PCRは、95℃3分間反応後、98℃20秒間、60℃15秒間、72℃3分間を25サイクル行った後、72℃1分間反応を行った。PCR産物はアガロースゲル電気泳動で確認後、Wizard SV Gel PCR Clean-Up System(Promega)で精製し30μlのDNA溶液として回収しTOPO KBioFlagHisINVを得た。 RNase-free water was added to 0.5 μl of pcDNA3.3 TOPO KBioFlagHis, 10 μl of KAPA HiFi HS RM, 0.6 μl of 10 μM F-Bio, and 0.6 μl of 10 μM TOPOKstartINV-R to make the total volume 20 μl, and a PCR reaction was performed. PCR was carried out by reacting at 95°C for 3 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 3 minutes, followed by reaction at 72°C for 1 minute. After confirming the PCR product by agarose gel electrophoresis, it was purified by Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 30 μl of DNA solution to obtain TOPO KBioFlagHisINV.
　Kstart-DOCK11-IF 1.0μl、TOPO KBioFlagHisINV 1.0μl、5x in fusion HD Enzyme premix 1.0μl (Takara) にRNase-Free水を添加して全体量を5μlとし、50℃、15分間反応させた。2.5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、クローンを得た。クローンの配列解析の結果、プラスミドpcDNA3.3 TOPO KDOCK11-BioFLAGHisであることを確認した。 Kstart-DOCK11-IF 1.0 μl, TOPO KBioFlagHisINV 1.0 μl, 5x infusion HD Enzyme premix 1.0 μl (Takara) was added with RNase-free water to make the total volume 5 μl, and reacted at 50°C for 15 minutes. 2.5 μl was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain clones. Sequence analysis of the clone confirmed the plasmid pcDNA3.3 TOPO KDOCK11-BioFLAGHis.
（3-2）ビオチン化C末側領域DOCK11の発現ベクター構築
　次にC末側領域のDOCK11遺伝子にビオチン化配列、Flag-tag、His-tag配列を付加したpcDNA3.3 TOPO KDOCK11(1516-2073)-BioFLAGHisベクターを作成した。 (3-2) Construction of expression vector for biotinylated C-terminal region DOCK11 )-BioFLAGHis vector was generated.
　DHR2ドメインを含むDOCK11のC末側領域のペプチド(aa1516-2073)にビオチン化配列、Flag-tag、His-tag配列を付加したDOCK11(1516-2073)-BioFLAGHis-pcDNAベクターを作成するためにKOD-Plus-Mutagenesis Kit（TOYOBO）でInverse PCRを行なった。pcDNA3.3 TOPO KDOCK11-BioFLAGHis プラスミド0.14μl、10×buffer for iPCR 5μl、2 mM dNTPs 5μl、forwardプライマー：DOCK11-1516-F(10 pmol/μl) 1.5μl、reverseプライマー：Kstart-R (10 pmol/μl) 1.5μl、KOD plus ポリメラーゼ1μlにRNase-Free水を添加し全体量を50μlとしてPCR反応させた。PCRは94℃、2分間反応後98℃、10秒間、68℃ 10分間を6サイクル行った。続いてPCR product 50μlに制限酵素Dpn I 12μlを加え37℃、1時間反応させ鋳型 Plasmid DNAを分解した。Dpn I処理済みproduct 2μl、Ligation high 5μl、T4 Polynucleotide Kinase 1μl を加え16℃、1時間反応させPCR 産物を自己環状化させた。Ligation product5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、クローンを得た。クローンの配列解析の結果、プラスミドpcDNA3.3 TOPO KDOCK11(1516-2073)-BioFLAGHisであることを確認した。 KOD to create DOCK11(1516-2073)-BioFLAGHis-pcDNA vector with biotinylated sequence, Flag-tag, and His-tag sequences added to DOCK11 C-terminal region peptide (aa1516-2073) containing DHR2 domain Inverse PCR was performed with the -Plus-Mutagenesis Kit (TOYOBO). pcDNA3.3 TOPO KDOCK11-BioFLAGHis plasmid 0.14 μl, 10×buffer for iPCR 5 μl, 2 mM dNTPs 5 μl, forward primer: DOCK11-1516-F (10 pmol/μl) 1.5 μl, reverse primer: Kstart-R (10 pmol/ μl) 1.5 μl, RNase-free water was added to 1 μl of KOD plus polymerase to bring the total volume to 50 μl, and PCR was performed. PCR was performed at 94°C for 2 minutes, followed by 6 cycles of 98°C for 10 seconds and 68°C for 10 minutes. Subsequently, 12 μl of restriction enzyme DpnI was added to 50 μl of the PCR product and allowed to react at 37° C. for 1 hour to degrade the template plasmid DNA. 2 μl of Dpn I-treated product, 5 μl of Ligation high, and 1 μl of T4 Polynucleotide Kinase were added and allowed to react at 16°C for 1 hour to self-circularize the PCR product. 5 μl of the ligation product was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain clones. Sequence analysis of the clone confirmed the plasmid pcDNA3.3 TOPO KDOCK11(1516-2073)-BioFLAGHis.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（3-3）ビオチン化全長DOCK11あるいはビオチン化C末側領域DOCK11の発現と精製
　ヒト胎児腎細胞293T細胞は10%FCS(ニチレイ)、DMEM(1.0g/l Glucose) with L-Gln and Sodium Pyruvate, liquid (ナカライ)培地で培養した。トランスフェクションの24時間前に6cmシャーレに(1:2)の割合でプレーティングした。1枚のシャーレに対しプラスミドpcDNA3.3 TOPO KDOCK11-BioFLAGHisあるいはpcDNA3.3 TOPO KDOCK11(1516-2073)-BioFLAGHisをOpti-MEM I 500μlに入れたものに、Lipofectamine 2000 20μlをOpti-MEM I 500μlに入れ室温で5分間置いたものを穏やかに加えた。混和物は室温で20分間放置後293T細胞のシャーレに加え37℃、5% CO2条件下１日培養した。 (3-3) Expression and Purification of Biotinylated Full-Length DOCK11 or Biotinylated C-Terminal Region DOCK11 , liquid (Nacalai) medium. 24 hours before transfection, the cells were plated on a 6 cm petri dish at a ratio (1:2). Plasmid pcDNA3.3 TOPO KDOCK11-BioFLAGHis or pcDNA3.3 TOPO KDOCK11(1516-2073)-BioFLAGHis was added to 500 μl of Opti-MEM I, and 20 μl of Lipofectamine 2000 was added to 500 μl of Opti-MEM I. What was left at room temperature for 5 minutes was gently added. The mixture was allowed to stand at room temperature for 20 minutes, then added to a petri dish of 293T cells and cultured for 1 day at 37°C and 5% CO 2 .
　シャーレの培地を除去後、冷PBS 2mlで細胞を洗った。RIPA buffer (25 mM Tris・HCl、150 mM NaCl、1% NP-40、1% sodium deoxycholate、0.1% SDS、pH 7.6）800μl、プロテアーゼ阻害剤、動物細胞抽出物用(Cat#25955-11) x50（フッ化4-(2-アミノエチル)、ベンゼンスルホニル塩酸塩(AEBSF)、アプロチニン、E-64、ロイペプチンヘミ硫酸塩一水和物、ベスタチン、ペプスタチン A）を16μl、0.1M PMSF 8μlを細胞に加え15分間氷上で放置した。セルスクレーパーで細胞を剥がしチューブに集め21Gの注射針でホモジネート後60分間氷上で放置した。12,300 rpm、30分間 4℃で遠心分離し、上清を新しいチューブに移し細胞画分とした。それぞれ全部で6cmシャーレ4枚をトランスフェクションし細胞画分を得た。これらを使って固定用のビオチン化全長DOCK11あるいはビオチン化C末側領域DOCK11を精製した。TALON magnetic beads (Clontech) 500μl(50%スラリー)をTBS buffer (ナカライ) 5.0 mlで3回洗浄したものに上記のそれぞれの細胞画分を加え、4℃で１晩ミニディスクローター（Bio craft）で回転攪拌した。ビーズをTBS buffer 5.0 mlで3回洗浄した。500mM imidazoleを含むTBS 500μlを加え4℃で１時間ミニディスクローターで回転攪拌した後上清を集めた（溶出画分 E1）。残ったビーズに500mM imidazoleを含むTBS 250μlを加え、同様の操作で溶出画分 E2を回収した。溶出画分 E1 15μlにサンプル緩衝液LDS(4x)、0.2mM DTT 6.8μlを加え70℃、10分間加熱後SDS-PAGEに供した。SDS-PAGEは4-12% Bis-Tris NuPAGE ゲル、MES電気泳動緩衝液 (Invitrogen)で200V, 400mA, 35分間電気泳動後、Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turboで転写した。膜はBlocking One Buffer：TBST（1：9）でブロッキングし、Blocking One Buffer：TBST(1：9)で2:3000に希釈したanti-Flag-HRP (Sigma: A8592)を反応させた。検出はECL(Enhanced ChemiLuminescence)を用い、ChemiDoc(BIORAD)で行った。ビオチン化全長DOCK11は分子量246,762 Daあるいはビオチン化C末側領域DOCK11は分子量73,404 Daのバンドとして確認することができた。 After removing the medium from the petri dish, the cells were washed with 2 ml of cold PBS. RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6) 800 μl, protease inhibitor, for animal cell extracts (Cat#25955-11) x50 16 μl of (4-(2-aminoethyl)fluoride, benzenesulfonyl hydrochloride (AEBSF), aprotinin, E-64, leupeptin hemisulfate monohydrate, bestatin, pepstatin A) and 8 μl of 0.1M PMSF were added to the cells. Left on ice for 15 minutes. The cells were scraped off with a cell scraper, collected in a tube, homogenized with a 21G injection needle, and left on ice for 60 minutes. After centrifugation at 4°C for 30 minutes at 12,300 rpm, the supernatant was transferred to a new tube and used as a cell fraction. A total of four 6 cm petri dishes were transfected to obtain cell fractions. Using these, biotinylated full-length DOCK11 or biotinylated C-terminal region DOCK11 for immobilization was purified. 500 μl (50% slurry) of TALON magnetic beads (Clontech) was washed 3 times with 5.0 ml of TBS buffer (Nacalai), and each of the above cell fractions was added. Rotate and stir. The beads were washed 3 times with 5.0 ml of TBS buffer. After adding 500 μl of TBS containing 500 mM imidazole and stirring with a mini disc rotor at 4° C. for 1 hour, the supernatant was collected (elution fraction E1). 250 μl of TBS containing 500 mM imidazole was added to the remaining beads, and the eluted fraction E2 was recovered by the same operation. Sample buffer LDS (4x) and 6.8 μl of 0.2 mM DTT were added to 15 μl of the eluted fraction E1, heated at 70° C. for 10 minutes, and subjected to SDS-PAGE. SDS-PAGE was performed on a 4-12% Bis-Tris NuPAGE gel with MES electrophoresis buffer (Invitrogen) at 200V, 400mA, 35 minutes, followed by Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turbo. transcribed. The membrane was blocked with Blocking One Buffer: TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer: TBST (1:9). Detection was performed using ChemiDoc (BIORAD) using ECL (Enhanced ChemiLuminescence). Biotinylated full-length DOCK11 was identified as a band with a molecular weight of 246,762 Da, and biotinylated C-terminal region DOCK11 was identified as a band with a molecular weight of 73,404 Da.
[実施例4]: DOCK11と結合するペプチドの選択
　DOCK11と結合するペプチドの選択実験を図４に示すような手順で行った。
（4-1）DOCK11の固定化
　ビアコアはビアコア3000システムを用い、センサーチップSAにビオチン化C末側領域DOCK11の固定化を行った。フローは緩衝液HBS-P(10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20) で10μl/minで行った。フローセル１～4への50mM NaOH, 1M NaClを含む溶液10μlのインジェクトを3回繰り返し行い、固定化の前処理を行った。ビオチン化C末側領域DOCK11（1nM）を用いて、フローセル１～４に固定化した。フローは緩衝液HBS-P、20μl/minで行った。23μlをマニュアルインジェクションしたところフローセル１～４に1267.4 RU（平均）結合した。センサーチップを洗浄する目的で緩衝液HBS-Pで10μl/min, 50% Isopropanol, 50mM NaOH, 1M NaClを用いてextra washを行った。 [Example 4]: Selection of peptides that bind to DOCK11 A selection experiment for peptides that bind to DOCK11 was performed according to the procedure shown in Fig. 4 .
(4-1) Immobilization of DOCK11 Biacore immobilized the biotinylated C-terminal region DOCK11 on the sensor chip SA using the Biacore 3000 system. Flow was performed at 10 μl/min in buffer HBS-P (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20). Pretreatment for immobilization was performed by injecting 10 μl of a solution containing 50 mM NaOH and 1 M NaCl into flow cells 1 to 4 three times. Biotinylated C-terminal region DOCK11 (1 nM) was used to immobilize to flow cells 1-4. Flow was in buffer HBS-P, 20 μl/min. Manual injection of 23 μl resulted in 1267.4 RU (average) binding to flow cells 1-4. For the purpose of washing the sensor chip, an extra wash was performed with a buffer solution HBS-P at 10 μl/min using 50% Isopropanol, 50 mM NaOH and 1M NaCl.
（4-2）DOCK11と結合するペプチドの選択
　調製したIVVライブラリー100 μlにfinal 20mMとなるように0.5M EDTA 4μlを加え、20分間室温で回転攪拌した。HBS-Pで膨潤ならびに平衡化させたSephadex G200 (Amersham Biosciences)ゲル1 mlをカラム（バイオラッド）に充填したものにIVVライブラリー溶液100μlを供し、2滴ずつ96穴プレートに集め１wellから10wellまでを回収した。Multi-detection Microplate Reader POWERSCAN HTで励起波長485nm、蛍光波長528nmで蛍光を検出し、3well目から7well目あたりに溶出したIVV画分を集めた。Strep Magne Sphae Paramagnetic Part (9013-20-1) 100μlをHBS-P 500μlで3回洗浄したものにIVV画分約200μlを加え、20分間室温で回転攪拌し、その上清をビアコアにインジェクトした（図4）。ビアコアでのセレクションは緩衝液HBS-Pで40μl/minで行い、160秒結合、5000秒解離させた。その後、センサーチップを機械より抜き取った。センサーチップの金膜の上にRNase-Free水55μlを静かに加え、20分間、365nmのUVを照射してスペーサーを切断してmRNA部を溶出・回収した。 (4-2) Selection of DOCK11-Binding Peptide To 100 μl of the prepared IVV library, 4 μl of 0.5 M EDTA was added to a final concentration of 20 mM, and the mixture was rotated and stirred at room temperature for 20 minutes. Apply 100 μl of the IVV library solution to a column (Bio-Rad) packed with 1 ml of Sephadex G200 (Amersham Biosciences) gel swollen and equilibrated with HBS-P, collect 2 drops in 96-well plates from 1 well to 10 wells. recovered. Fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Multi-detection Microplate Reader POWERSCAN HT, and IVV fractions eluted from the 3rd well to the 7th well were collected. About 200 μl of the IVV fraction was added to 100 μl of Strep Magne Sphae Paramagnetic Part (9013-20-1) washed three times with 500 μl of HBS-P, and the mixture was stirred at room temperature for 20 minutes, and the supernatant was injected into Biacore. (Fig. 4). Biacore selection was performed with buffer HBS-P at 40 μl/min, binding for 160 seconds and dissociation for 5000 seconds. After that, the sensor chip was extracted from the machine. 55 μl of RNase-free water was gently added to the gold film of the sensor chip, and UV light of 365 nm was applied for 20 minutes to cleave the spacer, and the mRNA part was eluted and collected.
（4-3）RT-PCRによるcDNAライブラリーの回収
　上記（4-2）の選択実験で回収した溶液55μl、5×RT緩衝液(TOYOBO) 20μl、(10 mM) dNTPs(TOYOBO) 10μl、及びreverseプライマー：Atail (R) (10pmol/μl) 5μlにRNase-Free水を添加し全体量を90μlとして混和させ、65℃で9分間反応後直ちに氷上に冷却し、2分間放置した後、ReverTra Ace(TOYOBO) 5μl及びRNase inhibitor(TOYOBO) 5μlを加え、50℃30分間、99℃、5分間逆転写反応させた。逆転写反応させた反応溶液100μl、10×KOD plus緩衝液(TOYOBO) 100μl、2 mM dNTPs(TOYOBO) 100μl、25mM MgSO4 40μl、forwardプライマー：GSP6-GFP-F (10 pmol/μl) 30μl、reverseプライマー：Atail (R) (10 pmol/μl) 30μl、及びKOD plus ポリメラーゼ(TOYOBO) 20μlにRNase-Free水を添加して全体量を1000μlとしチューブ１本につき100μl入れ合計１０本をPCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を20から40サイクル行った後、68℃で5分間反応を行った。 (4-3) Recovery of cDNA library by RT-PCR 55 μl of the solution recovered in the selection experiment of (4-2) above, 20 μl of 5×RT buffer (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and Reverse primer: Atail (R) (10 pmol/μl) Add RNase-free water to 5 μl to bring the total volume to 90 μl. 5 μl of (TOYOBO) and 5 μl of RNase inhibitor (TOYOBO) were added, and reverse transcription reaction was carried out at 50° C. for 30 minutes and 99° C. for 5 minutes. 100 μl of reverse transcription reaction solution, 100 μl of 10×KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO 4 , forward primer: GSP6-GFP-F (10 pmol/μl) 30 μl, reverse Primers: RNase-free water was added to 30 μl of Atail (10 pmol/μl) and 20 μl of KOD plus polymerase (TOYOBO) to make a total volume of 1000 μl, and 100 μl was added to each tube and a total of 10 tubes were subjected to PCR reaction. . PCR was carried out at 94°C for 5 minutes, followed by 20 to 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
　ビアコアを使った選択実験の選択圧は表3に示すように段階的にラウンドごとに選択圧を順次上げた。 The selection pressure in the selection experiment using Biacore was increased step by step for each round as shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
[実施例5]: クローニングと塩基配列の決定
（5-1）クローニングと塩基配列
　DOCK11と結合するペプチドライブラリーから in-fusionクローニング
ライブラリーのinsert作成
　８ラウンドの選択実験を行ったライブラリー　1μl、10×KOD plus緩衝液(TOYOBO) 100μl、2 mM dNTPs(TOYOBO) 100μl、25mM MgSO4 40μl、GFP-F in (10 pmol/μl) 30μl、His-S28-R in (10 pmol/μl) 30μl、及びKOD plus ポリメラーゼ(TOYOBO) 20μlにRNase-Free水を添加して全体量を1000μlとし、PCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を8サイクル行った後、68℃5分間反応を行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、50μlのDNA溶液として回収しGFP-DC-S8 inを得た。 [Example 5]: Cloning and nucleotide sequence determination (5-1) Cloning and nucleotide sequence Creation of an in-fusion cloning library insert from a peptide library that binds to DOCK11 Library subjected to 8 rounds of selection experiment 1 μl, 10×KOD plus buffer (TOYOBO) 100 μl, 2 mM dNTPs (TOYOBO) 100 μl, 25 mM MgSO 4 40 μl, GFP-F in (10 pmol/μl) 30 μl, His-S28-R in (10 pmol/μl) 30 μl, RNase-free water was added to 20 μl of KOD plus polymerase (TOYOBO) to bring the total volume to 1000 μl, followed by PCR reaction. PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 µl of DNA solution to obtain GFP-DC-S8in.
　kozak-GFP配列とFlagHisS28配列とstopコドンを含む pcDNA 3.3ベクター8μl、10×KOD plus緩衝液(TOYOBO) 80μl、2 mM dNTPs(TOYOBO) 80μl、25mM MgSO4 32μl、S28-F iv (10 pmol/μl) 24μl、3.3K-GFP-Riv (10 pmol/μl) 24μl、及びKOD plus ポリメラーゼ(TOYOBO) 16μlにRNase-Free水を添加して全体量を800μlとし、PCR反応させた。PCRは、94℃2分間反応後、98℃10秒間、68℃5分11秒間を25サイクル行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、40μlのDNA溶液として回収しKGFP-S28 3.3 vectorを得た。 8 μl of pcDNA 3.3 vector containing kozak-GFP sequence, FlagHisS28 sequence and stop codon, 80 μl of 10×KOD plus buffer (TOYOBO), 80 μl of 2 mM dNTPs (TOYOBO), 32 μl of 25 mM MgSO 4 , S28-F iv (10 pmol/μl ), 24 μl of 3.3K-GFP-Riv (10 pmol/μl), and 16 μl of KOD plus polymerase (TOYOBO) were added with RNase-free water to make the total volume 800 μl, and subjected to PCR reaction. PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 μl of DNA solution to obtain KGFP-S28 3.3 vector.
　GFP-DC-S8 in 0.08μl、KGFP-S28 3.3 vector 0.14μl、5x in fusion HD Enzyme premix 1.0μl (Takara) を混ぜ、RNase-Free水を添加し全体量を5μlとし50℃、15分間反応させた。反応後の溶液2.5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、KGFP-DCS8クローンを得た。得られたクローンの配列解析はユーロフィンDNAシーケンス受託サービス、ValueReadプレミックスにより行った。 Mix GFP-DC-S8 in 0.08 μl, KGFP-S28 3.3 vector 0.14 μl, 5x infusion HD Enzyme premix 1.0 μl (Takara), add RNase-free water to bring the total volume to 5 μl, and react at 50°C for 15 minutes. rice field. 2.5 μl of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain KGFP-DCS8 clones. Sequence analysis of the obtained clones was performed by Eurofin DNA sequence contract service, ValueRead Premix.
　GFP配列の上流に核移行シグナル配列遺伝子CCTGCTGCCAAGAGGGTCAAGTTGGAC（配列番号47）を導入し、stopコドンを含む pcDNA 3.3ベクター8μl、10×KOD plus緩衝液(TOYOBO) 80μl、2 mM dNTPs(TOYOBO) 80μl、25mM MgSO4 32μl、S28iv-25F (10 pmol/μl) 24μl、GFPiv-71R (10 pmol/μl) 24μl、及びKOD plus ポリメラーゼ(TOYOBO) 16μlにRNase-Free水を添加して全体量を800μlとし、PCR反応させた。PCRは、94℃2分間反応後、98℃10秒間、68℃5分11秒間を25サイクル行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、40μlのDNA溶液として回収しNLS-GFP-S28 3.3 vectorを得た。 A nuclear localization signal sequence gene CCTGCTGCCAAGAGGGGTCAAGTTGGAC (SEQ ID NO: 47) was introduced upstream of the GFP sequence, and 8 μl of pcDNA 3.3 vector containing a stop codon, 80 μl of 10×KOD plus buffer (TOYOBO), 80 μl of 2 mM dNTPs (TOYOBO), 25 mM MgSO. 4 Add RNase-free water to 32 μl, S28iv-25F (10 pmol/μl) 24 μl, GFPiv-71R (10 pmol/μl) 24 μl, and KOD plus polymerase (TOYOBO) 16 μl to make the total volume 800 μl, and perform PCR reaction. let me PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 μl of DNA solution to obtain NLS-GFP-S28 3.3 vector.
　KGFP-DCS8クローンDNA　1μl、10×KOD plus緩衝液(TOYOBO) 100μl、2 mM dNTPs(TOYOBO) 100μl、25mM MgSO4 40μl、GFPin-92F (10 pmol/μl) 30μl、S28in-7R (10 pmol/μl) 30μl、及びKOD plus ポリメラーゼ(TOYOBO) 20μlにRNase-Free水を添加して全体量を1000μlとし、PCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を8サイクル行った後、68℃5分間反応を行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、50μlのDNA溶液として回収しNLS-GFP-DC-S8 inを得た。 KGFP-DCS8 clone DNA 1 μl, 10×KOD plus buffer (TOYOBO) 100 μl, 2 mM dNTPs (TOYOBO) 100 μl, 25 mM MgSO 4 40 μl, GFPin-92F (10 pmol/μl) 30 μl, S28in-7R (10 pmol/μl ) and 20 μl of KOD plus polymerase (TOYOBO), RNase-free water was added to make the total volume 1000 μl, and PCR reaction was performed. PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of DNA solution to obtain NLS-GFP-DC-S8in.
　NLS-GFP-DC-S8 in 0.08μl、NLS-GFP-S28 3.3 vector 0.14μl、5x in fusion HD Enzyme premix 1.0μl (Takara) を混ぜ、RNase-Free水を添加し全体量を5μlとし50℃、15分間反応させた。反応後の溶液2.5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、NLS-GFP-DCS8クローンを得た。得られたクローンの配列解析はユーロフィンDNAシーケンス受託サービス、ValueReadプレミックスにより行った。 Mix NLS-GFP-DC-S8 in 0.08 μl, NLS-GFP-S28 3.3 vector 0.14 μl, 5x infusion HD Enzyme premix 1.0 μl (Takara), add RNase-Free water to bring the total volume to 5 μl, and heat at 50℃. React for 15 minutes. 2.5 μl of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain NLS-GFP-DCS8 clones. Sequence analysis of the obtained clones was performed by Eurofin DNA sequence contract service, ValueRead Premix.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（5-2）ライブラリーの配列解析
　ライブラリーの配列解析の結果、表５に示すように3ラウンド、5ラウンド、８ラウンドからそれぞれDOCK11結合ペプチドのクローンが得られた。なお、表５中のDCS8-42TN、DCS8-59R、DCS8-59Cは、後述するとおり、DCS8-42及びDCS8-59のホモロジー検索の結果に基づいて設計したペプチドである。 (5-2) Library Sequence Analysis As a result of library sequence analysis, as shown in Table 5, DOCK11-binding peptide clones were obtained from 3rd, 5th and 8th rounds, respectively. DCS8-42TN, DCS8-59R, and DCS8-59C in Table 5 are peptides designed based on the results of homology search of DCS8-42 and DCS8-59, as described later.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
[実施例6]: DOCK11結合ペプチドのクローンの活性評価
（6-1）蛋白質の調製
　インフレームのクローンはカルベニシリン20μg/mlを含むLB培地にマスタープレートよりコロニーを植菌し37℃、16時間培養した。菌体ペレットからPureLink HiPure Plasmid Maxiprep Kit (インビトロジェン)でプラスミドを精製した。 [Example 6]: Evaluation of activity of DOCK11-binding peptide clones (6-1) Preparation of protein In-frame clones were inoculated from a master plate onto LB medium containing 20 µg/ml of carbenicillin and cultured at 37°C for 16 hours. bottom. Plasmids were purified from cell pellets with the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen).
（6-2）蛋白質の調製
　ヒト胎児腎細胞293T細胞は10%FCS(ニチレイ)、DMEM(1.0g/l Glucose) with L-Gln and Sodium Pyruvate, liquid (ナカライ)培地で培養した。トランスフェクションの24時間前に6cm シャーレに(1:2)の割合でプレーティングした。6cm シャーレに対しプラスミドDNA 8μgをOpti-MEM I 500μlに入れたものに、Lipofectamine 2000 20μlあるいはPEI-MAX 20μlをOpti-MEM I 500μlに入れ室温で5分間置いたものを穏やかに加えた。混和物は室温で20分間放置後293T細胞の6cm シャーレに加え37℃、5% CO2条件下4日～６日培養した。 (6-2) Protein Preparation Human embryonic kidney 293T cells were cultured in 10% FCS (Nichirei), DMEM (1.0 g/l Glucose) with L-Gln and Sodium Pyruvate, liquid medium (Nacalai). Plated at a ratio (1:2) on a 6 cm petri dish 24 hours before transfection. 8 μg of plasmid DNA in 500 μl of Opti-MEM I, 20 μl of Lipofectamine 2000 or 20 μl of PEI-MAX in 500 μl of Opti-MEM I, and left at room temperature for 5 minutes were gently added to a 6 cm petri dish. The mixture was allowed to stand at room temperature for 20 minutes, and then added to a 6 cm petri dish of 293T cells and cultured for 4 to 6 days under conditions of 37°C and 5% CO 2 .
（6-3）プルダウン実験
　レジン(Streptavidin MagneSphere Paramagnetic Particles)(Promega) の洗浄はMagneSphere Technology Magnetic Separation Stand (tweleve-position)を使って行った。レジン40μlを1.5 mlチューブに取り、溶液を除去した後PBS 1 mlで3回洗浄を行った。新しいチューブにレジンを移し、ビオチン化全長DOCK11あるいはビオチン化C末側領域DOCK11 あるいはベイトなしは等量のPBSを加え、ミニディスクローター（Bio craft）で回転攪拌し4℃で1時間結合させた。溶液を除去後TBST (Tween20 0.05%) 1 mlで3回洗浄を行った。新しいチューブにレジンを移し、ELISA BSA緩衝液(ナカライテスク株式会社　TBS, 0.05%, Tween 20, 1% BSA) 1 mlで4℃1時間ブロッキングを行い、ELISA BSA緩衝液1 mlで3回洗浄を行った。新しいチューブにレジンを移し、DCS3あるいは DCS5あるいはDCS8を発現させた培養上清を1000μlずつ、ビオチン化全長DOCK11あるいはビオチン化C末側領域DOCK11あるいはベイトなしで処理したレジン40μlに混和させ、ミニディスクローター（Bio craft）で回転攪拌し、4℃で1時間30分結合させた。溶液を除去後TBST (Tween20 0.1%) 500μlで3回洗浄を行い、回収レジンをウエスタンブロットした。 (6-3) Pull-down experiment The resin (Streptavidin MagneSphere Paramagnetic Particles) (Promega) was washed using MagneSphere Technology Magnetic Separation Stand (twelve-position). 40 µl of the resin was placed in a 1.5 ml tube, the solution was removed, and then washed with 1 ml of PBS three times. The resin was transferred to a new tube, biotinylated full-length DOCK11 or biotinylated C-terminal domain DOCK11, or an equal volume of PBS without bait was added, and the mixture was stirred with a mini disc rotor (Bio craft) and allowed to bind at 4°C for 1 hour. After removing the solution, washing was performed three times with 1 ml of TBST (Tween20 0.05%). Transfer the resin to a new tube, block with 1 ml of ELISA BSA buffer (Nacalai Tesque Co., Ltd. TBS, 0.05%, Tween 20, 1% BSA) at 4°C for 1 hour, and wash 3 times with 1 ml of ELISA BSA buffer. gone. The resin was transferred to a new tube, and 1000 μl of the culture supernatant expressing DCS3, DCS5 or DCS8 was mixed with 40 μl of biotinylated full-length DOCK11, biotinylated C-terminal region DOCK11, or bait-free treated resin, and mixed in a mini disc rotor. (Bio craft) and combined at 4° C. for 1 hour 30 minutes. After removing the solution, washing was performed three times with 500 μl of TBST (0.1% Tween 20), and the recovered resin was subjected to Western blotting.
　各レジンに水とサンプル緩衝液LDS(4x)、0.2mM DTTを加え、70℃、10分間加熱後SDS-PAGEに供した。SDS-PAGEは4-12% Bis-Tris Gel、NuPAGE MES SDS電気泳動緩衝液 (Invitrogen)で200V, 400mA, 35分間電気泳動後、Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turboで転写した。膜はBlocking One Buffer：TBST（1：9）でブロッキングし、Blocking One Buffer：TBST(1：9)で2:3000に希釈したanti-Flag-HRP (Sigma: A8592)を反応させた。検出はECL(Enhanced ChemiLuminescence)を用い、ChemiDoc(BIORAD)で行った。 Water, sample buffer LDS (4x), and 0.2 mM DTT were added to each resin, heated at 70°C for 10 minutes, and subjected to SDS-PAGE. SDS-PAGE was performed with 4-12% Bis-Tris Gel, NuPAGE MES SDS electrophoresis buffer (Invitrogen) at 200V, 400mA for 35 minutes, followed by Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turbo. transcribed with The membrane was blocked with Blocking One Buffer: TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer: TBST (1:9). Detection was performed using ChemiDoc (BIORAD) using ECL (Enhanced ChemiLuminescence).
　DOCK11結合ペプチドによるDHR2領域を含むC末端DOCK11に対するプルダウンアッセイ（図５）を行った。3ラウンドの選択実験を行なったクローンの内3個を評価した結果、DCS3-1（配列番号1）、DCS3-2、DCS3-3がポジティブだった。 A pull-down assay (Fig. 5) was performed against C-terminal DOCK11 containing the DHR2 region by DOCK11-binding peptides. Three of the clones subjected to three rounds of selection experiments were evaluated and positive for DCS3-1 (SEQ ID NO: 1), DCS3-2 and DCS3-3.
　3ラウンド、5ラウンド、8ラウンドのクローンの内4つのクローンDCS3-1（配列番号1）、DCS5-15（配列番号4）、DCS8-42（配列番号9）並びにDCS8-42TN（配列番号10）についてDOCK11結合ペプチドによるDHR2領域を含むC末端DOCK11に対するプルダウンアッセイを行なった（図６）。ポジティブだったDCS3-1（配列番号1）とシグナル強度を比較した結果、８ラウンドから得られたDCS8-42（配列番号9）並びにDCS8-42TN（配列番号10）は強くDOCK11に結合していることがわかった。 4 clones DCS3-1 (SEQ ID NO: 1), DCS5-15 (SEQ ID NO: 4), DCS8-42 (SEQ ID NO: 9) and DCS8-42TN (SEQ ID NO: 10) among clones of 3 rounds, 5 rounds, and 8 rounds was subjected to a pull-down assay against the C-terminal DOCK11 containing the DHR2 region with a DOCK11-binding peptide (Fig. 6). As a result of comparing the signal intensity with DCS3-1 (SEQ ID NO: 1), which was positive, DCS8-42 (SEQ ID NO: 9) and DCS8-42TN (SEQ ID NO: 10) obtained from 8 rounds strongly bind to DOCK11. I understand.
[実施例7]: スクリーニングしたDOCK11結合ペプチドの抗HBV活性
（7-1）HepG2-NTCP-C4細胞におけるDOCK11G結合ペプチドの抗HBV活性
　DHR2領域を含むC末端DOCK11に対するプルダウン実験でポジティブだったクローンについて、HepG2-NTCP-C4細胞（HBVの受容体であるNTCPを過剰発現させたHepG2細胞、Iwamoto, M. et al. Biochem. Biophys. Res. Commun. 443:808-813, 2014）におけるDOCK11結合ペプチドの抗HBV活性を評価した（図７）。ペプチドを含むプラスミドには細胞内に発現するもの(DCS3-1)と、核移行シグナルPAAKRVKLD（配列番号40）を付加したもの(N-DCS3-1)を調整した。HBVは、HBV core上流に存在するpackaging signal（ε）を欠損させたヘルパープラスミドをHepG2細胞に安定発現させた細胞であるHepG2.2.15に由来するHBVを用いた。HepG2-NTCP-C4細胞にHepG2.2.15由来HBVを感染させた後、種々のペプチドをコードしたプラスミドをLipofectamine 3000でそれぞれトランスフェクションした。トランスフェクションから3-5日後にサンプルを回収し、HBV-DNA、cccDNAを評価した。 [Example 7]: Anti-HBV activity of screened DOCK11-binding peptide (7-1) Anti-HBV activity of DOCK11G-binding peptide in HepG2-NTCP-C4 cells Regarding clones that were positive in pull-down experiments against C-terminal DOCK11 containing DHR2 region , DOCK11-binding peptides in HepG2-NTCP-C4 cells (HepG2 cells overexpressing the HBV receptor NTCP, Iwamoto, M. et al. Biochem. Biophys. Res. Commun. 443:808-813, 2014) was assessed for anti-HBV activity (Fig. 7). A peptide-containing plasmid (DCS3-1) for intracellular expression and a plasmid (N-DCS3-1) added with a nuclear localization signal PAAKRVKLD (SEQ ID NO: 40) were prepared. The HBV used was HBV derived from HepG2.2.15 cells in which a helper plasmid lacking the packaging signal (ε) present upstream of the HBV core was stably expressed in HepG2 cells. After HepG2-NTCP-C4 cells were infected with HepG2.2.15-derived HBV, plasmids encoding various peptides were transfected with Lipofectamine 3000, respectively. Samples were collected 3-5 days after transfection and evaluated for HBV-DNA and cccDNA.
　プルダウン実験でポジティブだった２つのクローンDCS3-2、DCS3-3はコントロールと比較してHBV DNAのコピー数とcccDNAのコピー数を減少させることはなかったが、DCS3-1はコントロールと比較してHBV DNAのコピー数を減少させた。また、cccDNAのコピー数についても減少していた。 Two clones, DCS3-2 and DCS3-3, which were positive in pull-down experiments, did not decrease HBV DNA copy number and cccDNA copy number compared to the control, whereas DCS3-1 did Reduced the copy number of HBV DNA. In addition, the copy number of cccDNA was also decreased.
　5ラウンドの選択実験を行なったクローン6個についても同様にDOCK11結合ペプチドの抗HBV活性を評価した（図８）。DCS5-1、DCS5-4（配列番号2）、DCS5-5（配列番号3）、DCS5-15（配列番号4）、DCS5-7、DCS5-33のうち、DCS5-1、DCS5-7、DCS5-33はコントロールと比較してHBV DNAのコピー数とcccDNAのコピー数を減少させることはなかったが、DCS5-4（配列番号2）、DCS5-5（配列番号3）、DCS5-15（配列番号4）はコントロールと比較してHBV DNAのコピー数を減少させた。また、cccDNAのコピー数についても減少していた。 The anti-HBV activity of DOCK11-binding peptides was similarly evaluated for 6 clones subjected to 5 rounds of selection experiments (Fig. 8). DCS5-1, DCS5-7, DCS5 among DCS5-1, DCS5-4 (SEQ ID NO: 2), DCS5-5 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 4), DCS5-7, DCS5-33 -33 did not decrease HBV DNA copy number and cccDNA copy number compared to controls, but DCS5-4 (SEQ ID NO: 2), DCS5-5 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 3), DCS5-15 (SEQ ID NO: 3) number 4) decreased the copy number of HBV DNA compared to controls. In addition, the copy number of cccDNA was also decreased.
　さらに強い抗HBV活性を有するDOCK11結合ペプチドを選択するために、強力な選択圧をかけてIVV選択実験を行なった。8ラウンドの選択実験を行ない5個のクローンを得た。そのうち２つのクローンDCS8-42とDCS8-59はBLASTPにおいてホモロジー検索を行った結果、DCS8-42はTNK2（Ack1）と高いホモロジーを示し、DCS8-59はradixin、β-Centractinと高いホモロジーを示した。DCS8-42と相同配列を有するDCS8-42TN（DCS8-42と高いホモロジーを示したTNK2（Ack1）の部分領域からなるペプチド、配列番号10）、並びに、DCS8-59と相同配列を有するDCS8-59R（DCS8-59と高いホモロジーを示したradixinの部分領域からなるペプチド、配列番号11）、DCS8-59C（DCS8-59と高いホモロジーを示したβ-Centractinの部分領域からなるペプチド、配列番号12）を調製し、他の３つのクローンと合わせて抗HBV活性を検討した。２０日間HBVをHepG2-NTCP-C4細胞に感染させて評価したところ、DCS8-6（配列番号5）、DCS8-29（配列番号6）、DCS8-59（配列番号7）、DCS8-72（配列番号8）、DCS8-42TN（配列番号10）のいずれもHBV DNAのコピー数とcccDNAのコピー数についても減少させた（図９）。 In order to select DOCK11-binding peptides with even stronger anti-HBV activity, IVV selection experiments were performed under strong selective pressure. Eight rounds of selection experiments were performed and five clones were obtained. Two of these clones, DCS8-42 and DCS8-59, were subjected to BLASTP homology search, and DCS8-42 showed high homology with TNK2 (Ack1), and DCS8-59 showed high homology with radixin and β-Centractin. . DCS8-42TN having a sequence homologous to DCS8-42 (peptide consisting of a partial region of TNK2 (Ack1) showing high homology with DCS8-42, SEQ ID NO: 10), and DCS8-59R having a sequence homologous to DCS8-59 (peptide consisting of a partial region of radixin showing high homology with DCS8-59, SEQ ID NO: 11), DCS8-59C (peptide consisting of a partial region of β-Centractin showing high homology with DCS8-59, SEQ ID NO: 12) was prepared and examined for anti-HBV activity together with the other three clones. HepG2-NTCP-C4 cells were infected with HBV for 20 days and evaluated. No. 8) and DCS8-42TN (SEQ ID NO: 10) also decreased the HBV DNA copy number and the cccDNA copy number (Fig. 9).
　DOCK11は細胞質だけでなく核画分にも局在することから、核移行シグナル（N-と表記する）をC末端につけたペプチドについて、検討した。５日間HBVをHepG2-NTCP-C4細胞に感染させて評価したところ、N-DCS8-6、N-DCS8-29、N-DCS8-59、N-DCS8-72、N-DCS8-42TNのいずれもHBV DNAのコピー数とcccDNAのコピー数についても減少させた（図10）。19日間HBVをHepG2-NTCP-C4細胞に感染させて評価したところN-DCS8-59、N-DCS8-59C、N-DCS8-59R、N-DCS8-42TNのいずれもHBV DNAのコピー数とcccDNAのコピー数についても減少させた（図11）。 Since DOCK11 is localized not only in the cytoplasm but also in the nuclear fraction, we investigated peptides with a nuclear localization signal (denoted as N-) attached to the C-terminus. HepG2-NTCP-C4 cells were infected with HBV for 5 days and evaluated. HBV DNA copy number and cccDNA copy number were also reduced (Fig. 10). HepG2-NTCP-C4 cells were infected with HBV for 19 days and evaluated. also reduced the copy number of .
　血中のタンパク質の多くはシアル酸が付加された糖タンパク質であるが、劣化してくるとアシアロ糖タンパク質となり、主として肝臓で分解される。そのために肝臓には、血中のアシアロ糖タンパク質を特異的に認識し、取り込むためのアシアロ糖タンパク質受容体が存在する。抗アシアロ糖タンパク質受容体抗体も同様に受容体に結合した後肝臓細胞内に取り込まれると考えられる。これを利用した抗アシアロ糖タンパク質受容体抗体を用いたDOCK11結合ペプチドの肝細胞選択的送達法を確立するためにアシアロ糖タンパク質受容体に結合する一本鎖抗体の選択実験をIVV法を用いて行なった。 Most of the proteins in the blood are glycoproteins to which sialic acid has been added, but as they deteriorate, they become asialoglycoproteins, which are mainly decomposed in the liver. Therefore, the liver has asialoglycoprotein receptors for specifically recognizing and taking up asialoglycoprotein in the blood. Anti-asialoglycoprotein receptor antibodies are also thought to be taken up into liver cells after binding to the receptor. In order to establish a method for selectively delivering DOCK11-binding peptides to hepatocytes using anti-asialoglycoprotein receptor antibodies, selection experiments for single-chain antibodies that bind to asialoglycoprotein receptors were conducted using the IVV method. did.
[実施例8]: ビオチン化ASGRの調製
　初めに抗原となるビオチン化アシアロ糖タンパク質受容体を作成した。図12に示すようにアシアロ糖タンパク質受容体（ASGR）はN末端を細胞内、糖認識部位（CRDs）を細胞外に向けたII型の１回膜貫通タンパク質である。これをビアコアのセンサーチップ上に提示するために膜貫通ドメインを除き、ビオチン化配列等を付加したコンストラクトを作成した。ヒトの肝細胞には受容体ASGR1とASGR2が存在し、ヘテロオリゴマーを形成していることからそれぞれの細胞外ドメインにビオチンを付加した、ビオチン化ASGR1exとビオチン化ASGR2exを作成した。 [Example 8]: Preparation of biotinylated ASGR First, a biotinylated asialoglycoprotein receptor as an antigen was prepared. Asialoglycoprotein receptor (ASGR) is a type II single-pass transmembrane protein with the N-terminus directed intracellularly and the carbohydrate recognition sites (CRDs) directed extracellularly, as shown in FIG. In order to display this on a Biacore sensor chip, a construct was prepared by removing the transmembrane domain and adding a biotinylated sequence and the like. Since human hepatocytes have receptors ASGR1 and ASGR2 and form hetero-oligomers, we created biotinylated ASGR1ex and biotinylated ASGR2ex by adding biotin to their extracellular domains.
（8-1）ビオチンASGRの発現ベクター構築
　ASGRの細胞外ドメインにHis-tag、ビオチン化配列、Flag-tag、配列を付加したpcDNA3.3 TOPO HisBioFLAG-ASGR1exベクターとpcDNA3.3 TOPO HisBioFLAG-ASGR2exベクターを作成した。 (8-1) Construction of biotin ASGR expression vector pcDNA3.3 TOPO HisBioFLAG-ASGR1ex vector and pcDNA3.3 TOPO HisBioFLAG-ASGR2ex vector in which His-tag, biotinylated sequence, Flag-tag and sequence are added to the extracellular domain of ASGR It was created.
　図13に示すようにASGR1とASGR2のそれぞれの細胞外ドメインをクローニングした。
　cDNA Library, Human Liver (1 ng/μl) (タカラバイオ) 1μl、KAPA HiFi HS RM 10μl、10μM ASGR1-ex-if-F1 0.6μlあるいはASGR2-ex-if-F1 0.6μl、及び10μM ASGR1-if-R2 0.6μlあるいはASGR2-if-R2 0.6μlにRNase-Free水を添加して全体量を20μlとし、PCR反応を行った。PCRは、95℃5分間反応後、98℃20秒間、60℃15秒間、72℃1分間を25サイクル行った後72℃1分間反応を行った。PCR産物はアガロースゲル電気泳動でDNAのバンドを確認後、Wizard SV Gel PCR Clean-Up System(Promega)で精製し30μlのDNA溶液として回収しASGR1-ifあるいはASGR2-ifを得た。 The extracellular domains of ASGR1 and ASGR2 were cloned as shown in FIG.
cDNA Library, Human Liver (1 ng/μl) (Takara Bio) 1 μl, KAPA HiFi HS RM 10 μl, 10 μM ASGR1-ex-if-F1 0.6 μl or ASGR2-ex-if-F1 0.6 μl, and 10 μM ASGR1-if- RNase-free water was added to 0.6 μl of R2 or 0.6 μl of ASGR2-if-R2 to make the total volume 20 μl, and PCR reaction was performed. PCR was carried out at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 1 minute. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified by Wizard SV Gel PCR Clean-Up System (Promega) and collected as a 30 μl DNA solution to obtain ASGR1-if or ASGR2-if.
　図14に示すようなコンストラクトの発現プラスミドを作成するために、kozak配列-Hisタグ配列-ビオチン化配列-Flagタグ配列を含むベクターpcDNA3.3 TOPO KHisBioFlagを使ってASGR1-ifあるいはASGR2-ifをインフュージョンクローニングした。pcDNA3.3 TOPO KHisBioFlagINV 1.0μl、ASGR1-if 1.0μlあるいはASGR2-if 1.0μl、5x in fusion HD Enzyme premix 1.0μl (Takara) にRNase-Free水を添加して全体量を5μlとし、50℃、15分間反応させた。2.5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、クローンを得た。クローンの配列解析の結果、プラスミドpcDNA3.3 TOPO KHisBioFlag-ASGR1exあるいはpcDNA3.3 TOPO KHisBioFlag-ASGR2exであることを確認した。 ASGR1-if or ASGR2-if was imported using the vector pcDNA3.3 TOPO KHisBioFlag containing the kozak sequence-His tag sequence-biotinylation sequence-Flag tag sequence to generate an expression plasmid for the construct shown in Figure 14. Fusion cloned. pcDNA3.3 TOPO KHisBioFlagINV 1.0 μl, ASGR1-if 1.0 μl or ASGR2-if 1.0 μl, 5x infusion HD Enzyme premix 1.0 μl (Takara), add RNase-free water to make the total volume 5 μl, heat at 50℃ for 15 minutes. reacted for a minute. 2.5 μl was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain clones. Sequence analysis of the clones confirmed the plasmid pcDNA3.3 TOPO KHisBioFlag-ASGR1ex or pcDNA3.3 TOPO KHisBioFlag-ASGR2ex.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（8-2）ビオチン化細胞外ドメインASGR1exあるいはビオチン化細胞外ドメインASGR2exの発現と精製
　ヒト胎児腎細胞293T細胞を用いてプラスミドpcDNA3.3 TOPO KHisBioFlag-ASGR1exあるいはpcDNA3.3 TOPO KHisBioFlag-ASGR2exをトランスフェクションした。37℃ 5% CO2 48時間培養後得られた細胞をCell lysis buffer (Tris-HCl pH 7.4　25 mM、NaCl　137 mM、KCl　2.68 mM、Triton X-100　1%)で抽出した。得られた細胞抽出液に、TBSで洗浄したM2アガロースビーズ(ANTI-FLAG(登録商標) M2 Affinity Gel, Sigma, A2220)を加え、4℃ 16時間混合した。TBST(10x TBS-t 1%Tween-20, ナカライ, 12749-21)で3回洗浄した後、TBSで希釈した150μg/mL 3x FLAG peptide(Sigma, F4799)で競合溶出した。 (8-2) Expression and Purification of Biotinylated Extracellular Domain ASGR1ex or Biotinylated Extracellular Domain ASGR2ex Human embryonic kidney 293T cells were transfected with plasmid pcDNA3.3 TOPO KHisBioFlag-ASGR1ex or pcDNA3.3 TOPO KHisBioFlag-ASGR2ex. bottom. The cells obtained after culturing at 37°C and 5% CO 2 for 48 hours were extracted with Cell lysis buffer (Tris-HCl pH 7.4 25 mM, NaCl 137 mM, KCl 2.68 mM, Triton X-100 1%). TBS-washed M2 agarose beads (ANTI-FLAG (registered trademark) M2 Affinity Gel, Sigma, A2220) were added to the obtained cell extract and mixed at 4°C for 16 hours. After washing three times with TBST (10x TBS-t 1% Tween-20, Nakarai, 12749-21), competitive elution was performed with 150 µg/mL 3x FLAG peptide (Sigma, F4799) diluted with TBS.
　SAビーズ(Streptavidin MagneSphere(登録商標) Paramagnetic Particles, Promega)20 μLをTBST(10x TBS-T 1%Tween-20, ナカライ) 200μLで3回洗浄し、FLAG精製済みタンパク質を以加えた。1時間室温で回転させて混合した後TBST 200μLで3回洗浄した。ビーズをTBS 10μLに懸濁させて70℃ 10 min処理し、SDS-PAGEとFlag抗体を用いてウエスタンブロットで検出したところ。KHisBioFlag-ASGR1exは分子量35,597Da、KHisBioFlag-ASGR2exは分子量35,708Daのバンドとして確認することができた。 20 μL of SA beads (Streptavidin MagneSphere (registered trademark) Paramagnetic Particles, Promega) were washed three times with 200 μL of TBST (10x TBS-T 1% Tween-20, Nacalai), and FLAG-purified protein was added. After mixing by rotating at room temperature for 1 hour, the cells were washed with 200 μL of TBST three times. Beads were suspended in 10 μL of TBS, treated at 70° C. for 10 min, and detected by Western blot using SDS-PAGE and Flag antibody. KHisBioFlag-ASGR1ex was confirmed as a band with a molecular weight of 35,597 Da, and KHisBioFlag-ASGR2ex was confirmed as a band with a molecular weight of 35,708 Da.
　同様の方法で得たHisBioFLAG-ASGR1exの細胞抽出液をSAビーズに結合し、洗浄した後EKMax buffer (500 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 1% Tween-20)に交換した。EKMaxTM Enterokinase （Thermo Fisher Scientific）を加え37℃ O/N (16.5h) 回転させEKMax digestionを行ない、マグネットスタンドでSAビーズを吸着させて上清回収した。回収した上清に前処理したEK Away resin を加え、室温15min 回転させEKMaxを除去した。遠心して上清を回収 (5000rcf 2min x2)しSDS-PAGEとFlag抗体を用いてウエスタンブロットで検出したところASGR1ex　分子量26394 Daバンドとして確認することができた。 A cell extract of HisBioFLAG-ASGR1ex obtained in a similar manner was bound to SA beads, washed, and then exchanged with EKMax buffer (500 mM Tris-HCl, pH 8.0, 10 mM CaCl 2 , 1% Tween-20). EKMax ™ Enterokinase (Thermo Fisher Scientific) was added, EKMax digestion was performed by rotating at 37°C O/N (16.5h), SA beads were adsorbed on a magnet stand, and the supernatant was recovered. Pretreated EK Away resin was added to the recovered supernatant, and the mixture was rotated at room temperature for 15 minutes to remove EKMax. After centrifugation, the supernatant was recovered (5000 rcf 2 min x 2) and detected by Western blotting using SDS-PAGE and Flag antibody, and an ASGR1ex molecular weight 26394 Da band could be confirmed.
[実施例9]: 一本鎖抗体cDNAライブラリーの作成
（9-1）DNAライブラリーデザイン
　抗体の抗原認識はH鎖、L鎖のN末端側のドメインが担っており、可変領域 (Variable region) と呼ばれる。一方、その他のドメインはクラスごとに一定で、定常領域 (Constant region) と呼ばれる (図15左)。 VH鎖、VL鎖はともに3つのCDR (Complementarity-determining regions) と4つのFR (Framework regions) から構成されている (図15右)。CDRは抗体ごとに多様なアミノ酸配列からなり、様々な抗原との結合を可能にしている。人工的な抗体断片としては、VH鎖とVL鎖をペプチドリンカーで連結させた一本鎖抗体 (Single-chain antibody : scFv) がある (図16)。VH鎖、VL鎖を15アミノ酸からなるペプチドリンカーがドメイン間の会合を妨げないよう、二次構造が強制されない配列としてグリシンと親水性のセリンを含むGGGGSユニットを3回繰り返したリンカー（配列番号48）が多く利用されている。 [Example 9]: Construction of single-chain antibody cDNA library (9-1) DNA library design ). On the other hand, other domains are constant for each class and are called constant regions (Fig. 15 left). Both the V H chain and V L chain are composed of three CDRs (Complementarity-determining regions) and four FRs (Framework regions) (Fig. 15 right). CDRs are composed of a variety of amino acid sequences for each antibody, enabling them to bind to various antigens. Artificial antibody fragments include single-chain antibodies (scFv), in which VH and VL chains are linked by a peptide linker (Fig. 16). A linker ( sequence 48) is widely used.
[実施例10]: マウス由来一本鎖抗体cDNAライブラリーの作成
　マウス由来一本鎖抗体cDNAライブラリーの作成は図17に示すようにマウス脾臓Poly A+ RNAを出発原料に行った。H鎖DNA溶液の調製、L鎖DNA溶液の調製、H鎖とL鎖の一本化PCRを行なった。(Nucleic Acids Research, 2009, Vol. 37, No. 8 e64)。 [Example 10]: Construction of mouse-derived single-chain antibody cDNA library As shown in Fig. 17, a mouse-derived single-chain antibody cDNA library was constructed using mouse spleen Poly A+ RNA as a starting material. Preparation of H chain DNA solution, preparation of L chain DNA solution, and unification PCR of H chain and L chain were carried out. (Nucleic Acids Research, 2009, Vol. 37, No. 8 e64).
（10-1）H鎖DNA溶液の調製
　一本鎖抗体cDNAライブラリーの作成は、始めにH鎖DNA溶液の調製を行った。マウス脾臓Poly A+ RNA (5 μg/μl)（DEPC-処理水）(CLONTECH社)をRNase-Free水で100倍に希釈したものを 11μl、5×RT緩衝液(TOYOBO) 22μl、(10 mM) dNTPs(TOYOBO) 11μl、forwardプライマー MulgG1/2 (1pmol/μl) 27.5μl、forwardプライマー MulgG3 (1pmol/μl) 27.5μl、を混和させ65℃ 9分間反応後直ちに4℃に冷却し4℃ 2分間放置した後、ReverTra Ace(TOYOBO) 5.5μl、RNase inhibitor(TOYOBO) 5.5μlを加え50℃ 30分間、99℃、5分間反応させcDNA-Hを合成した。cDNA-H溶液 5μlに、HBプライマーに示した各HBプライマー (1pmol/μl) 各2.5μl、10×PCR緩衝液(TOYOBO) 2.5μl、forwardプライマー MulgG1/2 (1pmol/μl) 1.25μl、forwardプライマー MulgG3 (1pmol/μl) 1.25μl、KOD DASH ポリメラーゼ(TOYOBO) 0.25μl、とRNase-Free水を添加し全体量を 25μl、としてそれぞれPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間、50℃ 30秒間、72℃ 1分間を25サイクル行った後72℃ 5分間反応を行った。増幅した遺伝子は2%アガロースゲル電気泳動によりそれぞれ500-900bpのバンドを確認し、フェノール/クロロホルム処理を行った。すなわち同量のフェノール:クロロホルム:イソアミルアルコール（25:24:1）を加えよく混和し4℃で13,200rpm、5分間遠心し、水層部のみを新しいチューブに移しもう一度同量のフェノール:クロロホルム:イソアミルアルコール（25:24:1）を加えよく混和し4℃で13,200rpm、5分間遠心し、水層部のみを新しいチューブに移した。得られた溶液についてエタノール沈殿を行った。約15分間遠心乾燥した後、各DNA(19種)をRNase-free水20μlに溶解した。合成した各DNA溶液(19種) 各1μlに、HBプライマーに示した対応する各HBプライマー (10pmol/μl) 各2μl、10×PCR緩衝液(TOYOBO) 10μl、(2 mM) dNTPs(TOYOBO) 10μl、HFプライマーに示したVH forwardプライマー HF1:HF2:HF3:HF4(1:1:1:1)混合液 (10pmol/μl) 2μl、KOD DASH ポリメラーゼ(TOYOBO) 0.5μl、とRNase-Free水を添加し全体量を 100μl、としてそれぞれPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間、50℃ 30秒間、72℃ 1分間を20サイクル行った後72℃ 5分間反応を行った。増幅した遺伝子は2%アガロースゲル電気泳動によりそれぞれ500-900bpのバンドを確認し、フェノール/クロロホルム処理及びエタノール沈殿を行った。その後、約15分間遠心乾燥した後、各DNA(19種)をRNase-free水10μlに溶解した。得られた19種のDNAを2%低融点アガロースゲル(Sigma)電気泳動しそれぞれのバンドを切り出した。各DNA(19種)はRNase-free水10μlに溶解した。各DNA溶液について260nmの吸収を測定しHB1:HB2:HB3:HB4:HB5: HB6:HB7:HB8:HB9:HB10:HB11:HB12:HB13: HB14:HB15:HB16:HB17:HB18:HB19(8:9:4:4:12:4:1:4:12:4:4:2:2:4:4:8:6:1:1)の比率で混和させ合計0.5pmolのH鎖DNA溶液とした。 (10-1) Preparation of H-chain DNA solution For preparation of a single-chain antibody cDNA library, first, an H-chain DNA solution was prepared. 11 μl of mouse spleen Poly A+ RNA (5 μg/μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-free water, 22 μl of 5×RT buffer (TOYOBO), (10 mM) Mix 11 μl of dNTPs (TOYOBO), 27.5 μl of forward primer MulgG1/2 (1 pmol/μl), and 27.5 μl of forward primer MulgG3 (1 pmol/μl), react at 65°C for 9 minutes, then immediately cool to 4°C and leave at 4°C for 2 minutes. After that, 5.5 μl of ReverTra Ace (TOYOBO) and 5.5 μl of RNase inhibitor (TOYOBO) were added and reacted at 50° C. for 30 minutes and 99° C. for 5 minutes to synthesize cDNA-H. To 5 μl of cDNA-H solution, 2.5 μl of each HB primer (1 pmol/μl) shown in HB primer, 2.5 μl of 10× PCR buffer (TOYOBO), 1.25 μl of forward primer MulgG1/2 (1 pmol/μl), forward primer 1.25 μl of MulgG3 (1 pmol/μl), 0.25 μl of KOD DASH polymerase (TOYOBO), and RNase-free water were added to make the total volume 25 μl, and PCR reaction was carried out. PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. Bands of 500-900 bp were confirmed for each amplified gene by 2% agarose gel electrophoresis, and treated with phenol/chloroform. That is, add the same amount of phenol:chloroform:isoamyl alcohol (25:24:1), mix well, centrifuge at 13,200 rpm at 4℃ for 5 minutes, transfer only the aqueous layer to a new tube, and add the same amount of phenol:chloroform: Isoamyl alcohol (25:24:1) was added, mixed well, centrifuged at 13,200 rpm at 4°C for 5 minutes, and only the aqueous layer was transferred to a new tube. Ethanol precipitation was performed on the resulting solution. After centrifugation for about 15 minutes, each DNA (19 types) was dissolved in 20 μl of RNase-free water. 1 μl of each synthesized DNA solution (19 types), 2 μl of each corresponding HB primer (10 pmol/μl) shown in HB primer, 10 μl of 10×PCR buffer (TOYOBO), 10 μl of (2 mM) dNTPs (TOYOBO) , VH forward primer HF1:HF2:HF3:HF4 (1:1:1:1) mixture (10pmol/μl) 2μl, KOD DASH polymerase (TOYOBO) 0.5μl, and RNase-free water were added to HF primer. The total volume was 100 μl, and each was subjected to PCR reaction. PCR was performed at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. The amplified gene was subjected to 2% agarose gel electrophoresis to confirm bands of 500-900 bp, followed by phenol/chloroform treatment and ethanol precipitation. After centrifugation for about 15 minutes, each DNA (19 types) was dissolved in 10 μl of RNase-free water. The obtained 19 DNAs were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and respective bands were excised. Each DNA (19 species) was dissolved in 10 μl of RNase-free water. HB1: HB2: HB3: HB4: HB5: HB6: HB7: HB8: HB9: HB10: HB11: HB12: HB13: HB14: HB15: HB16: HB17: HB18: HB19 (8: 9:4:4:12:4:1:4:12:4:4:2:2:4:4:8:6:1:1) with a total of 0.5 pmol H-chain DNA solution and bottom.
（10-2）L鎖DNA溶液の調製
　マウス脾臓Poly A+ RNA (5 μg/μl)（DEPC-処理水）(CLONTECH社)をRNase-Free水で100倍に希釈したものを 10μl、5×RT緩衝液(TOYOBO) 20μl、(10 mM) dNTPs(TOYOBO) 10μl、forwardプライマー MuCK (1pmol/μl) 50μl、を混和させ65℃ 9分間反応後直ちに4℃に冷却し4℃ 2分間放置した後、ReverTra Ace(TOYOBO) 5μl、RNase inhibitor(TOYOBO) 5μl、を加え50℃ 30分間、99℃、5分間反応させcDNA-Lを合成した。cDNA-L溶液 5μlに、LBプライマーに示した各LBプライマー (1pmol/μl) 各2.5μl、10×PCR緩衝液(TOYOBO) 2.5μl、forwardプライマー MuCK (1pmol/μl) 2.5μl、KOD DASH ポリメラーゼ(TOYOBO) 0.25μl、とRNase-Free水を添加し全体量を 25μlとしてそれぞれPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間、48℃ 30秒間、72℃ 1分間を25サイクル行った後72℃ 5分間反応を行った。増幅した遺伝子は2%アガロースゲル電気泳動によりそれぞれ500-900bpのバンドを確認し、フェノール/クロロホルム処理を行った。得られた溶液についてエタノール沈殿を行った。その後、約15分間遠心乾燥した後、各DNA(18種)をRNase-free水20μlに溶解した。合成した各DNA溶液(18種) 各1μlに、LBプライマーに示した対応する各LBプライマー (10pmol/μl) 各2μl、10×PCR緩衝液(TOYOBO) 10μl、(2 mM) dNTPs(TOYOBO) 10μl、LFプライマーに示したLH forwardプライマー LF1:LF2:LF3:LF4:LFλ(1:1:1:1)混合液 (10pmol/μl) 2μl、KOD DASH ポリメラーゼ(TOYOBO) 0.5μl、とRNase-Free水を添加し全体量を 100μl、としてそれぞれPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間、48℃ 30秒間、72℃ 1分間を20サイクル行った後72℃5分間反応を行った。増幅した遺伝子は2%アガロースゲル電気泳動によりそれぞれ500-900bpのバンドを確認し、フェノール/クロロホルム処理及びエタノール沈殿を行った。その後、約15分間遠心乾燥した後、各DNA(18種)をRNase-free水10μlに溶解した。得られた18種のDNAを2%低融点アガロースゲル(Sigma)電気泳動しそれぞれのバンドを切り出した。各DNA(18種)はRNase-free水10μlに溶解した。各DNA溶液について260nmの吸収を測定しLB1:LB2:LB3:LB4:LB5:LB6:LB7:LB8: LB9:LB10:LB11: LB12:LB13:LB14:LB15:LB16:LB17:LBλ(2:4:8:8:8:16:12:4:4:8:16:16:12:4:4:2:2:1)の比率で混和させ合計0.5pmolのL鎖DNA溶液とした。 (10-2) Preparation of L-chain DNA solution Mouse spleen Poly A+ RNA (5 μg/μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-free water was diluted to 10 μl, 5×RT. 20 μl of buffer solution (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and 50 μl of forward primer MuCK (1 pmol/μl) were mixed, reacted at 65° C. for 9 minutes, immediately cooled to 4° C. and left at 4° C. for 2 minutes. 5 μl of ReverTra Ace (TOYOBO) and 5 μl of RNase inhibitor (TOYOBO) were added and reacted at 50° C. for 30 minutes and at 99° C. for 5 minutes to synthesize cDNA-L. To 5 μl of cDNA-L solution, 2.5 μl of each LB primer (1 pmol/μl) shown in LB primer, 2.5 μl of 10×PCR buffer (TOYOBO), 2.5 μl of forward primer MuCK (1 pmol/μl), KOD DASH polymerase ( TOYOBO) (0.25 μl) and RNase-free water were added to make the total volume 25 μl, and PCR reaction was carried out. PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. Bands of 500-900 bp were confirmed for each amplified gene by 2% agarose gel electrophoresis, and treated with phenol/chloroform. Ethanol precipitation was performed on the resulting solution. After centrifugation for about 15 minutes, each DNA (18 types) was dissolved in 20 μl of RNase-free water. 1 μl of each synthesized DNA solution (18 types), 2 μl of each corresponding LB primer (10 pmol/μl) shown in LB primer, 10 μl of 10×PCR buffer (TOYOBO), 10 μl of (2 mM) dNTPs (TOYOBO) , LH forward primer LF1:LF2:LF3:LF4:LFλ(1:1:1:1) mixture (10pmol/μl) 2μl, KOD DASH polymerase (TOYOBO) 0.5μl, and RNase-Free water was added to make the total volume 100 μl, and the PCR reaction was carried out respectively. PCR was performed at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. The amplified gene was subjected to 2% agarose gel electrophoresis to confirm bands of 500-900 bp, followed by phenol/chloroform treatment and ethanol precipitation. After centrifugation for about 15 minutes, each DNA (18 types) was dissolved in 10 μl of RNase-free water. The obtained 18 DNAs were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and respective bands were excised. Each DNA (18 species) was dissolved in 10 μl of RNase-free water. LB1:LB2:LB3:LB4:LB5:LB6:LB7:LB8:LB9:LB10:LB11:LB12:LB13:LB14:LB15:LB16:LB17:LBλ(2:4: 8:8:8:16:12:4:4:8:16:16:12:4:4:2:2:1) to give a total L-chain DNA solution of 0.5 pmol.
（10-3）H鎖とL鎖を一本化するためのPCR
　合成したH鎖DNA溶液 0.5pmol、L鎖DNA溶液 0.5pmol、5'UTR (1pmol/μl) 0.5μl、McD-Linker+ (1pmol/μl) 0.5μl、McD 3'UTR (His Tag) (1pmol/μl) 0.5μl、10×PCR緩衝液(TOYOBO) 5μl、(2 mM) dNTPs(TOYOBO) 5μl、KOD DASH ポリメラーゼ(TOYOBO) 0.25μl、とRNase-Free水を添加し全体量を 45.75μl、としてPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間続いて5分間のスロープで58℃とし58℃30秒間、72℃1分間を10サイクル行った後72℃5分間反応を行った。続いて、PCR反応溶液 45.75μl、McD-F (1pmol/μl) 2μl、McD-R (His Tag) (1pmol/μl) 2μl、KOD DASH ポリメラーゼ(TOYOBO) 0.25μl、を加えさらにPCR反応させた。PCRは96℃、5分間反応後96℃、30秒間、58℃30秒間、72℃ 1分間を15サイクル行った後72℃ 5分間反応を行った。得られたDNAを1%低融点アガロースゲル(Sigma)電気泳動しそれぞれのバンドを切り出した。DNAはRNase-free水10μlに溶解し、マウス由来一本鎖抗体ライブラリーDNAを得た。 (10-3) PCR to integrate H and L chains
Synthesized H chain DNA solution 0.5 pmol, L chain DNA solution 0.5 pmol, 5'UTR (1 pmol/μl) 0.5 μl, McD-Linker+ (1 pmol/μl) 0.5 μl, McD 3'UTR (His Tag) (1 pmol/μl ) 0.5 μl, 10× PCR buffer (TOYOBO) 5 μl, (2 mM) dNTPs (TOYOBO) 5 μl, KOD DASH polymerase (TOYOBO) 0.25 μl, and RNase-free water were added to bring the total volume to 45.75 μl. let me PCR was carried out at 96°C for 5 minutes, followed by 96°C for 30 seconds, followed by 5 minutes slope to 58°C for 10 cycles of 58°C for 30 seconds and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. Subsequently, 45.75 µl of the PCR reaction solution, 2 µl of McD-F (1 pmol/µl), 2 µl of McD-R (His Tag) (1 pmol/µl), and 0.25 µl of KOD DASH polymerase (TOYOBO) were added and further PCR-reacted. PCR was performed at 96°C for 5 minutes, followed by 15 cycles of 96°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, followed by reaction at 72°C for 5 minutes. The resulting DNA was electrophoresed on a 1% low melting point agarose gel (Sigma) and each band was excised. The DNA was dissolved in 10 µl of RNase-free water to obtain mouse-derived single-chain antibody library DNA.
[実施例11]: ヒト由来一本鎖抗体cDNAライブラリーの作成
　ヒト骨髄由来のリンパ球からの一本鎖抗体cDNAライブラリーの作成は図18に示すようにヒト骨髄由来Poly A+ RNAを出発原料に行った。マウスの場合と同様に複数の特異的プライマーを用いてH鎖DNA溶液の調製、L鎖DNA溶液の調製、H鎖とL鎖の一本化PCRを行うことでIVV選択実験用のヒト一本鎖抗体cDNAライブラリーを作成した。(J. Mol. Biol. 1991, 222, 581-597、Nature Biotechnology, 2005, 23, 344-348、Antibody Engineering, Springer Lab. Manual (2001) 93-108)。 [Example 11]: Construction of human-derived single-chain antibody cDNA library As shown in Fig. 18, human bone-marrow-derived Poly A+ RNA was used as a starting material for construction of a single-chain antibody cDNA library from human bone marrow-derived lymphocytes. went to Using multiple specific primers as in the case of mice, H-chain DNA solution preparation, L-chain DNA solution preparation, and H- and L-chain unification PCR were performed to obtain a single human sample for IVV selection experiments. A chain antibody cDNA library was constructed. (J. Mol. Biol. 1991, 222, 581-597, Nature Biotechnology, 2005, 23, 344-348, Antibody Engineering, Springer Lab. Manual (2001) 93-108).
　さらに図17に示すように調製したマウスH鎖DNAならびにマウスL鎖DNAあるいはヒトH鎖DNAならびにヒトL鎖DNAをそれぞれMutazyme IIを使ってError-prone PCRによりランダムな突然変異導入させた。マウス変異H鎖DNAならびにマウス変異L鎖DNAをH鎖とL鎖を一本化PCRによりマウス変異一本鎖抗体のcDNAライブラリーを作成した。ヒト変異H鎖DNAならびにヒト変異L鎖DNAをH鎖とL鎖の一本化PCRによりヒト変異一本鎖抗体のcDNAライブラリーを作成した。 Furthermore, mouse H-chain DNA and mouse L-chain DNA or human H-chain DNA and human L-chain DNA prepared as shown in Fig. 17 were each subjected to random mutagenesis by error-prone PCR using Mutazyme II. A cDNA library of mouse mutated single-chain antibodies was prepared by PCR that unifies the H and L chains of mutated mouse H-chain DNA and mutated mouse L-chain DNA. A cDNA library of human mutated single-chain antibodies was prepared by PCR for unifying H and L chains of human mutated H-chain DNA and human mutated L-chain DNA.
[実施例12]: ASGRと結合する一本鎖抗体の選択
　ASGRと結合する一本鎖抗体の選択実験を図19に示すような手順で行った。 [Example 12]: Selection of single-chain antibodies that bind to ASGR A selection experiment for single-chain antibodies that bind to ASGR was performed according to the procedure shown in Fig. 19 .
（12-1）一本鎖抗体IVVライブラリーの調製
（12-1-1）一本鎖抗体ライブラリーの転写
　各一本鎖抗体ライブラリーDNA 2pmol、5×SP6緩衝液 8μl、ATP (100mM) 2μl、CTP (100mM) 2μl、UTP (100mM) 2μl、GTP (10mM) 4μl、キャップアナログ(m7G(5')PPP(5')G) (Invitrogen) (40mM) 5μl、エンザイムミックスSP6RNA ポリメラーゼ（Promega） 4μl、RNase-Free水を添加し全体量を 40μl、37℃、3時間反応後、RQ1 RNase-Free DNase（Promega） 10μl、を添加しさらに37℃、1時間反応させた。
　得られたRNAはRNeasy Mini kit (Qiagen)により精製した。すなわち転写反応液に、RNase-Free水を添加し全体量を 100μlとし、RLT緩衝液(Qiagen) 350μl、2-メルカプトエタノール 5μl、(100%) エタノール 250μl、を加えRNeasy ミニスピンカラムに供し、4℃、12,000 rpm、15秒間遠心後排出された溶液を除去し、RPE緩衝液(Qiagen)500μlを同カラムに加え、4℃、12,000 rpm、15秒間遠心後排出された溶液を除去し、さらにRPE緩衝液(Qiagen)500μlを同カラムに加え、4℃、12,000 rpm、2分間遠心後排出された溶液を除去し、同カラムを新しいチューブに差し替え、4℃、12,000 rpm、1分間遠心し、再び同カラムを新しいチューブに差し替え同カラムに、RNase-Free水を33μl添加し、10分間氷上で放置後、4℃、14000 rpm、1分間遠心しRNA溶液として回収した。 (12-1) Preparation of single-chain antibody IVV library (12-1-1) Transcription of single-chain antibody library 2 pmol of each single-chain antibody library DNA, 8 μl of 5×SP6 buffer, ATP (100 mM) 2 μl CTP (100 mM) 2 μl UTP (100 mM) 2 μl GTP (10 mM) 4 μl Cap analog (m7G(5')PPP(5')G) (Invitrogen) (40 mM) 5 μl Enzyme mix SP6 RNA polymerase (Promega) After adding 4 μl of RNase-free water and reacting the total amount to 40 μl at 37° C. for 3 hours, 10 μl of RQ1 RNase-Free DNase (Promega) was added and further reacted at 37° C. for 1 hour.
The obtained RNA was purified by RNeasy Mini kit (Qiagen). That is, RNase-free water was added to the transcription reaction solution to bring the total volume to 100 μl, and 350 μl of RLT buffer (Qiagen), 5 μl of 2-mercaptoethanol, and 250 μl of (100%) ethanol were added and applied to an RNeasy mini spin column. Remove the discharged solution after centrifugation at 12,000 rpm for 15 seconds at ℃, add 500 μl of RPE buffer (Qiagen) to the same column, remove the discharged solution after centrifugation at 4 ℃, 12,000 rpm for 15 seconds, and further remove the RPE Add 500 μl of buffer solution (Qiagen) to the column, centrifuge at 12,000 rpm for 2 minutes at 4°C, remove the discharged solution, replace the column with a new tube, centrifuge at 12,000 rpm for 1 minute at 4°C, and repeat. The same column was replaced with a new tube, 33 μl of RNase-free water was added to the same column, left on ice for 10 minutes, centrifuged at 4° C., 14000 rpm for 1 minute, and collected as an RNA solution.
（12-1-2）PEGスペーサーとのライゲーション
　精製した各RNA溶液 32μl、T4 ligation 10×緩衝液 5μl、(0.1 M) DTT 1μl、(40 mM) ATP 0.5μl、(100%) DMSO 5μl、(0.1%) BSA 1μl、RNase inhibitor(TOYOBO) 1μl、ピューロマイシン付きPEGスペーサー (特開2002-176987) (10 nmol) 0.5μl、ポリエチレングリコール（PEG） 2000 （日本油脂）(30 nmol) 1μl、T4 RNA リガーゼ (Takara)(250 U/μl)  3μl、遮光条件下15℃、15時間反応させた。得られたスペーサー分子が結合したRNAはRNeasy Mini kit (Qiagen)により精製した。 (12-1-2) Ligation with PEG spacer Each purified RNA solution 32 μl, T4 ligation 10× buffer 5 μl, (0.1 M) DTT 1 μl, (40 mM) ATP 0.5 μl, (100%) DMSO 5 μl, ( 0.1%) BSA 1 μl, RNase inhibitor (TOYOBO) 1 μl, PEG spacer with puromycin (JP 2002-176987) (10 nmol) 0.5 μl, polyethylene glycol (PEG) 2000 (NOF) (30 nmol) 1 μl, T4 RNA 3 μl of ligase (Takara) (250 U/μl) was allowed to react at 15° C. under light-shielding conditions for 15 hours. The resulting spacer-molecule-bound RNA was purified using the RNeasy Mini kit (Qiagen).
（12-1-3）IVVライブラリーの調製
　ヒト一本鎖抗体PEG-RNAライブラリー、ヒト変異一本鎖抗体PEG-RNAライブラリー、マウス一本鎖抗体PEG-RNAライブラリー、マウス変異一本鎖抗体PEG-RNAライブラリーを1:1:1:1の比で混合したPEG-RNA 10 pmol、小麦胚芽抽出液（Promega）20μl、クレアチンキナーゼ (40μg/μl)（Roche） 2μl、RNase inhibitor(TOYOBO) 1.6μl、5×翻訳緩衝液(Promega) 20μl、RNase-Free水を添加し全体量を 100μlとし、遮光条件下26℃、1時間30分反応させ翻訳を行いIVVライブラリーを調製した。 (12-1-3) Preparation of IVV library Human single-chain antibody PEG-RNA library, human mutant single-chain antibody PEG-RNA library, mouse single-chain antibody PEG-RNA library, mouse mutation 10 pmol of PEG-RNA mixed in a 1:1:1:1 ratio with the chain antibody PEG-RNA library, 20 μl of wheat germ extract (Promega), 2 μl of creatine kinase (40 μg/μl) (Roche), RNase inhibitor ( 1.6 μl of TOYOBO), 20 μl of 5× translation buffer (Promega) and RNase-free water were added to bring the total volume to 100 μl, and the mixture was allowed to react at 26° C. under light shielding conditions for 1 hour and 30 minutes for translation to prepare an IVV library.
（12-2）ASGRの固定化
　ビアコアはビアコア3000システムを用い、センサーチップSAに固定化を行った。フローは緩衝液HBS-P(10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20) で10μl/minで行った。フローセル１～4への50mM NaOH, 1M NaClを含む溶液10μlのインジェクトを3回繰り返し行い、固定化の前処理を行った。初めにビオチン化細胞外ドメインASGR1exを用いて、フローセル１～４にベイトを固定化した。フローは緩衝液HBS-P、20μl/minで行った。ベイトの固定量は表7に示した。続いてビオチン化細胞外ドメインASGR2exを用いて、フローセル3～４に固定化した。フローは緩衝液HBS-P、20μl/minで行った。ベイトの固定量は表7に示した。センサーチップを洗浄する目的で緩衝液HBS-Pで10μl/min, 50% Isopropanol, 50mM NaOH, 1M NaClを用いてextra washを行った。 (12-2) Immobilization of ASGR Biacore immobilized on the sensor chip SA using the Biacore 3000 system. Flow was performed at 10 μl/min in buffer HBS-P (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20). Pretreatment for immobilization was performed by injecting 10 μl of a solution containing 50 mM NaOH and 1 M NaCl into flow cells 1 to 4 three times. Bait was first immobilized on flow cells 1-4 using the biotinylated extracellular domain ASGR1ex. Flow was in buffer HBS-P, 20 μl/min. The fixed amount of bait is shown in Table 7. The biotinylated extracellular domain ASGR2ex was then used to immobilize to flow cells 3-4. Flow was in buffer HBS-P, 20 μl/min. The fixed amount of bait is shown in Table 7. For the purpose of washing the sensor chip, an extra wash was performed with a buffer solution HBS-P at 10 μl/min using 50% Isopropanol, 50 mM NaOH and 1M NaCl.
（12-3）ASGRと結合する一本鎖抗体の選択
　調製した一本鎖抗体IVVライブラリー100 μlにfinal 20mMとなるように0.5M EDTA 4μlを加え、20分間室温で回転攪拌した。HBS-Pで膨潤ならびに平衡化させたSephadex G200 (Amersham Biosciences)ゲル1 mlをカラム（バイオラッド）に充填したものにIVVライブラリー溶液100μlを供し、2滴ずつ96穴プレートに集め１wellから10wellまでを回収した。Multi-detection Microplate Reader POWERSCAN HTで励起波長485nm、蛍光波長528nmで蛍光を検出し、3well目から7well目あたりに溶出したIVV画分を集めた。Strep Magne Sphae Paramagnetic Part (9013-20-1) 100μlをHBS-P 500μlで3回洗浄したものにIVV画分約200μlを加え、20分間室温で回転攪拌し、その上清をビアコアにインジェクトした。ビアコアでのセレクション条件は表7に示した。図20に示すように１ラウンドから３ラウンドの選択実験では洗浄工程の終了後、センサーチップを機械より抜き取り、センサーチップの金膜の上にRNase-Free水55μlを静かに加え、20分間、365nmのUVを照射してスペーサーを切断してmRNA部を溶出・回収した。また４ラウンドから５ラウンドの選択実験では洗浄工程の終了後、ASGR1exを用いて競合溶出を行ないIVV画分を回収した。 (12-3) Selection of ASGR-Binding Single-Chain Antibody To 100 μl of the prepared single-chain antibody IVV library, 4 μl of 0.5 M EDTA was added to a final concentration of 20 mM, and the mixture was stirred at room temperature for 20 minutes. Apply 100 μl of the IVV library solution to a column (Bio-Rad) packed with 1 ml of Sephadex G200 (Amersham Biosciences) gel swollen and equilibrated with HBS-P, collect 2 drops in 96-well plates from 1 well to 10 wells. recovered. Fluorescence was detected at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Multi-detection Microplate Reader POWERSCAN HT, and IVV fractions eluted from the 3rd well to the 7th well were collected. About 200 μl of the IVV fraction was added to 100 μl of Strep Magne Sphae Paramagnetic Part (9013-20-1) washed three times with 500 μl of HBS-P, and the mixture was stirred at room temperature for 20 minutes, and the supernatant was injected into Biacore. . Table 7 shows the selection conditions in Biacore. As shown in Figure 20, in the 1st to 3rd round selection experiments, after the washing process, the sensor chip was pulled out from the machine, and 55 µl of RNase-free water was gently added to the gold film of the sensor chip, followed by washing at 365 nm for 20 minutes. The spacer was cleaved by UV irradiation, and the mRNA portion was eluted and collected. In addition, in the 4th to 5th round selection experiments, after the washing step was completed, competitive elution was performed using ASGR1ex to collect the IVV fraction.
（12-4）RT-PCRによるcDNAライブラリーの回収
　選択実験で回収した溶液55μl、5×RT緩衝液(TOYOBO) 20μl、(10 mM) dNTPs(TOYOBO) 10μl、及びreverseプライマー：Flag His tag A02 (10pmol/μl) 5μlにRNase-Free水を添加し全体量を90μlとして混和させ、65℃で9分間反応後直ちに氷上に冷却し、2分間放置した後、ReverTra Ace(TOYOBO) 5μl及びRNase inhibitor(TOYOBO) 5μlを加え、50℃30分間、99℃、5分間逆転写反応させた。逆転写反応させた反応溶液100μl、10×KOD plus緩衝液(TOYOBO) 100μl、2 mM dNTPs(TOYOBO) 100μl、25mM MgSO4 40μl、forwardプライマー：SP6 Omega (10 pmol/μl) 30μl、reverseプライマー：Flag His tag A02 (10 pmol/μl) 30μl、及びKOD plus ポリメラーゼ(TOYOBO) 20μlにRNase-Free水を添加して全体量を1000μlとしチューブ１本につき100μl入れ合計１０本をPCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を20から40サイクル行った後、68℃で5分間反応を行った。 (12-4) Recovery of cDNA library by RT-PCR 55 μl of the solution recovered in the selection experiment, 20 μl of 5×RT buffer (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and reverse primer: Flag His tag A02 (10 pmol/μl) RNase-free water was added to 5 μl to make the total volume 90 μl, mixed, reacted at 65°C for 9 minutes, immediately cooled on ice, left for 2 minutes, then added 5 μl of ReverTra Ace (TOYOBO) and RNase inhibitor. (TOYOBO) 5 μl was added and reverse transcription reaction was carried out at 50° C. for 30 minutes and 99° C. for 5 minutes. 100 μl of reverse transcription reaction solution, 100 μl of 10×KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO 4 , forward primer: SP6 Omega (10 pmol/μl) 30 μl, reverse primer: Flag RNase-free water was added to 30 μl of His tag A02 (10 pmol/μl) and 20 μl of KOD plus polymerase (TOYOBO) to make a total volume of 1000 μl, and 100 μl was added to each tube and a total of 10 tubes were subjected to PCR reaction. PCR was carried out at 94°C for 5 minutes, followed by 20 to 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes.
　ビアコアを使って選択実験の選択圧は表7に示すように段階的にラウンドごとに選択圧を順次上げた。 　Biacore was used to increase the selection pressure in the selection experiment step by step as shown in Table 7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
[実施例13]: クローニングと塩基配列の決定
（13-1）クローニングと塩基配列
　ASGRと結合する一本鎖抗体ライブラリーから in-fusionクローニング
　ライブラリーのinsert作成を行なった。3ラウンドの選択実験を行ったライブラリー1μl、10×KOD plus緩衝液(TOYOBO) 100μl、2 mM dNTPs(TOYOBO) 100μl、25mM MgSO4 40μl、T7-long-F in (10 pmol/μl) 30μl、Flag(Histag) in R (10 pmol/μl) 30μl、及びKOD plus ポリメラーゼ(TOYOBO) 20μlにRNase-Free水を添加して全体量を1000μlとし、PCR反応させた。PCRは、94℃5分間反応後、94℃30秒間、58℃30秒間、68℃2分間を8サイクル行った後、68℃5分間反応を行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、50μlのDNA溶液として回収しT7-ASGR3を得た。 [Example 13]: Cloning and base sequence determination (13-1) Cloning and base sequence An in-fusion cloning library was inserted from a single-chain antibody library that binds to ASGR. 1 μl of library subjected to three rounds of selection experiments, 100 μl of 10×KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO 4 , 30 μl of T7-long-F in (10 pmol/μl), RNase-free water was added to 30 μl of Flag (Histag) in R (10 pmol/μl) and 20 μl of KOD plus polymerase (TOYOBO) to bring the total volume to 1000 μl, and a PCR reaction was performed. PCR was carried out by reacting at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reaction at 68°C for 5 minutes. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of DNA solution to obtain T7-ASGR3.
　kozak-Igk-T7tag配列とHis配列とstopコドンを含む pcDNA 3.3ベクター8μl、10×KOD plus緩衝液(TOYOBO) 80μl、2 mM dNTPs(TOYOBO) 80μl、25mM MgSO4 32μl、His-stop-topo F (10 pmol/μl) 24μl、Igk-T7-R (10 pmol/μl) 24μl、及びKOD plus ポリメラーゼ(TOYOBO) 16μlにRNase-Free水を添加して全体量を800μlとし、PCR反応させた。PCRは、94℃2分間反応後、98℃10秒間、68℃5分11秒間を25サイクル行った。cDNAライブラリーはWizard SV Gel PCR Clean-Up System（Promega）で精製し、40μlのDNA溶液として回収しKIgk-stop 3.3 vectorを得た。 8 μl of pcDNA 3.3 vector containing kozak-Igk-T7tag sequence, His sequence and stop codon, 80 μl of 10×KOD plus buffer (TOYOBO), 80 μl of 2 mM dNTPs (TOYOBO), 32 μl of 25 mM MgSO 4 , His-stop-topo F ( RNase-free water was added to 24 μl of Igk-T7-R (10 pmol/μl), 24 μl of Igk-T7-R (10 pmol/μl), and 16 μl of KOD plus polymerase (TOYOBO) to bring the total volume to 800 μl, followed by PCR reaction. PCR was carried out by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds. The cDNA library was purified with Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 40 μl of DNA solution to obtain KIgk-stop 3.3 vector.
　T7-ASGR3 0.08μl、KIgk-stop 3.3 vector 0.14μl、5x in fusion HD Enzyme premix 1.0μl (Takara) を混ぜ、RNase-Free水を添加し全体量を5μlとし50℃、15分間反応させた。反応後の溶液2.5μlをOne Shot TOP10 competent cellにトランスフォーメーションし37℃１晩培養し、KIgk-ASGR3クローンを得た。得られたクローンの配列解析はユーロフィンDNAシーケンス受託サービス、ValueReadプレミックスにより行った。 　T7-ASGR3 0.08 μl, KIgk-stop 3.3 vector 0.14 μl, 5x infusion HD Enzyme premix 1.0 μl (Takara) were mixed, RNase-Free water was added to make the total volume 5 μl, and the mixture was reacted at 50°C for 15 minutes. 2.5 μl of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37° C. to obtain a KIgk-ASGR3 clone. Sequence analysis of the obtained clones was performed by Eurofin DNA sequence contract service, ValueRead Premix.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
（13-2）ライブラリーの配列解析
　ライブラリーの配列解析の結果、表9に示すように3ラウンド4ラウンド5ラウンドからそれぞれASGR結合一本鎖抗体のクローンが得られた。 (13-2) Sequence Analysis of Library As a result of sequence analysis of the library, as shown in Table 9, ASGR-binding single-chain antibody clones were obtained from each of 3 rounds, 4 rounds, and 5 rounds.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
[実施例14]: ASGR結合一本鎖抗体のクローンの活性評価
（14-1）タンパク質の調製
　クローンはカルベニシリン20μg/mlを含むLB培地にマスタープレートよりコロニーを植菌し37℃、16時間培養した。菌体ペレットからPureLink HiPure Plasmid Maxiprep Kit (インビトロジェン)でプラスミドを精製した。 [Example 14]: Activity evaluation of ASGR-binding single-chain antibody clone (14-1) Protein preparation Clones were inoculated from a master plate onto an LB medium containing 20 µg/ml of carbenicillin and cultured at 37°C for 16 hours. bottom. Plasmids were purified from cell pellets with the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen).
（14-2）タンパク質の調製
　ヒト胎児腎細胞293T細胞は10%FCS(ニチレイ)、DMEM(1.0g/l Glucose) with L-Gln and Sodium Pyruvate, liquid (ナカライ)培地で培養した。トランスフェクションの24時間前に6cm シャーレに(1:2)の割合でプレーティングした。6cm シャーレに対しプラスミドDNA 8μgをOpti-MEM I 500μlに入れたものに、Lipofectamine 2000 20μlあるいはPEI-MAX 20μlをOpti-MEM I 500μlに入れ室温で5分間置いたものを穏やかに加えた。混和物は室温で20分間放置後293T細胞の6cm シャーレに加え37℃、5% CO2条件下4日～６日培養した。 (14-2) Preparation of Protein Human embryonic kidney 293T cells were cultured in 10% FCS (Nichirei), DMEM (1.0 g/l Glucose) with L-Gln and Sodium Pyruvate, liquid (Nacalai) medium. Plated at a ratio (1:2) on a 6 cm petri dish 24 hours before transfection. 8 μg of plasmid DNA in 500 μl of Opti-MEM I, 20 μl of Lipofectamine 2000 or 20 μl of PEI-MAX in 500 μl of Opti-MEM I, and left at room temperature for 5 minutes were gently added to a 6 cm petri dish. The mixture was allowed to stand at room temperature for 20 minutes, and then added to a 6 cm petri dish of 293T cells and cultured for 4 to 6 days under conditions of 37°C and 5% CO 2 .
（14-3）プルダウン実験
　レジン(Streptavidin MagneSphere Paramagnetic Particles)(Promega) の洗浄はMagneSphere Technology Magnetic Separation Stand (tweleve-position)を使って行った。レジン40μlを1.5 mlチューブに取り、溶液を除去した後PBS 1 mlで3回洗浄を行った。新しいチューブにレジンを移し、ビオチン化細胞外ドメインASGR1exあるいはビオチン化細胞外ドメインASGR2ex あるいはベイトなしは等量のPBSを加え、ミニディスクローター（Bio craft）で回転攪拌し4℃で1時間結合させた。溶液を除去後TBST (Tween20 0.05%) 1 mlで3回洗浄を行った。新しいチューブにレジンを移し、ELISA BSA緩衝液(ナカライテスク株式会社　TBS, 0.05%, Tween 20, 1% BSA) 1 mlで4℃1時間ブロッキングを行い、ELISA BSA緩衝液1 mlで3回洗浄を行った。新しいチューブにレジンを移し、ASGR3-10M、ASGR3-39D、ASGR4-70DあるいはASGR5-24Mを発現させた培養上清を1000μlずつ、ビオチン化細胞外ドメインASGR1exあるいはビオチン化細胞外ドメインASGR2exあるいはベイトなしで処理したレジン40μlに混和させ、ミニディスクローター（Bio craft）で回転攪拌し、4℃で1時間30分結合させた。溶液を除去後TBST (Tween20 0.1%) 500μlで3回洗浄を行い、回収レジンをウエスタンブロットした。 (14-3) Pull-down experiment Resin (Streptavidin MagneSphere Paramagnetic Particles) (Promega) was washed using MagneSphere Technology Magnetic Separation Stand (twelve-position). 40 µl of the resin was placed in a 1.5 ml tube, the solution was removed, and then washed with 1 ml of PBS three times. The resin was transferred to a new tube, biotinylated ectodomain ASGR1ex or biotinylated ectodomain ASGR2ex or without bait was added with an equal volume of PBS, and the mixture was rotated with a mini disc rotor (Bio craft) and allowed to bind at 4°C for 1 hour. . After removing the solution, washing was performed three times with 1 ml of TBST (Tween20 0.05%). Transfer the resin to a new tube, block with 1 ml of ELISA BSA buffer (Nacalai Tesque Co., Ltd. TBS, 0.05%, Tween 20, 1% BSA) at 4°C for 1 hour, and wash 3 times with 1 ml of ELISA BSA buffer. gone. The resin was transferred to a new tube, and 1000 μl of the culture supernatant expressing ASGR3-10M, ASGR3-39D, ASGR4-70D or ASGR5-24M was added without biotinylated extracellular domain ASGR1ex or biotinylated extracellular domain ASGR2ex or without bait. It was mixed with 40 μl of treated resin, rotated with a mini disc rotor (Biocraft) and allowed to bind at 4° C. for 1 hour and 30 minutes. After removing the solution, washing was performed three times with 500 μl of TBST (0.1% Tween 20), and the recovered resin was subjected to Western blotting.
　各レジンに水とサンプル緩衝液LDS(4x)、0.2mM DTTを加え、70℃、10分間加熱後SDS-PAGEに供した。SDS-PAGEは4-12% Bis-Tris Gel、NuPAGE MES SDS電気泳動緩衝液 (Invitrogen)で200V, 400mA, 35分間電気泳動後、Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turboで転写した。膜はBlocking One Buffer：TBST（1：9）でブロッキングし、Blocking One Buffer：TBST(1：9)で2:3000に希釈したanti-Flag-HRP (Sigma: A8592)を反応させた。検出はECL(Enhanced ChemiLuminescence)を用い、ChemiDoc(BIORAD)で行った。 Water, sample buffer LDS (4x), and 0.2 mM DTT were added to each resin, heated at 70°C for 10 minutes, and subjected to SDS-PAGE. SDS-PAGE was performed with 4-12% Bis-Tris Gel, NuPAGE MES SDS electrophoresis buffer (Invitrogen) at 200V, 400mA for 35 minutes, followed by Mini Format, 0.2μm PVDF, Single application (BIORAD) Trans-Blot Turbo. transcribed with The membrane was blocked with Blocking One Buffer: TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer: TBST (1:9). Detection was performed using ChemiDoc (BIORAD) using ECL (Enhanced ChemiLuminescence).
　ASGR結合一本鎖抗体によるプルダウンアッセイ（図20、21）を行った。3ラウンドの選択実験を行なったクローン、ASGR3-10M、ASGR3-39Dは、コントロールと比べて抗原を強く認識した。ASGR3-10Mは両方の抗原を認識し、ASGR3-39Dの方はASGR1抗原を特に強く認識した。4ラウンドと５ラウンドの選択実験を行なったクローン、ASGR4-70D、ASGR5-24Mは、コントロールと比べて抗原を認識した。ASGR4-70D、ASGR5-24MはASGR1抗原を特に強く認識した。 A pull-down assay (Figs. 20, 21) was performed using an ASGR-binding single-chain antibody. Clones, ASGR3-10M, ASGR3-39D, subjected to three rounds of selection experiments, recognized the antigen more strongly than the control. ASGR3-10M recognized both antigens, and ASGR3-39D recognized the ASGR1 antigen particularly strongly. Clones ASGR4-70D, ASGR5-24M subjected to 4 and 5 rounds of selection experiments recognized the antigen compared to controls. ASGR4-70D and ASGR5-24M particularly strongly recognized the ASGR1 antigen.
　図22に、抗ASGR一本鎖抗体を肝細胞内送達用担体として用いたDOCK11結合ペプチドの肝細胞選択的送達法の概要図を示した。抗ASGR抗体を連結したDOCK11結合ペプチドは、受容体に結合後、エンドサイトーシスにより初期エンドソームに内包される。DOCK11結合ペプチドの上流にはエンドソーム内の酵素により切断を受ける切断配列を組み込んでおくことでそれが切断を受けペプチドが解離する。DOCK11結合ペプチドの下流には膜透過促進ペプチドを付加し、それにより細胞質にペプチドが放出される。標的であるDOCK11は、細胞内あるいは核画分に局在するタンパク質であることから、核に送達させる場合は核移行シグナルを付加させる。 Fig. 22 shows a schematic diagram of a method for selectively delivering DOCK11-binding peptides to hepatocytes using an anti-ASGR single-chain antibody as a carrier for intrahepatocyte delivery. DOCK11-binding peptides conjugated with anti-ASGR antibodies are packaged into early endosomes by endocytosis after binding to receptors. By incorporating a cleavage sequence that is cleaved by an enzyme in the endosome upstream of the DOCK11-binding peptide, it is cleaved and the peptide is dissociated. A membrane permeabilization peptide is added downstream of the DOCK11-binding peptide, thereby releasing the peptide into the cytoplasm. Since the target DOCK11 is a protein localized in cells or nuclear fractions, a nuclear localization signal is added when delivered to the nucleus.
　IVV法による選択実験の結果、DOCK11結合ペプチドとASGR結合一本鎖抗体はそれぞれ複数個得られた。まずは図23に示すように抗アシアロ糖タンパク質受容体一本鎖抗体はASGR3-10M、DOCK11結合ペプチドはDCS8-42TNを使って融合体を作成した。それぞれ発現ベクターに組み込まれた遺伝子に対し必要な機能性ペプチドをインフュージョンクローニングで各コンストラクトを作成した。本実施例では、切断配列として内因性プロテアーゼFurinが認識する配列を、膜透過促進ペプチドとして膜融合促進ペプチドS28又はS39を、核移行シグナルとしてPAAKRVKLD（配列番号４０）を利用した。 As a result of the selection experiment by the IVV method, multiple DOCK11-binding peptides and multiple ASGR-binding single-chain antibodies were obtained. First, as shown in FIG. 23, a fusion was prepared using ASGR3-10M as an anti-asialoglycoprotein receptor single-chain antibody and DCS8-42TN as a DOCK11-binding peptide. Each construct was prepared by infusion cloning of functional peptides necessary for the genes incorporated into the respective expression vectors. In this example, the sequence recognized by the endogenous protease Furin was used as the cleavage sequence, the membrane fusion promoting peptide S28 or S39 as the membrane permeation promoting peptide, and PAAKRVKLD (SEQ ID NO: 40) as the nuclear localization signal.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　DOCK11結合ペプチドDCS8-42TNは、トランスフェクション法でプラスミドを細胞内に導入しタンパク質を発現させた場合、アクチンの形態変化を生じる。図23Aに示すコンストラクトの融合体をHepG2細胞へ直接細胞外より取り込ませた場合にも同様にアクチンの形態変化を生じるかどうかを検証した。また、Furinにより切断配列が切断され機能しているかについても同時に検証した。図23Bのコンストラクトを使用し、膜融合促進ペプチドが機能しているかどうかを検証した。図23Cのコンストラクト使用し、核移行シグナルが機能しているかどうかを検証した。これらはDOCK11結合ペプチドDCS8-42TNの上流に付加したGFPの蛍光を利用し、共焦点顕微鏡を観察することにより明らかにした。図23Dのコンストラクトは、PXB細胞を使った抗HBV効果の評価、あるいはPXBマウスを用いたin vivoでの評価に用いた。またこのコンストラクトはN-10M-D42TNと呼称した。N-10M-D42TNのうち、切断配列＋DCS8-42TNペプチド＋S28＋核移行シグナルの部分のアミノ酸配列を配列番号90に示す。配列番号90のアミノ酸配列中、第21番～第28番アミノ酸はFlagタグ、第29番～第35番アミノ酸はHisタグである。 The DOCK11-binding peptide DCS8-42TN causes actin morphology changes when the plasmid is introduced into cells by the transfection method and the protein is expressed. It was verified whether or not the fusion of the construct shown in FIG. 23A would also cause a similar morphological change in actin when it was directly extracellularly incorporated into HepG2 cells. At the same time, it was also verified whether the cleaved sequence was cleaved by Furin and functioned. Using the construct of FIG. 23B, it was verified whether the membrane fusion-promoting peptide was functioning. Using the construct in Figure 23C, it was verified whether the nuclear localization signal was functioning. These were clarified by confocal microscopy using the fluorescence of GFP added upstream of the DOCK11-binding peptide DCS8-42TN. The construct in Figure 23D was used for evaluation of anti-HBV effects using PXB cells or in vivo evaluation using PXB mice. This construct was also called N-10M-D42TN. SEQ ID NO:90 shows the amino acid sequence of the cleavage sequence+DCS8-42TN peptide+S28+nuclear localization signal portion of N-10M-D42TN. In the amino acid sequence of SEQ ID NO:90, the 21st to 28th amino acids are the Flag tag, and the 29th to 35th amino acids are the His tag.
　DCS8-42TNペプチド有り、無しのコンストラクトで一本鎖抗体-ペプチド融合体をHepG2細胞に加えた。図24Aに示す手順で処理し共焦点顕微鏡で観察したところ、ペプチド有りをかけた細胞はアクチンの断片化が起きていたのに対し、ペプチド無しはアクチンの断片化は見られなかった。またペプチドの局在を示すGFPのシグナルは細胞質に見られ、Furinにより切断配列が切断され機能し、また膜融合促進ペプチドが機能していることがわかった。 A single-chain antibody-peptide fusion was added to HepG2 cells with or without the DCS8-42TN peptide. When the cells treated with the procedure shown in FIG. 24A were observed under a confocal microscope, actin fragmentation occurred in the cells with the peptide applied, whereas actin fragmentation was not observed in the cells without the peptide. In addition, the GFP signal indicating the localization of the peptide was observed in the cytoplasm, and it was found that the cleavage sequence was cleaved by Furin and functioned, and the fusion-promoting peptide functioned.
　図25Aに示すように膜融合促進ペプチドが機能しているかどうかを検証した。エンドソームはインキュベーション時間に伴い大きくなっていた。また同じ位置にGFP（ペプチド）が観察された。それに対し細胞質中にGFPはほとんど観察出来ないだけでなくアクチンの断片化も見られなかった。膜融合促進ペプチドS28が無いと、ペプチドは細胞質に放出されずエンドソームに蓄積しているようだった。 　As shown in Fig. 25A, it was verified whether the membrane fusion-promoting peptide was functioning. Endosomes grew larger with incubation time. GFP (peptide) was also observed at the same position. In contrast, GFP was hardly observed in the cytoplasm, and actin fragmentation was not observed. Without the fusion-promoting peptide S28, the peptide appeared to accumulate in endosomes rather than being released into the cytoplasm.
　図25Bに示すように膜融合促進ペプチドの種類に応じて機能に違いかあるかどうかを検証した。HepG2細胞に取り込ませた抗体-ペプチド融合体より核画分を抽出し、anti-FLAG M2 magnetic beadsでプルダウンさせ比較した。その結果、膜融合促進ペプチドS28よりも膜融合促進ペプチドS39のバンド強度が濃かったことから膜融合促進ペプチドS39の方がエンドソームからの離脱の効率がよいことがわかった。 As shown in Fig. 25B, it was verified whether there was a difference in function depending on the type of membrane fusion-promoting peptide. Nuclear fractions were extracted from antibody-peptide fusions incorporated into HepG2 cells and compared by pulling down with anti-FLAG M2 magnetic beads. As a result, the intensity of the band of fusion-promoting peptide S39 was higher than that of fusion-promoting peptide S28, indicating that fusion-promoting peptide S39 was more efficient in leaving the endosome.
　図26に示すように核移行シグナルが機能しているかどうかを検証した。抗体-ペプチド融合体を加えないコントロール(PBS)の場合は核内アクチンは網目状に見え、断片化は起こっていなかった。これに対し、核移行シグナル有り-ペプチド融合体をかけた細胞ではペプチド(GFP)が核内に局在し核内アクチンの断片化が起きていた。ペプチド(GFP)は核内アクチンに沿って点々と見られた。アクチンに重なっているか、あるいは近接しているようだった。 　As shown in Fig. 26, it was verified whether the nuclear localization signal was functioning. In the control (PBS) to which the antibody-peptide fusion was not added, nuclear actin appeared mesh-like and fragmentation did not occur. On the other hand, in cells with nuclear localization signal-peptide fusion, the peptide (GFP) was localized in the nucleus and nuclear actin fragmentation occurred. A peptide (GFP) was spotted along the nuclear actin. It appeared to overlap or be close to actin.
[実施例15]: PXB細胞におけるDOCK11結合ペプチドの抗HBV活性
　PXBマウス（ヒト肝細胞キメラマウス）から分離された新鮮ヒト肝細胞であるPXB細胞は、ヒト型の肝機能が高く維持されたヒト肝細胞であり、B型肝炎ウイルス（HBV）が持続感染する。そこで、PXB細胞を使用してDOCK11結合ペプチドの抗HBV活性を評価した。DOCK11結合ペプチドは細胞の外から加えるため抗ASGR一本鎖抗体-ペプチド融合体N-10M-D42TNとし、PXB細胞でのHBVアッセイに供した。 [Example 15]: Anti-HBV activity of DOCK11-binding peptide in PXB cells Liver cells, persistently infected with hepatitis B virus (HBV). Therefore, we evaluated the anti-HBV activity of DOCK11-binding peptides using PXB cells. Since the DOCK11-binding peptide was added from the outside of the cell, the anti-ASGR single-chain antibody-peptide fusion N-10M-D42TN was used and subjected to HBV assay in PXB cells.
　図27A、図27BにPXB細胞におけるDOCK11結合ペプチドの抗HBV活性を示した。N-10M-D42TNはHBV-DNAおよびcccDNAのコピー数を顕著に減少させ、濃度依存的に高い抗HBV活性を示した。非常に高い抗HBV活性を示すことが明らかになった。また図27Bに示すように再現性の良い結果が得られた。 Figures 27A and 27B show the anti-HBV activity of DOCK11-binding peptides in PXB cells. N-10M-D42TN markedly decreased the copy numbers of HBV-DNA and cccDNA, and showed high anti-HBV activity in a dose-dependent manner. It was found to exhibit very high anti-HBV activity. In addition, results with good reproducibility were obtained as shown in FIG. 27B.
　DOCK11結合ペプチドDCS8-42TNは非受容体型チロシンキナーゼAck1（NM_001387713.1、配列番号49、50）の674-689残基の配列である。そのため、DOCK11とAck1が細胞内で相互作用するかどうかを共免疫沈降実験により調べた。細胞に投与するにあたり、ペプチドDCS8-42TNを抗ASGR一本鎖抗体と融合させ、細胞内に取り込まれるようにした。以下、DCS8-42TNと一本鎖抗体の融合体をN-10M-D42TNと呼ぶ。293T細胞内でHisBioFLAG-DOCK11およびT7-Ack1-Hisを共発現させ、一本鎖抗体-DOCK11結合ペプチド融合体N-10M-42TNで24時間処理したところ、HisBioFLAG-DOCK11およびT7-Ack1-Hisは細胞内で結合し、この結合はN-10M-D42TNにより阻害された（図28A）。 The DOCK11-binding peptide DCS8-42TN is the sequence of residues 674-689 of non-receptor tyrosine kinase Ack1 (NM_001387713.1, SEQ ID NOS: 49, 50). Therefore, we examined whether DOCK11 and Ack1 interact in cells by co-immunoprecipitation experiments. For administration to cells, the peptide DCS8-42TN was fused with an anti-ASGR single-chain antibody so that it was taken up into cells. The fusion of DCS8-42TN and a single chain antibody is hereinafter referred to as N-10M-D42TN. When HisBioFLAG-DOCK11 and T7-Ack1-His were co-expressed in 293T cells and treated with the single-chain antibody-DOCK11-binding peptide fusion N-10M-42TN for 24 h, HisBioFLAG-DOCK11 and T7-Ack1-His were It bound intracellularly and this binding was inhibited by N-10M-D42TN (Fig. 28A).
　DOCK11はDOCK-Dサブファミリーのひとつであり、グアニンヌクレオチド交換因子（guanine nucleotide exchange factor, GEF）としてCdc42を非活性型（GDP結合型）から活性型（GTP結合型）へと置換することが知られているが、他のGEFタンパク質と異なり活性型Cdc42にも結合してさらに活性化し、ポジティブフィードバックをもたらすことがわかっている (Lin, Q. et al. J. Biol. Chem., 281, 35253-35262, 2006; Nishikimi, A. et al. Exp Cell Res 319, 2343-2349, 2013)。また、Ack1は活性型Cdc42結合タンパク質であり、GTP結合型Cdc42と特異的に結合して活性化される(Prieto-Echague, V., Miller, J., Signal Transduct, 1-9, 2011)。 DOCK11 is a member of the DOCK-D subfamily and is known to be a guanine nucleotide exchange factor (GEF) that converts Cdc42 from an inactive (GDP-bound) to an active (GTP-bound) form. However, unlike other GEF proteins, it is known that it also binds to active Cdc42 and activates it further, resulting in positive feedback (Lin, Q. et al. J. Biol. Chem., 281, 35253 -35262, 2006; Nishikimi, A. et al. Exp Cell Res 319, 2343-2349, 2013). Ack1 is an active Cdc42-binding protein and is activated by binding specifically to GTP-bound Cdc42 (Prieto-Echague, V., Miller, J., Signal Transduct, 1-9, 2011).
　Hisタグ精製したHisBioFLAG-DOCK11およびT7-Ack1-Hisを用いて、DCS8-42TNおよびCdc42存在下でプルダウンアッセイを行ったところ、共免疫沈降実験の結果と同様に、T7-Ack1-HisはHisBioFLAG-DOCK11に結合し、この結合はDCS8-42TNにより阻害された。同様に、この結合は、野生型のGST-Cdc42濃度依存的（図28B）、あるいは活性型（Q61L）および非活性型（T17N）GST-Cdc42によっても阻害された（図28C）。以上の結果から、Cdc42とAck1は競合的にDOCK11に結合することが示唆される。 His-tag purified HisBioFLAG-DOCK11 and T7-Ack1-His were used in pull-down assays in the presence of DCS8-42TN and Cdc42. Binds DOCK11 and this binding is inhibited by DCS8-42TN. Similarly, this binding was inhibited by wild-type GST-Cdc42 concentration-dependently (FIG. 28B), or by active (Q61L) and non-active (T17N) forms of GST-Cdc42 (FIG. 28C). These results suggest that Cdc42 and Ack1 competitively bind to DOCK11.
　次いで、DCS8-42TNがDOCK11のGEF活性に与える影響について調べた。DOCK11はN末端のDHR2ドメインでCdc42と相互作用すると予測されていることから(Lin, Q. et al., J. Biol. Chem., 281, 35253-35262, 2006)、固定したDOCK11のDHR2ドメインにDCS8-42TNおよびGDP結合型Cdc42を反応させた後、Mant-GTPを加えたところ、DCS8-42TN濃度依存的にGDPからGTPへの置換が阻害された（図29）。この結果から、DCS8-42TNは濃度依存的にDOCK11のGEF活性を阻害することが示唆される。 Next, we investigated the effect of DCS8-42TN on the GEF activity of DOCK11. Since DOCK11 is predicted to interact with Cdc42 at its N-terminal DHR2 domain (Lin, Q. et al., J. Biol. Chem., 281, 35253-35262, 2006), the DHR2 domain of anchored DOCK11 was reacted with DCS8-42TN and GDP-bound Cdc42, and Mant-GTP was added to inhibit the displacement of GDP to GTP in a DCS8-42TN concentration-dependent manner (Fig. 29). This result suggests that DCS8-42TN dose-dependently inhibits the GEF activity of DOCK11.
　Ack1はWASPをリン酸化し、アクチン重合を促進することが知られている(Yokoyama, N., Lougheed, J., and Miller, W.T. (2005). Phosphorylation of WASP by the Cdc42-associated Kinase ACK1. Journal of Biological Chemistry 280, 42219-42226.)。DOCK11およびAck1を標的としたsiRNAをHepG2細胞にトランスフェクションし、蛍光ファロイジンでアクチンフィラメントを染色したところ、細胞質におけるアクチンフィラメントの断片化および微小突起の増加が見られた（図30A-C）。同様に、HepG2細胞をN-10M-D42TNで24-48時間処理すると、処理時間依存的に細胞質のアクチンフィラメントが断片化した（図31A,B）。24時間処理後に洗浄し、さらに24-72時間培養したところ、アクチンフィラメントは処理前と同様の長さに回復したことから、N-10M-D42TNによるアクチンフィラメントへの影響には可塑性があることがわかった。また、HepG2細胞にEYFP-NLS-Actinをトランスフェクションして核内のアクチンフィラメントへの影響も調べたところ、GFPタグ付きのN-10M-D42TN(N-10M-GFP-D42TN)で処理した細胞では核内アクチンの断片化が起きた（図30C）。ペプチドは核内に局在し、核内アクチンに沿って点々と見られた。これに対しペプチドで処理しない場合は核内アクチンは網目状に見え、断片化は起こらなかった。以上の結果から、N-10M-D42TNは細胞質および核内においてアクチンフィラメントを断片化することがわかった。 Ack1 is known to phosphorylate WASP and promote actin polymerization (Yokoyama, N., Lougheed, J., and Miller, W.T. (2005). Phosphorylation of WASP by the Cdc42-associated Kinase ACK1. Journal of Biological Chemistry 280, 42219-42226.). Transfection of HepG2 cells with siRNAs targeting DOCK11 and Ack1 and staining of actin filaments with fluorescent phalloidin showed fragmentation of actin filaments and increased microprojections in the cytoplasm (FIGS. 30A-C). Similarly, when HepG2 cells were treated with N-10M-D42TN for 24 to 48 hours, cytoplasmic actin filaments were fragmented in a treatment time-dependent manner (Fig. 31A, B). After treatment for 24 hours, washing, and culturing for 24-72 hours, the length of actin filaments recovered to that before treatment, suggesting that N-10M-D42TN exerts plasticity on actin filaments. have understood. In addition, when HepG2 cells were transfected with EYFP-NLS-Actin and the effect on actin filaments in the nucleus was investigated, cells treated with GFP-tagged N-10M-D42TN (N-10M-GFP-D42TN) fragmentation of nuclear actin occurred (Fig. 30C). The peptide localized to the nucleus and was spotted along nuclear actin. In contrast, when not treated with peptides, nuclear actin appeared mesh-like and was not fragmented. These results indicated that N-10M-D42TN fragmented actin filaments in the cytoplasm and nucleus.
　HBVは細胞膜上で受容体NTCPと結合した後、上皮成長因子受容体EGFRと共にエンドサイトーシスによって細胞内へ侵入することがわかっている (Iwamoto, M. et al., Proc Natl Acad Sci USA, 116, 8487-8492, 2019)。Cdc42により活性化されたAck1はEGF刺激に応じてEGFRと相互作用し、EGFRのエンドサイトーシスに寄与することから（Shen, F. et al., Mol. Biol. Cell, 18, 732-742, 2007; Lin, Q. et al., J. Biol. Chem. 281, 35253-35262, 2010）、D42TNペプチドがEGFRのエンドサイトーシスを阻害することでHBVの侵入を阻害している可能性が示唆される。図32Aにこの機序の模式図を示す。すなわち、EGF刺激によってEGFRが活性化されると、リン酸化およびユビキチン化を受ける。Ack1はDOCK11によって活性化されたCdc42と結合して活性化し、EGFRと結合してリン酸化される。その結果、EGFR-Ack1複合体はともにエンドサイトーシスされて分解される。一方で、N-10M-D42TNがDOCK11の機能を阻害すると、Ack1の活性化が阻害されるため、Ack1とリン酸化EGFRはエンドサイトーシスされず分解されない。また、Ack1によってリン酸化されるWASPのリン酸化も阻害されると考えられる。Huh7細胞をN-10M-D42TNで処理後にEGF刺激したところ、EGF刺激によるAck1のリン酸化が抑制されるとともに、EGFRの分解が抑制された（図32B）。このときEGFRのリン酸化は阻害されなかったことから、EGFRはエンドサイトーシスの段階で阻害されたことが示唆され、N-10M-D42TNによってAck1の活性化が阻害されたためと考えられる。また、N-10M-D42TN処理によってWASPのリン酸化も阻害されたことから、アクチンフィラメントの断片化はWASPの阻害によることが示唆される。また、HepG2細胞をEGF処理すると初期エンドソームにおけるAck1の局在が増加するが、N-10M-D42TN処理によってこの局在変化が抑制された（図33A,B）。以上の結果から、N-10M-D42TNはEGFRのリン酸化には影響せず、Ack1の活性化を阻害することで、Ack1およびEGFRのエンドサイトーシスを阻害することが示唆される。 It is known that HBV binds to the receptor NTCP on the cell membrane and then enters the cell by endocytosis together with the epidermal growth factor receptor EGFR (Iwamoto, M. et al., Proc Natl Acad Sci USA, 116 , 8487-8492, 2019). Ack1 activated by Cdc42 interacts with EGFR in response to EGF stimulation and contributes to EGFR endocytosis (Shen, F. et al., Mol. Biol. Cell, 18, 732-742, 2007; Lin, Q. et al., J. Biol. Chem. 281, 35253-35262, 2010), suggesting that D42TN peptide may inhibit HBV entry by inhibiting EGFR endocytosis be done. Figure 32A shows a schematic of this mechanism. That is, when EGFR is activated by EGF stimulation, it undergoes phosphorylation and ubiquitination. Ack1 binds to and activates DOCK11-activated Cdc42, and binds to and phosphorylate EGFR. As a result, both EGFR-Ack1 complexes are endocytosed and degraded. On the other hand, inhibition of DOCK11 function by N-10M-D42TN inhibits Ack1 activation, preventing Ack1 and phosphorylated EGFR from being endocytosed and degraded. It is also thought that the phosphorylation of WASP, which is phosphorylated by Ack1, is also inhibited. When Huh7 cells were treated with N-10M-D42TN and then EGF-stimulated, EGF-stimulated Ack1 phosphorylation and EGFR degradation were suppressed (FIG. 32B). At this time, EGFR phosphorylation was not inhibited, suggesting that EGFR was inhibited at the stage of endocytosis, possibly because N-10M-D42TN inhibited Ack1 activation. In addition, N-10M-D42TN treatment also inhibited the phosphorylation of WASP, suggesting that fragmentation of actin filaments is due to inhibition of WASP. In addition, EGF treatment of HepG2 cells increased the localization of Ack1 in early endosomes, but N-10M-D42TN treatment suppressed this localization change (Fig. 33A, B). These results suggest that N-10M-D42TN does not affect EGFR phosphorylation and inhibits Ack1 activation, thereby inhibiting Ack1 and EGFR endocytosis.
　HBVがrcDNAからcccDNAを合成する際には宿主のDNA修復機構、特にATRシグナル伝達経路を利用することが知られている(Luo, J. et al. mBio, 11, e03423-19, 2020)。ATRはDNA損傷部位へのアクチン集積に応じてリクルートされることから(Wang, Y-H. et al. Nat Commun, 8, 2118-2133, 2017)、DOCK11がアクチン重合を通じてATRシグナル伝達経路に影響し、cccDNA合成に寄与している可能性が考えられる。HepG2細胞にUV照射したところDOCK11のmRNAが増加したことから（図34A）、DOCK11がDNA修復機構に寄与することが示唆された。また、DOCK11のノックダウンによってDNA修復時のChk1の活性化が抑制されたことから（図34B,C）、DOCK11はATRシグナル伝達経路の活性化に必要であることがわかった。 　HBV is known to utilize the host's DNA repair mechanism, especially the ATR signaling pathway, when synthesizing cccDNA from rcDNA (Luo, J. et al. mBio, 11, e03423-19, 2020). Since ATR is recruited according to actin accumulation at sites of DNA damage (Wang, Y-H. et al. Nat Commun, 8, 2118-2133, 2017), DOCK11 affects the ATR signaling pathway through actin polymerization. Possibility of contributing to cccDNA synthesis is conceivable. UV irradiation of HepG2 cells increased DOCK11 mRNA (FIG. 34A), suggesting that DOCK11 contributes to the DNA repair mechanism. In addition, knockdown of DOCK11 suppressed the activation of Chk1 during DNA repair (Fig. 34B,C), indicating that DOCK11 is required for the activation of the ATR signaling pathway.
　DOCK11のノックダウンと同様に、N-10M-D42TNは処理時間依存的にChk1のリン酸化を抑制したことから（図34D,E）、DOCK11の機能を阻害することでATRシグナル伝達経路を阻害することが示唆された。抗pChk1抗体を用いたHepG2細胞の免疫染色によって、DOCK11のノックダウンによって核内のChk1のリン酸化が抑制された（図35A）。また、N-10M-D42TN処理においても同様に、核内でのChk1のリン酸化が抑制された（図35B）。これらの結果からも、DOCK11が核内でのATRシグナル伝達経路の活性化に必須であり、N-10M-D42TNがそれを阻害することが示唆される。次いで、抗DOCK11抗体を用いてヒト肝細胞PXB細胞を免疫染色し、DNA修復時のDOCK11の局在を調べた（図36A,B）。その結果、DOCK11はUV照射によって核内で凝集して点状の局在を示し、N-10M-D42TNでの処理はそれを阻害することがわかった。抗γH2AX抗体を用いた免疫染色によって、DNA修復時のDOCK11の局在はγH2AXと一致することがわかった（図37A,B）。さらに、DOCK11を標的としたsiRNAのトランスフェクションによってDOCK11の発現が減少すると（図37C）、γH2AXの発現も同様に減少し（図37D）、わずかに発現したDOCK11とγH2AXの局在は一致した（図37E）。γH2AXはリン酸化されたヒストンH2AXであり、DNA損傷部位のマーカーであることから、DOCK11がDNA損傷部位に蓄積し、ATRシグナル伝達経路を活性化することが示唆される。N-10M-D42TNはDOCK11のノックダウンと同様にγH2AXの発現を減少させたが（図37D）、このときγH2AXはDOCK11と共局在しなかった（図37F,G; Wang, Y-H. et al. Nat Commun, 8, 2118, 2017）。このことから、N-10M-D42TNはDNA損傷部位におけるDOCK11のATRシグナル伝達経路活性化を阻害していることが示唆される。すなわち、N-10M-D42TNで処理した細胞ではHBVがrcDNAからcccDNAを合成する際にDNA修復機構を利用できず、感染が抑制されると考えられる。 Similar to knockdown of DOCK11, N-10M-D42TN suppressed Chk1 phosphorylation in a time-dependent manner (Fig. 34D,E), indicating that inhibiting DOCK11 function inhibits the ATR signaling pathway. It has been suggested. Immunostaining of HepG2 cells with anti-pChk1 antibody showed that knockdown of DOCK11 suppressed the phosphorylation of Chk1 in the nucleus (FIG. 35A). In addition, phosphorylation of Chk1 in the nucleus was similarly suppressed by N-10M-D42TN treatment (Fig. 35B). These results also suggest that DOCK11 is essential for activation of the ATR signaling pathway in the nucleus and that N-10M-D42TN inhibits it. Next, human hepatocyte PXB cells were immunostained using an anti-DOCK11 antibody to examine the localization of DOCK11 during DNA repair (FIGS. 36A,B). As a result, it was found that DOCK11 was aggregated in the nucleus by UV irradiation and showed punctate localization, and treatment with N-10M-D42TN inhibited it. Immunostaining with an anti-γH2AX antibody revealed that the localization of DOCK11 during DNA repair was consistent with γH2AX (FIGS. 37A,B). Furthermore, when DOCK11 expression was reduced by transfection of DOCK11-targeted siRNA (Fig. 37C), γH2AX expression was similarly reduced (Fig. 37D), and the localization of weakly expressed DOCK11 and γH2AX coincided (Fig. 37D). Figure 37E). γH2AX is a phosphorylated histone H2AX and a marker for sites of DNA damage, suggesting that DOCK11 accumulates at sites of DNA damage and activates the ATR signaling pathway. N-10M-D42TN reduced the expression of γH2AX similarly to DOCK11 knockdown (Fig. 37D), but γH2AX did not co-localize with DOCK11 (Fig. 37F,G; Wang, Y-H. et al. Nat Commun, 8, 2118, 2017). This suggests that N-10M-D42TN inhibits DOCK11 activation of the ATR signaling pathway at sites of DNA damage. In other words, in N-10M-D42TN-treated cells, HBV cannot utilize the DNA repair mechanism when synthesizing cccDNA from rcDNA, and it is thought that infection is suppressed.
　以上の結果から、DOCK11結合ペプチドN-10M-D42TNはDOCK11とAck1の結合を阻害し、DOCK11のGEF活性を阻害することがわかった。またAck1の活性化を阻害してアクチン重合を阻害するとともに、EGFRのエンドサイトーシスを阻害してHBVの細胞内侵入を阻害する可能性も示唆された。また、アクチンフィラメントがエンドサイトーシスに寄与することが知られているので(Toshima, JY. et al., eLife 5, e10276, 2016)、N-10M-D42TNはアクチン重合を阻害することによりHBVの侵入を阻害している可能性もある。また、核内においても、DOCK11結合ペプチドN-10M-D42TNはDNA損傷部位に集合・蓄積したDOCK11によるATRシグナル伝達経路の活性化を阻害し、rcDNAからcccDNAへの修復過程を阻害する可能性が考えられる。すなわち、DOCK11結合ペプチドN-10M-D42TNは、EGFRのエンドサイトーシスおよびrcDNAからcccDNAへの修復過程を阻害することで、HBV感染を抑制すると考えられる。 From the above results, it was found that the DOCK11-binding peptide N-10M-D42TN inhibited the binding of DOCK11 and Ack1 and inhibited the GEF activity of DOCK11. In addition, it was suggested that it may inhibit actin polymerization by inhibiting Ack1 activation and HBV entry into cells by inhibiting EGFR endocytosis. In addition, since it is known that actin filaments contribute to endocytosis (Toshima, JY. et al., eLife 5, e10276, 2016), N-10M-D42TN inhibits actin polymerization to promote HBV activation. It may be blocking intrusions. In the nucleus, the DOCK11-binding peptide N-10M-D42TN may also inhibit the activation of the ATR signaling pathway by DOCK11 assembled and accumulated at sites of DNA damage, thus inhibiting the repair process from rcDNA to cccDNA. Conceivable. Thus, the DOCK11-binding peptide N-10M-D42TN is thought to suppress HBV infection by inhibiting EGFR endocytosis and the repair process from rcDNA to cccDNA.
[実施例16]: HBV感染PXBマウスを用いた薬効試験
　PXBマウスにHBVを感染させ、被験物質（N-10M-D42TN）投与による薬効を確認することを目的として下記の実験を行なった。 [Example 16]: Efficacy test using HBV-infected PXB mice PXB mice were infected with HBV, and the following experiment was performed for the purpose of confirming the efficacy of administration of the test substance (N-10M-D42TN).
（16-1） 被験物質、溶媒および調製方法
薬剤 D : N-10M-D42TN
　　性状：PBSに溶解
　　量：21.6mL （1800μL x 12本）
　　保存：冷蔵
　　調製方法：初回投与（Day 0）及び2回目投与（Day 3）は、1倍希釈で300μL/匹に投与した。3回目投与（Day 5）以降は、900μLを分取し、PBS 900μLと混合（2倍希釈）し、300μL/匹に投与した。
溶媒
　　名称：PBS
　　入手先：Thermo Fisher Scientific (Life) 10010049 PBS pH 7.4 
　　保存：冷蔵 (16-1) Test substance, solvent and preparation method Drug D: N-10M-D42TN
Properties: dissolved in PBS Volume: 21.6mL (1800μL x 12 bottles)
Storage: refrigerated Preparation method: The first administration (Day 0) and the second administration (Day 3) were administered at 300 μL/animal at 1-fold dilution. After the third administration (Day 5), 900 µL was taken, mixed with 900 µL of PBS (2-fold dilution), and administered at 300 µL/animal.
Solvent Name: PBS
Available from: Thermo Fisher Scientific (Life) 10010049 PBS pH 7.4
Storage: Refrigerate
（16-2）使用ウイルス
　使用ウイルスは株式会社フェニックスバイオら分与されたHBVを用いた。
ウイルス名：Hepatitis B Virus
株名：PBB004 (Genotype C)
BSL：2
ウイルス力価：1.1E+09 copies/mL（10μL/tubeを2本）
保存：超低温冷凍庫で保存
調製方法：凍結保存したウイルス液1本を解凍し、生理食塩液（株式会社大塚製薬工場）を用いて1.0E+06 copies/mLに調製する。 (16-2) Virus used The virus used was HBV provided by Phoenix Bio Co., Ltd.
Virus name: Hepatitis B virus
Strain name: PBB004 (Genotype C)
BSL: 2
Viral titer: 1.1E+09 copies/mL (2 tubes of 10 μL/tube)
Storage: Store in an ultra-low temperature freezer Preparation method: Thaw one bottle of frozen virus solution and adjust to 1.0E+06 copies/mL using physiological saline (Otsuka Pharmaceutical Factory Co., Ltd.).
（16-3）使用動物
動物種：マウス
系統：PXBマウス（cDNA-uPAwild/+/SCIDマウスにヒト肝細胞を導入したなかで，マウス血中h-Alb濃度に基づいて計算したヒト肝細胞予想置換率が70%以上のマウス）
入手先：株式会社フェニックスバイオ
性別：雄
週齢：12週齢以上（入荷時）
ヒト肝細胞移植後；9週以上
使用匹数：10匹
動物種選択理由： HBV感染モデルとして一般的に使用されている。 (16-3) Animal species used: Mouse strain: PXB mouse mice with a substitution rate of 70% or more)
Source: Phoenix Bio Co., Ltd. Gender: Male Age: 12 weeks or older (at the time of arrival)
After transplantation of human hepatocytes; more than 9 weeks Number of animals used: 10 Reason for species selection: Generally used as an HBV infection model.
（16-4）動物管理条件
16-4.1 飼育条件（SOP/環境/504）
　室温24 ± 3 ℃、湿度50 ± 20 %、換気（10～25回/1時間）、照明12時間（8:00～20:00） (16-4) Animal care conditions
16-4.1 Husbandry Conditions (SOP/Environment/504)
Room temperature 24 ± 3 ℃, humidity 50 ± 20%, ventilation (10 to 25 times/hour), lighting 12 hours (8:00 to 20:00)
16-4.2 飼料（SOP/飼育/206、SOP/飼育/512）
　MF（オリエンタル酵母工業株式会社）にPS-A（オリエンタル酵母工業株式会社）を2:1で混合した飼料を自由摂取。 16-4.2 Feed (SOP/Freed/206, SOP/Freed/512)
Ad libitum feed mixed with MF (Oriental Yeast Co., Ltd.) and PS-A (Oriental Yeast Co., Ltd.) at a ratio of 2:1.
16-4.3 飲料水（SOP/飼育/206、SOP/飼育/512）
　オートクレーブ滅菌した市水に次亜塩素酸ナトリウム（終濃度：14.4 ppm）を添加した飲水を自由摂取。 16-4.3 Drinking Water (SOP/Husband/206, SOP/Husband/512)
Ad libitum drinking water containing sodium hypochlorite (final concentration: 14.4 ppm) was added to autoclave-sterilized city water.
16-4.4 ケージ（SOP/動物/301）
　オートクレーブ滅菌したケージを使用。1～5匹/ケージ飼育とする。
　ケージ交換、給餌（補充）および給水瓶交換は、微生物的汚染のリスク低減のために同時期に実施する。 16-4.4 Cage (SOP/animal/301)
Use autoclaved cages. 1 to 5 animals/cage should be reared.
Cage changes, feeding (replenishment) and water bottle changes should be done at the same time to reduce the risk of microbial contamination.
16-4.5 検疫および馴化（SOP/動物/301）
　動物入荷後から検疫および馴化終了日まで、一般状態を毎日観察する。また、入荷日ならびに検疫および馴化終了日に体重を測定する。 16-4.5 Quarantine and Habituation (SOP/Animals/301)
Observe daily clinical signs from the time animals are received until the end of quarantine and acclimatization. Body weights are also measured on the day of arrival and the end of quarantine and acclimatization.
16-4.6 群分け（SOP/試験/002）
　ウイルス接種6週間目に採血を実施し、その血中HBV DNA量をもとにウイルス接種8週目（被験物質初回投与日）に各群にマウスを振り分ける。 16-4.6 Grouping (SOP/Study/002)
Six weeks after virus inoculation, blood is collected, and based on the amount of HBV DNA in the blood, mice are sorted into groups at eight weeks after virus inoculation (day of first administration of the test substance).
16-4.7 動物の識別(SOP/試験/001)
　背側尾部へ油性インクを用いて個体識別を行う。ケージに試験番号、動物番号、試験期間、試験群、試験責任者名等を記載したラベルを付ける。 16-4.7 Animal Identification (SOP/Trials/001)
Individual identification is performed using oil-based ink on the dorsal tail. Label the cage with the test number, animal number, test period, test group, name of the investigator, etc.
（16-5）試験群構成および投与スケジュール
　下記表11の通り。 (16-5) Study group composition and administration schedule As shown in Table 11 below.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
ウイルス接種
　HBVを1.0E+05 copies/100 μL/bodyでマウス尾静脈より接種する。 Virus Inoculation HBV is inoculated into the mouse tail vein at 1.0E+05 copies/100 μL/body.
被験物質の投与
　ウイルス接種8週間目（Day 0）から標本採取前日（Day 27）まで300μL/bodyで2～3日（月、水、金）に1回、マウスに腹腔内投与する。 Administration of Test Substance From the 8th week after virus inoculation (Day 0) to the day before sample collection (Day 27), 300 μL/body is intraperitoneally administered to mice once every 2 to 3 days (Mon, Wed, Fri).
採血
　ウイルス接種6週目（初回投与2週間前）および初回投与前（Day 0：ウイルス接種8週目）からDay 21まで1週間に1回、鎖骨下静脈から約70μLの血液を採取した。剖検時（Day 28）にはイソフルラン吸入麻酔下で、腹大静脈もしくは心臓より採取可能全量（400μL以上）の血液を採取した。採血終了後に血中h-Alb濃度測定として2μLの血液を200μLの生理食塩液と混合し、390 ×g、室温、10分間の遠心分離を行った後、1.5mLチューブ（INA・OPTIKA, RC-0150）に上清を入れて、送付するまで凍結保存（-60℃以下）した。残りの血液は6000 rpm、15分間、4℃遠心分離を行い、血清（経時採血時（HBV関連因子測定用）：30μL（5μL×2本、20μL×1本）、剖検時：160μL以上（5μL×2本、20μL×1本、残余1本）を分離し、下記に送付するまで凍結保存（-60℃以下）した。 Blood collection About 70 µL of blood was collected from the subclavian vein once a week from 6 weeks after virus inoculation (2 weeks before the first administration) and before the first administration (Day 0: 8 weeks after virus inoculation) to Day 21. At the time of necropsy (Day 28), under isoflurane inhalation anesthesia, the total amount of blood (400 μL or more) that can be collected was collected from the abdominal vena cava or heart. After the blood collection is completed, 2 μL of blood is mixed with 200 μL of physiological saline to measure h-Alb concentration in the blood. 0150) and stored frozen (below −60° C.) until shipment. The rest of the blood was centrifuged at 6000 rpm for 15 minutes at 4°C. × 2 bottles, 20 μL × 1 bottle, and the remaining 1 bottle) were separated and stored frozen (below -60°C) until sent to the following.
剖検
　Day28に安楽死後、肝臓を採取し、重量を測定する。肝臓はHBV DNAおよびcccDNAのために、一部を凍結保存し残りを病理組織学的検査用にホルマリン固定する。また肺、脾臓および腎臓はホルマリン固定する。 Necropsy After euthanasia on Day 28, livers are collected and weighed. Livers are partially cryopreserved and the rest formalin-fixed for histopathological examination for HBV DNA and cccDNA. Lungs, spleens and kidneys are also fixed in formalin.
HBsAg、HBeAgおよびHBcrAgの測定
　血清中HBsAg濃度測定は、株式会社エスアールエル（東京）によって実施された。測定には、化学発光酵素免疫測定法（ChemiLuminescence Enzyme ImmunoAssay，CLEIA）を利用するLumipulse(登録商標) Presto II（富士レビオ株式会社，東京）を用いた。測定範囲は0.005 から 150 IU/mLであった。当試験での被測定試料の希釈倍率は30倍とし、同希釈倍率での測定範囲は0.15 から 4500 IU/mLとした。血清中HBsAg濃度測定は、株式会社エスアールエル（東京）によって実施された。測定には、化学発光酵素免疫測定法（ChemiLuminescence Enzyme ImmunoAssay，CLEIA）を利用するLumipulse(登録商標) Presto II（富士レビオ株式会社，東京）を用いた。測定範囲は0.1から1590 C.O.I.であった。当試験での被測定試料の希釈倍率は30倍とし、同希釈倍率での測定範囲は3から47700 C.O.I.とした。血清中HBc-rAg濃度測定は，株式会社エスアールエル（東京）によって実施された。測定には、化学発光酵素免疫測定法（ChemiLuminescence Enzyme ImmunoAssay，CLEIA）を利用するLUMIPULSE HBcrAg, LUMIPULSE F（富士レビオ株式会社，東京）を用いた。測定下限は3.0 log U/mLであった。当試験での被測定試料の希釈倍率は300倍とし、同希釈倍率での測定下限は5.5 log U/mLとした。 Measurement of HBsAg, HBeAg and HBcrAg Serum HBsAg concentration measurement was performed by SRL Co., Ltd. (Tokyo). For the measurement, Lumipulse (registered trademark) Presto II (Fujirebio Co., Ltd., Tokyo) using ChemiLuminescence Enzyme ImmunoAssay (CLEIA) was used. The measurement range was 0.005 to 150 IU/mL. The sample to be measured in this test was diluted 30 times, and the measurement range at the same dilution was 0.15 to 4500 IU/mL. Serum HBsAg concentrations were measured by SRL Co., Ltd. (Tokyo). For the measurement, Lumipulse (registered trademark) Presto II (Fujirebio Co., Ltd., Tokyo) using ChemiLuminescence Enzyme ImmunoAssay (CLEIA) was used. The measurement range was from 0.1 to 1590 COI. The sample to be measured in this test was diluted 30 times, and the measurement range at the same dilution was 3 to 47,700 COI. Serum HBc-rAg concentrations were measured by SRL Co., Ltd. (Tokyo). For the measurement, LUMIPULSE HBcrAg, LUMIPULSE F (Fujirebio Co., Ltd., Tokyo) using ChemiLuminescence Enzyme ImmunoAssay (CLEIA) was used. The lower limit of measurement was 3.0 log U/mL. The dilution ratio of the sample to be measured in this test was 300 times, and the lower limit of measurement at the same dilution ratio was 5.5 log U/mL.
h-Albの測定
　株式会社フェニックスバイオにて以下の操作を実施した。
　2μLの血液を200μLの生理食塩液で希釈し、390×g，室温の条件で10分間の遠心分離を行った。血中h-Alb濃度は、ラテックス凝集免疫比濁法（LZテスト‘栄研’U-ALB，栄研化学株式会社，東京）を用いて，自動分析装置BioMajestyTM（JCA-BM6050，JEOL，東京）で測定した。 Measurement of h-Alb The following operations were carried out at Phoenix Bio Co., Ltd.
2 μL of blood was diluted with 200 μL of physiological saline and centrifuged at 390×g at room temperature for 10 minutes. Blood h-Alb concentration was measured using a latex agglutination immunoturbidimetric assay (LZ test 'Eiken' U-ALB, Eiken Chemical Co., Ltd., Tokyo) using an automatic analyzer BioMajestyTM (JCA-BM6050, JEOL, Tokyo). measured in
ALTの測定
　剖検時の血清を用いてフェニックスバイオにて測定した。
　血漿中ALT活性は、採取した血漿10μLを利用して測定した。測定対象はジアリールイミダゾールロイコ色素（ピルビン酸オキシダーゼにより発生する過酸化水素とペルオキシダーゼによりジアリールイミダゾールロイコ色素を青色に発色）であり、測定にはドライケム7000/NX500sVを利用した。 Measurement of ALT ALT was measured by PhoenixBio using serum at autopsy.
Plasma ALT activity was measured using 10 μL of collected plasma. The object to be measured is a diarylimidazole leuco dye (diarylimidazole leuco dye is colored blue by hydrogen peroxide generated by pyruvate oxidase and peroxidase), and DRYCHEM 7000/NX500sV was used for the measurement.
肝臓中HBV DNAの測定
　RNAlater浸漬肝臓試料からDNeasy(登録商標) Blood & Tissue Kits（株式会社キアゲン，東京）を用いてDNA抽出を行い、DNAをNuclease-free waterに溶解した。DNA濃度をBioPhotometer(登録商標) 6131（エッペンドルフ株式会社）で測定した後、Nuclease-free waterを用いて最終濃度を20 ng/μLに調製した。PCR反応液は，溶解したDNA原液もしくは希釈したDNAを5 μLとTaqMan(登録商標) Fast Advanced Master Mixを用いて調製した。また、PCR反応と解析にはCFX96 TouchTM Real-Time PCR Detection Systemを用いた。PCR反応は、50℃ 2分→95℃ 20秒→（95℃ 3秒→60℃ 32秒）×53サイクルで行った。肝臓中HBV DNA濃度は，2ウエルの平均で算出した。使用したプライマーおよびプローブの配列は、以下の表12に記した。なお、当定量方法による検出下限は、50 copies/100ng DNAである。HBV DNAスタンダードにはHBV感染PXBマウスから得られた血清を使用した。この血清中に含まれるHBV DNA濃度はデジタルPCRにより定量した。 Measurement of HBV DNA in Liver DNA was extracted from RNAlater-immersed liver samples using DNeasy (registered trademark) Blood & Tissue Kits (Qiagen Co., Ltd., Tokyo), and the DNA was dissolved in Nuclease-free water. After DNA concentration was measured with BioPhotometer (registered trademark) 6131 (Eppendorf Co., Ltd.), the final concentration was adjusted to 20 ng/μL using Nuclease-free water. A PCR reaction solution was prepared using 5 μL of dissolved DNA stock solution or diluted DNA and TaqMan (registered trademark) Fast Advanced Master Mix. In addition, the CFX96 Touch ™ Real-Time PCR Detection System was used for PCR reaction and analysis. The PCR reaction was carried out as follows: 50°C 2 minutes → 95°C 20 seconds → (95°C 3 seconds → 60°C 32 seconds) × 53 cycles. HBV DNA concentration in liver was calculated by averaging 2 wells. The sequences of the primers and probes used are listed in Table 12 below. The lower limit of detection by the quantitation method is 50 copies/100 ng DNA. The HBV DNA standard used serum obtained from HBV-infected PXB mice. The HBV DNA concentration contained in this serum was quantified by digital PCR.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
cccDNAの測定
　肝臓中HBV DNAの測定時に精製したDNA原液もしくは希釈したDNAを5 μLとTaqMan(登録商標 Fast Advanced Master Mixを用いて調製した。また、PCR反応と解析にはCFX96 TouchTM Real-Time PCR Detection Systemを用いた。PCR反応は、50℃ 2分→95℃ 20秒→（95℃ 3秒→60℃ 32秒）×55サイクルで行った。肝臓中HBV cccDNA濃度は、2ウエルの平均で算出した。使用したプライマー（タカラバイオ株式会社，滋賀）及びプローブ（タカラバイオ株式会社）の配列は、以下の表13の通りである。なお、当定量方法による検出下限は、1.0×102 copies/100 ng DNAであった。また、HBV cccDNAスタンダードにはHBVの全ゲノム配列を含むプラスミドを利用した。 Measurement of cccDNA When measuring HBV DNA in liver, 5 μL of purified DNA stock solution or diluted DNA was prepared using TaqMan® Fast Advanced Master Mix. Using the PCR Detection System, the PCR reaction was performed as follows: 50°C for 2 minutes → 95°C for 20 seconds → (95°C for 3 seconds → 60°C for 32 seconds) × 55 cycles.The HBV cccDNA concentration in the liver was the average of 2 wells. The sequences of the primers (Takara Bio Inc., Shiga) and the probes (Takara Bio Inc.) used are shown in Table 13. The lower limit of detection by the quantification method is 1.0×10 2 . copies/100 ng DNA, and the HBV cccDNA standard utilized a plasmid containing the entire HBV genome sequence.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
病理組織学的検査（HBsAg免疫染色、HBcAg免疫染色）
　肝臓組織は、10%中性緩衝ホルマリン液固定後に70%エタノールに置換し、これらの試料を奈良病理研究所（奈良）に依頼して常法によりパラフィン包埋ブロックを作製し、その後薄切標本を得た。
　パラフィン切片を脱パラフィン処理した後にマイクロウェーブにより抗原賦活化を施した。一次抗体（HBsAg (anti-HBsAg antibody, Code: OBT0990, Bio-Rad AbD Serotec Limited, Oxford, UK)もしくはHBcAg (anti-HBcAg antibody, Code: PAB14506, Abnova, Taipei City, Taiwa)）を4℃で一晩反応させた。一次抗体をビオチン-アビジン-ペルオキシダーゼ複合体と反応させた後、DABにより発色させた。細胞核をヘマトキシリンにて染色した後、これらの切片を脱水・透徹し、封入した。その後、ハムリー株式会社で光学顕微鏡を用いて鏡検を実施した。 Histopathological examination (HBsAg immunostaining, HBcAg immunostaining)
Liver tissue was fixed in 10% neutral buffered formalin solution and then replaced with 70% ethanol. These samples were requested to Nara Pathological Research Institute (Nara) to prepare paraffin-embedded blocks by standard methods, and then sliced. got
After deparaffinization of paraffin sections, they were subjected to antigen retrieval by microwave. Primary antibody (HBsAg (anti-HBsAg antibody, Code: OBT0990, Bio-Rad AbD Serotec Limited, Oxford, UK) or HBcAg (anti-HBcAg antibody, Code: PAB14506, Abnova, Taipei City, Taiwa)) was incubated at 4°C. reacted overnight. The primary antibody was reacted with a biotin-avidin-peroxidase complex and then developed with DAB. After staining cell nuclei with hematoxylin, these sections were dehydrated, cleared and mounted. After that, a microscopic examination was performed using an optical microscope at Hamley Co., Ltd.
（16-6）観察・測定項目
　サンプルの投与は、図38に示すスケジュールに従った。HBsAg（図39）、HBeAg（図40）およびHBcrAg（図41）に関してN-10M-D42TNとcontrol (PBS)を比較した結果、いずれの場合もN-10M-D42TNが抑制的に働いていた。血中HBV DNA（図42）についてもN-10M-D42TNが抑制的に働いていた。h-Albの測定（図43）の結果、キメラマウスにおいて十分なヒトアルブミン量が得られた。本アッセイはヒト肝臓細胞での評価として優れていた。ALT（図44）の測定の結果、N-10M-D42TNはcontrol (PBS)と比較して、肝炎を悪化させることはなかった。剖検して得た肝臓より肝臓中のHBV-DNA（図45）の量を測定した結果、N-10M-D42TNはコントロール投与群と比較し、HBV DNAのコピー数を減少させていた。同様に肝臓中のcccDNA（図46）のコピー数を測定した結果、N-10M-D42TNはコントロール投与群と比較し、HBV DNAのコピー数を減少させた。 (16-6) Observation/measurement items The administration of samples was according to the schedule shown in FIG. As a result of comparing N-10M-D42TN and control (PBS) with respect to HBsAg (Fig. 39), HBeAg (Fig. 40) and HBcrAg (Fig. 41), N-10M-D42TN acted suppressively in all cases. N-10M-D42TN also inhibited HBV DNA in blood (Fig. 42). As a result of h-Alb measurement (Fig. 43), a sufficient amount of human albumin was obtained in the chimeric mice. This assay was excellent for evaluation in human liver cells. As a result of measuring ALT (Fig. 44), N-10M-D42TN did not exacerbate hepatitis compared to control (PBS). As a result of measuring the amount of HBV-DNA in the liver (Fig. 45) from the liver obtained by necropsy, N-10M-D42TN reduced the copy number of HBV DNA compared to the control administration group. As a result of similarly measuring the copy number of cccDNA in the liver (Fig. 46), N-10M-D42TN decreased the copy number of HBV DNA compared to the control administration group.
　以上の結果から、DOCK11結合ペプチド(N-10M-D42TN)はHBVを感染させたヒト肝細胞キメラマウスを用いた動物実験において、肝臓中のHBV-DNAおよびcccDNAを減少させ、明確な抗HBV作用を有することが証明された。また、in vitroおよびin vivoアッセイ系において、HBVを十分に感染させた後にDOCK11結合ペプチド(N-10M-D42TN)を投与しているので、DOCK11結合ペプチド(N-10M-D42TN)はHBV粒子が細胞の外に一度分泌された後、再度細胞内に侵入する再感染の過程を阻止している可能性が高い。 Based on the above results, DOCK11-binding peptide (N-10M-D42TN) reduced HBV-DNA and cccDNA in the liver in animal experiments using HBV-infected human hepatocyte chimeric mice, demonstrating a clear anti-HBV effect. has been proven to have Moreover, in the in vitro and in vivo assay systems, DOCK11-binding peptide (N-10M-D42TN) was administered after sufficient HBV infection. After being secreted outside the cell once, it is highly likely that it prevents the process of re-infection, in which it reenters the cell.
　実施例15の結果と併せると、DOCK11結合ペプチドは、rcDNAからcccDNAへの修復過程の阻害、並びにEGFRのエンドサイトーシスの阻害等によるHBV粒子の細胞内への再侵入の阻害により、抗HBV効果を発揮することが示唆される。 Combined with the results of Example 15, the DOCK11-binding peptide exhibited an anti-HBV effect by inhibiting the repair process from rcDNA to cccDNA and inhibiting re-entry of HBV particles into cells, such as by inhibiting EGFR endocytosis. It is suggested that

Claims (16)

  1. 　DOCK11に結合し、DOCK11の機能を阻害する物質を有効成分として含む、抗B型肝炎ウイルス剤。 An anti-hepatitis B virus agent containing as an active ingredient a substance that binds to DOCK11 and inhibits the function of DOCK11.
  2. 　前記物質は、DOCK11の第1516番～第2073番残基の領域に結合する、請求項１記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to claim 1, wherein the substance binds to the region of residues 1516 to 2073 of DOCK11.
  3. 　DOCK11の機能の阻害が、DOCK11とAck1の結合の阻害によるAck1活性化の阻害、DOCK11のグアニンヌクレオチド交換因子活性の阻害、及びDOCK11によるATRシグナル伝達経路の活性化の阻害から選択される少なくとも１種である、請求項１又は２記載の抗B型肝炎ウイルス剤。 Inhibition of DOCK11 function is at least one selected from inhibition of Ack1 activation by inhibition of binding between DOCK11 and Ack1, inhibition of guanine nucleotide exchange factor activity of DOCK11, and inhibition of activation of the ATR signaling pathway by DOCK11 The anti-hepatitis B virus agent according to claim 1 or 2, which is
  4. 　前記物質が、下記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドである、請求項１～３のいずれか１項に記載の抗B型肝炎ウイルス剤。
    (1)　IITPGTEVLNSDLQAS（配列番号1）の配列のポリペプチド。
    (2)　HNVLSVYNPAWGKYFH（配列番号2）の配列のポリペプチド。
    (3)　NFPPNPMHNTDSCICA（配列番号3）の配列のポリペプチド。
    (4)　TEKRRLMKPVLLTYNP（配列番号4）の配列のポリペプチド。
    (5)　IICPGAEVLNGDLVAS（配列番号5）の配列のポリペプチド。
    (6)　TEYRRCVTPVLLTYNN（配列番号6）の配列のポリペプチド。
    (7)　TEEHRGLLPVLMTYNV（配列番号7）の配列のポリペプチド。
    (8)　TEFCRWTWPVLCTYNA（配列番号8）の配列のポリペプチド。
    (9)　TEQARPTPPPVLDTYNL（配列番号9）の配列のポリペプチド。
    (10)　PEQARPPPPLEDNLFL（配列番号10）の配列のポリペプチド。
    (11)　HEEHRGMLREDSMMEYLK（配列番号11）の配列のポリペプチド。
    (12)　AEEHRGLLTIRYPMEH（配列番号12）の配列のポリペプチド。
    (13)　PEQARPPPPLEDNLFL（配列番号10）の領域を含むAck1の部分ポリペプチド。
    (14)　HEEHRGMLREDSMMEYLK（配列番号11）の領域を含むラディキシンの部分ポリペプチド。
    (15)　AEEHRGLLTIRYPMEH（配列番号12）の領域を含むβ-セントラクチンの部分ポリペプチド。
    (16)　(1)～(15)のいずれかと８０％以上１００％未満の配列同一性を有するポリペプチド。     The anti-hepatitis B virus agent according to any one of claims 1 to 3, wherein the substance is at least one polypeptide selected from the following polypeptides (1) to (16).
    (1) A polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1).
    (2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
    (3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
    (4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4).
    (5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5).
    (6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
    (7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
    (8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
    (9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
    (10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (14) A partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (15) A partial polypeptide of β-centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (16) A polypeptide having 80% or more and less than 100% sequence identity with any of (1) to (15).
  5. 　前記ポリペプチドは、肝細胞内への送達のための担体分子と連結した形態にある、請求項４記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to claim 4, wherein the polypeptide is in a form linked to a carrier molecule for delivery into hepatocytes.
  6. 　前記担体分子が、アシアロ糖タンパク質受容体に結合する抗体、抗体断片又は一本鎖抗体である、請求項５記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to claim 5, wherein the carrier molecule is an antibody, antibody fragment or single-chain antibody that binds to the asialoglycoprotein receptor.
  7. 　前記抗体、抗体断片又は一本鎖抗体が、
    　配列番号１３、１９、２５若しくは３１に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR1と、
    　配列番号１４、２０、２６若しくは３２に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR2と、
    　配列番号１５、２１、２７若しくは３３に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR3と、
    　配列番号１６、２２、２８若しくは３４に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR1と、
    　配列番号１７、２３、２９若しくは３５に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR2と、
    　配列番号１８、２４、３０若しくは３６に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR3と
    を有する、請求項６記載の抗B型肝炎ウイルス剤。     wherein said antibody, antibody fragment or single chain antibody is
    A heavy chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
    A heavy chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    A heavy chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
    a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    a light chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    light chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 18, 24, 30 or 36, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has 80% or more identity with the amino acid sequence 7. The anti-hepatitis B virus agent according to claim 6.
  8. 　前記抗体、抗体断片又は一本鎖抗体が、内因性酵素により切断される切断配列を介して前記ポリペプチドと連結する、請求項６又は７記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to claim 6 or 7, wherein said antibody, antibody fragment or single-chain antibody is linked to said polypeptide via a cleavage sequence that is cleaved by an endogenous enzyme.
  9. 　前記ポリペプチドは、細胞膜透過促進分子と連結した形態にある、請求項４～８のいずれか１項に記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to any one of claims 4 to 8, wherein the polypeptide is in a form linked to a cell membrane permeation promoting molecule.
  10. 　細胞膜透過促進分子が、配列番号３８又は３９に示すアミノ酸配列のポリペプチドである、請求項９記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to claim 9, wherein the cell membrane permeation promoting molecule is a polypeptide having the amino acid sequence shown in SEQ ID NO: 38 or 39.
  11. 　前記ポリペプチドは、核移行シグナルと連結した形態にある、請求項４～１０のいずれか１項に記載の抗B型肝炎ウイルス剤。 The anti-hepatitis B virus agent according to any one of claims 4 to 10, wherein the polypeptide is in a form linked to a nuclear localization signal.
  12. 　下記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドの、DOCK11結合ペプチドとしての使用。
    (1)　IITPGTEVLNSDLQAS（配列番号1）の配列のポリペプチド。
    (2)　HNVLSVYNPAWGKYFH（配列番号2）の配列のポリペプチド。
    (3)　NFPPNPMHNTDSCICA（配列番号3）の配列のポリペプチド。
    (4)　TEKRRLMKPVLLTYNP（配列番号4）の配列のポリペプチド。
    (5)　IICPGAEVLNGDLVAS（配列番号5）の配列のポリペプチド。
    (6)　TEYRRCVTPVLLTYNN（配列番号6）の配列のポリペプチド。
    (7)　TEEHRGLLPVLMTYNV（配列番号7）の配列のポリペプチド。
    (8)　TEFCRWTWPVLCTYNA（配列番号8）の配列のポリペプチド。
    (9)　TEQARPTPPPVLDTYNL（配列番号9）の配列のポリペプチド。
    (10)　PEQARPPPPLEDNLFL（配列番号10）の配列のポリペプチド。
    (11)　HEEHRGMLREDSMMEYLK（配列番号11）の配列のポリペプチド。
    (12)　AEEHRGLLTIRYPMEH（配列番号12）の配列のポリペプチド。
    (13)　PEQARPPPPLEDNLFL（配列番号10）の領域を含むAck1の部分ポリペプチド。
    (14)　HEEHRGMLREDSMMEYLK（配列番号11）の領域を含むラディキシンの部分ポリペプチド。
    (15)　AEEHRGLLTIRYPMEH（配列番号12）の領域を含むβ-セントラクチンの部分ポリペプチド。
    (16)　(1)～(15)のいずれかと８０％以上１００％未満の配列同一性を有するポリペプチド。     Use of at least one polypeptide selected from the following polypeptides (1) to (16) as a DOCK11-binding peptide.
    (1) A polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1).
    (2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
    (3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
    (4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4).
    (5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5).
    (6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
    (7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
    (8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
    (9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
    (10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (14) A partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (15) A partial polypeptide of β-centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (16) A polypeptide having 80% or more and less than 100% sequence identity with any of (1) to (15).
  13. 　下記(1)～(16)のポリペプチドから選択される少なくとも１種のポリペプチドからなる、DOCK11結合ペプチド。
    (1)　IITPGTEVLNSDLQAS（配列番号1）の配列のポリペプチド。
    (2)　HNVLSVYNPAWGKYFH（配列番号2）の配列のポリペプチド。
    (3)　NFPPNPMHNTDSCICA（配列番号3）の配列のポリペプチド。
    (4)　TEKRRLMKPVLLTYNP（配列番号4）の配列のポリペプチド。
    (5)　IICPGAEVLNGDLVAS（配列番号5）の配列のポリペプチド。
    (6)　TEYRRCVTPVLLTYNN（配列番号6）の配列のポリペプチド。
    (7)　TEEHRGLLPVLMTYNV（配列番号7）の配列のポリペプチド。
    (8)　TEFCRWTWPVLCTYNA（配列番号8）の配列のポリペプチド。
    (9)　TEQARPTPPPVLDTYNL（配列番号9）の配列のポリペプチド。
    (10)　PEQARPPPPLEDNLFL（配列番号10）の配列のポリペプチド。
    (11)　HEEHRGMLREDSMMEYLK（配列番号11）の配列のポリペプチド。
    (12)　AEEHRGLLTIRYPMEH（配列番号12）の配列のポリペプチド。
    (13)　PEQARPPPPLEDNLFL（配列番号10）の領域を含むAck1の部分ポリペプチド。
    (14)　HEEHRGMLREDSMMEYLK（配列番号11）の領域を含むラディキシンの部分ポリペプチド。
    (15)　AEEHRGLLTIRYPMEH（配列番号12）の領域を含むβ-セントラクチンの部分ポリペプチド。
    (16)　(1)～(15)のいずれかと８０％以上１００％未満の配列同一性を有するポリペプチド。     A DOCK11-binding peptide comprising at least one polypeptide selected from the following polypeptides (1) to (16).
    (1) A polypeptide of the sequence IITPGTEVLNSDLQAS (SEQ ID NO: 1).
    (2) A polypeptide of the sequence HNVLSVYNPAWGKYFH (SEQ ID NO:2).
    (3) A polypeptide of the sequence NFPPNPMHNTDSCICA (SEQ ID NO:3).
    (4) A polypeptide of the sequence TEKRRLMKPVLLTYNP (SEQ ID NO:4).
    (5) A polypeptide of the sequence IICPGAEVLNGDLVAS (SEQ ID NO:5).
    (6) A polypeptide of the sequence TEYRRCVTPVLLTYNN (SEQ ID NO:6).
    (7) A polypeptide of the sequence TEEHRGLLPVLMTYNV (SEQ ID NO:7).
    (8) A polypeptide of the sequence TEFCRWTWPVLCTYNA (SEQ ID NO:8).
    (9) A polypeptide of the sequence TEQARPTPPPVLDTYNL (SEQ ID NO:9).
    (10) A polypeptide of sequence PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (11) A polypeptide of sequence HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (12) A polypeptide of the sequence AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (13) A partial polypeptide of Ack1 containing the region of PEQARPPPPLEDNLFL (SEQ ID NO: 10).
    (14) A partial polypeptide of radixin containing the region of HEEHRGMLREDSMMEYLK (SEQ ID NO: 11).
    (15) A partial polypeptide of β-centractin containing the region of AEEHRGLLTIRYPMEH (SEQ ID NO: 12).
    (16) A polypeptide having 80% or more and less than 100% sequence identity with any of (1) to (15).
  14. 　配列番号１３、１９、２５若しくは３１に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR1と、
    　配列番号１４、２０、２６若しくは３２に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR2と、
    　配列番号１５、２１、２７若しくは３３に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む重鎖CDR3と、
    　配列番号１６、２２、２８若しくは３４に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR1と、
    　配列番号１７、２３、２９若しくは３５に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR2と、
    　配列番号１８、２４、３０若しくは３６に示すアミノ酸配列、又は該アミノ酸配列において一部の残基が置換され、該アミノ酸配列と８０％以上の同一性を有するアミノ酸配列を含む軽鎖CDR3と
    を有する、アシアロ糖タンパク質受容体に結合する抗体、抗体断片又は一本鎖抗体。     A heavy chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 13, 19, 25 or 31, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
    A heavy chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 14, 20, 26 or 32, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    A heavy chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 15, 21, 27 or 33, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has an identity of 80% or more with the amino acid sequence;
    a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 16, 22, 28 or 34, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    a light chain CDR2 comprising an amino acid sequence shown in SEQ ID NO: 17, 23, 29 or 35, or an amino acid sequence in which some residues are substituted and having 80% or more identity with the amino acid sequence;
    light chain CDR3 comprising an amino acid sequence shown in SEQ ID NO: 18, 24, 30 or 36, or an amino acid sequence in which some residues are substituted in the amino acid sequence and has 80% or more identity with the amino acid sequence , an antibody, antibody fragment or single-chain antibody that binds to the asialoglycoprotein receptor.
  15. 　請求項１４記載の抗体、抗体断片又は一本鎖抗体を含む、肝細胞内に薬物を送達するための薬物送達用担体。 A drug delivery carrier for delivering a drug into hepatocytes, comprising the antibody, antibody fragment or single-chain antibody according to claim 14.
  16. 　請求項１５記載の薬物送達用担体と肝細胞内に送達すべき薬物との複合体を含む、医薬組成物。 A pharmaceutical composition comprising a complex of the drug delivery carrier according to claim 15 and a drug to be delivered into hepatocytes.
PCT/JP2022/042642 2021-11-18 2022-11-17 Anti-hepatitis b virus agent targeting host factor dock11 WO2023090381A1 (en)

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Citations (2)

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WO2017082202A1 (en) * 2015-11-09 2017-05-18 Ｉｄａｃセラノスティクス株式会社 Antiviral drug
KR20200101075A (en) * 2019-02-19 2020-08-27 대한민국(관리부서 질병관리본부장) Composition for treating hepatitis B comprising inhibitor of HBV enhancer, ACK1

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WO2017082202A1 (en) * 2015-11-09 2017-05-18 Ｉｄａｃセラノスティクス株式会社 Antiviral drug
KR20200101075A (en) * 2019-02-19 2020-08-27 대한민국(관리부서 질병관리본부장) Composition for treating hepatitis B comprising inhibitor of HBV enhancer, ACK1

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