WO2022065344A1 - 抗ｈｔｌｖ－１抗体 - Google Patents

抗ｈｔｌｖ－１抗体 Download PDF

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WO2022065344A1
WO2022065344A1 PCT/JP2021/034714 JP2021034714W WO2022065344A1 WO 2022065344 A1 WO2022065344 A1 WO 2022065344A1 JP 2021034714 W JP2021034714 W JP 2021034714W WO 2022065344 A1 WO2022065344 A1 WO 2022065344A1
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amino acid
acid sequence
seq
antibody
acid residues
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勇悦 田中
千草 吉村
肇 菅原
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国立大学法人琉球大学
第一三共株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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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
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention is an antibody that binds to and neutralizes HTLV-1, an antibody that prevents HTLV-1 infection, prevents or treats the onset of a disease caused by HTLV-1 infection, and a pharmaceutical composition containing the antibody. Regarding.
  • Human T-cell leukemia virus type 1 (Human T-cell Leukemia virus type 1: HTLV-1) belongs to the deltaretrovirus and causes CD4 positive T cells to become cancerous. It is estimated that 10 to 20 million people are infected with HTLV-1 worldwide, and it is often found in central and southern Africa, north-central South America, the Caribbean region, central Australia, Papua New Guinea, and Japan (Non-Patent Document 1). .. A characteristic of HTLV-1 is that T cells already infected with HTLV-1 are infected by contact with uninfected T cells. In other words, for human-to-human infection, it is necessary for cells derived from the infected person to enter the body and make cell-to-cell contact. Regarding the transmission route, although horizontal transmission due to sexual activity has increased in recent years, most of them are vertical transmission between mother and child via breast milk (Non-Patent Document 2).
  • Non-Patent Document 3 T-cell leukemia
  • HAM HTLV-1-related myelopathy
  • HAM HTLV-1-related uveitis
  • Non-Patent Document 4 HTLV-1 assisted Uveitis and the like develop.
  • ATL is known to develop 40 to 50 years after infection. It has been nearly 40 years since HTLV-1 was known to be the epidemiological causative virus of ATL (Non-Patent Document 4), but there have been no reports of effective vaccine development for preventing HTLV-1 infection to date.
  • Non-Patent Document 5 there is a report on a neutralizing antibody (LAT-27) against HTLV-1 (Non-Patent Document 5), and by administering this neutralizing antibody to peripheral blood humanized mice before infection, it is possible to counteract HTLV-1. We have confirmed the prevention of infection (Non-Patent Documents 6 and 7).
  • the anti-HTLV-1 antibody can be expected to be used as a drug for preventing infection with HTLV-1 and for preventing or treating diseases caused by HTLV-1 such as ATL.
  • the present invention relates to an anti-HTLV-1 antibody that prevents or treats infection of a non-HTLV-1 infected person or a disease caused by HTLV-1 of an HTLV-1 infected person, and a drug containing the antibody as an active ingredient. For the purpose of provision.
  • the present inventors obtained a rat-derived anti-HTLV-1 antibody using gp62, which is an enveloped protein of HTLV-1, as an antigen in order to impart neutralizing activity to HTLV-1.
  • the humanized anti-HTLV-1 antibody obtained by humanizing the variable region of the antibody maintains its affinity for the antigenic peptide as compared with the pre-humanized rat-derived anti-HTLV-1 antibody. It was confirmed.
  • the subclass of the antibody was set to human IgG1.
  • [6] The antibody of [5], wherein the neutralizing activity is syncytial formation inhibitory activity, and the syncytial formation inhibitory activity is exhibited at a concentration of 4 ⁇ g / mL or less.
  • the antibody of [6] which exhibits syncytial formation inhibitory activity at a concentration of 2 ⁇ g / mL or less.
  • the antibody of [6] or [7] which exhibits syncytial formation inhibitory activity at a concentration of 1 ⁇ g / mL or less.
  • a light chain comprising, and (d) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7.
  • a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 or (aii) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15 and having binding activity to HTLV-1, as well as a light chain variable region.
  • a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 19 or (bii) HTLV-1 envelope protein gp46, which comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19 and contains a heavy chain variable region having binding activity to HTLV-1.
  • a monoclonal antibody that recognizes and binds to.
  • the humanized anti-HTLV-1 antibody according to any one of the following (1) to (24): (1) An antibody consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 32; (2) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 32.
  • the humanized anti-HTLV-1 antibody according to any one of (1) to (24) below: (1) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 32. An antibody that recognizes and binds to the envelope protein gp46; (2) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 32.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (3) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 34.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (5) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 36.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (7) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 38.
  • An antibody that recognizes and binds to the envelope protein gp46 (8) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 38.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (9) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 40.
  • An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1 which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (11) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 42.
  • An antibody that recognizes and binds to the envelope protein gp46 (12) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 42.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (13) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 32.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence;
  • HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 34.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (17) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 36.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (19) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 38.
  • An antibody that recognizes and binds to the envelope protein gp46 (20) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 38.
  • An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1 which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence;
  • HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 40.
  • An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1 which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (23) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 42.
  • the humanized anti-HTLV-1 antibody according to any one of (1) to (8) below: (1) An antibody consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 56 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 54; (2) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 56 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 54.
  • An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1 which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence;
  • An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1 which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (7) An antibody consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 52; and ( 8) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and an amino acid sequence having 95% or more sequence identity, and an amino acid consisting of amino acid residues 21 to 240 of SEQ ID NO: 52.
  • the humanized anti-HTLV-1 antibody according to any one of the following (1) to (8): (1) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 56 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 54.
  • An antibody that recognizes and binds to the envelope protein gp46 (2) A heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 56 and an amino acid sequence having 95% or more sequence identity, and amino acid residues 21 to 240 of SEQ ID NO: 54.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (3) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 48.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (5) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 50.
  • HTLV-1 An antibody that recognizes and binds to the envelope protein gp46 of HTLV-1, which consists of a light chain consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence; (7) HTLV-1 consisting of a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 52.
  • [25] The antibody of [24] in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains.
  • [26] The antibody of [24] or [25], wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.
  • [27] A polynucleotide consisting of a nucleotide sequence encoding the amino acid sequences of the heavy chain and the light chain of any of the antibodies of [1] to [26].
  • [28] A vector containing the polynucleotide of [27].
  • [29] A host cell containing the polynucleotide of [27] or the vector of [28].
  • [30] The method for producing an antibody according to any one of [1] to [26], which comprises culturing the host cells of [29] and purifying the antibody from the culture.
  • [31] The antibody produced by the method of [30].
  • [32] A pharmaceutical composition comprising any of the antibodies of [1] to [26] and [31] as an active ingredient.
  • the pharmaceutical composition of [33], wherein the infection is horizontal transmission or vertical transmission between mother and child.
  • HTLV-1 infected individuals For HTLV-1 infected individuals, HTLV- selected from the group consisting of adult T-cell leukemia / lymphoma (ATLL), HTLV-1-related myelopathy (HAM) and HTLV-1 uveitis (HU).
  • ATLL adult T-cell leukemia / lymphoma
  • HAM HTLV-1-related myelopathy
  • HU HTLV-1 uveitis
  • the pharmaceutical composition of [32] for the prevention and treatment of the onset of the disease caused by 1.
  • a pharmaceutical composition comprising an antibody according to any one of [1] to [26] and [31], and at least one anticancer agent, simultaneously, separately or continuously, administered to an individual.
  • [38] Adult T, comprising administering to an HTLV-1 infected person an antibody of any of [1]-[26] and [31], a pharmaceutical composition of any of [32]-[37].
  • a method for preventing or treating the onset of a disease caused by HTLV-1 selected from the group consisting of cell leukemia / lymphoma (ATLL), HTLV-1-related myelopathy (HAM) and HTLV-1 uveitis (HU).
  • ATLL cell leukemia / lymphoma
  • HAM HTLV-1-related myelopathy
  • HU HTLV-1 uveitis
  • the anti-HTLV-1 antibody of the present invention strongly interacts with gp46, which is an enveloped protein of HTLV-1, and exhibits a neutralizing effect on HTLV-1 to prevent infection, and ADCC in vitro. Since it has a function, it is expected to eliminate infected cells by cell-killing activity against HTLV-1-infected cells.
  • Binding activity to gp62-expressing cells flow cytometry.
  • A is a microscopic observation image used as a reference for determining the activity
  • B shows the syncytial formation inhibitory activity of each antibody.
  • A shows the amino acid sequence of the peptide used
  • B shows the binding activity of LAT-27
  • C shows the binding activity of rMAb1
  • D shows the binding activity of hMAb1_H1L12.
  • ADCC activity of humanized anti-HTLV-1 antibody (hMAb1_H1L12).
  • Nucleotide sequence encoding rMAb98 light chain variable region and amino acid sequence of rMAb98 light chain variable region Nucleotide sequence encoding rMAb98 heavy chain variable region and amino acid sequence of rMAb98 heavy chain variable region. Nucleotide sequence encoding cMAb1 light chain and amino acid sequence of cMAb1 light chain. Nucleotide sequence encoding cMAb1 heavy chain and amino acid sequence of cMAb1 heavy chain. Nucleotide sequence encoding cMAb98 light chain and amino acid sequence of cMAb98 light chain. Nucleotide sequence encoding cMAb98 heavy chain and amino acid sequence of cMAb98 heavy chain.
  • the present invention is an anti-HTLV-1 antibody that recognizes and binds to the envelope protein gp46 of HTLV-1.
  • Human T-cell Leukemia virus type 1 (Human T-cell Leukemia Virus type 1: HTLV-1) is a gag gene encoding a structural protein, a pol gene encoding a reverse transcription enzyme, and an envelope gene encoding a virus surface envelope protein.
  • a plurality of regulatory gene groups such as Tax gene and HBZ gene are encoded in the pX region.
  • the env gene encodes gp62, which is cleaved by a protease into two regions, gp46, which is exposed on the surface and binds to the receptor, and gp21, which is a transmembrane domain.
  • amino acid sequence of the envelope protein gp62 of HTLV-1 is described in UniProt Accession No. : Disclosed on P03381, gp46 corresponds to amino acid sequences 1-312.
  • the monoclonal antibody used in the present invention includes mammals such as mice, rats, rabbits, hamsters, guinea pigs, horses, monkeys, dogs, pigs, cows, goats, sheep, camels, alpaca and lyama, chickens, ostriches, sharks, carp and the like.
  • mammals such as mice, rats, rabbits, hamsters, guinea pigs, horses, monkeys, dogs, pigs, cows, goats, sheep, camels, alpaca and lyama, chickens, ostriches, sharks, carp and the like.
  • Hybridomas can be produced by known methods.
  • Gp46 as an immunogen can be chemically synthesized based on sequence information, or can be obtained as a recombinant protein by a known method based on the DNA sequence information encoding the protein. Further, as an immunogen, DNA encoding HTLV-1, mRNA, a viral vector incorporating them, animal cells transfected with them, or the like can be used.
  • the antibody can be screened by any method, but preferably, it may be screened by flow cytometry using animal cells transfected with DNA encoding GLUT1 which is a receptor molecule of gp62 and HTLV-1.
  • GLUT1 which is a receptor molecule of gp62 and HTLV-1.
  • the amino acid sequences of gp62 and GLUT1 are shown in SEQ ID NOs: 60 and 63 of the sequence listing.
  • the anti-HTLV-1 antibody of the present invention has neutralizing activity against HTLV-1.
  • the anti-HTLV-1 antibody has an effector function and binds to an Fc receptor such as an Fc ⁇ receptor of a phagocytic cell such as a natural killer cell or a macrophage
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • Human IgG1 is known to have strong effector functions such as CDC (Complement-Dependent Cytotoxicity) activity and ADCC activity via complement binding in the human IgG subclass. It exhibits a therapeutic effect by promoting cell death induction of HTLV-1-infected cells by cellular cytotoxicity (J. Exp. Med. (1987), 166, p. 1351-1361).
  • the neutralizing activity against HTLV-1 can be expressed, for example, by the syncytial formation inhibitory activity, and the antibody of the present invention has a concentration of 4 ⁇ g / mL or less, preferably 2 ⁇ g / mL or less, and more preferably 1 ⁇ g / mL. It has syncytial formation inhibitory activity at a concentration of mL or less.
  • the binding dissociation constant (KD) of the antibody of the present invention is, for example, a KD value for HTLV-1 gp46 peptide 180 to 204 as measured by the SPR method, and is 50 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM. Hereinafter, it is 20 nM or less, 15 nM or less, or 10 nM or less.
  • the antibody of the present invention retains the binding activity to the mutant peptide in which the amino acid sequences 191 to 196 of the envelope protein gp46 of HTLV-1 are replaced with Ala one residue at a time. It means that such an antibody can also bind to an HTLV-1 mutant strain in which one or more amino acids of any one of the epitope sites are replaced with other amino acids and can exert neutralizing activity. That is, the antibody of the present invention is expected to be able to prevent the infection of the HTLV-1 mutant strain and also prevent or treat the onset of the disease caused by the HTLV-1 mutant strain infection.
  • the antibody heavy chain constant region consists of CH1, hinge, CH2 and CH3 regions, CH1 has EU indexes 118 to 215, hinges have EU indexes 216 to 230, CH2 has EU indexes 231 to 340, and CH3 has EU indexes 341 to 446. Defined.
  • the present invention also includes an antibody lacking the lysine residue at the heavy chain carboxyl terminal.
  • the present invention also includes an antibody lacking one or several amino acids at the heavy chain carboxyl terminus. The number is 1, 2, 3, 4, 5, 5, 6, 7, 8 or 9.
  • the anti-HTLV-1 antibodies of the invention also include human chimeric antibodies and humanized antibodies modified to reduce heterologous antigenicity to humans. Humanized antibodies are also referred to as CDR transplant antibodies.
  • Human chimeric antibody is an antibody consisting of a light chain variable region and a heavy chain variable region of an antibody of a non-human animal and a light chain constant region and a heavy chain constant region of a human antibody.
  • the cDNA encoding the light chain variable region and the cDNA encoding the heavy chain variable region are collected from the hybrid dome that produces the anti-HTLV-1 antibody, and the light chain constant region and the heavy chain constant region of the human antibody are encoded. It can be prepared by inserting it into an expression vector having cDNA to construct a human chimeric antibody expression vector, introducing it into a host cell, and expressing it.
  • the heavy chain constant region is composed of three domains CH1, CH2 and CH3.
  • the human heavy chain constant region of the chimeric antibody is the heavy chain constant region of the IgG1 subclass.
  • the light chain constant region may belong to human Ig and is a ⁇ or ⁇ constant region.
  • human chimeric antibodies of the anti-HTLV-1 antibody of the present invention include human chimeric antibodies, cMAb1 and cMAb98, which have variable regions of rat anti-HTLV-1 monoclonal antibodies MAb1 and MAb98. These two antibodies are highly binding antibodies to HTLV-1.
  • cMAb1 which is an antibody having high activity, is preferable.
  • Antibody cMAb1 The nucleotide sequence of the cDNA encoding the light chain variable region of cMAb1 is the nucleotide sequence represented by SEQ ID NO: 13 in the sequence listing (FIG. 8), and the amino acid sequence of the light chain variable region of cMAb1 is the SEQ ID NO: SEQ ID NO: It is an amino acid sequence represented by 15 (FIG. 8).
  • the nucleotide sequence of cDNA encoding the heavy chain variable region of cMAb1 is the nucleotide sequence represented by SEQ ID NO: 17 in the sequence listing (FIG. 9), and the amino acid sequence of the heavy chain variable region of cMAb1 is the amino acid sequence of the sequence listing. It is an amino acid sequence represented by SEQ ID NO: 19 (FIG. 9).
  • the anti-HTLV-1 antibody of the present invention comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 19. It is an anti-HTLV-1 antibody that binds.
  • nucleotide sequence represented by SEQ ID NO: 13 or the nucleotide sequence represented by SEQ ID NO: 17 and CLUSTAL W (alignment tool) or the like for example, default, that is, the default parameter.
  • a DNA consisting of a nucleotide sequence having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more.
  • DNA encoding a protein having the activity of the light chain variable region or the heavy chain variable region of the antibody, that is, the binding activity to HTLV-1 is also included in the DNA encoding the light chain variable region or the heavy chain variable region of the antibody of the present invention. Is done.
  • DNA encoding a protein having light chain variable region or heavy chain variable region activity, that is, binding activity to HTLV-1, is also included in the DNA encoding the light chain variable region or heavy chain variable region of the present invention.
  • the nucleotide sequence portion encoding the CDR of the antibody is preferably the same as the nucleotide sequence represented by SEQ ID NO: 13 or the nucleotide sequence represented by SEQ ID NO: 17.
  • the light chain variable region or heavy chain variable region is not limited to the light chain variable region or heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 or the amino acid sequence represented by SEQ ID NO: 19. From an amino acid sequence in which one or several amino acids, for example 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid are deleted, substituted, or added. It also includes the activity of the heavy or light chain variable region of the antibody, i.e., the light chain or heavy chain variable region consisting of a protein having binding activity to HTLV-1.
  • the amino acid represented by SEQ ID NO: 15 is an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 15 or the amino acid sequence represented by SEQ ID NO: 19.
  • CLUSTAL W alignment tool
  • Preferred are those having 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more sequence identity.
  • Such a protein having an amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 19 is represented by SEQ ID NO: 15. It is substantially the same as the protein having the amino acid sequence represented by the amino acid sequence or the amino acid sequence represented by SEQ ID NO: 19.
  • the CDR sequence portion of the antibody is preferably the same as the amino acid sequence represented by SEQ ID NO: 15 or the amino acid sequence represented by SEQ ID NO: 19.
  • cMAb1 is CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 1 (KSSQTLLYSGNQKNYLA) and the amino acid sequence represented by SEQ ID NO: 2 (WASTRQS) as CDRs (complementarity determining regions) of the light chain variable region. ), CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 3 (QQYFATPIT), and CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7 (GFTFGGFPMA) as the CDR of the heavy chain variable region. It contains CDRH2 consisting of the amino acid sequence represented by No. 8 (TISPGAVNTY) and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 9 (EGYGFSSFEY) (FIG. 30).
  • the anti-HTLV-1 antibody of the present invention has CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 3.
  • the CDR of the heavy chain variable region comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9. It is an antibody containing CDRH3.
  • Each of the above CDRs also includes a CDR consisting of an amino acid sequence in which one or several, preferably one or two, more preferably one amino acid is deleted, substituted or added in the amino acid sequence represented therein. ..
  • Antibody cMAb98 The nucleotide sequence of the cDNA encoding the light chain variable region of cMAb98 is the nucleotide sequence represented by SEQ ID NO: 14 in the sequence listing (FIG. 10), and the amino acid sequence of the light chain variable region of cMAb98 is the SEQ ID NO: SEQ ID NO: It is an amino acid sequence represented by 16 (FIG. 10).
  • the nucleotide sequence of the cDNA encoding the heavy chain variable region of cMAb98 is the nucleotide sequence represented by SEQ ID NO: 18 in the sequence listing (FIG. 11), and the amino acid sequence of the heavy chain variable region of cMAb98 is the amino acid sequence of the sequence listing. It is an amino acid sequence represented by SEQ ID NO: 20 (FIG. 13).
  • the anti-HTLV-1 antibody of the present invention comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16 and a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 20. It is an anti-HTLV-1 antibody that binds.
  • nucleotide sequence represented by SEQ ID NO: 14 or the nucleotide sequence represented by SEQ ID NO: 18 and CLUSTAL W (alignment tool) or the like for example, default, that is, the default parameter.
  • a DNA consisting of a nucleotide sequence having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more.
  • DNA encoding a protein having the activity of the light chain variable region or the heavy chain variable region of the antibody, that is, the binding activity to HTLV-1 is also included in the DNA encoding the light chain variable region or the heavy chain variable region of the antibody of the present invention. Is done.
  • DNA capable of hybridizing under stringent conditions with a DNA having a sequence complementary to the nucleotide sequence represented by SEQ ID NO: 14 or the nucleotide sequence represented by SEQ ID NO: 18 and which is an antibody.
  • DNA encoding a protein having light chain variable region or heavy chain variable region activity, that is, binding activity to HTLV-1, is also included in the DNA encoding the light chain variable region or heavy chain variable region of the present invention.
  • the nucleotide sequence portion encoding the CDR of the antibody is preferably the same as the nucleotide sequence represented by SEQ ID NO: 14 or the nucleotide sequence represented by SEQ ID NO: 18.
  • the light chain variable region or heavy chain variable region is not limited to the light chain variable region or heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 20. From an amino acid sequence in which one or several amino acids, for example 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid are deleted, substituted, or added. It also includes the activity of the heavy or light chain variable region of the antibody, i.e., the light chain or heavy chain variable region consisting of a protein having binding activity to HTLV-1.
  • the amino acid represented by SEQ ID NO: 16 is an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 20.
  • CLUSTAL W alignment tool
  • Preferred are those having 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more sequence identity.
  • Such a protein having an amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 20 is represented by SEQ ID NO: 16. It is substantially the same as the protein having the amino acid sequence represented by the amino acid sequence or the amino acid sequence represented by SEQ ID NO: 20.
  • the CDR sequence portion of the antibody is preferably the same as the amino acid sequence represented by SEQ ID NO: 16 or the amino acid sequence represented by SEQ ID NO: 20.
  • cMAb98 is represented by SEQ ID NO: 4, as CDRs of the light chain variable region, CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4 (KSSQSLLFSGYQKNYLA), CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5 (WASTRQS), and SEQ ID NO: 6.
  • CDRL3 consisting of the amino acid sequence (QQYFDTPLT) to be added
  • CDRH1 consisting of the amino acid sequence (GFTFSGSPMG) represented by SEQ ID NO: 10
  • the amino acid sequence represented by SEQ ID NO: 11 TISPTGATTY
  • the anti-HTLV-1 antibody of the present invention has CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 6.
  • the CDR of the heavy chain variable region comprises CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and the amino acid sequence represented by SEQ ID NO: 12. It is an antibody containing CDRH3.
  • Each of the above CDRs also includes a CDR consisting of an amino acid sequence in which one or several, preferably one or two, more preferably one amino acid is deleted, substituted or added in the amino acid sequence represented therein. ..
  • Humanized antibody A humanized antibody (CDR transplanted antibody) is a suitable combination of the CDR amino acid sequences of the light chain variable region and the heavy chain variable region of a non-human animal antibody into the light chain variable region and the heavy chain variable region of a human antibody. An antibody transplanted to a position.
  • the humanized anti-HTLV-1 antibody of the present invention can be expressed and produced by introducing a humanized antibody expression vector constructed by the following method into animal cells. That is, the amino acid sequences of the CDRs of the light chain variable region and the heavy chain variable region of the antibody of a non-human animal produced from a hybridoma that produces a monoclonal antibody that binds to and neutralizes HTLV-1 can be lightened by any human antibody. Construct a cDNA encoding the variable region transplanted into the framework region of the chain variable region and the heavy chain variable region.
  • a humanized antibody expression vector can be constructed by inserting the cDNA into an expression vector for animal cells having a gene encoding a light chain constant region and a heavy chain constant region of a human antibody.
  • a DNA sequence designed to link the CDR of MAb1 or MAb98 to the framework region of a human antibody may be synthesized.
  • the framework region of the human antibody linked via the CDR is selected so that the CDR forms a good antigen binding site.
  • amino acids in the framework region in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen binding site.
  • the production of a humanized antibody transplanted with CDR can be performed by a known CDR graphing technique.
  • Humanized MAb1 antibody The heavy chain of a humanized antibody having the CDRs of the heavy chain variable region of MAb1 (three CDRs consisting of the amino acids shown in SEQ ID NOs: 7 to 9) by the above method, and the framework region of the variable region.
  • the heavy chain in which some amino acids are substituted include the humanized antibody heavy chain hMAb1_H1 and the humanized antibody heavy chain hMAb1_H2.
  • it is a light chain of a humanized antibody having a CDR of the light chain variable region of MAb1 (three CDRs consisting of the amino acids shown in SEQ ID NOs: 1 to 3), and some amino acids in the framework region of the variable region are substituted.
  • Examples of the light chain obtained include a humanized antibody light chain hMab1_L11, a humanized antibody light chain hMAb1_L12, a humanized antibody light chain hMAb1_L13, a humanized antibody light chain hMAb1_L41, a humanized antibody light chain hMAb1_L42, and a humanized antibody light chain hMAb1_L43. ..
  • the full-length nucleotide sequence of the humanized antibody heavy chain hMAb1_H1 is shown in SEQ ID NO: 43, and the amino acid sequence is shown in SEQ ID NO: 44. Further, the full-length nucleotide sequence of the humanized antibody heavy chain hMAb1_H2 is shown in SEQ ID NO: 45, and the amino acid sequence is shown in SEQ ID NO: 46.
  • nucleotide sequence consisting of the 1st to 57th nucleotides is the signal sequence
  • nucleotide sequence consisting of the 58th to 414th nucleotides is the variable region
  • nucleotide sequence consisting of the 415th to 1404th nucleotides is the constant region.
  • amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence
  • amino acid sequence consisting of the 20th to 138th amino acid residues is from the variable region and the 139th to 468th amino acid residues.
  • the amino acid sequence is a constant region amino acid sequence.
  • the anti-HTLV-1 antibody of the present invention comprises an antibody having a heavy chain variable region consisting of amino acid residues 20 to 138 of SEQ ID NO: 44 or 46 and a heavy chain constant region consisting of amino acid residues 139 to 468. ..
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb1_L11 is shown in SEQ ID NO: 31, and the amino acid sequence is shown in SEQ ID NO: 32.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb1_L12 is shown in SEQ ID NO: 33, and the amino acid sequence is shown in SEQ ID NO: 34.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb1_L13 is shown in SEQ ID NO: 35, and the amino acid sequence is shown in SEQ ID NO: 36.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb1_L41 is shown in SEQ ID NO: 37, and the amino acid sequence is shown in SEQ ID NO: 38.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb1_L42 is shown in SEQ ID NO: 39, and the amino acid sequence is shown in SEQ ID NO: 40.
  • the full-length nucleotide sequence of the humanized antibody hMAb1_L43 is shown in SEQ ID NO: 41, and the amino acid sequence is shown in SEQ ID NO: 42.
  • nucleotide sequence consisting of the 1st to 60th nucleotides is the signal sequence
  • nucleotide sequence consisting of the 61st to 402nd nucleotides is the variable region
  • the 403rd to 720th nucleotides encodes a constant region.
  • amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence
  • amino acid sequence consisting of the 21st to 134th amino acid residues is a variable region, 135 to 240.
  • the amino acid sequence consisting of the second amino acid residue is the amino acid sequence of the constant region.
  • the anti-HTLV-1 antibody of the present invention comprises a variable region consisting of amino acid residues 21 to 134 of SEQ ID NOs: 32, 34, 36, 38, 40 and 42 and a light chain constant consisting of amino acid residues 135 to 240. Contains antibodies with regions.
  • the heavy chain constant region of a humanized antibody is a heavy chain constant region of the IgG1 subclass.
  • an antibody having binding property to HTLV-1 an antibody consisting of a humanized antibody heavy chain hMAb1_H1 and a humanized antibody light chain hMAb1_L11 (hMAb1_H1L11), an antibody consisting of a humanized antibody heavy chain hMAb1_H1 and a humanized antibody light chain hMAb1_L12 (hMAb1_H1L12).
  • An antibody consisting of a humanized antibody heavy chain hMAb1_H1 and a humanized antibody light chain hMAb1_L13 (hMAb1_H1L13)
  • an antibody consisting of a humanized antibody heavy chain hMAb1_H1 and a humanized antibody light chain hMAb1_L41 (hMAb1_H1L41), a humanized antibody heavy chain hMAb1_H1 and a human.
  • the antibody consisting of the humanized antibody light chain hMAb1_L42 (hMAb1_H1L42), the humanized antibody heavy chain hMAb1_H1 and the humanized antibody light chain hMAb1_L43 (hMAb1_H1L43), the humanized antibody heavy chain hMAb1_H2 and the humanized antibody light chain hMAb1_L11 (hMAb1_L11).
  • An antibody consisting of a humanized antibody heavy chain hMAb1_H2 and a humanized antibody light chain hMAb1_L12 (hMAb1_H2L12), an antibody consisting of a humanized antibody heavy chain hMAb1_H2 and a humanized antibody light chain hMAb1_L13 (hMAb1_H2L13), a humanized antibody heavy chain hMAb1_H2 and a human.
  • the antibody consisting of the humanized antibody light chain hMAb1_L41 (hMAb1_H2L41), the humanized antibody heavy chain hMAb1_H2 and the humanized antibody light chain hMAb1_L42 (hMAb1_H2L42), the humanized antibody heavy chain hMAb1_H2 and the humanized antibody light chain hMAb1_L43 (hMAb1_L43). ).
  • HMAb1_H1L11 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 32.
  • HMAb1_H1L12 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 34.
  • HMAb1_H1L13 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 36.
  • HMAb1_H1L41 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 38.
  • HMAb1_H1L42 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 40.
  • HMAb1_H1L43 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 44 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 42.
  • HMAb1_H2L11 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 32.
  • HMAb1_H2L12 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 34.
  • HMAb1_H2L13 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 36.
  • HMAb1_H2L41 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 38.
  • HMAb1_H2L42 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 40.
  • HMAb1_H2L43 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 46 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 42.
  • Humanized MAb98 antibody A heavy chain of a humanized antibody having a CDR of the heavy chain variable region of MAb98 (three CDRs consisting of the amino acids shown in SEQ ID NOs: 10 to 12), and an amino acid of a part of the framework region of the variable region.
  • Examples of the heavy chain substituted with the above include the humanized antibody heavy chain hMAb98_H2 and the humanized antibody heavy chain hMAb98_H3.
  • it is a light chain of a humanized antibody having a CDR of the light chain variable region of MAb98 (three CDRs consisting of the amino acids shown in SEQ ID NOs: 4 to 6), and some amino acids in the framework region of the variable region are substituted.
  • Examples of the light chain obtained include a humanized antibody light chain hMab98_L11, a humanized antibody light chain hMAb98_L13, a humanized antibody light chain hMAb98_L41, and a humanized antibody light chain hMAb98_L43.
  • the full-length nucleotide sequence of the humanized antibody heavy chain hMAb98_H2 is shown in SEQ ID NO: 55, and the amino acid sequence is shown in SEQ ID NO: 56. Further, the full-length nucleotide sequence of the humanized antibody heavy chain hMAb98_H3 is shown in SEQ ID NO: 57, and the amino acid sequence is shown in SEQ ID NO: 58.
  • nucleotide sequence consisting of the 1st to 57th nucleotides is the signal sequence
  • nucleotide sequence consisting of the 58th to 414th nucleotides is the variable region
  • nucleotide sequence consisting of the 415th to 1404th nucleotides is the constant region.
  • amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence
  • amino acid sequence consisting of the 20th to 138th amino acid residues is from the variable region and the 139th to 468th amino acid residues.
  • the amino acid sequence is a constant region amino acid sequence.
  • the anti-HTLV-1 antibody of the present invention comprises an antibody having a heavy chain variable region consisting of amino acid residues 20 to 138 of SEQ ID NO: 56 or 58 and a heavy chain constant region consisting of amino acid residues 139 to 468. ..
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb98_L11 is shown in SEQ ID NO: 47, and the amino acid sequence is shown in SEQ ID NO: 48.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb98_L13 is shown in SEQ ID NO: 49, and the amino acid sequence is shown in SEQ ID NO: 50.
  • the full-length nucleotide sequence of the humanized antibody light chain hMAb98_L41 is shown in SEQ ID NO: 51, and the amino acid sequence is shown in SEQ ID NO: 52.
  • nucleotide sequence of the humanized antibody light chain hMAb98_L43 is shown in SEQ ID NO: 53
  • amino acid sequence is shown in SEQ ID NO: 54.
  • nucleotide sequence consisting of the 1st to 60th nucleotides is the signal sequence
  • nucleotide sequence consisting of the 61st to 402nd nucleotides is the variable region
  • nucleotide sequence consisting of the 403rd to 720th nucleotides Each sequence encodes a constant region.
  • amino acid sequence consisting of the 1st to 20th amino acid residues is the signal sequence
  • amino acid sequence consisting of the 21st to 134th amino acid residues is the variable region
  • amino acid residue of the 135th to 240th positions is the amino acid sequence of the constant region.
  • the anti-HTLV-1 antibody of the present invention has a variable region consisting of amino acid residues 21 to 134 of SEQ ID NOs: 48, 50, 52 and 54 and a light chain constant region consisting of amino acid residues 135 to 240. including.
  • the heavy chain constant region of a humanized antibody is a heavy chain constant region of the IgG1 subclass.
  • an antibody having binding property to HTLV-1 an antibody consisting of a humanized antibody heavy chain hMAb98_H2 and a humanized antibody light chain hMAb98_L43 (hMAb98_H2L43), an antibody consisting of a humanized antibody heavy chain hMAb98_H3 and a humanized antibody light chain hMAb98_L11 (hMAb98_H3L11).
  • HMAb98_H2L43 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 56 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 54.
  • HMAb98_H3L11 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 48.
  • HMAb98_H3L13 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 50.
  • HMAb98_H3L41 is an antibody having a heavy chain consisting of amino acid residues 20 to 468 of SEQ ID NO: 58 and a light chain consisting of amino acid residues 21 to 240 of SEQ ID NO: 52.
  • the antibody of the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. Fragments of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin, or by modifying the antibody gene by a genetic engineering technique and expressing it in a suitable cultured cell. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be referred to as an antigen-binding fragment of the antibody.
  • Functions of the antibody generally include antigen-binding activity, activity of neutralizing the activity of the antigen, activity of enhancing the activity of the antigen, ADCC activity, ADCP activity and CDC activity. The function retained by the antigen-binding fragment of the antibody in the present invention is the binding activity to HTLV-1.
  • Fab fragments
  • F (ab') 2 variable region
  • scFv single chain Fv
  • diabodies diabodies
  • Fab' which is a monovalent fragment of the variable region of the antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antibody fragment.
  • the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens. Normally, such molecules bind to two types of antigens (ie, bispecific antibodies), but the "multispecific antibodies" in the present invention are more (eg, three types). It includes antibodies having specificity against the antigen of.
  • the multispecific antibody of the present invention may be a full-length antibody or a fragment of such an antibody (eg, an F (ab') bibispecific antibody).
  • Bispecific antibodies can be produced by binding the heavy and light chains of two types of antibodies, or by fusing hybridomas that produce different monoclonal antibodies to produce bispecific antibody-producing fused cells. This can also be made (Nature (1983), 305, p. 537-540).
  • the antibody of the present invention may be a single-chain antibody (also referred to as scFv).
  • the single-chain antibody is obtained by linking the heavy chain variable region and the light chain variable region of the antibody with a linker of a polypeptide (The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore ed., Springer Verlag, New). , P.269-315 (1994), Nature Biotechnology (2005), 23, p.1126-1136).
  • a BiscFv fragment prepared by binding two scFvs with a polypeptide linker can also be used as a bispecific antibody.
  • the heavy chain variable region and the light chain variable region are linked via a linker that does not form a conjugate, preferably a polypeptide linker (Proc. Natl. Acad. Sci. USA). 1988), 85, p. 5879-5883).
  • the heavy chain variable region and the light chain variable region in scFv may be derived from the same antibody or may be derived from different antibodies.
  • the polypeptide linker linking the variable region for example, any single-stranded peptide consisting of 12 to 19 residues is used.
  • the DNA encoding scFv is a DNA encoding a heavy chain or a heavy chain variable region of the antibody, and a DNA encoding a light chain or a light chain variable region, all of the sequences thereof or a desired amino acid sequence.
  • the DNA portion encoding is used as a template, amplified by the PCR method using a primer pair defining both ends thereof, and then the DNA encoding the polypeptide linker portion and both ends thereof are linked to the heavy chain and the light chain, respectively. It is obtained by amplifying by combining the primer pairs specified in 1.
  • an expression vector containing them and a host transformed by the expression vector can be obtained according to a conventional method, and by using the host, it is usual.
  • ScFv can be obtained according to the method.
  • These antibody fragments can be obtained and expressed in the same manner as described above, and can be produced by the host.
  • the antibody of the present invention may be increased in quantity to increase the affinity for the antigen.
  • the antibody to be increased in quantity may be one type of antibody or a plurality of antibodies recognizing a plurality of epitopes of the same antigen.
  • Examples of the method for increasing the amount of antibody include binding of the IgG CH3 domain to two scFvs, binding to streptavidin, and introduction of a helix-turn-helix motif.
  • the antibody of the present invention may be a polyclonal antibody, which is a mixture of a plurality of types of anti-HTLV-1 antibodies having different amino acid sequences.
  • a polyclonal antibody a mixture of a plurality of types of antibodies having different CDRs can be mentioned.
  • a mixture of cells producing different antibodies can be cultured, and an antibody purified from the culture can be used (see WO2004 / 061104).
  • the antibody of the present invention also includes a modified antibody.
  • the modified product means an antibody of the present invention that has been chemically or biologically modified.
  • Chemical modifications include the binding of chemical moieties to the amino acid skeleton, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
  • Biological modifications are post-translational modifications (eg, N-linked or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation). It also includes those having a methionine residue added to the N-terminal by expression using a prokaryotic host cell.
  • those labeled to enable the detection or isolation of the antibody or antigen of the present invention for example, an enzyme-labeled substance, a fluorescent-labeled substance, and an affinity-labeled substance are also included in the meaning of such modified substances.
  • Such a modified version of the antibody of the present invention is useful for improving the stability and retention of the antibody, reducing the antigenicity, detecting or isolating the antibody or the antigen, and the like.
  • an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • WO1999 / 54342, WO2000 / 61739, WO2002 / 31140, and the like are known as techniques for regulating the sugar chain modification of an antibody, but the technique is not limited thereto.
  • the antibody of the present invention also includes an antibody in which the sugar chain modification has been adjusted.
  • the antibody of the present invention may be an antibody in which these antibodies and other drugs form a conjugate (Immunoconjugate).
  • immunoconjugate examples include those in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p. 1137-1146).
  • the antibody of the present invention may be an antibody having a single heavy chain variable region and not having a light chain sequence.
  • Such antibodies are called single domain antibodies (sdAb) or Nanobodies, and are actually observed in camels or llamas, and it has been reported that antigen-binding ability is retained. (Protein Eng. (1994), 7, p. 1129-1135; Nature (1993), 363, p. 446-448).
  • the above antibody can also be interpreted as a kind of antigen-binding fragment of the antibody in the present invention.
  • a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region is inserted into an expression vector, and a host cell for expression is transformed with the vector to host. By culturing the cells, the cells can be produced as recombinant antibodies.
  • a DNA encoding a heavy chain is obtained by linking a DNA encoding a heavy chain variable region and a DNA encoding a heavy chain constant region, and further, a DNA encoding a light chain variable region and a light chain are obtained. By ligating the DNA encoding the chain constant region, the DNA encoding the light chain is obtained.
  • the DNA encoding the heavy chain and the DNA encoding the light chain are inserted into an expression vector, the host cell is transformed with the vector, and the host cell is cultured. Can be produced.
  • the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector, and the host cell may be transformed with the vector, or the DNA encoding the heavy chain may be light.
  • the DNA encoding the strand may be inserted into separate vectors and the two vectors may be used to transform the host cell.
  • the DNA encoding the heavy chain variable region and the light chain variable region may be introduced into the vector into which the DNA encoding the heavy chain constant region and the DNA encoding the light chain constant region have been introduced in advance.
  • the vector may also contain DNA encoding a signal peptide that promotes antibody secretion from the host cell.
  • the DNA encoding the signal peptide and the DNA encoding the antibody are linked in an in-frame. By removing the signal peptide after the antibody is produced, the antibody can be obtained as a mature protein.
  • the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and the light chain variable region are encoded.
  • the DNA in which the DNA and the DNA encoding the light chain constant region are linked may be functionally linked to an element such as a promoter, an enhancer, or a polyadenylation signal.
  • functionally connected means that elements are connected so as to perform their functions.
  • the expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, a bacterium, or yeast, and examples thereof include known plasmids and phages.
  • Examples of the vector used for constructing the expression vector include pcDNA (trademark) (Thermo Fisher SCIENTIFIC), Flexi (registered trademark) vector (Promega), pUC19 (New England Biolabs), and pGEX-4T (Cytiva). (Manufactured by Clontech), pMAM-neo (manufactured by Clontech) and the like.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used.
  • animal cells HEK293 cells, Chinese hamster ovary (CHO) cells, which are human fetal kidney cell lines, and the like may be used.
  • the expression vector may be introduced into a host cell by a known method to transform the host cell. For example, an electroporation method, a calcium phosphate precipitation method, a DEAE-dextran transfection method and the like can be mentioned.
  • the antibody produced can be purified using the separation and purification methods used for conventional proteins. For example, affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis and the like may be appropriately selected and combined.
  • the present invention Since the present invention has a neutralizing activity against HTLV-1, it is expected to be used as an antibody drug for preventing horizontal transmission of HTLV-1 and vertical transmission between mother and child.
  • the present invention specifically binds to HTLV-1-infected cells and has ADCC and / or ADCP activity, thereby having cell-killing activity against the cells. Therefore, a person infected with HTLV-1.
  • it is expected to be used as a preventive agent and a therapeutic agent for the onset of diseases caused by HTLV-1.
  • Diseases caused by HTLV-1 include adult T-cell leukemia / lymphoma (ATLL), HTLV-1-related myelopathy (HAM), HTLV-1 uveitis (HU), and the like.
  • ADCC recognizes antibodies bound on target cells by non-specific cytotoxic cells expressing Fc ⁇ receptors (eg, natural killer cells, neutrophils, macrophages, etc.) and subsequently causes lysis of the target cells.
  • Fc ⁇ receptors eg, natural killer cells, neutrophils, macrophages, etc.
  • a cell-mediated reaction Natural killer cells, which are the primary cells responsible for ADCC, express Fc ⁇ RIIC and Fc ⁇ RIIIA, and monocytes express Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIC, and Fc ⁇ RIIIA.
  • ADCP refers to a cell-mediated reaction in which phagocytic cells expressing the Fc receptor (for example, macrophages, neutrophils, etc.) recognize an antibody bound on the target cell and then phagocytose the target cell into the cell. ..
  • Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIC and Fc ⁇ RIIIA are expressed in monocytes, which are the primary
  • the present invention can include therapeutically effective amounts of anti-HTLV-1 antibodies and pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, auxiliaries and the like.
  • pharmaceutically acceptable carrier and the like can be appropriately selected from a wide range according to the type of the target disease and the administration form of the drug.
  • the method for administering the antitumor agent of the present invention can be appropriately selected, and for example, it can be administered by injection, such as local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusate injection, etc. Can be adopted.
  • the solution for injection can be formulated using a carrier consisting of a salt solution, a glucose solution, a mixture of salt water and a glucose solution, various buffer solutions, and the like. Further, it may be formulated in a powder state and mixed with the liquid carrier at the time of use to prepare an injection solution.
  • oral liquids powders, pills, capsules, tablets and the like can be applied.
  • oral liquid preparations such as suspending agents and syrups, water, shoe cloth, sorbitol, saccharides such as fructose, glycols such as polyethylene glycol, sesame oil, soybean oil and the like. It can be produced by using oils, preservatives such as alkylparahydroxybenzoate, flavors such as strawberry flavor and peppermint, and the like.
  • Powders, pills, capsules and tablets include excipients such as lactose, glucose, shoe cloth and mannitol, disintegrants such as starch and sodium arginate, lubricants such as magnesium ester and talc, and polyvinyl alcohol. , Hydroxypropyl cellulose, a binder such as gelatin, a surface active agent such as a fatty acid ester, a plasticizer such as glycerin, and the like can be used for the formulation. Tablets and capsules are preferred unit dosage forms in the compositions of the invention in that they are easy to administer. When producing tablets and capsules, a solid production carrier is used.
  • the amount of antibody effective for treatment varies depending on the nature of the medical condition to be treated, the age and condition of the patient, and is ultimately decided by the doctor. For example, it is 0.0001 mg to 100 mg per 1 kg of body weight at a time.
  • the predetermined dose may be administered once every 1 to 180 days, or may be administered in divided doses of 2 times, 3 times, 4 times or more per day at appropriate intervals.
  • the antibody of the present invention when used as a prophylactic or therapeutic agent for a disease, it can be used in combination with other agents.
  • the other agents used in combination when the antibody of the present invention is used for the prevention or treatment of adult T-cell leukemia / lymphoma are not particularly limited as long as they are anticancer agents, and are preferably chemotherapeutic agents and molecular target agents.
  • topoisomerase inhibitors include irinotecan, topotecan, etoposide and the like.
  • Preferable examples of the microtubule inhibitor include paclitaxel, docetaxel, vincristine, vinblastine, vindesine, eribulin, paclitaxel-encapsulating micelle NK105 and the like.
  • platinum preparation include oxalplatin, carboplatin, cisplatin, nedaplatin, cisplatin-encapsulating micelle NC-6004 and the like.
  • the other agents used in combination are therapeutic antibodies such as steroids, immunosuppressants, interferon ⁇ , and mogamurizumab (anti-CCR4 antibody). That is not particularly limited, and preferred examples of the steroid include corticosteroids such as prednisolone, methylprednisolone, dexamethasone, betamethasone and hydrocortisone, and immunosuppressive agents include azathiopurine and mofetil mycophenolate.
  • immunosuppressants such as misolibin, carcinulinin inhibitors such as cyclosporin and tacrolimus, JAK inhibitors such as tofasotinib and tacrolimus, and mTOR such as everolimus.
  • the other agent used in combination when the antibody of the present invention is used for the prevention or treatment of HTLV-1 vasculitis (HU) is not particularly limited as long as it is a steroid, a therapeutic antibody, etc., and is preferably used as a steroid. , Prednisolone, Methylprednisolone, Dexamethasone, Betamethasone, Hydrocortisone and other corticosteroids.
  • the antibody of the present invention may be administered at the same time as the above-mentioned other agents or separately. In addition, it may be administered sequentially.
  • the antibody of the present invention may be administered before or after administration of the above-mentioned other agent. When administered simultaneously, they can be administered as separate formulations or as a single pharmaceutical composition containing both.
  • the other agent may be administered 1 to 24 hours before or after the administration of the antibody, or 1 to 30 days before or after the administration of the antibody.
  • the administration interval between the antibody of the present invention and the other agent may be the same or different.
  • the present invention includes an antibody to be used in combination with the above-mentioned other agent, and further includes an antibody to be combined with the above-mentioned other agent.
  • Example 1 Preparation of rat-derived anti-HTLV-1 antibody 1
  • HTLV-1 expression vector Amino acid sequence of extracellular region (SEQ ID NOs: 60, 1-312) of HTLV-1 envelope protein gp62 (UniProt ACCESSION number: P03381)
  • the cDNA encoding (SEQ ID NOs: 59-936) is inserted between the cleavage sites of the restriction enzymes EcoRI and EcoRV of the pcDNA3.1 (+) (Thermo Fisher SCIENTIFIC) vector according to a method known to those skilled in the art.
  • EndoFree Plasmamid Giga Kit manufactured by QIAGEN was used for the large-scale preparation of the vector expressing HTLV-1.
  • Hybridoma production Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells were fused by electric cells using LF301-Cell Fusion Unit (manufactured by BEX), and ClonaCell- The cells were diluted with HY Selection Medium D (manufactured by StemCell Technologies) and cultured. Monoclone hybridomas were produced by collecting the hybridoma colonies that appeared. Each of the recovered hybridoma colonies was cultured, and the obtained hybridoma culture supernatant was used to screen for anti-HTLV-1 antibody-producing hybridomas.
  • Example 2 Evaluation of rat-derived anti-HTLV-1 antibody 2) -1 Evaluation of binding activity by Cell-ELISA method 2) -1-1 Preparation of GLUT1 expression vector
  • GLUT1 amino acid sequence of GLUT1 protein (GLUT1) (NCBI Reference Sequence ACCESS No .: NM_006516)
  • GLUT1 is expressed by inserting the encoding cDNA (SEQ ID NO: 62) between the restriction enzymes EcoRI and the cleavage site of XbaI of the cDNA3.1 (+) (ThermoFiser SCIENTIFIC) vector according to a method known to those skilled in the art.
  • a vector was prepared. EndoFree Plasmamid Giga Kit (manufactured by QIAGEN) was used for the large-scale preparation of the vector expressing GLUT1.
  • pcDNA3.1 (+) (manufactured by Thermo Fisher SCIENTIFIC) as a vector or control expressing GLUT1 which is a receptor of GLUT1 and dispensing 100 ⁇ L each into 96-well plate (manufactured by Corning), DMEM medium containing 10% FBS. The cells were cultured in the medium at 37 ° C. and 5% CO 2 for 24-27 hours. The obtained introduced cells were used for Cell-ELISA in an adhered state.
  • OPD color-developing solution OPD lysate (0.05M sodium citrate, 0.1M sodium hydrogen phosphate, 12 water pH 4.5)
  • OPD color-developing solution OPD lysate (0.05M sodium citrate, 0.1M sodium hydrogen phosphate, 12 water pH 4.5)
  • the color reaction was carried out with occasional stirring, and 1M HCl was added at 100 ⁇ L / well to stop the color reaction, and then the absorbance at 490 nm was measured with a plate reader ENVISION (manufactured by PerkinElmer).
  • a plate reader ENVISION manufactured by PerkinElmer
  • vector introductions expressing HTLV-1 and GLUT1 were compared with control pcDNA3.1-introduced HEK293 cells.
  • Hybridomas that produce a culture supernatant that exhibits higher absorbance in HEK293 cells were selected as positive for anti-HTLV-1 antibody production.
  • pcDNA3.1 (+) (manufactured by Thermo Fisher SCIENTIFIC) was introduced into 293T cells as a vector expressing HTLV-1 and a vector expressing GLUT1 or as a control, respectively, using Lipofectamine 2000 (manufactured by Thermo Fisher SCIENTIFIC) 37. The cells were further cultured overnight at ° C. and 5% CO 2 . The next day, the expression vector-introduced 293T cells were treated with TrypLE Express (manufactured by Thermo Fisher SCIENTIFIC), the cells were washed with DMEM containing 10% FBS, and then suspended in PBS containing 5% FBS. The resulting cell suspension was used for flow cytometric analysis.
  • Example 2 Flow cytometry analysis The specific binding reproducibility of the antibody produced by the hybridoma positive in Cell-ELISA of Example 2) -1 to HTLV-1 was confirmed by the flow cytometry method.
  • Example 2 The suspension of transiently expressed 293T cells prepared in 2-1 was centrifuged, the supernatant was removed, and then a hybridoma culture supernatant was added to each suspension to suspend the cells, and the suspension was suspended at 4 ° C. for 1 hour. It was left still.
  • Goat Anti-Rat IgG Antibody and FITC Conjugate (manufactured by SIGMA-ALDRICH) diluted 500 times with PBS containing 5% FBS were added and suspended, and suspended at 4 ° C. for 1 hour. It was left still. After washing twice with PBS containing 5% FBS, it was resuspended in PBS containing 5% FBS containing 2 ⁇ g / mL 7-aminoactinomycin D (manufactured by Molecular Probes), and detected with a flow cytometer FC500 (manufactured by Beckman Coulter). went. Data analysis was performed by Flowjo (manufactured by TreeStar).
  • a histogram of FITC fluorescence intensity of live cells was prepared.
  • the hybridoma that produces an antibody in which the histogram of the vector-introduced 293T cells expressing HTLV-1 and GLUT1 is shifted to the strong fluorescence intensity side with respect to the fluorescence intensity histogram of the control pcDNA3.1-introduced 293T cells is specific to HTLV-1.
  • 76 clones were selected as antibody-producing hybridomas that bind to the cells.
  • Example 2 2) -3 Syncytial formation inhibitory activity
  • the syncytial formation inhibitory activity was confirmed for 76 clones selected by flow cytometric analysis in Example 2) -2.
  • YT # 1 cells which are HTLV-1 infected cells derived from healthy humans, were prepared in 20 U / mL IL-2, 10% FBS-containing RPMI1640 medium (manufactured by SIGMA-ALDRICH) so as to be 2 ⁇ 10 6 cells / mL. Then, 25 ⁇ L was dispensed into 96-well plate.
  • Antibodies contained in the two hybridoma culture supernatants that could completely inhibit syncytial formation as in the positive control LAT-27 were selected as the syncytial formation inhibitory positive anti-HTLV-1 antibody. ..
  • the anti-HTLV-1 monoclonal antibody was purified from the culture supernatant of rat hybridoma.
  • a rat anti-HTLV-1 monoclonal antibody-producing hybridoma was grown to a sufficient amount in ClonaCell-HY Selection Medium E (manufactured by StemCell Technologies), and Ultra Low IgG FBS (manufactured by Thermo Fisher SCIENTIFIC) was added to 20% After changing the medium, hybridomas of 8-9 ⁇ 107 cells were inoculated in a 1272 cm 2 flask (manufactured by Corning) and cultured for 7 days. The culture supernatant was collected by centrifugation, passed through a 0.8 ⁇ m filter, and then sterilized through a 0.45 ⁇ m filter (manufactured by Corning).
  • IgG antibody As an isotype control IgG antibody is added from a diluted series solution of 10 ⁇ g / mL in 3 series with a 10-fold common ratio, and Anti-Rat IgG FITC Conjugate (manufactured by SIGMA-ALDRICH) is reacted to cause a flow cytometer FC500 (Beckman Coulter). Detected by). Data analysis was performed by Flowjo (manufactured by TreeStar), and a histogram of FITC fluorescence intensity of living cells was created.
  • SIGMA-ALDRICH Anti-Rat IgG FITC Conjugate
  • Histogram of vector-introduced 293T cells expressing HTLV-1 and GLUT1 is strongly fluorescent against fluorescence intensity histogram of pcDNA3.1-introduced 293T cells, which is a control only in cells supplemented with rat anti-HTLV-1 monoclonal purified antibody and LAT-27. It was confirmed that it was shifted to the strength side.
  • FIG. 1 shows the median fluorescence intensity according to the dilution rate of the antibody specifically bound to HTLV-1-expressing cells.
  • Example 2 HTLV-1 which is an antigen of rMAb1 whose specific binding property to HTLV-1-expressing cells could be confirmed in Example 2) -6-1.
  • the binding activity to gp46 peptides 180 to 204 was immobilized using Biacore T200 (manufactured by Cytiva) using an antibody as a ligand, and the antigen was measured as an analysis.
  • HBS-EP + manufactured by Cytiva
  • Series S Sensor Chip CM5 manufactured by Cytiva
  • rMAb1 diluted to 5 ⁇ g / mL with AcOH 5.0 (manufactured by Cytiva) was added at 10 ⁇ L / min for 360 seconds to solidify the chip.
  • a diluted series solution of the antigen (8 series from 3 ⁇ g / mL to 3 times the common ratio) was added at a flow rate of 10 ⁇ L / min for 1200 seconds to obtain a response at the time of binding.
  • 3M magnesium chloride (manufactured by Cytiva) was added at a flow rate of 20 ⁇ L / min for 30 seconds.
  • the KD value calculated using the affinity analysis was 3.49 nM.
  • Example 3 Preparation of human chimeric antibody 3) -1 Determination of nucleotide sequence of variable region of rMAb1 3) -1-1 Preparation of total RNA of rMAb1 production hybridoma TRIzol Reagent (Ambion) from rMAb1-producing hybridoma to amplify cDNA of variable region of rMAb1 Total RNA was prepared using (manufactured by).
  • Example 3 1-2 5'-RACE PCR amplification of cDNA in the light chain variable region of rMAb1 and determination of sequence
  • the amplification of the cDNA in the variable region of the light chain was performed in Example 3) -total RNA prepared in 1-1. It was carried out using about 1 ⁇ g of the above and SMARTer RACE 5'/3'Kit (manufactured by Clontech). From the sequences of UPM (Universal Primer A Mix: SMARTer RACE 5'/3'Kit) and known rat light chain constant regions as primers for amplifying the cDNA of the variable region of the light chain of rMAb1 by PCR. The designed primer was used.
  • the cDNA of the variable region of the light chain amplified by 5'-RACE PCR was cloned into a plasmid, and then the nucleotide sequence of the cDNA of the variable region of the light chain was sequenced.
  • nucleotide sequence of the cDNA in the variable region of the light chain of rMAb1 determined is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 15.
  • Example 3 3) -1-3 5'-RACE PCR amplification of cDNA in the heavy chain variable region of rMAb1 and determination of sequence
  • the amplification of the cDNA in the heavy chain variable region was performed in Example 3) -total RNA prepared in 1-1. It was carried out using about 1 ⁇ g of the above and SMARTer RACE 5'/3'Kit (manufactured by Clontech).
  • UPM included with Universal Primer A Mix: SMARTer RACE 5'/3'Kit
  • the designed primer was used as a primer for amplifying the cDNA of the variable region of the heavy chain of rMAb1 by PCR.
  • the cDNA of the variable region of the heavy chain amplified by 5'-RACE PCR was cloned into a plasmid, and then the nucleotide sequence of the cDNA of the variable region of the heavy chain was sequenced.
  • nucleotide sequence of the cDNA of the determined variable region of the heavy chain of rMAb1 is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 19.
  • 3) -2 Determining the nucleotide sequence of the cDNA in the variable region of rMAb98 The procedure was the same as in Example 3) -1.
  • the nucleotide sequence of the cDNA in the variable region of the light chain of the determined rMAb98 is shown in SEQ ID NO: 14, and the amino acid sequence is shown in SEQ ID NO: 16.
  • the nucleotide sequence of the cDNA in the variable region of the heavy chain of the determined rMAb98 is shown in SEQ ID NO: 18, and the amino acid sequence is shown in SEQ ID NO: 20.
  • PCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.
  • a cMAb1 light chain expression vector was constructed by inserting a synthesized DNA fragment into a site where pCMA-LK was cleaved with the restriction enzyme BsiWI using an In-Fusion HD PCR cloning kit (manufactured by Clontech).
  • the amino acid sequence of the cMAb1 light chain is shown in SEQ ID NO: 25.
  • Example 3) -8 Purification of human chimeric anti-HTLV-1 antibody
  • the antibody was purified from the culture supernatant obtained in Example 3) -7 by a one-step process of rProtein A affinity chromatography at 4 to 6 ° C.
  • the buffer replacement step after rProteinA affinity chromatography purification was performed at 4-6 ° C.
  • the culture supernatant was applied to HiTrap Protein A HP (Cytiva) equilibrated with 50 mM NaCl, 1.5 M NaCl, 10 mM EDTA, 0.1 M L-arginine hydrochloride, pH 7.0. After all the culture was in the column, the column was washed with equilibrated buffer at least twice the column volume.
  • Example 4 Design of humanized anti-HTLV-1 antibody 4) -1 Molecular modeling of variable region
  • a method known as homology modeling (Methods in Enzyme (1991), 203, p.121-153) was used. .. Using the structure registered in Protein Data Bank (Nuc. Acid Res. (2007), 35, p. D301-D303), which has high sequence homology to the variable region, as a template, a commercially available protein three-dimensional structure analysis program BioLuminate. (Manufactured by Schrodinger) was used.
  • Donor residues to be transferred onto the acceptor are described in Queen et al. (Proc. Natl. Acad. Sci. USA (1989), 86, p.10029-10033) analyzes the three-dimensional model with reference to the criteria, etc., and designs it independently according to each sequence. did.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 32 is set forth in SEQ ID NO: 31.
  • the full-length amino acid sequence of hMAb1_L12 is set forth in SEQ ID NO: 34.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34 is set forth in SEQ ID NO: 33.
  • the full-length amino acid sequence of hMAb1_L13 is set forth in SEQ ID NO: 36.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36 is set forth in SEQ ID NO: 35.
  • the full-length amino acid sequence of hMAb1_L41 is set forth in SEQ ID NO: 38.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 38 is set forth in SEQ ID NO: 37.
  • the full-length amino acid sequence of hMAb1_L42 is set forth in SEQ ID NO: 40.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 40 is set forth in SEQ ID NO: 39.
  • the full-length amino acid sequence of hMAb1_L43 is set forth in SEQ ID NO: 42.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 42 is set forth in SEQ ID NO: 41.
  • the sequences of SEQ ID NO: 31 and SEQ ID NO: 32 are shown in FIG.
  • sequences of SEQ ID NO: 33 and SEQ ID NO: 34 are shown in FIG. 17, and the sequences of SEQ ID NO: 35 and SEQ ID NO: 36 are shown in FIG.
  • sequence of SEQ ID NO: 38 is shown in FIG. 19
  • sequences of SEQ ID NO: 39 and SEQ ID NO: 40 are shown in FIG. 20
  • sequences of SEQ ID NO: 41 and SEQ ID NO: 42 are shown in FIG. 21, respectively.
  • hMAb1_H1 and hMAb1_H2 were named hMAb1_H1 and hMAb1_H2, respectively.
  • the full-length amino acid sequence of hMAb1_H1 is set forth in SEQ ID NO: 44.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44 is set forth in SEQ ID NO: 43.
  • the full-length amino acid sequence of hMAb1_H2 is set forth in SEQ ID NO: 46.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46 is set forth in SEQ ID NO: 45.
  • the sequences of SEQ ID NO: 43 and SEQ ID NO: 44 are shown in FIG. 22, and the sequences of SEQ ID NO: 45 and SEQ ID NO: 46 are shown in FIG. 23, respectively.
  • the full-length amino acid sequence of hMAb98_L13 is set forth in SEQ ID NO: 50.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 50 is set forth in SEQ ID NO: 49.
  • the full-length amino acid sequence of hMAb98_L41 is set forth in SEQ ID NO: 52.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 is set forth in SEQ ID NO: 51.
  • the full-length amino acid sequence of hMAb98_L43 is set forth in SEQ ID NO: 54.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 54 is set forth in SEQ ID NO: 53.
  • sequences of SEQ ID NO: 47 and SEQ ID NO: 48 are shown in FIG. 24, the sequences of SEQ ID NO: 49 and SEQ ID NO: 50 are shown in FIG. 25, and the sequences of SEQ ID NO: 51 and SEQ ID NO: 52 are shown in FIG. 26.
  • the sequences of numbers 54 are also shown in FIG. 27, respectively.
  • hMAb98_H2 A humanized antibody heavy chain in which the ⁇ chain constant region of human IgG1 was connected to the designed humanized antibody heavy chain variable region of MAb98 was designed and named hMAb98_H2 and hMAb98_H3, respectively.
  • the full-length amino acid sequence of hMAb98_H2 is described in 56.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 56 is set forth in SEQ ID NO: 55.
  • the full-length amino acid sequence of hMAb98_H3 is described in 58.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 58 is set forth in SEQ ID NO: 57.
  • the sequences of SEQ ID NO: 55 and SEQ ID NO: 56 are shown in FIG. 28, and the sequences of SEQ ID NO: 57 and SEQ ID NO: 58 are shown in FIG. 29, respectively.
  • hMAb1_H1L11 the antibody composed of hMAb1_H1 and hMAb1_L11 is referred to as "hMAb1_H1L11".
  • the antibody consisting of hMAb1_H1 and hMAb1_L12 is referred to as "hMAb1_H1L12".
  • the antibody consisting of hMAb1_H1 and hMAb1_L13 is referred to as "hMAb1_H1L13".
  • the antibody consisting of hMAb1_H1 and hMAb1_L41 is referred to as "hMAb1_H1L41".
  • the antibody consisting of hMAb1_H1 and hMAb1_L42 is referred to as "hMAb1_H1L42".
  • the antibody consisting of hMAb1_H1 and hMAb1_L43 is referred to as "hMAb1_H1L43".
  • the antibody consisting of hMAb1_H2 and hMAb1_L11 is referred to as "hMAb1_H2L11".
  • the antibody consisting of hMAb1_H2 and hMAb1_L12 is referred to as "hMAb1_H2L12".
  • the antibody consisting of hMAb1_H2 and hMAb1_L13 is referred to as "hMAb1_H2L13".
  • the antibody consisting of hMAb1_H2 and hMAb1_L41 is referred to as "hMAb1_H2L41".
  • the antibody consisting of hMAb1_H2 and hMAb1_L42 is referred to as "hMAb1_H2L42".
  • the antibody consisting of hMAb1_H2 and hMAb1_L43 is referred to as "hMAb1_H1L43".
  • hMAb98_H2L43 an antibody composed of hMAb98_H2 and hMAb1_L43 is referred to as "hMAb98_H2L43".
  • the antibody consisting of hMAb98_H3 and hMAb98_L11 is referred to as "hMAb98_H3L11”.
  • the antibody consisting of hMAb98_H3 and hMAb98_L13 is referred to as "hMAb98_H3L13".
  • the antibody consisting of hMAb98_H3 and hMAb98_L41 is referred to as "hMAb98_H3L41".
  • Example 5 Preparation of humanized anti-HTLV-1 antibody 5) -1 Construction of light chain expression vector for humanized anti-HTLV-1 antibody 5) -1-1 Construction of hMAb1_L11 expression vector Nucleotide sequence of hMAb1_L11 shown in SEQ ID NO: 31. The DNA fragments shown in 37 to 420 were synthesized (manufactured by GENEART). An hMAb1_L11 expression vector was constructed in the same manner as in Example 3) -5-1.
  • hMAb1_L12 expression vector The DNA fragment shown in nucleotide numbers 37 to 420 of the nucleotide sequence of hMAb1_L12 shown in SEQ ID NO: 33 was synthesized (manufactured by GENEART). An hMAb1_L12 expression vector was constructed in the same manner as in Example 3) -5-1.
  • hMAb1_L13 expression vector The DNA fragment shown in nucleotide numbers 37 to 420 of the nucleotide sequence of hMAb1_L13 shown in SEQ ID NO: 35 was synthesized (manufactured by GENEART). An hMAb1_L13 expression vector was constructed in the same manner as in Example 3) -5-1.
  • hMAb1_L41 expression vector The DNA fragment shown in nucleotide numbers 37 to 420 of the nucleotide sequence of hMAb1_L41 shown in SEQ ID NO: 37 was synthesized (manufactured by GENEART). An hMAb1_L41 expression vector was constructed in the same manner as in Example 3) -5-1.
  • hMAb1_L43 expression vector The DNA fragment shown in nucleotide numbers 37 to 420 of the nucleotide sequence of hMAb1_L43 shown in SEQ ID NO: 41 was synthesized (manufactured by GENEART). An hMAb1_L43 expression vector was constructed in the same manner as in Example 3) -5-1.
  • hMAb1_H1 expression vector The DNA fragment shown in nucleotide numbers 36 to 431 of the nucleotide sequence of hMAb1_H1 shown in SEQ ID NO: 43. Synthesized (manufactured by GENEART). An hMAb1_H1 expression vector was constructed in the same manner as in Example 3) -5-2.
  • hMAb1_H2 expression vector The DNA fragment shown in nucleotide numbers 36 to 431 of the nucleotide sequence of hMAb1_H2 shown in SEQ ID NO: 45 was synthesized (manufactured by GENEART). An hMAb1_H2 expression vector was constructed in the same manner as in Example 3) -5-2.
  • hMAb98_H2 expression vector The DNA fragment shown in nucleotide numbers 36 to 431 of the nucleotide sequence of hMAb98_H2 shown in SEQ ID NO: 55 was synthesized (manufactured by GENEART). An hMAb98_H2 expression vector was constructed in the same manner as in Example 3) -5-2.
  • hMAb98_H3 expression vector The DNA fragment shown in nucleotide numbers 36 to 431 of the nucleotide sequence of hMAb98_H3 shown in SEQ ID NO: 57 was synthesized (manufactured by GENEART). An hMAb98_H3 expression vector was constructed in the same manner as in Example 3) -5-2.
  • Example 5 -4 Small-scale purification of humanized anti-HTLV-1 antibody Using the culture supernatant obtained in Example 5) -3 using a MonoSpin ProA column (manufactured by GL Sciences), load the culture supernatant onto the column according to the manual. It was washed and eluted.
  • Example 5 -5 Evaluation of spore formation inhibitory activity of humanized anti-HTLV-1 antibody Similar to Example 2) -3, it contains 20 U / mL IL-2, 10% FBS, 100 U / mL penicillin, and 100 ⁇ g / mL streptomycin. In the presence of RPMI1640 medium (manufactured by SIGMA-ALDRICH), 25 ⁇ L of YT # 1 cells, which are HTLV-1-infected CD4-positive T cells derived from healthy humans at 2 ⁇ 10 6 cells / mL, and humans prepared in Example 5) -4.
  • RPMI1640 medium manufactured by SIGMA-ALDRICH
  • hMAb1_H1L11 four types of hMAb1_H1L11, hMAb1_H1L12, hMAb1_H1L42, and hMAb1_H1L43 were selected from the degree of substitution of the human sequence and the type of light chain acceptor, and hMAb98_H3L11 and hMAb98 were selected from hMAb98_H3L11 and hMAb98 with the same criteria as hMAb1.
  • hMAb1_H1L43, hMAb98_H3L11, and hMAb98_H3L41 were produced.
  • FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) were subcultured and cultured according to the manual. 1.2 ⁇ 10 9 FreeStyle 293F cells in the logarithmic growth phase were seeded in a 3L Fernbach Erlenmeyer Flask (Corning), diluted with FreeStyle 293 Expression Medium (Thermo Fisher 2.0) SCIEN. Prepared to / mL. 1.8 mg of Polyethylenimine (manufactured by Polyscience) was added to 20 mL of Opti-Pro SFM medium (manufactured by Thermo Fisher SCIENTIFIC).
  • Example 5 -7
  • Purification of humanized anti-HTLV-1 antibody The antibody was purified from the culture supernatant obtained in Example 5) -6 by a two-step step of rProtein A affinity chromatography and ceramic hydroxyapatite. After applying the culture supernatant to a column (manufactured by Cytiva) filled with MabSelect SuRe equilibrated with PBS, the column was washed with PBS at least twice the column volume. The antibody was then eluted with 2ML-arginine hydrochloride solution (pH 4.0).
  • 2ML-arginine hydrochloride solution pH 4.0
  • the fraction containing the antibody was buffer-replaced with PBS by dialysis using Slide-A-Lyzer Dialysis Cassette (manufactured by Thermo Fisher SCIENTIFIC), and diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0. Then, it was applied to Bio-Scale CHT Type-1 Hydroxyapatite Color (manufactured by Nippon Biorad Co., Ltd.) equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. A linear concentration gradient elution with sodium chloride was performed and fractions containing the antibody were collected.
  • the fraction was buffer-substituted with HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis using a Slide-A-Lyzer Diarysis Cassette (manufactured by Thermo Fisher Scientific).
  • Antibodies were concentrated with Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut off 10 kDa, manufactured by Sartorius) to adjust the IgG concentration to 50 mg / mL or higher. Finally, it was filtered through a Minisart-Plus Filter (manufactured by Sartorius) to prepare a purified sample.
  • Example 6 Evaluation of in vitro activity of humanized anti-HTLV-1 antibody 6) -1 Evaluation of cell formation inhibitory activity 20U / mL IL-2, 10% FBS, 100U / mL penicillin, 100 ⁇ g / mL streptomycin-containing RPMI1640 medium (SIGMA-ALDRICH) In the presence of 2 ⁇ 10 6 cells / mL, 25 ⁇ L of HTLV-1-infected CD4-positive T cells YT # 1 cells derived from healthy individuals and a diluted series solution of humanized anti-HTLV-1 antibody (mixed with CEM cells).
  • SIGMA-ALDRICH streptomycin-containing RPMI1640 medium
  • HMAb1_H1L12 After suspending 50 ⁇ L of (4 concentration series of 1.5, 3, 6, 12 ⁇ g / mL) for 5 minutes on a 96-well plate, add 25 ⁇ L of 2 ⁇ 10 6 cells / mL HTLV-1 uninfected Jarkat cells. , 37 ° C., 5% CO 2 for 16 hours. Syncytial formation was confirmed under a microscope. The results are shown in FIG. HMAb1_H1L12, which showed syncytial formation inhibition at a concentration of 3 ⁇ g / mL, was identified as the antibody with the strongest syncytial formation inhibitory activity.
  • the syncytial formation inhibitory activity of hMAb1_H1L12 and LAT-27 was evaluated by the same experimental procedure.
  • the concentration of the diluted series solution was set to 0.4, 0.8, 1.5, 3, 6 ⁇ g / mL.
  • the results are shown in Table 2.
  • LAT-27 showed inhibitory activity only at 6 ⁇ g / mL
  • hMAb1_H1L12 showed inhibitory activity even at a concentration of 0.8 ⁇ g / mL, so hMAb1_H1L12 was stronger than LAT-27. It was confirmed that it has syncytium formation inhibitory activity.
  • T-PBS PBS containing 0.05% Tween
  • 100 ⁇ L of T-PBS containing 1% gelatin was added, and the mixture was allowed to stand at room temperature for 1 hour.
  • 5 ⁇ L of anti-HTLV-1 antibody 50 ⁇ g / ml was added, and the mixture was allowed to stand at room temperature for 2 hours.
  • the antibody was diluted with PBS (abbreviated as AAD) containing 0.2% BSA and 0.05% Tween20.
  • HRP Goat Anti-Rat IgG Minimal X-Reactiveity
  • HRP Donkey Anti-Human IgG Minibily-Human IgG diluted 500-fold with AAD after washing twice with T-PBS.
  • T-PBS Tetra-PBS
  • the color reaction was carried out with occasional stirring, and 50 ⁇ L / well of 2M HCl was added to stop the color reaction, and then the absorbance at 450 nm was measured with a plate reader (manufactured by Bio-Rad).
  • the results are shown in FIG. Nos. 192 to 196 of gp46 are epitopes because no binding activity is observed for the partial peptide having the amino terminus of 193 or later and the binding activity is observed for the partial peptide having the carboxyl terminus of 196 or later. It was confirmed.
  • the measured value of 51Cr released by treating ATL-040 (1-1) cells labeled with 51Cr with Triton X-100 is the Maximum release value
  • the measured value of 51Cr released from the antibody-treated cells to which PBMC is not added is the measured value.
  • As the Spontaneus release value % spectral release was calculated from the following formula and summarized in FIG.
  • a negative control sample a sample treated with human IgG derived from a non-infected person at 10 ⁇ g / ml was similarly measured and shown together. The measurement was carried out in triplicate, and the average value and standard deviation were calculated and shown together.
  • % Specific release (Total release-Spontaneus release) / Maximum release
  • Example 7 In vivo evaluation of anti-HTLV-1 antibody 7) -1 Infection prevention evaluation of humanized anti-HTLV-1 antibody using humanized mice transplanted with peripheral blood mononuclear cells 7) -1-1 HTLV- using flow cytometry Confirmation of expression of 1-derived Tax gene
  • NOD-SCID / ⁇ cmul manufactured by In vivo Science
  • the mice were fed a sterilized solid feed (FR-2, manufactured by Funabashi Farm) and fed with sterilized tap water (5-15 ppm sodium hypochlorite solution was added).
  • Blood mononuclear cells (manufactured by Lonza) were intraperitoneally administered in 0.5 mL increments. Two weeks later, 5 mL of 10% FCS-added RPM11640 medium containing 5 U / mL heparin was injected into the abdominal cavity of NOG mice euthanized with carbon dioxide gas, the mice were shaken up and down, and the abdominal cavity lavage cells were collected with a syringe. .. The obtained cells were washed twice by adding RPMI1640 medium containing 10% FBS, and then placed in RPMI1640 medium containing 50 U / mL IL-2 (manufactured by Milteny) so as to be 1 ⁇ 10 6 cells / mL.
  • the cultured cells were centrifuged at 4 ° C. for 5 minutes at 1200 rpm to remove the supernatant, washed with 200 ⁇ L / well FACS buffer (0.2% BSA, 0.1% PBS containing sodium azide), and then 100 ⁇ L / well.
  • the cells were suspended in PBS containing 1% paraformaldehyde and fixed at room temperature for 10 minutes. Then, the cells were washed with a 200 ⁇ L / well FACS buffer, suspended in a 100 ⁇ L / well 0.5% saponin-containing FACS buffer, and 5 ⁇ L / well PE-labeled anti-human CD4 antibody (OKT-4, manufactured by BioLegend).
  • an anti-HTLV-1 Tax antibody labeled with a 5 ⁇ L / well Hilyte Flow 647 label kit (manufactured by Dojin Kagaku Kenkyusho) was added, and the mixture was allowed to stand at room temperature for 30 minutes. Then, it was washed twice with a 200 ⁇ L / well FACS buffer. It was suspended in PBS containing 300 ⁇ L / well of 1% paraformaldehyde, and measured by flow cytometry FACSCalibur (manufactured by Becton Dickinson). The results are shown in FIG. The Tax antigen was not detected in the Mock cell group and the hMAb1_H1L12 administration group, while the Tax antigen was detected in the control antibody administration group, confirming the prevention of HTLV-1 infection by the anti-HTLV-1 antibody.
  • HTLV-1 provirus using PCR Using QIAamp DNA Mini Kit (manufactured by QIAGEN) from 1 ⁇ 10 6 cells / mL abdominal cavity washed cells recovered by 1-1. And the genomic DNA was extracted. PCR using two primers to detect the pX region of HTLV-1 shown in SEQ ID NOs: 64 and 65, or two primers to detect the ⁇ -actin region for endogenous control shown in SEQ ID NOs: 66 and 67. was done.
  • the obtained PCR product was subjected to agarose gel electrophoresis and stained with GelRed Nucleic Acid Gel Stein (manufactured by Biotium). The results are shown in FIG. Bands in the pX region were not detected in the Mock cells and the hMAb1_H1L12 administration group, but they were detected in the control antibody administration group. Confirmed prevention.
  • HTLV-1 infection can be prevented and diseases caused by HTLV-1 infection, that is, adult T-cell leukemia (ATL), HTLV-1-related spinal cord. It is possible to prevent or treat the onset of illness (HTLV-1 Associated Myelopathy: HAM), HTLV-1-related uveitis (HTLV-1 Associated Uveitis: HU), and the like.
  • ATL adult T-cell leukemia
  • HAM HTLV-1 Associated Myelopathy
  • HTLV-1-related uveitis HTLV-1 Associated Uveitis
  • SEQ ID NO: 1 Amino acid sequence of MAb1 CDRL1 SEQ ID NO: 2: Amino acid sequence of MAb1 CDRL2 SEQ ID NO: 3: Amino acid sequence of MAb1 CDRL3 Amino acid sequence number 4: MAb98 Amino acid sequence of CDRL1 SEQ ID NO: 5: MAb98 Amino acid sequence number 6: MAb98 Amino acid sequence of CDRL3 SEQ ID NO: 7: MAb1 Amino acid sequence of CDRH1 SEQ ID NO: 8: MAb1 Amino acid sequence of CDRH2 SEQ ID NO: 9: MAb1 Amino acid sequence of CDRH3 SEQ ID NO: 10: MAb98 Amino acid sequence of CDRH1 SEQ ID NO: 11: Amino acid sequence of CARH2 No.
  • hMAb1_H2 Amino acid SEQ ID NO: 48: amino acid sequence No. 49 of hMAb98_L11: amino acid sequence No. 50 of hMAb98_L13: amino acid sequence No. 51 of hMAb98_L13: amino acid sequence No. 53 of nucleotide sequence No. 52: hMAb98_L41 encoding hMAb98_L41. : Amino acid sequence No. 54 encoding hMAb98_L43: Amino acid sequence No. 55 of hMAb98_L43: Amino acid sequence No. 56 of hMAb98_H2: Amino acid sequence No.
  • SEQ ID NO: 71 Sequence of gp46 partial peptide (12 residues of 188 to 199) SEQ ID NO: 193 H replaced with Ala SEQ ID NO: 72: sequence of gp46 partial peptide (12 residues of 188 to 199) SEQ ID NO: 73: gp46 partial peptide (12 residues of 188 to 199) in which S of 194 is replaced with Ala SEQ ID NO: 74: gp46 partial peptide (188 to 199) in which N of 195 is replaced with Ala. Sequence of No. 12 residue) Sequence in which L of No. 196 is replaced with Ala All publications, patents and patent applications cited herein are incorporated herein by reference as they are.

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MINEKI SAITO;REIKO TANAKA;HIDEKI FUJII;AKIRA KODAMA;YOSHIAKI TAKAHASHI;TOSHIO MATSUZAKI;HIROSHI TAKASHIMA;YUETSU TANAKA: "The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/?cnull (NOG) mice", RETROVIROLOGY, vol. 11, no. 1, 28 August 2014 (2014-08-28), GB , pages 74, XP021198629, ISSN: 1742-4690, DOI: 10.1186/s12977-014-0074-z *
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TANAKA YUETSU, LEE ZENG, HIROSHI SHIRAKI, HISATOSHI SHIDA , HIDEKI TOZAWA: "Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization", THE JOURNAL OF IMMUNOLGY, vol. 147, no. 1, 1 January 1991 (1991-01-01), pages 354 - 360, XP000608451, ISSN: 0022-1767 *
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