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WO2024253420A1 - Human antibody specifically binding to human trem2 protein, and use thereof - Google Patents

Human antibody specifically binding to human trem2 protein, and use thereof Download PDF

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Publication number
WO2024253420A1
WO2024253420A1 PCT/KR2024/007692 KR2024007692W WO2024253420A1 WO 2024253420 A1 WO2024253420 A1 WO 2024253420A1 KR 2024007692 W KR2024007692 W KR 2024007692W WO 2024253420 A1 WO2024253420 A1 WO 2024253420A1
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seq
amino acid
acid sequence
sequence represented
light chain
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French (fr)
Korean (ko)
Inventor
임현호
황준모
박소라
유빈
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Daegu Gyeongbuk Institute of Science and Technology
Osong Medical Innovation Foundation
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Daegu Gyeongbuk Institute of Science and Technology
Osong Medical Innovation Foundation
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Priority claimed from KR1020240073131A external-priority patent/KR20240174995A/en
Application filed by Daegu Gyeongbuk Institute of Science and Technology, Osong Medical Innovation Foundation filed Critical Daegu Gyeongbuk Institute of Science and Technology
Publication of WO2024253420A1 publication Critical patent/WO2024253420A1/en
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a human antibody that specifically binds to human TREM2 (Triggering receptor expressed on myeloid cells 2) protein and uses thereof.
  • TREM2 Triggering receptor expressed on myeloid cells 2
  • TREM2 Triggering receptor expressed on myeloid cells 2 is a receptor that transmits signals through an adapter protein called DAP12. It is known to regulate myeloid cell function and suppresses inflammatory responses by inhibiting the expression of cytokines, confirming that TREM2 is involved in immune regulation.
  • the TREM2 protein is mainly present in microglia and has been found to play an important role in phagocytosis of dementia target factors, amyloid-beta, processing of damaged neurons, and maintaining immune cell homeostasis.
  • TREM2 is a cell membrane protein, so it is difficult to purify it in its original intact form within the cell while maintaining its original function within the cell as much as possible, and antibodies that can specifically detect TREM2 protein are also insufficiently developed.
  • Phage display technology is a technology that produces a human antibody library and displays it on the surface of a bacteriophage in the form of antibody fragments (Fab, ScFv) to select antibody clones for a specific antigen. It has been suggested that almost all types of human recombinant monoclonal antibodies that specifically react with an antigen can be selected from a single pot antibody library system, which means that if phage display antibody technology is utilized, various antibody fragments (in the form of Fab or ScFv) that can be applied to in vivo diagnosis or treatment can be obtained.
  • Fab antibody fragments
  • An object of the present invention is to provide a human antibody or an antigen-binding fragment thereof that specifically binds to human TREM2 protein.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid molecule, and an isolated cell transformed with the recombinant expression vector.
  • Another object of the present invention is to provide a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an effective ingredient.
  • Another object of the present invention is to provide a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention comprises a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 8, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 9, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence represented by SEQ ID NO: 20, and an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1,
  • the present invention also provides a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof.
  • the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
  • the present invention provides an isolated cell transformed with the recombinant expression vector.
  • the present invention provides a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention relates to a human antibody specifically binding to human TREM2 protein and a use thereof, and a human monoclonal antibody specifically binding to TREM2 was discovered through a bio-panning technique using a human synthetic antibody phage display library. The antigen-binding ability and biological activity of the discovered antibody were confirmed.
  • the present invention enables the development of an antibody-based diagnostic system capable of diagnosing TREM2 as a biomarker for various Alzheimer's dementias because it has high affinity for TREM2.
  • most of the existing Alzheimer's treatment drugs developed are symptom alleviators due to temporary regulation of neurotransmitters, it is thought that the development of an antibody targeting TREM2 will lead to the development of a promising new drug with a practical therapeutic effect.
  • Figure 1 shows the results of screening scFvs that bind to human TREM2 protein through phage display panning.
  • FIG. 2 shows the results of SDS-PAGE gel analysis and SE-HPLC analysis of TREM2 binding antibody protein.
  • Figure 3 shows the results of evaluating the binding affinity of TREM2-binding antibodies to human TREM2.
  • Figure 4 shows the results of the efficacy verification of human antibodies.
  • the present invention relates to a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 8, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 9, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence represented by SEQ ID NO: 20, and an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 6, an amino
  • a heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 23 and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence represented by SEQ ID NO: 36, an amino acid sequence represented by SEQ ID NO: 38, and an amino acid sequence represented by SEQ ID NO: 42, a light chain CDR1 selected from the group consisting of a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, an amino acid sequence
  • the human antibody or antigen-binding fragment thereof that specifically binds to the human TREM2 protein comprises: 1) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 9, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 10, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 31, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 11, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 14, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 15, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 36, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 16, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 17, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 38, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 18, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 20, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented
  • a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7;
  • a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid
  • the present invention may be, but is not limited to, a human antibody or an antigen-binding fragment thereof, characterized in that it comprises a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30.
  • the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and examples thereof may include a monoclonal antibody, a polyclonal antibody, a full-length antibody, and an antibody fragment.
  • the term “antibody” may include a bivalent or dual specific molecule (e.g., a bispecific antibody), a diabody, a triabody, or a tetrabody.
  • the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit single binding affinity and binding specificity for a specific epitope, unlike polyclonal antibodies that can bind to multiple epitopes.
  • the term “full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types and has gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1), and alpha 2 ( ⁇ 2) as subclasses.
  • the light chain constant region has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.
  • human antibody means a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody, including the complementarity determining region and the structural region, are composed of the amino acid sequence of human immunoglobulin.
  • Human antibodies are commonly used in the treatment of human diseases, and may have at least three potential advantages. First, they can interact better with the human immune system, and thus can more efficiently destroy target cells, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, there is an advantage in that the human immune system does not recognize the antibody as foreign. Third, there is an advantage in that the half-life in the human circulation is similar to that of naturally occurring antibodies, even when a smaller amount or less frequent administration of the drug is performed.
  • the term “heavy chain” may include both a full-length heavy chain and fragments thereof, comprising a variable region VH and three constant regions CH1, CH2 and CH3, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • the term “light chain” in the present invention may include both a full-length light chain and fragments thereof, comprising a variable region VL and a constant region CL, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • fragment In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody.
  • exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.
  • the antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the antibodies described herein, but also biological equivalents thereof, to the extent that they can exhibit the ability to specifically bind to human TREM2.
  • additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody.
  • Such changes may include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody.
  • Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Accordingly, based on this advantage, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
  • the present invention also provides a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof.
  • nucleic acid molecule has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified.
  • sequence of a nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.
  • the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
  • vector means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).
  • the “expression vector” means a recombinant DNA molecule including a target coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism.
  • the expression vector may preferably include one or more selectable markers.
  • the markers are nucleic acid sequences having characteristics that can be selected, typically by a chemical method, and include all genes that can distinguish transformed cells from non-transformed cells. Examples thereof include, but are not limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol, and can be appropriately selected by those skilled in the art.
  • any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the promoter and enhancer of CMV, the LTR of retroviruses, the lac system, the trp system, the TAC or TRC systems, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of such phosphatases, e.g. Pho5, the promoter of the yeast alpha-mating system, and any other sequence of structure and inducibility known to control the expression of genes of prokaryotes or eukaryotes or their viruses, and various combinations thereof.
  • the vector expressing the antibody of the present invention can be a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation.
  • Co-transformation is a method in which each vector DNA encoding the light chain and the heavy chain is simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected.
  • Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), to finally select cells expressing both the light chain and the heavy chain.
  • the present invention provides an isolated cell transformed with a recombinant expression vector.
  • the cell capable of stably and continuously cloning and expressing the vector of the present invention can be any host cell known in the art, including but not limited to, prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylocus carnosus).
  • prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylocus carnosus).
  • the culture of transformed cells can be carried out according to appropriate media and culture conditions known in the relevant technical field.
  • Such culture process can be easily adjusted and used by a person skilled in the art according to the selected strain.
  • Cell culture is divided into suspension culture and attachment culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method.
  • the medium used for culture must appropriately satisfy the requirements of a specific strain.
  • the present invention provides a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.
  • Panning against human TREM2 protein was performed using synthetic human antibody phage display library (KFab-I, KFab-II, KscFv-I).
  • Human TREM2 protein (10 ⁇ g/ml) was immobilized in an immunotube, and ⁇ 1.0 ⁇ 10 ⁇ 12 cfu/ml of phage library was added. After binding at 37°C and the reaction was completed, unbound phage was removed by washing three times with phosphate-buffered saline (PBST) containing 0.05% Tween-20.
  • PBST phosphate-buffered saline
  • the immunotube was treated with 1 mL of 100 mM trimethylamine for 10 minutes to harvest (elution) phage bound to human TREM2 protein.
  • a PBS control panning without immobilized human TREM2 protein was also performed in parallel, and the output titers of each round were compared, and the results are shown in Fig. 1 through the elution titer ratio (the value obtained by dividing the output titer by the output titer of the control group).
  • the phage titer (the number of infectious particles contained in the phage solution) increased by at least 1,000-fold and up to 15,000-fold compared to the control group.
  • a monoclonal phage ELISA and a soluble ELISA using the periplasmic fraction containing Fab and scFv through osmotic shock were performed on the clones obtained in the 3rd or 4th round of panning.
  • the completed panning round clones were used to prepare for phage-antibody elution.
  • the phage ELISA was performed on a 96-well ELISA plate coated with 2 ⁇ g/ml of human TREM2 protein, and after reacting with anti-M13-HRP (1:5,000) antibody, the color was developed by treating with TMB (3,3', 5,5'-Tetramethylbenzidine) substrate, and the reaction was stopped by treating with 2N H 2 SO 4 .
  • TMB 3,3', 5,5'-Tetramethylbenzidine
  • the periplasmic fraction containing Fab and scFv was secured by the osmotic shock method using TES solution (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0).
  • the soluble ELISA was performed on a 96-well ELISA plate coated with 2 ⁇ g/ml of human TREM2 protein, and the antibody anti-VK-HRP (1:10,000) was reacted, followed by treatment with TMB substrate to develop the color, and then the reaction was stopped by treatment with 2N H 2 SO 4 .
  • the absorbance (A450 nm) of the phage-antibody was more than twice the absorbance (A450 nm) of the periplasmic fraction containing Fab and scFv for human TREM2 protein, the clones were considered to bind specifically, and the corresponding clones were finally selected.
  • variable region sequences for eight new human antibodies and cloned them into the animal cell expression vector pcDNATM 3.4 TOPO vector (ThermoFisher Scientific, Cat. A14697) together with constant region sequences (P01857, P01834).
  • each plasmid DNA was transfected into Expi293 cells as described in the Thermo Fisher manufacturer's protocol (ExpiFectamine 293 transfection kit, ThermoFisher Scientific). Briefly, 6 ⁇ 10 6 cells/ml Expi293 cells were transfected with DNA and ExpiFectamine complexes and cultured at 37°C under 8% CO 2 conditions, and the expression enhancer was added 24 h later. On the second day after transfection, the cells were cultured at 32°C for 6 days, and the culture supernatant containing TREM2-binding antibody proteins was collected.
  • a Protein A affinity chromatography column (GE Healthcare) was equilibrated with PBS (pH 7.4). The culture supernatant containing each TREM2-binding antibody protein was filtered through a 0.2 ⁇ m filter and loaded onto the Protein A affinity chromatography column. After washing the column with PBS (pH 7.4), the TREM2-binding antibody protein was eluted with a 100 mM glycine (pH 3.4) solution. The eluate was neutralized by adding 1 M Tris solution and the buffer was exchanged with PBS.
  • TREM2-binding antibody was confirmed by SDS-PAGE gel analysis (Invitrogen) and SE-HPLC analysis, and the protein concentration was quantified using a Nanodrop 2000C spectrophotometer (Thermo Scientific). Except for TREM2-3-B6, which had a monomer ratio of 88.57% based on SE-HPLC, the remaining seven antibodies were confirmed to have a monomer ratio of more than 90% (Fig. 2).
  • the binding affinity to the antigen was measured as the K D value for each antibody using Biacore.
  • the system and sample buffer used were HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Polysorbate 20 (v/v)).
  • the antigen tagged with His was immobilized on the surface of the CM5 chip by performing the amine coupling procedure according to the manufacturer's protocol of the His capture kit (Cytiva).
  • the antibody protein diluted in HBS-EP buffer was flowed through the CM5 chip with the antibody immobilized at a flow rate of 30 ⁇ L/min for 2 minutes for binding and 5 minutes for dissociation.
  • the antibody protein bound to the antigen was washed by reacting with 10 mM glycine-HCl (pH 1.5) for 60 seconds.
  • the sensorgrams of the antibodies were analyzed using Biacore evaluation software. As a result, it was confirmed that all eight types bound to the human TREM2 antigen protein at sub-nanomolar levels or higher (Fig. 3).
  • TREM2 loses its function when the outer part of the cell membrane is cleaved by sheddase such as ADAM10/17, and at the same time, the amount of cleaved soluble TREM2 (sTREM2) present in the cell culture medium increases.
  • the antibody specified in the present invention can inhibit shedding by binding to TREM2 exposed to the outer part of the cell membrane, and this protective effect was evaluated as a decrease in the amount of sTREM2 present in the culture medium and internal TREM2 (iTREM2) present in the cell, and an increase in the protein amount of membrane TREM2 (mTREM2).
  • HEK2393T cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) were transfected with polyethylenimine (PEI) to express human TREM2-His and DAP12-V5. After culturing for 8 hours, the cells were replaced with fresh culture medium containing the control (C2 and human IgG produced from mouse) or each human antibody at a concentration of 20 ug/mL, and further cultured for 16 hours.
  • pure culture medium and cell fraction samples total, internal, membrane fraction
  • the whole cell fraction was prepared by dissolving the cells obtained by centrifugation in the lysate (1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC)), rotating them in a refrigerator (4°C) for 1 hour, and then centrifuging them to remove the remaining debris to secure all the intracellular and cell membrane fraction proteins.
  • lysate 1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC)
  • the intracellular fraction was prepared by dissolving the cells obtained by centrifugation in a storage solution (20 mM Tris, pH 7.0, 1 mM EDTA/EGTA, 1X PIC), keeping them on ice for 30 minutes to fill the inside of the cells with the buffer, and then freezing them in an ultra-low temperature freezer (-80°C) and thawing them to rupture the cells, followed by centrifugation to separate the intracellular fraction from the cell membrane fraction.
  • the proteins of the remaining cell membrane fraction were eluted from the cell membrane using the lysate, and then separated/secured from the debris through centrifugation.
  • PVDF polyvinylidene difluoride
  • B2, B7, and E10 showed excellent inhibitory effects compared to the shedding inhibition of mouse-derived antibody C2, and B1 and F3 showed inhibitory effects similar to the mouse-derived antibody C2.
  • B1 and F3 showed inhibitory effects similar to the mouse-derived antibody C2.
  • A9, G11, and H4 showed somewhat reduced inhibitory effects compared to the shedding inhibition of mouse-derived antibody C2 (Table 2).

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Abstract

The present invention relates to a human antibody specifically binding to a human TREM2 protein, and use thereof. The human monoclonal antibody specifically binding to TREM2 has been discovered through biopanning using a human synthetic antibody phage display library, and the antigen binding ability and biological activity of the discovered antibody have been identified. The present invention has a high affinity for TREM2, and thus enables the development of an antibody-based diagnosis system capable of diagnosing TREM2 as a biomarker of various Alzheimer's dementia. In addition, since a conventional therapeutic agent for Alzheimer's disease is mostly a symptom reliever through the temporary regulation of neurotransmitters, it is considered that the development of antibodies targeting TREM2 can lead to the development of promising new drugs having substantial therapeutic effects.

Description

인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 및 이의 용도Human antibodies specifically binding to human TREM2 protein and uses thereof

본 발명은 인간 TREM2(Triggering receptor expressed on myeloid cells 2) 단백질에 특이적으로 결합하는 인간 항체 및 이의 용도에 대한 것이다. The present invention relates to a human antibody that specifically binds to human TREM2 (Triggering receptor expressed on myeloid cells 2) protein and uses thereof.

TREM2(Triggering receptor expressed on myeloid cells 2)는 DAP12라는 어댑터 단백질을 통해 신호전달을 수행하는 수용체로서 골수 세포(myeloid cell) 기능을 조절하는 것으로 알려져 있으며, 사이토카인의 발현을 억제하여 염증반응을 억제하는 것으로 나타나 TREM2가 면역조절에 관여하는 것으로 확인되었다. TREM2 단백질은 주로 신경아교세포(microglia)에 존재하며 치매 표적인자, 아밀로이드- 베타의 식세포작용과 손상된 신경세포 처리 및 면역세포 항상성 유지와 같은 중요한 역할을 하는 것으로 밝혀져 있다.TREM2 (Triggering receptor expressed on myeloid cells 2) is a receptor that transmits signals through an adapter protein called DAP12. It is known to regulate myeloid cell function and suppresses inflammatory responses by inhibiting the expression of cytokines, confirming that TREM2 is involved in immune regulation. The TREM2 protein is mainly present in microglia and has been found to play an important role in phagocytosis of dementia target factors, amyloid-beta, processing of damaged neurons, and maintaining immune cell homeostasis.

최근에는 TREM2의 돌연변이체가 알츠하이머 발병율을 3배 증진시킨다는 연구 보고가 발표되면서 알츠하이머성 치매와 같은 뇌질환 연구를 위한 바이오 마커 또는 새로운 뇌질환 치료제 타겟으로 더욱 부각되고 있다. 그러나 TREM2는 세포막 단백질로서 세포 내 고유의 기능을 최대한 그대로 유지하면서 세포 내 고유의 온전한 형태로의 정제가 어려울 뿐만 아니라 TREM2 단백질을 특이적으로 검출할 수 있는 항체 또한 개발이 미비한 실정이다.Recently, as a research report was published that a TREM2 mutant increases the incidence of Alzheimer's disease by three times, it is gaining more attention as a biomarker for studying brain diseases such as Alzheimer's disease or as a target for new brain disease treatment. However, TREM2 is a cell membrane protein, so it is difficult to purify it in its original intact form within the cell while maintaining its original function within the cell as much as possible, and antibodies that can specifically detect TREM2 protein are also insufficiently developed.

파지 디스플레이 기술은 인간 항체 라이브러리(library)를 제조하여 항체 절편(Fab, ScFv)의 형태로 박테리오파지의 표면에 발현시켜 특정 항원에 대한 항체 클론들을 선별하는 기술이다. 항원과 특이적으로 반응하는 거의 모든 종류의 인간 재조합 단일 클론 항체를 단일 pot 항체 라이브러리 시스템으로부터 선별해낼 수 있는 가능성이 제안되었으며, 이는 파지 디스플레이 항체 기술을 활용할 경우 체내 진단이나 치료에 응용할 수 있는 다양한 항체 단편들(Fab 또는 ScFv 형태)을 획득할 수 있다.Phage display technology is a technology that produces a human antibody library and displays it on the surface of a bacteriophage in the form of antibody fragments (Fab, ScFv) to select antibody clones for a specific antigen. It has been suggested that almost all types of human recombinant monoclonal antibodies that specifically react with an antigen can be selected from a single pot antibody library system, which means that if phage display antibody technology is utilized, various antibody fragments (in the form of Fab or ScFv) that can be applied to in vivo diagnosis or treatment can be obtained.

본 발명의 목적은 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편을 제공하는 데에 있다.An object of the present invention is to provide a human antibody or an antigen-binding fragment thereof that specifically binds to human TREM2 protein.

본 발명의 다른 목적은 상기 인간 항체 또는 이의 항원 결합 단편을 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 재조합 발현벡터 및 상기 재조합 발현벡터로 형질전환되어 분리된 세포를 제공하는 데에 있다. Another object of the present invention is to provide a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid molecule, and an isolated cell transformed with the recombinant expression vector.

본 발명의 또 다른 목적은 상기 인간 상기 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 인간 TREM2 항원 검출용 조성물을 제공하는 데에 있다. Another object of the present invention is to provide a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an effective ingredient.

본 발명의 또 다른 목적은 상기 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 알츠하이머병 진단용 조성물을 제공하는 데에 있다. Another object of the present invention is to provide a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.

상기 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열, 서열번호 8로 표시되는 아미노산 서열 및 서열번호 13으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 4로 표시되는 아미노산 서열, 서열번호 9로 표시되는 아미노산 서열, 서열번호 11로 표시되는 아미노산 서열, 서열번호 14로 표시되는 아미노산 서열, 서열번호 16으로 표시되는 아미노산 서열, 서열번호 18로 표시되는 아미노산 서열, 서열번호 20으로 표시되는 아미노산 서열 및 서열번호 22로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 10으로 표시되는 아미노산 서열, 서열번호 12로 표시되는 아미노산 서열, 서열번호 15로 표시되는 아미노산 서열, 서열번호 17로 표시되는 아미노산 서열, 서열번호 19로 표시되는 아미노산 서열, 서열번호 21로 표시되는 아미노산 서열 및 서열번호 23으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열, 서열번호 31로 표시되는 아미노산 서열, 서열번호 36으로 표시되는 아미노산 서열, 서열번호 38로 표시되는 아미노산 서열 및 서열번호 42로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 27로 표시되는 아미노산 서열, 서열번호 32로 표시되는 아미노산 서열 및 서열번호 34로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 29로 표시되는 아미노산 서열, 서열번호 33으로 표시되는 아미노산 서열, 서열번호 35로 표시되는 아미노산 서열, 서열번호 37로 표시되는 아미노산 서열, 서열번호 39로 표시되는 아미노산 서열, 서열번호 40으로 표시되는 아미노산 서열 및 서열번호 41로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하는, 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편을 제공한다.In order to achieve the above object, the present invention comprises a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 8, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 9, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence represented by SEQ ID NO: 20, and an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 10, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 17 ...2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: A heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 19, an amino acid sequence represented by SEQ ID NO: 21, and an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence represented by SEQ ID NO: 36, an amino acid sequence represented by SEQ ID NO: 38, and an amino acid sequence represented by SEQ ID NO: 42, a light chain CDR1 selected from the group consisting of a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, an amino acid sequence represented by SEQ ID NO: 29, an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 40, and an amino acid sequence represented by SEQ ID NO: 41, and a light chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 29, an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 40, and an amino acid sequence represented A human antibody or antigen-binding fragment thereof that specifically binds to human TREM2 protein is provided, comprising a light chain variable region comprising a light chain FR4 consisting of an amino acid sequence represented by 30.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.The present invention also provides a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof.

또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.Additionally, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

또한, 본 발명은 상기 재조합 발현벡터로 형질전환되어 분리된 세포를 제공한다.In addition, the present invention provides an isolated cell transformed with the recombinant expression vector.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 인간 TREM2 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 알츠하이머병 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.

본 발명은 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 및 이의 용도에 관한 것으로서, 인간 합성 항체 파지 디스플레이 라이브러리를 이용한 바이오 패닝 기법을 통해 TREM2에 특이적으로 결합하는 인간 단일클론 항체를 발굴하였고. 발굴한 항체의 항원 결합능과 생물학적 활성을 확인하였다. 본 발명은 TREM2에 대한 고친화도를 가지고 있어 다양한 알츠하이머성 치매의 바이오마커로서 TREM2를 진단할 수 있는 항체 기반 진단 시스템 개발이 가능하다. 또한, 기존 알츠하이머 치료제 개발이 신경전달 물질의 일시적 조절로 인한 증상 완화제가 대부분이므로, TREM2를 타겟팅하는 항체를 개발함으로써 실질적으로 치료효과를 갖는 유망한 신약개발로 이어질 수 있을 것이라고 사료된다.The present invention relates to a human antibody specifically binding to human TREM2 protein and a use thereof, and a human monoclonal antibody specifically binding to TREM2 was discovered through a bio-panning technique using a human synthetic antibody phage display library. The antigen-binding ability and biological activity of the discovered antibody were confirmed. The present invention enables the development of an antibody-based diagnostic system capable of diagnosing TREM2 as a biomarker for various Alzheimer's dementias because it has high affinity for TREM2. In addition, since most of the existing Alzheimer's treatment drugs developed are symptom alleviators due to temporary regulation of neurotransmitters, it is thought that the development of an antibody targeting TREM2 will lead to the development of a promising new drug with a practical therapeutic effect.

도 1은 파지 디스플레이 패닝 (panning)을 통한 인간 TREM2 단백질과 결합하는 scFv 선별 결과를 나타낸다Figure 1 shows the results of screening scFvs that bind to human TREM2 protein through phage display panning.

도 2는 TREM2 결합 항체 단백질의 SDS-PAGE gel 분석 및 SE-HPLC 분석 결과를 나타낸다Figure 2 shows the results of SDS-PAGE gel analysis and SE-HPLC analysis of TREM2 binding antibody protein.

도 3은 TREM2 결합 항체의 인간 TREM2에 대한 결합력 평가 결과를 나타낸다.Figure 3 shows the results of evaluating the binding affinity of TREM2-binding antibodies to human TREM2.

도 4는 인간 항체의 효능 검증 결과를 나타낸다.Figure 4 shows the results of the efficacy verification of human antibodies.

본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열, 서열번호 8로 표시되는 아미노산 서열 및 서열번호 13으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 4로 표시되는 아미노산 서열, 서열번호 9로 표시되는 아미노산 서열, 서열번호 11로 표시되는 아미노산 서열, 서열번호 14로 표시되는 아미노산 서열, 서열번호 16으로 표시되는 아미노산 서열, 서열번호 18로 표시되는 아미노산 서열, 서열번호 20으로 표시되는 아미노산 서열 및 서열번호 22로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 10으로 표시되는 아미노산 서열, 서열번호 12로 표시되는 아미노산 서열, 서열번호 15로 표시되는 아미노산 서열, 서열번호 17로 표시되는 아미노산 서열, 서열번호 19로 표시되는 아미노산 서열, 서열번호 21로 표시되는 아미노산 서열 및 서열번호 23으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열, 서열번호 31로 표시되는 아미노산 서열, 서열번호 36으로 표시되는 아미노산 서열, 서열번호 38로 표시되는 아미노산 서열 및 서열번호 42로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 27로 표시되는 아미노산 서열, 서열번호 32로 표시되는 아미노산 서열 및 서열번호 34로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 29로 표시되는 아미노산 서열, 서열번호 33으로 표시되는 아미노산 서열, 서열번호 35로 표시되는 아미노산 서열, 서열번호 37로 표시되는 아미노산 서열, 서열번호 39로 표시되는 아미노산 서열, 서열번호 40으로 표시되는 아미노산 서열 및 서열번호 41로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하는, 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편을 제공한다.The present invention relates to a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 8, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 9, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence represented by SEQ ID NO: 20, and an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 10, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 17, and an amino acid sequence represented by SEQ ID NO: 19. A heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 23 and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence represented by SEQ ID NO: 36, an amino acid sequence represented by SEQ ID NO: 38, and an amino acid sequence represented by SEQ ID NO: 42, a light chain CDR1 selected from the group consisting of a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, an amino acid sequence represented by SEQ ID NO: 29, an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 40, and an amino acid sequence represented by SEQ ID NO: 41, and a light chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 26, an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 29, an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 40, and an amino acid sequence represented A human antibody or antigen-binding fragment thereof that specifically binds to human TREM2 protein is provided, comprising a light chain variable region comprising a light chain FR4 consisting of an amino acid sequence represented by 30.

바람직하게는, 상기 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편은, 1) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 27로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 29로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,Preferably, the human antibody or antigen-binding fragment thereof that specifically binds to the human TREM2 protein comprises: 1) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 29, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or

2) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 9로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 10으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 31로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 33으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,2) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 9, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 10, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 31, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or

3) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 11로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 12로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 35로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,3) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 11, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or

4) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 13로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 14로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 15로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 36으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 37로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,4) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 14, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 15, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 36, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 37, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30, or

5) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 16으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 17로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 38로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 39로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,5) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 16, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 17, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 38, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or

6) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 18로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 19로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 40으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,6) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 18, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or

7) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 20으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 21로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 41로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,7) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 20, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 41, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30, or

8) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 22로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 23으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 42로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 35로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하는 것을 특징으로 하는 인간 항체 또는 이의 항원 결합 단편일 수 있으나, 이에 한정되는 것은 아니다.8) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; And a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30. The present invention may be, but is not limited to, a human antibody or an antigen-binding fragment thereof, characterized in that it comprises a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30.

본 발명에서 용어, “항체”는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하는, 항원을 특이적으로 인식하는 수용체 역할을 하는 단백질 분자를 의미하며, 그 예로, 단일 클론 항체, 다클론 항체, 전장항체(full-length antibody) 및 항체 단편을 모두 포함할 수 있다. 또한 상기 용어, “항체”는 이가(bivalent) 또는 이중 특이성 분자(예컨대, 이중특이성 항체), 디아바디, 트리아바디 또는 테트라바디를 포함할 수 있다.As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and examples thereof may include a monoclonal antibody, a polyclonal antibody, a full-length antibody, and an antibody fragment. In addition, the term “antibody” may include a bivalent or dual specific molecule (e.g., a bispecific antibody), a diabody, a triabody, or a tetrabody.

본 발명에서 용어, “단일 클론 항체”는 실질적으로 동일한 항체 집단에서 수득한 단일 분자 조성의 항체 분자를 지칭하고, 이러한 단일 클론 항체는 다클론 항체가 여러 개의 에피토프에 결합할 수 있는 것과 달리, 특정 에피토프에 대해 단일 결합성 및 친화도를 나타낸다. 본 발명에서 용어, “전장항체”는 2 개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다. IgG는 서브타입(subtype)으로, IgG1, IgG2, IgG3 및 IgG4를 포함한다.As used herein, the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit single binding affinity and binding specificity for a specific epitope, unlike polyclonal antibodies that can bind to multiple epitopes. As used herein, the term “full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) as subclasses. The light chain constant region has kappa (κ) and lambda (λ) types. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

본 발명에서 용어, "인간 항체"는 인간 면역글로불린으로부터 유래하는 분자로서, 상보성 결정영역, 구조 영역을 포함한 항체를 구성하는 모든 아미노산 서열 전체가 인간 면역글로불린의 아미노산 서열로 구성되어 있는 것을 의미한다. 인간 항체는 통상적으로 인간의 질병의 치료에 사용되는데, 이는 3가지 이상의 잠재적인 장점을 가질 수 있다. 먼저, 이는 인간 면역 체계와 보다 양호하게 상호작용하여, 예를 들어 보체-의존성 세포독성(complement-dependent cytotoxicity, CDC) 또는 항체-의존성 세포성 세포독성(antibody-dependent cell-mediated cytotoxicity, ADCC)에 의하여 목적 세포를 보다 효율적으로 파괴시킬 수 있다. 둘째로, 인간 면역 체계가 상기 항체를 외래의 것으로 인식하지 않는 이점이 있다. 셋째로, 더 적은 양, 보다 적은 빈도의 약물을 투여하였을 때에도 인간 순환계 내 반감기가 천연 발생 항체와 유사하다는 이점이 있다.In the present invention, the term "human antibody" means a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody, including the complementarity determining region and the structural region, are composed of the amino acid sequence of human immunoglobulin. Human antibodies are commonly used in the treatment of human diseases, and may have at least three potential advantages. First, they can interact better with the human immune system, and thus can more efficiently destroy target cells, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, there is an advantage in that the human immune system does not recognize the antibody as foreign. Third, there is an advantage in that the half-life in the human circulation is similar to that of naturally occurring antibodies, even when a smaller amount or less frequent administration of the drug is performed.

본 발명에서 용어, “중쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VH 및 3 개의 불변 영역 CH1, CH2 및 CH3를 포함하는 전체길이 중쇄 및 이의 단편을 모두 포함할 수 있다. 또한, 본 발명에서 용어, “경쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VL 및 불변 영역 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 포함할 수 있다.In the present invention, the term “heavy chain” may include both a full-length heavy chain and fragments thereof, comprising a variable region VH and three constant regions CH1, CH2 and CH3, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. In addition, the term “light chain” in the present invention may include both a full-length light chain and fragments thereof, comprising a variable region VL and a constant region CL, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

본 발명에서 용어, “단편”, “항체 단편” 및 “항원 결합 단편”은 항체의 항원결합 기능을 보유하는 본 발명의 항체의 임의의 단편을 지칭하는 것으로 호환적으로 사용된다. 예시적인 항원 결합 단편은 Fab, Fab', F(ab')2 및 Fv 등을 포함하나, 이에 제한되지 않는다.In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

본 발명의 항체 또는 이의 항원 결합 단편은 인간 TREM2에 특이적으로 결합하는 능력을 나타낼 수 있는 범위 내에서, 본 명세서에 기재된 항체의 서열뿐만 아니라, 이의 생물학적 균등물도 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 예를 들어, 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 리신과 히스티딘은 모두 양전하를 띈 잔기이고; 알라닌, 글리신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 티로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이점에 기초하여, 아르기닌, 라신과 히스티딘; 알라닌, 글리신과 세린; 그리고 페닐알라닌, 트립토판과 티로신은 생물학적으로 기능 균등물이라 할 수 있다.The antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the antibodies described herein, but also biological equivalents thereof, to the extent that they can exhibit the ability to specifically bind to human TREM2. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such changes may include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of the amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Accordingly, based on this advantage, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.The present invention also provides a nucleic acid molecule encoding the human antibody or an antigen-binding fragment thereof.

본 명세서에서 사용되는 용어, “핵산 분자”는 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성단위인 뉴클레오티드는 자연의 뉴클레오티드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명의 중쇄 및 경쇄 가변 영역을 코딩하는 핵산 분자의 서열은 변형될 수 있으며, 상기 변형은 뉴클레오티드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.As used herein, the term “nucleic acid molecule” has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified. The sequence of a nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.Additionally, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

본 발명에 있어서, "벡터"는 클론유전자(또는 클론 DNA의 다른 조각)를 운반하는데 사용되는 스스로 복제되는 DNA 분자를 의미한다.In the present invention, "vector" means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

본 발명에서 있어서, “발현 벡터”는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질 전환된 세포를 비 형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 앰피실린(Ampicillin), 카나마이신(Kanamycin), 제네티신(Geneticin; G418), 블레오마이신(Bleomycin), 하이그로마이신(Hygromycin), 클로람페니콜(Chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니며, 당업자에 의해 적절히 선택 가능하다.In the present invention, the “expression vector” means a recombinant DNA molecule including a target coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism. The expression vector may preferably include one or more selectable markers. The markers are nucleic acid sequences having characteristics that can be selected, typically by a chemical method, and include all genes that can distinguish transformed cells from non-transformed cells. Examples thereof include, but are not limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol, and can be appropriately selected by those skilled in the art.

본 발명의 DNA 서열을 발현시키기 위하여, 매우 다양한 발현 조절 서열 중 어느 것이라도 벡터에 사용될 수 있다. 유용한 발현 조절서열의 예에는, 예를 들어, SV40 또는 아데노바이러스의 초기 및 후기 프로모터들, CMV의 프로모터와 인핸서, 레트로바이러스의 LTR, lac 시스템, trp 시스템, TAC 또는 TRC 시스템, T3 및 T7 프로모터들, 파지 람다의 주요 오퍼레이터 및 프로모터 영역, fd 코드 단백질의 조절 영역, 3-포스포글리세레이트 키나제 또는 다른 글리콜분해 효소에 대한 프로모터, 상기 포스파타제의 프로모터들, 예를 들어 Pho5, 효모 알파-교배 시스템의 프로모터 및 원핵세포 또는 진핵 세포 또는 이들의 바이러스의 유전자의 발현을 조절하는 것으로 알려진 구성과 유도의 기타 다른 서열 및 이들의 여러 조합이 포함될 수 있다.Any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the promoter and enhancer of CMV, the LTR of retroviruses, the lac system, the trp system, the TAC or TRC systems, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of such phosphatases, e.g. Pho5, the promoter of the yeast alpha-mating system, and any other sequence of structure and inducibility known to control the expression of genes of prokaryotes or eukaryotes or their viruses, and various combinations thereof.

본 발명의 항체를 발현하는 벡터는, 경쇄와 중쇄가 하나의 벡터에서 동시에 발현되는 벡터 시스템이거나 또는 경쇄와 중쇄를 각각 별도의 벡터에서 발현시키는 시스템 모두 가능하다. 후자의 경우, 두 벡터는 동시 형질전환(co-transfomation) 및 표적 형질전환(targeted transformation)을 통하여 숙주세포로 도입된다. 동시 형질전환은 경쇄 및 중쇄를 코딩하는 각각의 벡터 DNA를 동시에 숙주세포로 도입한 뒤 경쇄와 중쇄를 모두 발현하는 세포를 선별하는 방법이다. 표적 형질전환은 경쇄(또는 중쇄)를 포함하는 벡터로 형질전환 된 세포를 선별하고 경쇄를 발현하는 선별된 세포를 중쇄(또는 경쇄)를 포함하는 벡터로 다시 형질전환 하여 경쇄 및 중쇄 모두를 발현하는 세포를 최종적으로 선별하는 방법이다.The vector expressing the antibody of the present invention can be a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which each vector DNA encoding the light chain and the heavy chain is simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), to finally select cells expressing both the light chain and the heavy chain.

또한, 본 발명은 재조합 발현벡터로 형질전환되어 분리된 세포를 제공한다. In addition, the present invention provides an isolated cell transformed with a recombinant expression vector.

본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 세포는 관련 기술 분야에 공지된 임의의 숙주 세포일 수 있으며, 예컨대, 에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주 세포를 포함하나, 이에 제한되는 것은 아니다.The cell capable of stably and continuously cloning and expressing the vector of the present invention can be any host cell known in the art, including but not limited to, prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylocus carnosus).

상기 항체 또는 이의 항원 결합 단편의 제조 방법에서 형질전환 세포의 배양은 관련 기술 분야에 공지된 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양과정은 통상의 기술자라면 선택되는 균주에 따라 용이하게 조정하여 사용할 수 있다. 세포 배양은, 세포의 성장 방식에 따라 현탁배양과 부착배양, 배양방법에 따라 회분식, 유가식 및 연속배양식의 방법으로 구분된다. 배양에 사용되는 배지는 특정한 균주의 요구조건을 적절하게 만족시켜야 한다.In the above method for producing an antibody or an antigen-binding fragment thereof, the culture of transformed cells can be carried out according to appropriate media and culture conditions known in the relevant technical field. Such culture process can be easily adjusted and used by a person skilled in the art according to the selected strain. Cell culture is divided into suspension culture and attachment culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method. The medium used for culture must appropriately satisfy the requirements of a specific strain.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 인간 TREM2 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting human TREM2 antigen comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.

또한, 본 발명은 상기 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 알츠하이머병 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing Alzheimer's disease comprising the human antibody or an antigen-binding fragment thereof as an active ingredient.

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

<실시예 1> TREM2 결합 항체의 제조<Example 1> Preparation of TREM2 binding antibody

1. 파지 디스플레이 1. Phage display 패닝Panning (panning)을 통한 인간 Human through (panning) TREM2TREM2 단백질과 결합하는 scFv 선별Selection of scFv binding to protein

합성 인간항체 파지디스플레이 라이브러리 (KFab-I, KFab-II, KscFv-I)를 이용하여 인간 TREM2 단백질에 대한 패닝을 수행하였다. 인간 TREM2 단백질 (10μg/ml)이 고정된 면역튜브(immunotube)에 ~1.0 × 10^12 cfu/ml의 파지 라이브러리를 첨가한 후, 37℃에서 결합시키고 반응이 종료된 후에는 0.05% tween-20가 포함된 PBS (PBST)로 3회 세척하여 비결합 파지를 제거하였다. 1mL의 100 mM trimethylamine 을 상기 면역튜브에 10분간 처리하여 인간 TREM2 단백질에 결합된 파지를 수확 (elution)하였다. 수확한 파지에 Tris-Glycine buffer (pH 7.5)를 첨가하여 중화 (neutralization)시킨 후, mid-log phase (OD600 = 0.5-1.0) 상태로 배양한 E.coli TG1를 첨가하여 감염되도록 유도하였다. 일부는 산출 적정 (output titration)에 이용하고, 나머지는 배양 후 세포 펠렛을 모아 2-YT 배지 플레이트에 접종하였다. 다음 날 배양된 콜로니를 모아 다음 라운드의 패닝을 위해 파지를 제조하였다. 후속 패닝 라운드가 진행될수록 인간 TREM2 단백질 코팅 농도를 낮추고, PBS-T 를 이용한 증가된 세척 횟수를 적용하여 항원에 특이적 파지를 증폭 및 농축하였다. 인간 TREM2 단백질이 고정되지 않은 PBS 대조군 패닝도 함께 병행하여 각각의 산출 역가 (output titer)를 각 라운드마다 비교함으로써, 용리 역가 비율 (elution titer ratio, 산출 역가를 대조군의 산출 역가로 나눈 값)을 통해 그 결과를 도 1 에 나타내었다. 3, 4 라운드에서 파지 역가 (파지액에 포함된 감염성 입자수)가 대조군에 비하여 최소 1,000배에서 최대 15,000배 가량 증가하였다.Panning against human TREM2 protein was performed using synthetic human antibody phage display library (KFab-I, KFab-II, KscFv-I). Human TREM2 protein (10 μg/ml) was immobilized in an immunotube, and ~1.0 × 10^12 cfu/ml of phage library was added. After binding at 37°C and the reaction was completed, unbound phage was removed by washing three times with phosphate-buffered saline (PBST) containing 0.05% Tween-20. The immunotube was treated with 1 mL of 100 mM trimethylamine for 10 minutes to harvest (elution) phage bound to human TREM2 protein. Tris-Glycine buffer (pH 7.5) was added to the harvested phage to neutralize it, and E. coli TG1 cultured at mid-log phase (OD600 = 0.5-1.0) was added to induce infection. Some were used for output titration, and the rest were collected as cell pellets after culture and inoculated onto 2-YT medium plates. The cultured colonies were collected the next day to prepare phage for the next round of panning. As the subsequent panning rounds progressed, the human TREM2 protein coating concentration was lowered and the number of washes using PBS-T was increased to amplify and concentrate the antigen-specific phage. A PBS control panning without immobilized human TREM2 protein was also performed in parallel, and the output titers of each round were compared, and the results are shown in Fig. 1 through the elution titer ratio (the value obtained by dividing the output titer by the output titer of the control group). In rounds 3 and 4, the phage titer (the number of infectious particles contained in the phage solution) increased by at least 1,000-fold and up to 15,000-fold compared to the control group.

2. 인간 TREM2 단백질에 특이적으로 결합하는 단일클론 파지 항체 선별2. Screening of monoclonal phage antibodies that specifically bind to human TREM2 protein

상기 패닝을 통해 확보한 파지들로부터 인간 TREM2 단백질에 특이적으로 결합하는 단일클론항체를 선별하기 위해, 패닝 3 라운드 또는 4 라운드에서 얻은 클론들을 상대로 단일클론 파지 ELISA (monoclonal phage ELISA)와 삼투압 충격법 (osmotic shock)을 통하여 Fab 및 scFv를 포함하고 있는 주변 세포질 (periplasmic fraction)을 이용한 soluble ELISA를 수행하였다. 완료된 패닝 라운드 클론을 이용하여 파지-항체가 용출되도록 준비하였다. 인간 TREM2 단백질이 2 μg/ml로 코팅된 96well ELISA 플레이트에 상기 파지 ELISA를 수행하였고, Anti-M13-HRP (1:5,000) 항체를 반응시킨 다음 TMB (3,3', 5,5’-Tetramethylbenzidine) 기질을 처리하여 발색시킨 후 2N 농도의 H2SO4를 처리하여 반응을 중지시켰다. 인간 TREM2 단백질에 대해 흡광도 (A450 nm) cut-off를 0.4 이상으로 정하고 음성 대조군인 PBS control에 대해 흡광도 (A450 nm) cut-off를 0.15 이하인 클론들의 경우 특이적으로 결합하는 클론들로 선택하였다.In order to select a monoclonal antibody that specifically binds to human TREM2 protein from the phages obtained through the above panning, a monoclonal phage ELISA and a soluble ELISA using the periplasmic fraction containing Fab and scFv through osmotic shock were performed on the clones obtained in the 3rd or 4th round of panning. The completed panning round clones were used to prepare for phage-antibody elution. The phage ELISA was performed on a 96-well ELISA plate coated with 2 μg/ml of human TREM2 protein, and after reacting with anti-M13-HRP (1:5,000) antibody, the color was developed by treating with TMB (3,3', 5,5'-Tetramethylbenzidine) substrate, and the reaction was stopped by treating with 2N H 2 SO 4 . For human TREM2 protein, the absorbance (A450 nm) cut-off was set to 0.4 or higher, and for the negative control, PBS control, the clones with an absorbance (A450 nm) cut-off of 0.15 or lower were selected as specifically binding clones.

인간 TREM2 단백질에 친화도가 높은 클론들을 선별하기 TES 용액 (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0)을 이용한 삼투압 충격법을 통하여 Fab 및 scFv를 포함하고 있는 주변 세포질 (periplasmic fraction)을 확보하였다. 인간 TREM2 단백질을 2 μg/ml로 코팅된 96well ELISA 플레이트에 상기 soluble ELISA를 수행하였고, Anti-VK-HRP (1:10,000) 항체를 반응시킨 다음 TMB 기질을 처리하여 발색시킨 후 2N 농도의 H2SO4를 처리하여 반응을 중지시켰다. 인간 TREM2 단백질에 대해 Fab 및 scFv를 포함하고 있는 주변 세포질 발현양의 흡광도 (A450 nm) 대비 파지-항체의 흡광도 (A450 nm) 가 2배 이상일 경우 특이적으로 결합하는 것으로 간주하여 해당 클론들을 최종으로 선택하였다. To select clones with high affinity for human TREM2 protein, the periplasmic fraction containing Fab and scFv was secured by the osmotic shock method using TES solution (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0). The soluble ELISA was performed on a 96-well ELISA plate coated with 2 μg/ml of human TREM2 protein, and the antibody anti-VK-HRP (1:10,000) was reacted, followed by treatment with TMB substrate to develop the color, and then the reaction was stopped by treatment with 2N H 2 SO 4 . When the absorbance (A450 nm) of the phage-antibody was more than twice the absorbance (A450 nm) of the periplasmic fraction containing Fab and scFv for human TREM2 protein, the clones were considered to bind specifically, and the corresponding clones were finally selected.

최종적으로 선별된 항체의 CDR 및 Framework 아미노산 서열은 아래 표 1에 기재하였다.The CDR and framework amino acid sequences of the finally selected antibodies are listed in Table 1 below.

IDID FHR1FHR1 CDRCDR -H1-H1 FHR2FHR2 CDRCDR -H2-H2 FHR3FHR3 CDRCDR -H3-H3 FHR4FHR4 A9A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) NYAMH
(서열번호 2)NYAMH
(sequence number 2) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) AISGSGGNTYYADSVKG
(서열번호 4)AISGSGGNTYYADSVKG
(sequence number 4) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HERTWGSQYMDH
(서열번호 6)HERTWGSQYMDH
(sequence number 6) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) AATTATGCAATGCAC
(서열번호 44)AATTATGCAATGCAC
(Sequence number 44) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) GCAATCTCTGGTTCTGGTGGTAATACTTACTATGCCGATTCAGTGAAGGGT
(서열번호 46)GCAATCTCTGGTTCTGGTGGTAATACTTACTATGCCGATTCAGTGAAGGGT
(Sequence number 46) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATGAACGTACTTGGGGTTCTCAGTACATGGATCAT
(서열번호 48)CATGAACGTACTTGGGGTTCTCAGTACATGGATCAT
(Sequence number 48) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(Sequence number 49) B1B1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) SYAMH
(서열번호 8)SYAMH
(sequence number 8) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) SIKSSGSYTYYADSVKG
(서열번호 9)SIKSSGSYTYYADSVKG
(sequence number 9) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HYYTSMDH
(서열번호 10)HYYTSMDH
(sequence number 10) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) TCTTATGCAATGCAC
(서열번호 50)TCTTATGCAATGCAC
(sequence number 50) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) TCTATCAAATCTTCTGGTTCTTACACTTACTATGCCGATTCAGTGAAGGGT
(서열번호 51)TCTATCAAATCTTCTGGTTCTTACACTTACTATGCCGATTCAGTGAAGGGT
(Sequence number 51) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATTACTACACTTCTATGGATCAT
(서열번호 52)CATTACTACACTTCTATGGATCAT
(Sequence number 52) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(Sequence number 49) B2B2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) SYAMH
(서열번호 8)SYAMH
(sequence number 8) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) AISSDGGNKYYADSVKG
(서열번호 11)AISSDGGNKYYADSVKG
(Sequence number 11) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HEYSSGWQYMDV
(서열번호 12)HEYSSGWQYMDV
(Sequence number 12) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGCGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGCGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) TCTTATGCAATGCAC
(서열번호 50)TCTTATGCAATGCAC
(sequence number 50) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) GCAATCTCTTCTGATGGTGGTAATAAATACTATGCCGATTCAGTGAAGGGT
(서열번호 53)GCAATCTCTTCTGATGGTGGTAATAAATACTATGCCGATTCAGTGAAGGGT
(Sequence number 53) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATGAATACTCTTCTGGTTGGCAGTACATGGATGTT
(서열번호 54)CATGAATACTCTTCTGGTTGGCAGTACATGGATGTT
(Sequence number 54) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(Sequence number 49) B7B7 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) DYAMH
(서열번호 13)DYAMH
(Sequence number 13) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) SISSTGSSTYYADSVKG
(서열번호 14)SISSTGSSTYYADSVKG
(Sequence number 14) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HYYTAMDV
(서열번호 15)HYYTAMDV
(Sequence number 15) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) GATTATGCAATGCAC
(서열번호 55)GATTATGCAATGCAC
(sequence number 55) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) TCTATCTCTTCTACTGGTTCTTCTACTTACTATGCCGATTCAGTGAAGGGT
(서열번호 56)TCTATCTCTTCTACTGGTTTCTTCTACTTACTATGCCGATTCAGTGAAGGGT
(Sequence number 56) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATTACTACACTGCAATGGATGTT
(서열번호 57)CATTACTACACTGCAATGGATGTT
(Sequence number 57) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(Sequence number 49) E10E10 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) NYAMH
(서열번호 2)NYAMH
(sequence number 2) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) GISGSGGTTGYADSVKG
(서열번호 16)GISGSGGTTGYADSVKG
(Sequence number 16) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) VRGRYSSQYFDI
(서열번호 17)VRGRYSSQYFDI
(Sequence number 17) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) AATTATGCAATGCAC
(서열번호 44)AATTATGCAATGCAC
(Sequence number 44) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) GGTATCTCTGGTTCTGGTGGTACTACTGGTTATGCCGATTCAGTGAAGGGT
(서열번호 58)GGTATCTCTGGTTCTGGTGGTACTACTGGTTATGCCGATTCAGTGAAGGGT
(Sequence number 58) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) GTTCGTGGTCGTTACTCTTCTCAGTACTTCGATATC
(서열번호 59)GTTCGTGGTCGTTACTCTTCTCAGGTACTTCGATATC
(Sequence number 59) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(Sequence number 49) F3F3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) NYAMH
(서열번호 2)NYAMH
(sequence number 2) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) AISGDGGNTYYADSVKG
(서열번호 18)AISGDGGNTYYADSVKG
(Sequence number 18) RFTISRNNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRNNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HHVGQGVQVFDI
(서열번호 19)HHVGQGVQVFDI
(Sequence number 19) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) AATTATGCAATGCAC
(서열번호 44)AATTATGCAATGCAC
(Sequence number 44) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) GCAATCTCTGGTGATGGTGGTAATACTTACTATGCCGATTCAGTGAAGGGT
(서열번호 60)GCAATCTCTGGTGATGGTGGTAATACTTACTATGCCGATTCAGTGAAGGGT
(sequence number 60) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATCATGTTGGTCAGGGTGTTCAGGTTTTCGATATC
(서열번호 61)CATCATGTTGGGTCAGGGTGTTCAGGTTTTCGATATC
(Sequence number 61) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCG
(Sequence number 49) G11G11 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) SYAMH
(서열번호 8)SYAMH
(sequence number 8) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) SISQSGSTKYYADSVKG
(서열번호 20)SISQSGSTKYYADSVKG
(sequence number 20) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HFYTAADY
(서열번호 21)HFYTAADY
(Sequence number 21) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) TCTTATGCAATGCAC
(서열번호 50)TCTTATGCAATGCAC
(sequence number 50) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) TCTATCTCTCAGTCTGGTTCTACTAAATACTATGCCGATTCAGTGAAGGGT
(서열번호 62)TCTATCTCTCAGTCTGGTTCTACTAAATACTATGCCGATTCAGTGAAGGGT
(Sequence number 62) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATTTCTACACTGCAGCAGATTAC
(서열번호 63)CATTTCTACACTGCAGCAGATTAC
(Sequence number 63) TGGGGTCAGGGTACTCTGGTTACTGTATCATCG
(서열번호 49)TGGGGTCAGGGTACTCTGGTTACTGTATCATCG
(Sequence number 49) H4H4 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(서열번호 1)EVQLVESGGGLVQPGGSLRLSCAASGFTFS
(sequence number 1) SYAMH
(서열번호 8)SYAMH
(sequence number 8) WVRQAPGKGLEWVS
(서열번호 3)WVRQAPGKGLEWVS
(sequence number 3) SISSSGGTTYYADSVKG
(서열번호 22)SISSSGGTTYYADSVKG
(Sequence number 22) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(서열번호 5)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
(sequence number 5) HYRTAMDY
(서열번호 23)HYRTAMDY
(Sequence number 23) WGQGTLVTVSS
(서열번호 7)WGQGTLVTVSS
(sequence number 7) GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(서열번호 43)GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT
(Sequence number 43) TCTTATGCAATGCAC
(서열번호 50)TCTTATGCAATGCAC
(sequence number 50) TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(서열번호 45)TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA
(Sequence number 45) TCTATCTCTTCTTCTGGTGGTACTACTTACTATGCCGATTCAGTGAAGGGT
(서열번호 64)TCTATCTCTTCTTCTGGTGGTACTACTTACTATGCCGATTCAGTGAAGGGT
(Sequence number 64) CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(서열번호 47)CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA
(Sequence number 47) CATTACCGTACTGCAATGGATTAC
(서열번호 65)CATTACCGTACTGCAATGGATTAC
(sequence number 65) TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(서열번호 49)TGGGGTCAGGGCACTTTAGTGACCGTCTCATCG
(Sequence number 49) IDID FLR1FLR1 CDRCDR -L1-L1 FLR2FLR2 CDRCDR -L2-L2 FLR3FLR3 CDRCDR -L3-L3 FLR4FLR4 A9A9 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISNYLN
(서열번호 25)RASQSISNYLN
(sequence number 25) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) ATSSLQS
(서열번호 27)ATSSLQS
(Sequence number 27) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(Sequence number 28) QQAYSSPWT
(서열번호 29)QQAYSSPWT
(Sequence number 29) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66)
 CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(서열번호 67)CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(Sequence number 67) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(Sequence number 68) GCAACTTCCTCTCTGCAGTCT
(서열번호 69)GCAACTTCCTCTCTGCAGTCT
(Sequence number 69) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAAGCATACTCTTCTCCGTGGACG
(서열번호 71)CAGCAAGCATACTCTTCTCCGTGGACG
(Sequence number 71) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) B1B1 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISNWLN
(서열번호 31)RASQSISNWLN
(Sequence number 31) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASRLQS
(서열번호 32)AASRLQS
(Sequence number 32) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(Sequence number 28) QQSYSLPWT
(서열번호 33)QQSYSLPWT
(Sequence number 33) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGTCTATCTCTAATTGGCTGAAC
(서열번호 73)CGCGCTAGCCAGTCTATCTCTAATTGGCTGAAC
(Sequence number 73) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(sequence number 68) GCAGCATCCCGTCTGCAGTCT
(서열번호 74)GCAGCATCCCGTCTGCAGTCT
(Sequence number 74) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACTCTCTGCCGTGGACG
(서열번호 75)CAGCAATCTTACTCTCTGCCGTGGACG
(sequence number 75) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) B2B2 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISNYLN
(서열번호 25)RASQSISNYLN
(sequence number 25) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASNLQS
(서열번호 34)AASNLQS
(Sequence number 34) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(sequence number 28) QQSYSSPWT
(서열번호 35)QQSYSSPWT
(Sequence number 35) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(서열번호 67)
CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(Sequence number 67)
 TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(Sequence number 68) GCAGCATCCAATCTGCAGTCT
(서열번호 76)GCAGCATCCAATCTGCAGTCT
(Sequence number 76) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACTCTTCTCCGTGGACG
(서열번호 77)CAGCAATCTTACTCTTCTCCGTGGACG
(Sequence number 77) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) B7B7 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISSYLN
(서열번호 36)RASQSISSYLN
(Sequence number 36) WYQQKPGKAPKLLIY
(서열번호 26)
WYQQKPGKAPKLLIY
(Sequence number 26)
 AASNLQS
(서열번호 34)AASNLQS
(Sequence number 34) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(Sequence number 28) QQSYSTPLT
(서열번호 37)QQSYSTPLT
(Sequence number 37) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66)
CGCGCTAGCCAGTCTATCTCTTCTTACCTGAAC
(서열번호 78)
CGCGCTAGCCAGTCTATCTCTTCTTACCTGAAC
(Sequence number 78)
 TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(sequence number 68) GCAGCATCCAATCTGCAGTCT
(서열번호 76)GCAGCATCCAATCTGCAGTCT
(Sequence number 76) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)
GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70)
 CAGCAATCTTACTCTACTCCGCTGACG
(서열번호 79)CAGCAATCTTACTCTACTCCGCTGACG
(Sequence number 79) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) E10E10 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQDIGRYLN
(서열번호 38)RASQDIGRYLN
(Sequence number 38) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASRLQS
(서열번호 32)AASRLQS
(Sequence number 32) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(Sequence number 28) QQSYTSPLT
(서열번호 39)QQSYTSPLT
(Sequence number 39) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGGATATCGGTCGTTACCTGAAC
(서열번호 80)CGCGCTAGCCAGGATATCGGTCGTTACCTGAAC
(sequence number 80) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(Sequence number 68) GCAGCATCCCGTCTGCAGTCT
(서열번호 74)GCAGCATCCCGTCTGCAGTCT
(Sequence number 74) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACACTTCTCCGCTGACG
(서열번호 81)CAGCAATCTTACACTTCTCCGCTGACG
(Sequence number 81) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) F3F3 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISNYLN
(서열번호 25)RASQSISNYLN
(sequence number 25) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASNLQS
(서열번호 34)AASNLQS
(Sequence number 34) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(sequence number 28) QQSYSFPFT
(서열번호 40)QQSYSFPFT
(sequence number 40) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(서열번호 67)CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(Sequence number 67) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(Sequence number 68) GCAGCATCCAATCTGCAGTCT
(서열번호 76)GCAGCATCCAATCTGCAGTCT
(Sequence number 76) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACTCTTTTCCGTTTACG
(서열번호 82)CAGCAATCTTACTCTTTTCCGTTTACG
(Sequence number 82) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) G11G11 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24) RASQSISNYLN
(서열번호 25)RASQSISNYLN
(sequence number 25) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASRLQS
(서열번호 32)AASRLQS
(Sequence number 32) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(sequence number 28) QQSYSTPWT
(서열번호 41)QQSYSTPWT
(Sequence number 41) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(서열번호 67)CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC
(Sequence number 67) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(sequence number 68) GCAGCATCCCGTCTGCAGTCT
(서열번호 74)GCAGCATCCCGTCTGCAGTCT
(Sequence number 74) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACTCTACTCCGTGGACG
(서열번호 83)CAGCAATCTTACTCTACTCCGTGGACG
(Sequence number 83) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72) H4H4 DIQMTQSPSSLSASVGDRVTITC
(서열번호 24)
DIQMTQSPSSLSASVGDRVTITC
(Sequence number 24)
 RASQSIRNYLN
(서열번호 42)RASQSIRNYLN
(Sequence number 42) WYQQKPGKAPKLLIY
(서열번호 26)WYQQKPGKAPKLLIY
(Sequence number 26) AASRLQS
(서열번호 32)AASRLQS
(Sequence number 32) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(서열번호 28)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
(Sequence number 28) QQSYSSPWT
(서열번호 35)QQSYSSPWT
(Sequence number 35) FGQGTKVEIK
(서열번호 30)FGQGTKVEIK
(sequence number 30) GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(서열번호 66)GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT
(Sequence number 66) CGCGCTAGCCAGTCTATCCGTAATTACCTGAAC
(서열번호 84)CGCGCTAGCCAGTCTATCCGTAATTACCTGAAC
(Sequence number 84) TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(서열번호 68)TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC
(sequence number 68) GCAGCATCCCGTCTGCAGTCT
(서열번호 74)GCAGCATCCCGTCTGCAGTCT
(Sequence number 74) GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(서열번호 70)GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT
(sequence number 70) CAGCAATCTTACTCTTCTCCGTGGACG
(서열번호 77)CAGCAATCTTACTCTTCTCCGTGGACG
(Sequence number 77) TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(서열번호 72)TTCGGGCAGGGAACTAAAGTGGAAATTAAA
(Sequence number 72)

3. TREM2 결합 항체 단백질의 생산3. Production of TREM2 binding antibody protein

신규 인간 항체 8종에 대한 가변영역서열을 확보하고, 불변영역 서열(P01857, P01834)과 함께 동물세포 발현벡터인 pcDNA™ 3.4 TOPO vector (ThermoFisher Scientific, Cat.A14697)에 클로닝 하였다.We obtained variable region sequences for eight new human antibodies and cloned them into the animal cell expression vector pcDNA™ 3.4 TOPO vector (ThermoFisher Scientific, Cat. A14697) together with constant region sequences (P01857, P01834).

TREM2 결합 항체 단백질을 발현시키기 위하여 각 플라스미드 DNA를 Thermo Fisher 제조업체의 프로토콜 (ExpiFectamine 293 transfection kit, ThermoFisher Scientific)에 설명된 대로 Expi293 세포에 형질도입하였다. 간단하게, 6×106 cells/ml Expi293 세포를 DNA 및 ExpiFectamine 복합체로 형질감염 후 37℃, 8% CO2 조건에서 배양하였고, 24시간 후에 발현 증강제 (Enhancer)를 첨가하였다. 형질감염 후 2 일차에 세포를 32℃에서 6일간 배양한 후, TREM2 결합 항체 단백질을 포함하는 배양 상등액을 회수하였다.To express TREM2-binding antibody proteins, each plasmid DNA was transfected into Expi293 cells as described in the Thermo Fisher manufacturer's protocol (ExpiFectamine 293 transfection kit, ThermoFisher Scientific). Briefly, 6 × 10 6 cells/ml Expi293 cells were transfected with DNA and ExpiFectamine complexes and cultured at 37°C under 8% CO 2 conditions, and the expression enhancer was added 24 h later. On the second day after transfection, the cells were cultured at 32°C for 6 days, and the culture supernatant containing TREM2-binding antibody proteins was collected.

Protein A 친화크로마토그래피 칼럼 (GE Healthcare)을 PBS (pH 7.4)로 평형화하였다. 각 TREM2 결합 항체 단백질을 포함하는 배양 상등액을 0.2 μm 필터로 여과한 후, Protein A 친화크로마토그래피 칼럼에 로딩하였다. PBS (pH 7.4)로 칼럼을 세척한 후 100 mM 글리신 (pH 3.4) 용액으로 TREM2 결합 항체 단백질을 용출하였다. 용출 액에 1 M Tris 용액을 첨가하여 중화한 다음 PBS로 버퍼를 교환하였다. 정제된 TREM2 결합 항체는 SDS-PAGE gel (Invitrogen) 분석과 SE-HPLC 분석을 통해 확인하였으며, 단백질 농도는 Nanodrop 2000C 분광광도계(Thermo Scientific)를 사용하여 정량화하였다. SE-HPLC 기준 monomer 비율이 88.57% TREM2-3-B6을 제외한 나머지 7종의 항체는 90% 이상의 monomer 비율을 갖는 것을 확인하였다(도 2). A Protein A affinity chromatography column (GE Healthcare) was equilibrated with PBS (pH 7.4). The culture supernatant containing each TREM2-binding antibody protein was filtered through a 0.2 μm filter and loaded onto the Protein A affinity chromatography column. After washing the column with PBS (pH 7.4), the TREM2-binding antibody protein was eluted with a 100 mM glycine (pH 3.4) solution. The eluate was neutralized by adding 1 M Tris solution and the buffer was exchanged with PBS. The purified TREM2-binding antibody was confirmed by SDS-PAGE gel analysis (Invitrogen) and SE-HPLC analysis, and the protein concentration was quantified using a Nanodrop 2000C spectrophotometer (Thermo Scientific). Except for TREM2-3-B6, which had a monomer ratio of 88.57% based on SE-HPLC, the remaining seven antibodies were confirmed to have a monomer ratio of more than 90% (Fig. 2).

<실시예 2> TREM2 결합 항체의 인간 TREM2에 대한 결합력 평가<Example 2> Evaluation of binding affinity of TREM2 binding antibody to human TREM2

실시예 1에서 제조된 TREM2 결합 항체의 항원에 대한 결합력을 정량적으로 분석하기 위해 Biacore를 통해 각 항체에 대한 KD 값으로 항원에 대한 결합친화도 (affinity)를 측정하였다. 시스템 및 샘플 버퍼는 HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Polysorbate 20 (v/v))를 사용하였다. His capture kit (Cytiva)의 제조업체 프로토콜에 따라 아민 커플링 절차를 수행한 CM5칩 표면에 his tagging이 되어있는 항원을 고정화하였다. HBS-EP 완충액에 희석된 항체 단백질을 30 μL/min의 유속으로 결합을 위해 2분 동안, 해리를 위해 5분 동안 항체가 고정화된 CM5 칩에 흘려주었다. 다음으로, 10mM 글리신-HCl(pH 1.5)을 60초 동안 반응시켜 항원에 결합된 항체 단백질을 세척하였다. Biacore 평가 소프트웨어를 사용하여 항체의 센서그램을 분석하였다. 그 결과, 8종 모두 인간 TREM2 항원 단백질에 sub-nanomolar 수준 이상으로 결합함을 확인하였다(도 3). In order to quantitatively analyze the binding affinity of the TREM2-binding antibody manufactured in Example 1 to the antigen, the binding affinity to the antigen was measured as the K D value for each antibody using Biacore. The system and sample buffer used were HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Polysorbate 20 (v/v)). The antigen tagged with His was immobilized on the surface of the CM5 chip by performing the amine coupling procedure according to the manufacturer's protocol of the His capture kit (Cytiva). The antibody protein diluted in HBS-EP buffer was flowed through the CM5 chip with the antibody immobilized at a flow rate of 30 μL/min for 2 minutes for binding and 5 minutes for dissociation. Next, the antibody protein bound to the antigen was washed by reacting with 10 mM glycine-HCl (pH 1.5) for 60 seconds. The sensorgrams of the antibodies were analyzed using Biacore evaluation software. As a result, it was confirmed that all eight types bound to the human TREM2 antigen protein at sub-nanomolar levels or higher (Fig. 3).

<실시예 3> 인간 항체의 효능 검증<Example 3> Verification of the efficacy of human antibodies

TREM2는 ADAM10/17과 같은 sheddase에 의해 세포막외부가 잘려지게 되어 그 기능을 잃게 되고 이와 동시에 세포배양액에 존재하는 잘려진 soluble TREM2 (sTREM2)의 양이 증가하게 된다. 본 발명에 명시된 항체는 세포막 외부에 노출된 TREM2에 결합함으로써 shedding을 억제할 수 있는데 이러한 보호 효과를 배양액에 존재하는 sTREM2의 양과 세포내에 존재하는 internal TREM2 (iTREM2)는 줄어들고, membrane TREM2 (mTREM2)의 단백질 양은 증가하는 것으로 평가하였다.TREM2 loses its function when the outer part of the cell membrane is cleaved by sheddase such as ADAM10/17, and at the same time, the amount of cleaved soluble TREM2 (sTREM2) present in the cell culture medium increases. The antibody specified in the present invention can inhibit shedding by binding to TREM2 exposed to the outer part of the cell membrane, and this protective effect was evaluated as a decrease in the amount of sTREM2 present in the culture medium and internal TREM2 (iTREM2) present in the cell, and an increase in the protein amount of membrane TREM2 (mTREM2).

본 발명에서 명시된 인간 항체에 의한 TREM2 shedding 억제 효과를 평가하기 이를 확인하기 위하여 10% fetal bovine serum (FBS)가 포함된 Dulbecco’s Modified Eagle Medium (DMEM)에서 배양된 HEK2393T 세포주를 polyethylenimine (PEI)를 이용하여 인간 TREM2-His와 DAP12-V5 발현하도록 형질전환시켰다. 8시간 동안 배양한 후 대조군 (마우스에서 생산된 C2 및 인간 IgG) 혹은 인간 항체 각각이 20 ug/mL의 농도로 포함된 새로운 배양액으로 바꿔주고 16 시간 동안 추가 배양하였다. 배양액에 포함된 sTREM2의 양을 확인하여 위하여 원심분리로 세포를 제거한 순수 배양액과 세포 분획 (전체 세포 용출액 (total), 세포내 분획 (internal), 세포막 분획 (membrane)) 샘플을 확보하였다. 전체 세포 분획은 원심분리로 획득한 세포를 용출액 (1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC))에 풀고 1 시간 동안 냉장 (4℃)에서 회전시키며 반응한 후 원심분리를 통해 용출되고 남은 찌꺼기를 제거함으로써 세포내 및 세포막 분획 단백질 전체를 확보하였다. 세포내 분획은 원심분리로 획득한 세포를 저장액 (20 mM Tris, pH 7.0, 1 mM EDTA/EGTA, 1X PIC)에 풀고 얼음에서 30 분 동안 유지하여 세포 내부를 버퍼로 가득 채운 후 초저온 냉동고 (-80℃)에서 동결시켰다가 녹이는 방법으로 세포를 터뜨린 후 원심분리하여 세포내 분획을 세포막 분획으로부터 분리하였다. 남은 세포막 분획의 단백질들은 용출액을 사용하여 세포막으로부터 용출시킨 후 원심분리를 통해 찌꺼기로부터 분리/확보하였다. 확보된 각 분획은 환원제가 포함된 lithium dodecyl sulfate (LDS) 버퍼와 섞어주어 단백질 3차 구조를 풀어준 후, Bolt™ 4-12% Bis-Tris Plus Gels (ThermoFisher 사)에서 크기별로 분리하고 polyvinylidene difluoride (PVDF) 막에 옮겨주었다. PVDF 막은 TREM2 항체 (Abcam #ab209814, 0.4 ug/mL)와 이에 결합하는 2차 항체 (Rockland #18-8816-31, 1 ug/mL, horseradish peroxidase (HRP)가 결합된 형태)가 포함된 버퍼에 차례로 반응시킨 후 루미놀 용액을 처리하여 인간 TREM2의 양을 확인하였다. 단백질 양에 따른 루미놀 신호의 강도를 ImageJ 프로그램을 이용하여 분석하여 단백질의 양을 수치화한 후, shedding억제 효과를 다음의 수식으로 평가하였다(도 4).To evaluate the TREM2 shedding inhibition effect by the human antibodies specified in the present invention, HEK2393T cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) were transfected with polyethylenimine (PEI) to express human TREM2-His and DAP12-V5. After culturing for 8 hours, the cells were replaced with fresh culture medium containing the control (C2 and human IgG produced from mouse) or each human antibody at a concentration of 20 ug/mL, and further cultured for 16 hours. To confirm the amount of sTREM2 contained in the culture medium, pure culture medium and cell fraction samples (total, internal, membrane fraction) from which cells were removed were obtained by centrifugation. The whole cell fraction was prepared by dissolving the cells obtained by centrifugation in the lysate (1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC)), rotating them in a refrigerator (4°C) for 1 hour, and then centrifuging them to remove the remaining debris to secure all the intracellular and cell membrane fraction proteins. The intracellular fraction was prepared by dissolving the cells obtained by centrifugation in a storage solution (20 mM Tris, pH 7.0, 1 mM EDTA/EGTA, 1X PIC), keeping them on ice for 30 minutes to fill the inside of the cells with the buffer, and then freezing them in an ultra-low temperature freezer (-80°C) and thawing them to rupture the cells, followed by centrifugation to separate the intracellular fraction from the cell membrane fraction. The proteins of the remaining cell membrane fraction were eluted from the cell membrane using the lysate, and then separated/secured from the debris through centrifugation. Each fraction obtained was mixed with lithium dodecyl sulfate (LDS) buffer containing a reducing agent to release the protein 3D structure, then separated by size on Bolt™ 4-12% Bis-Tris Plus Gels (ThermoFisher) and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was reacted in sequence with a buffer containing TREM2 antibody (Abcam #ab209814, 0.4 ug/mL) and a secondary antibody (Rockland #18-8816-31, 1 ug/mL, horseradish peroxidase (HRP)-conjugated form), and then treated with a luminol solution to confirm the amount of human TREM2. The intensity of the luminol signal according to the amount of protein was analyzed using the ImageJ program to quantify the protein amount, and the shedding inhibition effect was evaluated using the following formula (Fig. 4).

[membrane TREM2 ÷ (soluble TREM2 × internal TREM2)][membrane TREM2 ÷ (soluble TREM2 × internal TREM2)]

본 발명에서 명시된 인간 항체 중 B2, B7, E10은 마우스 유래항체 C2의 Shedding 억제 대비 우수한 억제효과를 보였으며, B1, F3는 마우스 유래항체 C2와 유사한 억제 효과를 보였다. 하지만, A9, G11, H4는 마우스 유래항체 C2의 Shedding 억제 대비 다소 감소한 억제효과를 보였다(표 2).Among the human antibodies specified in the present invention, B2, B7, and E10 showed excellent inhibitory effects compared to the shedding inhibition of mouse-derived antibody C2, and B1 and F3 showed inhibitory effects similar to the mouse-derived antibody C2. However, A9, G11, and H4 showed somewhat reduced inhibitory effects compared to the shedding inhibition of mouse-derived antibody C2 (Table 2).

항체Antibody -- IgGIgG C2C2 A9A9 B1B1 B2B2 B7B7 E10E10 F3F3 G11G11 H4H4 평균average 0.500.50 0.420.42 1.001.00 0.770.77 0.960.96 1.161.16 1.231.23 1.281.28 0.930.93 0.750.75 0.670.67 표준오차Standard error 0.140.14 0.160.16 0.030.03 0.010.01 0.030.03 0.110.11 0.100.10 0.240.24 0.170.17 0.170.17 0.200.20

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred implementation examples and that the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열, 서열번호 8로 표시되는 아미노산 서열 및 서열번호 13으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 4로 표시되는 아미노산 서열, 서열번호 9로 표시되는 아미노산 서열, 서열번호 11로 표시되는 아미노산 서열, 서열번호 14로 표시되는 아미노산 서열, 서열번호 16으로 표시되는 아미노산 서열, 서열번호 18로 표시되는 아미노산 서열, 서열번호 20으로 표시되는 아미노산 서열 및 서열번호 22로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 10으로 표시되는 아미노산 서열, 서열번호 12로 표시되는 아미노산 서열, 서열번호 15로 표시되는 아미노산 서열, 서열번호 17로 표시되는 아미노산 서열, 서열번호 19로 표시되는 아미노산 서열, 서열번호 21로 표시되는 아미노산 서열 및 서열번호 23으로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및A heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence represented by SEQ ID NO: 8, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 9, an amino acid sequence represented by SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence represented by SEQ ID NO: 16, an amino acid sequence represented by SEQ ID NO: 18, an amino acid sequence represented by SEQ ID NO: 20, and an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence represented by SEQ ID NO: 10, an amino acid sequence represented by SEQ ID NO: 12, an amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 17, and an amino acid represented by SEQ ID NO: 19. A heavy chain variable region comprising a heavy chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 21 and an amino acid sequence represented by SEQ ID NO: 23 and a heavy chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 7; and 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열, 서열번호 31로 표시되는 아미노산 서열, 서열번호 36으로 표시되는 아미노산 서열, 서열번호 38로 표시되는 아미노산 서열 및 서열번호 42로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 27로 표시되는 아미노산 서열, 서열번호 32로 표시되는 아미노산 서열 및 서열번호 34로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 29로 표시되는 아미노산 서열, 서열번호 33으로 표시되는 아미노산 서열, 서열번호 35로 표시되는 아미노산 서열, 서열번호 37로 표시되는 아미노산 서열, 서열번호 39로 표시되는 아미노산 서열, 서열번호 40으로 표시되는 아미노산 서열 및 서열번호 41로 표시되는 아미노산 서열로 이루어진 그룹에서 선택된 어느 하나의 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하는, 인간 TREM2(Triggering receptor expressed on myeloid cells 2) 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편.A light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence represented by SEQ ID NO: 31, an amino acid sequence represented by SEQ ID NO: 36, an amino acid sequence represented by SEQ ID NO: 38, and an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 27, an amino acid sequence represented by SEQ ID NO: 32, and an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 29, an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence represented by SEQ ID NO: 35, an amino acid sequence represented by SEQ ID NO: 37, an amino acid sequence represented by SEQ ID NO: 39, an amino acid sequence represented by SEQ ID NO: 40, and an amino acid sequence represented by SEQ ID NO: 41, and a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 30. A human antibody or antigen-binding fragment thereof that specifically binds to human TREM2 (Triggering receptor expressed on myeloid cells 2) protein, comprising a light chain variable region comprising a light chain FR4 consisting of the amino acid sequence indicated. 제1항에 있어서, 상기 인간 TREM2 단백질에 특이적으로 결합하는 인간 항체 또는 이의 항원 결합 단편은,In the first paragraph, the human antibody or antigen-binding fragment thereof that specifically binds to the human TREM2 protein is, 1) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 27로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 29로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,1) A heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 29, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 2) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 9로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 10으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 31로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 33으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,2) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 9, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 10, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 31, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 3) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 11로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 12로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 35로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,3) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 11, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 12, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 4) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 13로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 14로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 15로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 36으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 37로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,4) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 13, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 14, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 15, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 36, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 37, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30, or 5) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 16으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 17로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 38로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 39로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,5) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 16, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 17, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 38, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 39, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 6) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 18로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 19로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 34로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 40으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,6) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 18, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 19, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 34, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 40, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 7) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 20으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 21로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 25로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 41로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하거나,7) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 20, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 21, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 41, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 30, or 8) 서열번호 1로 표시되는 아미노산 서열로 이루어진 중쇄 FR1, 서열번호 8로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 3으로 표시되는 아미노산 서열로 이루어진 중쇄 FR2, 서열번호 22로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 FR3, 서열번호 23으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3 및 서열번호 7로 표시되는 아미노산 서열로 이루어진 중쇄 FR4를 포함하는 중쇄 가변 영역; 및 서열번호 24로 표시되는 아미노산 서열로 이루어진 경쇄 FR1, 서열번호 42로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 26으로 표시되는 아미노산 서열로 이루어진 경쇄 FR2, 서열번호 32로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2, 서열번호 28로 표시되는 아미노산 서열로 이루어진 경쇄 FR3, 서열번호 35로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3 및 서열번호 30으로 표시되는 아미노산 서열로 이루어진 경쇄 FR4를 포함하는 경쇄 가변 영역을 포함하는 것을 특징으로 하는 인간 항체 또는 이의 항원 결합 단편.8) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 22, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 23, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7; A human antibody or antigen-binding fragment thereof, characterized in that it comprises a light chain variable region comprising a light chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 42, a light chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 32, a light chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 28, a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 35, and a light chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 30. 제1항 또는 제2항의 인간 항체 또는 이의 항원 결합 단편을 코딩하는 핵산 분자.A nucleic acid molecule encoding a human antibody or an antigen-binding fragment thereof according to claim 1 or 2. 제3항의 핵산 분자를 포함하는 재조합 발현벡터.A recombinant expression vector comprising the nucleic acid molecule of claim 3. 제4항의 재조합 발현벡터로 형질전환되어 분리된 세포.A cell isolated by transformation with the recombinant expression vector of clause 4. 제1항 또는 제2항의 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 인간 TREM2 항원 검출용 조성물.A composition for detecting human TREM2 antigen, comprising the human antibody or antigen-binding fragment thereof of claim 1 or 2 as an active ingredient. 제1항 또는 제2항의 인간 항체 또는 이의 항원 결합 단편을 유효성분으로 포함하는 알츠하이머병 진단용 조성물.A composition for diagnosing Alzheimer's disease, comprising the human antibody or antigen-binding fragment thereof of claim 1 or 2 as an active ingredient.
PCT/KR2024/007692 2023-06-09 2024-06-05 Human antibody specifically binding to human trem2 protein, and use thereof Pending WO2024253420A1 (en)

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KR20200033794A (en) * 2017-08-03 2020-03-30 알렉터 엘엘씨 Anti-TREM2 antibodies and methods of use
US20200140545A1 (en) * 2018-10-15 2020-05-07 Novartis Ag Trem2 stabilizing antibodies
US20220177576A1 (en) * 2020-01-13 2022-06-09 Denali Therapeutics Inc. Anti-trem2 antibodies and methods of use thereof
WO2023039612A1 (en) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Trem2 antigen binding proteins and uses thereof

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