WO2024253420A1 - Anticorps humain se liant de manière spécifique à la protéine trem2 humaine et son utilisation - Google Patents

Anticorps humain se liant de manière spécifique à la protéine trem2 humaine et son utilisation Download PDF

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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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Korean (ko)
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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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    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], 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

La présente invention concerne un anticorps humain se liant de manière spécifique à une protéine TREM2 humaine et son utilisation. L'anticorps monoclonal humain se liant de manière spécifique à TREM2 a été découvert par criblage biologique à l'aide d'une banque d'affichage sur phage d'anticorps synthétique humain, et la capacité de liaison à l'antigène et l'activité biologique de l'anticorps découvert ont été identifiées. La présente invention a une affinité élevée pour TREM2, et permet ainsi le développement d'un système de diagnostic à base d'anticorps capable de diagnostiquer TREM2 en tant que biomarqueur de diverses démence d'Alzheimer. De plus, étant donné qu'un agent thérapeutique classique pour la maladie d'Alzheimer est principalement un symptôme de soulagement par la régulation temporaire des neurotransmetteurs, il est considéré que le développement d'anticorps ciblant TREM2 peut conduire au développement de nouveaux médicaments prometteurs ayant des effets thérapeutiques importants.
PCT/KR2024/007692 2023-06-09 2024-06-05 Anticorps humain se liant de manière spécifique à la protéine trem2 humaine et son utilisation Pending WO2024253420A1 (fr)

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KR10-2023-0074263 2023-06-09
KR20230074263 2023-06-09
KR1020240073131A KR20240174995A (ko) 2023-06-09 2024-06-04 인간 trem2 단백질에 특이적으로 결합하는 인간 항체 및 이의 용도
KR10-2024-0073131 2024-06-04

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200033794A (ko) * 2017-08-03 2020-03-30 알렉터 엘엘씨 항-trem2 항체 및 이의 사용 방법
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
US20220204611A1 (en) * 2017-04-21 2022-06-30 Amgen Inc. Trem2 antigen binding proteins and uses thereof
WO2023039612A1 (fr) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Protéines de liaison à l'antigène trem2 et leurs utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220204611A1 (en) * 2017-04-21 2022-06-30 Amgen Inc. Trem2 antigen binding proteins and uses thereof
KR20200033794A (ko) * 2017-08-03 2020-03-30 알렉터 엘엘씨 항-trem2 항체 및 이의 사용 방법
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 (fr) * 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Protéines de liaison à l'antigène trem2 et leurs utilisations

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