TREM2 Stabilizing Antibody

Abstract

This invention provides an antibody that binds to and stabilizes the trigger receptor 2 (TREM2) protein expressed in human myeloid cells. [Solution] The present invention provides a nucleic acid molecule or a set of nucleic acid molecules encoding an anti-TREM2 antibody or its antigen-binding fragment, comprising three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) containing specific amino acid sequences, as defined by the Kabat numbering system, Chothia numbering system, IMGT numbering system, and combined numbering system. Furthermore, the present invention provides a vector containing the nucleic acid molecule, cells containing the vector, and a method for producing an anti-TREM2 antibody or its antigen-binding fragment, comprising culturing the cells.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, and the entire listing is included in the reference. This is incorporated herein by reference. The aforementioned ASCII was created on September 16, 2019. The copy is named PAT058251_ST25.txt and has a size of 147,551 bytes. It is Ito.

[0002] This invention relates to the trigger receptor 2 (TREM2) protein expressed in human myeloid cells. This document provides antibodies that bind to and stabilize a substance, as well as methods for using these antibodies. [Background technology]

[0003] The trigger receptor or "TREM" expressed in myeloid cells is used by macrophages and dendritic cells. Cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, cutaneous Langerhans cells A group of transmembrane glucose molecules expressed in various types of myeloid cells, including cytoplasm, Kupffer cells, and neutrophils. It is a protein (Takaki, R. et al., Immunol. Rev., 20 06,214:118-29). TREM has immunoglobulin (I) in its extracellular domain. It has a type g) folding and therefore belongs to the immunoglobulin superfamily (IgSF). The TREM receptor contains a short intracellular domain, but it is a signaling mediator. It lacks a activating motif, and cell activation requires DAP12 (12kDa DNAX activity). Adapter proteins such as sexualizing proteins are required. For TREM, there are two Members TREM1 and TREM2 have been reported, and all of them relate to immunity and inflammation. Plays an important role in the reaction. The gene encoding human TREM is TREM 1, a gene cluster encoding TREM2, TREM3, TREM4 and TREM5 , and is located on chromosome 6p21.1 carrying TREM-like genes.

[0004] TREM2 is a glycoprotein of about 40 kDa, which decreases to 26 kDa after N-deglycosylation. The entire TREM2 protein consists of a leading signal peptide (amino acids 1-18), a single V-type IgSF extracellular region (amino acids 19-132), a stalk region (amino acids 133-172), a positively charged transmembrane domain (amino acids 173-197), and a cytosolic tail (amino acids 198-230) (Kober et al., El ife 5(2016); Kober et al., J. Mol. Biol. 429( 2017)l607-1629). The extracellular region encoded by exon 2 consists of a single V-type IgSF domain containing three potential N-glycosylation sites. The putative transmembrane region contains charged lysine residues. The cytoplasmic tail of TREM2 lacks a signal transduction motif and is thought to send signals through the signal transduction adapter molecule DAP12 / TRYROBP.

[0005] TREM2 physically associates with DAP12, and DAP12 functions as a signal transduction adapter protein for TREM2 and several other cell surface receptors. The cytoplasmic domain of DAP12 contains an immunoreceptor activation tyrosine motif (ITAM) (Wunderlic h, J. Biol. Chem. 288, 33027-33036, 2013). This interaction After activation of the activating receptor, DAP12 receives conserved ITAM cells via Src kinase. Phosphorylation of rosine residues occurs. This is followed by the recruitment and activation of Syk protein kinase. mitogen-activated protein kinase (MAPK), PI3K, NFκB and phospho This triggers downstream signaling pathways, including the activation of lipase Cγ (PLCγ).

[0006] TREM2 is composed of lipopolysaccharide (LPS), heat shock protein 60, neurite fragments, and other materials. Bacteria, apolipoprotein E, and a wide range of anionic and zwitterionic lipids, for example, phosphate Phatidic acid (PA), phosphatidylglycerol (PG), phosphatidylserine ( PS) Phosphatidylinositol (PI), phosphatidylcholine (PC), cardio It can be activated by olipin and sphingomyelin. When TREM2 is activated, Increased phagocytic activity of chlorocyria and macrophages, and decreased release of pro-inflammatory cytokines. Furthermore, TLR signaling is suppressed. TREM2 is CSF-1 receptor signaling Through a synergistic effect with plexin, it maintains the survival of microglia. Furthermore, TREM2 is plexin It interacts with A1 to regulate cell adhesion and motility. TREM2 also interacts with Aβ plaques. It is densely present in the surface region of microglia cells that come into contact with nerve cell fragments (Yuan et al.) al., Neuron 90(2016)724-739). Recently, this environment Some ligands sensed by TREM2, such as phospholipids and myelinlipids (P oliani et al.,J.Clin.Invest.125(2015):21 61-2170) and ApoE (Atagi et al., J. Biol. Chem .290(2015):26043-26050;Bailey et al.,JB iol.Chem.290(2015):26033-26042) has been identified. To give another ligand, TREM2 contributes to the uptake of Aβ into microglia. Therefore, it may be Aβ and plaque-associated neuronal fragments (Xiang et al., EMB O Mol.Med.8(2016):992-1004). TREM2 also has an apothecary. -Cystic cells (Takahashi et al., J.Exp.Med.201(200 5), 647-657), myelin fragments (Poliani et al., J. Clin Invest.125(2015):2161-2170) and bacterial beads (Cen To exclude et al., Am.J.Respir.188(2013)201-212) It has also been shown to play a role in the uptake of the receptors. It promotes the breakdown of food and is critically important for lipid metabolism, myelin uptake, and intracellular degradation. .

[0007] TREM2 is a sequential process of ectodomain shedding and intramembrane protein degradation. Proteins undergo protein decomposition processing (Wunderlich, J. Biol. Chem.) 288, 33027-33036, 2013). During ectodomain shedding, A DAM (disintegrin and metalloproteinase domain-containing protein) or B T is cleaved by proteases such as members of the ACE (beta-site APP cleavage enzyme) family. The ectodomain of REM2 is released (Kleinberger, Sci.Trans l.Med.2014;6(243):243ra86).

[0008] After the ectodomain is removed, the fragments held in the remaining membrane are γ-secretase. Further processing is carried out by mediated intramembrane protein degradation. Ectodomain sched The soluble fragment of TREM2 (sTREM2) produced by the process is used in dendritic cell cultures. The supernatant, as well as the plasma and CSF (cerebrospinal fluid) of patients with non-inflammatory neurological diseases and multiple sclerosis. It has been observed in samples (Kleinberger, 2014). Sheath in human CSF The modified TREM2 ectodomain, i.e., sTREM2, is a potential Alzheimer's disease. It is valued as a biomarker for Imer's disease (AD), and generally increases during the aging process. This has been shown (Suarez-Calvet, EMBO Mol. Med. 8, 466) (-476, 2016). Detailed analysis of the course of AD showed that sTREM2 is in the early stages of AD. It increases before clinical symptoms appear, peaks in MCI-AD, and rises in AD-type dementia. However, it was revealed that the level remained lower compared to the MCI-AD stage. Suarez-Calvet, 2016).

[0009] Increased TREM2 expression at the peak of a disease promotes its resolution (e.g., peritonitis, wound healing). Healing) (Turnbull, 2006; Gawish, 2015). Chronic conditions such as neuroinflammation. Under inflammatory conditions, TREM2 is constantly shedding, and microglia and macrophages It cannot exert its signal transduction function at the cell surface. Therefore, TR Stabilizing and / or preventing EM2 shedding will improve microglia and macrophages. In this stage, TREM2 expression, which has functional signaling ability, will be restored.

[0010] In human genetics research, it is not so much the absence of sTREM2, but rather the loss of surface TREM2. It has been pointed out that this influences disease risk. For example, the TREM2 protein, for example The amino acid mutation from R to H at position 47 of SEQ ID NO: 1 results in a slight decrease in cell surface expression. Kleinberger 2014), and decreased ligand binding ability of TREM2 (Wan This causes (g 2015, Atagi 2015, Bailey 2015). The amino acid mutation T66M in M2 leads to a deficiency in TREM2 expression on the cell surface. (Kleinberger 2014), and consequently, soluble TREM2 is no longer produced. The H157Y mutation at the TREM2 cleavage site enhances sTREM2 expression and complete... It reduces full-length membrane-bound TREM2 and is associated with an increased risk of AD (Thornt (Schlepckow 2017). Therefore, from these genetic studies Both the decrease in sTREM2 and the increase in cell membrane-bound TREM2 are related to TREM2 on the cell surface. This suggests that stabilization is desirable.

[0011] Haass et al. (International Publication No. 18015573) presented the story of TREM2. A 10-amino acid peptide (AHVEHS) located in the C region, spanning amino acids 152-161. An antibody was generated that binds to ISRS (Sequence ID 132) and inhibits TREM2 cleavage. Antibodies such as these cleave TREM2 by direct binding and blocking the cleavage site. To hinder. Schwabe et al. (International Publication No. 17062672 pamphlet) stated that TREM The disclosure includes antibodies that bind to 2. However, none of these disclosed antibodies No stabilizing effect has been noted for this either. Conversely, International Publication No. 17062672 Pan Some of the antibodies listed in the frets have been reported to have an destabilizing effect (implementation) Example 15). [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, it is possible to stabilize TREM2 and activate TREM2-related functions and / or It can promote, has good developable properties, and is beneficial for neurological changes in which TREM2 stabilization is advantageous. It is necessary to identify and develop hTREM2 antibodies suitable for the treatment of patients suffering from sexually transmitted diseases. It is being done. [Means for solving the problem]

[0013] Previously published literature has shown that when generating antibodies to stabilize TREM2, the cutting of ADAM17 Because the fracture site is located within the stalk region, the stalk region of TREM2 is targeted. In fact, for large molecules such as antibodies (or their bound fragments), schedase is stoker's ratio It is expected that this will sterically hinder contact with a relatively small region (amino acids 133-172). Therefore, previous attempts to produce stabilized antibodies against TREM2 have been successful. It is not surprising that they have targeted the stalk region of REM2. Region (Amino acids 19-132 of any one of sequence numbers 1, 2, or 3 are cleavage sites (H15 7) It is located further away and is part of the ectodomain. TREM2 IgSF Antibodies against the region do not sterically impair the contact of schedase with the TREM2 cleavage site. Because it is expected to be funneled, the IgSF region has so far been considered an antibody that can stabilize TREM2. It had not been considered a potential target.

[0014] Surprisingly, the inventors have found that the antibody disclosed herein binds to the IgSF region. We found that it has the ability to effectively stabilize TREM2 on the cell surface. Furthermore, such antibodies exhibit TREM2-dependent phagocytosis in human M2A macrophages. It was also shown that it has the ability to exhibit functional downstream effects, such as promoting [something]. Furthermore, it was demonstrated that such [something] The body also increases the phagocytic ability of microglia or macrophages in the brain, for example. Furthermore, TREM2-dependent functions may also be enhanced in vivo.

[0015] Therefore, this specification specifically refers to the IgSF domain of human TREM2 (hTREM2). An antibody or its antigen-binding fragment that binds to and stabilizes the hTREM2 protein, for example, Monoclonal antibodies or antigen-binding fragments thereof are provided. Such antibodies are described herein. This is referred to as "hTREM2 antibody or its antigen-binding fragment." The antigen-binding fragment (i) reduces or inhibits the shedding of the TREM2 ectodomain. (ii) Stabilize the TREM2 protein on the cell surface; and / or (iii) Binding to cognitive ligands, intracellular signaling, increased phagocytosis, and degradation of phagocytic substances. Maintain or enhance TREM2 functions such as promotion, and increase TREM2-dependent downstream regulatory functions. It can be advanced. Dysfunction of TREM2 or deficiency of surface TREM2 is associated with human neuroinflammation. Because it is related to sex and neurodegenerative lesions, the hTREM2 antibody or its anti- The primordial binding fragment is associated with Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. Chemical disease, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor Somatic encephalitis, autism, cerebral lupus (NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN) ), postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Gila GBS, inclusion body myositis, lysosomal storage disorders, for example, sphingomyelia Lymphipidosis (Niemann-Pick disease type C) and mucopolysaccharidosis type II / IIIB, metachromatic white leukemia Multifocal motor dystrophy, multifocal motor neuropathy, myasthenia gravis, neurological Behçet's disease, Neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen encephalitis, Rett syndrome Syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial myelitis It can be used in the treatment, prevention, or diagnosis of neuroinflammatory or neurodegenerative diseases such as fasciitis. The hTREM2 antibody or its antigen-binding fragment described herein also broadens the TREM2 Excessive proteolytic cleavage or expression of abnormal or mutated TREM2 receptor variants Autoimmune disorders, inflammatory disorders, or malignant disorders mediated or associated with cells It is also suitable for treating, preventing, or diagnosing harm. In some preferred embodiments, as described herein The hTREM2 antibody or its antigen-binding fragment described is used in Alzheimer's disease and frontotemporal dementia. A disease selected from dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease. It can be used for treatment, prevention, or diagnosis. Furthermore, this specification discloses... TREM2-binding antibodies or antigen-binding fragments are used to diagnose and / or diagnose TREM2-related diseases. Methods for treating it are also provided.

[0016] In one embodiment, this specification describes a method that specifically binds to the IgSF domain of the TREM2 protein. An antibody or its antigen-binding fragment that stabilizes the TREM2 protein is provided. In several preferred embodiments, the antibody or its antigen-binding fragment is used by macrophages, dendritic cells, etc. Cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, cutaneous Langerhans cells TR on the cell surface of TREM2-expressing cells such as Kupffer cells, neutrophils, or hepatocellular carcinoma cells The EM2 protein is stabilized. In some embodiments, these antibodies or their antigens are used. The binding fragment enables proteolytic shedding of the ectodomain of the TREM2 protein. Reduce.

[0017] In some embodiments, this specification describes a specific approach to the IgSF domain of human TREM2. An antibody or an antigen-binding fragment thereof that binds to an antigen is provided. For example, such an antibody or its anti The original binding fragment consists of amino acid residues 19-132 of SEQ ID NO: 1 and amino acid residue 19 of SEQ ID NO: 2. Human TREM2 IgSF containing amino acid residues ~132, or amino acid residues 19~132 of SEQ ID NO: 3 It binds to the domain. In some embodiments, the TREM2 antibody is human or a humanized antibody. In some embodiments, the antigen-binding fragment is Fab, F(ab')2, Fv fragment. It is an scFv, mini-body, or diamond-body.

[0018] In some embodiments, the TREM2 antibody is a bispecific antibody. In this state, the bispecific antibody specifically binds to human TREM2 and DAP12.

[0019] In some embodiments, the TREM2 antibody includes an Fc region. In some embodiments, The Fc region has one or more mutations when compared to the parent antibody and is associated with antibody-dependent cell cytotoxicity (A Modified IgG1 F with reduced DCC or complement-dependent cell-mediated cytotoxicity (CDC) activity. This is the c region. In some embodiments, the Fc region is the IgG2 Fc region, IgG4 The region is selected from either an Fc region or an IgG2 / IgG4 hybrid Fc region.

[0020] In some embodiments, the hTREM2 antibody or its antigen-binding fragment is monoclonal. Yes. This specification codes for such monoclonal antibodies or their antigen-binding fragments. Nucleic acids, and nucleic acids that encode such monoclonal antibodies or antigen-binding fragments thereof. A vector and host cells are provided.

[0021] In another embodiment, this specification includes the TREM2 antibody or its antigen conjugate as described herein. A fragment, or a nucleic acid encoding such an antibody or antigen-binding fragment, or containing such nucleic acid A pharmaceutical composition is provided comprising one or more cells and a pharmaceutically acceptable carrier.

[0022] In another embodiment, this specification includes the TREM2 antibody or its antigen conjugate as described herein. By administering a therapeutically effective dose of any of the fragments to the target population, treatment can be performed on subjects requiring treatment. A method is provided for treating diseases associated with TREM2 loss of function. Such a method is The following steps: (1) The TREM2 level on the cell surface of the sample obtained from the subject is aggregated. Steps to be taken: (2) Select subjects in which the cell surface TREM2 level is lower than the reference level. This step involves setting the reference level to the cell surface TREM of a sample obtained from a healthy subject. (3) A two-level step; and (4) specific to the IgSF domain of the TREM2 protein. The therapeutically effective amount of an antibody or its antigen-binding fragment that binds to and stabilizes the TREM2 protein. The procedure may include one or more steps of administering to the target. In some embodiments, Such methods further include administering a second drug to the subject. The cell surface TR of the sample EM2 levels can be determined by flow cytometry, immunohistochemistry, Western blotting, and immunotherapy. Epidemic fluorescence assay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELI) Select from SA, homogeneous time-resolved fluorescence (HTRF), or positron emission tomography (PET). This can be determined by the assay performed. In some embodiments, the sample is taken from the brain vertebrae. Includes cerebrospinal fluid and its cellular components. In some embodiments, it relates to TREM2 loss of function. The diseases include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis autism, cerebral lupus (NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN), vein Postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Bass syndrome Reye's syndrome (GBS), inclusion body myositis, lysosomal storage disorders, for example, sphingomyelin lysosomal storage disorders. Pydosis (Niemann-Pick disease type C) and mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy Trophy, multifocal motor neuropathy, myasthenia gravis, neurological Behçet's disease, optic nerve Myelitis (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen encephalitis, Rett syndrome, Stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis Which neuroinflammatory or neurodegenerative disease is it? In some preferred embodiments, TREM2 Diseases associated with loss of function include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and muscle disorders. It is a neurodegenerative disease selected from atrophic lateral sclerosis or Nasu-Hakola disease. In some embodiments, the disease is Alzheimer's disease. In some embodiments, TREM 2. Antibodies or their antigen-binding fragments are used on macrophages, dendritic cells, osteoclasts, microglia, Mast cells, monocytes, lung epithelial cells, cutaneous Langerhans cells, Kupffer cells, neutrophils, or liver The TREM2 protein on the cell surface of TREM2-expressing cells selected from cellular cancer cells is stabilized. To stabilize. In some embodiments, the TREM2 antibody or its antigen-binding fragment is taken orally. It is administered to the target patient via intravenous, intracranial, intrathecal, subcutaneous, or intranasal route.

[0023] In another aspect, this specification describes the use in the treatment of diseases related to TREM2 loss of function. A TREM2 antibody or its antigen-binding fragment is provided for this purpose. Several preferred implementations In this state, this antibody or its antigen-binding fragment is the IgSF domain of the TREM2 protein ( In other words, amino acid residues 19-132 of SEQ ID NO: 1, and amino acid residues 19-13 of SEQ ID NO: 2 2. Specifically binds to amino acid residues 19-132 of SEQ ID NO: 3 and the TREM2 protein To stabilize the substance. In some preferred embodiments, the antibody or its antigen-binding fragment is Macrophages, dendritic cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, TRE selected from cutaneous Langerhans cells, Kupffer cells, neutrophils, or hepatocellular carcinoma cells. To stabilize the TREM2 protein on the cell surface of M2-expressing cells. Several embodiments So, diseases associated with TREM2 function loss include Alzheimer's disease, frontotemporal dementia, and pa Kinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis ALS, anti-NMDA receptor encephalitis, autism, cerebral lupus (NP-SLE), chemotherapy CIPN (induced peripheral neuropathy), postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy Chi (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion body myositis, lysosomal storage Severe cases, such as sphingomyelin lipidosis (Niemann-Pick disease type C) and mucopolycythemia. Diabetes II / IIIB, Metachromatic Leukodystrophy, Multifocal Motor Neuropathy, Myasthenia Gravis Myositis, neurological Behçet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis Rasmussen encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, These are neuroinflammatory or neurodegenerative diseases such as viral encephalitis or bacterial meningitis. In a preferred embodiment, diseases associated with TREM2 loss of function include Alzheimer's disease, pre- Choose from cranial-temporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease. It is a neurodegenerative disease. In a more preferred embodiment, the disease is Alzheimer's disease. In some embodiments, the TREM2 antibody or its antigen-binding fragment is used on macrophages. Dendritic cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, cutaneous Langerhan cells TREM2-expressing cells selected from styloid cells, Kupffer cells, neutrophils, or hepatocellular carcinoma cells It stabilizes the TREM2 protein on the surface of the cell. [Brief explanation of the drawing]

[0024] [Figure 1A] Figure 1: Figure 1A shows exemplary amino acid sequence alignments of human TREM2 isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2), and isoform 3 (SEQ ID NO: 3). Figure 1B illustrates the structure of TREM2 and its interaction with the signaling adapter protein DAP12. Mature TREM2 contains a single immunoglobulin (IgSF) domain, a stalk region, a transmembrane (TM) domain, and a cytoplasmic domain. [Figure 1B] (As stated above.) [Figure 2] This demonstrates the stabilization of TREM2 in CHO-hDAP12-hTREM2 cells by antibody treatment. [Figure 3] This shows the binding of the TREM2 antibody to human M2A macrophages before and after treatment with PMA. [Figure 4A] Figure 4: Shows antibody binding to WT-TREM2 (Figure 4A) and TREM2-TREM1 chimeric protein recombinantly expressed in CHO-hDAP12 cells (Figures 4B, 4C). [Figure 4B] (As stated above.) [Figure 4C] (As stated above.) [Figure 5] This shows the stabilization of TREM2 on the cell surface of hM2A by a TREM2 antibody. [Figure 6A] Figure 6: This shows that antibodies that stabilize TREM2 on the cell surface also increase the phagocytic activity of human M2A. Statistical values ​​were calculated using Student's t-test. Compared to isotype controls, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. [Figure 6B] (As stated above.) [Figure 7A] Figure 7: Shows the determination of the minimum effective dose in the phagocytosis assay in hM2A. Statistics were calculated using Student's t-test. Compared to isotype controls, *P-value < 0.05, **P-value < 0.01, and ***P-value < 0.001. [Figure 7B] (As stated above.) [Figure 8] This study demonstrates that plate-bound TREM2 antibodies induce TREM2-dependent NFAT promoter-dependent gene transcription. [Figure 9A] Figure 9: Shows that the TREM2 antibody increases Syk phosphorylation in human M2A macrophages. (B) shows the quantification of pSyk under various conditions in correlation with total Syk in the Western blot shown in (A). [Figure 9B] (As stated above.) [Figure 10]This shows the phagocytosis of Staphylococcus aureus (S. aureus) bioparticles by human M2A macrophages over a period of 0-3 hours. [Figure 11] This shows the phagocytosis of SH-SY5Y cells by human M2A macrophages over a period of 3 to 12 hours. [Figure 12] This study demonstrates that the TREM2 antibody promotes the chemotaxis of human M2A macrophages. [Figure 13] Using apoptotic pHrodo-labeled SH-SY5Y cells and cumulative phagocytosis levels as readouts, we demonstrate that the TREM2 antibody promotes phagocytosis in human iPS-derived microglia. [Figure 14] This study demonstrates that the TREM2 antibody promotes chemotaxis in human iPS-derived microglia. [Figure 15A] Figure 15: Results of prophylaxis / combination therapy and therapeutic treatment with TREM2 antibodies in the cuprizon model. [Figure 15B] (As stated above.) [Figure 15C] (As stated above.) [Figure 15D] (As stated above.) [Figure 15E] (As stated above.) [Figure 16A] Figure 16: Image analysis (A) results of the MPTP model in humanized TREM2 mice and representative microscopic images (B) of each group are shown. [Figure 16B] (As stated above.) [Figure 17A] Figure 17: Results of cross-blockage experiments between Fab MOR041877, MOR041895, and MOR042596 and full-length IgG MOR041877, MOR41895, MOR042596, MOR044698, and MOR03207 in CHO-hDAP12-hTREM2 cells. [Figure 17B] (As stated above.) [Figure 17C] (As stated above.) [Figure 18] This shows the increased phagocytic activity of hM2A after treatment with MOR042596. [Figure 19]This shows the TREM2 epitope bound to MOR042596 as determined by X-ray crystallography. The TREM2 protein backbone is shown schematically, with the side chains of TREM2 residues within a 5 Å distance from Fab shown as sticks. The Fab heavy chain is shown as a dark gray surface, and the light chain is shown as a light gray surface. [Figure 20] A magnified view of the TREM2-Fab junction (see Figure 19) is shown, comparing MOR042596 (crystal structure) and MOR044698 (homology model). TREM2 residues within 5 Å of Fab are shown as sticks, and residues proximal to LCDR3 (D39-K42) are marked. The heavy chain (dark gray, upper right) and the light chains LCDR1 and LCDR2 (light gray, upper left) are identical for both Fabs. LCDR3 has several important shared residues (positions 90, 93, 95) that maintain the same skeletal loop conformation of LCDR3 in both Fabs. There are differences at positions 89, 91, 92, 94, and 96, and MOR044698 has one additional insertion (S95a). The overall epitope is conserved between MOR042596 and MOR044698. [Figure 21] This shows the TREM2 epitope bound to MOR041877 as determined by X-ray crystallography. The TREM2 protein backbone is shown schematically, with the side chains of TREM2 residues within a 5 Å distance from Fab shown as sticks. The Fab heavy chain is shown as a dark gray surface, and the light chain is shown as a light gray surface. [Figure 22A] Figure 22: The cerebral cortex of hTREM2-KI mice stained for TREM2 and Iba1 and treated with either Cuprizone and MOR044698-mu or an isotype control antibody is shown alongside a naive control mouse (A). A quantitative analysis of the normalized hTREM2-positive area is provided (B). [Figure 22B] (As stated above.) [Modes for carrying out the invention]

[0025] This specification describes a method that specifically binds to the extracellular domain of human TREM2 and modifies the TREM2 protein. An antibody that stabilizes the substance and its antigen-binding fragment are provided. This TREM2 antibody and its Antigen-binding fragments can reduce or inhibit the shedding of the TREM2 ectodomain. It can stabilize the TREM2 protein on the cell surface; and optionally, it can stabilize the TREM2 protein on the cell surface. Binding to cognitive ligands, intracellular signaling, increased phagocytosis, and phagocytic substances It can maintain or improve TREM2 functions such as promoting decomposition. Dysfunctional TRE Since M2 or surface TREM2 deficiency is associated with human neuroinflammatory and neurodegenerative lesions, The TREM2-stabilized antibody and its antigen-binding fragment described in the document are used for Alzheimer's disease, pre- Cranotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis Amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, cerebral lupus (NP- SLE, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, chronic inflammatory bowel syndrome Myelin polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion bodies Myositis, lysosomal storage disorders, for example, sphingomyelin lipidosis (Niemann-Pick). Disease type C, mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuron Lopathy, myasthenia gravis, neurological Behçet's disease, neuromyelitis optica (NMO), optic neuritis, Polymyositis, dermatomyositis, Rasmussen encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic Neuroinflammatory or neurodegenerative diseases such as brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis. It can be used for the treatment, prevention, or diagnosis of disease. TREM2 bonds as described herein The combined antibody and its antigen-binding fragment also exhibit extensive proteolytic cleavage or abnormality of TREM2. or mediated by cells expressing a mutated TREM2 receptor variant or It is also suitable for the treatment, prevention, or diagnosis of related autoimmune disorders, inflammatory disorders, or malignant disorders. Yes. In some preferred embodiments, the hTREM2 antibody described herein or so The antigen-binding fragment is associated with Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and muscular atrophy. It is used for the treatment, prevention, or diagnosis of diseases selected from fibrous sclerosis or Nasu-Hakola disease. This can be done. Furthermore, this specification discloses TREM2-binding antibodies and their anti- Methods for diagnosing and / or treating TREM2-related diseases using the original binding fragment are also provided.

[0026] TREM2 mediates the phagocytosis of non-inflammatory bacteria and dying cells, thereby reducing the inflammatory response. Homozygous loss of function of EM2 leads to Nasu-Hakola disease (lipid membrane polysclerosing leukoencephalopathy). Cystic dysplasia of the bone ("PLOSL"), or frontotemporal dementia (FTD)-like syndrome, bone Diseases characterized by cysts, neuroinflammation, progressive neurodegeneration, and presenile dementia are caused by cysts. It is also caused by the TREM2 heterozygous loss-of-function mutation R47H, which is associated with late-onset Alzheimer's disease. It is an important risk factor for Mer's disease (AD), and is comparable to the apolipoprotein E ε4 allele. This is the effect size. TREM2 is expressed in microglia found in the white matter, hippocampus, and neocortex. This is in part consistency with the pathological features reported in the brain of AD, and TREM is linked to the etiology of AD. This supports the possibility that 2 is involved. Currently, genetic screening is also showing that In addition to AD, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal type Heterozygous missense mutations in TREM2 are a risk factor for premature dementia (FTD). It has been identified (Kleinberger, Sci Transl Med. 2014) Jul 2;6(243):243ra86). Therefore, severe cognitive impairment and dementia Functional TREM2 is essential to prevent age-related neuroinflammatory and neurodegenerative diseases. It is essential.

[0027] Due to alternative splicing, humans have three TREM2 isoforms. Isoform 1 is the longest isoform. Figure 1A shows the human TREM2 isoform. Form 1 (SEQ ID NO: 1), Human TREM2 Isoform 2 (SEQ ID NO: 2), and Human Provides amino acid sequence alignment for TREM2 isoform 3 (SEQ ID NO: 3). Figure 1B shows the structure of TREM2 and its relationship with the signal transduction adapter protein DAP12. The interaction between them is illustrated in the diagram.

[0028] definition When used in the specification and claims, the singular forms "a," "an," and "the" are used. Unless explicitly indicated otherwise in the context, this includes multiple references. For example, "one (a) detail The term "cell" refers to a group of cells that contain a mixture of those cells.

[0029] All numerical values, such as pH, temperature, time, concentration, and molecular weight, including ranges, are 0.1 It is an approximate value that fluctuates (+) or (-) in increments of . It is not always explicitly stated. However, it should be understood that the term "approximately" is preceded by all numerical notations. Regarding X, the term "approximately" means, for example, X ± 15% (including all values ​​within that range). It tastes good. Also, although not always explicitly stated, the reagents described in this specification are simple. This is an example, and it should be understood that equivalent reagents are known in the art.

[0030] Throughout this specification and the following claims, unless otherwise specifically required by the context, the phrase " "~ to include (comprise)", and "~ to include (comprises)" and "~ Variations such as "comprising" are included in this specification unless otherwise noted. In writing, it is used in a non-restrictive sense, in its open-ended form.

[0031] As used herein, "consisting of" means "aspects" , any elements, steps, or components not specified in the embodiments and / or claimed elements The minutes are also excluded. When used herein, "consistent" means "to be essentially derived from". g essentially of) Materials or steps that do not substantially affect the new features are not excluded.

[0032] When used herein, "TREM2" (also known as "trigger expression in myeloid cells") is used to mean "trigger expression in myeloid cells." Receptor 2 (TREM2, TREM2a, TREM2b, or TREM2c) is immune This refers to transmembrane glycoproteins belonging to the globulin superfamily (IgSF). The entire M2 protein (SEQ ID NO: 1) is a leading signal peptide (amino acids 1-1) 8) Single type V IgSF extracellular domain (amino acids 19-132), stalk domain (amino acids) 133-172), positively charged transmembrane domain (amino acids 173-197), and cytosol The tail consists of amino acids 198-230 (Feuerbach et al., Neu Rosci. Lett. 660 (2017): 109-114). Human TREM2 gene. It is located at chromosome position 6p21.1, and the genome sequence of the TREM2 gene is Gen You can refer to the Bank (gene ID: 54209). For alternative splicing Consequently, humans have three TREM2 isoforms (ENSEMBL IDs ENSP00000362205, ENSP00000342651, and EN (Protein sequence available in SP00000362214). The term "TREM2" is T This term is used to refer collectively to all isoforms of REM2. The longest isoform is human TREM2. The isoform protein and mRNA sequences are as follows: Trigger receptor 2 precursor isoform 1 precursor expressed in myeloid cells [Human (Ho mo sapiens)](NP_061838.1) [ka] Trigger receptor 2 (TRE) expressed in human (Homo sapiens) myeloid cells M2), transcription variant 1, mRNA (NCBI reference sequence: NM_018965.3) [ka]

[0033] Human TREM2 isoform 2 (SEQ ID NO: 2) and isoform 3 (SEQ ID NO: 3) The amino acid sequence is shown in Figure 1A. When used herein, it refers to the human TREM2 protein. It also contains at least about 70 sequences of sequence numbers 1, 2, or 3 over its full length. %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 This also includes proteins with 00% sequence identity, and such proteins are ligands. Binding, intracellular signaling, promotion of phagocytosis and degradation of phagocytic substances, and other functions of TREM2. It still possesses the regulatory function of mice and cynomolgus monkeys (crab The TREM2 protein sequences of quinoa monkeys (cyno) and other animals are relevant to this technology. It is publicly known (for example, regarding the mouse TREM2 protein, see NP_112544.1 (and NP_001259007.1).

[0034] The term "extracellular domain" refers to the extracellular domain of a transmembrane protein located on the extracellular side of the cell's lipid bilayer. This refers to the exposed portion. Methods for determining the ectodomain of a protein are available in this technology. It is publicly known (Singer (1990); High et al. (1993), and (McVector software, Oxford Molecular). For example. The extracellular domain of the human TREM2 protein is amino acid residues 19-17 of SEQ ID NO: 1. It may include 2.

[0035] The term "ectodomain" in TREM2 refers to the extracellular domain of TREM2, specifically the sheath This refers to the portion released after enzyme cleavage. The cleavage site is between amino acids H157 and S158. It has been reported that (Feuerbach et al., Neurosci. Lett) .660(2017):109-114). Therefore, the ectodomain of hTREM2 is It will consist of amino acids 19-157 of any one of sequence numbers 1, 2, or 3.

[0036] The term "IgSF domain" refers to the extracellular domain of TREM2, specifically the immunoglobulin domain. This includes the Ig-type folding, and consequently, a portion of the immunoglobulin superfamily. It refers to. In humans, for example, the IgSF domain is one of sequence numbers 1, 2, and 3. It consists of amino acid residues 19 to 132.

[0037] The term "Stork region" in TREM2 refers to the extracellular domain of TREM2, specifically V This refers to the region that connects the IgSF (isoglobulin-type) domain and the transmembrane domain. The stalk region of the human TREM2 isoform 1 protein is the amino acid of SEQ ID NO: 1. It may contain acids 133-172.

[0038] The term "transmembrane domain" refers to the portion of a transmembrane protein that spans the lipid bilayer of a cell. This refers to minutes. Methods for determining the transmembrane domain of a protein are well known in the art. (Elofsson et al.,Annu.Rev.Biochem.76(200 7):125-140; Bernsel et al., Protein Science e 14(2005):1723-1728).

[0039] The terms "cytoplasmic domain" and "cytoplasmic tail" are used synonymously, and refer to the transmembrane protein. It refers to the cytoplasmic portion of the lipid bilayer of a cell. It determines the cytoplasmic tail of a protein. The method is publicly known in the art (Elofsson et al. (2007)). And Bernsel et al. (2005).

[0040] The term "stabilize" as used herein refers, for example, to inflammatory diseases or neurological disorders. In healthy subjects without sexually transmitted diseases, the process of reaching the TREM2 level in corresponding TREM2-expressing cells is as follows: This refers to the maintenance, restoration, or increase of TREM2 cell surface levels in TREM2-expressing cells. For example, by reducing or inhibiting the shedding of the TREM2 ectodomain, This can be achieved by increasing TREM2 cell surface expression. Bell uses flow cytometry / FACS or TREM2 cell surface immunoprecipitation. This can be evaluated by the decrease in soluble TREM2 over time. TREM2 cell surface expression can also be detected by enzyme-linked immunosorbent assay (ELISA), radioimmuno No-assay (RIA), bioassay (e.g., increased phagocytosis), Western blot Immunossay, flow cytometry, immunohistochemistry, immunofluorescence assay, homogeneous time-resolved fluorescence ( It can also be detected by HTRF or positron emission tomography (PET).

[0041] In this specification, the term "activate" refers, for example, to healthy subjects or appropriate TREM2-dependent subjects. TREM2 expression cells in individuals with inflammatory or neurodegenerative diseases whose activity is impaired. This refers to the induction or maintenance of downstream signaling of TREM2 expressed on the cell surface in cells. This is not limited to, but can be achieved by phosphorylation of TREM2-related DAP12 or DAP10. This can occur, and this can lead to Syk phosphorylation through various intracellular signaling cascades. Enhancement, phagocytosis, increased directional cell motility (chemotaxis) towards targets, increased cell survival, Regulation of cytokine or chemokine release in cells expressing TREM2, and intracellular phagocytic materials. This can lead to increased degradation or altered gene expression. TREM2-dependent DAP12 or Syk Increased phosphorylation can be detected by Western blot, ELISA, or flow cytometry / FAC. It can be evaluated by S. Directional motility of cells, such as chemotaxis, is bio- This can be evaluated by enzyme binding immunoadsorption. Regulation of cytokine release is achieved through enzyme binding immunoadsorption. ELISA, radioimmunoassay (RIA), or flow cytometry / FA Changes in gene expression can be evaluated by CS. By RT-PCR, or by Western blotting or flow saturation at the protein level. It can be evaluated using iteometry.

[0042] The term "promote" in this specification means increasing disease-induced impaired TREM2-dependent activity. This refers to strong or restorative activity, such as phagocytosis and directional cell motility (chemotaxis) towards a target. Increased (sex), increased cell survival, cytokines or chemokines of cells expressing TREM2 Regulation of release, increased degradation of intracellular phagocytic material, and adjacent cells (astrocytes / neurons) This may include the regulation of cellular responses or changes in gene expression.

[0043] As used herein, the term “antibody” refers to an immunoglobulin that specifically binds to an antigen. This refers to a protein or polypeptide sequence derived from a molecule. Antibodies are polyclonal or molecular. They may be noclonal, multiple, single-stranded, or intact immunoglobulins, and may be from natural sources or complexes. It may originate from alternative sources. Naturally occurring "antibodies" are interconnected by disulfide bonds. It is a glycoprotein containing at least two linked heavy (H) chains and two light (L) chains. Each heavy chain consists of a variable heavy chain region (abbreviated as VH in this specification) and a constant heavy chain region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. It consists of a light chain variable region (abbreviated as VL in this specification) and a light chain steady region. The region contains one domain CL. The VH and VL regions are called complementarity-determining regions (CDRs). This can be further subdivided into areas of extreme variability, which are called framework areas (FR). More conserved regions are scattered throughout. Each VH and VL is from the amino terminus in the following order. It consists of three CDRs and four FRs arranged at the carboxyl terminus: FR1, C DR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains are anti- It contains a binding domain that interacts with the progenitor. The constant region of the antibody interacts with various cells of the immune system (e.g., Host tissue or factors containing effector cells and the first component (C1q) of the classical complement system. It can mediate the binding of immunoglobulins to the antibody. Antibodies are monoclonal antibodies, human antibodies. The antibody may be an antibody, a humanized antibody, a camelized antibody, or a chimeric antibody. The antibody may be any isotype Ip (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses It is possible. Throughout this document, the terms “antibody” or “antibody molecule” are also used in context as specifically indicated. This also includes any fragments thereof and any derivatives thereof, unless otherwise specified.

[0044] The term "antibody fragment" or "antigen-binding fragment" refers to a fragment that specifically interacts with an antigen's epitope. Antibodies that retain their ability (for example, through binding, steric hindrance, stabilization / destabilization, and spatial distribution) This refers to at least a part of it. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv. Fragments, scFv antibody fragments, disulfide-bonded Fvs (sdFv), VH and CH1 domains. Fd fragments consisting of , linear antibodies, single domains such as sdAb (either VL or VH) The antibody, camel VHH domain, and two components linked by disulfide crosslinks in the hinge region Multispecific antibodies formed from antibody fragments such as bivalent fragments containing Fab fragments, and antibodies This includes, but is not limited to, isolated CDRs or other epitope-binding fragments. The proto-binding fragment is also a single-domain antibody, maxibody, minibody, nanobody, and intra Labodi, Diabodi, Triabodi, Tetrabodi, v-NAR and Bis-scFv It can be incorporated into (for example, Hollinger and Hudson, Nature) (See Biotechnology 23:1126-1136, 2005) The antigen-binding fragment is also based on polypeptides such as fibronectin type III (Fn3). It can be transplanted onto a scaffold (US patent describing fibronectin polypeptide minibodies) (See Specification No. 6,703,199). The term "scFv" refers to the variable region of the light chain. an antibody fragment containing at least one antibody fragment containing a variable region of the heavy chain and This refers to a fusion protein containing the light chain and heavy chain variable regions, for example, a synthetic linker, for example They are continuously linked via short, flexible polypeptide linkers, forming a single-strand polypeptide. It can be expressed, and the scFv retains the specificity of the intact antibody from which it originates. When used in a specification, unless otherwise specified, scFv is, for example, N of polypeptide With respect to the terminal and C-terminus, the VL and VH variable regions may be present in either order. scFv may contain VL-linker-VH, or VH-linker-VL. You can stay like that.

[0045] The terms “complementarity-determining region” or “CDR” are used herein to mean antigen-specific regions. This refers to the sequence of amino acids within the antibody variable region that confers isomerism and binding affinity. For example, generally , with three CDs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) R, and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3) There exists. The precise amino acid sequence boundary of a given CDR is, Kabat et al. (1 991), “Sequences of Proteins of Immunolog ical Interest”,5th Ed.Public Health Serv ice,National Institutes of Health,Bethes da,MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering) (G scheme), or a combination thereof, and ImMunoGenTics (IMGT) Barring (Lefranc, M.-P., The Immunologist, 7, 13) 2-136(1999);Lefranc,M.-P.et al.,Dev.Comp .Immunol.,27,55-77(2003);Lefranc et al., (2015) Nucleic Acids Res.43,D413-422)(``I Using any of the many well-known schemes, including the "MGT" numbering scheme It can be determined. Given CDR regions (e.g., HCDR1, HCDR2, HCDR3, LC) Combining Kabat and Chothia for DR1, LCDR2, or LCDR3 In the combined numbering scheme, in some embodiments, CDR is Choth Along with amino acid residues defined as part of the ia CDR, it is part of the Kabat CDR. Corresponds to an amino acid residue defined as such. When used herein, "Chothi CDRs, defined according to the "a" number scheme, are sometimes also called "hypervariable loops." Under IMGT, the CDR region of the antibody is programmed with IMGT / DomainGap Al This can be determined using ign. Generally, unless otherwise specified, antibody molecules are one or more This may include any combination of Kabat CDRs and / or Chothia CDRs.

[0046] The term "epitope" refers to something that can specifically bind to an immunoglobulin, or It contains any protein determinants that can interact with the molecule. Epitope determinants are Generally, chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains. Therefore, it can have unique three-dimensional structural properties and unique charge properties. The loop can be "linear" or "conformal". Conformal and linear An epitope, for example, loses its binding to the former in the presence of a denaturing solvent, but loses its binding to the latter. They are distinguished by the fact that they do not break.

[0047] "~binds to the same epitope" means using the same epitope mapping technique for comparing antibodies. When used, the antibody, antibody fragment, or other antigenic antibody that binds to the same epitope as the example antibody is used. This refers to the binding ability of the binding portion to the specific antigen. Examples of antibodies and other antibody epitopes are shown. This can be determined using epitope mapping techniques. The law is well known in the art. For example, stereoepitopes include, for example, hydrogen / deuterium. Spatial conformation of amino acids by methods such as exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. It can be easily identified by determining the composition.

[0048] In another embodiment, the present disclosure relates to antibodies or antibody fragments that cross-compete with the antibodies listed in Table 1. Regarding.

[0049] In one embodiment, the present disclosure relates to an antibody or antibody fragment, wherein the antibody or antibody fragment is One or more of the antibodies listed in Table 1 are Kabat, Chothia, IMGT, or Kab. An antibody containing six CDRs defined by either the at / Chothia combination method or It cross-competes with antibody fragments.

[0050] In another embodiment, the present disclosure relates to the same epitope as one of the antibodies listed in Table 1 (for example) If so, an antibody or antibody that binds to it and / or stabilizes it. Regarding fragments.

[0051] In further embodiments, the antibody or its antigen-binding fragment is any of the antibodies listed in Table 1. One Kabat, Chothia, IMGT, or Kabat / Chothia combination Epitope of an antibody or antibody fragment containing six CDRs defined by one of the methods To overlap with an epitope (for example, to bind to it and / or stabilize it) (By) they are joined.

[0052] As used herein, the term "monovalent antibody" refers to an antibody that binds to a single epitope on a target molecule. It refers to antibodies.

[0053] The term "bivalent antibody," as used herein, refers to at least two identical targets. This refers to an antibody that binds to two epitopes on a molecule. A bivalent antibody also binds to two epitopes on a target molecule. A "bivalent antibody" can bridge two different epithelial cells on at least two identical target molecules. It also refers to antibodies that bind to the substance.

[0054] The term "multivalent antibody" refers to a single binding molecule that has two or more binding valencies, and here "binding" is used. The "valency" is described as the number of antigen-binding sites present per molecule of the antibody construct. A single binding molecule can bind to two or more binding sites on the target molecule. Examples of antibodies include bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, and bispecific antibodies. Examples include, but are not limited to, biparatopic antibodies. For example, TREM2 Regarding this, multivalent antibodies (e.g., TREM2 biparatopic antibodies) are each TRE It has a binding portion to two domains of M2.

[0055] The term "multivalent antibody" refers to an antibody that has two or more antigen-binding moieties for two separate target molecules. This also refers to a single binding molecule. For example, an antibody that binds to TREM2 and a non-TREM2 antibody. Two target molecules. In one embodiment, a polyvalent antibody is tetravalent, having four epitope-binding domains. It is an antibody. The tetravalent molecule has bispecificity and is bivalent for each binding site on its target molecule. It is possible.

[0056] The term "bispecific antibody," as used herein, refers to two or more different epidermal antibodies. This refers to an antibody that binds to a tope. In some embodiments, a bispecific antibody is two different antibodies. It binds to the target. In some embodiments, the bispecific antibody binds to two targets on a single target molecule. It binds to different epitopes. It binds to two different epitopes on a single target molecule. These antibodies are also known as "biparatopic antibodies."

[0057] The terms "monoclonal antibody" or "monoclonal antibody composition" used herein refer to... The phrases refer to antibodies that have substantially the same amino acid sequence or originate from the same gene source. This term refers to polypeptides, including bispecific antibodies. It also refers to antibodies with a single molecular composition. Includes molecular preparations. Monoclonal antibody compositions contain a single antibody against a specific epitope. It exhibits specificity and affinity.

[0058] As used herein, the term "human antibody" refers to the framework area and the CDR area. Both contain antibodies with variable regions derived from human sequences. The constant region also contains human sequences. For example, a human germline sequence, or a variant version of a human germline sequence, or, for example, K nappik et al.,(2000.J Mol Biol 296,57-86 As described in ), the consensus framework derived from human framework sequence analysis Derived from antibodies containing the '-' sequence. The structure of immunoglobulin variable domains, such as CDRs, and The location is based on a well-known numbering scheme, for example, the Kabat numbering scheme, Ch Chothia numbering scheme, or combination of Kabat and Chothia, and Im It can be defined using MunoGenTics (IMGT) numbering (for example, S sequences of Proteins of Immunological In terest,USDepartment of Health and Huma n Services(1991), eds.Kabat et al.;Al Laz ikani et al.,(1997)J.Mol.Bio.273:927 948 );Kabat et al.,(1991)Sequences of Protei ns of Immunological Interest,5th edit.,N IH Publication no.91-3242 USDepartment of Health and Human Services;Chothia et al.,(1987)J.Mol.Biol.196:901-917;Chothi a et al., (1989) Nature 342:877-883; and Al-L azikani et al.,(1997)J.Mal.Biol.273:927- 948 and Lefranc, M.-P., The Immunologist, 7 ,132-136(1999);Lefranc,M.-P.et al.,Dev.C omp.Immunol.,27,55-77(2003);Lefranc et a See l., (2015) Nucleic Acids Res. 43, D413-422. I want to be illuminated.

[0059] The human antibody of the present invention contains amino acid residues not encoded by human sequences (e.g., inv By random or site-directed mutagenesis in Toro, or by somatic mutation in vivo This may include mutations introduced, or conservative substitutions to promote stability or production. However, the term "human antibody" as used herein refers to antibodies from another mammal, such as a mouse. This includes antibodies in which a CDR sequence derived from the germline of a species is transplanted onto a human framework sequence. This is not the intention.

[0060] As used herein, the term "recombinant human antibody" refers to a human immunoglobulin gene. From animals (e.g., mice) that are transgenic or transchromosomes A released antibody, or a hybridoma prepared therefrom, is phenotyped to express a human antibody. Converted host cells, e.g., antibodies isolated from transfectomas, recombinant, combi Antibodies isolated from a natural human antibody library, and the entire human immunoglobulin gene. The procedure is performed by any other means, including splicing of part or a portion of the sequence to another DNA sequence. Prepared, expressed, produced, or isolated by recombinant means such as antibodies produced, expressed, manufactured, or isolated. This includes all recombinant human antibodies. Such recombinant human antibodies are included in the framework and CDR. The region has a variable region derived from a human germline immunoglobulin sequence. However, In certain embodiments, such recombinant human antibodies are used for in vitro mutagenesis (or human Ig mutations). When animal transgenic cells are used for the row, they are subjected to in vivo somatic mutagenesis. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are human germline VH and It originates from and is related to the VL sequence, but in vivo it is naturally present within the human antibody germline repertoire. This is an impossible sequence.

[0061] The term "Fc region," as used herein, refers to the constant state of CH3, CH2, and the antibody. Refers to a polypeptide that includes at least a portion of the main hinge region. In some cases, the Fc region. It can contain the CH4 domain present in several antibody classes. The Fc region is It may include the entire hinge region of the constant domain of the antibody. In one embodiment, the present invention relates to the F of the antibody. The c region and the CH1 region are included. In one embodiment, the present invention relates to the Fc region and CH3 region of the antibody. Includes. In another embodiment, the present invention includes an Fc region, CH1 region, derived from the constant domain of the antibody. and includes the Cκ / λ region. In one embodiment, the binding molecule of the present invention has a constant region (e.g., heavy It includes a chain steady region. In one embodiment, such a steady region is compared to the wild-type steady region. It is modified by the following. That is, the polypeptide of the present invention disclosed herein is a three-heavy chain One or more of the normal domains (CH1, CH2, or CH3) and / or the light chain constant domain ( This may include changes or modifications to the CL. Examples of modifications include changes to one or more domains. This includes the addition, deletion, or substitution of one or more amino acids. Such changes are used in effectors. This may be included to optimize performance, half-life, etc.

[0062] As used herein, the term "affinity" refers to the relationship between an antibody and an antigen at a single antigen site. This refers to the strength of the interaction between them. At each antigen site, the variable region of the antibody is numerous. It interacts with the antigen at the site via weak non-covalent bonding forces; the more interactions there are, the greater the affinity. The properties become stronger. When used herein, IgG antibodies or fragments thereof (e.g., Fab fragments) The term "high affinity" in relation to the target antigen is 10 -8 M or less, 10 -9 More than M bottom, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, or 10 -13 M or less This refers to antibodies that have affinity. However, high affinity binding is associated with other antibody isotypes. It can change. For example, high affinity binding to IgM isotypes is 10 -7 M or less , or 10 -8Refers to an antibody having an affinity of less than M.

[0063] As used herein, the terms "Kassoc", "Ka", or "K" on are intended to refer to the association rate of a specific antibody-antigen interaction, while the terms "Kdis", "K" d", or "K" off are intended to refer to the dissociation rate of a specific antibody-antigen interaction In one embodiment, the term "KD" (or "K" D ) is intended to refer to the dissociation constant determined from the ratio of K d to Ka (i.e., Kd / Ka) as used herein, expressed as molar concentration (M). The KD value of an antibody can be determined using methods well established in the art . Methods for determining the KD of an antibody can be by using surface plasmon resonance or by using a biosensor system such as a Biacore® system.

[0064] As used herein, the term "binding activity" refers to a useful measure of the overall stability or strength of an antibody-antigen complex. This is controlled by three main factors: antibody epitope affinity; the valence of both the antigen and the antibody; and the structural arrangement of the interacting parts. Ultimately , these factors define the specificity of the antibody, i.e., the likelihood that a particular antibody is binding to the correct antigen epitope.

[0065] As used herein, the terms "binding specificity" or "specific binding" refer to the ability of an individual antibody binding site to react with one antigen determinant rather than different antigen determinants. The binding site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains It occurs. The binding affinity of an antibody is the reaction between a single antigenic determinant and a single binding site on the antibody strength. It is the sum of the attractive and repulsive forces acting between the antigenic determinant and the binding site of the antibody calculation.

[0066] The terms "treating" and "treatment" mean therapeutic treatment, which is to slow down an undesirable physiological change or disorder in a subject. For the purposes of the present invention, a beneficial or desired clinical outcome is , whether detectable or not, including but not limited to symptom relief, reduction in the degree of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, alleviation or mitigation of the disease state, and remission (partial or complete). "Treatment" may also mean an extension of the survival period compared to the expected survival period if the subject had not received treatment [[ID=1​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0069] "Effective dose" refers to an amount sufficient to produce a beneficial or desired result. For example, therapeutic dose This is the amount that achieves the desired therapeutic effect. This amount is the amount that prevents the onset of the disease or disease symptoms. The effective preventive dose, which is the amount needed, may be the same as or different from the effective preventive dose. The effective dose is administered one or more times. It can be administered in the specified dosage, application, or dose. The "therapeutic effective amount" of the therapeutic compound (i.e.) The effective dose depends on the selected therapeutic compound. The composition is administered at least once a day. It can be administered at least once a week, including every other day. Those skilled in the art will know the severity of the disease or disability. This includes the degree, previous treatment, the subject's general health and / or age, and any other existing medical conditions. , and not limited to these, certain factors are necessary for the treatment of the subject effectively. It will be recognized that this may affect the dosage and timing. Furthermore, as described herein The therapeutically effective dose of the listed therapeutic compound may be used for a single treatment or a series of treatments. It is possible.

[0070] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleotides in single-stranded or double-stranded form. This refers to ribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. It is not particularly limited to these. To the extent that this term is used, it refers to a substance that has similar binding properties to a reference nucleic acid and is a naturally occurring nucleotide. This includes nucleic acids, including known analogues of native nucleotides that are metabolized in a similar manner. Unless otherwise specified, a particular nucleic acid sequence will also have its conservatively modified variants (e.g., degenerate Codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as explicitly indicated. It implicitly includes sequences that are included. Specifically, a degenerate codon substitution includes one or more selected (and In all codons, the third position is replaced by a mixed base and / or a deoxyinosine residue. This can be achieved by generating a sequence (Batzer et al., Nucleo). eic Acid Res.19:5081(1991);Ohtsuka et al. .,J.Biol.Chem.260:2605-2608(1985);and Ross olini et al.,Mol.Cell.Probes 8:91-98(199 4)).

[0071] The terms "peptide," "polypeptide," and "protein" are used interchangeably. This refers to a compound consisting of amino acid residues covalently linked by butyl bonds. A peptide must contain at least two amino acids, and is either a protein or a peptide. There is no limit to the maximum number of amino acids that can be included in the sequence. A polypeptide is a peptide bond. Any peptide or protein containing two or more amino acids linked together by a compound Includes. As used herein, this term is commonly used in the art, for example, peptides. Short chains, also called oligopeptides and oligomers, and proteins, which are commonly referred to in this technology. It refers to both the long chains, also known as the subsequences, and there are many different types. Among the various types of polypeptides, in particular, For example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodiplex Merges, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, It contains fusion proteins. The polypeptide is a natural peptide, a recombinant peptide, or something similar. This includes combinations of these.

[0072] The term "conservative sequence modification" refers to a significant modification of the binding properties of an antibody or antibody fragment containing an amino acid sequence. Refers to amino acid modifications that do not affect or change it. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be made to the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain . Families of amino acid residues having similar side chains are defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid ), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine , tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues within the antibodies or antigen-binding fragments of the present invention can be replaced with other amino acid residues of the same side chain family, and the altered antibodies or antigen-binding fragments can be tested using the functional assays described in the present specification . . .

[0073] The terms "homologous" or "identity" refer to subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules . The subunit positions of both molecules are the same monomeric subunit When occupied by nit, for example, the respective positions of two DNA molecules are adenine If occupied by, they are homologous or identical in their position. Sameness is a direct function of the number of identical or homologous positions; for example, half the number of positions in two arrays ( For example, if the five positions in a polymer of length 10 subunits are homologous, then the two configurations The columns are 50% homologous; 90% of the positions (e.g., 9 out of 10) are identical or homologous. In this case, the two sequences are 90% homologous. The percentage of "sequence identity" is compared to This can be determined by comparing two optimally aligned sequences across the window. The amino acid sequence fragments in the comparison window serve as a reference for optimal alignment of the two sequences. Compared to a sequence (without additions or deletions), additions or deletions (e.g., gaps or o May include bar hangs. The percentage is calculated by both sides to produce the number of matching positions. Determine the number of positions in a column where the same amino acid residue exists, and compare the number of matching positions. Divide by the total number of positions in the window to obtain the percentage of sequence identity. It can be calculated by multiplying by 100. The output is the parsing of the target sequence with respect to the query sequence. It is identity.

[0074] The term "isolated" means modified or removed from its natural state. However, nucleic acids or peptides that are naturally present in living animals are "not isolated," The same nucleic acid or peptide, partially or completely isolated from naturally occurring coexisting substances, is "isolated". "The isolated nucleic acids or proteins exist in a substantially purified form." It can be isolated, or it can exist in a non-natural environment such as a host cell. The antibody contains substantially no other antibodies with different antigen specificities (e.g., TREM2). Isolated antibodies that specifically bind to TREM2 are antibodies that specifically bind to antigens other than TREM2. (Essentially does not contain). However, isolated antibodies that specifically bind to target molecules are other It may exhibit cross-reactivity to the same antigen originating from the same species (for example, it may bind specifically to TREM2). The isolated antibodies can bind to TREM2 molecules from other species. The isolated antibody may be a monoclonal antibody. The isolated antibody is a recombinant monoclonal antibody. It may be present. Furthermore, isolated antibodies substantially affect other cellular material and / or chemical substances. It doesn't have to be included.

[0075] Unless otherwise defined, all technical and scientific terms used herein are defined in accordance with the present invention. This has the same meaning as generally understood by those skilled in the art. The present invention can be carried out using methods and materials similar to or equivalent to those used, however The necessary methods and materials are described below. All publications, patent applications, patents, and this specification Other references mentioned are incorporated herein by reference in their entirety. In some cases, this specification, including definitions, shall prevail. In addition, materials, methods, and examples are provided for reference only. These are merely examples and not limitations.

[0076] Details of one or more embodiments of the present invention are shown in the accompanying drawings and the following description. Other features, purposes, and advantages of the invention are evident from the detailed description and drawings and the claims. It is.

[0077] Antibodies that stabilize functional TREM2 This specification provides antibodies and antigen-binding fragments thereof that stabilize TREM2 on the cell surface. This antibody or its antigen-binding fragment interferes with the proteolytic cleavage of TREM2 and / or by reducing the shedding of the ectodomain of the TREM2 protein Stabilization can be achieved. In some preferred embodiments, the antibody or its anti The proto-binding fragment is the IgSF domain of human TREM2, for example, SEQ ID NOs: 1, 2, or 3. It specifically binds to one of the amino acid residues, from 19 to 132.

[0078] Because a decrease or deficiency of cell surface TREM2 is associated with human neuroinflammatory and neurodegenerative lesions. The TREM2-stabilized antibodies or antigen-binding fragments described herein are used in Alzheimer's disease. Diseases, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple dementia Sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, lupus ( NP-SLE, chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, chronic inflammation Symptomatic demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), Inclusion body myositis, lysosomal storage disorders, for example, sphingomyelin lipidosis (Niemann's disease). Pick's disease type C, mucopolysaccharidosis type II / IIIB, metachromatic leukodystrophy, multifocal motility Neuropathy, myasthenia gravis, neurological Behçet's disease, neuromyelitis optica (NMO), optic nerve Menstrual inflammation, polymyositis, dermatomyositis, Rasmussen encephalitis, Rett syndrome, stroke, transverse myelitis, Neuroinflammatory and neurological conditions such as traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis. It can be used for the treatment, prevention, or diagnosis of degenerative diseases. Some preferred embodiments Therefore, the hTREM2 antibody or its antigen-binding fragment described herein is for Alzheimer's disease. From diseases such as frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease It can be used for the treatment, prevention, or diagnosis of selected diseases.

[0079] Due to its pharmacological profile, the hTREM2 antibody described herein or The antigen-binding fragment is used in CNS-related diseases, PNS-related diseases, systemic inflammation, and other inflammation-related diseases. , pain and various diseases or illnesses such as withdrawal symptoms caused by the abuse of chemicals It is useful in treating the condition. Diseases or disorders related to the CNS include generalized anxiety disorder and cognitive disorders. Disabilities, learning and memory impairments and dysfunctions, Alzheimer's disease (mild, moderate and severe), note Attention Deficit Hyperactivity Disorder, Parkinson's Disease, Parkinsonian Dementia, Huntington's Disease, ALS , prion neurodegenerative disorders such as Creutzfeldt-Jakob disease and Kuru disease, Gilles de Jakob Tourette syndrome, psychosis, depression and depressive disorders, mania, bipolar disorder, schizophrenia, Psychosynchronic cognitive deficit, obsessive-compulsive disorder, panic disorder, eating disorder, narcolepsy, pain perception, etc. Iz dementia, senile dementia, age-related mild cognitive impairment (MCI), age-related memory impairment, Autism, dyslexia, tardive dyskinesia, epilepsy, convulsive disorders, and post-traumatic stress disorder. Transient oxygen deficiency, pseudodementia, premenstrual syndrome, late luteal phase syndrome, chronic fatigue syndrome, and Jet lag is one possible cause.

[0080] The hTREM2 antibodies or their antigen-binding fragments described herein are, in particular, TREM2 Extensive proteolytic cleavage or abnormal or mutated TREM2 receptor variants Autoimmune disorders, inflammatory disorders, or malignancies mediated or associated with the cells presenting It is suitable for the treatment, prevention, or diagnosis of disorders. Examples of autoimmune diseases include, without limitation, joints. Inflammation (for example, rheumatoid arthritis, chronic progressive arthritis) (progestion) and osteoarthritis, as well as inflammatory conditions and bone loss. Rheumatic diseases, including rheumatic diseases, inflammatory pain, and spondyloarthritis, including ankylosing spondylitis. (spondyloarhropathy), Reiter's syndrome, reactive arthritis, psoriatic arthritis Arthritis, enterophatic arthritis, hypersensitivity This includes conditions (including both airway hypersensitivity and skin hypersensitivity) and allergies. Autoimmune diseases Patients include autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, pure red blood cell anemia). (including hematopoietic and idiopathic thrombocytopenia), systemic lupus erythematosus, inflammatory myopathy, polycystic soft tissue Osteitis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active liver Inflammation, myasthenia gravis, psoriasis, Stevens-Johnson syndrome Son syndrome, idiopathic sprue, endocrine eye disorders, Graves' disease, sarco syndrome Idosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (type 1 diabetes), grapes Conjunctivitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis and glomerulonephritis (with and without nephrotic syndrome, e.g., gout, Langerhans syndrome) Cellular histiocytosis, idiopathic nephrotic syndrome or minimal change nephropathy, tumors, inflammatory skin and Corneal diseases, myositis, loosening of bone implants, metabolic disorders, such as atherosclerosis. Examples include diabetes and dyslipidemia.

[0081] The hTREM2 antibody or its antigen-binding fragment described herein is also used for asthma, bronchitis , pneumoconiosis, emphysema, and other idiopathic pulmonary fibrosis or obstructive or inflammatory airways including COPD It is also useful for treating, preventing, or improving diseases.

[0082] The hTREM2 antibody or its antigen-binding fragment described herein is used for acute myeloid leukemia. Chronic myeloid leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, paroxysmal nocturnal hemorrhagic disease Globinuria, Fanconi anemia, severe thalassemia, Viscott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, and other hematopoietic or It can be used to treat malignant disorders of the hematopoietic system.

[0083] The hTREM2 antibody or its antigen-binding fragment described herein is abnormal TREM2 activity To be used to treat any disease or disorder directly or indirectly related to sex and / or manifestation. This is possible. TREM2-related disorders include immune disorders, especially inflammatory disorders (e.g., bacterial infections). Infections, fungal infections, viral infections, protozoan or other parasitic infections, psoriasis, sepsis, cerebral malignancy Lariasis, inflammatory bowel disease, arthritis such as rheumatoid arthritis, folliculitis, impetigo, granuloma, lipoid Pneumonia, vasculitis, and osteoarthritis), autoimmune disorders (e.g., rheumatoid arthritis, Hashimoto's thyroiditis and Graves' disease and other types of thyroiditis, insulin-resistant diabetes, pernicious anemia, Addison's disease, pemphigus Acne, vitiligo, ulcerative colitis, systemic lupus erythematosus (SLE), Sjögren's syndrome, multiple Sclerosis, dermatomyositis, mixed connective tissue disease, scleroderma, polymyositis, allograft rejection, etc. Graft rejection, T-cell damage (e.g., AIDS), allergic inflammatory disorders (e.g., Allergic rhinitis, asthma, psoriasis (and other skin and / or mucosal allergies), neurological disorders, Directly or indirectly related to eye disorders, fetal abnormalities, or abnormal TREM2 activity and / or expression Any other disorder that is accompanied by (e.g., tumors, cancer, leukemia, myeloid diseases, and trauma) It can be listed.

[0084] In some embodiments, TREM2-related disorders include asthma, encephalitis, inflammatory bowel disease, and chronic bowel dysfunction. Obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spine For arthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, or chronic viral or bacterial infections. Therefore, it is selected from the chronic inflammations that result.

[0085] In some embodiments, TREM2-related disorders are associated with dementia, frontotemporal dementia, and Alzheimer's disease. Heimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus Amyotrophic lateral sclerosis, Huntington's disease, taupathies (ease), Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic stroke Enteritis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential Tremor, central nervous system lupus, Behçet's disease, Parkinson's disease, Lewy body dementia, multiple System atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute dispersal Intracellular encephalomyelitis, granulomatous disorder, Lucoidosis, age-related diseases, seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, green Internal eye disease, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, rheumatism Puss, arthritis, multiple sclerosis, low bone density, osteoporosis, bone formation, osteopetrosis, Paget's bone The disease is selected from cancer. In some preferred embodiments, TREM2-related disorders are Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu • Selected from a list of Hakora diseases.

[0086] In some preferred embodiments, TREM2-related disorders include dementia, frontotemporal dementia. The choice is made from Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis. In some embodiments, TREM2-related disorders include frontotemporal dementia, Alzheimer's disease, and hematological disorders. This includes dementias such as vascular dementia, semantic dementia, or Lewy body dementia. In one embodiment, the TREM2-related disorder is Alzheimer's disease. In another preferred embodiment... The disorder is Parkinson's disease.

[0087] Antibodies that specifically bind to human TREM2 and their antigen-binding fragments In one embodiment, this specification specifically describes the IgSF domain of the human TREM2 protein. The antibody to bind or its antigen-binding fragment, for example, a monoclonal antibody or its antigen-binding fragment ( "hTREM2 antibody or its antigen-binding fragment" is provided. This antibody or antigen-binding fragment is , stabilize the TREM2 protein on the cell surface, and / or the TREM2 protein This can reduce ectodomain shedding.

[0088] In some embodiments, the hTREM2 antibody or its antigen binding cleavage provided herein is described herein. The components are heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HC DR3), as well as light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain Includes CDR3 (LCDR3). In some embodiments, the hTR provided herein The EM2 antibody or antigen-binding fragment contains a heavy chain variable region including CDR1, CDR2, and CDR3. (VH), and including the light chain variable region (VL) which includes CDR1, CDR2, and CDR3. In some embodiments, the hTREM2 antibody or antigen-binding fragment provided herein is It includes full-length heavy chain sequences (HC) and full-length light chain sequences (LC).

[0089] Table 1 shows exemplary TREM2 antibodies or antigens that specifically bind to the human TREM2 protein. The arrangement of the combined fragments is shown. Throughout this application, refer to the documents of this specification (e.g., Table 1). In the event of any discrepancy between this specification and the sequence listing, the document in this specification shall prevail.

[0090] [Table 1]

[0091] [Table 2]

[0092] [Table 3]

[0093] [Table 4]

[0094] [Table 5]

[0095] [Table 6]

[0096] Table 7

[0097] Table 8

[0098] Table 9

[0099] Table 10

[0100] Table 11

[0101] Table 12

[0102] Table 13

[0103] Table 14

[0104] Table 15

[0105] Table 16

[0106] Table 17

[0107] Table 18

[0108] Table 19

[0109] Table 20

[0110] Table 21

[0111] Table 22

[0112] Table 23

[0113] Table 24

[0114] Table 25

[0115] Table 26

[0116] Table 27

[0117] [Table 28]

[0118] In some embodiments, the hTREM2 antibody or its antigen-binding fragment is as shown in Table 1. Contains a VH domain having any VH domain amino acid sequence. Other suitable hTRE The M2 antibody or its antigen-binding fragment is mutated, but in the VH domain, as shown in Table 1. The VH region shown in the listed sequence and at least 80, 85, 90, 95, 96, 97, 9 This may contain 8 or 99 percent identical amino acids in certain embodiments. The disclosure includes an antibody or antibody fragment that specifically binds to human ENTPD2 (e.g., an antigen-binding fragment). ) also provides an antibody or its antigen-binding fragment, which can be any one of the HCDRs listed in Table 1. The present invention includes VH CDR having a noacid sequence. In certain embodiments, the present invention is as listed in Table 1. One, two, three, four, five or more having any one amino acid sequence of HCDR Contains (or consists of) VH CDRs exceeding a certain level, which specifically bind to human TREM2. The present invention provides antibodies or antibody fragments (e.g., antigen-binding fragments).

[0119] In some embodiments, the hTREM2 antibody or antibody fragment (e.g., antigen-binding fragment) is It includes a VL domain having any of the VL domain amino acid sequences listed in Table 1. The appropriate anti-human TREM2 antibody or antibody fragment (e.g., antigen-binding fragment) is mutated. However, in the VL domain, the VL region shown in the sequence listed in Table 1 and at least 8 Amino acids having 0, 85, 90, 95, 96, 97, 98, or 99 percent identity This disclosure may include antibodies or antibody fragments that specifically bind to human TREM2 (e.g., Antigen-binding fragments are also provided, and antibodies or antibody fragments (e.g., antigen-binding fragments) are listed in Table 1. The present invention includes a VL CDR having any one amino acid sequence of the LCDR. In particular, the present invention is One, two, three, or any one amino acid sequence of the LCDRs listed in Table 1. Contains (or consists of) VL CDRs exceeding this amount, specifically bound to human TREM2. The present invention provides a binding antibody or antibody fragment (e.g., an antigen-binding fragment).

[0120] Other anti-human TREM2 antibodies or antibody fragments disclosed herein (e.g., antigen-binding fragments) Although it is mutated, in the CDR region, the CDR region shown in the sequence listed in Table 1 Region and at least 80, 85, 90, 95, 96, 97, 98, or 99 percent identity It contains amino acids having the following characteristics. In some embodiments, it is shown in the sequence listed in Table 1. Compared to the CDR area, the CDR area has 1, 2, 3, 4 or 5 or fewer pixels. It contains a mutant amino acid sequence in which the no acid has been mutated.

[0121] Antibodies that specifically bind to human TREM2 and their antigen-binding fragments (VH, VL, and full-length heavy chains) Nucleic acid sequences encoding the full-length light chain, such as the nucleic acid sequences in Table 1, are also provided herein. Such nucleic acid sequences are most suitable for expression in the target host cell, e.g., mammalian cells. It can be optimized.

[0122] Other anti-human TREM2 antibodies disclosed herein may be amino acids or amino acid-encoding antibodies. The nucleic acid is mutated, but at least the sequence shown in Table 1 is different from the sequence shown in Table 1. Those having 80, 85, 90, 95, 96, 97, 98, or 99 percent identity Includes. In some embodiments, the antibody or its antigen-binding fragment exhibits substantially the same therapeutic activity. It is retained, but compared to the variable region shown in the sequence in Table 1, in the variable region It contains a mutant amino acid sequence in which 1, 2, 3, 4, or 5 or fewer amino acids are mutated.

[0123] Each antibody provided binds to human TREM2, so VH, VL, full-length light chain, and full-length The heavy chain sequence (the amino acid sequence encoded by the amino acid sequence and nucleotide sequence) is: By "mixing and matching," other TREM2-binding antibodies disclosed herein can be produced. A "mixed and matched" TREM2-binding antibody like the one described above is a known binding assay in the art. Testing can be performed using (for example, ELISA, the assay described in the examples). When the strands are mixed and matched, the VH sequence derived from a particular VH / VL pairing is structurally It should replace a VH sequence similar to the above. Long heavy chain sequences should be replaced by structurally similar full-length heavy chain sequences. Specific VH / VL VL sequences derived from pairing should be replaced by structurally similar VL sequences. The full-length light chain arrangement derived from heavy chain / full-length light chain pairings is replaced by structurally similar full-length light chain arrangements. It should be bad.

[0124] Therefore, in one embodiment, the present invention is selected from any one of sequence numbers 13 and 50. A heavy chain variable region (VH) containing the amino acid sequence and any one of SEQ ID NOs. 24 and 61 Isolated monoclonal having a light chain variable region (VL) containing a selected amino acid sequence. A monoantibody or its antigen-binding fragment; the antibody specifically binds to human TREM2. The present invention provides a clonal antibody or its antigen-binding fragment.

[0125] In another embodiment, the present invention includes: (i) Sequence IDs 15, 29, 33, 35, 37, 39, Total weight containing any one of 52, 66, 70, 72, 74, or 76 amino acids The full-length chain (HC); and an amino acid selected from any one of SEQ ID NOs: 26 and 63. (ii) an isolated functional protein having a light chain (LC); or (ii) an antigen-binding fragment thereof. We provide monoclonal antibodies.

[0126] In another embodiment, this disclosure relates to the heavy chain CDR1, CDR2, and CDR3 listed in Table 1. Human TREM2 including light chains CDR1, CDR2, and CDR3, or combinations thereof. The present invention provides a conjugated antibody or an antibody fragment thereof. The amino acid sequence of the HCDR1 of the antibody is SEQ ID NO: 4 The amino acid sequence of the antibody HCDR2 is shown in 7, 8, 10, 41, 44, 45, and 47. This is shown in SEQ ID NOs. 5, 9, 11, 41, 45, and 47. The amino acids of the antibody HCDR3. The sequences are shown in SEQ ID NOs: 6, 12, 43, and 49. The amino acid sequence of the antibody LCDR1 is: This is shown in SEQ ID NOs: 17, 20, 23, 54, 57, and 60. The amino acid of the antibody's LCDR2. The acid sequences are shown in SEQ ID NOs: 18, 21, 55, and 58. The amino acid sequence of the antibody LCDR3 is shown below. The columns are shown in sequence numbers 19, 22, 56, 59, and 56.

[0127] Each antibody binds to human TREM2, and its antigen-binding specificity is primarily CDR1, C Provided by the DR2 and CDR3 regions, and the VH CDR1, CDR2, and CDR3 sequences. Assuming this is the case, the VL CDR1, CDR2, and CDR3 sequences can be "mixed and matched". (That is, CDRs derived from different antibodies can be mixed and matched), but each antibody is, To produce other human TREM2-binding antibodies disclosed in the document, VH CDR1, CD It must include R2, CDR3, and VL CDR1, CDR2, and CDR3. Such "mixed and matched" TREM2-binding antibodies are known in the art and described in the examples. It can be tested using the listed binding assay (e.g., ELISA). VH C When DR sequences are mixed and matched, CDR1, CDR2 and / or CDR1 are derived from a specific VH sequence. The CDR3 sequence should be replaced by a structurally similar CDR sequence. Similarly, VL When CDR sequences are mixed and matched, CDR1, CDR2 and / or CDR1 derived from a specific VL sequence are used. Alternatively, the CDR3 sequence should be replaced by a structurally similar CDR sequence. Novel VH and The VL sequence contains one or more VH and / or VL CDR region sequences, monoclonal according to the present invention. The NAL antibody is substituted with a structurally similar sequence derived from the CDR sequence shown herein. It will be readily apparent to those skilled in the art that it can be manufactured by doing so.

[0128] Therefore, this disclosure is a group consisting of Sequence IDs 4, 7, 8, 10, 41, 44, 45, and 47. Heavy chain CDR1 containing selected amino acid sequences; SEQ ID NOs: 5, 9, 11, 42, 46, 4 Heavy chain CDR2 containing an amino acid sequence selected from a group of 8; SEQ ID NOs: 6, 12, 43 , heavy chain CDR3 containing an amino acid sequence selected from a group of 49; SEQ ID NOs: 17, 20 Light chain CDR1 containing an amino acid sequence selected from the group consisting of 23, 54, 57, and 60; Light chain CD containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 21, 55, and 58. R2; and amino acid combinations selected from the group consisting of SEQ ID NOs: 19, 22, 56, 59, and 56. Provides an isolated monoclonal antibody or its antigen-binding region containing a light chain CDR3 including a column. The antibody specifically binds to human TREM2.

[0129] In certain embodiments, the antibody that specifically binds to human TREM2 is the antibody listed in Table 1. It is either a whole body or an antibody fragment (e.g., an antigen-binding fragment).

[0130] In some embodiments, an antibody or its antigen-binding region specifically binds to human TREM2. The region is the heavy chain complementarity determination region 1 (HC) containing the amino acid sequence of SEQ ID NOs: 4, 7, 8, or 10. DR1); Heavy chain complementarity determination region 2 (HC) containing the amino acid sequence of SEQ ID NO: 5, 9, or 11 DR2); Heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 6 or 12 ); SEQ ID NOs: 17, 20, or 23 Light chain complementarity determination region 1 (LCDR1); SEQ ID NOs: 18 or light chain complementarity determination region 2 (LCDR2) containing 21 amino acid sequences; and SEQ ID NO: 19 Alternatively, it may include a light chain complementarity determination region 3 (LCDR3) containing a sequence of 22 amino acids.

[0131] In some embodiments, an antibody or its antigen-binding region specifically binds to human TREM2. The region contains the amino acid sequence of SEQ ID NO: 41, 44, 45, or 47 of HCDR1; SEQ ID NO: HCDR2 containing amino acid sequences 42, 46, or 48; amino acid sequence number 43 or 49 HCDR3 containing an acid sequence; LCDR containing the amino acid sequence of SEQ ID NOs. 54, 57, or 60. 1;LCDR2 containing the amino acid sequence of SEQ ID NO: 55 or 58; and SEQ ID NO: 56 or 59 It contains LCDR3, which has the amino acid sequence of the following.

[0132] In some embodiments, an antibody or its antigen-binding region specifically binds to human TREM2. The region is the amino acid sequence of SEQ ID NO: 13 (or at least approximately 90%, 95%, 99%). This refers to the same and / or one, two, three or more substitutions, insertions, or deletions beyond that. The heavy chain variable region (VH) containing a sequence that is either a modified or otherwise modified sequence, and the amino acid sequence of SEQ ID NO: 24 (or at least about 90%, 95%, 99%, or more than that, identical, and / or Light chains containing sequences having one, two, three or more substitutions, insertions, deletions or alterations. Includes a variable region (VL).

[0133] In some embodiments, an antibody or its antigen-binding region specifically binds to human TREM2. The range is SEQ ID NO: 50 (or at least approximately 90%, 95%, 99%, or more than that). Having the same, and / or one, two, three or more substitutions, insertions, deletions or alterations A heavy chain variable region (VH) containing the amino acid sequence of (or a similar sequence), and SEQ ID NO: 61 (or a similar sequence). At least 90%, 95%, 99%, or more than that, identical, and / or one, two, three Light chain containing an amino acid sequence (a sequence having one or more substitutions, insertions, deletions, or modifications) Includes a variable region (VL).

[0134] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 15 Amino acid sequence (or at least approximately 90%, 95%, 99%, or more identical thereto) , and / or sequences having one, two, three or more substitutions, insertions, deletions or alterations ) including a heavy chain, and the amino acid sequence of SEQ ID NO: 26 (or at least about 90%, 95%) %, 99% or more identical, and / or one, two, three or more substitutions Includes a light chain containing a sequence that has been inserted, deleted, or modified.

[0135] In some embodiments, the antibody that specifically binds to human TREM2 is the antibody of SEQ ID NO: 29 Amino acid sequence (or at least approximately 90%, 95%, 99%, or more identical thereto) , and / or sequences having one, two, three or more substitutions, insertions, deletions or alterations ) including a heavy chain, and the amino acid sequence of SEQ ID NO: 26 (or at least about 90%, 95%) %, 99% or more identical, and / or one, two, three or more substitutions Includes a light chain containing a sequence that has been inserted, deleted, or modified.

[0136] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 33( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 26 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0137] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 35( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 26 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0138] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 37( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 26 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0139] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 39( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 26 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0140] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 52( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 63 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0141] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 66( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 63 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0142] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 70( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 63 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0143] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 72( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 63 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0144] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 74( Or at least about 90%, 95%, 99%, or more than that, identical, and / or 1 amino acid sequences having one, two, three or more substitutions, insertions, deletions or modifications Heavy chains including columns, and SEQ ID NO: 63 (or at least about 90%, 95%, 99% or Beyond that, identical, and / or one, two, three or more substitutions, insertions, deletions or It includes a light chain containing an amino acid sequence (which has been modified).

[0145] In some embodiments, the antibody that specifically binds to human TREM2 is SEQ ID NO: 76 Amino acid sequence (or at least approximately 90%, 95%, 99%, or more identical thereto) , and / or sequences having one, two, three or more substitutions, insertions, deletions or alterations ) including the heavy chain, and the amino acid sequence of SEQ ID NO: 63 (or at least about 90%, 95%) %, 99% or more identical, and / or one, two, three or more substitutions Includes a light chain containing a sequence that has been inserted, deleted, or modified.

[0146] In some embodiments, the present invention measures, for example, by surface plasmon resonance (SPR). Then, the dissociation constant (K) is less than 200 pM. D ), for example, less than 150 pM, less than 120 pM, Less than 100 pM, less than 90 pM, less than 70 pM, less than 50 pM, less than 40 pM, 30 pM K is less than 20 pM, or less than 10 pM. D The TREM2 protein IgSF domain The present invention provides an antibody or antigen-binding fragment thereof that binds to an antigen. In some preferred embodiments, The antibodies or antigen-binding fragments provided herein have a dissociation constant (K) of less than 50 pM. D ) with TR It binds to the IgSF domain of the EM2 protein. In some preferred embodiments, The antibody or antigen-binding fragment provided in the specification has a dissociation constant (K) of less than 5 pM. D ) TREM It binds to the IgSF domain of protein 2.

[0147] Once the desired epitope on the antigen has been determined, for example, using the technique described in this invention It becomes possible to generate antibodies against that epitope. Alternatively, during the discovery process, Antibody generation and characterization can elucidate information about the desired epitope. Subsequently, this information The report suggests that it is possible to competitively screen antibodies for binding to the same epitope. This is the result. An approach to achieve this is to use antibodies that bind competitively to each other, for example, anti This involves conducting cross-competition tests to identify antibodies that compete for binding to the original substance. A high-throughput process for "binning" antibodies based on cross-competition has been filed for international patent application. It is described in the international publication no. 2003 / 48731. The epitope is an antibody. It may contain residues to which it binds.

[0148] Generally, antibodies specific to a particular target antigen are complex mixtures of proteins and / or macromolecules. It preferentially recognizes epitopes on target antigens within a material.

[0149] A given region of polypeptide containing an epitope can be any number of epitopes known in the art. It can be identified using Epitope Mapp. For example, Epitope Mapp ing Protocols in Methods in Molecular Bi ology, Vol. 66 (Glenn E. Morris, Ed., 1996, Hum See ANA Press, Totowa, NJ. For example, linear epitopes. For example, a large number of peptides (peptides corresponding to parts of protein molecules) are supported by a solid support. The peptide is synthesized simultaneously, and while the peptide is still bound to the support, the antibody reacts with the peptide. This can be determined by doing so. Such techniques are known in the art, for example, U.S. Patent No. 4,708,871; Geysen et al., (1984)P roc.Natl.Acad.Sci.USA 8:3998-4002;Geysen et al.,(1985)Proc.Natl.Acad.Sci.USA 82: 78-182;Geysen et al.,(1986)Mol.Immunol.2 It is described in 3:709-715. Similarly, conformational epitopes are, for example Furthermore, by methods such as X-ray crystallography and two-dimensional nuclear magnetic resonance, the spatial conformation of amino acids can be determined. It can be easily identified by determining the epitope mapping. For example, the epitope mapping described above See Rotol. The antigenic region of the protein also has standard antigenic and hydrophobic properties. For example, O available from Oxford Molecular Group. Using calculations performed with the MIGA version 1.0 software program, etc. This allows for identification. This computer program helps in determining the antigenic profile. Therefore, see the Hopp / Woods method, Hopp et al., (1981) Proc.Natl.Acad.Sci USA 78:3824-3828; and hydrophobic Regarding sex plots, see the Kyte-Doolittle technique, Kyte Use et al., (1982) J.Mol.Biol.157:105-132; In some embodiments, the anti-TREM2 antibody is the IgSF domain of human TREM2. It specifically binds to the epitope located there. For example, anti-TREM2 antibodies, SEQ ID NOs: 1, 2, Or, specifically target an epitope located within the range of 19-132 amino acid residues in any one of the three categories. They can be combined.

[0150] The antibody molecule may be a polyclonal or monoclonal antibody. This is done by hybridoma technology, or by phage display or combinatorics. It can be manufactured by other methods such as the Lu method.

[0151] Phage display and combinatorial methods for generating antibodies are described in the Art of the Art. This is known (for example, Ladner et al., U.S. Patent No. 5,223,409). Detailed notes; Kang et al. International Publication No. 92 / 18619 pamphlet; Dower et al. International Publication No. 91 / 17271 Pamphlet; Winter et al. International Publication No. 92 / 20791 pamphlet; Markland et al. International Publication Brochure No. 92 / 15679; Breitling et al. International Publication No. 9 Pamphlet No. 3 / 01288; McCafferty et al. International Publication No. 92 / Pamphlet No. 01047; Garrard et al. International Publication No. 92 / 09690 Pamphlet No.; Ladner et al. International Publication No. 90 / 02809 Pamphlet Fuchs et al. (1991) Bio / Technology 9:137 0-1372;Hay et al.(1992)Hum Antibod Hybri domas 3:81-85;Huse et al.(1989)Science 2 46:1275-1281;Griffths et al.(1993)EMBO J 12:725-734; Hawkins et al. (1992) J Mol Bi ol 226:889-896;Clackson et al.(1991)Natu re 352:624-628;Gram et al.(1992)PNAS 89: 3576-3580;Garrad et al.(1991)Bio / Technol ogy 9:1373-1377;Hoogenboom et al.(1991)N uc Acid Res 19:4133-4137; and Barbas et al. (As described in PNAS 88:7978-7982, 1991).

[0152] In one embodiment, the antibody is a human antibody (for example, an antibody produced from a human immunoglobulin sequence). Antibodies produced in genetically modified mice that have been genetically engineered to do so, or non-Hyperbolic antibodies. Antibodies (e.g., rodents (mice or rats), goats, primates (e.g., monkeys), camels) It is an antibody.

[0153] Chimeric and / or humanized antibodies are produced in non-human subjects or are derived from non-human antibodies. The genes are engineered to minimize the immune response by human patients to antibodies derived from gene expression. This is possible. Chimeric antibodies contain a variable region of a non-human animal antibody and a constant region of a human antibody. These antibodies retain the epitope-binding specificity of the original monoclonal antibody, but when administered to humans... In this case, the immunogenicity is lower, and therefore, the patient is more likely to tolerate it. For example, one or all of the variable regions of the light chain of a mouse antibody (e.g., a mouse monoclonal antibody) (For example, one, two, or three), and / or one or all of the variable regions of the heavy chain (e.g.) (For example, one, two, or three) each represent a human constant region, for example, non-limitingly IgG1 Although it can be linked to the human constant region, chimeric monoclonal antibodies are recombinant antibodies known in this art. It can be produced by DNA technology. For example, genes that encode the constant region of a non-human antibody molecule. The gene can be replaced with a gene encoding a human constant region (Robinson et al. l., PCT Patent Publication No. PCT / US86 / 02269; Akira, et a l., European Patent Application No. 184, 187; or Taniguchi, M., European (See Patent Application No. 171,496). In addition, to generate chimeric antibodies Other suitable techniques that may be used for this purpose are, for example, U.S. Patent No. 4,816,567; Specification No. 4,978,775; Specification No. 4,975,369; and Specification No. 4,81 It is described in Specification No. 6,397.

[0154] Chimeric antibodies replace the variable region that is not involved in antigen binding with an equivalent portion derived from the human variable region. It can be further "humanized" by replacing it with a minute. Humanized antibodies are heavy chain and / or This is the non-human (e.g., mouse, rat, or hamster) complementarity-determining region (CDR) of the light chain. The variable region includes one or more human framework regions. Several embodiments Therefore, humanized antibodies contain sequences that are completely human except for the CDR region. Humanized antibodies are, Typically, compared to non-humanized antibodies, they exhibit lower immunogenicity against humans, and therefore, in certain conditions... In this context, it offers therapeutic advantages. Humanized TREM2 antibodies are used in methods known in the art. It can be generated using the following method. For example, Hwang et al., Methods 36: 35,2005;Queen et al.,Proc.Natl.Acad.Sci. USA86:10029-10033,1989;Jones et al.,N ature 321:522-25,1986;Riechmann et al.,N ature 332:323-27,1988;Verhoeyen et al.,S science 239:1534-36,1988;Orlandi et al.,P roc.Natl.Acad.Sci.USA86:3833-3837,198 9; U.S. Patent No. 5,225,539; U.S. Patent No. 5,530,101; U.S. Patent No. 5 ,585,089 specification; 5,693,761 specification; 5,693,762 Specification No. 6,180,370; and International Publication No. 90 / 07861 Please refer to the brochure.

[0155] Human TREM2 antibodies can be produced using methods known in the art. For example, human Humaneering technology is used to convert non-human antibodies into engineered human antibodies. It was used. U.S. Patent Application Publication No. 20050008625 describes the use of non-human antibodies. The variable region is replaced with the human variable region in the antibody, while maintaining the same binding properties as that of a non-human antibody. To maintain or provide better binding characteristics compared to those of non-human antibodies The in vitro method is described below. This method involves epitoting the variable region of a non-human reference antibody with a fully human antibody. It depends on inducible substitution. The resulting human antibody is generally structurally different from the reference non-human antibody. Although unrelated, it binds to the same epitope on the same antigen as the reference antibody. In short, it binds to the same epitope. The epitope-induced complementary substitution approach is a reporter that responds to the binding of the test antibody to the antigen. In the presence of the system, a variety of "competitors" and reference antibodies are needed to bind to a limited amount of antigen. By setting up competition in cells between the brid library ("test antibody") This becomes possible. The competitor may be a reference antibody or its derivative, such as a single-stranded Fv fragment. The competitor is also a natural or artificial ligand of the antigen that binds to the same epitope as the reference antibody. It is possible. The only requirement for a competitor is that it binds to the same epitope as the reference antibody, and This is because it competes with the reference antibody for antigen binding. The test antibody is a non-human reference antibody. They share a common antigen-binding V region and a diverse range of human antibody repertoire libraries. It has other V regions randomly selected from the source. The common V region from the reference antibody is guide It functions as a selector, placing the test antibody on the same epitope on the antigen in the same orientation. It exhibits a bias towards the highest antigen-binding fidelity relative to the reference antibody.

[0156] Many types of reporter systems are used to detect desired interactions between test antibodies and antigens. This can be done, for example, by binding complementary reporter fragments to the antigen and test antibody, respectively. By doing so, the reporter activity via fragment complementation occurs only when the test antibody binds to the antigen. Sexualization can be made to occur. The test antibody and antigen-reporter fragment fusion compete. When co-expressed with the body, reporter activation is proportional to the affinity of the test antibody for the antigen. This becomes dependent on the ability of the test antibody to compete with other competitors. Other reporter systems that can be used This includes the specification of U.S. Patent Application No. 10 / 208,730 (Publication No. 20030198971). Reactivation of the self-inhibitory reporter reactivation system (RAIR) as disclosed in the specification. Factor, or U.S. Patent Application No. 10 / 076,845 (Publication No. 2003015757) This includes the competitive activation systems disclosed in Specification No. 9.

[0157] Using a serial epitope-induced complementary substitution system, selection is performed to identify competitors, antigens, and reports. Identify cells that express a single test antibody along with the ter component. In these cells, each The test antibody competes one-to-one with competitors for binding to a limited amount of antigen. The activity of the test antibody is proportional to the amount of antigen bound to the test antibody, and this is then proportional to the amount of antigen the test antibody has against the antigen. It is proportional to the affinity of the body and the stability of the test antibody. The test antibody is expressed as a test antibody. Initially, the selection is based on their activity relative to the activity of the reference antibody. Wound's results are a set of "hybrid" antibodies, each derived from a reference antibody. It consists of the same non-human V region and human V regions from the library, each of which is referenced The antibody binds to the same epitope on the antigen as the antibody. Hybrid antibody selected in the first round. One or more of these are parental to antigens with affinity comparable to or higher than that of the reference antibody. It has a harmonious nature.

[0158] In the second V-region replacement step, the human V-region selected in the first step is replaced with that of a congener human. Selection of human substitutions for the remaining non-human reference antibody V region, which has a diverse library of V region libraries. Use as a guide for selection. The hybrid antibodies selected in the first round are also It can be used as a competitor for the second round of selection. The result of the second round of selection is, A fully human antibody that is structurally different from the reference antibody but competes with the reference antibody for binding to the same antigen. It is a set of body antibodies. Some of the selected human antibodies are the same epidermal antigen as the reference antibody. It binds to the tope. Among these selected human antibodies, one or more have the affinity of the reference antibody. It binds to the same epitope with comparable or higher affinity.

[0159] In some embodiments, the present invention binds to the human TREM2 protein, and TREM It promotes 2-dependent physiological activity, for example, phagocytosis (for example, in hM2A macrophages). or in human iPS-derived microglia-like cells, or in microglia / macro in the brain Enhances phages and increases chemotaxis in human iPS-derived microglia-like cells. It strengthens, increases NFAT-driven reporter gene activity in human monocytic cell lines, and This refers to antibodies that increase Syk phosphorylation in hM2A macrophages or their antigen binding cleavage. Provides a piece. This enhancement and / or boost is, for example, at least 10%, at least 20% %, at least 30%, at least 40%, at least 50%, at least 60%, less It could be at least 70%, at least 80%, or at least 90%.

[0160] Manipulated and modified antibodies The antibodies of the present invention are further modified using the starting materials listed in Table 1. It may be prepared using an antibody having one or more VH and / or VL sequences, and this modified antibody is The antibody may have altered properties from the starting antibody. The antibody may have one or both variable regions (i.e., V Within H and / or VL), for example, within one or more CDR regions and / or one or more frames It can be manipulated by modifying one or more residues within the work region. In addition, Instead, the antibody, for example, alters the effector function of the antibody, in the constant region. This can be manipulated by altering residues within the region.

[0161] One type of variable region manipulation that can be performed is CDR implantation. The antibody is 6 Targeted antibodies primarily through amino acid residues located in the heavy and light chain complementarity-determining regions (CDRs). It interacts with the progenitor. For this reason, the amino acid sequence inside the CDR is different from the sequence outside the CDR. It is more diverse among individual antibodies than CDR sequences. CDR sequences are involved in most antibody-antigen interactions. Therefore, the specially implanted framework sequences from different antibodies having different properties are given By constructing an expression vector containing a CDR sequence from heterologous natural antibodies, specific natural It is possible to express recombinant antibodies that mimic the characteristics of an antibody (for example, Riechm ann, L. et al., 1998 Nature 332:323-327;Jon es,P.et al.,1986 Nature 321:522-525;Quee n,C.et al.,1989 Proc.Natl.Acad.,USA86 :10029-10033; U.S. Patent No. 5,225,539 to Winter, Also, U.S. Patent No. 5,530,101 to Queen et al.; and No. 5,585,08 See Specification No. 9; Specifications No. 5,693,762 and No. 6,180,370. .)

[0162] Such framework sequences include germline antibody gene sequences or reconstituted antibody sequences. This can be obtained from public DNA databases or published references. For example, human heavy chain and The germline DNA sequences of light chain variable region genes are from the "VBase" human germline sequence data. Base (www.mrc-cpe.cam.ac.uk / vbase on the internet) (Available at) and Kabat, EA, et al., 1991 Sequenc es of Proteins of Immunological Interest ,Fifth Edition,USDepartment of Health and Human Services,NIH Publication No.91 -3242;Tomlinson,IM,et al.,1992 J.fol.B iol.227:776-798; and Cox, JP Let al., 1994. Eur.J Immunol.24:827-836, each referred to herein by reference. Explicitly incorporated; for example, germline DNA of human heavy chain and light chain variable region genes. The sequence and rearranged antibody sequences are from the "IMGT" database (www.imgt.org). Available online; Lefranc, MP et al., 1999 Nucle Please refer to eic Acids Res.27:209-212, each of which is a reference. (More explicitly incorporated herein) can be found in:

[0163] Examples of framework sequences for use in the antibody and antigen-binding fragment of the present invention are: , framework sequence used by the selected antibody and its antigen-binding fragment of the present invention , for example, consensus sequences used by the monoclonal antibody of the present invention and / or It is structurally similar to the framework sequence. VH CDR1, 2, and 3 sequences, and Furthermore, the VL CDR1, 2, and 3 sequences are derived from germline immunoglobulins (GDLs). It can be transplanted onto a framework region having the same sequence as the sequence found in the brin gene. The CDR sequence is a framework region containing one or more mutations compared to the germline sequence. It can be transplanted onto the region. For example, in some cases, by mutating residues within the framework region. It has been found that maintaining or enhancing the antigen-binding ability of antibodies is beneficial (for example, U.S. Patent No. 5,530,101 to Queen et al.; U.S. Patent No. 5,585,089 to Queen et al. See details; specifications No. 5,693,762 and No. 6,180,370).

[0164] Another type of variable region modification is VH and / or VL CDR1, CDR2 and / or C By mutating amino acid residues within the DR3 region, a process known as "affinity maturation" is achieved. The objective is to improve one or more binding properties (e.g., affinity) of the target antibody. Mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and antibodies The effects on bonding, or other functional properties of interest, are described herein and provided in the examples. It can be evaluated in vitro or in vivo assay. Conservative modification (above Mutations such as those described above can be introduced. Mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically, one, two, three, four, or five or fewer residues within the CDR region are modified. It will be changed.

[0165] The resulting polypeptide contains at least one binding region that specifically binds to TREM2. As long as it is possible, a wide variety of antibody / immunoglobulin frameworks or scaffolds can be used. This framework or scaffolding is based on the five major idioms of human immunoglobulins. The type, comprising its antigen-binding fragment, preferably having a humanized aspect, is immune to other animal species. It contains disease globulins. Single heavy-chain antibodies, such as those identified in camelids, are particularly interesting in this respect. Deeply flavorful. Novel frameworks, scaffolding, and fragments continue to be discovered and developed by those skilled in the art. It is.

[0166] In one embodiment, the present invention provides a non-immunoglobulin scaffold on which the CDR of the present invention can be implanted. This describes a method for generating non-immunoglobulin-based antibodies using the target TREM2 tangent. Known or future non-immunoglobulin frameworks, insofar as they include protein-specific binding regions. And scaffolding can be used. Known non-immunoglobulin frameworks or scaffolding include , Fibronectin (Compound Therapeutics, Inc., Wal Tham, Mass.), Ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibody (Domantis, Ltd.) , Cambridge, Mass., and Ablynx nv., Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freis (ing, Germany), small modular immunotherapy drug (Trubion Pharmac euticals Inc., Seattle, Wash.), Maxi Body (Avid ia, Inc., Mountain View, Calif.), Protein A (Aff ibody AG (Sweden), and affilin (γ-crystallin or ubiquitin) (SciI Proteins GmbH, Halle, Germany) is included, but , but not limited to these.

[0167] The fibronectin scaffold consists of fibronectin type III domains (for example, fibronectin Based on the 10th module of tin type III (10 Fn3 domain). The type III domain has 7 or 8 β-chains distributed between two β-sheets, and these These components then pack together to form the protein core, and furthermore, their β-chains are linked to each other. It includes a loop (similar to a CDR) that is exposed to the solvent. Each edge of the β-sheet sandwich has a small amount There are at least three such loops, where the edges are protein boundaries perpendicular to the direction of the β chain. This is the world (see U.S. Patent No. 6,818,418). These fibronectin The scaffolding of the hyphen is not immunoglobulin, but the overall folding is based on camel and llama IgG. The smallest functional antibody fragment containing the entire antigen recognition unit, the folding and tightness of the variable region of the heavy chain. It is related to the connection. Due to this structure, non-immunoglobulin antibodies have different properties and affinity. These scaffolds mimic similar antigen-binding properties. These scaffolds improve the affinity of antibodies in vivo. In in vitro loop randomization and shuffling strategies similar to the maturation process These fibronectin-based molecules have a standard loop region. It can be used as a scaffold that can be replaced with the CDR of the present invention using cloning technology.

[0168] Camelid antibodies Camels and dromedary camels (two-humped camels (Camelus bactrianus) ) and the men of the dromedary camel (Calelus dromaderius) family Obtained from bar, Llama species (alpaca (Lama paccos), Lama (Lama New world men such as the genera *glama* and *Lama vicugna*. Antibody proteins containing bars are related to size, structural complexity, and antigenicity in relation to human subjects. It is characterized by a specific Ig derived from this mammalian family found in nature. G antibodies lack light chains, and therefore, antibodies from other animals have two heavy chains and two light chains. It is structurally different from a typical four-chain quaternary structure with chains. (PCT / EP93 / 02214) See International Publication No. 94 / 04678 (published March 3, 1994). I want to.

[0169] The region of camelid antibodies, identified as a small single variable domain called VHH, is a genetically modified domain. It produces small proteins obtained through genetic manipulation that have high affinity for the target, As a result, low molecular weight antibody-derived proteins known as "camelid nanobodies" are produced. See U.S. Patent No. 5,759,808, issued June 2, 1998. tijlemans,B.et al.,2004 J Biol Chem 279: 1256-1261;Dumoulin,M.et al.,2003 Nature 424:783-788;Pleschberger, M. et al.2003 Bi oconjugate Chem 14:440-448;Cortez-Retamo zo,V.et al.2002 Int J Cancer 89:456-62; Lauwereys, M. et al.1998 EMBO J 17:3512-3 See also 520. Manipulated libraries of camel antibodies and antibody fragments are, for example, A It is commercially available from blynx, Ghent, and Belgium. Other antibodies of non-human origin and Similar to the antigen-binding fragments of bison, the amino acid sequence of camelid animal antibodies is recombinantly modified, By obtaining a sequence that is more closely similar to the human sequence, it is possible to "humanize" the nanobody. Therefore, the natural low antigenicity of camelid antibodies against humans can be further enhanced. It can be reduced.

[0170] The nanobodies of camelid animals have a molecular weight about one-tenth that of human IgG molecules, and are proteins. The particles have a physical diameter of only a few nanometers. One result of their small size is Pilum nanobodies bind to functionally invisible antigen sites on larger antibody proteins. The ability to combine, that is, camelid nanobodies, using classical immunological techniques And, using possible therapeutic agents, as reagents to detect the otherwise potential antigen Therefore, it is useful. Thus, yet another result of the small size is that camelid nanobodies are targeted. Inhibition as a result of binding to specific sites in grooves or narrow cracks of target proteins. Therefore, it is possible to achieve this, and thus the function is more closely related to that of classical low molecular weight drugs than to that of classical antibodies. It can be useful in the ability to imitate.

[0171] Furthermore, the low molecular weight and compact size make the camelid nanobodies extremely thermally stable. It is stable against extreme pH and proteolytic digestion, and has poor antigenicity. As a result, camelid nanobodies can easily move from the circulatory system to tissues. It can even cross the blood-brain barrier and treat damage affecting nerve tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. 20 Refer to U.S. Patent Application Publication No. 20040161738, published on August 19, 2004. These characteristics, combined with low antigenicity to humans, offer significant therapeutic potential. These molecules demonstrate the potential for interaction with prokaryotic cells such as E. coli. It can be fully expressed in and expressed as a fusion protein with a bacteriophage, and It is capable.

[0172] Therefore, a feature of the present invention is a camelid antibody having high affinity for TREM2 or This is a nanobody. In one embodiment of this specification, in camelid animals, camelid antibodies Alternatively, nanobodies are naturally produced, i.e., the techniques described herein for other antibodies. Using the technique, camelids are immunized with TREM2 or its peptide fragments. It is produced. Alternatively, TREM2-bound camelid nanobodies are manipulated, i.e., for example. For example, as described in the examples of this specification, panning using TREM2 as a target Using the sequence, we present appropriately mutagenic camelid nanobody proteins. It is produced by selection from the library of elements. The manipulated nanobody is then processed by the recipe. In the target population, genetic manipulation was performed to give the half-life of 45 minutes to 2 weeks. It can be tamized. In certain embodiments, the camelid antibody or nanobody is, for example, P As described in CT / EP93 / 02214, the heavy or light chain of the human antibody of the present invention Grafting the CDR sequence onto a nanobody or single-domain antibody framework sequence. It is obtained by [method].

[0173] Bispecific molecules and polyvalent antibodies In another aspect, the present invention relates to a bispecificity comprising the TREM2-binding antibody or a fragment thereof. Alternatively, it is characterized by a multispecific molecule. The antibody of the present invention, or its antigen-binding region, is at least To generate bispecific molecules that bind to two different binding sites or target molecules, another mechanism Potential molecules, for example, another peptide or protein (for example, another antibody against a receptor or The antibodies of the present invention can be derivatized or linked to a ligand. To generate a multispecific molecule that binds to the binding site and / or target molecule, two or more other Such multispecific molecules can be derivatized or linked to functional molecules; also, according to this specification It is intended to be encompassed by the term "bispecific molecule" as used in this context. In order to generate a bispecific molecule, the antibody of the present invention is designed to produce a bispecific molecule. , functioning with one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic. They can be coupled (e.g., by chemical coupling, genetic fusion, non-covalent bonding, or other methods). (by).

[0174] Therefore, the present invention provides at least one first binding specificity to TREM2 and a second It includes a bispecific molecule that has a second binding specificity to a target epitope. For example, the second The target epitope is a different epitope of TREM2, distinct from the first target epitope. .

[0175] In addition, in the present invention, where the bispecific molecule is multispecific, the molecule is first and second In addition to the target epitope, a third binding specificity may be included.

[0176] In one embodiment, the bispecific molecule of the present invention has at least one antibody as a binding specificity. The body, or its antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, or single-stranded) Antibodies also include light chain or heavy chain dimers, or their smallest fragments, for example. Fv, or as described in U.S. Patent No. 4,946,778 by Ladner et al. It may also be a single-chain structure like that.

[0177] Diabody is characterized by the expression of VH and VL domains on a single polypeptide chain, on the same chain The two domains above are linked by a linker that is too short to enable matching between them. It is a bispecific molecule. The VH and VL domains pair with complementary domains on other chains, This creates two antigen-binding sites (for example, Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448 ;Poijak et al.,1994 Structure 2:1121-112 See section 3). The diamond bodies are VHA-VLB and VHB-VLA (VH-VL (structure), or having either VLA-VHB and VLB-VHA (VL-VH structure) It can be produced by expressing two polypeptide chains within the same cell. The portion can be expressed in a soluble form in bacteria. The single-stranded diabody (scDb) is The two diabody-forming polypeptide chains are linked by a linker of approximately 15 amino acid residues. Produced by (Holliger and Winter, 1997 Can cer Immunol.Immunother.,45(3-4):128-30;W u et al.,1996 Immunotechnology,2(1):21-3 See 6). scDb can be expressed in bacteria in a soluble active monomer form. (Holliger and Winter,1997 Cancer Immunol .Immunother.,45(34):128-30;Wu et al.,199 6 Immunotechnology,2(1):21-36;Pluckthun and Pack,1997 Immunotechnology,3(2):83-1 05;Ridgway et al.,1996 Protein Eng.,9(7) (See 617-21). By fusing the Diabody with Fc, "The Diabody" is created. It can generate " (Lu et al., 2004 J. Biol. Chem. See 279(4):2856-65.

[0178] Other antibodies that can be used in the bispecific molecule of the present invention include mouse, chimeric, and It is a humanized monoclonal antibody.

[0179] The bispecific molecules of the present invention utilize methods known in the art to achieve binding specificity of their constituent elements. It can be prepared by conjugating properties. For example, a bispecific molecule Each binding specificity can be generated separately and then combined with each other. When it is a protein or peptide, various coupling agents or crosslinking agents are used for covalent bonding. It is possible. Examples of crosslinking agents include protein A, carbodiimide, and N-succinimidyl. -5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzo) (DTNB) fragrant acid, o-phenylenedimaleimide (oPDM), N-succinimidyl -3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimid 4(N-maleimidomethyl)cyclohaxane-1-carb Xylates (sulfo-SMCC) are included (e.g., Karpovsky et al.) .,1984 J.Exp.Med.160:1686;Liu, MA et al. See Proc.Natl.Acad.Sci.USA 82:8648, 1985. (I want to do this). Other methods include Paulus, 1985 Behring Ins. Mitt .No.78,118-132;Brennan et al.,1985 Scien ce 229:81-83), and Glennie et al., 1987 J.Im This includes those described in munol.139:2367-2375). The conjugate is SATA and sulfo-SMCC, both of which are Pierce Chemical C It is available from o. (Rockford, Ill.).

[0180] If the binding specificity is that of an antibody, they are sulfohydrates in the C-terminal hinge region of the two heavy chains. It can be conjugated by a rill bond. In certain embodiments, the hinge region is conjugated The molecule is modified to include an odd number of sulfhydryl residues, for example, one, before the eugate.

[0181] Alternatively, both binding specificities may be encoded in the same vector and expressed in the same host cell. This can then be assembled. This method involves a bispecific molecule consisting of mAb X mAb, mAb X Fab, Fab XF(ab')2, or ligand X Fab fusion protein It is particularly useful in certain cases. The bispecific molecule of the present invention contains one single-chain antibody and a binding determinant. It may be a single-stranded molecule containing two binding determinants, or a single-stranded bispecific molecule containing two binding determinants. Isomer molecules may contain at least two single-chain molecules. Methods for preparing bispecific molecules are as follows: For example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030 Detailed Statement; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405 U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Japanese Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. This is described in the specification of Japanese National Patent No. 5,482,858.

[0182] The binding of bispecific molecules to their specific targets is, for example, used in enzyme-linked immunosorbent assays. (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (examples) For example, it can be confirmed by growth inhibition or by Western blot assay. Each of these methods generally uses a labeling reagent (e.g., an antibody) that is specific to the target complex. This allows for the detection of the presence of a specific protein-antibody complex.

[0183] In another embodiment, the present invention relates to the antibody of the present invention that binds to TREM2 and its antigen-binding fragment. The present invention provides a polyvalent compound comprising at least two identical or different antigen-binding moieties. The components can be linked together via protein fusion or covalent or non-covalent bonds. The bonding method is described for bispecific molecules. For example, tetravalent compounds are... The antibody of the present invention and its antigen-binding fragment are used in the constant region of the antibody of the present invention and its antigen-binding fragment ( For example, by crosslinking with an antibody or antigen-binding fragment that binds to the Fc or hinge region. It is possible to obtain it.

[0184] Trimerized domains are described, for example, in European Patent No. 1012280B1. The pentamerization module is described, for example, in PCT / European Patent No. 97 / 05897. It is stated.

[0185] In some embodiments, the TREM2-binding molecule binds to both TREM2 and DAP12. It is a bispecific antibody that binds. In some embodiments, the TREM2 binding molecule is the first It is a bispecific antibody that recognizes the first antigen and the second antigen. In some embodiments, the first The antigen is human TREM2 or a naturally occurring variant thereof. Several embodiments So, the second antigen is human DAP12 or its naturally occurring variant. In this embodiment, the second antigen is human DAP10 or Siglec (sialic acid-conjugated immunoglobulin). It is a phosphorus-type lectin. In some embodiments, the second antigen is amyloid-beta or so Fragments of tau, IAPP, α-synuclein, TDP-43, FUS protein, plio Proteins, PrPSc, huntingtin, calcitonin, superoxide dismutase Ze, ataxin, Lewy bodies, atrial natriuretic factor, pancreatic islet amyloid polypeptide Insulin, apolipoprotein AI, serum amyloid A, mezin, prolactin, Lancethyretin, lysozyme, β2-microglobulin, gelzolin, keratoepiterine Cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-related non-ATG (RAN) Translation product, dipeptide repeat (DPR) peptide, glycine-alanine (G A) Repeat peptide, glycine-proline (GP) repeat peptide, glycine-al Guine (GR) repeat peptide, proline-alanine (PA) repeat peptide, and Disease-causing proteins selected from proline-arginine (PR) repeat peptides In some embodiments, the second antigen is a transferrin receptor, an insulin receptor. Body, insulin-like growth factor receptor, LRP-1, and blood-brain barrier selected from LRP1. Targeted proteins; or ligands and / or proteins expressed on immune cells, C D40, OX40, ICOS, CD28, CD137 / 4-1BB, CD27, GITR , PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM BTLA, KIR, GAL9, TIM3, A2AR, LAG, and phosphatidyl ceramide A ligand and / or protein selected from the group consisting of , or a second antigen. This may be a protein expressed in one or more tumor cells.

[0186] Antibodies with extended half-lives The present invention relates to an antibody that specifically binds to TREM2 and has an extended half-life in vivo. provide.

[0187] Many factors can affect the half-life of a protein in vivo. For example, renal filtration Metabolism in the liver, degradation by proteases, and immunogenicity Responses (e.g., protein neutralization by antibodies, and removal by macrophages and dendritic cells) (Inclusion). Various strategies are employed to extend the half-life of the antibody and its antigen-binding fragment of the present invention. It can be used. For example, polyethylene glycol (PEG), reCODE P EG, antibody scaffold, polysialic acid (PSA), hydroxyethyl starch (HES), alpha By chemical binding to bumin-binding ligands and carbohydrate shields; albumin, IgG, Gene fusion to proteins that bind to serum proteins such as FcRn; nanobody serum proteins such as Fab, DARPin, Avimer, Afibody, and Antikalin By (genetic or chemical) binding to other binding sites that bind to the substance; rPEG, albumin This is due to genetic fusion of the albumin domain, albumin-binding protein, and Fc. ; or by integration into nanocarriers, sustained-release formulations, or medical devices.

[0188] To prolong the serum circulation of antibodies in vivo, inactive polymers such as high molecular weight PEG are used. The molecule is transmitted via site-specific binding of PEG to the N-terminus or C-terminus of the antibody, or via lysine residues. Through the ε-amino group present above, with or without a multifunctional linker, an antibody or It can be attached to the fragment. To pegylate the antibody, the antibody and its antigen-binding fragment are used. Typically, this occurs under conditions in which one or more PEG groups adhere to the antibody or antibody fragment. PEG reactive esters or aldehyde derivatives of polyethylene glycol (PE React with G). PEGylation involves reacting with a reactive PEG molecule (or a similar reactive water-soluble polymer). This can be carried out by an acylation or alkylation reaction. In addition, the term "polyethylene glycol" refers to mono(C1-C10)alkoxy- or Aryloxy polyethylene glycol or polyethylene glycol maleimide, etc. This includes any form of PEG that has been used to derivatize other proteins. The intention is to do so. In one embodiment, the antibody to be pegylated is an aglycosylated antibody. Linear or branched polymer derivatization is used to minimize the loss of biological activity. The degree of conjugation ensures proper conjugation of the PEG molecule to the antibody. Therefore, it can be closely monitored by SDS-PAGE and mass spectrometry. Unreacted PEG The antibody-PEG conjugate is obtained by size exclusion or ion exchange chromatography. It can be separated from. PEG-derivative antibodies have binding activity and in vivo efficacy. Using methods well known to those skilled in the art, for example, by the immunoassay described herein It can be tested. Methods for pegyrating proteins are known in the art and the anti- It can be applied to the body and its antigen-binding fragments. For example, Nishimura et al. European Patent No. 0154316 and European Patent No. 04013 by Ishikawa et al. Please refer to Specification No. 84.

[0189] Other modified pegylation techniques involve reconstituted tRNA synthetase and tRNA. Reconstitution chemical orthogonal manipulation, which incorporates chemically identified side chains into biosynthetic proteins via a system. Includes the ReCODE PEG technology. This technology allows for the removal of E. coli and yeast. In the mother, it was possible to incorporate more than 30 new amino acids into biosynthetic proteins within mammalian cells. tRNA can incorporate a standard amino acid at any location where an amber codon is located, and Convert the bar from a stop codon to something that signals the incorporation of a chemically identified amino acid. .

[0190] Recombinant pegylation technology (rPEG) can also be used to extend the serum half-life. This technology The unstructured protein tail of 300-600 amino acids is genetically modified to an existing pharmaceutical protein. This includes fusion. The apparent molecular weight of such unstructured protein chains is Because it is approximately 15 times larger than its actual molecular weight, the serum half-life of the protein is significantly increased. In contrast to conventional PEGylation, which requires chemical bonding and repurification, the manufacturing process is significantly different. The products are simplified and uniform.

[0191] Polysialylation uses natural polymer polysialic acid (PSA) to extend the activity life. Furthermore, it is another technology that improves the stability of therapeutic peptides and proteins. PSA is a shear It is a polymer of lic acid (sugar). It is used for protein and therapeutic peptide drug delivery. In this case, polysialic acid provides a protective microenvironment for binding. This is because during circulation The active lifespan of therapeutic proteins increases, and they become unrecognized by the immune system. PSA polymers, It is found naturally in the human body. It is produced by specific bacteria that have evolved over millions of years. It was then used to coat those walls. Then these natural polysiallylated The bacteria, through molecular mimicry, were able to replicate the body's defense system. PSA, an extreme stealth technology, can be extracted in large quantities from such bacteria, and possesses specific physical properties. It can be easily produced. Bacterial PSA is chemically identical to PSA in the human body. Therefore, even when bound to a protein, it is completely non-immunogenic.

[0192] Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. It contains. HES is a modified natural polymer derived from waxy corn starch, and is beneficial to the body. It can be metabolized by enzymes. HES solutions typically replace deficient blood volume and blood rheo It is administered to improve the logy properties. Molecular stability is improved by hesylation of the antibody. By increasing renal clearance and decreasing renal clearance, it is possible to extend the circulating half-life. This increases biological activity by changing different parameters such as the molecular weight of HES. This allows for the customization of a wide range of HES antibody conjugates.

[0193] Antibodies with increased half-life in vivo also have an increased IgG constant domain or its FcRn binding. One or more amino acid modifications (i.e., It can be generated by introducing (substitution, insertion, or deletion). For example, International Publication No. 98 / 232 Pamphlet No. 89; International Publication No. 97 / 34631; and U.S. 6, Please refer to specifications No. 277 and 375.

[0194] Furthermore, in order to make antibodies or antibody fragments more stable in vivo, or to make them longer in vivo Because they have a long half-life, antibodies can be conjugated to albumin. The technology is well known in this field, for example, in International Publication No. 93 / 15199. International publication no. 93 / 15200, and international publication no. 01 / 77137 See Fret and European Patent No. 413,622.

[0195] Strategies to increase half-life include nanobodies and fibronectin-based binders. , and is particularly useful in other antibodies or proteins where an increase in in vivo half-life is desired. .

[0196] Antibody conjugate This invention relates to an antibody that specifically binds to the IgSF domain of TREM2 or to the antigen binding thereof. A fragment is provided, and this antibody or antigen-binding fragment is a heterologous protein or polypeptide (or its antibody). Original binding fragments, preferably at least 10, at least 20, at least 30, at 40, at least 50, at least 60, at least 70, at least 80, at least Recombinantly fused to a polypeptide of 90 or at least 100 amino acids, or chemically They are conjugated (including both covalent and non-covalent bonds) to form a fusion protein. To generate. In particular, the present invention relates to antigen-binding fragments of antibodies described herein (e.g., Fab fragments). Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain or VL CDR) and fusion proteins containing heterologous proteins, polypeptides or peptides To provide quality. To fuse proteins, polypeptides, or peptides to antibodies or antibody fragments. Methods for conjugation are known in the art. For example, U.S. Patent No. 5,3 Specification No. 36,603, Specification No. 5,622,929, Specification No. 5,359,046 Detailed Statement, Specification No. 5,349,053, Specification No. 5,447,851, and the same No. 5 , Specification No. 112,946; Specification No. 307,434 and No. 367, Specification No. 166; International Publication No. 96 / 04388 and International Publication No. 91 / 06 Pamphlet No. 570; Ashkenazi et al., 1991, Proc. Na tl.Acad.Sci.USA 88:10535-10539;Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil e t al.,1992,Proc.Natl.Acad.Sci.USA 89:113 See 37-11341.

[0197] Further fusion proteins are involved in gene-shuffling, motif-shuffling, and Xon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling") DNA shuffling can be generated through a technique called "fling." DNA shuffling is the present invention. It can be used to modify the activity of antibodies and their antigen-binding fragments (for example, higher parentage) Antibodies and antigen-binding fragments having a symmetry and lower dissociation rate. Generally, in the United States. Specification No. 5,605,793, Specification No. 5,811,238, Specification No. 5,830,72 Specification No. 1, Specification No. 5,834,252, and Specification No. 5,837,458; Patten et al.,1997,Curr.Opinion Biotechn. ol.8:724-33;Harayama,1998,Trends Biotech nol.16(2):76-82;Hansson,et al.,1999,J.Mo l.Biol.287:265-76; and Lorenzo and Blasco,1 998, Biotechniques 24(2):308-313 (These patents and See the references to each publication (each of which is incorporated in its entirety within this specification by reference). Antibodies and their antigen-binding fragments, or encoded antibodies and their antigen-binding fragments, before recombination. Random mutagenesis is then induced by error-prone PCR, random nucleotide insertion, or other methods. It can be modified by exposure to light. It specifically binds to the IgSF region of TREM2. The polynucleotide encoding the antibody-antigen binding fragment is one or more heterologous molecules. It can be rearranged with components, motifs, sections, parts, domains, fragments, etc.

[0198] Furthermore, the antibody and its antigen-binding fragment are treated with a marker sequence (for example) to facilitate purification. If so, it can be fused to a peptide. In one embodiment, the marker amino acid sequence is, in particular, Hexahistidine peptides, e.g., pQE vectors (QIAGEN, Inc., 9259) Eton Avenue, Chatsworth, Calif., 91311) These are tags provided by [company name], and many others are commercially available. l.,1989,Proc.Natl.Acad.Sci.USA 86:821-82 As described in section 4, for example, hexahistidine is used for the simple purification of fusion proteins. It provides other peptide tags useful for purification, such as influenza hemagglutinin tag. Hemagglutinin ("HA") tags corresponding to protein-derived epitopes (Wilson et al.) al., 1984, Cell 37:767), and the "FLAG" tag (Hopp e tal., Bio / Technology6(1988):1204-1210) is cited. These are possible, but are not limited to these.

[0199] In one embodiment, the antibody of the present invention is conjugated with a diagnostic agent or a detectable drug. Such antibodies are part of clinical testing procedures that determine the effectiveness of a particular treatment. to monitor or predict the onset, occurrence, progression, and / or severity of a disease or disorder. It may be useful for such diagnosis and detection, but is not limited to horseradish peroxy. β-galactosidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase Various enzymes such as -ase; but not limited to streptavidin / biotin and avidin / Prosthetic group molecules such as biotin; but not limited to, umbelliferone, fluorescein, flu Olecein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein , fluorescent substances such as dansyl chloride or phycoerythrin; but not limited to luminols. Any luminescent substance; but not limited to, luciferase, luciferin, and aequorin. Bioluminescent substances; not limited to, but including iodine (131I, 125I, 123I, and 121I) ) Carbon (14C), sulfur (35S), tritium (3H), indium (115In, 1 13In, 112In, and 111In), technetium (99Tc), thallium (20 1Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum ( 99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 1 59Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 18 6Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Z n, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 11 Radioactive materials such as 3Sn and 117Tin; and various positron emission tomography techniques Antibodies are attached to detectable substances containing positron-emitting metals and non-radioactive paramagnetic metal ions. This can be achieved by doing so.

[0200] Furthermore, the antibody or antigen-binding fragment is conjugated to the therapeutic or drug portion. The therapeutic or drug portion should be interpreted as being limited to classical chemotherapy drugs. There is no such thing. For example, the drug portion is a protein, peptide, or having the desired biological activity. It may be a polypeptide. Examples of such proteins include abrin and lysine A. , toxins such as Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, combination Proteins such as plasminogen activators, apoptotic agents, and anti-angiogenic agents; Alternatively, examples include biological response modifiers such as lymphokines.

[0201] Furthermore, radioactive metal ions, such as α-emitters of 213Bi, or 131In, 1 Radioactive metals including, but not limited to, 31LU, 131Y, 131Ho, and 131Sm. Therapies such as macrocyclic chelating agents useful for conjugating ions into polypeptides Antibodies can be conjugated to the target portion. In one embodiment, a macrocyclic chelating agent 1,4,7,10-tetraazacyclodopropyl can bind to antibodies via a linker molecule. This is can-N,N',N",N'"-tetraacetic acid (DOTA). Such linker molecules are This is generally known in the art, as stated by Denardo et al., 1998, Clin. Cancer Res.4(10):2483-90;Peterson et al. ,1999, Bioconjug. Chem. 10(4):553-7; and Zimme rman et al.,1999,Nucl.Med.Biol.26(8):943 -50 (each of which is incorporated in its entirety by reference).

[0202] Techniques for conjugating the therapeutic portion into an antibody are well known, for example, Amon et al., “Monoclonal Antibodies For Immuno targeting Of Drugs In Cancer Therapy,”i n Monoclonal Antibodies And Cancer Thera py, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antib odies For Drug Delivery”,in Controlled D rug Delivery(2nd Ed.),Robinson et al.(ed. s.),pp.623-53(Marcel Dekker,Inc.1987);Th orpe,“Antibody Carriers Of Cytotoxic Age nts In Cancer Therapy:A Review”,in Monoc lonal Antibodies 84:Biological And Clini cal Applications,Pinchera et al.(eds.),p p.475-506(1985);“Analysis,Results,And Fu ture Prospective Of Therapeutic Use Of Radiolabeled Antibody In Cancer Thera py”,in Monoclonal Antibodies For Cancer Detection And Therapy,Baldwin et al.(eds ), pp.303-16 (Academic Press 1985), and Th See Orpe et al., 1982, Immunol. Rev. 62:119-58. I want to be illuminated.

[0203] Antibodies are also conjugated to solid supports, which are particularly useful for immunoassays or purification of target antigens. This is possible. Examples of such solid supports include glass, cellulose, polyacrylamide, and Examples include, but are not limited to, iron, polystyrene, polyvinyl chloride, or polypropylene. It is not determined.

[0204] nucleic acids that encode antibodies The nucleic acids encoding antibodies or antigen-binding fragments thereof described herein are also described herein. Such nucleic acids are provided. These nucleic acids are the hTREM2 antibody or its antigenic antibody as described herein. It may encode polypeptides containing segments or domains of composite fragments. Such nucleic acids or The polynucleotides are derived from the heavy or light chain of the hTREM2 antibody described herein. It can encode at least one CDR area, and usually all three CDR areas. Such a nucleus Acids or polynucleotides may also be the heavy chain and / or of the ENTPD2 antibody described herein. This can encode all or substantially all of the variable region sequence of the light chain. Such nucleic acids or poly Nucleotides can also encode both the variable and constant regions of an antibody. Genetic coding Due to degeneracy, various nucleic acid sequences encode each of the immunoglobulin amino acid sequences. For example, the present invention provides an hT selected from one or more antibodies disclosed herein. This encodes the heavy chain variable region and light chain variable region of the REM2 antibody or its antigen-binding fragment, respectively. It is characterized by the first and second nucleic acids. The nucleic acids have the nucleotide sequences shown in Table 1, or This is a substantially identical sequence (for example, with respect to at least approximately 85%, 90%, 95%) Alternatively, sequences with 99% sequence identity, or sequences shown in Table 1 and sequences 3, 6, 15, 30, Or may contain sequences that differ by 45 nucleotides or less. Nucleic acids may consist of two or more of the sequences shown in Table 1. Nucleotide sequences (for example, light chain variable domain sequences and heavy chain variable domain sequences, or for example) For example, a light chain sequence or a heavy chain sequence, or a sequence substantially identical thereto (for example, in relation to the same) A sequence or presentation having at least approximately 85%, 90%, 95%, or 99% sequence identity. Includes the sequence shown in 1 and sequences that differ by 3, 6, 15, 30, or 45 nucleotides or less. obtain.

[0205] In certain embodiments, nucleic acids are derived from a heavy chain variable region having the amino acid sequence shown in Table 1. At least one, two, or three nucleotide sequences encoding CDRs or hypervariable loops A column, or an array substantially homologous thereto (for example, at least about 85%, 90%, 95%) Sequences having % or 99% sequence identity and / or one or more substitutions, for example, preserved It may include sequences having substitutions. In other embodiments, the nucleic acid may be the amine shown in Table 1. At least one, two, or three CDRs or superacid sequences from the light chain variable region having an anoacid sequence A nucleotide sequence or a sequence substantially homologous thereto that codes for a variable loop (for example, and sequences having at least approximately 85%, 90%, 95%, or 99% sequence identity and / Or it may include one or more substitutions (for example, sequences having preserved substitutions). In another embodiment, the nucleic acid is derived from heavy chain and light chain variable regions having the amino acid sequence shown in Table 1. at least one, two, three, four, five, or six CDRs or ultra-variable loops A nucleotide sequence that is substantially homologous thereto (for example, at least approximately Sequences having 85%, 90%, 95%, or 99% sequence identity and / or one or more It can include substitutions (for example, an array with a preserved substitution).

[0206] In certain embodiments, the nucleic acid is derived from a heavy chain variable region having the nucleotide sequence shown in Table 1. Nucleo encoding at least one, two, or three CDRs or hypervariable loops A cytoplasmic sequence, or a sequence substantially homologous thereto (for example, at least about 85%, 90%) It may include sequences having 95% or 99% sequence identity. Another implementation In this state, nucleic acids are derived from at least one nucleotide sequence from a light chain variable region having the nucleotide sequence shown in Table 1. Nucleotide sequences encoding one, two, or three CDRs or hypervariable loops, or A sequence that is substantially homologous to it (for example, at least about 85%, 90%, 95%, if It may include sequences having 99% sequence identity. In yet another embodiment, The nucleic acid is derived from at least one of the heavy chain and light chain variable regions having the nucleotide sequence shown in Table 1. Nucleo codes for two, three, four, five, or six CDRs or super-variable loops. rheotide sequences, or sequences substantially homologous thereto (e.g., at least about 85% homologous thereto, 9 It may include sequences with 0%, 95%, or 99% sequence identity.

[0207] Polynucleotide sequences are synthesized by de novo synthesis (e.g., solid-phase DNA synthesis), or by TR Produced by PCR mutagenesis of existing sequences encoding EM2 antibodies or their antigen-binding fragments. It is possible. Direct chemical synthesis of nucleic acids is described in Narang et al., 1979, M. eth.Enzymol.68:90;the phosphodiester met hod of Brown et al.,Meth.Enzymol.68:109, 1979,;Beaucage et al.,Tetra.Lett.,22:185 Diethylphosphoramidite method 9,1981; and U.S. Patent No. 4,458,066 This can be achieved by methods known in the art, such as the solid support method described in detail. The introduction of mutations into polynucleotide sequences by CR is, for example, PCR Technology. gy:Principles and Applications for DNA A mplification, H.A. Erlich (Ed.), Freeman Pre. ss, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. d.), Academic Press, San Diego, Calif., 1990 ;Mattila et al., Nucleic Acids Res.19:967 ,1991; and Eckert et al., PCR Methods and Ap This can be done as described in plications 1:17,1991.

[0208] The hTREM2 antibody or its antigen-binding fragment segment or domain described herein. A vector containing polynucleotides that encode a polypeptide containing (e.g., an expression vector) Vectors are also provided herein. Such vectors can be used, for example, in ex vivo cells. In or in vivo in cells, for example, in one or more tissues of an organism The hTREM2 antibody or antigen-binding fragment (for example, as described herein) is expressed. It can be used to produce and / or influence its expression. Using a vector, a polynucleotide encoding the hTREM2 antibody or its conjugated fragment is used. It can be expressed in cells, for example, mammalian cells, for the production of antibodies or their fragments. Both virus-based and non-viral expression vectors can be used. Viral vectors and systems include plasmids, episomal vectors, and typically This includes expression cassettes for expressing proteins or RNA, and human artificial staining. Examples include the body (e.g., Harrington et al., Nat Genet.). See 15:345,1997). Such nonviral vectors are used in this technology. Using transfection or transduction methods known in the field, for example, lipids (e.g.) For example, lipofectamine can be delivered to target cells using methods such as electroporation or mechanical cell membrane deformation. It may also be done. The term "expression vector" refers to a vector into which a desired coding sequence can be expressed in cells. This refers to a carrier nucleic acid molecule that can be inserted for the introduction of a vector. Vectors include DNA vectors and RNA vectors. This may be a vector, plasmid, cosmid, viral vector, or artificial chromosome. For example, Harrington et al., Nat Genet 15:345,1 See 997). For example, hTREM2 in mammalian (e.g., human) cells. Non-viral vectors useful for the expression of antibodies or their antigen-binding fragments include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invit rogen, San Diego, Calif.), MPSV vector, and protein This includes numerous other vectors known in the art for expressing quality. For example, one The vectors in this class include bovine papillomavirus, polyomavirus, adenovirus, and vaccine. Senior viruses, baculoviruses, retroviruses (Rous sarcoma virus, MMTV or It is a DNA element derived from animal viruses such as MOMLV or SV40 virus. Use. Other classes of vectors include Semlik Forest virus, Eastern Equine Encephalitis virus and It utilizes RNA elements derived from RNA viruses such as flaviviruses. Examples of virus vectors include retroviruses (e.g., lentiviruses) and lentiviruses. adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) Russ (HSV), SV40-based vector, papillomavirus, HBP Epstein-Bomb - Viruses, vaccinia virus, cymbis virus, influenza virus, Leo Viruses, Newcastle disease virus (NDV), measles virus, varicella stomatitis virus ( VSV, parvovirus, poliovirus, poxvirus, Seneca Valley virus Coxsackievirus, enterovirus, myxoma virus, marabavirus or cem One example is the Rikiforest virus (SFV). Brent et al., cited above; S mith, Annu. Rev. Microbiol. 49:807, 1995; and Ro See senfeld et al., Cell 68:143, 1992.

[0209] In some embodiments, the vector is a retroviral vector. In terms of application, the vector is a lentiviral vector. Lovirus-derived vectors enable long-term, stable integration of the transgene and daughter cells. Because it enables proliferation, it is a suitable tool for achieving long-term gene transfer. Mouse virus vectors are vectors derived from oncoretroviruses such as mouse leukemia virus. It has an additional advantage over tar in that it can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity. Retroviral vectors It could also be, for example, a gamma retrovirus vector. - For example, promoter, packaging signal (Ψ), primer binding site (P BS), one or more (e.g., two) long-terminal repeats (LTRs), and the target transgene, For example, it may contain a gene that codes for CAR. Gamma retrovirus vectors are gag They may lack viral structural genes such as pol and env. An example is gamma retrogravity. Virus vectors include mouse leukemia virus (MLV) and splenic focal formation virus (SFF). V), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom It is rare. Other gamma retrovirus vectors include, for example, Tobias Maetzig et al., “Gammaretroviral Vectors: Biology ,Technology and Application” Viruses.201 It is described in 1 Jun;3(6):677-713.

[0210] In some embodiments, the vector is an adeno-associated virus (AAV) vector, for example It is a recombinant AAV (rAAV) vector. "AAV" is an abbreviation for adeno-associated virus. This term may be used to refer to the virus itself or its derivatives. Unless otherwise specified, it includes both naturally occurring forms and recombinant forms. Abbreviation "rAA" "V" is a recombinant adenovirus, also known as a recombinant AAV vector (or "rAAV vector"). It refers to an associated virus. The term "AAV" is used, for example, to refer to AAV1 type (AAV1), AA V2 type (AAV2), AAV3 type (AAV3), AAV4 type (AAV4), AAV5 type ( AAV5, AAV6, AAV7, AAV8 ), AAV9 type (AAV9), AAV10 type (including AAV10 and AAVrh10), A AV12 type (AAV12), bird AAV, cattle AAV, dog AAV, horse AAV, primates Includes AAV, non-primate AAV, and sheep AAV. "Primate AAV" infects primates. "AAV" refers to AAV that infects non-primate mammals, and "non-primate AAV" refers to AAV that infects non-primate mammals. "Bovine AAV" refers to AAV that infects bovine mammals, for example.

[0211] The genome sequences of various serotypes of AAV, as well as native reverse end repeats (ITRs), Rep The sequences of the protein and capsid subunit are known in the art. The sequence can be found in literature or in public databases such as GenBank. GenBank acceptance numbers NC-002077(AAV1), AF063497(AA V1), NC-001401(AAV2), AF043303(AAV2), NC-00 1729(AAV3), NC-001829(AAV4), U89790(AAV4), NC-006152(AAV5), AF513851(AAV7), AF513852( AAV8), and NC-006261(AAV8); or International Publication No. 200503332 Publications such as Pamphlet No. 1 (AAV1-9) (these disclosures are referred to in this Specified Reference). See (which is incorporated into). For example, Srivistava et al. (19 83) J. Virology 45:555; Chiorini et al. (199 8) J. Virology 71:6823; Chiorini et al. (199 9) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939;Xiao et al. (199) 9) J. Virology 73:3994; Muramatsu et al. (19 96) Virology 221:208;Shade et al.,(1986)J .Virol.58:921;Gao et al.(2002)Proc.Nat.A cad.Sci.USA 99:11854;Moris et al.(2004)V Irology 33:375-383; International Publication No. 00 / 28061 pamphlet. International Publication No. 99 / 61601 pamphlet, International Publication No. 98 / 11244 pamphlet See also U.S. Patent No. 6,156,303.

[0212] The term "rAAV vector" as used herein refers to a polynucleotide not of AAV origin. Sequences (i.e., heterogeneous polynucleotides in AAV), typically used in cellular genetic transmutation. This refers to an AAV vector containing the target sequence for exchange. In some embodiments, heterogeneous poly Nucleotides contain at least one, sometimes two, AAV reverse terminal repeat (ITR) sequences. They may be adjacent. The term rAAV vector refers to rAAV vector particles and rAAV vectors It includes both terplasmids. rAAV vectors are single-stranded (ssAAV) or self-plasmids. It may be either complementary (scAAV). "AAV virus" or "rAAV virus" A "Russian particle" or "rAAV vector particle" contains at least one AAV capsid protein Capsid proteins (typically, capsid proteins of one or more wild-type AAVs or derived from them) (by all of the above) and viral particles consisting of a capsidized polynucleotide rAAV vector It refers to offspring. The particle is a heterogeneous polynucleotide (i.e., an introduced gene delivered to a mammalian cell). If it contains polynucleotides other than the wild-type AAV genome (such as rA), it is typically referred to as "rA These are called "AV vector particles" or simply "rAAV vectors". Therefore, the rAAV particles Production is inevitable because such vectors are contained within the rAAV particles, and therefore the rAAV vector Includes the production of tar.

[0213] In some embodiments, the vector is an hTRE as described herein, for example. This may be a recombinant DNA molecule containing a nucleic acid encoding an M2 antibody or its antigen-binding fragment. As used herein, “recombinant” refers to vectors, polynucleotides, polypeptides or When cells undergo cloning, restriction, or ligation steps (for example, poly(P) contained within them) (related to nucleotides or polypeptides), and / or different from naturally occurring products This means that the construct is the product of various combinations of other procedures that produce it. Recombinant viruses Alternatively, a vector is a viral particle containing recombinant polynucleotides. This term refers to that These include copies of the original polynucleotide construct and offspring of the original viral construct.

[0214] A recombinant vector is typically a single vector operably ligated to the nucleic acid sequence to be expressed. The above regulatory sequences are included. The term "regulatory sequence" refers to promoters, enhancers, and other promoters. It includes a current control element (e.g., a polyadenylation signal). The regulatory sequence is a nucleoty This includes constitutive expression of D sequences, as well as those indicating tissue-specific regulatory sequences and / or inductive sequences. The expression vector also provides stability and translationability of messenger RNA in host cells. Elements designed to optimize functionality, and / or as described herein, for example. Establish permanent, stable cell clones that express the hTREM2 antibody or its antigen binding. This may also include drug selection markers for this purpose. The design of the expression vector is determined by the host to be transformed. This can depend on factors such as cell selection and the desired protein expression level. Common methods for generating recombinant expression vectors are, among others, known in the art. Sambrook and Russell eds. (2001) Molecul ar Cloning:A Laboratory Manual,3rd edition on;the series Ausubel et al.eds.(2007 wi th updated through 2010)Current Protocol It can be found in Molecular Biology.

[0215] A specific start signal may also be required for efficient translation of the code sequence. The signal contains the ATG start codon or an adjacent sequence. It may be necessary to provide an exogenous translation control signal. Those skilled in the art will find this easy. This will determine and provide the necessary signals. The start codon is the entire insertion. To ensure translation, the reading frame of the desired code array and "in frame" It is well known that this must be the case. Exogenous translation control signals and start codons are heaven It can be either natural or synthetic. Expression efficiency depends on the appropriate transcription enhancer element. It can be enhanced by including it.

[0216] Expression can be transmitted to any suitable host cell known in the art, e.g., mammalian host cells, bacteria. Host cells, such as yeast host cells and insect host cells, can be used. Both prokaryotes and eukaryotes are supported. Expression systems are widely available. In some embodiments, the expression system is a CHO cell expression system. Which mammalian cell expression is it? In some embodiments, nucleic acids are expressed in the desired host cell. Codon optimization can be performed to facilitate expression. Selected cell type for expression, Ganellus and promoters that effectively direct the expression of DNA segments in living organisms. It would be important to use a smear and / or enhancer. Those skilled in molecular biology will generally know that Knowing how to use combinations of promoters, enhancers, and cell types for protein expression (See, for example, Sambrook et al. (2001)).

[0217] Most transcribed eukaryotic RNA molecules undergo RNA splicing, and are primary Remove introns from the transcript. A vector containing a genomic eukaryotic sequence can be used to generate proteins. To ensure proper processing of the transcript for the present, donors and / or acceptors May require a splicing site (Chandler et al., 1997, See Proc.Natl.Acad.Sci.USA,94(8):3596-601. I want to be treated that way.

[0218] The vectors or constructs of this disclosure generally include at least one termination signal. A "termination signal" or "terminator" is a specific signal of an RNA transcript produced by RNA polymerase. It consists of DNA sequences involved in the termination of RNA transcription. Therefore, in certain embodiments, RNA transcription A termination signal is intended to stop the production of a substance. The Terminator is intended to deliver a desired message. It may be necessary in vivo to achieve the level. In eukaryotes, termination The ter region also allows for site-specific cleavage of the novel transcript to expose polyadenylated sites. It may contain a specific DNA sequence that enables this. This may leave approximately 200 Å residue at the 3' end of the transcript. A signal is sent to a special endogenous polymerase to add stretch to the base (polyA). RNA molecules modified with this poly(A) tail are more stable and more efficiently translated. It appears that in other embodiments, including eukaryotes, the terminator is RNA Preferably, it includes a signal for termination, and the terminator signal is the message point It is more preferable to promote rear denylation. Terminator and / or polyadenylation. Part elements are used to enhance the message level and / or other sequences from the cassette. This may help minimize the read to the terminology used in this disclosure. Terminator is any known terminator of any transcription described herein or known to those skilled in the art. —including, for example, but not limited to, bovine growth hormone terminator — A gene termination sequence like, or a viral termination like, for example, the SV40 terminator Includes a sequence. In certain embodiments, the termination signal is transcribed, for example, by sequence cleavage or This may be due to the absence of a translatable sequence.

[0219] In expression, particularly in eukaryotic expression, in order to ensure proper polyadenylation of transcripts, Typically, it includes a polyadenylation signal. The nature of the polyadenylation signal is described herein. It is not considered important for the successful implementation of and / or any such sequence is used A preferred embodiment involves an SV40 polyadenylation signal and / or a bovine growth hormone. It contains a monpoliadenylation signal, is convenient, and / or works in various target cells. It is known to function well. Polyadenylation may increase the stability of the transcript. Alternatively, it may promote cytoplasmic transport.

[0220] In order to propagate the vector within the host cell, a specific nucleic acid sequence is used to initiate replication. It may contain more than one origin of replication (often called "ori"). Or it may be a host cell. If it is yeast, a self-replicating sequence (ARS) can be used.

[0221] In certain embodiments of this disclosure, cells containing the nucleic acid construct of this disclosure are marked in an expression vector. By including a marker, it can be identified in vitro or in vivo. - This imparts identifiable changes to cells, enabling easy identification of cells containing expression vectors. Generally, selection markers are those that confer properties that enable selection. Positive selection markers Kerr's choice is made possible by the presence of a marker, while the negative choice marker is Its existence hinders that selection. An example of a positive selection marker is a drug resistance marker. That is the case.

[0222] Typically, the inclusion of drug selection markers is helpful in the cloning and identification of transformants, for example. Neomycin, puromycin, hygromycin, DHFR, GPT, zeosin and Genes that confer resistance to histidinol are useful selection markers. In addition to markers that confer phenotypes that enable the identification of transformants based on the process, the basis The foundation includes other types of markers that can be screened using colorimetric analysis, such as GFP. It is also intended to be herpes simplex virus thymidine kinase (HSV-tk) or Screening capabilities such as loramphenicol acetyltransferase (CAT) Enzymes may also be used. Those skilled in the art will also likely use immunological methods in combination with FACS analysis. They know how to use markers. The markers used are those that produce genes. As long as it can be expressed simultaneously with nucleic acids that code for substances, it is not considered important. Further examples of markers and screenable markers are well known to those skilled in the art.

[0223] The selection of an expression vector involves choosing a target cell that will express one or more components of the vector. It depends. Typically, the vector encodes the hTREM2 antibody or its antigen-binding fragment. A promoter and other regulatory sequences (e.g., E) operably linked to a polynucleotide. It contains one or more regulatory sequences, such as a cyanohypenser.

[0224] A "promoter" is a region within a nucleic acid sequence that controls the initiation and rate of transcription. This is a sequence. This is a regulatory protein and a fraction of RNA polymerase and other transcription factors. The offspring may contain genetic elements that can be combined. The phrases "operationally arranged" and "operationally linked" are used. "Under control," "Under transcriptional control" means that the promoter controls the nucleic acid sequence. The correct functional location and / or direction for controlling the transcription initiation and / or expression of the sequence. It means that it is in the mood. The promoter is an "enhancer" (this is the transcription activity of the nucleic acid sequence). It may be used in conjunction with (referring to a cis-acting regulatory element involved in sexualization), or not. That's fine.

[0225] The promoter is a 5' non-coded element located upstream of the code segment and / or exon. As may be obtained by isolating the sequence, the gene or sequence is naturally related It may also be something like that. Such promoters can be called "endogenous." The enhancer is also naturally related to the nucleic acid sequence, either downstream or upstream of the sequence. It may be located at [location]. Alternatively, the coding nucleic acid segment may be recombinant or heterologously promo The promoter (which usually refers to a promoter that is not associated with the nucleic acid sequence in its natural environment) By bringing it under control, certain advantages can be obtained. Recombinant or heterogeneous enhancers —This also usually refers to enhancers that are not related to the nucleic acid sequence in their natural environment. The promoter or enhancer may be a promoter or enhancer of another gene, and and promoters or e-cells isolated from any other prokaryotic cell, viral cell, or eukaryotic cell. Enhancers, and promoters or enhancers that do not "naturally exist," i.e., different This includes mutations that alter the expression of different elements and / or components in the transcriptional regulatory region. To obtain. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences are , recombinant cloning and / or nucleic acid amplification technologies related to the compositions disclosed herein, For example, it may be produced using PCR (U.S. Patent No. 4,683,202, U.S. Patent). See Specification No. 5928906. Furthermore, mitochondria, chloroplasts, etc., nucleus Regulatory sequences that lead to the transcription and / or expression of sequences within other organelles can also be used in a similar manner. This is intended.

[0226] The promoters used are constitutive, inducible, synthetic, tissue-specific, or cell-specific. , and / or under appropriate conditions, to lead to high levels of expression of the introduced DNA segment It is intended to be advantageous in the large-scale production of recombinant proteins and / or peptides. It can be such. In addition, it binds to the human TREM2 protein (i.e., hTREM2). To improve the expression of nucleic acids encoding antibodies, for example, enhancers, ribosomal bonds Other regulatory elements, such as merging sites and transcription termination sequences, may also be incorporated.

[0227] In some embodiments, the steady expression of the hTREM2 antibody or its antigen-binding fragment is performed. Constitutive promoters are used to achieve this. Examples of constitutive promoters include: However, the early cytomegalovirus (CMV) promoter and Simian virus are not involved. 40 (SV40) initial promoter, mouse mammary cancer virus (MMTV) promoter, To immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV Romordant leukemia virus promoter, Epstein-Barr virus pre-early pro Motors, Rouss sarcoma virus promoters, and, but not limited to, actin promo promoter, myosin promoter, elongation factor-1α promoter, hemoglobin promoter Examples include human gene promoters such as the creatine kinase promoter.

[0228] In one embodiment, in order to prevent the inserted sequence from being expressed under conditions other than induction conditions, an inducible p A motor is used. When an inductive promoter is used, it is linked in an operable manner. The expression of the polynucleotide sequence is turned on when such expression is desired, or This provides a molecular switch that allows expression to be turned off when it is not desired. Examples of inductive promoters include, but are not limited to, metallothionein (metall Othionine promoters are one example.

[0229] Inducible promoters are not limited to these, but for example, the arabinose promoter. lacZ promoter, tetracycline promoter, metallothionein promoter - Glucocorticoid promoter, progesterone promoter, or heat shock promoter Romotor is one example.

[0230] In addition to the promoter, for the efficient expression of the hTREM2 antibody or its antigen-binding fragment. Other adjustment elements may also be necessary or desired. Such elements include These include the ATG start codon and adjacent ribosome binding sites or other sequences. In addition, Expression efficiency can be enhanced by including the appropriate enhancer in the cell line being used (e.g.) For example, Scharf et al.,Results Probl.Cell Diff er.20:125,1994; and Bittner et al., Meth.Enz See YMOL., 153:516, 1987. For example, SV40 enhancer Alternatively, using a CMV enhancer may increase expression in mammalian host cells.

[0231] In some embodiments, the expression of the hTREM2 antibody or its antigen-binding fragment is controlled to specific tissues. Alternatively, tissue-specific or cell-specific promoters are used to deliver the effect only to cells. Identity of tissue-specific or cell-specific promoters or elements, and their activity Assays for characterizing sex are well known to those skilled in the art. For example, human LIMK2 assays Genetics (Nomoto et al. 1999, Gene, 236(2):259-271) ), somatostatin receptor 2 gene (Kraus et al., 1998, FEES Lett., 428(3):165-170), mouse epididymal retinoic acid-binding gene (Lareyre et al.,1999,J.Biol.Chem.,274(12 ):8282-8290), human CD4 (Zhao-Emonet et al.,19 98,Biochirn.Biophys.Acta,1442(2-3):109-1 19) Mouse α2(XI) collagen (Tsumaki, et al., 1998, J Biol. Chem., 273(36):22861-22864), D1A dopamine Receptor gene (Lee, et al., 1997, J. Auton. Nerv. Syst .,74(2-3):86-90), insulin-like growth factor II (Wu et al., 1997,Biochem.Biophys.Res.Commun.,233(1): 221-226), Human platelet endothelial cell adhesion molecule-1 (Almendro et al. ,1996,J.Immunol.,157(12):5411-5421), Muscle Crea Tin kinase (MCK) promoter (Wang et al., Gene Ther.) (2008 Nov;15(22):1489-99) is one example.

[0232] In some embodiments, the expression of the hTREM2 antibody or its antigen-binding fragment is brought about by Therefore, synthetic promoters are used. Synthetic promoters have the transcriptional capacity of natural promoters. This can be significantly exceeded. For example, when activity is blocked or reduced by endogenous cellular mechanisms or factors. A synthetic promoter can be selected that does not have a built-in one. The synthetic promoter improves transcription efficiency. Therefore, other elements, including the trans-acting factor binding site and enhancers, are inserted. It may be done. A synthetic promoter may be both a synthetic promoter and a biological promoter. It can be rationally designed and chemically synthesized to combine the best features of each. Through several processes, the synthetic oligo is annealed and ligated, thereby A fully-length promoter is synthesized scientifically. The synthetic promoter is inducible or short-term. A cytoplasm-specific promoter may also be used.

[0233] In a preferred embodiment, the vector is an adeno-associated vector (AAV). The AAV vector is, for example, an hTREM2 antibody or the like as described herein. It contains a polynucleotide encoding an antigen-binding fragment. In a typical embodiment, AAV vector The antibody is an hTREM2 antibody with reverse-terminal repeat (ITR) sequences adjacent to one or both sides. It contains a polynucleotide that codes for its binding fragment. This polynucleotide also contains For example, promoters, enhancers, one or more intron sequences, poly(A) sequences and These may also include one or more additional elements, such as combinations of these. In terms of form, the vector is encapsulated in an AAV capsid, as described herein, for example. Polynucleotides containing the hTREM2 antibody or its antigen-binding fragment Contains nucleotide AAV vector plasmids.

[0234] In some embodiments, the vector contains at least one target cell compatibility regulatory sequence, e.g. For example, an hTR operably connected to a promoter as described herein. It contains a polynucleotide encoding an EM2 antibody or its antigen-binding fragment.

[0235] In some embodiments, the ITR in the AAV vector originates from the same AAV serotype. In some embodiments, the ITRs in the AAV vector are derived from different AAV serotypes. In some embodiments, the ITR in AAV particles is the same. In embodiments, AAV particles The ITRs for children and middle school students are different.

[0236] In some embodiments, the ITR in the AAV vector is the same as the AAV capsid. It is derived from the serotype. In the embodiment, the ITR in the AAV vector is different from that of the AAV capsid. It is derived from the serotype. In one embodiment, the ITR is derived from AAV2 and the AAV capsid is A It originates from serotypes other than AV2, such as AAV9.

[0237] The expression vector can also be activated by the inserted hTREM2 antibody or its antigen-binding fragment sequence. In order to form a fusion protein with the encoded polypeptide, the secretion signal sequence location is also It can be provided. In many cases, the insertion sequence of the hTREM2 antibody or its antigen-binding fragment is vector The signal sequence is ligated before being included in the ter. hTREM2-binding antibody light chain and heavy chain variable The vector used to accept the main encoding sequence is a constant region or a part thereof. It may also encode a constant region as a fusion protein. This enables the expression of variable regions, thereby producing intact antibodies and their antigen-binding fragments. To guide life. Typically, such a steady state is human.

[0238] The generation of an expression vector involves multiple cloning processes, which are nucleic acid regions containing multiple restriction enzyme sites. A vector containing a site (MCS) can be used, and any one of them can be used in a standard recombinant form. It can be used in combination with other technologies to digest vectors. t al., 1999, Levenson et al., 1998, and Coce See a, 1997. "Restriction enzyme digestion" functions only at specific locations within nucleic acid molecules. This refers to the catalytic cleavage of nucleic acid molecules by enzymes. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Often, the vector is MC Linearization is performed using restriction enzymes that cleave within S to allow the exogenous sequence to be linked to the vector. It is fragmented. "Linking" is the process of forming a phosphodiester bond between two nucleic acid fragments. This refers to the fact that these may or may not be adjacent to each other. Restriction enzymes and ligation reactions The technology including the response is well known to those skilled in the art of recombinant technology.

[0239] A method for introducing an expression vector containing the target polynucleotide sequence into a cell host is used. It varies depending on the type. For example, calcium chloride transfection is generally used in prokaryotic cells. It is used for other purposes, while calcium phosphate treatment or electroporation is used for other cell colonies. It can be primarily used (see Sambrook et al., above for general information). Other methods include For example, electroporation, calcium phosphate treatment, liposome-mediated plasmapheresis. Exchange, injection and microinjection, ballistic / gene gun, virosom, immunoliposcopy Polycations: nucleic acid conjugates, naked DNA, artificial virions, herpes viruses Fusion to the structural protein VP22, drug enhancement uptake of DNA, ex vivo transduction, Protoplast fusion, retroviral transduction, viral transfection, lipid fusion Examples include transfection of the protoplast or other prior art. The cells are grown in culture medium and screened for appropriate activity. Recombinant protein For long-term high-yield production, stable expression is often desired. For example, polypeptide Cell lines that stably express the virus are the origin of viral replication or endogenous expression element and selected It can be prepared using an expression vector containing the maker gene. After introducing the vector, the details Before switching the cells to the selective medium, they may be grown in concentrated medium for 1-2 days. Selective marker The purpose of this is to confer resistance to selection, and its presence leads to the development of this in the selective medium. This enables the proliferation of cells that successfully express the introduced sequence. It also promotes resistance and stable transfection. The selected cells can be proliferated using tissue culture techniques appropriate to the cell type. Methods and conditions for culturing infected cells and recovering the produced antibodies are provided by our company. The specific expression vectors and mammalian hosts used are known to the user and are described herein. It can be modified or optimized depending on the cell.

[0240] Cells containing any one of the expression vectors described herein are also provided herein. In some embodiments, the present disclosure provides a host cell containing the nucleic acid molecules described herein. These cells are characterized by being either host cells or therapeutic cells. The “recombinant host cells” used interchangeably in this specification are specific target cells. Furthermore, it refers to the offspring or potential offspring of such cells, either due to mutation or environmental influence. Therefore, certain modifications can occur in the next generation, so such offspring are, in fact, parent It may not be identical to a cell, but it is still within the scope of the terminology used herein. It is included in.

[0241] In one embodiment, the host cell has a nucleus encoding the hTREM2 antibody or its antigen-binding fragment. It is genetically engineered to contain acid. In one embodiment, the host cell uses an expression cassette. Genetic manipulation is performed by doing so. The term "expression cassette" refers to a nucleotide sequence. Effective gene expression is possible in hosts that are compatible with such sequences. The set is an open reading phrase with or without a promoter and intron. This may include m and termination signals. Additional factors necessary or useful to achieve expression, for example Alternatively, an inductive promoter may be used.

[0242] Host cells that possess and express the hTREM2 antibody or its antigen-binding fragment chain are This can be a eukaryotic cell, or a prokaryotic cell such as a bacterial cell, insect cell, or human cell. These are not limited to the present invention. Escherichia coli (E. coli) is a clonoid of the polynucleotide of the present invention. It is a useful prokaryotic host for expression and growth. Other suitable microbial hosts include , rod-shaped bacteria such as Bacillus subtilis, and salmon Nera (Salmonella), Serratia, and various Pseudomonas This includes other intestinal bacterial species such as Pseudomonas. In prokaryotic hosts, typically, the expression of regulatory sequences compatible with the host cell (e.g., replication) is performed. It is also possible to create expression vectors that include the starting point. Furthermore, lactose promoter systems, Tryptophan (trp) promoter system, β-lactamase promoter system, or phage λ There are any number of well-known promoters, such as promoter systems from these sources. The promoter typically controls expression using an operator sequence, It has ribosome binding site sequences for initiating and completing transcription and translation. Other microorganisms also express the hTREM2 antibody or its antigen-binding fragment according to the present invention. It can be used for this purpose. Insect cells combined with baculovirus vectors can also be used. Appropriate Examples of insect cells include Sf9 cells, but the study is not limited to them.

[0243] In one embodiment, mammalian host cells undergo cleavage of the hTREM2 antibody of the present invention or its antigen binding. They are used to express and produce fragments. For example, they are used for endogenous immunoglobulin genes. Hybridoma cell lines that express offspring (e.g., 1D6.C9 myeloma hybridoma cells) or mammalian cell lines having an exogenous expression vector (e.g., SP2 / 0 myeloma cells) These may include any normal death or normal or abnormal immortal animal or Human cells are included. For example, CHO cell lines, various Cos cell lines, HeLa cells, and bone marrow. It can secrete intact immunoglobulins, including tumor cell lines, transformed B cells, and hybridomas. Numerous suitable host cell lines have been developed for polypeptide expression. The use of fibrous cell cultures is generally seen, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987 This will be discussed. Expression vectors for mammalian host cells are based on the origin of replication and promoter. , and enhancers (e.g., Queen, et al., Immunol. Rev. 8) Expression regulatory sequences such as 9:49-68, 1986, and ribosome binding sites, RNA The necessary processes such as the splice site, polyadenylation site, and transcription terminator sequence These expression vectors may contain a single information processing site. These expression vectors are usually derived from mammalian genes or It includes promoters derived from mammalian viruses. Appropriate promoters are constitutive, cell-type. It may be specific, stage-specific, and / or modifiable or modifiable. Useful promotion The promoters include the metallothionein promoter and the major late promoter of constitutive adenovirus. Dexamethasone-inducible MMTV promoter, SV40 promoter, MRP pol III promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoter Lomotors (such as the human early CMV promoter), constitutive CMV promoter, and This includes, but is not limited to, promoter-enhancer combinations known in the technical field. do not have.

[0244] Host cells produce antibodies that bind to the human TREM2 protein (i.e., hTREM2). or it may be used to express hTREM using host cells. Therefore, this disclosure also relates to the use of host cells to hTREM The present invention is characterized by a method for producing an antibody or its antigen-binding fragment. In one embodiment, the present invention is characterized by The method involves the host cell (antibody) being converted so that the hTREM2 antibody or its antigen-binding fragment is produced. This includes culturing the recombinant expression vector (in which the gene is introduced) in an appropriate culture medium. In another embodiment, the method further includes isolating the antibody from the culture medium or host cells. Examples of eukaryotic cells include Vero cells, HeLa cells, COS cells, CHO cells, and HEK cells. Examples include, but are not limited to, 293 cells, BHK cells, and MDCKII cells. The method for introducing an expression vector containing the target polynucleotide sequence varies depending on the type of cell host. No, they are different. For example, calcium chloride transfection is commonly used in prokaryotic cells, Alternatively, calcium phosphate treatment or electroporation may be used for other cell hosts (generally, See Sambrook, et al., cited above. Other methods include, for example, Electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection Infection, gene gun law, virosom, immunoliposome, polycation: nucleic acid conjugate Naked DNA, artificial virions, herpesvirus structural protein VP22 fusion (Elliot and O'Hare, Cell 88:223, 1997), medicine Examples include enhancing DNA uptake by agents and exovivo transduction. Recombinant proteins To produce it in high yield over a long period, stable expression is often required. For example, cell lines that stably express hTREM2 antibody chains or antigen-binding fragments can replicate viruses. The present invention's expression vector comprising a starting or endogenous expression element and a selectable marker gene. It can be prepared using the following method: After vector introduction, before switching cells to the selective medium. The plants may be grown in fortified medium for 1-2 days. The purpose of the selectable marker is to ensure resistance to selection. This involves conferring a specific gene, and its presence allows cells to successfully express the introduced sequence in the selective medium. It becomes possible to grow them. Resistant, stably transfected cells are Cells can be propagated using tissue culture techniques appropriate to their cell type.

[0245] Regulatory array Those skilled in the art will know that regulatory sequences are required for the expression of one or more components of the vector in target cells. One can recognize what one has gained.

[0246] In one embodiment, the AAV vector plasmid is as described herein, for example. It contains a regulatory sequence that efficiently regulates the expression of the hTREM2 antibody or its antigen-binding fragment.

[0247] In one embodiment, the AAV vector plasmid drives expression in target cells. It includes an efficient regulatory sequence.

[0248] In one embodiment, the AAV vector plasmid is, but is not limited to, a promoter, etc. It includes regulatory sequences. As a non-limiting example, the promoter is (1) CMV promoter, (2) CBA promoter, (3) FRDA or FXN promoter, (4) UBC promoter Motor, (5) GUSB promoter, (6) NSE promoter, (7) Synapsin (8) MeCP2 promoter, (9) GFAP promoter, (10) HI promoter, (11) U6 promoter, (12) NFL promoter, (13) NFH promoter, (14) SCN8A promoter, or (15) PGK promoter —That's fine.

[0249] promoter Those skilled in the art will know, for example, how to cleave the hTREM2 antibody or its antigen binding as described herein. For a compound to be expressed in target cells, it must be species-specific, inducible, tissue-specific, or Specific promoters may be necessary, including those specific to the cell cycle. Recognizable (Parr et al., Nat. Med. 3:1145-9 (1997)) (This content is incorporated herein by reference in its entirety.)

[0250] In one embodiment, the AAV vector plasmid is as described herein, for example. It contains a promoter that is efficient for the expression of the hTREM2 antibody or its antigen-binding fragment.

[0251] In one embodiment, the AAV vector plasmid drives expression in target cells. It includes an efficient promoter.

[0252] In one embodiment, the promoter is used in target tissue, such as nervous system tissue, but is not limited to this embodiment. For a certain period of time, for example, an hTREM2 antibody or the one described herein This results in the expression of an antigen-binding fragment. For example, the hTREM2 antibody as described herein. Or the expression of the antigen-binding fragment occurs at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and 7 hours. Hours: 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours 1st, 2nd, 3rd, 4th, 5th, 6th, 1 week, 8th, 9th, 10th, 11th, 12th 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days , 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 1 month 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 Year, Year 9, Year 10, Year 11, Year 12, Year 13, Year 14, Year 15, Year 16, Year 17, Year 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 It can be obtained over a period of 49, 50, 55, 60, 65, or more than 65 years. For example. The expression of the hTREM2 antibody or its antigen-binding fragment as described herein is 1 to 5 Hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1- 2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5 years 10 years, or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50 years It can be obtained over 55 years, or 55-60 years, or 60-65 years.

[0253] In one embodiment, the AAV vector plasmid is as described herein, for example. Approximately 5kb upstream from the first exon of the encoded hTREM2 antibody or its antigen-binding fragment. Includes a region located at; more specifically, to be expressed with the FRDA promoter, Located approximately 4.9kb upstream from the first exon of the frataxin gene, at 17bp There is a region (for example, Puspasari et al. Long Range Reg ulation of Human FXN Gene Expression,PLO See S ONE, 2011; this content is entirely referenced herein. (to be used).

[0254] In one embodiment, the promoter is less than 1kb. The promoter is 200, 210 , 220, 230, 240, 250, 260, 270, 280, 290, 300, 310 , 320, 330, 340, 350, 360, 370, 380, 390, 400, 410 , 420, 430, 440, 450, 460, 470, 480, 490, 500, 510 , 520, 530, 540, 550, 560, 570, 580, 590, 600, 610 , 620, 630, 640, 650, 660, 670, 680, 690, 700, 710 , 720, 730, 740, 750, 760, 770, 780, 790, 800 or 80 Lengths greater than 0 are also acceptable. Promoters are 200-300, 200-400, 200 ~500, 200~600, 200~700, 200~800, 300~400, 300 ~500, 300~600, 300~700, 300~800, 400~500, 400 ~600, 400~700, 400~800, 500~600, 500~700, 500 The length may be ~800, 600~700, 600~800, or 700~800.

[0255] In one embodiment, the promoter is, but is not limited to, CMV and CBA, the same or This may be a combination of two or more components, regions, or sequences of different promoters. Each component is , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 , 300, 310, 320, 330, 340, 350, 360, 370, 380, 381 ,382,383,384,385,386,387,388,389,390,400 , 410, 420, 430, 440, 450, 460, 470, 480, 490, 500 , 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 , 610, 620, 630, 640, 650, 660, 670, 680, 690, 700 , 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 Alternatively, the length may exceed 800. Each component is 200-300, 200-400, 20 0-500, 200-600, 200-700, 200-800, 300-400, 30 0-500, 300-600, 300-700, 300-800, 400-500, 40 0-600, 400-700, 400-800, 500-600, 500-700, 50 Lengths of 0-800, 600-700, 600-800, or 700-800 are not specified. .

[0256] In one embodiment, the promoter is a CMV enhancer sequence, for example, a pre-initial CMV enhancer sequence. Enhancer sequences (e.g., 382-nucleotide CMV enhancer sequences) and chicken β-acid Chin (CBA) promoter sequence (e.g., 260-nucleotide CBA promoter sequence) This is the combination.

[0257] In one embodiment, the promoter is a 280-nucleotide fragment of the CMV enhancer sequence. and combination with a 266-nucleotide fragment of the chicken β-actin (CBA) promoter sequence In some embodiments, the CMV enhancer sequence is [ka] It includes, for example, consists of.

[0258] In some embodiments, the CBA promoter sequence is [ka] It includes, for example, consists of.

[0259] In one embodiment, the AAV vector is an array: [ka] This includes, for example, a hybrid CMV enhancer / chicken β-actin probiotic. Includes motor.

[0260] In one embodiment, the AAV vector plasmid contains a ubiquitous promoter. Non-exclusive examples of motors include CMV, CBA (including derivatives such as CAG and CBh), EF-1a, PGK, UBC, GUSB (hGBp), and UCOE (HNRPA2B1 -CBX3 promoter) is one example. In one embodiment, Yu, Soderblom , Gill, Husain, Passini, Xu, Drews or Raymond Any of the promoters taught as such may be used in the present invention. Yu et al (Molecular Pain 2011, 7:63; This content is used in this specification.) (The entire text is referenced) Using a lentiviral vector in rat DRG cells And in primary DRG cells under the CAG, EF-1a, PGK, and UBC promoters eGFP expression was evaluated, and UBC showed weaker expression compared to the other three promoters. In all of these promoters, only 10-12% glial expression was observed. They discovered that.

[0261] Soderblom et al. (E. Neuro 2015; This content is as specified herein) (The entire text is referenced in this document) CMV and UBC prostaglandins are injected into the motor cortex. eGFP expression in AAV8 containing the motor and AAV2 containing the CMV promoter The following was evaluated: Intranasal administration of plasmids containing UBC or EF-1a promoter was used in CM It showed higher sustained airway expression than expression induced by the V promoter (e.g., Gill e t al.,Gene Therapy 2001,Vol.8,1539-1546 See reference; this content is incorporated herein by reference in its entirety. n et al. (Gene Therapy 2009; This content is used in this specification.) (The entire text is referenced) is an HβH construct containing the hGUSB promoter, HSV -1Evaluated the LAT promoter and NSE promoter to construct HβH in mouse brain. They found that the substance showed weaker expression than NSE. Passini and Wolf e(J.Virol.2001, 12382-12392, this content is as specified herein) (The entire text is referenced) HβH vector after intraventricular injection in neonatal mice We evaluated the long-term effects and found that there was sustained expression for at least one year. et al. (Gene Therapy 2001, 8, 1323-1332; of these According to the (which is incorporated by reference in this specification) NF-L and NF-H When using a motor, CMV-lacZ, CMV-luc, EF, GFAP, hE NK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8 Compared to kb and NSE (1.8kb+wpre), low expression was found in all brain regions. Xu et al. found that promoter activity was NSE (1.8kb), EF, and NSE (0.3kb). We found that GFAP, CMV, hENK, PPE, NFL, and NFH were the highest in that order. NFL is a 650 nucleotide promoter, and NFH is a 920 nucleotide promoter. They are motors, and neither is found in the liver, but NFH is proprioceptive sensory neurons, brain It is abundant in the spinal cord, and NFH is present in the heart. SCN8A is a 470-nucleotide progenitor. It is a motor, expressed throughout the DRG, spinal cord, and brain, and especially in hippocampal neurons and High expression is observed in cerebellar Purkinje cells, cortex, thalamus, and hypothalamus (e.g., Drews et al.Identification of evolutionary co nserved, functional noncoding elements in the promoter region of the sodium chann el gene SCN8A, Mamm Genome(2007)18:723-73 1; and Raymond et al. Expression of Alternat ively Spliced ​​Sodium Channel a-subunit g enes,Journal of Biological Chemistry(200 See 4)279(44)46234-4624; the contents of each of these are as follows: (The entire text is referenced in the details.)

[0262] In one embodiment, the AAV vector plasmid includes a non-cell-specific promoter. In one embodiment, the promoter is, for example, hTREM2 as described herein. A weak promoter (R) for sustained expression of an antibody or its antigen-binding fragment in nerve tissue. Its affinity for NA polymerase and / or sigma factor and its affinity for other promoters (Classified based on sex). In one embodiment, the promoter is, but is not limited to, two To sustainably express frataxin in nervous system tissues such as neuronal and glial tissues. It is a weak promoter.

[0263] In one embodiment, the AAV vector plasmid is a pro-Friedreich's ataxia (FRDA) pro- Includes motor.

[0264] In one embodiment, the AAV vector plasmid has a ubiquitin c (UBC) promoter. Includes. The UBC promoter can be 300-350 nucleotides in size. Non-limiting. For example, the UBC promoter has 332 nucleotides.

[0265] In one embodiment, the AAV vector plasmid is a β-glucuronidase (GUSB) protoplasm. Includes a motor. The GUSB promoter can be 350-400 nucleotides in size. As a non-limiting example, the GUSB promoter is 378 nucleotides long. As an example, the AAV vector plasmid is a 5'-promoter-CMV / globin inhibitor It may also be ron-hFXN-RBG-3', where the AAV vector plasmid is self The capsid may be complementary, and the capsid may be of the DJ serotype.

[0266] In one embodiment, the AAV vector plasmid is a neurofilament (NFL) promo Includes a motor. The NFL promoter can be 600-700 nucleotides in size. As a non-limiting example, the NFL promoter is 650 nucleotides. The AAV vector plasmid is 5'-promoter-CMV / globin intron- It may also be hFXN-RBG-3, where the AAV vector plasmid is self-complementary. It may be present, and the capsid may be of the DJ serotype.

[0267] In one embodiment, the AAV vector plasmid is a neurofilament heavy chain (NFH) plasmid. Includes a morphometer. The NFH promoter can be 900-950 nucleotides in size. As an unrestricted example, the NFH promoter is 920 nucleotides. For example, the AAV vector plasmid is a 5'-promoter-CMV / globin intro It may also be n-hFXN-RBG-3', where the AAV vector plasmid is autophase It may be complementary, and the capsid may be of the DJ serotype.

[0268] In one embodiment, the AAV vector plasmid contains the SCN8A promoter. The 8A promoter can be 450-500 nucleotides in size. (As a non-limiting example...) The SCN8A promoter is 470 nucleotides long. As a non-restrictive example, AAV The vector plasmid is d'-promoter-CMV / globinintron-hFXN-R It may also be BG-3, and the AAV vector plasmid may be self-complementary. The capsid may also be of the DJ serotype.

[0269] In one embodiment, the AAV vector plasmid contains a frataxin (FXN) promoter. include.

[0270] In one embodiment, the AAV vector plasmid is phosphoglycerate kinase 1 (PGK ) Includes promoters.

[0271] In one embodiment, the AAV vector plasmid is a chicken β-actin (CBA) promo Includes a motor.

[0272] In one embodiment, the AAV vector plasmid is a pre-early cytomegalovirus (CMV) Includes promoters.

[0273] In one embodiment, the AAV vector plasmid contains an H1 promoter.

[0274] In one embodiment, the AAV vector plasmid contains a U6 promoter.

[0275] In one embodiment, the AAV vector plasmid is a liver promoter or a skeletal muscle promoter. This includes non-limiting examples of liver promoters, such as hAAT and TBG. Non-exclusive examples of muscle promoters include desmin, MCK, and C5-12. ru.

[0276] In one embodiment, the AAV vector plasmid is a manipulated promoter, for example, Includes promoters derived from, but not identical to, those described in the specification. .

[0277] Enhancement element In one embodiment, the AAV vector plasmid contains at least one enhancer and / or It may contain an expression element. The enhancer or expression element is a regulatory sequence (e.g., p It may be used in combination with a motor. Mido contains a transgene enhancer, promoter, and / or 5'UTR intron. In this specification, the term "enhancer" is used to refer to a transgene enhancer, and is not limited to this definition. However, CMV enhancer (or fragment thereof, for example, as described herein) You may do so. Promoters are not limited to CMV, CBA, UBC, GUSB, NSE, synapsin, MeCP2, and GFAP promoter may also be present. 5'UT The R / intron may be, but is not limited to, SV40 and CBA-MVM.

[0278] In one embodiment, the AAV vector includes an intron, which optionally is a promoter - Element and hTREM2 antibody or its antigen-binding fragment (for example, as described herein) It is placed between the polynucleotides that code for the (as shown). Constrained by theory However, if the 5' intron is included, then the hTREM2 antibody or its antigen-binding fragment (for example, The levels and steady state of mRNA encoding (as described herein) are enhanced. It has been shown that, in the embodiment, the enhancer is derived from SV40 5' int It is a ron. In one embodiment, the SV40 intron is arranged as follows: [ka] It includes, for example, consists of.

[0279] In one embodiment, the AAV vector plasmid (e.g., one of the AAV vector) is limited Although not yet confirmed, (1) CMV enhancer, CMV promoter, SV40 5'UT (2) CMV enhancer, CBA promoter, SV40 5'UTR intron (for example, as described herein) (3) CMV enhancer, CBA promoter, CBA-MVM 5'U Enhancers, promo, etc., such as TR introns (for example, as described herein). Includes combinations of otrons and / or introns.

[0280] Enhancement of introduced genes In one embodiment, the AAV vector plasmid increases the target specificity and expression of the transgene. It includes at least one transgene enhancer element that can enhance (for example) , Powell et al.Viral Expression Cassette Elements to Enhance Transgene Target Spe significance and expression in Gene Therapy, See 2015; this content is incorporated herein by reference in its entirety. Non-limiting transgene enhancer elements that enhance the target specificity and expression of the transgene. Typical examples include promoters, endogenous miRNAs, and post-transcriptional regulatory elements (PREs). Polyadenylated (PolyA) signal sequences and upstream enhancers (USE), CMV enhancers Examples include sensors and introns.

[0281] In one embodiment, the AAV vector plasmid is at least a CMV enhancer. It contains one transgene enhancer element. In one embodiment, the AAV vector plastic Sumid contains at least one transgene enhancer element that acts as a promoter. nothing.

[0282] In one embodiment, the AAV vector plasmid contains at least one intron that is a guide Contains an input gene enhancer element.

[0283] In one embodiment, the AAV vector plasmid is at least one endogenous miRNA. It contains two introduced gene enhancer elements.

[0284] In one embodiment, the AAV vector plasmid is a post-transcriptional regulatory element (PRE). It includes at least one transgene enhancer element.

[0285] In one embodiment, the AAV vector plasmid is polyadenylated (poly-A) signaling It includes at least one transgene enhancer element which is a column. In an embodiment, A The AV vector plasmid contains a growth hormone polyA signaling molecule. Morphologically, the growth hormone polyA signaling pathway is linked to the bovine growth hormone (BGH) polyA signaling pathway. It originates from. An example of a BGH polyA signal sequence is, [ka] That is the case.

[0286] In embodiments, the AAV vector plasmid is, for example, as described herein. Downstream of a polynucleotide encoding a TREM2 antibody or its antigen-binding fragment (for example, The 3' side contains a poly-A signal (e.g., a BGH poly-A signal).

[0287] In one embodiment, the AAV vector plasmid is a small upstream enhancer (USE). It contains at least one transgene enhancer element.

[0288] Tissue-specific expression In one embodiment, the vector genome is an hTREM as described herein, for example. 2. Tissue-specific expression enhances the expression of antibodies or their antigen-binding fragments in tissues and / or cells. It may include rement. As a non-limiting example, promoter is not limited to, but human growth Long factor LA subunit (EF-1a), early cytomegalovirus (CMV), Chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB) The tissue-specific expression elements may include ubiquitin C (UBC) and others.

[0289] In one embodiment, the vector genome expresses neurons, astromorphogenetic neurons, etc., but is not limited to these. Nervous system promo that can be used to restrict sites or oligodendrocytes Tissue-specific expression vectors, such as ter, can be used to restrict expression to specific cell types. It may include ment.

[0290] In one embodiment, the vector genome is, but is not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β ), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca<2 +> / Calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate Acid receptor 2 (mGluR2), NFL, NFH, ηβ2, PPE, Enk, and EAAT It may include tissue-specific expression elements for neurons, such as a 2-promoter.

[0291] In one embodiment, the vector genome is, but is not limited to, glial fibrillary acidic protein Tissue-specific expression of astrocytes, such as the GFAP and EAAT2 promoters. It may contain elements.

[0292] In one embodiment, the vector genome is, but is not limited to, myelin basic protein ( Tissue-specific expression elements for oligodendrocytes, such as MBP promoters. It may include.

[0293] Intron In one embodiment, the AAV vector plasmid contains one or more introns or portions thereof. It includes at least one element that enhances the expression of the introduced gene.

[0294] In one embodiment, the payload structure is a guide such as one or more introns or a portion thereof. It includes at least one element that enhances input gene expression.

[0295] Non-restrictive examples of introns include MVM (67-97bp) and FIX truncated Type 1 intron (300 bp), β-globin SD / immunoglobulin heavy chain splice Scepter (250bp), adenovirus splice donor / immunoglobulin (immu noglobin) Splice acceptor (500bp), SV40 late model spliced Ner / splice acceptor (19S / 16S) (180bp) and hybrid acceptor One example is the novirus splice donor / IgG splice acceptor (230 bp). ru.

[0296] In one embodiment, the intron or intronic portion is 100 to 500 nucleotides long. It is also acceptable. The introns are 80, 90, 100, 110, 120, 130, 140, 1 50, 160, 170, 171, 172, 173, 174, 175, 176, 177, 1 78, 179, 180, 190, 200, 210, 220, 230, 240, 250, 2 60, 270, 280, 290, 300, 310, 320, 330, 340, 350, 3 60, 370, 380, 390, 400, 410, 420, 430, 440, 450, 4 The length may be 60, 470, 480, 490, or 500. The intron is 80~ 100, 80-120, 80-140, 80-160, 80-180, 80-200, 8 0-250, 80-300, 80-350, 80-400, 80-450, 80-500 , 200~300, 200~400, 200~500, 300~400, 300~500 Alternatively, it may be 400-500 in length.

[0297] In one embodiment, the AAV vector includes an intron, which optionally is a promoter - Element and hTREM2 antibody or its antigen-binding fragment (for example, as described herein) It is placed between the polynucleotides that code for the (as shown). Constrained by theory However, if the 5' intron is included, then the hTREM2 antibody or its antigen-binding fragment (for example, The levels and steady state of mRNA encoding (as described herein) are enhanced. It has been shown that, in the embodiment, the enhancer is derived from SV40 5' int It is a ron. In one embodiment, the SV40 intron is arranged as follows: [ka] It includes, for example, consists of.

[0298] In one embodiment, the AAV vector plasmid of the AAV vector is (1) CMV enhanced (1) Sequencer (e.g., SEQ ID NO: 134), (2) CBA promoter (e.g., SEQ ID NO: 134) 5) (3) SV40 intron (e.g., SEQ ID NO: 137), (4) hTREM2 antibody or polynucleations encoding the antigen-binding fragment (e.g., as described herein) (5) A rheotide and a BGH polyA signal (e.g., SEQ ID NO: 138). Morphologically, elements (1) to (5) are located on the AAV vector plasmid from 5' to 3'. They are placed. In the embodiment, elements (1) to (5) are ITRs (for example, 5'ITR and It is placed on an AAV vector plasmid further containing (3'ITR). In embodiments, I TR stands for AAV2 ITR.

[0299] In one embodiment, the AAV vector plasmid is a self-complementary AAV vector plasmid. Yes. "Self-complementarity" or the abbreviation "sc" refers to the state of being self-complementary. "AAV" or "scAAV" refers to a coding region supported by a recombinant AAV nucleic acid sequence. This refers to a construct designed to form an intramolecular double-stranded DNA template. Although not bundled, during infection, rather than waiting for cell-mediated second-chain synthesis, these scAAVs The two complementary halves of a double-stranded DNA molecule combine to form a single double-stranded DNA molecule that is immediately ready for replication and transcription. This will result in the formation of (dsDNA) units. For example, DM McCarty et al. l,“Self-complementary recombinant adeno- associated virus(scAAV)vectors promote e fficient transduction independently of D NA synthesis”,Gene Therapy,(August 2001) Vol 8, Number 16, Pages 1248-1254 (The entire document is referenced) See (referenced). For self-complementary AAV, see, for example, U.S. Article 6, Specification Nos. 596, 535; Specification Nos. 7,125, 717; and Specification Nos. 7,456, 68 As described in Specification No. 3 (each of which is incorporated herein by reference in whole): For example, if the 5'ITR is mutated by, for example, the deletion of a terminal degradation site, the genome This makes it possible to form hairpins.

[0300] Capsids and capsid serotypes In some embodiments, the AAV particles of the present invention are packaged in a capsid structure. A may also be a capsid-free product. Regarding viral vector donor and / or acceptor sequences such as AV, for example, National Patent Application Publication No. 2014 / 0107186 Specification (This entire content is referenced by reference) It is described in (the document).

[0301] In some embodiments, the AAV particles produced by the present invention are used in the central nervous system. Enhanced transduction to the target specific cell type, sustained transgene expression and / or safety This may include hybrid serotypes with sex profiles. Hybrid serotypes are trans Capsidation, bispecific antibodies, capsid surface, mosaic capsid, and texture It may be generated by adsorption to the racapsid and / or by other capsid protein modifications. .

[0302] In some embodiments, the AAV particles of the present invention are used for specific therapeutic applications, such as capsids. Rational mutagenesis of proteins (e.g., Pulicherla et al, Mol See Ther, 201 1, 19:1070-1078), pephate to capsid Tydoligands, for example, peptides derived from NMDA receptor agonists to enhance retrograde transport Incorporation of (Xu et al., Virology, 2005, 341:203-21) 4) and novel AAV barriers by orthogonal evolution to increase CNS transduction, for example. It may be further modified by the production of ions.

[0303] In some embodiments, the AAV particles produced by the present invention are, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ / 8 capsid serotypes, or their variants (e.g., A Including AV3A and AAV3B, either naturally occurring or / or recombinant forms It may contain various capsid proteins. In the present invention, one or more AAV capsid proteins are useful. The nucleic acid sequence encoding the protein is described in International Publication No. 2015191508 (this pamphlet). The contents of this document are disclosed in this specification (which are incorporated by reference in their entirety).

[0304] In some embodiments, the AAV particles of the present invention are any natural or recombinant AAV serotype It may contain or be derived from the following. According to the present invention, AAV particles are AAV1, AA V2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, A AV4-4、AAV5、AAV6、AAV6.1、AAV6.2、AAV6.1.2、A AV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV 9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV 9.68、AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AA V16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-lb、 AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42- 5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AA V42-11、AAV42-12、AAV42-13、AAV42-15、AAV42- aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AA V43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、 AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV22 3.5、AAV223.6、AAV223.7、AAVl-7 / rh.48、AAVl- 8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2- 4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-l l / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29 .5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AA V127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu. 44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145. 5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、A AV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu. 15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu. 20、AAV58.2 / hu.25、AAV A3.3、AAV A3.4、AAV A 3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AA V-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8 、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi .2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、A AVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu .12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu. 3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / h u.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5Rl、A AVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5Rl 、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、A AVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、A AVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11 、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAV hu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.2 3.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、 AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAV hu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.4 0、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AA Vhu.44Rl、AAVhu.44R2、AAVhu.44R3、AAVhu.45、 AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48Rl、AA Vhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AA Vhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu. 57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、A AVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAV hu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8 R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、A AVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh .20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、 AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVr h.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37 R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、A AVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48 .2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、A AVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh .61、AAVrh.64、AAVrh.64Rl、AAVrh.64R2、AAVrh .67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A5 86R variant、AAVrh8R R533A variant、AAAV、BAAV、ヤギAAV、 ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVh Er1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、A AVhEr1.7、AAVhEr1.36、AAVhEr2.29、AAVhEr2.4 、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr 2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PA EC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、A AV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-L K09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、 AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV- LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PA EC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-P AEC12, AAV-2-premiRNA-lOl, AAV-8h, AAV-8b, AA Vh, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle e 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP6 1 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh .43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV5 4.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AA V54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28 , AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttA AV), UPEN AAV10 and / or Japanese AAV10 serotype, and this A serotype selected from any of these variants may be used, or may be based on one of them. .

[0305] As a non-limiting example, the capsid of recombinant AAV virus is AAV2. For example, the capsid of recombinant AAV virus is AAVrh10. (This is a non-restrictive example.) The capsid of recombinant AAV virus is AAV9(hul4). As a non-specific example... The capsid of recombinant AAV virus is AAV-DJ. As a non-limiting example, recombinant The capsid of the AAV virus is AAV9.47. As a non-specific example, recombinant AA The capsid of the V virus is AAV-DJ8.

[0306] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV1 (USA). (Sequences 6 and 64 of Patent Application Publication No. 20030138772), AAV2 (USA) National Patent Application Publication No. 20030138772, Sequence IDs 7 and 70), AAV3( (Sequences 8 and 71 of U.S. Patent Application Publication No. 20030138772), AAV4 (Sequence ID 63 of U.S. Patent Application Publication No. 20030138772), AAV5 (U.S. National Patent Application Publication No. 20030138772, Sequence ID No. 114), AAV6 (United States) (Sequence ID 65 of Patent Application Publication No. 20030138772), AAV7 (U.S. Patent) (Sequences 1-3 of the specification published in Patent Application No. 20030138772), AAV8 (U.S. Patent Application Publication No. (Sequence IDs 4 and 95 of Patent Application Publication No. 20030138772), AAV9 (U.S. Patent) (Sequence IDs 5 and 100 of the specification published in Patent Application No. 20030138772), AAV10 (USA) (Sequence ID 117 of the specification in National Patent Application Publication No. 20030138772), AAV11 (USA) (Sequence ID 118 of National Patent Application Publication No. 20030138772), AAV12 (USA) National Patent Application Publication No. 20030138772, Sequence ID No. 119), AAVrhlO (Amino acids 1-7 of SEQ ID NO: 81 in U.S. Patent Application Publication No. 20030138772) 38) AAV16.3 (United States Patent Application Publication No. 20030138772, Sequence No.) (No. 10), AAV29.3 / bb. l (U.S. Patent Application Publication No. 20030138772) Specification (Sequence ID 11), AAV29.4 (U.S. Patent Application Publication No. 20030138772) Specification No. (Sequence ID 12), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030) Patent No. 138772 (Sequence ID 13), AAV1.3 (U.S. Patent Application Publication No. 20030) Patent No. 138772 (Sequence ID 14), AAV13.3 (U.S. Patent Application Publication No. 2003) Specification No. 0138772 (Sequence ID 15), AAV24.1 (U.S. Patent Application Publication No. 200) Patent No. 30138772 (Sequence ID 16), AAV27.3 (U.S. Patent Application Publication No. 20) Specification No. 030138772 (Sequence ID 17), AAV7.2 (U.S. Patent Application Publication No. 20) Specification No. 030138772, Sequence ID No. 18), AAVC1 (U.S. Patent Application Publication No. 200) Specification No. 30138772 (Sequence ID 19), AAVC3 (U.S. Patent Application Publication No. 2003) Specification No. 0138772 (Sequence ID 20), AAVC5 (U.S. Patent Application Publication No. 20030) Patent No. 138772 (Sequence ID 21), AAVF1 (U.S. Patent Application Publication No. 200301) Patent No. 38772 (Sequence ID 22), AAVF3 (U.S. Patent Application Publication No. 2003013) Specification No. 8772, Sequence ID No. 23), AAVF5 (U.S. Patent Application Publication No. 20030138) Specification No. 772 (Sequence ID 24), AAVH6 (U.S. Patent Application Publication No. 200301387) Specification No. 72, Sequence ID No. 25), AAVH2 (U.S. Patent Application Publication No. 2003013877) Specification No. 2, Sequence ID No. 26), AAV42-8 (U.S. Patent Application Publication No. 200301387) Specification No. 72, Sequence ID No. 27), AAV42-15 (U.S. Patent Application Publication No. 2003013) Patent No. 8772 (Sequence ID 28), AAV42-5b (U.S. Patent Application Publication No. 20030) Patent No. 138772 (Sequence ID 29), AAV42-lb (U.S. Patent Application Publication No. 200) Patent No. 30138772 (Sequence ID 30), AAV42-13 (U.S. Patent Application Publication No. 2) Patent No. 0030138772 (Sequence ID 31), AAV42-3a (U.S. Patent Application Publication) Patent No. 20030138772 (Sequence ID 32), AAV42-4 (U.S. Patent Application Publication No. 20030138772), Patent Publication No. 20030138772 (Sequence ID 33), AAV42-5a (U.S. Patent Publication No. Patent Application Publication No. 20030138772 (Sequence ID 34), AAV42-10 (US Patent) Application Publication No. 20030138772 Specification, Sequence ID No. 35), AAV42-3b (USA) National Patent Application Publication No. 20030138772, Specification (Sequence ID 36), AAV42-11 (US Patent Application Publication No. 20030138772, Specification Sequence ID No. 37), AAV42- 6b (US Patent Application Publication No. 20030138772, Sequence ID No. 38), AAV4 3-1 (US Patent Application Publication No. 20030138772, Sequence ID No. 39), AAV 43-5 (US Patent Application Publication No. 20030138772, Sequence ID No. 40), AA V43-12 (US Patent Application Publication No. 20030138772, Sequence ID No. 41), AAV43-20 (US Patent Application Publication No. 20030138772, Sequence ID No. 42) ), AAV43-21 (United States Patent Application Publication No. 20030138772, Sequence ID No. 43) AAV43-23 (United States Patent Application Publication No. 20030138772) (Number 44), AAV43-25 (U.S. Patent Application Publication No. 20030138772) Sequence ID No. 45), AAV44.1 (U.S. Patent Application Publication No. 20030138772) Sequence ID No. 46), AAV44.5 (U.S. Patent Application Publication No. 20030138772) (Sequence ID 47), AAV223.1 (U.S. Patent Application Publication No. 20030138772) Specification (Sequence ID 48), AAV223.2 (U.S. Patent Application Publication No. 2003013877) Specification No. 2, Sequence ID No. 49, AAV223.4 (U.S. Patent Application Publication No. 20030138) Specification No. 772 (Sequence ID 50), AAV223.5 (U.S. Patent Application Publication No. 200301) Patent No. 38772 (Sequence ID 51), AAV223.6 (U.S. Patent Application Publication No. 2003) Specification No. 0138772 (Sequence ID 52), AAV223.7 (U.S. Patent Application Publication No. 20) Specification No. 030138772 (Sequence ID 53), AAV A3.4 (U.S. Patent Application Publication No. Patent No. 20030138772 (Sequence ID 54), AAV A3.5 (U.S. Patent Application Publication No. 54), Patent Publication No. 20030138772 (Sequence ID 55), AAV A3.7 (U.S. Patent Publication No. Patent Application Publication No. 20030138772 (Sequence ID 56), AAV A3.3 (US Patent) Patent Application Publication No. 20030138772, Specification Sequence ID No. 57), AAV42.12 (USA) National Patent Application Publication No. 20030138772 Specification, Sequence ID No. 58), AAV44.2( U.S. Patent Application Publication No. 20030138772, Specification (Sequence ID 59), AAV42-2 (US Patent Application Publication No. 20030138772, Sequence ID No. 9), or these Riant, et al., U.S. Patent Application Publication No. 20030138772 (This content is the same as the present specification) The arrangement may be as described in the book (which is entirely referenced by reference) or It may contain or be derived from an AAV serotype that may have it.

[0307] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV2 (USA). (Sequences 7 and 23 of Patent Application Publication No. 20150159173), rh20 (US) National Patent Application Publication No. 20150159173, Sequence ID No. 1), rh32 / 33 (USA) SEQ ID NO. 2) of the specification of National Patent Application Publication No. 20150159173, rh39 (US Patent Specification No. 20150159173, Sequence IDs 3, 20 and 36), rh46( SEQ ID NOs. 4 and 22 of U.S. Patent Application Publication No. 20150159173, rh73 (Sequence ID 5 of U.S. Patent Application Publication No. 20150159173), rh74 (United States) SEQ ID NO. 6) of the specification of Patent Application Publication No. 20150159173, AAV6.1 (US Patent Sequence number 29 of the specification of Patent Application Publication No. 20150159173, rh.8 (U.S. Patent Publication No. SEQ ID NO. 41) of the specification published in Patent Application No. 20150159173, rh.48.1 (US Patent). SEQ ID NO. 44 of the specification published in Patent Application No. 20150159173, hu.44 (U.S. Patent Publication No. (Sequence ID 45 of the specification in Publication No. 20150159173), hu.29 (U.S. Patent Application) Sequence ID 42 of Publication No. 20150159173, hu.48 (U.S. Patent Application Publication No. SEQ ID NO. 38 of the specification No. 20150159173, rh54 (US Patent Application Publication No. Sequence ID No. 49 of Specification No. 20150159173, AAV2 (U.S. Patent Application Publication No. 20 SEQ ID NO. 7 of Specification No. 150159173, cy.5 (U.S. Patent Application Publication No. 20150 (Sequence IDs 8 and 24 of Specification No. 159173), rh.10 (U.S. Patent Application Publication No. 201) (Sequence IDs 9 and 25 of Patent No. 50159173), rh.13 (U.S. Patent Application Publication No. 2) (Sequence IDs 10 and 26 of Patent No. 0150159173), AAV1 (Published U.S. Patent Application) Sequence IDs 11 and 27 of Patent No. 20150159173, AAV3 (U.S. Patent Application (Sequences 12 and 28 of Publication No. 20150159173), AAV6 (U.S. Patent) (Sequence IDs 13 and 29 of the specification in Publication No. 20150159173), AAV7 (United States) (Sequences 14 and 30 of the specification of Patent Application Publication No. 20150159173), AAV8( (Sequences 15 and 31 of U.S. Patent Application Publication No. 20150159173), hu. 13 (Sequences 16 and 32 of U.S. Patent Application Publication No. 20150159173), hu.26 (Sequences 17 and 3 of U.S. Patent Application Publication No. 20150159173) 3) hu.37 (Sequence ID 18 of U.S. Patent Application Publication No. 20150159173) and 34), hu.53 (array number of U.S. Patent Application Publication No. 20150159173) (Articles 19 and 35), rh.43 (U.S. Patent Application Publication No. 20150159173) (Sequence IDs 21 and 37), rh2 (U.S. Patent Application Publication No. 20150159173) (Sequence ID 39), rh.37 (United States Patent Application Publication No. 20150159173) Sequence ID No. 40), rh.64 (Distribution of U.S. Patent Application Publication No. 20150159173) Column number 43), rh.48 (arrangement of the specification of U.S. Patent Application Publication No. 20150159173) Number 44), ch.5 (Sequence number of the specification of U.S. Patent Application Publication No. 20150159173) 46), rh.67 (Sequence ID 4 of U.S. Patent Application Publication No. 20150159173) 7) rh.58 (Sequence ID 48 of U.S. Patent Application Publication No. 20150159173) ), or variants thereof, but not limited to Cy5Rl, Cy5R2, Cy5 R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.4 8.1, rh.48.2, rh.48.1.2, hu.44Rl, hu.44R2, hu .44R3, hu.29R, ch.5R1, rh64R1, rh64R2, AAV6.2 AAV6.1, AAV6.12, hu.48Rl, hu.48R2, and hu.48R 3, including the specification of U.S. Patent Application Publication No. 20150159173 (this content is specified herein) The arrangement may be as described in (the whole is referenced in) or This may include, or be derived from, an AAV serotype that may contain this.

[0308] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV9 (USA). (Sequence IDs 1-3 of Japanese Patent No. 7198951), AAV2 (US Patent No. 719895) Sequence ID 4 of U.S. Patent No. 1, AAV1 (Sequence ID 5 of U.S. Patent No. 7198951) ), AAV3 (Sequence ID 6 of U.S. Patent No. 7198951), and AAV8 (United States Patent No. See Sequence ID No. 7198951, for example, U.S. Patent No. 7198951. The arrangement is as described in (this content is incorporated by reference in its entirety in this specification). It may contain or may be derived from an AAV serotype that may have or possess such a serotype.

[0309] In some embodiments, the AAV vector is, but is not limited to, AAV9.9, AA V9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AA V9.47, AAV9.61, AAV9.68, AAV9.84, etc., N Pulich Erla et al. (Molecular Therapy 19(6):1070 -1078 (2011, which is incorporated herein by reference in its entirety) AAV blood may have or may contain mutations in the AAV9 sequence as described. It contains or is derived from a clear form. In some embodiments, the AAV capsid is a blood-brain It contains one or more sequences that have been manipulated to deliver the vector across the barrier (e.g., B. E.Deverman et al,Nature Biotech,Vol.34,N o.2, pp. 204-211 (published online February 1, 2016) and Caltech press release, A. Wetherston, www.neurolog y-cenfrd.com / 2016 / 02 / 10 / successfd / brain- See barrier; also see International Publication No. 2016 / 0492301 pamphlet. See also the U.S. Patent No. 8,734,809 (the contents of each of these are (The entire text is referenced.)

[0310] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV3B (USA). (Sequences 1 and 10 of the specification of Japanese Patent No. 6156303), AAV6 (US Patent No. 615) (Sequence IDs 2, 7, and 11 of Patent No. 6303), AAV2 (US Patent No. 6156303) Sequence numbers 3 and 8 of the specification, AAV3A (Sequence numbers 3 and 8 of U.S. Patent No. 6,156,303) (Items 4 and 9), or derivatives thereof, etc., as specified in U.S. Patent No. 6,156,303 (this content Even if the arrangement is as described herein (which is entirely referenced herein), It may contain or be derived from good or potentially good AAV serotypes.

[0311] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV8 (USA). Sequence ID 1) of the specification of Patent Application Publication No. 20140359799, AAVDJ (U.S. Patent) See Sequence IDs 2 and 3 of the specification published in Patent Application No. 20140359799, or their respective barriers. For example, the details of U.S. Patent Application Publication No. 20140359799 (this content is specified herein) The arrangement may be as described in (where the whole is referenced) or It may contain or be derived from AAV serotypes that may have [specific characteristics].

[0312] In some embodiments, AAV particles are Grimm et al. (Journal of Virology 82(12):5887-5911(2008), Specified As described in (the whole is referenced in [the text]), it is not limited to, Serotypes such as AAVDJ or AAVDJ8 (or AAV-DJ8) and its variants It may contain a capsid from. The amino acid sequence of AAVDJ8 is the heparin-binding domain (H It may include two or more mutations to remove BD. As a non-limiting example, U.S. 7 , Specification No. 588,772 (The contents of this specification are incorporated herein by reference in their entirety) The AAV-DJ sequence described as sequence number 1 contains two mutations: (1) amino acid 587 R587Q and (2) The amino acid arginine (R;Arg) of amino acid 590 is changed to threonine (T;Thr) It may include R590T. Another non-limiting example is three mutations: (1) amino acid 4 K406R, in which lysine (K;Lys) of 06 is changed to arginine (R;Arg), 2) The amino acid arginine (R; Arg) at amino acid 587 is converted to glutamine (Q; Gln) R587Q and (3) amino acid 590, arginine (R;Arg), are present, and threonine (T This may include R590T which has changed to ;Thr).

[0313] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV4 (International International Publication No. 1998011244 (Sequences 1-20, etc.) Pamphlet No. 98011244 (This content is incorporated in its entirety by reference in this specification.) The AAV4 sequence may be as described in ( ) or may have such a sequence. It may contain or be derived from serotype V.

[0314] In some embodiments, the AAV particles of the present invention are described in International Publication No. 2014144229. AAV2G as described in the brochure and incorporated herein by reference in its entirety The AAV2 sequence may have a mutation that results in 9, or may have such a mutation. It may contain or be derived from AAV serotypes.

[0315] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV3-3( International Publication No. 2005033321, Sequence ID No. 217), AAV1 (International Publication Pamphlet No. 2005033321, Sequence IDs 219 and 202), AAV106 .1 / hu.37 (Sequence number 10 in International Publication No. 2005033321), AAV114.3 / hu.40 (Arrangement of the pamphlet International Publication No. 2005033321) Number 11), AAV127.2 / hu.41 (International Publication No. 2005033321 pamphlet) Lett's Sequence IDs 6 and 8, AAV128.3 / hu.44 (International Publication No. 200503) Pamphlet No. 3321, Sequence ID No. 81), AAV130.4 / hu.48 (International Publication No. (Sequence number 78 in pamphlet No. 2005033321), AAV145.1 / hu.53 (Sequence IDs 176 and 177 of International Publication No. 2005033321), AA V145.6 / hu.56 (Sequence number of the brochure International Publication No. 2005033321) 168 and 192), AAV16.12 / hu.11 (International Publication No. 2005033321) (Sequence numbers 153 and 57 of the pamphlet, AAV16.8 / hu.10 (International Publication No.) (Sequence numbers 156 and 56 in pamphlet No. 2005033321), AAV161.10 / hu.60 (Sequence ID 170 in International Publication No. 2005033321), A AV161.6 / hu.61 (Allocation number for International Publication No. 2005033321) (No. 174), AAV1-7 / rh.48 (International Publication No. 2005033321 pamphlet) (Sequence ID 32), AAVl-8 / rh.49 (International Publication No. 2005033321) (Sequence numbers 103 and 25 of the Script), AAV2 (International Publication No. 2005033321) Pamphlet Sequence IDs 211 and 221, AAV2-15 / rh.62 (International Publication No. (Sequence numbers 33 and 114 in pamphlet No. 2005033321), AAV2-3 / rh .61 (Sequence number 21 in International Publication No. 2005033321), AAV2- 4 / rh.50 (Sequence IDs 23 and 1 of International Publication No. 2005033321) 08), AAV2-5 / rh.51 (from the brochure International Publication No. 2005033321) Sequence IDs 104 and 22), AAV3.1 / hu.6 (International Publication No. 2005033321) (Sequence numbers 5 and 84 in the pamphlet), AAV3.1 / hu.9 (International Publication No. 2005) (Sequence numbers 155 and 58 in pamphlet No. 033321), AAV3-11 / rh.5 3 (Sequence IDs 186 and 176 of International Publication No. 2005033321), A AV3-3 (Sequence ID 200 of International Publication No. 2005033321), AA V33.12 / hu.17 (Sequence number of the brochure International Publication No. 2005033321) 4) AAV33.4 / hu.15 (from the brochure International Publication No. 2005033321) Sequence ID 50), AAV33.8 / hu.16 (International Publication No. 2005033321) Frets (sequence number 51), AAV3-9 / rh.52 (International Publication No. 200503332) (Sequence numbers 96 and 18 of Pamphlet No. 1), AAV4-19 / rh.55 (International Publication No. (Sequence number 117 of pamphlet No. 2005033321), AAV4-4 (International release No. 2) (Sequence IDs 201 and 218 in pamphlet number 005033321), AAV4-9 / rh .54 (Sequence ID 116 in International Publication No. 2005033321), AAV5 (Sequence numbers 199 and 216 of International Publication No. 2005033321), AA V52.1 / hu.20 (Sequence number 6 of the international release pamphlet No. 2005033321) 3) Distribution of AAV52 / hu. l9 (International Publication No. 2005033321) Column No. 133), AAV5-22 / rh.58 (International Publication No. 2005033321) Frets (sequence number 27), AAV5-3 / rh.57 (International Publication No. 200503332) (Sequence ID 105 in Pamphlet No. 1), AAV5-3 / rh.57 (International Publication No. 2005) Pamphlet No. 033321, Sequence ID 26), AAV58.2 / hu.25 (International release) Pamphlet No. 2005033321, Sequence ID No. 49), AAV6 (International Publication No. 200 (Sequence numbers 203 and 220 of pamphlet No. 5033321), AAV7 (International Publication No. 2) (Sequence numbers 222 and 213 of pamphlet number 005033321), AAV7.3 / hu .7 (Sequence ID 55 of International Publication No. 2005033321), AAV8 (Country (Sequence numbers 223 and 214 of the brochure published internationally, No. 2005033321), AAVH -l / hu.1 (Sequence ID 46 of International Publication No. 2005033321), A AVH-5 / hu.3 (Sequence ID 44 in International Publication No. 2005033321) ), AAVhu.1 (Sequence ID 144 of the international publication brochure No. 2005033321) ), AAVhu.10 (Sequence number 15 of the brochure International Publication No. 2005033321) 6) AAVhu.11 (Sequence ID 1 of the international publication pamphlet No. 2005033321) 53), AAVhu.12 (International Publication No. 2005033321 Pamphlet Sequence Number 5) 9) AAVhu.13 (Sequence ID 1 of the international publication pamphlet No. 2005033321) 29) AAVhu.14 / AAV9 (International Publication No. 2005033321 brochure) (Sequences 123 and 3), AAVhu.15 (International Publication No. 2005033321) Frets (sequence number 147), AAVhu.16 (International Publication No. 2005033321) (Sequence ID 148 of the 'Nfret'), AAVhu.17 (International Publication No. 2005033321) Pamphlet Sequence Number 83), AAVhu.18 (International Publication No. 2005033321) Pamphlet Sequence Number 149), AAVhu.19 (International Publication No. 2005033321) (Sequence number 133 of the pamphlet), AAVhu.2 (International Publication No. 2005033321) (Sequence number 143 of the pamphlet), AAVhu.20 (International Publication No. 200503332) (Sequence number 134 of Pamphlet No. 1), AAVhu.21 (International Publication No. 20050333) (Sequence number 135 of pamphlet No. 21), AAVhu.22 (International Publication No. 2005033) (Sequence number 138 of pamphlet No. 321), AAVhu.23.2 (International Publication No. 2005) Pamphlet No. 033321, Sequence ID No. 137), AAVhu.24 (International Publication No. 200 Pamphlet No. 5033321, Sequence ID No. 136), AAVhu.25 (International Publication No. 20) Pamphlet No. 05033321, Sequence ID No. 146), AAVhu.27 (International Publication No. 2) Pamphlet No. 005033321, Sequence ID No. 140), AAVhu.29 (International Publication No. Pamphlet No. 2005033321, Sequence ID No. 132), AAVhu.3 (International Publication No. (Sequence number 145 in pamphlet No. 2005033321), AAVhu.31 (International release) Pamphlet No. 2005033321, Sequence ID No. 121), AAVhu.32 (International Public Pamphlet No. 2005033321, Sequence ID 122), AAVhu.34 (International) Pamphlet No. 2005033321, Sequence ID 125), AAVhu.35 (Country) (Sequence number 164 of the brochure published in 2005033321), AAVhu.37( International Publication No. 2005033321, Sequence ID 88), AAVhu.39( International Publication No. 2005033321, Sequence ID 102), AAVhu.4( International Publication No. 2005033321, Sequence ID No. 141), AAVhu.40 (Sequence ID 87 of International Publication No. 2005033321), AAVhu.41 (Sequence number 91 of International Publication No. 2005033321), AAVhu.42 (Sequence ID 85 of International Publication No. 2005033321), AAVhu.43 (Sequence number 160 of International Publication No. 2005033321), AAVhu.4 4 (Sequence ID 144 of International Publication No. 2005033321), AAVhu. 45 (Sequence ID 127 of International Publication No. 2005033321), AAVhu .46 (Sequence ID 159 in International Publication No. 2005033321), AAVh u.47 (Sequence ID 128 in International Publication No. 2005033321), AAV hu.48 (Sequence ID 157 in International Publication No. 2005033321), AA Vhu.49 (Sequence ID 189 in International Publication No. 2005033321), A AVhu.51 (Sequence ID 190 in International Publication No. 2005033321), AAVhu.52 (Sequence ID 191 in International Publication No. 2005033321) AAVhu.53 (Sequence ID 186 of International Publication No. 2005033321) ), AAVhu.54 (Sequence ID 18 of the International Publication No. 2005033321 brochure) 8) AAVhu.55 (Sequence ID 1 of the international publication pamphlet 2005033321) 87), AAVhu.56 (Sequence number of International Publication No. 2005033321 brochure) 192), AAVhu.57 (arrangement number of the international publication no. 2005033321) No. 193), AAVhu.58 (arrangement of brochure international publication No. 2005033321) Number 194), AAVhu.6 (arrangement of brochure international publication no. 2005033321) Number 84), AAVhu.60 (arrangement of brochure international publication No. 2005033321) Number 184), AAVhu.61 (Distribution of brochure internationally published no. 2005033321) Column number 185), AAVhu.63 (from the brochure International Publication No. 2005033321) Sequence ID 195), AAVhu.64 (International Publication No. 2005033321 pamphlet) (Sequence number 196), AAVhu.66 (International Publication No. 2005033321 pamphlet) (Sequence ID 197), AAVhu.67 (International Publication No. 2005033321 pamphlet) (Sequence number 198 of the bot), AAVhu.7 (International Publication No. 2005033321 pamphlet) (Sequence number 150 of the bot), AAVhu.8 (International Publication No. 2005033321 pamphlet) (Sequence number 12), AAVhu.9 (International Publication No. 2005033321 brochure) (Sequence ID 155), AAVLG-10 / rh.40 (International Publication No. 2005033321) (Sequence number 14 of the pamphlet), AAVLG-4 / rh.38 (International Publication No. 20050) Pamphlet No. 33321, Sequence ID No. 86), AAVLG-4 / rh.38 (International Publication No. Pamphlet No. 2005033321, Sequence ID 7), AAVN721-8 / rh.43 (Sequence ID 163 of International Publication No. 2005033321), AAVN721 -8 / rh.43 (Sequence number 43 in International Publication No. 2005033321), AAVpi. l (International Publication No. 2005033321, Brochure Sequence Number 28), A AVpi.2 (International Publication No. 2005033321, Brochure Sequence Number 30), AAV pi.3 (International Publication No. 2005033321, Brochure Sequence Number 29), AAVrh .38 (Sequence ID 86 in International Publication No. 2005033321), AAVrh .40 (Sequence ID 92 in International Publication No. 2005033321), AAVrh .43 (Sequence ID 163 in International Publication No. 2005033321), AAVr h.44 (International Publication No. 2005033321, Brochure Sequence Number 34), AAVrh .45 (International Publication No. 2005033321, Brochure Sequence Number 41), AAVrh. 47 (International Publication No. 2005033321, Brochure Sequence Number 38), AAVrh.4 8 (Sequence ID 115 of International Publication No. 2005033321), AAVrh. 49 (Sequence ID 103 of International Publication No. 2005033321), AAVrh .50 (Sequence ID 108 in International Publication No. 2005033321), AAVr h.51 (Sequence ID 104 in International Publication No. 2005033321), AAV rh.52 (Sequence number 96 in International Publication No. 2005033321), AAV r h.53 (Sequence ID 97 in International Publication No. 2005033321), AAVr h.55 (International Publication No. 2005033321, Pamphlet Sequence Number 37), AAVrh .56 (Sequence ID 152 in International Publication No. 2005033321), AAVr h.57 (Sequence ID 105 in International Publication No. 2005033321), AAV rh.58 (Sequence ID 106 in International Publication No. 2005033321), AA Vrh.59 (International Publication No. 2005033321, Brochure Sequence Number 42), AAV rh.60 (International Publication No. 2005033321, Brochure Sequence Number 31), AAVr h.61 (Sequence ID 107 in International Publication No. 2005033321), AAV rh.62 (Sequence ID 114 in International Publication No. 2005033321), AA Vrh.64 (Sequence ID 99 in International Publication No. 2005033321), AA Vrh.65 (International Publication No. 2005033321, Brochure Sequence Number 35), AAV rh.68 (International Publication No. 2005033321, Brochure Sequence Number 16), AAVr h.69 (International Publication No. 2005033321, Pamphlet Sequence Number 39), AAVrh .70 (International Publication No. 2005033321, Brochure Sequence Number 20), AAVrh. 72 (International Publication No. 2005033321, Brochure Sequence Number 9), or these variants Ants, etc., are not limited to AAVcy.2, AAVcy.3, AAVcy.4, A AVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.1 8, AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AA Vrh.24, AAVrh.25, AAVrh.25 / 42 15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVr International Publication No. 200503332, including h.36, AAVrh.37, and AAVrhl4. The contents of Pamphlet No. 1 (which are incorporated herein by reference in their entirety) AAV serotypes may include or have the sequence as shown, or It may originate from this. An unrestricted example of a variant is International Publication No. 2005033321. Sequence ID 1 of the brochure (the entire contents of which are incorporated herein by reference) 3, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62 , 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 1 09-113, 118-120, 124, 126, 131, 139, 142, 151, 1 54, 158, 161, 162, 165~183, 202, 204~212, 215, 2 Numbers 19, 224-236 are examples.

[0316] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAVrh8R (Sequence number 9 of International Publication No. 2015168666), AAVrh8R A 586R mutant (Sequence ID 10 in International Publication No. 2015168666), A AVrh8R R533A variant (distribution of International Publication No. 2015168666) Column number 11), or variants thereof, etc., International Publication No. 2015168666 As stated in Lett (this content is incorporated by reference in its entirety in this specification) AAV serotypes may include or may have the sequence of AAV serotypes, or may be derived from AAV serotypes. ru.

[0317] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAVhu68 (For example, Sequence ID 2 of International Publication No. 2018160582), or its Riant, et al., International Publication No. 2018 / 160582 (This content is as specified in this specification) The arrangement may be as described in (the whole is referenced in) or This may include, or be derived from, an AAV serotype that may contain this.

[0318] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAVhE1. 1 (Sequence ID 44 of U.S. Patent No. 9,233,131), AAVhErl.5 (U.S. Patent No. 9,233,131) (Sequence ID 45 of Patent No. 9233131), AAVhER1.14 (U.S. Patent No. 92 (Sequence ID 46 of Patent No. 33131, AAVhErl.8 (U.S. Patent No. 9233131) (Sequence ID 47 of the specification), AAVhEr1.16 (U.S. Patent No. 9233131) Sequence No. 48 of (US Patent No. 9233131), AAVhEr1.18 (Sequence No. 48 of (US Patent No. 9233131) (No. 49), AAVhEr1.35 (Sequence ID 50 of U.S. Patent No. 9,233,131) , AAVhEr1.7 (Sequence ID 51 of U.S. Patent No. 9,233,131), AAVh Er1.36 (Sequence ID 52 of U.S. Patent No. 9,233,131), AAVhEr2. 29 (Sequence ID 53 of U.S. Patent No. 9,233,131), AAVhEr2.4 (United States Patent No. (Sequence ID 54 of Japanese Patent No. 9233131), AAVhEr2.16 (US Patent No. 9 (Sequence ID 55 of Patent No. 233131), AAVhEr2.30 (U.S. Patent No. 92331) (Sequence ID 56 of Specification No. 31), AAVhEr2.31 (U.S. Patent No. 9233131) (Detailed reference, Sequence ID 58), AAVhEr2.36 (Partial reference of U.S. Patent No. 9233131) Column number 57), AAVhER1.23 (Sequence ID 5 of U.S. Patent No. 9,233,131) 3) AAVhEr3.1 (Sequence ID 59 of U.S. Patent No. 9,233,131), AA V2.5T (Sequence ID 42 of U.S. Patent No. 9,233,131), or these barriers For example, U.S. Patent No. 9,233,131 (this content is referenced in its entirety in this specification). A may have the arrangement as described in (referenced by) It may contain or be derived from AV serotypes.

[0319] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV-PAE C (Sequence ID 1 of U.S. Patent Application Publication No. 20150376607), AAV-LK Ol (Sequence ID 2 of U.S. Patent Application Publication No. 20150376607), AAV-L K02 (Sequence ID 3 of U.S. Patent Application Publication No. 20150376607), AAV- LK03 (Sequence ID 4 in U.S. Patent Application Publication No. 20150376607), AAV -LK04 (Sequence ID 5 of U.S. Patent Application Publication No. 20150376607), AA V-LK05 (Sequence ID 6 of U.S. Patent Application Publication No. 20150376607), A AV-LK06 (Sequence ID 7 in U.S. Patent Application Publication No. 20150376607), AAV-LK07 (Sequence ID 8 in U.S. Patent Application Publication No. 20150376607) AAV-LK08 (Sequence ID 9 of U.S. Patent Application Publication No. 20150376607) ), AAV-LK09 (Sequence number of US Patent Application Publication No. 20150376607) 10) AAV-LK10 (arrangement of U.S. Patent Application Publication No. 20150376607) Number l), AAV-LK11 (United States Patent Application Publication No. 20150376607) Column number 12), AAV-LK12 (U.S. Patent Application Publication No. 20150376607) (Sequence ID 13), AAV-LK13 (U.S. Patent Application Publication No. 20150376607) See Sequence ID No. 14, AAV-LK14 (US Patent Application Publication No. 20150376607) (Sequence ID 15 of the specification), AAV-LK15 (U.S. Patent Application Publication No. 201503766) (Sequence ID 16 of Specification No. 07), AAV-LK16 (U.S. Patent Application Publication No. 2015037) SEQ ID NO. 6607, Sequence ID No. 17; AAV-LK17 (U.S. Patent Application Publication No. 20150) SEQ ID NO: 18 of Patent No. 376607, AAV-LK18 (U.S. Patent Application Publication No. 201) SEQ ID NO: 19 of Patent No. 50376607, AAV-LK19 (Publication No. 2 of the U.S. Patent Application). (Sequence ID 20 of Specification No. 0150376607), AAV-PAEC2 (U.S. Patent Application Publication No. 0150376607) SEQ ID NO. 20150376607, Sequence ID NO. 21, AAV-PAEC4 (US Patent) (Sequence ID 22 of Patent Application Publication No. 20150376607), AAV-PAEC6 (USA) National Patent Application Publication No. 20150376607, Sequence ID No. 23), AAV-PAEC 7 (Sequence ID 24 of U.S. Patent Application Publication No. 20150376607), AAV-P AEC8 (Sequence ID 25 of U.S. Patent Application Publication No. 20150376607), AA V-PAEC11 (Sequence ID 26 of U.S. Patent Application Publication No. 20150376607) ), AAV-PAEC12 (arrangement of the specification of U.S. Patent Application Publication No. 20150376607) Patent No. 27), or variants thereof, etc., U.S. Patent Application Publication No. 20150376607 As described in the specification (which is incorporated herein by reference in its entirety) AAV serotypes may include or may have the following sequence, or may contain such a sequence, or are derived from such a sequence. obtain.

[0320] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV-2-p miRNA-101 (Sequence ID 1, U.S. Patent No. 9163261), or its variant Riant, etc., U.S. Patent No. 9,163,261 (This content is not entirely as described herein) The arrangement may be as described (as referenced by reference) or may have such an arrangement. It may contain or be derived from the AAV serotype.

[0321] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV-8h( Sequence ID 6) of the specification of U.S. Patent Application Publication No. 20150376240, AAV-8b (U.S. National Patent Application Publication No. 20150376240, Sequence ID No. 5), AAV-h (US Patent SEQ ID NO. 2) of the specification of Patent Application Publication No. 20150376240, AAV-b (U.S. Patent Publication No. Sequence ID 1) of the Specification Publication No. 20150376240, or variants thereof, etc. , U.S. Patent Application Publication No. 20150376240 (This content is fully described herein) The arrangement may be as described in (the body is referenced by) or may have the same arrangement It may also contain or be derived from other AAV serotypes.

[0322] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAV SM 10-2 (Sequence ID 22 of U.S. Patent Application Publication No. 20160017295), AA V Shuffle 100-1 (U.S. Patent Application Publication No. 20160017295) (Sequence ID 23), AAV Shuffle 100-3 (U.S. Patent Application Publication No. 2016) (Sequence ID 24 of Patent No. 0017295), AAV Shuffle 100-7 (United States) Sequence ID 25) of Patent Application Publication No. 20160017295, AAV Shuff le 10-2 (Sequence ID 34 of U.S. Patent Application Publication No. 20160017295) AAV Shuffle 10-6 (U.S. Patent Application Publication No. 20160017295) See Sequence ID No. 35 in the detailed document, AAV Shuffle 10-8 (U.S. Patent Application Publication No. 201) (Sequence ID 36 of Patent No. 60017295), AAV Shuffle 100-2 (USA) National Patent Application Publication No. 20160017295, Sequence ID No. 37), AAV SM 1 0-1 (Sequence ID 38 of U.S. Patent Application Publication No. 20160017295), AAV SM 10-8 (Sequence ID 39 of U.S. Patent Application Publication No. 20160017295) ), AAV SM 100-3 (United States Patent Application Publication No. 20160017295) Sequence ID No. 40), AAV SM 100-10 (U.S. Patent Application Publication No. 201600172) See Sequence ID No. 95, Sequence ID No. 41), or any variant thereof, U.S. Patent Application Publication No. 20 Specification No. 160017295 (This content is incorporated herein by reference in its entirety) AAV serotypes may include or may have the sequence as described in ). or may derive from it.

[0323] In some embodiments, the AAV particles of the present invention are, but are not limited to, BNP61 A AV (Sequence ID 1 of U.S. Patent Application Publication No. 20150238550), BNP62 AAV (Sequence ID 3 of U.S. Patent Application Publication No. 20150238550), BNP 63 AAV (Sequence ID 4 of U.S. Patent Application Publication No. 20150238550), and These variants, etc., are described in U.S. Patent Application Publication No. 20150238550 (this The contents were arranged as described herein (which are incorporated by reference in their entirety). It may contain or be derived from AAV serotypes that may or may have them.

[0324] In some embodiments, the AAV particles of the present invention are, but are not limited to, AAVrh.5 0 (Sequence ID 108 of U.S. Patent Application Publication No. 20150315612), AAVr h.43 (Sequence ID 163 of U.S. Patent Application Publication No. 20150315612), A AVrh.62 (Sequence ID 114 of U.S. Patent Application Publication No. 20150315612) ), AAVrh.48 (Sequence number of the specification of U.S. Patent Application Publication No. 20150315612) 115), AAVhu.19 (the specification of U.S. Patent Application Publication No. 20150315612) (Column No. 133), AAVhu.ll (U.S. Patent Application Publication No. 20150315612) See Sequence ID No. 153, AAVhu.53 (US Patent Application Publication No. 2015031561) (Sequence ID 186 of Specification No. 2), AAV4-8 / rh.64 (U.S. Patent Application Publication No. 201) SEQ ID NO. 50315612, SEQ ID NO. 15, AAVLG-9 / hu.39 (US Patent Publication No. 50315612) Application Publication No. 20150315612, Sequence ID No. 24, AAV54.5 / hu.2 3 (Sequence ID 60 of U.S. Patent Application Publication No. 20150315612), AAV54 .2 / hu.22 (Sequence ID 67 of U.S. Patent Application Publication No. 20150315612) ), AAV54.7 / hu.24 (U.S. Patent Application Publication No. 20150315612) (Sequence ID 66), AAV54.1 / hu.21 (U.S. Patent Application Publication No. 20150315) (Sequence ID 65 of Specification No. 612), AAV54.4R / hu.27 (U.S. Patent Application Publication No. Sequence ID 64 of Specification No. 20150315612, AAV46.2 / hu.28 (United States) (Sequence ID 68 of Patent Application Publication No. 20150315612), AAV 46.6 / h u.29 (Sequence ID 69 of U.S. Patent Application Publication No. 20150315612), AA V128.1 / hu.43 (Arrangement of the specification of U.S. Patent Application Publication No. 20150315612) Patent No. 80), or variants thereof, etc., U.S. Patent Application Publication No. 20150315612 As described in the specification (which is incorporated herein by reference in its entirety) AAV serotypes may include or may have the following sequence, or may contain such a sequence, or are derived from such a sequence. obtain.

[0325] In some embodiments, the AAV particles of the present invention are, but are not limited to, true AAVs. ttAAV) (Sequence number 2 of the international release pamphlet No. 2015121501), "UP enn AAV10 (Sequence ID 8 in International Publication No. 2015121501) "Japanese AAV10" (International Publication No. 2015121501 brochure) Sequence ID 9), or variants thereof, etc., International Publication No. 2015121501 PAN As stated in the frets (this content is incorporated by reference in its entirety in this specification) AAV serotypes may include or may have the following sequence, or may contain such a sequence, or are derived from such a sequence. obtain.

[0326] According to the present invention, AAV particles can be selected from or derived from various species of AA It may include V capsid serotypes. In one embodiment, AAV was tri-AAV (AAAV). It is acceptable. AAAV serotypes are not limited to AAAV (US 9,238,8 Sequence IDs 1, 2, 4, 6, 8, 10, 12, and 14 of Specification No. 00, or their respective barriers For example, U.S. Patent No. 9238800 (this content is referenced in its entirety in this specification). The sequence may be as described in (referenced by) or may have such a sequence. .

[0327] In one embodiment, the AAV particles may be or originate from bovine AAV (BAAV). The AAV capsid serotype may include, but is not limited to, BAAV (Sequences 1 and 6 of U.S. Patent No. 9,193,769), or their variants, U.S. Patent No. 9,193,769 (This content is referred to in its entirety herein) The sequence may be as described in (referenced), or may have the same sequence. BA AV serotypes are not limited to BAAV (as specified in U.S. Patent No. 7,427,396). Numbers 5 and 6), or variants thereof, etc., U.S. Patent No. 7,427,396 (of which The contents are arranged as described herein (the whole is referred to herein by reference) It is also acceptable, or may have it.

[0328] In one embodiment, the AAV particles may be or may be derived from goat AAV. May include psido serotypes. Goat AAV serotypes are not limited to, but may include goat AAV (USA Special). See Sequence ID 3) of Patent No. 7427396, or its variants, etc., U.S. Patent No. 7447396 The contents of Specification No. 27396 (which are incorporated herein by reference in their entirety) The arrangement may be as described, or may have such an arrangement.

[0329] In other embodiments, the AAV particles are hybrid AAVs derived from two or more parent serotypes. It may include AAV capsid serotypes that have been manipulated. In one embodiment, AAV is AAV2G9 may also include sequences from AAV2 and AAV9. AAV2G9 A The AV serotype is described in U.S. Patent Application Publication No. 20160017005 (this content is in this specification). The arrangement may be as described in the book (which is entirely referenced by reference), and They may have it.

[0330] In one embodiment, AAV particles are described by Pulicherla et al. (Molecule). Lar Therapy 19(6):1070-1078(2011) (This content is from the book) Amino acid 39 (as described in the specification, which is entirely incorporated by reference) AAV9 capsid library containing mutations from 0 to 627 (VP1 numbering) May include AAV capsid serotypes that may be formed. Serotype and corresponding nucleotides and Amino acid substitutions are not limited to AAV9.1(G1594C;D532H), A AV6.2 (T1418A and T1436X; V473D and I479K), AAV9. 3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F4 17S), AAV9.5(A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6(T1231A;F411I), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A150 0G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A 1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T ;N457H, T574S), AAV9.14(T1340A, T1362C, T156 0C, G1713A;L447H), AAV9.16(A1775T;Q592L), A AV9.24 (T1507C, T1521G; W503R), AAV9.26 (A133 7G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D5 56A), AAV9.34 (A1534G, C1794T; N512D), AAV9.3 5(A1289T, T1450A, C1494T, A1515T, C1794A, G18 16A;Q430L, Y484N, N98K, V606I), AAV9.40(A169 4T, E565V), AAV9.41(A1348T, T1362C; T450S), A AV9.44(A1684C、A1701T、A1737G;N562H、K567N) 、AAV9.45(A1492T、C1804T;N498Y、L602F)、AAV9 .46(G1441C、T1525C、T1549G;G481R、W509R、L51 7V)、9.47(G1241A、G1358A、A1669G、C1745T;S41 4N、G453D、K557E、T582I)、AAV9.48(C1445T、A17 36T;P482L、Q579L)、AAV9.50(A1638T、C1683T、T 1805A;Q546H、L602H)、AAV9.53(G1301A、A1405C 、C1664T、G1811T;R134Q、S469R、A555V、G604V)、 AAV9.54(C1531A、T1609A;L511I、L537M)、AAV9. 55(T1605A;F535L)、AAV9.58(C1475T、C1579A;T 492I、H527N)、AAV.59(T1336C;Y446H)、AAV9.61 (A1493T;N498I)、AAV9.64(C1531A、A1617T;L51 1I)、AAV9.65(C1335T、T1530C、C1568A;A523D)、 AAV9.68(C1510A;P504T)、AAV9.80(G1441A、;G4 81R)、AAV9.83(C1402A、A1500T;P468T、E500D)、 AAV9.87(T1464C、T1468C;S490P)、AAV9.90(A11 96T;Y399F)、AAV9.91(T1316G、A1583T、C1782G、 T1806C;L439R、K528I)、AAV9.93(A1273G、A1421 G, A1638C, C1712T, G1732A, A1744T, A1832T;S42 5G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F5 35L is also acceptable.

[0331] In one embodiment, the AAV particles of the present invention exhibit increased directional attraction to the brain, as described in International Publication No. 2014. Distribution of pamphlet No. 160092 (the entire contents of which are incorporated herein by reference) It may contain a capsid protein having sequences 1 and 3.

[0332] In one embodiment, the AAV particles of the present invention target oligodendrocytes in the central nervous system. It may contain a capsid protein. The capsid protein is described in International Publication No. 201405. Brochure No. 2789 (the contents of which are incorporated herein by reference in their entirety) AAV containing the AAV capsid coding sequence of SEQ ID NO: 1 or the amino acid sequences of SEQ ID NOs: 2-4 It may contain capsid proteins.

[0333] In one embodiment, the AAV particles of the present invention are specified in U.S. Patent No. 8,927,514 ( The contents of the CNS blood are disclosed as described in (the entire contents of which are incorporated herein by reference). It may contain capsid proteins with an increased ability to cross the blood-brain barrier. The amino acid sequence and nucleic acid sequence of the protein are not limited to, but include, U.S. 8,92 The following are examples of Sequence IDs 2-17 and 25-33 from Specification No. 7,514. can.

[0334] In some embodiments, the AAV particles of the present invention are AAV2 capsid protein or the May contain variants. AAV particles containing the AAV2 capsid protein may transmit genes to the brain. It has been shown to be effectively delivered to neurons in the retina and spinal cord. In one embodiment, AA V2 capsid protein is described in U.S. Patent No. 6,691,948 and No. 8,299. , Specification No. 215 (The contents of each of these are incorporated herein by reference in whole) As disclosed in ( ), AAV particles are targeted to the vascular endothelium of the brain. Further modifications may be made, such as adding a component to the capsid protein. The child is a functional hTREM2 antibody or its antigen binding cleavage as described herein, for example. It can be used for the delivery of individual items.

[0335] In some embodiments, the AAV particles of the present invention are AAV5 capsid protein or the May contain variants. AAV particles containing the AAV5 capsid protein are found in the cortex, hippocampus ( Various regions of the CNS, including the HPC, cerebellum, substantia nigra (SN), striatum, globus pallidus, and spinal cord. Neurons located in this area can be transduced (Burger C et al, Mol Ther.,2004,10(2):302-317;Liu G et al,Mo l Ther.2007,15(2):242-247; and Colle M et a l, Hum, Mol. Genet. 2010, 19(1):147-158). One implementation form In this state, A has increased transduction of CNS cells into AAV 5 capsid proteins. AV particles are specified in U.S. Patent No. 7,056,502 (this entire content is referred to in the present invention). It may also be a particle from (as referenced in the detailed document).

[0336] In some embodiments, the AAV particles of the present invention are AAV6 capsid protein or the It may contain variants. Recombinant AAV6 serotypes are transmitted via intraventricular (ICV) injection into the spinal cord. Motor neurons can be targeted (Dirren E et al., Hum Gene Ther., 2014, 25(2):109-120). In addition, San S Research by ebastian et al. showed that in rat brains, the AAV6 serotype is transmitted from the nerve cell body to the terminal It has been shown that it can be transported retrogradely to the end (San Sebastian et al, G en Ther.,2014,20(12):1178-1183).

[0337] In some embodiments, the AAV particles of the present invention are AAV8 capsid protein or the It may contain variants. AAV particles containing the AAV8 capsid protein may, for example, be found in the hippocampus. Neurons can be transduced (Klein RL et al, Mol Th er.,2006,13(3):517-527). In one embodiment, AAV8 capsid Proteins are described in U.S. Patent No. 8,318,480 (this content is fully described herein). The body may contain the amino acid sequence of Sequence ID No. 2 (which is referenced by the body).

[0338] In some embodiments, the AAV particles of the present invention are AAV9 capsid protein or the May contain variants. Gene delivery mediated by AAV9 capsid serotypes occurs in the brain. This has been observed along with efficient long-term expression of the transgene after intracellular injection into the CNS (Kle in RL et al,Eur J Neurosci.,2008,27:1615 -1625). AAV9 serotype is administered peripherally (e.g., intravenously) to neonates. Later, it became possible to produce robust, widespread neuronal phenotype throughout the CNS. Kiru (Foust KD et al, Nat. Biotechnol, 2009, 27 :59-65; and Duque S et al, Mol Ther., 2009, 17 :1187-1196). Intrathecal (intraciscomasal route) administration of AAV9 serotypes is also widespread. It can cause spinal cord expression. In one embodiment, the AAV9 serotype is a U.S. patent Specification No. 7,198,951 (the entire content of which is incorporated herein by reference) It may contain an AAV capsid protein having the amino acid sequence of SEQ ID NO: 2. In another embodiment, The AAV9 serotype is specified in U.S. Patent Application Publication No. 20130224836 (this content is Surface in an amino acid sequence, as disclosed in the entirety of which is incorporated herein by reference. In a compound where at least one of the exposed tyrosine residues is substituted with another amino acid residue It may contain the VP1 capsid protein of row number 2, 4, or 6. In the embodiment, AAV9 The AAV vector containing the psid is administered systemically, for example, intravenously. In the embodiment, A AAV vectors containing AV9 capsids are injected into the brain, spinal cord, or cerebrospinal fluid (CSF), for example, into the spinal cavity. It is administered orally.

[0339] In some embodiments, the AAV vector is manipulated to achieve specific properties. It contains variant capsid proteins. For more information on this method, see, for example, International Publication No. 2011038187 (this content) Such methods and vectors are described herein (the whole is incorporated herein by reference). For more information on this, see, for example, International Publication No. 2012112832 and International Brochure No. 2015054653 (The entire contents of this document are referenced in this specification) It is also described in (which is referenced in). Such variant capsids include, for example, Sequence ID 23 of International Publication No. 2015054653, or its variant For example, further variant capsids include International Publication No. 2017 / 01 The capsulphuration described in pamphlet No. 9994 (this content is entirely referenced) Examples include the AAV sequence. In one embodiment, the AAV vector is an Anc80 AAV capsid sequence. Column capsid (e.g., Sequence ID 1 in International Publication No. 2017019994) For example, Anc80L65 (for example, International Publication No. 2017019994 pamphlet) Sequence ID 23), or, for example, Anc110 (for example, International Publication No. 20170199) Includes sequence number 42 of pamphlet No. 94.

[0340] In some embodiments, the AAV particles of the present invention are AAVrh10 capsid protein or It may contain its variant. AAV particles containing the AAVrh10 capsid protein are Intrathecal (IT) administration can target neurons in the spinal cord, as well as other cells. In one embodiment, the AAVrh10 capsid protein is specified in European Patent No. 2341068. It may include the amino acid sequence of Sequence ID No. 81 as detailed below.

[0188] In some embodiments The AAV of this invention is AAVDJ capsid protein, AAVDJ / 8 capsid protein The quality, or may include these variants. Holehonnur et al. found that AAVDJ / 8 blood We demonstrated that the clear form can target neurons within the basal lateral amygdala (BLA) (Hole honnur R et al.,BMC Neurosci,2014,Feb 18 :15:28). In one embodiment, AAVDJ capsid protein and / or AAVDJ The / 8 capsid protein is derived from the first AAV serotype (e.g., AAV2). The region, the second region derived from the second AAV serotype (e.g., AAV8), and the third AA It may also include an amino acid sequence containing a third region derived from serotype V (e.g., AAV9). Here, the first, second, and third regions include any amino acid sequence disclosed herein. It is visible.

[0341] In one embodiment, the AAV particles of the present invention can transduce dorsal root ganglia (DRGs). It may contain capsid proteins that have been shown or are publicly known to do so.

[0342] In one embodiment, the AAV particles of the present invention have been shown to transduce motor neurons. It may contain capsid proteins that are present or are known to be present.

[0343] In one embodiment, the AAV particle contains a self-complementary (SC) vector genome.

[0344] In one embodiment, the AAV particles contain a single-stranded (SS) genome.

[0345] In one embodiment, by using AAV particles containing a self-complementarity (sc) vector, This can result in higher expression than AAV particles containing the corresponding single-stranded vector genome.

[0346] In one embodiment, the serotype of AAV particles described herein is used for desired distribution and transduction. This may depend on efficiency and the required cell targeting. Sorrentino et a l.(comprehensive map of CNS transduction by eight adeno-associated virus serotyp es upon cerebrospinal fluid administration on in pigs,Molecular Therapy accepted ar tickle preview online 07 December 2015;do i:10.1038 / mt.2015.212;This content is referenced in its entirety in this specification. As described by (and cited by) the AAV serotypes have different distributions and transductions. This resulted in improved efficiency and cell targeting. In order to achieve the desired efficacy, the target is Not only the cells themselves, but also the AAV serotype that best matches the desired transduction efficiency and distribution. You need to make a choice.

[0347] In one embodiment, the AAV vector is an AAV9 capsid (as described herein). (1) ITR derived from AAV2, (2) CMV enhancer (for example, sequence number (134), (3) CBA promoter (e.g., SEQ ID NO. 135), (4) SV40 (e.g., SEQ ID NO: 137), (5) hTREM2 antibody or its antigen-binding fragment ( For example, polynucleotides encoding (as described herein), and (6)B Includes an AAV vector plasmid containing a GH polyA signal (e.g., SEQ ID NO: 138). In one embodiment, elements (2) to (6) are placed on the AAV vector plasmid at 5' It is placed from 3'. In the embodiment, elements (2) to (6) are AAV vector planes It is placed between the 5'ITR and 3'ITR on the Smid. In one embodiment, the AAV vector - is an scAAV vector.

[0348] Production of monoclonal antibodies Monoclonal antibodies (mAbs) are a conventional monoclonal antibody methodology, for example, Koh Ler and Milstein, 1975, Nature 256:495, standard It can be produced by various technologies, including somatic cell hybridization techniques. Many techniques for producing ronal antibodies (e.g., viral or carcinogenic B lymphocytes) (Transformed form) may be used.

[0349] The animal strain used to prepare hybridomas is the mouse strain. Doma production is a well-established procedure. For isolation of immunized splenocytes for fusion. Immunization protocols and technologies are known in the art. Fusion partners (e.g., M) The myeloma cells and fusion procedure are also known.

[0350] In some embodiments, the antibody of the present invention is a humanized monoclonal antibody. The chimeric or humanized antibody and its antigen-binding fragment are prepared as a mouse monoclean. It can be prepared based on the sequence of ronal antibodies, encoding heavy and light chain immunoglobulins. DNA was obtained from the target mouse hybridoma using standard molecular biology techniques. They can be manipulated to include non-mouse (e.g., human) immunoglobulin sequences. For example, To produce chimeric antibodies, the mouse variable region is used with methods known in the art. It can be linked to a steady-state region (for example, Cabilly et al., U.S. Patent No. 4,816,56 (See Specification No. 7). In order to produce humanized antibodies, the mouse CDR region is used in this technology. It can be inserted into the human framework using methods known in the field. For example, Wi U.S. Patent No. 5,225,539 to nter, and U.S. Patent to Queen et al. Specification No. 5,530,101; Specification No. 5,585,089; No. 5,693,762 Please refer to the specifications of No. 6180370 and No. 6180370.

[0351] In some embodiments, the antibody of the present invention is a human monoclonal antibody. Such human monoclonal antibodies against 2 are derived from a part of the human immune system, not a mouse system. It can be produced using the transgenic or transchromosomal mice that are in possession. These transgenic and transchromosomal mice are described herein. These include mice referred to as HuMAb mice and KM mice, respectively, and are collectively referred to herein as It is called a "human Ig mouse".

[0352] HuMAb Mouse® (registered trademark) (Medarex, Inc.) is an unrearranged human mouse. Human immunoglobulin genes encoding heavy chain (μ and γ) and κ light chain immunoglobulin sequences Mini loci include target mutations that inactivate endogenous μ and κ chain loci (for example) Lonberg, et al., 1994 Nature 368(6474):8 See pages 56-859). Therefore, mice exhibit reduced expression of mouse IgM or K. This indicates that, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching. (Lo nberg,N.et al.,1994 supra;reviewed in Lo nberg, N., 1994 Handbook of Experimental P. harmacology 113:49-101;Lonberg, N. and Hus zar, D., 1995 Intern. Rev. Immunol. 13:65-93, And Harding, F. and Lonberg, N., 1995 Ann. NY Acad.Sci.764:536-546). Preparation and use of HuMAb mice, and Such genome modifications carried by mice were described by Taylor, L. et al. ,1992 Nucleic Acids Research 20:6287-629 5;Chen,J.et al.,1993 International Immun ology 5:647-656;Tuaillon et al.,1993 Pro c.Natl.Acad.Sci.USA 94:3720-3724;Choi et al.,1993 Nature Genetics 4:117-123;Chen ,J.et al.,1993 EMBO J.12:821-830;Tuaillo n et al.,1994 J.Immunol.152:2912-2920;Ta ylor, L. et al., 1994 International Immunol ogy 579-591; and Fishwild, D. et al., 1996 Nat Further details are available in Ure Biotechnology 14:845-851. All of these contents are incorporated herein by reference in their entirety. Furthermore, All of the above is in reference to U.S. Patent No. 5,545,806 to Lonberg and Kay; 5,56 Specification No. 9,825; Specification No. 5,625,126; Specification No. 5,633,425; Specification No. 5,789,650; Specification No. 5,877,397; No. 5,661,016 No. 5,814,318 Specification; No. 5,874,299 Specification; No. 5,77 U.S. Patent No. 0,429; U.S. Patent No. 5,545,807 to Surani et al.; all PCT Public Publication No. 92103918 to Lonberg and Kay. International Publication No. 93 / 12227 pamphlet, International Publication No. 94 / 25585 pamphlet International Publication No. 97113852 pamphlet, International Publication No. 98 / 24884 pamphlet Lett and International Publication No. 99 / ...

Claims

1. Human myeloid cell trigger expression 2 Session on myeloid cells 2) (hTREM2) protein The immunoglobulin superfamily (IgSF) domain binds to the hTREM2 domain. An antibody or its antigen-binding fragment that stabilizes an protein (e.g., SEQ ID NO: 1, 2, or 3).

2. hTREM2's D39, S40, M41, K42, W44, G45, R46, R47, H67, N68, L69, W70, L71, L72, F74, L75, R77, D87, A claim that binds to one or more residues selected from the group consisting of T88, L89, and G90. The antibody or antigen-binding fragment thereof as described in 1.

3. hTREM2's D39, S40, M41, K42, W44, G45, R46 and R47 One or more residues selected from the group consisting of H67, N68, L69, W70, L71, One or more residues selected from the group consisting of L72, F74, L75, and R77, and D Binds to one or more residues selected from the group consisting of 87, T88, L89, and G90, The antibody or antigen-binding fragment thereof according to claim 1.

4. hTREM2's D39, S40, M41, K42, W44, G45, R46 and R47 Five or more residues selected from the group consisting of H67, N68, L69, W70, L71, Four or more residues selected from the group consisting of L72, F74, L75, and R77, and D Binds to three or more residues selected from the group consisting of 87, T88, L89, and G90. The antibody or antigen-binding fragment thereof according to claim 1.

5. hTREM2's D39, S40, M41, K42, W44, G45, R46 and R47 Seven or more residues selected from the group consisting of H67, N68, L69, W70, L71, Four or more residues selected from the group consisting of L72, F74, L75, and R77, and D Binds to three or more residues selected from the group consisting of 87, T88, L89, and G90. The antibody or antigen-binding fragment thereof according to claim 1.

6. hTREM2's D39, S40, M41, K42, W44, G45, R46 and R47 Five or more residues selected from the group consisting of H67, N68, L69, W70, L7 1. All of L72, F74, L75 and R77, as well as D87, T88, L89 and G9 The antibody according to claim 1, or the antibody that binds to three or more residues selected from the group consisting of 0. Antigen-binding fragment.

7. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to one or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

8. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to two or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

9. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to three or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

10. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to four or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

11. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to five or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

12. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to six or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

13. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to seven or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

14. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to eight or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

15. hTREM2 S40, M41, W44, G45, W70, L71, L72, F74, The following is a claim of binding to nine or more residues selected from the group consisting of T88 and L89. An antibody or its antigen-binding fragment.

16. hTREM2 residues S40, M41, W44, G45, W70, L71, L72, F7 4. The antibody or antigen-binding fragment thereof according to claim 1, which binds to all of T88 and L89.

17. One or more selected from the group consisting of D39, K42, R46, and G90 of hTREM2 An antibody or antigen-binding fragment thereof according to any one of claims 7 to 16, which binds to a residue of the antibody.

18. Two or more selected from the group consisting of D39, K42, R46, and G90 of hTREM2 An antibody or antigen-binding fragment thereof according to any one of claims 7 to 16, which binds to a residue of the antibody.

19. Three or more selected from the group consisting of hTREM2 D39, K42, R46, and G90 An antibody or antigen-binding fragment thereof according to any one of claims 7 to 16, which binds to a residue of the antibody.

20. Claims 7 to bind to all of the residues D39, K42, R46 and G90 of hTREM2. An antibody or antigen-binding fragment thereof as described in any one of item 15.

21. hTREM2 residues S40, M41, W44, G45, W70, L71, L72, F7 4. Combined with all of T88, L89, D39, K42, R46 and G90, as per claim 1. The antibody or its antigen-binding fragment as described above.

22. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 16, which binds to one or more residues selected from the group. A body or its antigen-binding fragment.

23. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 16, which binds to two or more residues selected from the group. A body or its antigen-binding fragment.

24. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 16, which binds to three or more residues selected from the group. A body or its antigen-binding fragment.

25. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 16, which binds to four or more residues selected from the group. A body or its antigen-binding fragment.

26. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 16, which binds to five or more residues selected from the group. A body or its antigen-binding fragment.

27. hTREM2 consists of R47, H67, N68, L69, L75, R77 and D87. The antibody according to any one of claims 7 to 15, which binds to six or more residues selected from the group. A body or its antigen-binding fragment.

28. hTREM2 residues S40, M41, W44, G45, W70, L71, L72, F7 4. All of T88, L89, R47, H67, N68, L69, L75, R77 and D87 An antibody or antigen-binding fragment thereof according to claim 1, which binds to a certain part.

29. An antibody or the antibody according to any one of claims 1 to 28 that activates hTREM2 Original combined fragment.

30. To promote one or more hTREM2-dependent physiological activities in TREM2-expressing cells, An antibody or antigen-binding fragment thereof as described in any one of items 1 to 29.

31. a) For example, in hTREM2-expressing cells, phagocytosis is increased (for example, here before The TREM2-expressing cells are hM2A macrophages or human iPS-derived microglia-like cells. (is) b) For example, in hTREM2-expressing cells, increasing chemotaxis (for example, here the above TREM2-expressing cells are hM2a ​​macrophages or human iPS-derived microglia-like cells. be) c) Increase NFAT-driven reporter gene activity in human monocytic cell lines. d) For example, increasing Syk phosphorylation in hTREM2-expressing cells (for example, In this case, the TREM2-expressing cell is an hM2A macrophage, or e) Increase any combination of two or more of a) to d), An antibody or antigen-binding fragment thereof according to any one of claims 1 to 30.

32. For example, against hTREM2 on the cell surface, for example, FAC of hM2a ​​macrophages The half-effective concentration (EC) of 1 nM or less was measured by S analysis. ５０ Claims 1 to 3, which are joined by ) An antibody or antigen-binding fragment thereof as described in any one of item 1.

33. For example, against hTREM2 on the cell surface, for example, FAC of hM2a ​​macrophages The half-effective concentration (EC) of 0.59 nM or less was measured by S analysis. ５０ Claims to be joined by An antibody or antigen-binding fragment thereof as described in any one of items 1 to 32.

34. For hTREM2, for example, by measuring using surface plasmon resonance (SPR), the result was 150 pM. The following dissociation constant (K) Ｄ The antibody or so described in any one of claims 1 to 33, which is bound by ) Antigen-binding fragment.

35. For hTREM2, for example, by measuring by surface plasmon resonance (SPR), the value is less than 50 pM. The dissociation constant (K) below Ｄ The antibody or the antibody according to any one of claims 1 to 34 that is bound by ) Antigen-binding fragment.

36. To stabilize the hTREM2 protein on the cell surface of hTREM2-expressing cells, An antibody or antigen-binding fragment thereof as described in any one of the requests 1 to 35.

37. The hTREM2-expressing cells are macrophages, such as M2a macrophages, and dendritic cells. Cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, cutaneous Langerhans cells The antibody or antigen according to claim 36, which is a Kupffer cell, neutrophil, or hepatocellular carcinoma cell. Combined fragments.

38. In hM2A macrophages, the erythrocytes of the hTREM2 protein were found to be eluting at concentrations of 100 nM or less. An antibody according to any one of claims 1 to 37 that reduces ct domain shedding. It is the antigen-binding fragment.

39. The TREM2 cell surface expression increases by more than three times, as described in any one of claims 1 to 38. An antibody or its antigen-binding fragment.

40. The IgSF domain of hTREM2 is one of sequence numbers 1, 2, and 3. An antibody or the antibody according to any one of claims 1 to 39, comprising mino acid residues 19 to 132. Original combined fragment.

41. a) Including sequence number 41, sequence number 44, sequence number 45, or sequence number 47, for example The heavy chain variable region CDR1 consisting of the same; SEQ ID NO: 42 or SEQ ID NO: 46 or SEQ ID NO: 48 Including, for example, a heavy chain variable region CDR2 consisting thereof; including SEQ ID NO: 43 or SEQ ID NO: 49 For example, a heavy chain variable region CDR3 consisting of the same; sequence number 54 or sequence number 57 or sequence number For example, a light chain variable region CDR1 including number 60; Sequence ID 55 or Sequence ID For example, a light chain variable region CDR2 comprising 58; and Sequence ID 56 or Sequence ID A light chain variable region CDR3 including, for example, 59; b) Including, for example, sequence number 4, sequence number 7, sequence number 8, or sequence number 10 A heavy chain variable region CDR1; including SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 11, for example For example, a heavy chain variable region CDR2 consisting of the same; including SEQ ID NO: 6 or SEQ ID NO: 12, for example. Heavy chain variable region CDR3 consisting of; including SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23 For example, a light chain variable region CDR1 consisting of the same; including sequence number 18 or sequence number 21, for example For example, a light chain variable region CDR2 consisting of the same; and including SEQ ID NO: 19 or SEQ ID NO: 22, For example, the light chain variable region CDR3 consisting of that; c) Including, for example, sequence number 4, sequence number 7, sequence number 8, or sequence number 10. A heavy chain variable region CDR1; including, for example, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:

11. A heavy chain variable region CDR2 consisting thereof; including, for example, SEQ ID NO: 6 or SEQ ID NO:

12. A heavy chain variable region CDR3; including SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23, For example, a light chain variable region CDR1 consisting of the same; including sequence number 18 or sequence number 21, for example. if a light chain variable region CDR2 consisting thereof; and including sequence number 78 or sequence number 79, for example if a light chain variable region CDR3 consisting thereof; or d) Including sequence number 84, sequence number 87, sequence number 88, or sequence number 90, for example Heavy chain variable region CDR1 consisting of; including SEQ ID NO: 85, SEQ ID NO: 89, or SEQ ID NO: 91 For example, a heavy chain variable region CDR2 consisting of the same; including sequence number 86 or sequence number 92, For example, a heavy chain variable region CDR3 consisting of the same; Sequence ID 97 or Sequence ID 100 or Sequence Number Light chain variable region CDR1 including, for example, number 103; Sequence ID 98 or Sequence ID A light chain variable region CDR2 including, for example, 101; and sequence number 99 or sequence number A light chain variable region CDR3 including, for example, item 102. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 40, comprising:

42. An antibody or its antigen-binding fragment that binds to the IgSF domain of the hTREM2 protein. That is, a) Including sequence number 41, sequence number 44, sequence number 45, or sequence number 47, for example The heavy chain variable region CDR1 consisting of the following; sequence number 42 or sequence number 46 or sequence number 48 Includes, for example, a heavy chain variable region CDR2 consisting thereof; including SEQ ID NO: 43 or SEQ ID NO: 49 For example, a heavy chain variable region CDR3 consisting of the same; Sequence ID 54 or Sequence ID 57 or Sequence ID No. Light chain variable region CDR1 including, for example, number 60; Sequence ID 55 or Sequence ID 5 For example, a light chain variable region CDR2 comprising 8; and Sequence ID No. 56 or Sequence ID No. 5 A light chain variable region CDR3 including, for example, 9; b) Including, for example, sequence number 4, sequence number 7, sequence number 8, or sequence number 10. A heavy chain variable region CDR1; including, for example, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:

11. A heavy chain variable region CDR2 consisting thereof; including, for example, SEQ ID NO: 6 or SEQ ID NO:

12. A heavy chain variable region CDR3; including SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23, For example, a light chain variable region CDR1 consisting of the same; including sequence number 18 or sequence number 21, for example. For example, a light chain variable region CDR2 consisting of the same; and including sequence number 19 or sequence number 22. if the light chain variable region CDR3 consists of the same; c) Including, for example, sequence number 4, sequence number 7, sequence number 8, or sequence number 10. A heavy chain variable region CDR1; including, for example, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO:

11. A heavy chain variable region CDR2 consisting thereof; including, for example, SEQ ID NO: 6 or SEQ ID NO:

12. A heavy chain variable region CDR3; including SEQ ID NO: 17, SEQ ID NO: 20, or SEQ ID NO: 23, For example, a light chain variable region CDR1 consisting of the same; including sequence number 18 or sequence number 21, for example. if a light chain variable region CDR2 consisting thereof; and including sequence number 78 or sequence number 79, for example if a light chain variable region CDR3 consisting thereof; or d) Including sequence number 84, sequence number 87, sequence number 88, or sequence number 90, for example Heavy chain variable region CDR1 consisting of; including SEQ ID NO: 85, SEQ ID NO: 89, or SEQ ID NO: 91 For example, a heavy chain variable region CDR2 consisting of the same; including sequence number 86 or sequence number 92, For example, a heavy chain variable region CDR3 consisting of the same; Sequence ID 97 or Sequence ID 100 or Sequence Number Light chain variable region CDR1 including, for example, number 103; Sequence ID 98 or Sequence ID A light chain variable region CDR2 including, for example, 101; and sequence number 99 or sequence number A light chain variable region CDR3 including, for example, item 102. An antibody containing an antigen-binding fragment thereof.

43. a) A VH poly having at least 95% sequence identity with sequence number 13 or sequence number 50. The peptide sequence has at least 95% sequence identity with SEQ ID NO: 24 or SEQ ID NO:

61. VL polypeptide sequence; or b) A VH poly having at least 95% sequence identity with sequence number 13 or sequence number 93. The peptide sequence and sequence identity of at least 95% with SEQ ID NO: 80 or SEQ ID NO: 104 VL polypeptide sequence An antibody or antigen-binding fragment thereof according to any one of claims 1 to 42, comprising:

44. a) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 7; including SEQ ID NO: 5 , for example, a heavy chain variable region CDR2 comprising the same; for example, a heavy chain variable region comprising the same including SEQ ID NO: 6 Chain variable region CDR3; light chain variable region CDR1, for example, comprising sequence number 17; For example, a light chain variable region CDR2 comprising sequence number 18; and sequence number 19 For example, a light chain variable region CDR3 consisting of the same; b) Heavy chain variable region CDR1 including, for example, sequence number 44; sequence number 42 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 43, from A heavy chain variable region CDR3; for example, a light chain variable region CD comprising sequence number 54. R1; for example, a light chain variable region CDR2 comprising sequence number 55; and sequence number 5 A light chain variable region CDR3 including, for example, 6; c) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 7; including SEQ ID NO: 5 , for example, a heavy chain variable region CDR2 comprising the same; for example, a heavy chain variable region comprising the same including SEQ ID NO: 6 Chain variable region CDR3; light chain variable region CDR1, for example, comprising sequence number 17; Including, for example, the light chain variable region CDR2 comprising SEQ ID NO: 18; and including SEQ ID NO: 78 For example, a light chain variable region CDR3 consisting of the same; or d) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 87; SEQ ID NO: 85 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 86, from Heavy chain variable region CDR3; light chain variable region CD comprising, for example, SEQ ID NO:

97. R1; for example, a light chain variable region CDR2 comprising SEQ ID NO: 98; and SEQ ID NO: 9 A light chain variable region CDR3 including 9, for example, consisting of the same. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, comprising:

45. a) Heavy chain variable region CDR1 of SEQ ID NO: 8; for example, a heavy chain comprising SEQ ID NO:

9. Variable region CDR2; heavy chain variable region CDR3, for example, containing sequence number 6; sequence For example, a light chain variable region CDR1 including number 20; for example, including sequence number 21 for example, a light chain variable region CDR2 comprising the same; and a light chain variable region comprising the same including sequence number 22. Chain variable region CDR3; b) Heavy chain variable region CDR1 including, for example, sequence number 45; sequence number 46 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 43, from A heavy chain variable region CDR3; for example, a light chain variable region CD comprising SEQ ID NO:

57. R1; for example, a light chain variable region CDR2 comprising SEQ ID NO: 58; and SEQ ID NO: 5 A light chain variable region CDR3 including, for example, 9; c) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 8; including SEQ ID NO: 9 , for example, a heavy chain variable region CDR2 comprising the same; for example, a heavy chain variable region comprising the same including SEQ ID NO: 6 Chain variable region CDR3; light chain variable region CDR1, for example, comprising sequence number 20; Including, for example, the light chain variable region CDR2 comprising SEQ ID NO: 21; and including SEQ ID NO: 79 For example, a light chain variable region CDR3 consisting of the same; or d) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 88; SEQ ID NO: 89 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 86, from A heavy chain variable region CDR3; for example, a light chain variable region C comprising sequence number 100. DR1; light chain variable region CDR2 including, for example, sequence number 101; and sequence number A light chain variable region CDR3 including, for example, item 102. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, comprising:

46. a) A heavy chain variable region CDR1 including, for example, SEQ ID NO: 10; SEQ ID NO: 11 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, SEQ ID NO: 12, then A heavy chain variable region CDR3; for example, a light chain variable region CD comprising SEQ ID NO:

23. R1; a light chain variable region CDR2 comprising, for example, SEQ ID NO: 21; and SEQ ID NO: 1 A light chain variable region CDR3 including, for example, 9; b) Heavy chain variable region CDR1 including, for example, sequence number 47; sequence number 48 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 49, from A heavy chain variable region CDR3; for example, a light chain variable region CD comprising SEQ ID NO:

60. R1; for example, a light chain variable region CDR2 comprising SEQ ID NO: 58; and SEQ ID NO: 5 A light chain variable region CDR3 including, for example, 6; c) Heavy chain variable region CDR1 including, for example, sequence number 10; sequence number 11 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, SEQ ID NO: 12, then A heavy chain variable region CDR3; for example, a light chain variable region CD comprising SEQ ID NO:

23. R1; a light chain variable region CDR2 comprising, for example, SEQ ID NO: 21; and SEQ ID NO: 7 A light chain variable region CDR3 including, for example, 8; or d) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 90; SEQ ID NO: 91 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, SEQ ID NO: 92, from A heavy chain variable region CDR3; for example, a light chain variable region C comprising SEQ ID NO:

103. DR1; light chain variable region CDR2 including, for example, sequence number 101; and sequence number A light chain variable region CDR3 including, for example, number 99. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, comprising:

47. a) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 4; including SEQ ID NO: 5 , for example, a heavy chain variable region CDR2 comprising the same; for example, a heavy chain variable region comprising the same including SEQ ID NO: 6 Chain variable region CDR3; light chain variable region CDR1, for example, comprising sequence number 17; For example, a light chain variable region CDR2 comprising sequence number 18; and sequence number 19 For example, a light chain variable region CDR3 consisting of the same; b) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 41; SEQ ID NO: 42 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 43, from A heavy chain variable region CDR3; for example, a light chain variable region CD comprising sequence number 54. R1; for example, a light chain variable region CDR2 comprising sequence number 55; and sequence number 5 A light chain variable region CDR3 including, for example, 6; c) Heavy chain variable region CDR1 including, for example, SEQ ID NO: 4; including SEQ ID NO: 5 , for example, a heavy chain variable region CDR2 comprising the same; for example, a heavy chain variable region comprising the same including SEQ ID NO: 6 Chain variable region CDR3; light chain variable region CDR1, for example, comprising sequence number 17; Including, for example, the light chain variable region CDR2 comprising SEQ ID NO: 18; and including SEQ ID NO: 78 For example, a light chain variable region CDR3 consisting of the same; or d) Heavy chain variable region CDR1 including, for example, sequence number 84; sequence number 85 Includes, for example, a heavy chain variable region CDR2 consisting of the same; including, for example, sequence number 86, from Heavy chain variable region CDR3; light chain variable region CD comprising, for example, SEQ ID NO:

97. R1; for example, a light chain variable region CDR2 comprising SEQ ID NO: 98; and SEQ ID NO: 9 A light chain variable region CDR3 including 9, for example, consisting of the same. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, comprising:

48. a) VH including, for example, sequence number 13 and sequence number 24 including, for example VL consisting of the following; or b) VH including, for example, sequence number 50 and VH including, for example, sequence number 61 VL consisting of the following; or c) A sequence having at least 95% homology to sequence number 13, for example, For example, a sequence having at least 95% homology to VH and sequence number 24. Ranaru VL; or d) A sequence having at least 95% homology to sequence number 50, for example, For example, a sequence having at least 95% homology to VH and sequence number 61. Ranaru VL; or e) A sequence that differs from sequence number 13 by at least 1, 2, 3, 4, 5, or 6 amino acids Including, for example, VH and Sequence ID No. 24 and at least 1, 2, 3, 4, 5, or A VL consisting of, for example, a sequence that differs by only 6 amino acids; or f) A sequence that differs from sequence number 50 by at least 1, 2, 3, 4, 5, or 6 amino acids Including, for example, VH and Sequence ID No. 61 and at least 1, 2, 3, 4, 5, or A VL containing a sequence that differs by only 6 amino acids, for example, g) VH including, for example, SEQ ID NO: 13 and SEQ ID NO: 80, for example, VL consisting of the following; or h) VH including, for example, sequence number 93 and sequence number 104, for example VL consisting of the same; or i) A sequence having at least 95% homology to sequence number 13, for example, For example, a sequence having at least 95% homology to VH and sequence number 80. Ranaru VL; or j) A sequence having at least 95% homology to sequence number 93, for example, For example, it includes a sequence having at least 95% homology to VH and sequence number 104. VL consisting of; or k) A sequence that differs from sequence number 13 by at least 1, 2, 3, 4, 5, or 6 amino acids. Including, for example, VH and Sequence ID No. 80 and at least 1, 2, 3, 4, 5, or A VL consisting of, for example, a sequence that differs by only 6 amino acids; or l) A sequence that differs from sequence number 93 by at least 1, 2, 3, 4, 5, or 6 amino acids Including, for example, VH and sequence number 104 consisting of the same and at least 1, 2, 3, 4, 5, and For example, a VL consisting of a sequence that differs by only six amino acids. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 47, comprising:

49. a) Sequence ID 15, Sequence ID 29, Sequence ID 33, Sequence ID 35, Sequence ID 37, or For example, a heavy chain amino acid sequence comprising SEQ ID NO: 39, and SEQ ID NO: 26, For example, a light chain amino acid sequence consisting of such; b) Sequence ID 52, Sequence ID 66, Sequence ID 70, Sequence ID 72, Sequence ID 74, or For example, a heavy chain amino acid sequence comprising SEQ ID NO: 76, and SEQ ID NO: 63, For example, a light chain amino acid sequence consisting of such; c) Sequence ID 15, Sequence ID 29, Sequence ID 33, Sequence ID 35, Sequence ID 37, or A heavy chain amino acid sequence having at least 95% sequence identity with Sequence ID No. 39, and Sequence ID No. A light chain amino acid sequence having at least 95% sequence identity with 26; d) Sequence ID 52, Sequence ID 66, Sequence ID 70, Sequence ID 72, Sequence ID 74, or A heavy chain amino acid sequence having at least 95% sequence identity with Sequence ID No. 76, and Sequence ID No. A light chain amino acid sequence having at least 95% sequence identity with 63; e) A heavy chain amino acid sequence including, for example, SEQ ID NO: 15, and SEQ ID NO: 82 Includes, for example, a light chain amino acid sequence consisting thereof; f) A heavy chain amino acid sequence including, for example, SEQ ID NO: 95, and SEQ ID NO: 106 A light chain amino acid sequence including, for example, one consisting thereof; g) A heavy chain amino acid sequence having at least 95% sequence identity with Sequence ID No. 15, and A light chain amino acid sequence having at least 95% sequence identity with sequence number 82; or h) A heavy chain amino acid sequence having at least 95% sequence identity with Sequence ID No. 95, and A light chain amino acid sequence having at least 95% sequence identity with sequence number 106. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 48, comprising:

50. a) A heavy chain sequence including, for example, sequence number 15 and sequence number 26, for example a light chain sequence consisting of the same; b) A heavy chain sequence including, for example, SEQ ID NO: 29 and SEQ ID NO: 26, for example a light chain sequence consisting of the same; c) A heavy chain sequence including, for example, SEQ ID NO: 33 and SEQ ID NO: 26, for example a light chain sequence consisting of the same; d) A heavy chain sequence including, for example, SEQ ID NO: 35 and SEQ ID NO: 26, for example a light chain sequence consisting of the same; e) Including, for example, a heavy chain sequence comprising SEQ ID NO: 37 and SEQ ID NO: 26, for example a light chain sequence consisting of the same; f) A heavy chain sequence including, for example, sequence number 39 and sequence number 26, for example a light chain sequence consisting of the same; g) A heavy chain sequence including, for example, SEQ ID NO: 52 and SEQ ID NO: 63, for example a light chain sequence consisting of the same; h) A heavy chain sequence including, for example, sequence number 66 and sequence number 63, for example a light chain sequence consisting of the same; i) Including, for example, a heavy chain sequence comprising SEQ ID NO: 70 and SEQ ID NO: 63, for example a light chain sequence consisting of the same; j) Including, for example, a heavy chain sequence consisting of SEQ ID NO: 72 and including, for example, SEQ ID NO: 63 a light chain sequence consisting of the same; k) A heavy chain sequence including, for example, sequence number 74 and sequence number 63, for example a light chain sequence consisting of the same; l) A heavy chain sequence including, for example, sequence number 76 and sequence number 63, for example a light chain sequence consisting of the same; m) Including, for example, a heavy chain sequence consisting of, sequence number 15 and, for example, including, sequence number 82 a light chain sequence consisting thereof; or n) For example, a heavy chain sequence including sequence number 95 and sequence number 106, For example, the light chain sequence consisting of that Human myeloid cell trigger expression 2 (human triggering e Immunity to xpression on myeloid cells 2 (hTREM2) Antibodies that bind to globulin superfamily (IgSF) domains or their antigen binding cleavage. Piece.

51. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 50 and hTREM2 tan Antibodies or their antigen-binding fragments that compete for binding to a protein.

52. The antibody or the antigen-binding fragment thereof overlaps with the epitope described in any one of claims 1 to 51. An antibody or its antigen-binding fragment that binds to the epitope.

53. The claim states that the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. An antibody or antigen-binding fragment thereof as described in any one of items 1 to 52.

54. The antibody or its antigen-binding fragment comprises the IgG1 Fc region, the IgG2 Fc region, and IgG 4. An Fc region selected from the Fc region or the IgG2 / IgG4 hybrid Fc region. Including the antibody or antigen-binding fragment thereof according to any one of claims 1 to 52.

55. The antibody or its antigen-binding fragment exhibits reduced antibody-dependent cell-mediated cytotoxicity compared to the parent antibody. Includes a modified Fc region having ADCC or complement-dependent cell-mediated cytotoxicity (CDC) activity. or the antibody or antigen-binding fragment thereof according to any one of claims 1 to 49 or 51 to 54.

56. Claim 1, wherein the antibody or its antigen-binding fragment is a human or humanized antibody or its fragment. An antibody or antigen-binding fragment thereof as described in any one of items ~55.

57. The antibody according to any one of claims 1 to 56 is conjugated to a further portion. It is the antigen-binding fragment.

58. An antibody according to any one of claims 1 to 57, which is an IgG1 isotype.

59. The antibody according to any one of claims 1 to 56, which is a human or humanized antibody.

60. Select from Fab, F(ab')², Fv fragment, scFv, minibody, or diamondbody. An antigen-binding fragment of an antibody according to any one of claims 1 to 57, which is selected.

61. Monoclonal antibody or antigen-binding agent according to any one of claims 1 to 60 piece.

62. A multispecific antibody, for example, a bispecific antibody, as described in any one of claims 1 to 61. Antibodies of [the substance / product].

63. The multispecific antibody, for example, the bispecific antibody, is hTREM2 and human DAP12 The anti- body.

64. Any of claims 1 to 63, having an effector function altered by a mutation in the Fc region. The antibody described in item 1.

65. One of the antibodies or antigen-binding fragments described in any one of claims 1 to 64 A nucleic acid molecule or a set of nucleic acid molecules.

66. The nucleic acid molecule according to claim 65, wherein the nucleic acid is DNA.

67. Sequence numbers 14, 28, 51, 65, 16, 30, 34, 36, 38, 40, 53, 67 、７１、７３、７５、７７、２５、３１、６２、６８、２７、３２、６４、６９、１３、 Claim 65, which includes one or more of 93, 15, 95, 80, 104, 82, or 106. Or the nucleic acid molecule described in 66 or a set of nucleic acid molecules.

68. A vector comprising a nucleic acid molecule according to any one of claims 65 to 67.

69. The vector according to claim 68, which is a cloning vector or an expression vector.

70. Sequence IDs 14, 28, 51, 65, 16, and 3, which encode at least one CDR area. ０、３４、３６、３８、４０、５３、６７、７１、７３、７５、７７、２５、３１、６２ , 68, 27, 32, 64, 69, 13, 93, 15, 95, 80, 104, 82 or 1 The vector according to claim 68 or 69, comprising one or more of 06, or a fragment thereof.

71. Claims 68-70, which are capable of replicating in prokaryotic cells and / or eukaryotic cells. A vector as described in any one of the items.

72. From DNA vectors, RNA vectors, plasmids, cosmids, or viral vectors A vector selected according to any one of claims 68 to 71.

73. Any one of claims 68 to 72 further includes a detectable and / or selectable marker. The vector shown.

74. A set of two expression vectors according to any one of claims 68 to 73, wherein one of them The vector a) encodes at least one VH domain including sequence number 13 or 50 The other vector coats at least one VL domain containing sequence number 24 or 61. Do; or b) Encoding at least one VH domain including sequence number 13 or 93, and the other VH domain The constructor encodes at least one VL domain including sequence number 80 or 104, vector.

75. The vector is a viral vector, and optionally the viral vector is a wrench Viruses, adenoviruses, adeno-associated viruses (AAVs), herpes simplex virus (H2A) SV), parvovirus, retrovirus, vaccinia virus, Sindbisphenus (Sinb IS virus, influenza virus, reovirus, Newcastle disease virus ( NDV, measles virus, varicella-stomatitis virus (VSV), poliovirus, pox Viruses, Seneca Barley virus, Coxsackievirus, Enterovirus, Myxoma The vector according to any one of claims 68 to 74, which is an Virus or Marabavirus. 。

76. The vector according to claim 75, which is an AAV vector.

77. A vector according to any one of claims 68 to 76, further comprising a promoter.

78. AAV9 capsid, (1) ITR derived from AAV2, (2) CMV enhancer ( (3) CBA promoter (for example, including SEQ ID NO: 134) (4) SV40 introns (e.g., including sequence number 137), (5) hTR Encoding an EM2 antibody or its antigen-binding fragment (for example, as described herein) (6) a polynucleotide and a BGH polyA signal (for example, containing SEQ ID NO: 138) The method according to any one of claims 68 to 77, comprising an AAV vector plasmid containing ) vector.

79. Elements (2) to (6) are placed on the AAV vector plasmid from 5' to 3'. The vector according to claim 78.

80. Any of the elements (2) to (6) on the AAV vector plasmid is the 5 The vector according to claim 78 or 79, which is placed between the 'ITR' and the 3'ITR.

81. The AAV vector is a scAAV vector, according to any one of claims 78 to 80. The vector shown.

82. A lentiviral vector, as described in any one of claims 68 to 75.

83. The nucleic acid molecule according to any one of claims 64 to 66 or a set of nucleic acid molecules or claim 68 Cells containing the vector described in any one of items ~82.

84. A method for producing an hTREM2 antibody or its antigen-binding fragment, wherein (i) the method described in claim 83. (ii) culturing the cells and (ii) the hTREM2 antibody or its antigen from the culture. A method comprising isolating a bound fragment.

85. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 64, claims 65 to 67 A nucleic acid molecule or a set of nucleic acid molecules as described in any one of claims 68 to 82 A medical device comprising the vector described in claim 83, or the cells described in claim 83, and a pharmaceutically acceptable carrier. A pharmaceutical composition.

86. An antibody or antigen according to any one of claims 1 to 64, for use as a pharmaceutical. A binding fragment, a nucleic acid molecule according to any one of claims 65 to 67, or a set of nucleic acid molecules, claim A vector according to any one of claims 68 to 82, a cell according to claim 83, or claim 8 The pharmaceutical composition described in 5.

87. Claims 1 to 64 for use in the treatment of diseases associated with hTREM2 loss of function An antibody or antigen-binding fragment thereof as described in any one of claims 65 to 67 A nucleic acid molecule or a set of nucleic acid molecules, the vector according to any one of claims 68 to 82, The cells according to claim 83, or the pharmaceutical composition according to claim 85.

88. Claims 1 to 64 for the manufacture of a pharmaceutical product for treating diseases associated with hTREM2 loss of function An antibody or antigen-binding fragment thereof as described in any one of claims 65 to 67 A nucleic acid molecule or a set of nucleic acid molecules, the vector according to any one of claims 68 to 82, Use of the cells described in claim 83, or the pharmaceutical composition described in claim 85.

89. The disease is a neuroinflammatory or neurodegenerative disease, and the neuroinflammatory or neurodegenerative disease is selected at will. Sexually transmitted diseases include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor brain Inflammation, autism, cerebral lupus (NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN), Postherpetic neuralgia, chronic inflammatory Demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion Body myositis, lysosomal storage disorders, sphingomyelin lipidosis (Niemann-Pick disease type C) ), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy Myasthenia gravis, neurological Behçet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis Dermatomyositis, Rasmussen encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic brain injury, The antibody or the Antigen-binding fragments, nucleic acid molecules or sets of nucleic acid molecules, vectors, cells, or pharmaceutical compositions, or Use as described in item 88.

90. The antibody or antigen-binding fragment thereof according to claim 87, wherein the disease is Alzheimer's disease. A nucleic acid molecule or a set of nucleic acid molecules, a vector, a cell, or a pharmaceutical composition, or the description in claim 88. Use.

91. The antibody or antigen-binding fragment thereof according to claim 87, wherein the disease is Parkinson's disease, nucleus An acid molecule or a set of nucleic acid molecules, a vector, a cell, or a pharmaceutical composition, or the method described in claim 88. use.

92. The antibody or antigen-binding fragment thereof, nucleic acid according to claim 87, wherein the disease is multiple sclerosis. A molecule or a set of nucleic acid molecules, a vector, a cell, or a pharmaceutical composition, or the use described in claim 88. For.

93. The antibody or antigen-binding fragment thereof according to claim 87, wherein the disease is frontotemporal dementia. A nucleic acid molecule or a set of nucleic acid molecules, a vector, a cell, or a pharmaceutical composition, or the description in claim 88. Use.

94. The antibody or its antigen-binding fragment, the nucleic acid molecule or a set of nucleic acid molecules, the vector, The cells, or the pharmaceutical composition, are administered orally, intravenously, intracranially, intrathecally, subcutaneously, or intranasally via the following routes: The antibody or antigen-binding fragment thereof, nucleic acid molecule or the antibody according to claim 87, which is administered to the subject. A set of nucleic acid molecules, vectors, cells, or pharmaceutical compositions, or the use described in claim 88.

95. The antibody or its antigen-binding fragment, the nucleic acid molecule or a set of nucleic acid molecules, the vector, The cells, or the pharmaceutical composition, in combination with at least one additional therapeutic agent or procedure. The antibody according to claim 87, or its antigen-binding fragment, nucleic acid molecule, or set thereof, administered to the subject described above. The nucleic acid molecule, vector, cell, or pharmaceutical composition, or the use described in claim 88.

96. The antibody or its antigen-binding fragment, the nucleic acid molecule or a set of nucleic acid molecules, the vector, The cell, or the pharmaceutical composition, in combination with at least one additional therapeutic agent, to the subject The administered therapeutic agent is a BACE inhibitor, an anti-tau antibody, or a tau antisense oligonucleotide. Otide, anti-amyloid β antibody, fingolimod, BG12 or dimethyl fumarate, intero - The antibody according to claim 87, comprising one or more of feron β or tysabri. Antigen-binding fragments, nucleic acid molecules or sets of nucleic acid molecules, vectors, cells, or pharmaceutical compositions, or Use as described in item 88.

97. A method for treating diseases related to hTREM2 function loss in subjects requiring treatment, , the antibody or antigen-binding fragment thereof according to any one of claims 1 to 64, claims 65 to 67 A nucleic acid molecule or a set of nucleic acid molecules as described in any one of claims 68 to 82 Therapy of the vector described in claim 83, the cells described in claim 83, or the pharmaceutical composition described in claim 85 A method comprising administering an effective amount to the subject.

98. a) Assay the cell surface hTREM2 level of samples obtained from the subject; b) Selecting subjects in which the cell surface hTREM2 level is lower than the reference level. The aforementioned reference level is the hTREM2 level on the cell surface of a sample obtained from a healthy subject. to do; and c) The selected target is given the antibody or antigen according to any one of claims 1 to 64. A binding fragment, a nucleic acid molecule according to any one of claims 65 to 67, or a set of nucleic acid molecules, claim A vector according to any one of claims 68 to 82, a cell according to claim 83, or claim 8 Administer a therapeutically effective amount of the pharmaceutical composition described in item 5. The method according to claim 97, including the method described in claim 97.

99. The method according to claim 98, wherein the sample includes cerebrospinal fluid.

100. The TREM2 level on the cell surface of the aforementioned sample was determined by flow cytometry, immunohistochemistry, Western blotting, immunofluorescence assay, radioimmunoassay (RIA), enzyme Binding immunosorbent assay (ELISA), homogeneous time-resolved fluorescence (HTRF), or positron emission tomography. Determined by an assay selected from tomography (PET), according to claim 98 or 99. Method of description.

101. The disease associated with the loss of TREM2 function is a neuroinflammatory or neurodegenerative disease, and optional selections are made. The selection of the neuroinflammatory or neurodegenerative disease includes Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. N'Son's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis ( ALS, anti-NMDA receptor encephalitis, autism, cerebral lupus (NP-SLE), chemotherapy induction Ciprogenic peripheral neuropathy (CIPN), postherpetic neuralgia Uralgia, chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Guillain's disease Barre syndrome (GBS), inclusion body myositis, lysosomal storage disorders, sphingomyelin lipid Niemann-Pick disease type C, mucopolysaccharidosis type II / IIIB, metachromatic leukodystrophy - Multifocal motor neuropathy, myasthenia gravis, neurological Behçet's disease, neuromyelitis optica ( NMO, optic neuritis, polymyositis, dermatomyositis, Rasmussen encephalitis, Rett syndrome, stroke, If you have transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis, please contact us. The method described in any one of the requests 97 to 100.

102. The method according to any one of claims 97 to 101, wherein the disease is Alzheimer's disease. 。

103. The method according to any one of claims 97 to 101, wherein the disease is Parkinson's disease.

104. The method according to any one of claims 97 to 101, wherein the disease is multiple sclerosis.

105. The method according to any one of claims 97 to 101, wherein the disease is frontotemporal dementia. 。

106. The antibody or its antigen-binding fragment, the nucleic acid molecule or a set of nucleic acid molecules, the vector, The cells, or the pharmaceutical composition, are administered orally, intravenously, intracranially, intrathecally, subcutaneously, or intranasally via the following routes: The method according to any one of claims 97 to 105, administered to the target.

107. A method for stabilizing the target hTREM2 protein, any one of claims 1 to 64 or the antibody or antigen-binding fragment thereof as described in claim 1, the nucleus as described in any one of claims 65 to 67 Acid molecule or set of nucleic acid molecules, vector according to any one of claims 68 to 82, claim The cells described in 83, or the pharmaceutical composition described in claim 85, stabilize hTREM2. A method comprising administering an effective amount to the subject.

108. Administering at least one additional therapeutic agent to the subject, or at least one additional The person according to any one of claims 97 to 107, further comprising performing a therapeutic procedure. Law.

109. The aforementioned additional therapeutic agents include BACE inhibitors, anti-tau antibodies, and tau antisense oligonucleotides. Tide, anti-amyloid-beta antibody, fingolimod, BG12 or dimethyl fumarate, interopropyl alcohol The method according to claim 108, comprising one or more of feron-β or tysabri.

110. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 64, claims 65 to 67 A nucleic acid molecule or a set of nucleic acid molecules as described in any one of claims 68 to 82 The vector described in claim 83, the cells described in claim 85, or the pharmaceutical composition described in claim 85 The drug is administered simultaneously with, before, or after an additional therapeutic agent, as described in claim 108 or 109. The method.

111. The antibody or its antigen-binding fragment, the nucleic acid molecule or a set of nucleic acid molecules, the vector, Administering the cells or the pharmaceutical composition results in the following effects a to d: a) To increase TREM2-dependent cellular activity such as phagocytosis and chemotaxis, b) Inducing TREM2-dependent gene transcription, c) Enhancing the TREM2-dependent intracellular signaling pathway via Syk phosphorylation. 、 d) Increase the removal of cell debris. The method according to any one of claims 97 to 110, comprising one or more of the above.

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