TREM2 Stabilizing Antibody

Abstract

To provide antibodies that bind to and stabilize human Triggering Receptor Expressed on Myeloid cells 2 (hTREM2) protein and methods of using these antibodies.SOLUTION: The present invention provides an antibody or an antigen-binding fragment thereof that binds to the immunoglobulin superfamily (IgSF) domain of hTREM2 protein and stabilizes the hTREM2 protein. The present invention also provides the antibody or an antigen-binding fragment thereof having a specific amino acid sequence. These antibodies or vectors encoding the antibodies can be used for the treatment of neuroinflammatory or neurodegenerative diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a sequence listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII The copy was named PAT058251_ST25.txt and was 147,551 bytes in size. It is thread.

[0002] The present invention relates to the triggering receptor 2 (TREM2) protein expressed in human myeloid cells. The present invention provides antibodies that bind to and stabilize α- and β-blocking agents and methods of using these antibodies. [Background technology]

[0003] Triggering receptors expressed on myeloid cells or "TREM" interact with macrophages, dendritic cells, and cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, skin Langerhans cells A group of transmembrane glycoproteins expressed on various types of myeloid cells, including cytoplasmic leukocytes, Kupffer cells, and neutrophils. It is a protein (Takaki, R. et al., Immunol. Rev., 20 06,214:118-29). TREMs bind to immunoglobulins (I) in their extracellular domains. g) fold and therefore belongs to the immunoglobulin superfamily (IgSF) TREM receptors contain a short intracellular domain, but the domain of the signaling mediator is It lacks a binding motif and requires DAP12 (a 12 kDa DNAX activity) for cell activation. Adaptor proteins such as activating proteins are required for TREM. Two members, TREM1 and TREM2, have been reported, both of which are involved in immune and inflammatory processes. The gene encoding human TREM plays an important role in the TREM response. 1, a gene cluster encoding TREM2, TREM3, TREM4, and TREM5 , as well as located on chromosome 6p21.1, which carries a TREM-like gene.

[0004] TREM2 is a glycoprotein of approximately 40 kDa, which is converted to 26 kDa after N-deglycosylation. The whole TREM2 protein contains a leading signal peptide (amino acid sequence). a single V-type IgSF extracellular domain (amino acids 19-132), and a stalk domain (amino acids 133-172), a positively charged transmembrane domain (amino acids 173-197), and It consists of a cytosolic tail (amino acids 198–230) (Kober et al., El ife 5(2016);Kober et al.,J.Mol.Biol.429( 2017)1607-1629). The extracellular region encoded by exon 2 is It consists of a single V-type IgSF domain containing a potential N-glycosylation site. The transmembrane domain of TREM2 contains a charged lysine residue. The cytoplasmic tail of TREM2 contains a signaling motif They lack the signaling adaptor molecule DAP12 / TRYROBP and signal through It is believed that the message is sent

[0005] TREM2 physically associates with DAP12, and DAP12 mediates the expression of TREM2 and several It acts as a signaling adaptor protein for other cell surface receptors. The cytoplasmic domain contains an immunoreceptor tyrosine-based activation motif (ITAM) (Wunderlic h, J.Biol.Chem.288,33027-33036,2013). After activation of the agonist receptor, DAP12 is bound to the conserved ITAM chain by Src kinase. Phosphorylation of lysine residues occurs, followed by recruitment and activation of Syk protein kinase. , mitogen-activated protein kinase (MAPK), PI3K, NFκB and phospho Downstream signaling pathways are initiated, including activation of lipase Cγ (PLCγ).

[0006] TREM2 is involved in the regulation of lipopolysaccharide (LPS), heat shock protein 60, neurite debris, and cytoplasmic endothelial cells. Bacteria, apolipoprotein E and a wide range of anionic and zwitterionic lipids, e.g., phosphatidylcholinesterase inhibitors. Phatidic acid (PA), phosphatidylglycerol (PG), phosphatidylserine ( PS), phosphatidylinositol (PI), phosphatidylcholine (PC), cardiolipin It can be activated by olipin and sphingomyelin. Activation of TREM2 promotes myoglobin production. Increased phagocytic activity of chromosomes and macrophages and decreased release of pro-inflammatory cytokines TREM2 mediates CSF-1 receptor signaling and suppresses TLR signaling. Furthermore, TREM2 acts synergistically with plexin to maintain microglial survival. TREM2 also interacts with Aβ plaques and regulates cell adhesion and motility. is densely present on the surface of microglial cells in contact with neuronal debris (Yuan et al. al., Neuron 90(2016)724-739). Recently, Some ligands sensed by TREM2 in vivo, such as phospholipids and myelin lipids (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. As for other ligands, TREM2 contributes to the uptake of Aβ into microglia. Therefore, it may be Aβ and plaque-associated neuronal debris (Xiang et al., EMB O Mol.Med.8(2016):992-1004). TREM2 also inhibits apoptosis. sis cells (Takahashi et al., J. Exp. Med. 201 (200 5), 647-657), myelin debris (Poliani et al., J. Clin .Invest.125(2015):2161-2170) and bacterial beads (Cen et al., Am. J. Respir. 188 (2013) 201-212) TREM2 signaling has also been shown to play a role in the regulation of internalized target cells. Facilitates food breakdown and is crucial for lipid metabolism, myelin uptake and intracellular degradation .

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

[0008] After the ectodomain is removed, the remaining membrane-retained fragment is converted to γ-secretase Further processing is achieved by membrane-mediated proteolysis. A soluble fragment of TREM2 (sTREM2) produced by immunization with dendritic cell cultures was in the supernatants of patients with non-inflammatory neurological disorders and multiple sclerosis, as well as in plasma and CSF (cerebrospinal fluid) samples. It has been observed in human CSF samples (Kleinberger, 2014). The sTREM2 ectodomain, or sTREM2, is a potential Alzheimer's disease It has been evaluated as a biomarker for Alzheimer's disease (AD), and is generally known to increase during the aging process. (Suarez-Calvet, EMBO Mol. Med. 8, 466 -476, 2016). Detailed analysis of the progression of AD revealed that sTREM2 is involved in the early stages of AD. It increases before the onset of clinical symptoms, peaks in MCI-AD, and is elevated in AD dementia. However, it was revealed that the level remained low compared to the MCI-AD stage ( Suarez-Calvet, 2016).

[0009] Increased TREM2 expression at the peak of disease promotes resolution (e.g., peritonitis, wound healing). (Turnbull, 2006; Gawish, 2015). Chronic conditions such as neuroinflammation Under inflammatory conditions, TREM2 is constantly shedding and is expressed in microglia and macrophages. Therefore, TRIPs on the cell surface cannot exert their signaling function in the cytoplasm. Stabilizing and / or preventing EM2 shedding may promote the proliferation of microglia and macrophages. This restores functional signaling-competent TREM2 expression in the thymus.

[0010] Human genetic studies show loss of surface TREM2 rather than absence of sTREM2 It has been pointed out that the risk of disease is affected by the TREM2 protein, for example. The amino acid mutation from R to H at position 47 of SEQ ID NO: 1 resulted in a slight decrease in cell surface expression ( Kleinberger 2014), and a decrease in the ligand binding ability of TREM2 (Wan g 2015, Atagi 2015, Bailey 2015). The amino acid mutation T66M in M2 results in loss of TREM2 expression on the cell surface (Kleinberger 2014), which in turn prevents the production of soluble TREM2. The mutation H157Y at the cleavage site of TREM2 enhances sTREM2 expression and completes Reduces full-length membrane-bound TREM2 and is associated with increased risk of AD (Thornton on 2017, Schlepckow 2017). Therefore, from these genetic studies Both the decrease in sTREM2 and the increase in plasma membrane-bound TREM2 resulted in cell surface TREM2. This suggests that stabilization of

[0011] Haass et al. (International Publication No. WO 18015573) reported that the TREM2 story A 10-amino acid peptide spanning amino acids 152-161 (AHVEHS) located in the IL-1 region We generated antibodies that bind to the TREM2 ISRS (SEQ ID NO: 132) and inhibit TREM2 cleavage. Such antibodies inhibit TREM2 cleavage by direct binding and thereby blocking the cleavage site. Schwabe et al. (International Publication No. WO 17062672) reported that TREM However, none of these disclosed antibodies bind to 2. Conversely, the stabilizing effect of WO 17062672 is not pointed out. Some of the antibodies listed in FRET have been reported to have destabilizing effects (see Example 15). Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, it may stabilize TREM2 and activate TREM2-associated functions and / or These results suggest that TREM2 stabilization may be beneficial for neuronal alterations that have favorable developability properties and that could be promoted. There is a need to identify and develop hTREM2 antibodies that are suitable for treating patients with sexual disorders. It has been done. [Means for solving the problem]

[0013] Previously published studies have demonstrated that ADAM17 cleavage is essential for generating antibodies that stabilize TREM2. The stalk region of TREM2 is targeted because the break is located within the stalk. In fact, for large molecules such as antibodies (or their binding fragments), the sheddase acts at a ratio of stalk to stalk. It is expected that there is a steric barrier to accessing a relatively small region (amino acids 133-172). Therefore, previous attempts to generate stabilizing antibodies against TREM2 have not yielded results. It is not surprising that the stalk region of TREM2 has been targeted. The region (amino acids 19 to 132 of any one of SEQ ID NOs: 1, 2, or 3) is the cleavage site (H15 7) and is part of the ectodomain. Antibodies against this region do not sterically hinder access of the sheddase to the TREM2 cleavage site. Therefore, the IgSF region has not been identified as a key component of antibodies that can stabilize TREM2. It had never been considered a potential target.

[0014] Surprisingly, the inventors have found that antibodies as disclosed herein bind to the IgSF region, We found that it has the ability to effectively stabilize TREM2 on the cell surface. Furthermore, such antibodies inhibited TREM2-dependent phagocytosis in human M2A macrophages. Furthermore, we have shown that such anti- The body may also increase the phagocytic capacity of microglia or macrophages, for example, in the brain. It may also enhance TREM2-dependent functions in vivo.

[0015] Thus, herein, there is provided an antibody specific for the IgSF domain of human TREM2 (hTREM2). and stabilize the hTREM2 protein, e.g., an antibody or antigen-binding fragment thereof, Monoclonal antibodies or antigen-binding fragments thereof are provided. Such antibodies are referred to herein as The hTREM2 antibody or its antigen-binding fragment is referred to as an "hTREM2 antibody or its antigen-binding fragment." The antigen-binding fragment of (i) reduces or inhibits TREM2 ectodomain shedding. (ii) stabilize the TREM2 protein on the cell surface; and / or (iii) Binding to cognate ligands, intracellular signaling, increased phagocytosis, and degradation of phagocytosed substances Maintain or enhance TREM2 functions, such as promoting TREM2 expression, thereby enhancing TREM2-dependent downstream regulatory functions. Dysfunctional TREM2 or lack of surface TREM2 can promote human neuroinflammation. and neurodegenerative pathologies, the hTREM2 antibodies described herein or their anti- The original binding fragment is a protein that is involved in Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. dementia, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor Somatoencephalitis, autism, cerebral lupus (NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN) ), postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Glenn-Barré syndrome (GBS), inclusion body myositis, lysosomal storage diseases, e.g., sphingomyelinase Phospholipidosis (Niemann-Pick disease type C) and mucopolysaccharidosis II / IIIB, metachromatic albuginea Streptococcal dystrophy, multifocal motor neuropathy, myasthenia gravis, neuro-Behçet's disease, Neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett's syndrome syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial myelitis They can be used to treat, prevent, or diagnose neuroinflammatory or neurodegenerative diseases, such as meningitis. The hTREM2 antibodies or antigen-binding fragments thereof described herein also have the potential to bind to a wide range of TREM2 receptors. expressing aberrant or mutated TREM2 receptor variants or autoimmune, inflammatory, or malignant disorders mediated by or associated with cells In some preferred embodiments, the compounds described herein are also suitable for the treatment, prevention or diagnosis of disorders. The described hTREM2 antibodies or antigen-binding fragments thereof are used to treat Alzheimer's disease, frontotemporal dementia, and Diseases selected from dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease The present invention also provides a method for treating, preventing, or diagnosing a cancer. The TREM2-binding antibodies or antigen-binding fragments can be used to diagnose and / or treat TREM2-related disorders. Methods of treating are also provided.

[0016] In one aspect, the present disclosure provides a method for the treatment of rhesus malabsorption, comprising administering to a subject a therapeutic agent that specifically binds to the IgSF domain of a TREM2 protein. Thus, antibodies or antigen-binding fragments thereof that stabilize the TREM2 protein are provided. In some preferred embodiments, the antibody or antigen-binding fragment thereof binds to macrophages, dendritic cells, alveoli, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, skin Langerhans cells TREM2 expression on the cell surface of TREM2-expressing cells, such as Kupffer cells, neutrophils, or hepatocellular carcinoma cells In some embodiments, these antibodies or their antigens stabilize the EM2 protein. The binding fragment inhibits proteolytic shedding of the ectodomain of the TREM2 protein. Reduce.

[0017] In some embodiments, provided herein are antibodies specific for the IgSF domain of human TREM2. For example, an antibody or antigen-binding fragment thereof that binds to The original binding fragments are amino acid residues 19 to 132 of SEQ ID NO: 1 and amino acid residues 19 to 132 of SEQ ID NO: 2. or an IgSF of human TREM2 comprising amino acid residues 19 to 132 of SEQ ID NO: 3 In some embodiments, the TREM2 antibody is a human or humanized antibody. In some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fv fragment, scFv, minibody, or diabody.

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

[0019] In some embodiments, the TREM2 antibody comprises an Fc region. The Fc region has one or more mutations compared to the parent antibody and is capable of antibody-dependent cellular cytotoxicity (ADC). Modified IgG1 F with reduced DCC or complement-dependent cytotoxicity (CDC) activity In some embodiments, the Fc region is an IgG2 Fc region, an IgG4 ... The Fc region is selected from an IgG2 / IgG4 hybrid Fc region.

[0020] In some embodiments, the hTREM2 antibody or antigen-binding fragment thereof is monoclonal. Described herein are sequences encoding such monoclonal antibodies or antigen-binding fragments thereof. Nucleic acids encoding such monoclonal antibodies or antigen-binding fragments thereof. Vectors and host cells containing the vector are provided.

[0021] In another aspect, provided herein is a TREM2 antibody or antigen-binding thereof described herein. or a nucleic acid encoding such an antibody or antigen-binding fragment, or a nucleic acid containing such a nucleic acid. Pharmaceutical compositions are provided that include one or more of the cells comprising the medicament, a medicament, or a pharmaceutically acceptable carrier.

[0022] In another aspect, provided herein is a TREM2 antibody or antigen-binding thereof described herein. A therapeutically effective amount of any of the fragments is administered to the subject to provide a therapeutic effect in a subject in need of treatment. Methods for treating diseases associated with loss of TREM2 function are provided. Such methods include: The following steps are performed: (1) assaying cell surface TREM2 levels in a sample obtained from a subject; (2) selecting a subject whose cell surface TREM2 level is lower than a baseline level; wherein the reference level is determined by measuring cell surface TREM activity in a sample obtained from a healthy subject. and (3) specific to the IgSF domain of the TREM2 protein. a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to and stabilizes the TREM2 protein In some embodiments, the method may include one or more of the steps of administering to a subject Such methods may further comprise administering to the subject a second agent. EM2 levels were measured using flow cytometry, immunohistochemistry, Western blotting, and immunohistochemistry. immunofluorescence assay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELI) Choose from SA, Homogeneous Time-Resolved Fluorescence (HTRF), or Positron Emission Tomography (PET) In some embodiments, the sample is In some embodiments, the present invention relates to a method for treating a cerebrospinal fluid disorder associated with a loss of TREM2 function, including the treatment of cerebrospinal fluid and its cellular components. Diseases include Alzheimer's disease, frontotemporal 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), obinuclear Postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome Reay syndrome (GBS), inclusion body myositis, lysosomal storage diseases, e.g., sphingomyelin Niemann-Pick disease type C, mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy Trophy, multifocal motor neuropathy, myasthenia gravis, neuro-Behçet's disease, optic nerve Myelitis (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett syndrome, Stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis In some preferred embodiments, the TREM2 Diseases associated with loss of function include Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and myelopathy. The neurodegenerative disease is selected from amyotrophic lateral sclerosis, and Nasu-Hakola disease. In a preferred embodiment, the disease is Alzheimer's disease. 2. The antibody or antigen-binding fragment thereof is capable of binding to macrophages, dendritic cells, osteoclasts, microglia, Mast cells, monocytes, lung epithelial cells, skin Langerhans cells, Kupffer cells, neutrophils, or liver The TREM2 protein on the cell surface of TREM2-expressing cells selected from cancer cells was stabilized. In some embodiments, the TREM2 antibody or antigen-binding fragment thereof is administered orally, The drug is administered to the subject by intravenous, intracranial, intrathecal, subcutaneous, or intranasal routes.

[0023] In another aspect, the present disclosure provides a method for treating a disease associated with loss of TREM2 function. Some preferred embodiments of the present invention provide a TREM2 antibody or antigen-binding fragment thereof for In this embodiment, the antibody or antigen-binding fragment thereof binds to the IgSF domain (or domains) of the TREM2 protein. That is, amino acid residues 19 to 132 of SEQ ID NO: 1 and amino acid residues 19 to 13 of SEQ ID NO: 2 2, or amino acid residues 19 to 132 of SEQ ID NO: 3) and binds specifically to TREM2 protein. In some preferred embodiments, the antibody or antigen-binding fragment thereof: Macrophages, dendritic cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, TRE selected from skin Langerhans cells, Kupffer cells, neutrophils, and hepatocellular carcinoma cells In some embodiments, the TREM2 protein is stabilized on the cell surface of M2-expressing cells. Diseases associated with TREM2 loss of function include Alzheimer's disease, frontotemporal dementia, and encephalopathy. 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 Chronic inflammatory demyelinating polyneuropathy (CIPN), postherpetic neuralgia, chronic inflammatory demyelinating polyneuropathy Cisplatin-Induced Diabetes Mellitus (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion body myositis, lysosomal storage disorders, such as sphingomyelin lipidosis (Niemann-Pick disease type C) and mucopolysaccharide Glycosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, myasthenia gravis Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis , Rasmussen's encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, Neuroinflammatory or neurodegenerative diseases such as viral encephalitis or bacterial meningitis. In a preferred embodiment, the disease associated with TREM2 loss of function is Alzheimer's disease, Selected from: temporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease In a further preferred embodiment, the disease is Alzheimer's disease. In some embodiments, the TREM2 antibody or antigen-binding fragment thereof is targeted to macrophages, Dendritic cells, osteoclasts, microglia, mast cells, monocytes, lung epithelial cells, skin Langerhans cells A cell expressing TREM2 is selected from the group consisting of a liver cell, a Kupffer cell, a neutrophil, and a hepatocellular carcinoma cell. Stabilizes TREM2 protein on the cell surface. [Brief explanation of the drawings]

[0024] [Figure 1A] Figure 1: Figure 1A shows an exemplary alignment of the amino acid sequences 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 adaptor protein DAP12. Mature TREM2 contains a single immunoglobulin (IgSF) domain, a stalk region, a transmembrane (TM) domain, and a cytoplasmic domain. [Figure 1B] (As mentioned above.) [Figure 2] 1 shows stabilization of TREM2 in CHO-hDAP12-hTREM2 cells by antibody treatment. [Figure 3] Binding of TREM2 antibodies to human M2A macrophages before and after treatment with PMA is shown. [Figure 4A] Figure 4: Antibody binding to WT-TREM2 (Figure 4A) and TREM2-TREM1 chimeric protein recombinantly expressed in CHO-hDAP12 cells (Figures 4B and 4C). [Figure 4B] (As mentioned above.) [Figure 4C] (As mentioned above.) [Figure 5] 1 shows stabilization of TREM2 on the cell surface of hM2A by a TREM2 antibody. [Figure 6A] Figure 6: Antibodies that stabilize TREM2 on the cell surface also increase the phagocytic activity of human M2A. Statistics were calculated using Student's t-test. *P<0.05, **P<0.01, ***P<0.001 compared to isotype control. [Figure 6B] (As mentioned above.) [Figure 7A] Figure 7: Determination of the lowest effective dose in the phagocytosis assay for hM2A. Statistics were calculated using Student's T-test. *P<0.05, **P<0.01, ***P<0.001 compared to isotype control. [Figure 7B] (As mentioned above.) [Figure 8] 1 shows that plate-bound TREM2 antibodies induce TREM2-dependent NFAT promoter-dependent gene transcription. [Figure 9A] Figure 9: TREM2 antibody increases Syk phosphorylation in human M2A macrophages. (B) Quantification of pSyk under various conditions in correlation with total Syk in the Western blot shown in (A). [Figure 9B] (As mentioned above.) [Figure 10]Phagocytosis of Staphylococcus aureus (S. aureus) bioparticles by human M2A macrophages from 0 to 3 hours. [Figure 11] Phagocytosis of SH-SY5Y cells by human M2A macrophages over 3 to 12 hours. [Figure 12] 1 shows that TREM2 antibodies promote the chemotaxis of human M2A macrophages. [Figure 13] Using apoptotic pHrodo-labeled SH-SY5Y cells and cumulative phagocytosis as readouts, we show that TREM2 antibodies promote phagocytosis in human iPS-derived microglia. [Figure 14] 1 shows that TREM2 antibody promotes the chemotaxis of human iPS-derived microglia. [Figure 15A] FIG. 15: Results of preventive / combination and therapeutic treatment with TREM2 antibodies in the cuprizone model. [Figure 15B] (As mentioned above.) [Figure 15C] (As mentioned above.) [Figure 15D] (As mentioned above.) [Figure 15E] (As mentioned above.) [Figure 16A] Figure 16: Image analysis results of the MPTP model in humanized TREM2 mice (A) and representative microscopic images of each group (B) are shown. [Figure 16B] (As mentioned above.) [Figure 17A] Figure 17: Shows the results of a cross-blocking experiment between Fabs MOR041877, MOR041895 and MOR042596 and full-length IgGs MOR041877, MOR41895, MOR042596, MOR044698 and MOR03207 in CHO-hDAP12-hTREM2 cells. [Figure 17B] (As mentioned above.) [Figure 17C] (As mentioned above.) [Figure 18] 1 shows the increased phagocytic activity of hM2A after treatment with MOR042596. [Figure 19]The epitope of TREM2 bound by MOR042596 as determined by X-ray crystallography is shown. The protein backbone of TREM2 is shown in a schematic representation, with the side chains of TREM2 residues within a 5 Å distance from the Fab shown as sticks. The Fab heavy chain is shown as a dark grey surface, and the light chain is shown as a light grey surface. [Figure 20] A close-up of the TREM2-Fab interface (see Figure 19) is shown, comparing MOR042596 (crystal structure) and MOR044698 (homology model). TREM2 residues within 5 Å of the Fab are shown as sticks, and residues more proximal to LCDR3 (D39-K42) are marked. The heavy chain (dark gray, top right) and light chain LCDR1 and LCDR2 (light gray, top left) are identical for both Fabs. LCDR3 shares several key residues (positions 90, 93, and 95), maintaining the same backbone loop conformation for LCDR3 in both Fabs. Differences exist at positions 89, 91, 92, 94, and 96, with one additional insertion (S95a) in MOR044698. The overall epitope is conserved between MOR042596 and MOR044698. [Figure 21] The epitope of TREM2 bound by MOR041877 as determined by X-ray crystallography is shown. The protein backbone of TREM2 is shown in a schematic representation, with the side chains of TREM2 residues within a 5 Å distance from the Fab shown as sticks. The Fab heavy chain is shown as a dark grey surface, and the light chain is shown as a light grey surface. [Figure 22A] Figure 22: Cerebral cortices of hTREM2-KI mice treated with cuprizone and either MOR044698-mu or an isotype control antibody, along with naive mice, stained for TREM2 and Iba1 (A). Quantitative analysis of hTREM2-positive area after normalization is provided (B). [Figure 22B] (As mentioned above.) DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a method for producing a TREM2 protein by specifically binding to the extracellular domain of human TREM2. The present invention provides an antibody and an antigen-binding fragment thereof that stabilizes proteins. The antigen-binding fragment can reduce or inhibit TREM2 ectodomain shedding. can stabilize the TREM2 protein on the cell surface; and optionally, Binding to cognate ligands, intracellular signaling, increased phagocytosis, and the release of phagocytosed substances It can maintain or improve TREM2 functions such as promoting degradation. Because deficiency of M2 or surface TREM2 is associated with human neuroinflammatory and neurodegenerative pathologies, The TREM2 stabilization antibodies and antigen-binding fragments thereof described herein are useful for treating Alzheimer's disease, Ophthalmotemporal 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 degeneration Myelopathic myelopathic polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion bodies Myositis, lysosomal storage diseases, e.g., sphingomyelin lipidosis (Niemann-Pick) Mucopolysaccharidosis type C, mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuron syndrome rhopexy, myasthenia gravis, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, Polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic Neuroinflammatory or neurodegenerative diseases such as brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis The TREM2 binding proteins described herein can be used in the treatment, prevention, or diagnosis of diseases. The antibody and its antigen-binding fragments also show extensive proteolytic cleavage or abnormal expression of TREM2. or mediated by cells expressing mutated TREM2 receptor variants. It is also suitable for the treatment, prevention or diagnosis of autoimmune, inflammatory or malignant disorders associated therewith. In some preferred embodiments, the hTREM2 antibodies or their derivatives described herein are The antigen-binding fragment of this antibody has been shown to be effective in treating Alzheimer's disease, frontotemporal dementia, Parkinson's disease, and amyotrophic lateral sclerosis. and for use in the treatment, prevention, or diagnosis of a disease selected from the group consisting of sclerosing encephalopathy, cerebrospinal fluid disorder, and Nasu-Hakola disease. The present specification also provides the TREM2-binding antibodies and their antibodies disclosed herein. Methods of diagnosing and / or treating TREM2-associated diseases using the original binding fragments are also provided.

[0026] TREM2 mediates the phagocytosis of non-inflammatory bacteria and dying cells, dampening the inflammatory response. Homozygous loss of function of EM2 causes Nasu-Hakola disease (lipomembranous polyposis with sclerosing leukoencephalopathy). Procystic skeletal dysplasia syndrome (PLOSL), or frontotemporal dementia (FTD)-like syndrome, A disease characterized by cysts, neuroinflammation, progressive neurodegeneration, and presenile dementia. The heterozygous loss-of-function mutation R47H in TREM2 is also associated with late-onset Alzheimer's disease. is an important risk factor for AD, comparable to the apolipoprotein E ε4 allele TREM2 is expressed in microglia found in the white matter, hippocampus, and neocortex. This is in part consistent with the pathological features reported in AD brains, suggesting that TREM may be involved in the pathogenesis of AD. 2 may be involved. Genetic screening has also now revealed In addition to AD, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal sclerosis Heterozygous missense mutations in TREM2 as a risk factor for dementia (FTD) has been identified (Kleinberger, Sci Transl Med. 2014 Jul 2;6(243):243ra86). Therefore, severe cognitive impairment and dementia Functional TREM2 is required to prevent age-related neuroinflammatory and neurodegenerative diseases. It is essential.

[0027] Due to alternative splicing, there are three TREM2 isoforms in humans. The human TREM2 isoforms are shown in Figure 1A. Form 1 (SEQ ID NO: 1), human TREM2 isoform 2 (SEQ ID NO: 2), and human Provides an alignment of the amino acid sequence of TREM2 isoform 3 (SEQ ID NO: 3). Figure 1B shows the structure of TREM2 and its interaction with the signaling adaptor protein DAP12. Illustrate the interaction of

[0028] definition As used in the specification and claims, the singular forms "a," "an," and "the" includes plural references unless the context clearly dictates otherwise. For example, "one (a) detail The term "cell" includes a plurality of cells, including mixtures thereof.

[0029] All numerical values, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are given in 0.1 increments. It is an approximation that may vary (+) or (-) in increments of 1. Although not specified, it should be understood that all numerical designations are preceded by the term "about." The term "about" in reference to X means, for example, X ±15% (including all values ​​within that range). Also, although not necessarily specified, the reagents described herein may be used alone. It should also be understood that these are examples and that equivalent reagents are known in the art.

[0030] Throughout this specification and the claims that follow, unless the context otherwise requires, the phrase " "comprises" and "comprises" "comprising" and other variations are used throughout this specification unless otherwise noted. In this document, it is used in a non-limiting sense in its open-ended form.

[0031] As used herein, "consisting of" refers to an embodiment Any element, step, or component not specified in any embodiment and / or claim element. As used herein, "consisting essentially of" also excludes. "(g essentially of)" means the basic The present invention does not exclude materials or steps that do not materially affect the claimed novel characteristics.

[0032] As used herein, "TREM2" (also known as "trigger receptor 2 receptor expressed in myeloid cells") TREM2 receptor 2 (TREM2, TREM2a, TREM2b, or TREM2c) is a It refers to a transmembrane glycoprotein belonging to the immunoglobulin superfamily (IgSF). The entire M2 protein (SEQ ID NO: 1) contains a leading signal peptide (amino acids 1-1 8), a single V-type IgSF extracellular domain (amino acids 19-132), a stalk domain (amino acids 133–172), a positively charged transmembrane domain (amino acids 173–197), and the cytosol The tail (amino acids 198-230) (Feuerbach et al., Neu rosci.Lett.660(2017):109-114). Human TREM2 gene The TREM2 gene is located at chromosomal location 6p21.1. The genomic sequence of the TREM2 gene is available from Gen The Bank can be referenced (Gene ID: 54209). As a result, there are three TREM2 isoforms in humans (ENSEMBL IDs ENSP00000362205, ENSP00000342651, and EN (Protein sequence available at SP00000362214). The term "TREM2" refers to the This term refers collectively to all isoforms of REM2. The protein and mRNA sequences of the isoforms are as follows: Triggering receptor precursor 2 isoform 1 precursor [human (Ho mo sapiens)](NP_061838.1) [ka] Triggering receptor 2 (TRE) expressed in human (Homo sapiens) myeloid cells M2), transcript 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 of the human TREM2 protein is shown in Figure 1A. The sequence also has at least about 70 sequences identical to any of SEQ ID NOs: 1, 2, or 3 over its entire 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 Proteins with 0.000% sequence identity are also included, and such proteins are known as ligands. Binding, intracellular signaling, promotion of phagocytosis and degradation of phagocytosed substances, and other functions of TREM2 It still has the regulatory function of The sequences of TREM2 proteins from monkeys (cynodonts), as well as other animals, are known in the art. For example, NP_112544.1 for mouse TREM2 protein is known. and NP_001259007.1).

[0034] The term "extracellular domain" refers to the domain of a transmembrane protein that is located on the extracellular side of the lipid bilayer of a cell. The term refers to the exposed portion of a protein. Methods for determining the ectodomain of a protein are well known in the art. It is well known that and McVector software, Oxford Molecular). The extracellular domain of the human TREM2 protein is amino acid residues 19 to 17 of SEQ ID NO: 1. It may include 2.

[0035] The term "ectodomain" of TREM2 refers to the extracellular domain of TREM2 that is involved in shedding. 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 consists of amino acids 19 to 157 of any one of SEQ ID NOs: 1, 2, or 3.

[0036] The term "IgSF domain" refers to the extracellular domain of TREM2 that binds to immunoglobulins ( Ig) type fold, and thus part of the immunoglobulin superfamily. In humans, for example, the IgSF domain may be any one of SEQ ID NOs: 1, 2, and 3. It consists of amino acid residues 19 to 132.

[0037] The term "stalk region" of TREM2 refers to the V region of the extracellular domain of TREM2. It refers to the part that connects the IgSF domain and the transmembrane domain. For example, the stalk region of the human TREM2 isoform 1 protein corresponds to amino acid sequence 1 of SEQ ID NO:1. 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. Methods for determining the transmembrane domain of a protein are 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 interchangeably and refer to the domain of a transmembrane protein. This refers to the part of the lipid bilayer that is on the cytoplasmic side of the cell. It determines the cytoplasmic tail of a protein. Methods for this are known in the art (Elofsson et al. (2007) and Bernsel et al. (2005)).

[0040] The term "stabilize" as used herein refers to, for example, the treatment of an inflammatory disease or neurodegenerative disorder. to the TREM2 levels in corresponding TREM2-expressing cells of healthy subjects without sexual disorders. This refers to the maintenance, restoration or increase of TREM2 cell surface levels in TREM2-expressing cells. For example, by reducing or inhibiting TREM2 ectodomain shedding, or This can be achieved by increasing the cell surface expression of TREM2. Bell was analyzed by flow cytometry / FACS or by TREM2 cell surface immunoprecipitation. This can be assessed by measuring the level of soluble TREM2 in the blood or by measuring the decrease in soluble TREM2 over time. TREM2 cell surface expression was also measured by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Radioimmunoassay (RIA), bioassay (e.g., increased phagocytosis), Western blot assay, flow cytometry, immunohistochemistry, immunofluorescence assay, homogeneous time-resolved fluorescence ( It can also be detected by HTRF, or positron emission tomography (PET).

[0041] The term "activate" as used herein refers to, for example, a healthy subject or a suitable TREM2-dependent TREM2-expressing cells in individuals with inflammatory or neurodegenerative diseases in which the activity is impaired This refers to the initiation or maintenance of downstream signaling of TREM2 expressed on the cell surface in cells. This is achieved by, but not limited to, phosphorylation of the TREM2-associated DAP12 or DAP10. This may result in the phosphorylation of Syk through various intracellular signaling cascades. Enhancement, phagocytosis, increased directed cell motility (chemotaxis) towards targets, increased cell survival, Regulation of cytokine or chemokine release from TREM2-expressing cells, intracellular phagocytic material Increased degradation or altered gene expression. TREM2-dependent DAP12 or Syk Increased phosphorylation can be measured by Western blot, ELISA, or flow cytometry / FAC The directional motility of cells, e.g., chemotaxis, can be assessed by bioassays. Modulation of cytokine release can be assessed by enzyme-linked immunosorbent assay. ELISA, radioimmunoassay (RIA) or flow cytometry / FA Changes in gene expression can be assessed by quantitative analysis of mRNA levels. by RT-PCR or at the protein level by Western blot or flow cytometry. This can be assessed by cytometry.

[0042] The term "promote" as used herein refers to an increase in a TREM2-dependent activity that is impaired by a disease. Such activities include phagocytosis, directional cell motility (chemotaxis) toward targets, and Increased cell viability, increased cytokines or chemokines in cells expressing TREM2 Regulation of release, increased degradation of intracellular phagocytic material, and the release of neighboring cells (astrocytes / neurons) This may involve modulation of a cellular response or alteration of gene expression.

[0043] As used herein, the term "antibody" refers to an immunoglobulin that specifically binds to an antigen. Antibodies are proteins or polypeptide sequences derived from a molecule. They may be monoclonal, multi- or single-chain, or intact immunoglobulins, and may be of natural or synthetic origin. Naturally occurring "antibodies" are interlinked by disulfide bonds and may be derived from recombinant sources. It is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. The antibody comprises a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions contain one domain CL. The VH and VL regions are called complementarity-determining regions (CDRs). These regions can be further subdivided into regions of hypervariability known as framework regions (FR). Each VH and VL consists of the following regions from the amino terminus in the order: It consists of three CDRs and four FRs arranged at the carboxyl terminal: FR1, C DR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains are The constant region of an antibody contains a binding domain that interacts with various cells of the immune system (e.g., , effector cells) and host tissues or factors, including the first component of the classical complement system (C1q) The antibody can mediate the binding of immunoglobulins to the target. The antibody may be a humanized antibody, a camelized antibody, or a chimeric antibody. Type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), Class (e.g., , IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses Throughout this document, the terms "antibody" or "antibody molecule" may also be used unless the context indicates otherwise. Unless otherwise specified, it also includes any fragment thereof and any derivative thereof.

[0044] The term "antibody fragment" or "antigen-binding fragment" refers to an antibody that specifically interacts with an epitope of an antigen. Antibodies that retain their potency (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), VH and CH1 domains Single domain antibodies such as Fd fragments, linear antibodies, and sdAbs (either VL or VH) consisting of Antibody, camelid VHH domain, two linked by disulfide bridges in the hinge region Multispecific antibodies formed from antibody fragments such as bivalent fragments including Fab fragments of the Antibodies include, but are not limited to, isolated CDR or other epitope-binding fragments. Antibody-binding fragments also include single domain antibodies, maxibodies, minibodies, nanobodies, intracellular antibodies, and the like. Rabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005) Antigen-binding fragments can also be derived from polypeptides such as fibronectin type III (Fn3). fibronectin polypeptide minibodies can be implanted into scaffolds based on (See US Pat. No. 6,703,199.) The term "scFv" refers to the variable region of the light chain. and at least one antibody fragment comprising a variable region of a heavy chain. wherein the light and heavy chain variable regions are linked, for example, via a synthetic linker, e.g. , which are sequentially linked via a short flexible polypeptide linker to form a single polypeptide chain. The scFv can be expressed in a variety of ways, and the scFv retains the specificity of the intact antibody from which it is derived. As used herein, unless otherwise specified, scFv refers to, for example, the N of a polypeptide. and may have the VL and VH variable regions in either order with respect to the terminal and C-terminal ends, The scFv may comprise VL-linker-VH, or may comprise VH-linker-VL. It's okay to do that.

[0045] The term "complementarity determining region" or "CDR" as used herein refers to an antigen-specific region. It refers to the sequence of amino acids within the antibody variable region that confers isomeric and binding affinity. , 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) The exact amino acid sequence boundaries of a given CDR are determined by Kabat et al. 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 ing scheme), or a combination thereof, and ImMunoGenTics (IMGT) number Balling (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 A given CDR region (e.g., HCDR1, HCDR2, HCDR3, LC Combination of Kabat and Chothia for LCDR1, LCDR2 or LCDR3 In some embodiments, the CDRs are numbered as follows: A portion of the Kabat CDR, along with the amino acid residues defined as part of the ia CDR. As used herein, "Chothi" corresponds to the amino acid residues defined as The CDRs, defined according to the "a" numbering scheme, are sometimes also called "hypervariable loops." Under IMGT, the CDR regions of an antibody are identified using the program IMGT / DomainGap Alignment. Generally, unless otherwise indicated, an antibody molecule comprises one or more It may contain any combination of Kabat and / or Chothia CDRs.

[0046] The term "epitope" refers to a molecule capable of specific binding to an immunoglobulin or includes any protein determinant capable of interacting with a molecule. An epitopic determinant is , generally from chemically active surface groups on molecules such as amino acids, carbohydrates, or sugar side chains. They can have specific three-dimensional structural characteristics and specific charge characteristics. A group can be "linear" or "conformational." Conformational and Linear An epitope may, for example, have a binding affinity to the former but not to the latter in the presence of a denaturing solvent. It is distinguished by the fact that it is not

[0047] "Binds to the same epitope as" means that the antibody is compared using the same epitope mapping technique. When used, an antibody, antibody fragment, or other antigen-binding molecule that binds to the same epitope as the exemplified antibody is The term "antibody" refers to the ability of a binding moiety to bind to a specific antigen. can be determined using epitope mapping techniques. Methods are well known in the art. For example, conformational epitopes can be identified by, for example, hydrogen / deuterium Spatial conformation of amino acids, such as by exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance These are easily identified by determining the

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

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

[0050] In another embodiment, the present disclosure provides antibodies directed to the same epitope as one of the antibodies in Table 1 (e.g., an antibody or antibodies that bind (e.g., by binding to and / or stabilizing it) Concerning fragments.

[0051] In a further embodiment, the antibody or antigen-binding fragment thereof is any of the antibodies in Table 1. One Kabat, Chothia, IMGT, or Kabat / Chothia combination Epitope of an antibody or antibody fragment comprising six CDRs defined by any one of the methods to (e.g., bind to and / or stabilize) an epitope that overlaps with (by) joining.

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

[0053] The term "bivalent antibody" as used herein refers to an antibody that binds to at least two identical targets. A bivalent antibody is an antibody that binds to two epitopes on a molecule. A bivalent antibody also crosslinks target molecules together. A "bivalent antibody" is an antibody that can bind to two different epitopes on at least two identical target molecules. It also refers to an antibody that binds to a polypeptide.

[0054] The term "multivalent antibody" refers to a single binding molecule with two or more valencies, where "binding "Valency" is described as the number of antigen-binding moieties present per molecule of antibody construct. Thus, a single binding molecule can bind to more than one binding site on a target molecule. Examples of antibodies include bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, etc., as well as bispecific antibodies. and biparatopic antibodies. For example, TREM2 For example, a multivalent antibody (e.g., a TREM2 biparatopic antibody) can be used to target the TREM2 receptor. It has binding sites for two domains of M2.

[0055] The term "multivalent antibody" refers to an antibody having two or more antigen-binding moieties directed against two separate target molecules. It also refers to a single binding molecule. For example, an antibody that binds to TREM2 and a target molecule that does not bind to TREM2. In one embodiment, the multivalent antibody is a tetravalent antibody having four epitope-binding domains. A tetravalent molecule is bispecific and bivalent for each binding site on its target molecule. It is possible.

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

[0057] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a The phrase "antibodies" refers to antibodies that have substantially the same amino acid sequence or are derived from the same genetic source. The term also refers to polypeptides including antibodies of single molecular composition. A monoclonal antibody composition includes a preparation of a single molecule that binds to a particular epitope. The binding specificity and affinity are shown.

[0058] As used herein, the term "human antibody" refers to a human antibody that is composed of the framework and CDR regions. The present invention also includes antibodies having variable regions derived from sequences of human origin, constant regions derived from human sequences, For example, a human germline sequence, or a mutated version of a human germline sequence, or, for example, K nappik et al.,(2000.J Mol Biol 296,57-86 Consensus frameworks derived from human framework sequence analysis, as described in The immunoglobulin variable domains are derived from antibodies containing CDRs and The positions are numbered according to well-known numbering schemes, e.g., the Kabat numbering scheme, Ch othia numbering scheme, or a combination of Kabat and Chothia, and Im MunoGenTics (IMGT) numbering can be used to define the 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 antibodies of the invention may also contain amino acid residues that are not encoded by human sequences (e.g., in vivo). by random or site-directed mutagenesis in vitro or by somatic mutation in vivo or conservative substitutions to enhance stability or production) However, the term "human antibody" as used herein refers to antibodies derived from other mammals, such as mice. It includes antibodies in which CDR sequences derived from the germline of a species are grafted onto human framework sequences. It is not intended to be.

[0060] As used herein, the phrase "recombinant human antibody" refers to an antibody produced using human immunoglobulin genes. isolated from animals (e.g., mice) that are transgenic or transchromosomal Isolated antibodies, or hybridomas prepared therefrom, transformed to express human antibodies Antibodies isolated from transformed host cells, e.g., transfectants, recombinant, combinatorial Antibodies isolated from a national human antibody library and the entire human immunoglobulin gene or by any other means, including splicing of a portion or sequence of the fragment to another DNA sequence. prepared, expressed, produced or isolated by recombinant means, such as an antibody prepared, expressed, produced or isolated by recombinant means Such recombinant human antibodies include all human antibodies that have been engineered to contain the framework and CDR sequences. The variable regions are derived from human germline immunoglobulin sequences. In certain embodiments, such recombinant human antibodies are prepared by in vitro mutagenesis (or human Ig sequencing). For the sequence, animals are subjected to in vivo somatic mutagenesis (if transgenic) Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody can be determined from the human germline VH and VL regions. VL sequences derived from and related to, but not naturally occurring in vivo within the human antibody germline repertoire This is an array that cannot exist.

[0061] The term "Fc region" as used herein refers to the CH3, CH2, and constant domain regions of an antibody. A polypeptide comprising at least a portion of the main hinge region. Optionally, an Fc region. The Fc region can contain a CH4 domain, which is present in some antibody classes. In one embodiment, the present invention provides a method for the production of a F-terminal fragment of an antibody. In one embodiment, the present invention provides a method for producing an antibody comprising the Fc region CH3 region of an antibody. In another embodiment, the present invention provides a method for producing a polypeptide comprising a polypeptide comprising an Fc region, a CH1 region, a CH2 region, a CH3 region, a CH4 region, a CH5 region, a CH6 region, a CH7 region, a CH8 region, a CH9 region, a CH1 ... and Cκ / λ regions. In one embodiment, a binding molecule of the invention comprises a constant region (e.g., a heavy In one embodiment, such a constant region comprises a That is, the polypeptides of the present invention disclosed herein are modified by three heavy chain constants. One or more of the constant domains (CH1, CH2, or CH3) and / or the light chain constant region domain ( Examples of modifications include changes or alterations to the CL in one or more domains. These changes include the addition, deletion or substitution of one or more amino acids. These may be included to optimize efficacy, half-life, etc.

[0062] As used herein, the term "affinity" refers to the affinity of an antibody to an antigen at a single antigenic site. At each antigen site, the variable region of an antibody binds to a number of interacts with antigens at sites through weak non-covalent forces; the more interactions, the higher the affinity As used herein, an IgG antibody or a fragment thereof (e.g., a Fab fragment) The term "high affinity" for a target antigen is defined as -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 However, high affinity binding is not known for other antibody isotypes. For example, high affinity binding to the IgM isotype can vary between 10 -7 M or less , or 10 -8It refers to an antibody with an affinity of M or less.

[0063] As used herein, the terms "Kassoc," "Ka," or "K" on " is a specific whereas the terms "Kdis," "K d" or "K off " is intended to refer to the off-rate of a particular antibody-antigen interaction. In one embodiment, the term "KD" (or "K D ") as used herein refers to K It is intended to refer to the dissociation constant, which is determined by the ratio of d to Ka (i.e., Kd / Ka). KD values ​​for antibodies are well established in the art and are expressed as molar concentrations (M). The KD of an antibody can be determined using a method such as surface plasmon resonance. or by using a biosensor system such as a Biacore® system. By using a stem.

[0064] As used herein, the term "avidity" refers to the overall stability of the antibody-antigen complex. It refers to a useful measure of antibody affinity or strength. It is based on three main factors: antibody epitope affinity; antigen affinity; and the valency of both the molecule and the antibody; and the structural arrangement of the interacting moieties. These factors determine antibody specificity, i.e., the ability of a particular antibody to bind to a precise antigen epitope. This defines the possibility that

[0065] As used herein, the term "binding specificity" or "specific binding" refers to the binding of an individual antibody to a The binding site refers to the ability to react with one antigenic determinant rather than reacting with different antigenic determinants. The binding site of an antibody is located in the Fab portion of the molecule and is composed of the hypervariable regions of the heavy and light chains. The binding affinity of an antibody is determined by the reaction between a single antigenic determinant and a single binding site on the antibody. It is the strength of the attractive and repulsive forces acting between the antigenic determinant and the binding site of the antibody. This is the total.

[0066] The terms "treat" and "treatment" refer to therapeutic treatment, which is the treatment of a subject suffering from an undesired physiological condition. For purposes of this invention, a beneficial or desired clinical result is a slowing down of the change or disorder. , whether detectable or undetectable, alleviation of symptoms, reduction in the extent of disease, or improvement of the condition Stabilization (i.e., not worsening), delay or slowing of disease progression, relief or remission of symptoms, "Treatment" includes, but is not limited to, the treatment of a disease or condition, including the improvement of a disease or condition, and the improvement of a disease or condition (whether partial or complete). It can also mean an increase in survival compared with the expected survival if not receiving treatment. do.

[0067] The terms "prevention," "prevent" and "preventing" of any particular disease or disorder For example, a compound of the present invention may be administered to a subject before any symptoms of the disease or disorder appear. refers to prophylactic or preventive measures against vinegar.

[0068] The term "subject" refers to an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated. The term includes mammals, e.g., humans. , other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters These include, but are not limited to, cattle, horses, cats, dogs, sheep and goats. Typical subjects include humans, farm animals, and domestic pets such as cats and dogs. In some preferred embodiments, the subject is a human.

[0069] An "effective amount" refers to an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is This amount is the amount that achieves the desired therapeutic effect. The effective amount may be the same as or different from the prophylactically effective amount, which is the amount required for one or more A "therapeutically effective amount" (i.e., The effective dose will depend on the therapeutic compound selected. The composition may be administered one or more times per day. It can be administered one or more times per week, including every other day. the severity of the condition, previous treatments, the general health and / or age of the subject, and other diseases present, Certain factors, including but not limited to, the dosage required to effectively treat a subject, may be varied. It will be recognized that the dosage and timing may be influenced. Treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. It is possible.

[0070] The term "nucleic acid" or "polynucleotide" refers to a deoxyribonucleic acid in either single-stranded or double-stranded form. It refers to nucleic acids such as DNA (DNA) or ribonucleic acid (RNA) and their polymers. Unless otherwise specified, the term refers to a nucleotide that has similar binding properties to the reference nucleic acid and does not contain naturally occurring nucleotides. Also included are nucleic acids containing known analogues of natural nucleotides that are metabolized in a similar manner. Unless otherwise specified, a particular nucleic acid sequence also refers to conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as those explicitly indicated Specifically, degenerate codon substitutions are those that involve one or more selected (or The third position of the codon (or all of the codons) is substituted with mixed base and / or deoxyinosine residues. This can be achieved by generating sequences containing 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 and A compound consisting of amino acid residues covalently linked by peptide bonds. A peptide must contain at least two amino acids and may be a protein or peptide. There is no limit to the maximum number of amino acids that a polypeptide can contain. Any peptide or protein containing two or more amino acids linked together by bonds As used herein, the term includes those commonly used in the art, e.g., peptides. , short chains also referred to as oligopeptides and oligomers, and those commonly referred to in the art as proteins. The term "polypeptide" refers to both short and long chains of polypeptides, and there are many different types. For example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, dimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, The polypeptide may be a natural peptide, a recombinant peptide, or a combination thereof. This includes combinations of the above.

[0072] The term "conservative sequence modifications" refers to modifications that have a significant effect on the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Conservative modifications are amino acid modifications that do not affect or alter the amino acid sequence. Modifications include amino acid substitutions, additions, and deletions. Modifications can be made using site-directed mutagenesis and PCR. Antibodies or polypeptides of the invention can be engineered by standard techniques known in the art, such as nucleotide-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residues have similar side chains. A family of amino acid residues with similar side chains is used. These families include those with 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), non-polar side chains (e.g., alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains ( threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, It contains amino acids with the following structure: tetrahydrofuran, thiamin, thiamin, tryptophan, and histidine. One or more amino acid residues in an antibody or antigen-binding fragment thereof of the invention may be from the same side chain family. The amino acid residues of the nucleotides of the present invention can be replaced with other amino acid residues, and the altered antibody or antigen-binding fragment can be This can be tested using the functional assays described herein.

[0073] The terms "homology" or "identity" refer to the degree of similarity between two polymer molecules, e.g., two DNA between two nucleic acid molecules, such as two RNA molecules, or between two polypeptide molecules. This refers to subunit sequence identity. Two molecules have the same monomeric subunit at both subunit positions. For example, if each position on two DNA molecules is occupied by an adenine If the position is occupied by a nucleotide, then they are homologous or identical at that position. Identity is a direct function of the number of matching or homologous positions; for example, half of the positions in the two sequences ( For example, if two sequences are homologous at five positions in a polymer 10 subunits in length, The alignment is 50% homologous; 90% (e.g., 9 out of 10) of the positions are matched or homologous. If the two sequences are 90% identical, the percentage of "sequence identity" is calculated based on the comparison. It can be determined by comparing two optimally aligned sequences across a window. The fragments of amino acid sequences in the comparison window are used as references for optimal alignment of the two sequences. Additions or deletions (e.g., gaps or open gaps) compared to the sequence (which does not contain the additions or deletions) The percentage is calculated by comparing both sequences to yield the number of matched positions. Determine the number of positions in the sequence where identical amino acid residues exist, and compare the number of matched positions. Divide the result by the total number of positions in the window to yield a percentage of sequence identity. The output is the percentage of the subject sequence relative to the query sequence. It is the same entity.

[0074] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but The same nucleic acid or peptide, partially or completely separated from the coexisting materials of its natural state, is called "isolated" An isolated nucleic acid or protein exists in substantially purified form. The isolated cells may be present in a non-native environment, such as a host cell. The antibodies are substantially free of other antibodies with different antigen specificities (e.g., TREM2 The isolated antibody that specifically binds to TREM2 is an antibody that specifically binds to an antigen other than TREM2. However, an isolated antibody that specifically binds to a target molecule may be may have cross-reactivity to the same antigen from different species (e.g., specific binding to TREM2). (An isolated antibody may bind to TREM2 molecules from other species). The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. It does not have to be included.

[0075] Unless otherwise defined, all technical and scientific terms used herein are intended to be used in conjunction with the present invention. have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, Suitable methods and materials are described below. All publications, patent applications, patents, and other materials cited herein are incorporated by reference. All other references mentioned are incorporated herein by reference in their entirety. In some cases, the present specification, including definitions, will take precedence. In addition, the materials, methods, and examples are not to be construed as limiting the scope of the present invention. These are examples only and are not limiting.

[0076] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the detailed description and drawings, and from the claims. It is.

[0077] Antibodies that stabilize functional TREM2 Provided herein are antibodies and antigen-binding fragments thereof that stabilize TREM2 on the cell surface. The antibody or antigen-binding fragment thereof is capable of preventing the proteolytic cleavage of TREM2 and and / or by reducing shedding of the ectodomain of the TREM2 protein In some preferred embodiments, stabilization of the antibody or its anti- The original binding fragment may be the IgSF domain of human TREM2, e.g., any of SEQ ID NOs: 1, 2, or 3. It specifically binds to any one of amino acid residues 19 to 132.

[0078] Reduction or absence of cell surface TREM2 is associated with human neuroinflammatory and neurodegenerative pathologies The TREM2 stabilization antibodies or antigen-binding fragments thereof described herein are useful for treating Alzheimer's disease. disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, Nasu-Hakola disease, multiple sclerosis Sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, cerebral lupus ( NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia, chronic inflammation Complicated demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), Inclusion body myositis, lysosomal storage diseases, e.g., sphingomyelin lipidosis (Niemann- Pick's disease type C) and mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal movement disorder Neuropathy, myasthenia gravis, neuro-Behçet's disease, neuromyelitis optica (NMO), optic nerve Myelitis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett syndrome, stroke, transverse myelitis, Neuroinflammatory and neuropathic diseases such as traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis It can be used to treat, prevent, or diagnose degenerative diseases. Therefore, the hTREM2 antibodies or antigen-binding fragments thereof described herein are useful for treating Alzheimer's disease. from dementia, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu-Hakola disease They can be used in the treatment, prevention, or diagnosis of selected diseases.

[0079] Due to their pharmacological profiles, the hTREM2 antibodies or their The antigen-binding fragments of the present invention are useful for treating CNS-related diseases, PNS-related diseases, systemic inflammation, and other inflammation-related diseases. various illnesses or diseases such as pain and withdrawal symptoms caused by chemical abuse The diseases or disorders related to the CNS are useful for the treatment of generalized anxiety disorder, cognitive impairment, and the like. Disability, learning and memory deficits and dysfunction, Alzheimer's disease (mild, moderate and severe), Mind-deficit hyperactivity disorder, Parkinson's disease, Parkinson's disease-type dementia, Huntington's disease, ALS , prion neurodegenerative disorders such as Creutzfeldt-Jakob disease and kuru, Tourette's syndrome, psychosis, depression and depressive disorders, mania, bipolar disorder, schizophrenia, psychiatric disorders Schizophrenic cognitive deficits, obsessive-compulsive disorder, panic disorder, eating disorders, narcolepsy, pain sensation, Age-related dementia, senile dementia, mild cognitive impairment (MCI), age-related memory impairment, Autism, dyslexia, tardive dyskinesia, epilepsy and seizure disorders, post-traumatic stress disorder , transient anoxia, pseudodementia, premenstrual syndrome, late luteal phase syndrome, chronic fatigue syndrome and Jet lag is one example.

[0080] The hTREM2 antibodies or antigen-binding fragments thereof described herein are particularly Extensive proteolytic cleavage or abnormal or mutated TREM2 receptor variants autoimmune disorders, inflammatory disorders, or malignancies mediated by or associated with cells expressing Examples of autoimmune diseases include, but are not limited to, arthritis, osteoarthritis, osteoporosis ... Arthritis (e.g., rheumatoid arthritis, chronic progressive arthritis) progrediente) and osteoarthritis) and associated with inflammatory conditions and bone loss Rheumatic diseases, including rheumatic diseases, inflammatory pain, spondyloarthropathy, including ankylosing spondylitis (spondyloarhropathy), Reiter's syndrome, reactive arthritis, psoriatic arthritis Arthritis and enteropathic arthritis, irritability These include autoimmune diseases (including both airway and skin hyperresponsiveness) and allergies. Diseases include autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, pure red blood cell anemia) thrombocytopenia and idiopathic thrombocytopenia), systemic lupus erythematosus, inflammatory myopathy, multiple myelopathy, Osteitis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active liver disease inflammation, myasthenia gravis, psoriasis, Stevens-Johnson syndrome son syndrome), idiopathic sprue, endocrine ophthalmopathy, Graves' disease, sarcoidosis Idosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (type 1 diabetes), grapes Uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis and glomerulonephritis (with or without nephrotic syndrome, e.g., gout, Langerhans syndrome) nephrotic syndrome or minimal change nephropathy), tumors, inflammatory skin and corneal diseases, myositis, loosening of bone implants, metabolic disorders, e.g., atherosclerosis , diabetes, and dyslipidemia.

[0081] The hTREM2 antibodies or antigen-binding fragments thereof described herein may also be used to treat asthma, bronchitis, and other respiratory diseases. obstructed or inflamed airways, including pneumoconiosis, emphysema, and other idiopathic pulmonary fibrosis or COPD They are also useful in treating, preventing, or ameliorating diseases.

[0082] The hTREM2 antibodies or antigen-binding fragments thereof described herein can be used to treat acute myeloid leukemia, Chronic myeloid leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, paroxysmal nocturnal hemoglobinuria Globinuria, Fanconi anemia, thalassemia major, Hematopoietic or hematopoietic disorders such as Wiskott-Aldrich syndrome and hemophagocytic lymphohistiocytosis It can be used to treat hepatopoetic malignancies.

[0083] The hTREM2 antibodies or antigen-binding fragments thereof described herein inhibit aberrant TREM2 activity. For use in the treatment of any disease or disorder directly or indirectly related to the sex and / or expression of TREM2-associated disorders include immune disorders, particularly inflammatory disorders (e.g., bacterial infections). Infectious diseases, fungal infections, viral infections, protozoan or other parasitic infections, psoriasis, sepsis, cerebral palsy Laria, 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 thyroiditis such as Graves' disease, insulin-resistant diabetes, pernicious anemia, Addison's disease, pemphigus acne, vitiligo, ulcerative colitis, systemic lupus erythematosus (SLE), Sjogren's syndrome, Sclerosis, dermatomyositis, mixed connective tissue disease, scleroderma, polymyositis, allograft rejection, etc. transplant rejection), T-cell disorders (e.g., AIDS), allergic inflammatory disorders (e.g., allergic rhinitis, asthma, skin and / or mucous membrane allergies such as psoriasis), neurological disorders, Directly or indirectly associated with ocular disorders, embryonic disorders, or abnormal TREM2 activity and / or expression and any other disorder (e.g., tumors, cancer, leukemia, myeloid disorders, and trauma) that Examples include:

[0084] In some embodiments, the TREM2-associated disorder is asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, or the like. COPD, allergic disorders, septic shock, pulmonary fibrosis, undifferentiated vertebrae for osteoarthrosis, undifferentiated arthropathy, arthritis, inflammatory osteolysis, or chronic viral or bacterial infections chronic inflammation caused by

[0085] In some embodiments, the TREM2-associated disorder is dementia, frontotemporal dementia, Alzheimer's disease, or the like. Heimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus , amyotrophic lateral sclerosis, Huntington's disease, tauopathy disease ease), Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic large intestine Enteritis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential Tremors, central nervous system lupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multifocal System atrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticobasal degeneration, acute respiratory distress syndrome encephalomyelitis, granulomatous disorder, lucidosis, age-related diseases, stroke, spinal cord injury, traumatic brain injury, age-related macular degeneration, green Internal cataracts, retinitis pigmentosa, retinal degeneration, respiratory tract infections, sepsis, eye infections, systemic infections, Arthritis, multiple sclerosis, low bone density, osteoporosis, bone formation, osteopetrosis, Paget's disease In some preferred embodiments, the TREM2-associated disorder is selected from: Alzheimer's disease, frontotemporal dementia, Parkinson's disease, amyotrophic lateral sclerosis, or Nasu -Hakora disease is selected from the list.

[0086] In some preferred embodiments, the TREM2-associated disorder is dementia, frontotemporal dementia Some preferred diseases are selected from Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis. In a preferred embodiment, the TREM2-associated disorder is frontotemporal dementia, Alzheimer's disease, hematoma, or rheumatoid arthritis. Dementia such as vascular dementia, semantic dementia, or dementia with Lewy bodies. In an embodiment, the TREM2-associated disorder is Alzheimer's disease. The disorder is Parkinson's disease.

[0087] Antibodies that specifically bind to human TREM2 and antigen-binding fragments thereof In one aspect, the present invention provides a method for the production of antibodies specific to the IgSF domain of the human TREM2 protein. A binding antibody or antigen-binding fragment thereof, such as a monoclonal antibody or antigen-binding fragment thereof (e.g., The antibody or antigen-binding fragment thereof is stabilizing the TREM2 protein on the cell surface and / or Ectodomain shedding can be reduced.

[0088] In some embodiments, the hTREM2 antibodies or antigen-binding fragments thereof provided herein The fragments are heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HC DR3), and light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain In some embodiments, the hTR provided herein comprises CDR3 (LCDR3). The EM2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3. (VH), and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3. In some embodiments, the hTREM2 antibodies or antigen-binding fragments provided herein , including the full-length heavy chain sequence (HC) and the full-length light chain sequence (LC).

[0089] Table 1 lists exemplary TREM2 antibodies or antigens that specifically bind to human TREM2 protein. The sequences of the binding fragments are listed. Throughout the document of this application, the sequences of the binding fragments are listed in the document (e.g., Table 1). In the event of any discrepancy between this and the sequence listing, the present document shall control.

[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 antigen-binding fragment thereof is selected from the group consisting of antibodies, antibodies, and antibodies described in Table 1. Other suitable hTREs include VH domains having the amino acid sequence of any VH domain. The M2 antibody or antigen-binding fragment thereof is mutated, but contains the amino acid sequence shown in Table 1 in the VH domain. VH regions shown in the sequences provided herein and at least 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 In certain embodiments, the present invention may comprise amino acids having 8, 99, or 100 percent identity. The disclosure provides antibodies or antibody fragments (e.g., antigen-binding fragments) that specifically bind to human ENTPD2. ), wherein the antibody or antigen-binding fragment thereof has any one of the amino acids of the HCDRs listed in Table 1. In certain embodiments, the present invention provides a VH CDR having the amino acid sequence listed in Table 1. HCDRs having any one of the amino acid sequences. A VH CDR comprising (or consisting of) more than The present invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that bind to the antibody.

[0119] In some embodiments, the hTREM2 antibody or antibody fragment (e.g., antigen-binding fragment) , including a VL domain having the amino acid sequence of any VL domain listed in Table 1. Suitable anti-human TREM2 antibodies or antibody fragments (e.g., antigen-binding fragments) of However, in the VL domain, the VL region shown in the sequence listed in Table 1 and at least 8 Amino acids with 0, 85, 90, 95, 96, 97, 98, or 99 percent identity The present disclosure may include antibodies or antibody fragments that specifically bind to human TREM2 (e.g., Antibodies or antibody fragments (e.g., antigen-binding fragments) are also provided, and the antibodies or antibody fragments (e.g., antigen-binding fragments) may be any of those listed in Table 1. In particular, the present invention provides a VL CDR having the amino acid sequence of any one of the LCDRs. One, two, three or more LCDRs having the amino acid sequence of any one of the LCDRs listed in Table 1. and a VL CDR comprising (or consisting of) more than 100 VL CDRs, which specifically bind to human TREM2. The present invention provides antibodies or antibody fragments (e.g., antigen-binding fragments) that bind to the antibody.

[0120] Other anti-human TREM2 antibodies or antibody fragments (e.g., antigen-binding fragments) disclosed herein The CDR regions of the sequences shown in Table 1 are mutated but not in the CDR regions. At least 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the sequence In some embodiments, it comprises an amino acid having the sequence shown in Table 1. 1, 2, 3, 4, or 5 or fewer amino acids in the CDR regions compared to the corresponding CDR regions It includes variant amino acid sequences in which one or more amino acids are mutated.

[0121] VH, VL, and full-length heavy chains of antibodies and their antigen-binding fragments that specifically bind to human TREM2 and nucleic acid sequences encoding full-length light chains, e.g., the nucleic acid sequences of Table 1, are also provided herein. Such nucleic acid sequences are optimized for expression in a desired host cell, e.g., a mammalian cell. It can be optimized.

[0122] Other anti-human TREM2 antibodies disclosed herein may contain an amino acid or a sequence encoding an amino acid. The nucleic acid is mutated but at least partially mutated relative to the sequence shown in the sequence set forth in Table 1. Those with 80, 85, 90, 95, 96, 97, 98, or 99 percent identity In some embodiments, the antibody or antigen-binding fragment thereof exhibits substantially the same therapeutic activity. The sequences are retained, but have differences in the variable regions when compared to the variable regions shown in the sequences listed in Table 1. Variant amino acid sequences include those in which no more than 1, 2, 3, 4 or 5 amino acids are mutated.

[0123] Each antibody provided binds to human TREM2 and therefore includes a VH, VL, full-length light chain, and full-length The heavy chain sequence (amino acid sequence and amino acid sequence encoded by the nucleotide sequence) is These antibodies can be "mixed and matched" to create other TREM2-binding antibodies disclosed herein. Such "mixed and matched" TREM2 binding antibodies can be used in binding assays known in the art. can be tested using an assay (e.g., ELISA, as described in the examples). When chains are mixed and matched, the VH sequence resulting from a particular VH / VL pairing will The full-length heavy chain / full-length light chain pairing is replaced by a VH sequence similar to the full-length heavy chain / full-length light chain pairing. The long heavy chain sequence should replace the structurally similar full-length heavy chain sequence. The VL sequence resulting from the pairing should replace a structurally similar VL sequence. The full-length light chain sequence derived from the heavy chain / full-length light chain pairing is replaced with a structurally similar full-length light chain sequence. It must be bad.

[0124] Thus, in one embodiment, the present invention provides a method for the preparation of a nucleotide sequence selected from any one of SEQ ID NOs: 13 and 50. a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 61; An isolated monoclonal antibody having a light chain variable region (VL) comprising an amino acid sequence selected from a monoclonal antibody or antigen-binding fragment thereof; the antibody specifically binds to human TREM2; A clonal antibody or antigen-binding fragment thereof is provided.

[0125] In another embodiment, the present invention provides: (i) SEQ ID NOs: 15, 29, 33, 35, 37, 39, A full-length heavy chain comprising any one of 52, 66, 70, 72, 74, and 76 amino acids. chain (HC); and a full length comprising an amino acid selected from any one of SEQ ID NOs: 26 and 63 or (ii) an isolated protein having a functional protein comprising an antigen-binding fragment thereof. The present invention provides a monoclonal antibody.

[0126] In another embodiment, the present disclosure provides heavy chain CDR1, CDR2 and CDR3 as set forth in Table 1. and human TREM2 comprising light chain CDR1, CDR2 and CDR3, or a combination thereof. The amino acid sequence of HCDR1 of the antibody is SEQ ID NO:4. , 7, 8, 10, 41, 44, 45, 47. Amino acid sequence of HCDR2 of antibody are shown in SEQ ID NOs: 5, 9, 11, 41, 45, and 47. The sequences are shown in SEQ ID NOs: 6, 12, 43, and 49. The amino acid sequences of LCDR1 of the antibodies are , as shown in SEQ ID NOs: 17, 20, 23, 54, 57, and 60. The amino acid sequences are shown in SEQ ID NOs: 18, 21, 55 and 58. The sequences are shown in SEQ ID NOs: 19, 22, 56, 59, 56.

[0127] Each of the antibodies binds to human TREM2, and the antigen-binding specificity is determined primarily by CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR1 ... DR2 and CDR3 regions, provided by the VH CDR1, CDR2 and CDR3 sequences Given this, VL CDR1, CDR2 and CDR3 sequences can be "mixed and matched." (i.e., CDRs from different antibodies can be mixed and matched), but each antibody To generate other human TREM2-binding antibodies disclosed herein, VH CDR1, CD It is necessary to include CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3. Such "mixed and matched" TREM2 binding antibodies are known in the art and described in the Examples. The VH C binding assay can be tested using the binding assays described above (e.g., ELISA). When DR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 sequences from a particular VH sequence may be In this case, the CDR3 sequence should be replaced with a structurally similar CDR sequence. When CDR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 from a particular VL sequence will Alternatively, the CDR3 sequence should be replaced with a structurally similar CDR sequence. The VH and VL sequences may be prepared by combining one or more VH and / or VL CDR region sequences with the monoclonal antibodies of the present invention. The CDR sequences are replaced with structurally similar sequences derived from the CDR sequences shown herein for the native antibody. It will be readily apparent to those skilled in the art that the present invention can be produced by the above method.

[0128] Thus, the present disclosure provides a method for the preparation of a nucleic acid sequence comprising the group consisting of SEQ ID NOs: 4, 7, 8, 10, 41, 44, 45, 47. heavy chain CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 5, 9, 11, 42, 46, 4 heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 12, 43; heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 20, 49; a light chain CDR1 comprising an amino acid sequence selected from the group consisting of: 23, 54, 57, and 60; A light chain CD40 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 21, 55, and 58. R2; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 22, 56, 59, and 56. and an isolated monoclonal antibody or antigen-binding region thereof, comprising a light chain CDR3 comprising the sequence. The antibody specifically binds to human TREM2.

[0129] In certain embodiments, antibodies that specifically bind to human TREM2 are those listed in Table 1. The antibody may be an antibody or antibody fragment (e.g., an antigen-binding fragment).

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

[0131] In some embodiments, an antibody or antigen-binding region thereof that specifically binds to human TREM2. HCDR1 comprising the amino acid sequence of SEQ ID NO: 41, 44, 45, or 47; HCDR2 comprising the amino acid sequence of SEQ ID NO: 42, 46, or 48; HCDR3 comprising the amino acid sequence of SEQ ID NO: 54, 57, or 60; LCDR comprising the amino acid sequence of SEQ ID NO: 54, 57, or 60 LCDR2 comprising the amino acid sequence of SEQ ID NO: 55 or 58; and SEQ ID NO: 56 or 59 and an LCDR3 comprising the amino acid sequence:

[0132] In some embodiments, an antibody or antigen-binding region thereof that specifically binds to human TREM2. The region is a region having the amino acid sequence of SEQ ID NO: 13 (or at least about 90%, 95%, 99% or or more than one identical and / or one, two, three or more substitutions, insertions, deletions or a sequence having a modification), and the amino acid sequence of SEQ ID NO: 24. (or at least about 90%, 95%, 99% or more identical thereto, and / or a light chain containing a sequence having one, two, three or more substitutions, insertions, deletions or modifications Contains the variable region (VL).

[0133] In some embodiments, an antibody or antigen-binding region thereof that specifically binds to human TREM2. The region is SEQ ID NO: 50 (or at least about 90%, 95%, 99% or more thereof). Identical and / or with one, two, three or more substitutions, insertions, deletions or modifications a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 61 (or a sequence similar thereto); At least about 90%, 95%, 99% or more identical, and / or one, two, three a light chain comprising an amino acid sequence having one or more substitutions, insertions, deletions or modifications Contains the variable region (VL).

[0134] In some embodiments, an antibody that specifically binds human TREM2 has the sequence of SEQ ID NO: 15 The amino acid sequence (or at least about 90%, 95%, 99% or more identical thereto) , and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications 26 (or at least about 90%, 95%, or more thereof), %, 99% or more identical and / or 1, 2, 3 or more substitutions and light chains containing the nucleotide sequence (sequence having insertions, deletions or modifications).

[0135] In some embodiments, an antibody that specifically binds human TREM2 has the sequence of SEQ ID NO: 29 The amino acid sequence (or at least about 90%, 95%, 99% or more identical thereto) , and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications 26 (or at least about 90%, 95%, or more thereof), %, 99% or more identical and / or 1, 2, 3 or more substitutions and light chains containing the nucleotide sequence (sequence having insertions, deletions or modifications).

[0136] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 33 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 26 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0137] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 35 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 26 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0138] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 37 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 26 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0139] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 39 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 26 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0140] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 52 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 63 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0141] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 66 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 63 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0142] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 70 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 63 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0143] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 72 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 63 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0144] In some embodiments, an antibody that specifically binds human TREM2 is identified as SEQ ID NO: 74 ( or at least about 90%, 95%, 99% or more identical thereto, and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications a heavy chain comprising SEQ ID NO: 63 (or at least about 90%, 95%, 99%, or and / or one, two, three or more substitutions, insertions, deletions or The light chain comprises an amino acid sequence of:

[0145] In some embodiments, an antibody that specifically binds human TREM2 has the sequence of SEQ ID NO: 76 The amino acid sequence (or at least about 90%, 95%, 99% or more identical thereto) , and / or a sequence having one, two, three or more substitutions, insertions, deletions or modifications 63 (or at least about 90%, 95%, or more identical thereto). %, 99% or more identical and / or 1, 2, 3 or more substitutions and light chains containing the nucleotide sequence (sequence having insertions, deletions or modifications).

[0146] In some embodiments, the present invention provides a method for detecting plasmon resonance (SPR) in a cellular environment. and a dissociation constant (K) of less than 200 pM. D ), e.g., less than 150 pM, less than 120 pM, Less than 100pM, Less than 90pM, Less than 70pM, Less than 50pM, Less than 40pM, 30pM K less than, less than 20 pM, or less than 10 pM D The IgSF domain of the TREM2 protein In some preferred embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to a nucleotide sequence comprising: The antibodies or antigen-binding fragments provided herein have a dissociation constant (K D ) and TR In some preferred embodiments, the present invention binds to the IgSF domain of the EM2 protein. The antibodies or antigen-binding fragments provided herein have a dissociation constant (K D ) with TREM 2 protein binds to the IgSF domain.

[0147] Once the desired epitope on the antigen has been determined, it can be identified by, for example, using the techniques described in this invention. It is then possible to generate antibodies against that epitope. Alternatively, during the discovery process, The generation and characterization of antibodies can elucidate information about the desired epitope. This information makes it possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to use antibodies that bind competitively with each other, e.g. The next step is to perform a cross-competition study to find antibodies that compete for binding to the antigen. A high-throughput process for "binning" antibodies based on cross-competition is being applied for in an international patent application. The epitope is described in WO 2003 / 48731. may contain a residue to which

[0148] Generally, antibodies specific for a particular target antigen are produced by complex mixtures of proteins and / or macromolecules. The antibody preferentially recognizes an epitope on the target antigen in the antibody.

[0149] The region of a given polypeptide that contains an epitope may be any number of epitopes known in the art. These can be identified using epitope mapping techniques. ing Protocols in Methods in Molecular Bi ology, Vol. 66 (Glenn E. Morris, Ed., 1996, Hum See, e.g., Chromatin Press, Totowa, NJ. For example, a number of peptides (peptides corresponding to portions of a protein molecule) are attached to a solid support. The peptides are simultaneously synthesized on the support and the antibody is reacted with the peptide while the peptide is still bound to the support. Such techniques are known in the art and can be determined, for example, by 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 3:709-715. Similarly, conformational epitopes can be used to identify, for example, For example, the spatial conformation of amino acids can be determined by X-ray crystallography and two-dimensional nuclear magnetic resonance. These can be easily identified by determining the epitope of the target gene, for example, using the epitope mapping method described above. The antigenic region of a protein can also be identified using standard antigenic and hydrophobic protocols. For example, O available from Oxford Molecular Group. Calculated using the MIGA version 1.0 software program. This computer program is useful for determining antigenic profiles. For details, see Hopp / Woods method, Hopp et al. (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; and hydrophobic For sex plots, see the Kyte-Doolittle technique. et al., (1982) J. Mol. Biol. 157:105-132; In some embodiments, the anti-TREM2 antibody binds to the IgSF domain of human TREM2. For example, anti-TREM2 antibodies may specifically bind to epitopes in SEQ ID NOs: 1, 2, or 3, specifically an epitope within the range of amino acid residues 19 to 132. can be combined.

[0150] The antibody molecule may be a polyclonal or monoclonal antibody. can be achieved by hybridoma technology, or by phage display or combinatorial techniques. It can be prepared by other methods such as the cellulose method.

[0151] Phage display and combinatorial methods for generating antibodies are well known in the art. (See, for example, Ladner et al., U.S. Pat. No. 5,223,409). Kang et al. International Publication No. 92 / 18619; Dower et al. International Publication No. 91 / 17271; Winter et al. International Publication No. 92 / 20791; Markland et al. International Publication International Publication No. 92 / 15679; Breitling et al. 3 / 01288; McCafferty et al. International Publication No. 92 / Garrard et al. WO 92 / 09690 Ladner et al. International Publication No. 90 / 02809 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. (1991) PNAS 88:7978-7982).

[0152] In one embodiment, the antibody is a human antibody (e.g., an antibody produced from human immunoglobulin sequences). antibodies produced in genetically engineered mice (antibodies produced in mice genetically engineered to Antibodies (e.g., rodent (mouse or rat), goat, primate (e.g., monkey), camel) antibody).

[0153] Chimeric and / or humanized antibodies are antibodies produced in non-human subjects or derived from non-human antibody genes. engineered to minimize immune responses by human patients to antibodies derived from expression of the gene A chimeric antibody comprises a non-human animal antibody variable region and a human antibody constant region. The antibody retains the epitope binding specificity of the original monoclonal antibody but is suitable for administration to humans. When administered intravenously, they are less immunogenic and therefore more likely to be tolerated by patients. For example, one or the entire variable region of the light chain of a mouse antibody (e.g., a mouse monoclonal antibody) and / or one or all of the variable regions of the heavy chain (e.g., Each of the IgG1 and IgG2a variants (e.g., one, two, or three) is a human constant region, such as, but not limited to, an IgG1 Chimeric monoclonal antibodies can be produced by recombinant methods known in the art, although they may be linked to human constant regions. For example, the constant region of a non-human antibody molecule can be produced by DNA technology. 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 U.S. Patent Application No. 171,496.) In addition, to generate chimeric antibodies, Other suitable techniques that may be used for this purpose are described, for example, in U.S. Pat. No. 4,816,567; Nos. 4,978,775; 4,975,369; and 4,81 No. 6,397.

[0154] Chimeric antibodies are antibodies in which the portions of the variable region that are not involved in antigen binding have been replaced with equivalent portions derived from human variable regions. A humanized antibody can be further "humanized" by replacing the heavy and / or is a non-human (e.g., mouse, rat, or hamster) complementarity-determining region (CDR) of the light chain. and one or more human framework regions in the variable regions. In the present context, a humanized antibody contains sequences that are completely human except for the CDR regions. Typically, they are less immunogenic in humans than non-humanized antibodies and therefore less effective in certain situations. Humanized TREM2 antibodies can be produced by methods known in the art. 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 Nos. 5,225,539; 5,530,101; ,585,089 specification; 5,693,761 specification; 5,693,762 Nos. 6,180,370 and WO 90 / 07861 Please refer to the brochure.

[0155] Human TREM2 antibodies can be generated using methods known in the art. Humaneering technology is used to convert non-human antibodies into engineered human antibodies. US Patent Application Publication No. 20050008625 describes the potential for the production of non-human antibodies. The variable regions of a non-human antibody are replaced with human variable regions in the antibody, while retaining the same binding characteristics as those of the non-human antibody. In order to maintain or provide better binding characteristics compared to those of non-human antibodies, An in vivo method is described, which involves translating the variable regions of a non-human reference antibody into epitopes in a fully human antibody. The resulting human antibody generally has structural similarities to the reference non-human antibody. The antibody is unrelated to the sequence but binds to the same epitope on the same antigen as the reference antibody. The epitope-guided complementarity displacement approach generates a reporter that responds to the binding of a test antibody to an antigen. In the presence of a system, various high-density "competitors" and reference antibodies are tested for binding to a limited amount of antigen. By setting up a competition in cells between a library of hybrids ("test antibodies") The competitor can be the reference antibody or a derivative thereof, such as a single chain Fv fragment. A competitor may also be a natural or artificial ligand of an antigen that binds to the same epitope as the reference antibody. The only requirement for a competitor is that it binds to the same epitope as the reference antibody, and The test antibody competes with the reference antibody for antigen binding. The V region of each antibody is a common antigen-binding region of the human antibody family. The V regions from the reference antibodies are randomly selected from the same V region as the guideline. It acts as a guide, placing the test antibodies on the same epitope on the antigen in the same orientation, allowing for selection is biased towards the highest antigen binding fidelity relative to the reference antibody.

[0156] Many types of reporter systems can be used to detect the desired interaction between the test antibody and the antigen. For example, complementary reporter fragments can be bound to the antigen and test antibody, respectively. By allowing the test antibody to bind to the antigen, reporter activity due to fragment complementation is observed only when the test antibody binds to the antigen. The test antibody and the antigen-reporter fragment fusion compete for When co-expressed with the antibody, reporter activation is proportional to the affinity of the test antibody for the antigen. The reporter system used in this study will depend on the ability of the test antibody to compete with the competitor. In U.S. Patent Application No. 10 / 208,730 (Publication No. 20030198971), Reactivation of the autoinhibited reporter reactivation system (RAIR) as disclosed in Factors, or U.S. Patent Application No. 10 / 076,845 (Publication No. 2003015757 Examples of such competitive activation systems include those disclosed in US Pat. No. 6,239,999.

[0157] Using a sequential epitope-guided complementary substitution system, selection is performed to identify competitors, antigens, and reporters. Cells expressing a single test antibody along with the target component are identified. The test antibody competes head-to-head with the competitor for binding to a limited amount of antigen. The activity of the antibody is proportional to the amount of antigen bound to the test antibody, which in turn determines the binding activity of the test antibody to the antigen. The affinity of the test antibody to the target molecule is proportional to the affinity of the target molecule to the target molecule and the stability of the test antibody. In this case, the antibodies are initially selected based on their activity relative to the activity of a reference antibody. The result of this analysis is a set of "hybrid" antibodies, each of which is a hybrid of the reference antibody and and human V regions from a library, each of which is Hybrid antibodies selected in the first round bind to the same epitope on the antigen as the antibody. One or more of the antibodies have an affinity for the antigen that is comparable to or higher than that of the reference antibody. It has harmony.

[0158] In the second V-region replacement step, the human V-regions selected in the first step are replaced with cognate human V-regions. Selection of human replacements for the remaining non-human reference antibody V regions with a diverse library of V regions. The hybrid antibodies selected in the first round are also used as a guide for selection. The results of the second round of selection can be used as competitors for the second round of selection. A fully human antibody that is structurally different from a reference antibody but competes with the reference antibody for binding to the same antigen. Some of the selected human antibodies have the same epitope on the same antigen as the reference antibody. Among these selected human antibodies, one or more bind to the affinity of the reference antibody. binds to the same epitope with an affinity comparable to or higher than that of

[0159] In some embodiments, the present invention provides a method for detecting TREM2 by binding to the human TREM2 protein. promotes 2-dependent physiological activities, such as phagocytosis (e.g., in hM2A macrophages) or in human iPS-derived microglia-like cells, or in microglia / macroglia in the brain. Enhanced chemotaxis in human iPS-derived microglia-like cells by enhancing the activity of phages enhances NFAT-driven reporter gene activity in a human monocytic cell line; an antibody or its antigen-binding fragment that increases Syk phosphorylation in hM2A macrophages; This promotion and / or enhancement can be, for example, at least 10%, at least 20%, %, at least 30%, at least 40%, at least 50%, at least 60%, at least It may be at least 70%, at least 80%, or at least 90%.

[0160] Engineered and Modified Antibodies The antibodies of the present invention may also be used as starting materials for engineering modified antibodies, as described in Table 1. The modified antibody can be prepared using an antibody having one or more of the VH and / or VL sequences, The antibody may have altered properties from the starting antibody. H and / or VL), for example, within one or more CDR regions and / or one or more frames It can be engineered by modifying one or more residues within the work region. Alternatively, antibodies may contain constant region modifications, e.g., to alter the effector functions of the antibody. The amino acid sequence can be engineered by modifying residues within the region.

[0161] One type of variable region engineering that can be performed is CDR grafting. The target antigen is primarily determined through amino acid residues located in the heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are similar to the sequences outside the CDRs. CDR sequences are more diverse between individual antibodies than between the CDRs. Therefore, specific sequences grafted onto framework sequences from different antibodies with different properties can be used. A specific natural antibody can be produced by constructing an expression vector containing CDR sequences from the specific natural antibody. It is possible to express recombinant antibodies that mimic the properties of antibodies (see, 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; and U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,08 See Patent No. 9; Patent Nos. 5,693,762 and 6,180,370 .)

[0162] Such framework sequences include germline antibody gene sequences or rearranged antibody sequences. These sequences can be obtained from public DNA databases or published references. For example, human heavy chain and The germline DNA sequences of the variable and light chain genes are available from the "VBase" human germline sequence database. Base (available online at www.mrc-cpe.cam.ac.uk / vbase (Available at: http: / / www.kabat.com / ) and Kabat, EA, et al., 1991 Sequence 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, J.P. Let al., 1994 Eur. J Immunol. 24:827-836, each of which is incorporated herein by reference. For example, the germline DNA sequences of human heavy and light chain variable region genes are expressly incorporated herein. The sequence and rearranged antibody sequences are indexed in the "IMGT" database (www.imgt.org). Available on the Internet; Lefranc, MP et al., 1999 Nucl See eic Acids Res. 27:209-212, each of which is incorporated by reference. (which is expressly incorporated herein by reference).

[0163] Exemplary framework sequences for use in the antibodies and antigen-binding fragments thereof of the invention are: , framework sequences used by selected antibodies and antigen-binding fragments thereof of the present invention , e.g., consensus sequences used by the monoclonal antibodies of the invention and / or The VH CDR1, 2 and 3 sequences are structurally similar to the framework sequences. The VL CDR1, 2, and 3 sequences are derived from the germline immunoglobulin G1 and G2 framework sequences. It can be grafted onto framework regions having sequences identical to those found in the purine gene. or the CDR sequences have framework regions that contain one or more mutations compared to the germline sequence. For example, in some cases, residues within the framework regions can be mutated to It has been found to be beneficial to maintain or enhance the antigen-binding ability of antibodies (e.g., U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,089 to Queen et al. (See specifications; Patent Nos. 5,693,762 and 6,180,370).

[0164] Another type of variable region modification is the VH and / or VL CDR1, CDR2 and / or CDR3. Mutating amino acid residues within the DR3 region, thereby performing a process known as "affinity maturation" The goal is to improve one or more binding properties (e.g., affinity) of the antibody of interest. Direct mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations into the antibody. The effect on binding, or other functional properties of interest, are described herein and provided in the Examples. Conservative modifications (see above) can be evaluated in in vitro or in vivo assays. Mutations can be amino acid substitutions, additions or deletions. Furthermore, typically no more than one, two, three, four or five residues within the CDR regions are varied. It will be changed.

[0165] The resulting polypeptide contains at least one binding domain that specifically binds to TREM2. A wide variety of antibody / immunoglobulin frameworks or scaffolds can be used, as long as Such frameworks or scaffolds are composed of the five major idiotypes of human immunoglobulins. Immunoglobulins of other animal species, including antigen-binding fragments thereof, preferably with humanized aspects. Single heavy chain antibodies, such as those identified in camelids, are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art. are.

[0166] In one aspect, the present invention provides a non-immunoglobulin scaffold onto which the CDRs of the present invention can be grafted. and a method for generating non-immunoglobulin-based antibodies targeting TREM2. Any known or future non-immunoglobulin framework fragment may be used, provided it contains a binding domain specific for the protein. and scaffolds can be used. Known non-immunoglobulin frameworks or scaffolds include , fibronectin (Compound Therapeutics, Inc., Wal tham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd. , Cambridge, Mass., and Ablynx nv., Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freis ing, Germany), small modular immunopharmaceuticals (Trubion Pharmac euticals Inc., Seattle, Wash.), Maxibody (Avid ia, Inc., Mountain View, Calif.), protein A (Aff ibody AG, Sweden), and affilin (γ-crystallin or ubiquitin) (SciI Proteins GmbH, Halle, Germany) , but not limited to these.

[0167] The fibronectin scaffold may comprise a fibronectin type III domain (e.g., Based on the 10th module of the Fn3 domain. Type III domains have seven or eight β-strands distributed between two β-sheets, which They themselves pack together to form the core of the protein, and further connect the β-strands to each other. Each edge of the β-sheet sandwich contains a small loop (similar to a CDR) that is exposed to the solvent. There are at least three such loops, where the edges are at the protein boundaries perpendicular to the direction of the β-strands. These fibronectin vectors are Although the scaffold of the base is not an immunoglobulin, the overall fold is similar to that of camel and llama IgG. The smallest functional antibody fragment contains the entire antigen recognition unit, including the folding and compaction of the variable region of the heavy chain. Because of this structure, non-immunoglobulin antibodies are closely related to the antibody's properties and affinity. These scaffolds mimic the antigen binding properties of antibodies that are similar to those of antibodies in vivo. In vitro loop randomization and shuffling strategies that resemble the maturation process These fibronectin-based molecules can be used in which the loop regions of the molecule are standard. They can be used as scaffolds that can be substituted with the CDRs of the present invention using cloning techniques.

[0168] Camelidae antibodies Camels and dromedaries (Camelus bactrianus) ) and dromedary (Calelus dromaderius) family members Llama species (alpacas, llamas) obtained from the bar New World Men such as Lama glama and Vicugna Antibody proteins containing antibodies have been characterized in terms of size, structural complexity, and antigenicity for human subjects. Specific Ig antigens from this mammalian family found in nature have been characterized. G antibodies lack light chains and therefore have two heavy chains and two light chains, unlike antibodies from other animals. It is structurally different from the typical four-chain quaternary structure. See International Publication No. WO 94 / 04678, published March 3, 1994. I want to be.

[0169] The regions of camelid antibodies that are small single variable domains identified as VHHs are genetically It produces small proteins obtained by genetic engineering that have high affinity for their target, The result was the production of low molecular weight antibody-derived proteins known as "camelid nanobodies." 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. Engineered libraries of camelid antibodies and antibody fragments are available, for example, from A Other antibodies of non-human origin and and antigen-binding fragments thereof, the amino acid sequence of the camelid antibody may be recombinantly modified to It is possible to obtain sequences that more closely resemble human sequences, i.e., to "humanize" nanobodies. Thus, the naturally low antigenicity of camelid antibodies to humans can be further can be lowered.

[0170] Camelid nanobodies have a molecular weight approximately one-tenth that of human IgG molecules and are protein-specific. Proteins have physical diameters of just a few nanometers. One consequence of their small size is that Culid nanobodies bind to antigenic sites that are functionally invisible to larger antibody proteins. The ability to bind to camelid nanobodies is key to their ability to bind to antibodies using classical immunological techniques. as reagents to detect otherwise cryptic antigens, and with potential therapeutic agents. Thus, a further consequence of the small size is that camelid nanobodies are targets. inhibition as a result of binding to a specific site in a groove or narrow cleft of a target protein and thus more closely resembles the function of classical small molecular weight drugs than that of classical antibodies. It can be useful in a similar capacity.

[0171] The low molecular weight and compact size also make camelid nanobodies highly thermostable. It is stable to extreme pH and proteolytic digestion and has poor antigenicity. Another consequence is that camelid nanobodies are readily transported from the circulation to tissues, It can even cross the blood-brain barrier and treat disorders that affect nervous tissue. Nanobodies can further enhance drug transport across the blood-brain barrier.20 See U.S. Patent Application Publication No. 20040161738, published August 19, 2004. These features, combined with low antigenicity to humans, give rise to great therapeutic potential. Furthermore, these molecules have been shown to be effective in the growth of prokaryotic cells such as E. coli. It can be expressed entirely in vitro and as a fusion protein with a bacteriophage, and functions It is efficient.

[0172] Thus, a feature of the present invention is the use of camelid antibodies or antibodies having high affinity for TREM2. In one embodiment herein, in a camelid, a camelid antibody Alternatively, Nanobodies may be naturally occurring, i.e., may be synthesized using techniques described herein for other antibodies. by camelids after immunization with TREM2 or a peptide fragment thereof using the Alternatively, TREM2-binding camelid nanobodies can be engineered, i.e., e.g. For example, a panning procedure using TREM2 as a target, as described in the Examples herein, Using this procedure, we generated phagocytic markers that display appropriately mutagenized camel nanobody proteins. Engineered nanobodies are produced by selection from a library of recipient nanobodies. It has been further engineered to have a half-life of 45 minutes to 2 weeks in human subjects. In certain embodiments, the camelid antibody or nanobody may be personalized, e.g., P The heavy or light chains of the human antibodies of the present invention as described in CT / EP93 / 02214 Grafting the CDR sequences of the compound into nanobody or single domain antibody framework sequences is obtained by

[0173] Bispecific molecules and multivalent antibodies In another aspect, the present invention provides a bispecific antibody comprising a TREM2-binding antibody or fragment thereof of the present invention. The antibodies of the present invention, or antigen-binding regions thereof, are characterized as multispecific molecules. Another method is to generate bispecific molecules that bind to two different binding sites or target molecules. a functional molecule, such as another peptide or protein (e.g., another antibody or The antibodies of the present invention may actually be derivatized or linked to more than two different To generate multispecific molecules that bind to two or more other binding sites and / or target molecules, Such multispecific molecules may also be derivatized or linked to functional molecules of the present invention; These are intended to be encompassed by the term "bispecific molecule" as used herein. To generate a clear bispecific molecule, the antibodies of the invention may be fused to the antibody or antibody fragments in a manner that allows the bispecific molecule to be generated. , functional to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic can be linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, or other methods) by).

[0174] Thus, the present invention provides a method for the treatment of leukemia, encephalopathy, and encephalopathy with at least one first binding specificity for TREM2 and a second binding specificity for TREM2. Bispecific molecules include those that contain a second binding specificity for a target epitope. For example, The target epitope is another epitope of TREM2 that is different from the first target epitope. .

[0175] Additionally, for those embodiments in which the bispecific molecule is multispecific, the molecule may comprise a first and a second In addition to the target epitope, a third binding specificity may further be included.

[0176] In one embodiment, the bispecific molecule of the invention has as binding specificity at least one antibody or antibody fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, or single chain Antibodies also include light or heavy chain dimers, or minimal fragments thereof, e.g., Fv. , Fv, or as described in U.S. Pat. No. 4,946,778 to Ladner et al. It may also be a single-stranded construct such as

[0177] Diabodies are structures in which the VH and VL domains are expressed on a single polypeptide chain and are expressed on the same chain. Bivalents connected by a linker that is too short to allow pairing between the two domains above The VH and VL domains pair with the complementary domains of different chains, and This creates two antigen-binding sites (see, e.g., Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448 ;Poijak et al.,1994 Structure 2:1121-112 3). Diabodies are VHA-VLB and VHB-VLA (VH-VL structure), or VLA-VHB and VLB-VHA (VL-VH structure) It can be produced by expressing two polypeptide chains in the same cell. The portion can be expressed in soluble form in bacteria. Single-chain diabodies (scDbs) , which connects the two diabody-forming polypeptide chains with a linker of about 15 amino acid residues. (Holliger and Winter, 1997 Can cer Immunol.Immunother.,45(3-4):128-30;W u et al.,1996 Immunotechnology,2(1):21-3 6). The scDb can be expressed in bacteria in a soluble, active monomeric 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, e.g., 617-21). Diabodies can be fused to Fc to form "di-diabodies." " can be produced (Lu et al., 2004 J. Biol. Chem. ,279(4):2856-65).

[0178] Other antibodies that can be used in the bispecific molecules of the invention include murine, chimeric and and humanized monoclonal antibodies.

[0179] Bispecific molecules of the invention can be prepared by isolating the binding specificities of the components using methods known in the art. For example, bispecific molecules can be prepared by conjugating the Each binding specificity can be generated separately and then conjugated to one another. In the case of proteins or peptides, various coupling or cross-linking agents are used for covalent attachment. Examples of cross-linking agents include protein A, carbodiimide, and N-succinimidyl -5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoate) benzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl -3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4(N-Maleimidomethyl)cyclohaxane-1-carbohydrate and hydroxylates (sulfo-SMCC) (e.g., Karpovsky et al. .,1984 J.Exp.Med.160:1686;Liu, MA et al. See, 1985 Proc. Natl. Acad. Sci. USA 82:8648. 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 Coupling agents include those described in (Munol. 139:2367-2375). , SATA and sulfo-SMCC, both from Pierce Chemical o. (Rockford, Ill.).

[0180] When the binding specificity is an antibody, they bind to sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge region can be conjugated via a conjugate bond. Prior to conjugation, the peptide is modified to contain an odd number of sulfhydryl residues, e.g., one.

[0181] Alternatively, both binding specificities can be encoded in the same vector and expressed in the same host cell. This method allows the bispecific molecule to be synthesized from mAb X mAb, mAb X mAb, and mAb X mAb. X Fab, Fab XF(ab')2 or Ligand X Fab fusion protein The bispecific molecules of the invention are particularly useful in cases where one single chain antibody and one binding determinant are present. The bispecific molecule may be a single-chain molecule containing two binding determinants, or a single-chain bispecific molecule containing two binding determinants. The heteromeric molecule may comprise at least two single chain molecules. Methods for preparing bispecific molecules include: See, for example, U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030 Details; 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. Patent No. US Patent No. 5,013,653; US Patent No. 5,258,498; and US This is described in US Patent No. 5,482,858.

[0182] Binding of bispecific molecules to their specific targets can be assayed, for example, by enzyme-linked immunosorbent assay. (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g. These assays can be confirmed by, for example, growth inhibition, or Western blot assays. Each of these assays generally uses a labeled reagent (e.g., an antibody) specific for the complex of interest. by specifically detecting the presence of a protein-antibody complex of interest.

[0183] In another aspect, the present invention provides antibodies and antigen-binding fragments thereof of the present invention that bind to TREM2. Multivalent compounds are provided that contain at least two identical or different antigen-binding moieties. The moieties may be linked together via protein fusion or covalent or non-covalent bonds. Alternatively, methods of attachment are described for bispecific molecules. Tetravalent compounds can be prepared by, for example, The antibodies and antigen-binding fragments thereof of the present invention are prepared by combining the constant region ( by cross-linking with antibodies or antigen-binding fragments that bind to the Fc or hinge region can be obtained.

[0184] Trimerization domains are described, for example, in EP 1 012 280 B1. The pentamerization module is described, for example, in PCT / EP 97 / 05897. It is written.

[0185] In some embodiments, the TREM2 binding molecule binds to both TREM2 and DAP12. In some embodiments, the TREM2 binding molecule is a bispecific antibody that binds to a first In some embodiments, the antibody is a bispecific antibody that recognizes the first antigen and a second antigen. In some embodiments, the antigen is human TREM2 or a naturally occurring variant thereof. In some cases, the second antigen is human DAP12 or a naturally occurring variant thereof. In this embodiment, the second antigen is human DAP10 or Siglec (sialic acid-binding immunoglobulin). In some embodiments, the second antigen is amyloid beta or amyloid β. fragments of tau, IAPP, α-synuclein, TDP-43, FUS protein, plio Protein, PrPSc, huntingtin, calcitonin, superoxide dismutase Ze, ataxin, Lewy bodies, atrial natriuretic factor, islet amyloid polypeptide , insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, Transthyretin, lysozyme, β2 microglobulin, gelsolin, keratoepithelin , cystatin, immunoglobulin light chain AL, S-IBM protein, repeat-associated non-ATG (RAN) translation product, dipeptide repeat (DPR) peptide, glycine-alanine (G A) Repeat peptide, glycine-proline (GP) repeat peptide, glycine-al Gyneine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and The disease-causing protein is selected from proline-arginine (PR) repeat peptides. In some embodiments, the second antigen is a transferrin receptor, an insulin receptor a blood-brain barrier receptor selected from the group consisting of insulin-like growth factor receptor, LRP-1, and LRP1; targeting proteins; or ligands and / or proteins expressed on immune cells, 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 phosphatidylserine Alternatively, the second antigen is a ligand and / or protein selected from the group consisting of may be a protein expressed in one or more tumor cells.

[0186] Antibodies with extended half-lives The present invention provides 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, the kidney filtrate Permeation, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenicity responses (e.g., protein neutralization by antibodies and uptake by macrophages and dendritic cells) Various strategies can be used to extend the half-life of the antibodies and antigen-binding fragments thereof of the present invention. For example, polyethylene glycol (PEG), reCODE P EG, antibody scaffold, polysialic acid (PSA), hydroxyethyl starch (HES), alginate By chemical conjugation to albumin-binding ligands and carbohydrate shields; albumin, IgG, By genetic fusion to proteins that bind to serum proteins such as FcRn; nanobodies Serum proteins such as Fabs, DARPins, avimers, affibodies, and anticalins By conjugation (genetic or chemical) to other binding moieties that bind to proteins; rPEG, albumin by genetic fusion to ATP, domains of albumin, albumin-binding protein, and Fc or by incorporation into nanocarriers, sustained release formulations, or medical devices.

[0188] Inert polymers such as high molecular weight PEG to prolong serum circulation of antibodies in vivo The molecule can be attached via site-specific attachment of PEG to the N-terminus or C-terminus of the antibody, or via attachment of PEG to lysine residues. via the ε-amino group present on the antibody or To PEGylate an antibody, the antibody, an antigen-binding fragment thereof, or a fragment thereof can be attached to the antibody. under conditions that typically result in one or more PEG groups becoming attached to the antibody or antibody fragment. , polyethylene glycol (PE), such as a reactive ester or aldehyde derivative of PEG PEGylation is achieved by reacting the polymer with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "aromatic" refers to a carboxylic acid or a carboxylic acid derivative that is a carboxylic acid derivative of a carboxylic acid. In this case, the term "polyethylene glycol" refers to mono(C1-C10)alkoxy- or Aryloxy-polyethylene glycol or polyethylene glycol-maleimide, etc. This includes any of the forms of PEG that have been used to derivatize other proteins. In one embodiment, the antibody to be pegylated is an aglycosylated antibody. Linear or branched polymer derivatization that minimizes loss of biological activity is used. The degree of conjugation can be adjusted to ensure proper conjugation of PEG molecules to the antibody. The unreacted PEG can be closely monitored by SDS-PAGE and mass spectrometry to determine the amount of unreacted PEG. 4. Purification of Antibody-PEG Conjugates by Size Exclusion or Ion Exchange Chromatography PEG-derivatized antibodies can be isolated from the using methods well known to those skilled in the art, for example, by immunoassays described herein. Methods for pegylating proteins are known in the art and can be tested using the antibodies of the present invention. For example, the method by Nishimura et al. European Patent No. 0154316 and European Patent No. 04013 by Ishikawa et al. See specification No. 84.

[0189] Another modified PEGylation technique involves the use of a reconstituted PEG containing tRNA synthetase and tRNA. Reconstituted chemical orthogonal manipulation systems for incorporating chemically defined side chains into biosynthetic proteins This technology includes the ReCODE PEG technology. It is possible to incorporate over 30 new amino acids into biosynthetic proteins in mammalian cells. tRNA can incorporate a standard amino acid at any position where an amber codon is located, Converting the bar from a stop codon to one that signals the incorporation of a chemically specified amino acid .

[0190] Recombinant PEGylation technology (rPEG) can also be used to extend serum half-life. , genetically inserting unstructured protein tails of 300-600 amino acids into existing pharmaceutical proteins. The apparent molecular weight of such unstructured protein chains is Since the molecular weight of the protein is approximately 15 times larger than its actual molecular weight, the serum half-life of the protein is greatly increased. In contrast to traditional PEGylation, which requires chemical conjugation and re-purification, the manufacturing process is significantly It is simplified and the product is uniform.

[0191] Polysialylation extends active life using the natural polymer polysialic acid (PSA) PSA is another technique for improving the stability of therapeutic peptides and proteins. It is a polymer of hydroxylic acid (sugar). It is used for protein and therapeutic peptide drug delivery. When present, polysialic acid provides a protective microenvironment for binding. The active life of the therapeutic protein is increased and it becomes unrecognized by the immune system. It is found naturally in the human body by certain bacteria that have evolved over millions of years. These naturally occurring polysialylated hydroxybenzoates were then employed to coat their walls with them. The bacteria were able to foil the body's defense system through molecular mimicry. PSA, a novel stealth technology, can extract these bacteria in large quantities and with specific physical characteristics. Bacterial PSA is chemically identical to human PSA. Therefore, even when conjugated to a protein, it is completely non-immunogenic.

[0192] Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy corn starch, HES solutions usually replace the deficient blood volume and improve blood rheology. It is administered to improve the rheological properties of antibodies. Hexylating antibodies improves the stability of the molecule. By increasing the concentration of steroids and decreasing renal clearance, it is possible to extend the circulating half-life. By changing different parameters such as the molecular weight of HES, the biological activity increases. This allows customization of a wide range of HES antibody conjugates.

[0193] Antibodies with increased in vivo half-lives may also be derived from IgG constant domains or their FcRn binding. The fragment (preferably the Fc or hinge-Fc domain fragment) may have one or more amino acid modifications (i.e., For example, the method of WO 98 / 232 No. 89; WO 97 / 34631; and U.S. Pat. See US Pat. No. 2,777,375.

[0194] Furthermore, to make the antibody or antibody fragment more stable in vivo or to make it last longer in vivo, To have a short half-life, antibodies can be conjugated to albumin. This technique is well known in the art and is described, for example, in WO 93 / 15199. , International Publication No. 93 / 15200 Pamphlet, and International Publication No. 01 / 77137 Pan See Fret; and European Patent No. 413,622.

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

[0196] antibody conjugates The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to the IgSF domain of TREM2. and the antibody or antigen-binding fragment is a heterologous protein or polypeptide (or its antigen-binding fragment). The original binding fragments, preferably at least 10, at least 20, at least 30, 40, at least 50, at least 60, at least 70, at least 80, at least a polypeptide of at least 90 or at least 100 amino acids) or conjugated (including both covalent and non-covalent bonds) to form the fusion protein In particular, the present invention provides antigen-binding fragments of the 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 a heterologous protein, polypeptide, or peptide. Proteins, polypeptides, or peptides can be fused or combined with antibodies or antibody fragments. Methods for conjugation are known in the art. See, for example, U.S. Patent Nos. 5,345,523 and 5,545,526. Specification No. 36,603, Specification No. 5,622,929, Specification No. 5,359,046 The detailed description, specifications of Nos. 5,349,053, 5,447,851, and 5 ,112,946; EP 307,434 and EP 367, 166; WO 96 / 04388 and WO 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] Additional fusion proteins include gene-shuffling, motif-shuffling, and xon-shuffling and / or codon-shuffling (collectively referred to as "DNA shuffling") DNA shuffling can be generated through the technique of DNA shuffling (also called "shuffling"). can be used to modify the activity of antibodies and antigen-binding fragments thereof (e.g., to increase the activity of higher parental (Antibodies and antigen-binding fragments thereof with improved affinity and lower dissociation rates). Specification No. 5,605,793, Specification No. 5,811,238, Specification No. 5,830,72 Nos. 1, 5,834,252, and 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 each of which is incorporated herein by reference in its entirety). The antibodies and antigen-binding fragments thereof, or the encoded antibodies and antigen-binding fragments thereof, may be prepared from the following recombinant vectors: random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods It specifically binds to the IgSF domain of TREM2. The polynucleotide encoding the antibody antigen-binding fragment may be linked to one or more of the one or more heterologous molecules. It may be recombined with a component, motif, segment, portion, domain, fragment, etc. of

[0198] Additionally, antibodies and antigen-binding fragments thereof may contain marker sequences (e.g., In one embodiment, the marker amino acid sequence may be fused to, inter alia, Hexa-histidine peptide, e.g., pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311 Many others are commercially available. l.,1989,Proc.Natl.Acad.Sci.USA 86:821-82 For example, hexa-histidine allows for convenient purification of the fusion protein, as described in [4]. Other peptide tags useful for purification include influenza hemagglutinin protein (HHTP). A hemagglutinin ("HA") tag corresponding to an epitope derived from protein (Wilson et al. al., 1984, Cell 37:767), and the "FLAG" tag (Hopp et al., 1984, Cell 37:767). tal., Bio / Technology 6 (1988): 1204-1210) Examples include, but are not limited to:

[0199] In one embodiment, the antibody of the invention is conjugated to a diagnostic or detectable agent. Such antibodies may be used as part of clinical testing procedures, such as to determine the effectiveness of certain treatments. to monitor or predict the onset, occurrence, progression and / or severity of a disease or disorder Such diagnosis and detection may be useful in, but not limited to, horseradish peroxide. enzymes, alkaline phosphatase, β-galactosidase, or acetylcholinesterase various enzymes such as ribosomal enzymes; including but not limited to streptavidin / biotin and avidin / prosthetic groups such as biotin; including but not limited to, umbelliferone, fluorescein, fluorescein Fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein fluorescent substances such as fluorescein, dansyl chloride, or phycoerythrin; any luminescent substance; such as, but not limited to, luciferase, luciferin, and aequorin Bioluminescent materials, including but not limited to iodine (I, I, I, and I ) 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 conjugated to detectable substances, including positron-emitting metals and non-radioactive paramagnetic metal ions. This can be achieved by:

[0200] Additionally, the antibody or antigen-binding fragment may be conjugated to a therapeutic or drug moiety. Therapeutic moieties or drug moieties should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or It may be a polypeptide. Examples of such proteins include abrin and ricin 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, proteins such as tissue plasminogen activators, apoptotic agents, and anti-angiogenic agents; Or biological response modifiers such as lymphokines.

[0201] Additionally, radioactive metal ions, such as alpha emitters such as 213Bi, or 131In, 1 Radioactive metals, including but not limited to 31LU, 131Y, 131Ho, and 131Sm Therapeutics such as macrocyclic chelators useful for conjugating ions to polypeptides In one embodiment, the antibody can be conjugated to a target moiety. is a 1,4,7,10-tetraazacyclododecanol that can be attached to an antibody via a linker molecule. Such linker molecules are di-N,N',N",N'"-tetraacetic acid (DOTA). As generally known in the art, 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 by reference in its entirety).

[0202] Techniques for conjugating therapeutic moieties to antibodies are well known and include, for example, Ammon 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 may also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include glass, cellulose, polyacrylamide, Examples include, but are not limited to, iron, polystyrene, polyvinyl chloride, or polypropylene. Not determined.

[0204] Nucleic acid encoding the antibody Nucleic acids encoding the antibodies or antigen-binding fragments thereof described herein are also provided herein. Such nucleic acids may be used in combination with the hTREM2 antibodies or antigen binding sites thereof described herein. Such nucleic acids may encode polypeptides comprising segments or domains of the fragments. The polynucleotides may also comprise a small fragment derived from the heavy or light chain of an hTREM2 antibody described herein. Such nucleic acids may encode at least one CDR region, and usually all three CDR regions. The acid or polynucleotide may also be used to encode the heavy chain and / or the ENTPD2 antibody described herein. Such nucleic acids or polypeptides may encode all or substantially all of the light chain variable region sequence. The nucleotides may also code for both the variable and constant regions of an antibody. Due to the degeneracy of the sequence, a variety of nucleic acid sequences encode each of the immunoglobulin amino acid sequences. For example, the present invention provides an antibody against an hT selected from one or more of the antibodies disclosed herein. REM2 antibody or its antigen-binding fragment, respectively encoding the heavy chain variable region and the light chain variable region. The nucleic acids are characterized by first and second nucleic acids having a nucleotide sequence shown in Table 1, or a sequence substantially identical thereto (e.g., at least about 85%, 90%, 95%, or sequences with 99% sequence identity, or 3, 6, 15, 30, or 10% identity with a sequence shown in Table 1 The nucleic acid may comprise two or more sequences shown in Table 1 (or sequences that differ by no more than 45 nucleotides). Nucleotide sequences (e.g., light chain variable domain sequences and heavy chain variable domain sequences, or e.g., a light chain sequence or a heavy chain sequence), or a sequence substantially identical thereto (e.g., a sequence having at least about 85%, 90%, 95%, or 99% sequence identity with the (a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequence shown in obtain.

[0205] In certain embodiments, the nucleic acid comprises a small fragment from a heavy chain variable region having an amino acid sequence shown in Table 1. Nucleotide sequences encoding at least one, two, or three CDRs or hypervariable loops or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, % or 99% sequence identity and / or one or more substitutions, e.g., conservative substitutions. In other embodiments, the nucleic acid may comprise an amino acid sequence shown in Table 1. At least one, two, or three CDRs or supersets from a light chain variable region having the amino acid sequence Nucleotide sequences encoding variable loops or sequences substantially homologous thereto (e.g., and / or a sequence having at least about 85%, 90%, 95% or 99% sequence identity with or a sequence having one or more substitutions, e.g., conservative substitutions). In another embodiment, the nucleic acids are derived from heavy and light chain variable regions having the amino acid sequences shown in Table 1. At least one, two, three, four, five or six CDRs or hypervariable loops of a nucleotide sequence that encodes the nucleotide sequence or a sequence that is substantially homologous thereto (e.g., at least about sequences with 85%, 90%, 95% or 99% sequence identity and / or one or more Substitutions may be included, for example sequences with conservative substitutions.

[0206] In certain embodiments, the nucleic acid comprises a heavy chain variable region having a nucleotide sequence as shown in Table 1. Nucleotides encoding at least one, two, or three CDRs or hypervariable loops of a sequence substantially homologous thereto (e.g., at least about 85%, 90% homologous thereto) 95%, or 99% sequence identity). In some embodiments, the nucleic acid comprises at least one nucleotide sequence from a light chain variable region having a nucleotide sequence shown in Table 1. a nucleotide sequence encoding one, two, or three CDRs or hypervariable loops; a sequence substantially homologous thereto (e.g., at least about 85%, 90%, 95%, or In yet another embodiment, the sequence may comprise a sequence having at least 99% sequence identity. The nucleic acid comprises at least one nucleotide sequence from the heavy and light chain variable regions having the nucleotide sequence shown in Table 1. Nucleotides encoding one, two, three, four, five, or six CDRs or hypervariable loops The nucleic acid sequence, or a sequence substantially homologous thereto (e.g., at least about 85%, 90%, or The sequences may include sequences having 0%, 95%, or 99% sequence identity.

[0207] Polynucleotide sequences can be synthesized by de novo synthesis (e.g., solid-phase DNA synthesis) or by TR Generated by PCR mutagenesis of existing sequences encoding the EM2 antibody or its antigen-binding fragments Direct chemical synthesis of nucleic acids can be achieved by the method of 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 9,1981; and U.S. Pat. No. 4,458,066. This can be achieved by methods known in the art, such as the solid support methods described herein. Introduction of mutations into a polynucleotide sequence by PCR can be achieved, for example, by 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 Applications This can be done as described in Applications 1:17, 1991.

[0208] A segment or domain of an hTREM2 antibody or antigen-binding fragment thereof described herein A vector (e.g., an expression vector) containing a polynucleotide encoding a polypeptide containing the Such vectors can be used, for example, in ex vivo cell cultures. in vivo in a cell, e.g., in one or more tissues of interest in an organism. and expressing an hTREM2 antibody or antigen-binding fragment (e.g., as described herein). and / or may be used to produce or affect the expression of various A polynucleotide encoding an hTREM2 antibody or a binding fragment thereof is prepared using a vector. For the production of antibodies or fragments thereof in cells, e.g., mammalian cells, Both viral-based and non-viral expression vectors can be used. Viral vectors and systems include plasmids, episomal vectors, and typically containing an expression cassette for expressing a protein or RNA, and human artificial staining Examples include the body (e.g., Harrington et al., Nat Genet. 15:345, 1997). Such non-viral vectors are well known in the art. Transfection or transduction methods known in the art can be used to transduce the vector, for example, by introducing lipids (e.g. Delivery to target cells using methods such as lipofectamine, electroporation, and mechanical cell membrane deformation. The term "expression vector" refers to a vector that delivers a desired coding sequence to a cell where it can be expressed. A vector refers to a carrier nucleic acid molecule into which a gene can be inserted for the introduction of a gene. It may be a vector, a plasmid, a cosmid, or a viral vector, or an artificial chromosome ( For example, Harrington et al., Nat Genet 15:345,1 997). For example, hTREM2 in mammalian (e.g., human) cells Non-viral vectors useful for expressing antibodies or antigen-binding fragments thereof include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invit rogen, San Diego, Calif.), MPSV vectors, and proteins Many other vectors known in the art for expressing genes are included. For example, Classes of vectors include bovine papillomavirus, polyomavirus, adenovirus, and vaccine Senior virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or DNA elements derived from animal viruses such as MOMLV or SV40 virus Another class of vectors is Semliki Forest virus, Eastern equine encephalitis virus, and Uses RNA elements derived from RNA viruses such as flaviviruses. Virus vectors include retroviruses (e.g., lentiviruses), , adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus) HSV), SV40-based vectors, papillomavirus, HBP Epstein-Barr virus -virus, vaccinia virus, simbis virus, influenza virus, reovirus Virus, Newcastle disease virus (NDV), measles virus, vesicular stomatitis virus ( VSV), parvovirus, poliovirus, poxvirus, Seneca Valley virus , Coxsackievirus, Enterovirus, Myxoma virus, Maraba virus or Seme virus Examples include Liqui Forest virus (SFV). Brent et al., supra; 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 an embodiment, the vector is a lentiviral vector. Rovirus-derived vectors have been shown to provide long-term, stable integration of transgenes and proliferation to daughter cells. This makes it a suitable tool for achieving long-term gene transfer, as it allows for proliferation in vitro. Antiviral vectors are vectors derived from oncoretroviruses such as murine leukemia viruses. It has the added advantage over conventional transducers in that it can transduce non-proliferating cells such as hepatocytes. They also have the added advantage of low immunogenicity. It may also be, for example, a gamma retroviral vector. The gene contains, for example, a promoter, a packaging signal (Ψ), a primer binding site (P BS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest; For example, the gammaretroviral vector may contain a gene encoding a CAR. They may lack viral structural genes such as , pol, and env. Viral vectors include murine leukemia virus (MLV), spleen focus-forming virus (SFF), and V), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, by Tobias Maetzig et al., “Gammaretroviral Vectors: Biology ,Technology and Application” Viruses.201 1 Jun;3(6):677-713.

[0210] In some embodiments, the vector is an adeno-associated virus (AAV) vector, e.g., For example, recombinant AAV (rAAV) vectors. "AAV" is an abbreviation for adeno-associated virus. and may be used to refer to the virus itself or its derivatives. Except where noted, the term encompasses both naturally occurring and recombinant forms. "V" refers to a recombinant adenovirus vector, also called a recombinant AAV vector (or "rAAV vector"). The term "AAV" refers to an associated virus, e.g., AAV type 1 (AAV1), AA V2 type (AAV2), AAV3 type (AAV3), AAV4 type (AAV4), AAV5 type ( AAV5), AAV6 type (AAV6), AAV7 type (AAV7), AAV8 type (AAV8 ), AAV9 type (AAV9), AAV10 type (including AAV10 and AAVrh10), A AV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate "Primate AAV" refers to AAV that infects primates. "non-primate AAV" refers to an AAV that infects non-primate mammals; "Bovine AAV" refers to an AAV that infects bovine mammals, and so forth.

[0211] The genomic sequences of various AAV serotypes, as well as the natural inverted terminal repeats (ITRs), Rep The sequences of the proteins and capsid subunits are known in the art. The sequences can be found in the literature or in public databases such as GenBank. GenBank accession 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 the brochure No. 1 (AAV1-9), the disclosures of which are incorporated herein by reference. See, e.g., Srivistava et al. 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; WO 00 / 28061 pamphlet, International Publication No. 99 / 61601 Pamphlet, International Publication No. 98 / 11244 Pamphlet See also U.S. Pat. Nos. 6,156,303 and 6,156,303.

[0212] As used herein, a "rAAV vector" refers to a polynucleotide that is not of AAV origin. sequence (i.e., a polynucleotide heterologous to the AAV), typically used for genetic transformation of cells. In some embodiments, a heterologous polynucleotide is used to describe an AAV vector containing a sequence of interest for recombinant expression. The nucleotide sequence is a sequence of at least one, and sometimes two, AAV inverted terminal repeat (ITR) sequences. The term rAAV vector refers to both rAAV vector particles and rAAV vectors. rAAV vectors include both single-stranded (ssAAV) or self-transfected AAV vectors. The term "AAV virus" or "rAAV virus" may be used interchangeably with "scAAV" or "rAAV virus." A "virus particle" or "rAAV vector particle" is a particle that contains at least one AAV capsid protein. a protein (typically one or more capsid proteins of or derived from wild-type AAV) (by all of the above) and viral particles consisting of an encapsidated polynucleotide rAAV vector The particle refers to a heterologous polynucleotide (i.e., a transgene) being delivered to a mammalian cell. When the vector contains a polynucleotide other than the wild-type AAV genome, such as a polynucleotide other than the wild-type AAV genome, it is typically referred to as "rA Therefore, the rAAV particles are called "rAAV vector particles" or simply "rAAV vectors." Production of such vectors necessarily involves the production of rAAV vectors, since they are contained within rAAV particles. This includes the production of tar.

[0213] In some embodiments, the vector comprises an hTRE vector, e.g., as described herein. It may also be a recombinant DNA molecule containing a nucleic acid encoding the M2 antibody or an antigen-binding fragment thereof. As used herein, "recombinant" refers to a vector, polynucleotide, polypeptide, or The cells may be subjected to a cloning, restriction or ligation step (e.g., a polynucleotide contained therein). related to nucleotides or polypeptides), and / or different from the product found in nature Recombinant viruses are the products of various combinations of other procedures that result in constructs that Alternatively, a vector is a viral particle that contains a recombinant polynucleotide. Each comprises a replica of the original polynucleotide construct and a progeny of the original viral construct.

[0214] Recombinant vectors typically contain one or more nucleic acid sequences operably linked to a nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other gene expression regulators. Regulatory sequences include nucleotide sequences containing transcription control elements (e.g., polyadenylation signals). These sequences include those directing constitutive expression of gene sequences, as well as tissue-specific regulatory and / or inducible sequences. Expression vectors also contain components that ensure the stability and translatability of messenger RNA in host cells. Elements designed to optimize functionality, and / or Establishment of permanent stable cell clones expressing the hTREM2 antibodies or their antigen-binding fragments The design of the expression vector may also be influenced by the host being transformed. This may depend on factors such as the choice of cells, the level of expression of the desired protein, etc. General methods for generating recombinant expression vectors are known in the art, inter alia. 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 s in Molecular Biology.

[0215] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including those encoding the nucleotides of the nucleotide sequence of interest. The initiation codon will be determined to be the same as the entire insert and will provide the necessary signals. to ensure translation is "in frame" with the reading frame of the desired coding sequence. It is well known that exogenous translational control signals and initiation codons must be The efficiency of expression can be determined by the presence of appropriate transcription enhancer elements. This can be enhanced by including

[0216] Expression can be carried out in any suitable host cell known in the art, e.g., mammalian host cells, bacteria Host cells, yeast host cells, insect host cells, etc. may be used. Both prokaryotic and eukaryotic host cells Expression systems for such cells are widely available. In some embodiments, the expression system is a CHO cell expression system. In some embodiments, the nucleic acid is expressed in a desired host cell. The cell type selected for expression, or the gene for which the gene is expressed, may be codon-optimized to facilitate expression. A promoter and a gene that effectively direct the expression of a DNA segment in a gene or organism. It may be important to use a promoter and / or enhancer. Those skilled in the art of molecular biology generally understand that Know the use of promoter, enhancer, and cell type combinations for protein expression (See, for example, Sambrook et al. (2001)).

[0217] Most transcribed eukaryotic RNA molecules undergo RNA splicing to form primary Remove introns from transcripts. Vectors containing genomic eukaryotic sequences are used to express proteins. To ensure proper processing of the transcripts for expression, donor and / or acceptor may require a primary splice site (Chandler et al., 1997, See Proc. Natl. Acad. Sci. USA, 94(8):3596-601 (I want to be).

[0218] A vector or construct of the present disclosure generally includes at least one termination signal. A "binding signal" or "terminator" is a specific sequence for the RNA transcript by RNA polymerase. Thus, in certain embodiments, the RNA transcript is composed of DNA sequences involved in the selective termination of the RNA transcript. A terminator is a signal that terminates the production of a desired message. In eukaryotic systems, terminators may be required in vivo to achieve high levels of The target region also allows for site-specific cleavage of the nascent transcript to expose the polyadenylation site. This may contain a specific DNA sequence that allows for approximately 200 A residues at the 3' end of the transcript. It signals a special endogenous polymerase to add a stretch of base (polyA). RNA molecules modified with this poly(A) tail are more stable and are translated more efficiently. Therefore, in other embodiments, including eukaryotes, the terminator is It is preferred to include a signal for cleavage, and a terminator signal is provided at the end of the message. More preferably, it promotes polyadenylation. Site elements may be used to enhance message levels and / or to remove other sequences from the cassette. The terminators contemplated for use in this disclosure may be useful for minimizing read-through. The terminator may be any known terminator of transcription described herein or known to one of skill in the art. including, but not limited to, the bovine growth hormone terminator. or viral terminators such as the SV40 terminator. In certain embodiments, the termination signal is a transcriptional or It may also be the absence of translatable sequences.

[0219] In order to provide for proper polyadenylation of the transcript in expression, particularly in eukaryotic expression, Typically, it will contain a polyadenylation signal. The nature of the polyadenylation signal will be determined by the methods described in this disclosure. It is not believed to be critical to the successful performance of the method and / or any such sequence may be used. A preferred embodiment may include an SV40 polyadenylation signal and / or a bovine growth hormone. It contains a polyadenylation signal, is convenient and / or performs well in a variety of target cells. Polyadenylation is known to function in a variety of ways, including increasing the stability of the transcript. Or it may facilitate cytoplasmic transport.

[0220] A specific nucleic acid sequence from which replication begins in order to propagate a vector in a host cell. It may contain one or more origins of replication (often called "ori"). If the vector is yeast, an autonomously replicating sequence (ARS) can be used.

[0221] In certain embodiments of the present disclosure, cells comprising a nucleic acid construct of the present disclosure are tagged with an expression vector. Such markers can be identified in vitro or in vivo. - confers an identifiable change to the cells, allowing for easy identification of cells containing the expression vector Generally, a selectable marker is one that confers a property that allows for selection. A selectable marker is one in which the presence of the marker allows for its selection, whereas a negative selectable marker is , the presence of which prevents its selection. An example of a positive selectable marker is a drug resistance marker. is.

[0222] The inclusion of a drug selection marker usually aids in cloning and identification of transformants, e.g. For example, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and Genes that confer resistance to erythritol and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on their performance, Other types of markers, including colorimetric and screenable markers such as GFP Alternatively, herpes simplex virus thymidine kinase (HSV-tk) or Screenable enzymes such as loramphenicol acetyltransferase (CAT) Enzymes may also be utilized. Those skilled in the art will also be familiar with immunological assays, possibly in combination with FACS analysis. Know how to use the markers. The markers used are those that are The selection marker is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the product. Further examples of markers and screenable markers are well known to those of skill in the art.

[0223] The choice of expression vector depends on the type of cell in which one or more components of the vector are to be expressed. Typically, the vector encodes an hTREM2 antibody or antigen-binding fragment thereof. a promoter and other regulatory sequences (e.g., The gene contains one or more regulatory sequences, such as a transcription factor (TF) or a transcription factor (TFR).

[0224] A "promoter" is a regulatory region in a nucleic acid sequence at which the initiation and rate of transcription are controlled. This is the sequence of regulatory proteins and molecules such as RNA polymerase and other transcription factors. The phrases "operatively arranged," "operatively linked" and "operably linked" may include genetic elements to which the offspring may be attached. "Controlled by," "under the control of," and "under transcriptional control" refer to the expression of a promoter relative to a nucleic acid sequence. and the correct functional location and / or orientation to control transcription initiation and / or expression of the sequence. A promoter is an "enhancer" (which increases the transcriptional activity of a nucleic acid sequence). The term "regulatory sequence" may be used in conjunction with a cis-acting regulatory sequence (which refers to a cis-acting regulatory sequence involved in activation of a gene). That's fine.

[0225] A promoter is a 5' non-coding sequence located upstream of a coding segment and / or exon. a gene or sequence naturally associated with the gene or sequence, as may be obtained by isolating the sequence; Such a promoter may be called "endogenous." Enhancers also include those naturally associated with a nucleic acid sequence, either downstream or upstream of that sequence. Alternatively, the encoding nucleic acid segment may be located in a recombinant or heterologous promoter. promoter (which refers to a promoter not normally associated with a nucleic acid sequence in its natural environment) By placing it under control, certain advantages are conferred. Recombinant or heterologous enhancers The term "enhancer" also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Suitable promoters or enhancers include promoters or enhancers of other genes, and and any other prokaryotic, viral, or eukaryotic cell promoter or enzyme isolated therefrom. promoters or enhancers, and promoters or enhancers that are not "naturally occurring," i.e., different These include those containing different elements of the transcriptional regulatory region and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences can also be synthesized using recombinant DNA technology. , recombinational cloning and / or nucleic acid amplification techniques related to the compositions disclosed herein; For example, it may be produced using PCR (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5 ... (See US Pat. No. 5,928,906.) In addition, nuclear Regulatory sequences that direct transcription and / or expression of sequences in organelles other than the ribosomal protein may also be utilized. It is intended that:

[0226] The promoters utilized may be constitutive, inducible, synthetic, tissue-specific, or cell-specific. and / or under appropriate conditions, effective in directing high-level expression of the introduced DNA segment. and which are advantageous in the large-scale production of recombinant proteins and / or peptides. In addition, it may be capable of binding to the human TREM2 protein (i.e., hTREM2). To improve expression of a nucleic acid encoding an antibody, for example, enhancers, ribosome binding inhibitors, Other regulatory elements, such as binding sites, transcription termination sequences, etc., may also be incorporated.

[0227] In some embodiments, the method further comprises the step of administering to the subject a therapeutically effective amount of an hTREM2 antibody or antigen-binding fragment thereof. To achieve this, a constitutive promoter is used. Examples of constitutive promoters include Although not used, the immediate early cytomegalovirus (CMV) promoter and simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter motor, Rous sarcoma virus promoter, and, but not limited to, actin promoter promoter, myosin promoter, elongation factor-1α promoter, hemoglobin promoter and human gene promoters such as the creatine kinase promoter.

[0228] In one embodiment, an inducible promoter is used to prevent the inserted sequence from being expressed except under inducing conditions. When an inducible promoter is used, the promoter to which it is operably linked turning on expression of a polynucleotide sequence containing the desired gene when such expression is desired, or A molecular switch is provided that can turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein (MT) promoters. othionine promoter.

[0229] Inducible promoters include, but are not limited to, arabinose promoters. , lacZ promoter, tetracycline promoter, metallothionein promoter glucocorticoid promoter, progesterone promoter, or heat shock promoter A motor is one example.

[0230] In addition to the promoter, for efficient expression of the hTREM2 antibody or antigen-binding fragment thereof, Other regulatory elements may also be necessary or desirable. The sequence includes the ATG initiation codon and adjacent ribosome binding site or other sequences. The efficiency of expression may be enhanced by including enhancers appropriate for the cell system being used (e.g., For example, Scharf et al.,Results Probl.Cell Diff er.20:125,1994; and Bittner et al., Meth.Enz (See, for example, SV40 enhancer Alternatively, the CMV enhancer can be used to increase expression in mammalian host cells.

[0231] In some embodiments, the expression of an hTREM2 antibody or antigen-binding fragment thereof is determined by measuring the expression of the antibody or antigen-binding fragment thereof in a specific tissue. To deliver the gene only to certain cells, a tissue-specific or cell-specific promoter is utilized. The identity of tissue- or cell-specific promoters or elements, and their activity Assays for characterizing the sex of a gene are well known to those of skill in the art. Gene (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 genes (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).

[0232] In some embodiments, a method for producing expression of an hTREM2 antibody or antigen-binding fragment thereof is provided. Synthetic promoters are used to enhance the transcriptional ability of natural promoters. For example, the activity of endogenous cellular mechanisms or factors may be blocked or reduced. Synthetic promoters can be selected that do not contain nucleotides, and have the potential to improve transcription efficiency. To achieve this, other elements, including trans-acting factor binding sites and enhancers, are inserted. Synthetic promoters include both synthetic and biological promoters. They can be rationally designed and chemically synthesized to combine the best features of Synthetic oligos are annealed and ligated in several steps to form a synthetic Chemically synthesized full-length promoters are produced. Synthetic promoters can be inducible or It may also be a cell-type specific promoter.

[0233] In a preferred embodiment, the vector is an adeno-associated vector (AAV). In some embodiments, the AAV vector may be an hTREM2 antibody or its derivative, e.g., as described herein. In an exemplary embodiment, the AAV vector comprises a polynucleotide encoding an antigen-binding fragment. The target is an hTREM2 antibody or antibody flanked on one or both sides by inverted terminal repeat (ITR) sequences. The polynucleotide may further comprise a polynucleotide encoding the binding fragment. For example, a promoter, an enhancer, one or more intron sequences, a poly(A) sequence, and It may also include one or more additional elements, such as: In this form, the vector is packaged in an AAV capsid, e.g., as described herein. a polynucleotide encoding an hTREM2 antibody or an antigen-binding fragment thereof; Nucleotide AAV vector plasmids.

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

[0235] In some embodiments, the ITRs in the AAV vector are from the same AAV serotype. In some embodiments, the ITRs in the AAV vector are derived from different AAV serotypes. In some embodiments, the ITRs in the AAV particles are the same. The ITRs of the children are different.

[0236] In some embodiments, the ITRs in the AAV vector are the same as those in the AAV capsid. In embodiments, the ITRs in the AAV vector are derived from a different serotype than the AAV capsid. In one embodiment, the ITRs are derived from AAV2 and the AAV capsid is A It is derived from a serotype other than AV2, for example, AAV9.

[0237] The expression vector also contains the inserted hTREM2 antibody or antigen-binding fragment thereof sequence. To form a fusion protein with the encoded polypeptide, the secretory signal sequence position is also In many cases, the insert sequence of the hTREM2 antibody or antigen-binding fragment thereof can be provided as a vector. The hTREM2-binding antibody light and heavy chain variable domains are linked to a signal sequence before being included in the target. The vector used to receive the sequence encoding the main Such vectors may encode the Allows expression of the variable regions, thereby producing intact antibodies and antigen-binding fragments thereof. Typically, such constant regions are human.

[0238] The generation of expression vectors involves multiple cloning steps, which are nucleic acid regions containing multiple restriction enzyme sites. Vectors containing multiple conversion sites (MCS) are available, any one of which can be used in standard recombination experiments. This technique can be used in conjunction with other techniques to digest vectors. t al., 1999, Levenson et al., 1998, and Coce See, e.g., 1997. "Restriction enzyme digestion" functions only at specific locations in a nucleic acid molecule. This refers to the catalytic cleavage of nucleic acid molecules by enzymes that perform the cleavage. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Often, vectors are Linearize or fragment with a restriction enzyme that cuts within S to allow ligation of the exogenous sequence into the vector. "Ligation" is the process of forming phosphodiester bonds between two nucleic acid fragments. Restriction enzyme and ligation reactions Techniques involving the reaction are well known to those skilled in the art of recombinant technology.

[0239] Methods for introducing an expression vector containing a polynucleotide sequence of interest into a cellular host include: For example, calcium chloride transfection is commonly used in prokaryotic cells. whereas calcium phosphate treatment or electroporation are used to treat other cell host (See generally Sambrook et al., supra). For example, electroporation, calcium phosphate treatment, liposome-mediated transfection, transfection, injection and microinjection, ballistic / gene gun, virosomes, immunoliposomes Polycation:nucleic acid conjugates, naked DNA, artificial virions, herpes viruses fusion to the structural protein VP22, drug-enhanced uptake of DNA, ex vivo transduction, Protoplast fusion, retroviral transduction, viral transfection, lipid-based In the case of protoplast fusion, the following techniques are used: transfection of the vector with a lysate or other conventional techniques. The cells are grown in culture and screened for appropriate activity. For long-term, high-yield production of a polypeptide, stable expression is often desired. Stable expression cell lines can be constructed using viral origins of replication or endogenous expression elements and selection vectors. It can be prepared by using an expression vector containing a marker gene. Cells may be grown in enriched medium for 1-2 days before switching to selective medium. The purpose of the nucleotide sequence is to confer resistance to selection, and its presence prevents the nucleotide sequence from being induced in selective media. This allows the growth of cells that successfully express the introduced sequence. The transformed cells can be propagated using tissue culture techniques appropriate to the cell type. Methods and conditions for culturing the infected cells and recovering the produced antibodies are well known in the art. The particular expression vectors and mammalian hosts used are known to those skilled in the art and are not intended to be limiting unless otherwise specified. It can be varied or optimized depending on the cell.

[0240] Also provided herein are cells containing any one of the expression vectors described herein. In some embodiments, the present disclosure provides host cells comprising the nucleic acid molecules described herein. Such cells may be host cells or therapeutic cells. and "recombinant host cell" are used interchangeably herein and refer to a particular cell of interest. It refers not only to the original cell but also to the progeny or potential progeny of such a cell. Because of this, certain modifications can occur in subsequent generations, so that such offspring are, in fact, the offspring of the parent. Although they may not be identical to cells, they are still within the scope of the term as used herein. Included in.

[0241] In one embodiment, the host cell contains a nucleic acid encoding an hTREM2 antibody or antigen-binding fragment thereof. In one embodiment, the host cell is genetically engineered to contain the acid. The term "expression cassette" refers to a nucleotide sequence Such sequences allow for efficient expression of genes in compatible hosts. The set includes a promoter, an open reading frame with or without introns, Additional factors necessary or helpful in achieving expression, such as For example, an inducible promoter may be used.

[0242] Host cells for harboring and expressing the chains of an hTREM2 antibody or antigen-binding fragment thereof include: The cell may be a eukaryotic cell, or a prokaryotic cell such as a bacterial cell, an insect cell, or a human cell. E. coli is a suitable bacterial strain for cloning the polynucleotides of the present invention. Other suitable microbial hosts for use include Bacillus subtilis, as well as Salmonella Salmonella, Serratia and various Pseudomonas These include other enterobacterial species such as Pseudomonas species. In prokaryotic hosts, expression control sequences compatible with the host cell (e.g., replication It is also possible to construct expression vectors containing the lactose promoter system, Tryptophan (trp) promoter system, β-lactamase promoter system or phage λ There are any number of different well-known promoters, such as promoter systems from The promoter typically controls expression, optionally with an operator sequence, It contains a ribosome binding site sequence for initiating and completing transcription and translation. Such other microorganisms may also express the hTREM2 antibodies or antigen-binding fragments thereof of the invention. Insect cells in combination with baculovirus vectors can also be used. Suitable insect cells include, but are not limited to, Sf9 cells.

[0243] In one embodiment, the mammalian host cell is an hTREM2 antibody or antigen-binding fragment thereof of the invention. For example, they are used to express and produce endogenous immunoglobulin genes. A hybridoma cell line expressing the gene (e.g., 1D6.C9 myeloma hybridoma cells) or a mammalian cell line (e.g., SP2 / 0 myeloma cells) carrying an exogenous expression vector. These can be any normal mortal or normal or abnormal immortal animal or Human cells include, for example, CHO cell lines, various Cos cell lines, HeLa cells, bone marrow Any cell capable of secreting intact immunoglobulins, including tumor cell lines, transformed B cells, and hybridomas. A number of suitable host cell lines have been developed. The use of tissue cell cultures is generally described, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987 Expression vectors for mammalian host cells include an origin of replication, a promoter, and , and enhancers (e.g., Queen, et al., Immunol. Rev. 8 9:49-68, 1986), and ribosome binding sites, RNA Necessary processing sequences such as splice sites, polyadenylation sites, and transcription terminator sequences These expression vectors are usually derived from mammalian genes or Suitable promoters include those derived from mammalian viruses. They may be specific, stage-specific, and / or modifiable or regulatable. The promoters include the metallothionein promoter, the constitutive adenovirus major late promoter, , dexamethasone-inducible MMTV promoter, SV40 promoter, MRP pol III promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoter promoters (such as the human immediate early CMV promoter), the constitutive CMV promoter, and Promoter-enhancer combinations known in the art include, but are not limited to: do not have.

[0244] The host cells produce antibodies that bind to the human TREM2 protein (i.e., hTREM2). Thus, the present disclosure also provides host cells for the production of hTREM The present invention features a method for producing a 2-antibody or antigen-binding fragment thereof. In one embodiment, the method The method comprises culturing a host cell (such as a host cell expressing an antibody) to produce an hTREM2 antibody or an antigen-binding fragment thereof. The method involves culturing the cells (into which a recombinant expression vector has been introduced) in an appropriate medium. In another embodiment, the method further comprises isolating the antibody from the medium or the host cell. Suitable eukaryotic cells include Vero cells, HeLa cells, COS cells, CHO cells, and HEK cells. These include, but are not limited to, 293 cells, BHK cells, and MDCKII cells. The method for introducing an expression vector containing a polynucleotide sequence of interest varies depending on the type of cell host. For example, calcium chloride transfection is often used for prokaryotic cells, On the other hand, calcium phosphate treatment or electroporation may be used for other cellular hosts (generally (See Sambrook, et al., supra.) Other methods include, for example, Electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection transfection, gene gun method, virosomes, immunoliposomes, polycation:nucleic acid conjugates DNA fragments, naked DNA, artificial virions, and herpesvirus structural protein VP22 fusion (Elliot and O'Hare, Cell 88:223, 1997), medicine These include enhanced DNA uptake by recombinant proteins and ex vivo transduction. Long-term, high-yield production of a gene often requires stable expression. For example, cell lines stably expressing hTREM2 antibody chains or antigen-binding fragments may be used to inhibit viral replication. Expression vectors of the present invention comprising an origin or endogenous expression element and a selectable marker gene After the introduction of the vector, cells can be cultured in a selective medium. Growth in enriched medium for 1-2 days is acceptable. The purpose of the selectable marker is to confer resistance to selection. The objective of this invention is to confer a nucleotide sequence which, when present, will select cells for successful expression of the introduced sequence in a selective medium. Resistant, stably transfected cells are then allowed to grow. They can be grown using tissue culture techniques appropriate to the cell type.

[0245] Regulatory sequences Those skilled in the art will appreciate that regulatory sequences are necessary for expression of one or more components of the vector in the target cell. You can recognize that you can get it.

[0246] In one embodiment, the AAV vector plasmid is, for example, as described herein. It contains efficient regulatory sequences for expression of the hTREM2 antibody or antigen-binding fragment thereof.

[0247] In one embodiment, the AAV vector plasmid drives expression in the target cell. It contains regulatory sequences that are efficient for

[0248] In one embodiment, the AAV vector plasmid contains a sequence encoding a promoter, such as, but not limited to, a promoter. As non-limiting examples, the promoter may include the regulatory sequences of: (1) the CMV promoter; (2) CBA promoter, (3) FRDA or FXN promoter, (4) UBC promoter Motor, (5) GUSB promoter, (6) NSE promoter, (7) synapsin promoter, (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 - may be possible.

[0249] promoter Those skilled in the art will appreciate that certain antibodies to hTREM2, e.g., antibodies to hTREM2 or antigen-binding fragments thereof, as described herein, may be used in the manufacture of such antibodies. The fragment may be expressed in a target cell in a manner that is, but is not limited to, species-specific, inducible, tissue-specific, or Note that specific promoters, including promoters that are cell cycle specific, may be required. (Parr et al., Nat. Med. 3:1145-9 (1997) ; the contents of which are incorporated herein by reference in their entirety).

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

[0251] In one embodiment, the AAV vector plasmid drives expression in the target cell. The promoter contains a promoter that is efficient for

[0252] In one embodiment, the promoter is a promoter that is expressed in a target tissue, such as, but not limited to, nervous system tissue. and for a period of time, e.g., an hTREM2 antibody or its resulting in the expression of an antigen-binding fragment, e.g., an hTREM2 antibody as described herein. or antigen-binding fragment thereof, expression is measured at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 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 , 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days , 13th, 2 weeks, 15th, 16th, 17th, 18th, 19th, 20th, 3 weeks, 22nd , 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 , 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years. Expression of an hTREM2 antibody or antigen-binding fragment thereof as described herein may be achieved by administering to a subject in need thereof a therapeutically effective amount of 1-5 mg / mL of a human serum albumin (HLA). 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- 10 years, or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or is 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50- It can range from 55 years, or 55-60 years, or 60-65 years.

[0253] In one embodiment, the AAV vector plasmid is, for example, as described herein. Approximately 5 kb upstream from the first exon of the encoded hTREM2 antibody or antigen-binding fragment thereof More specifically, the gene is expressed under the FRDA promoter. A 17-bp fragment located approximately 4.9 kb upstream from the first exon of the frataxin gene There are regions (e.g., Puspasari et al. Long Range Reg ulation of Human FXN Gene Expression,PLO See SONE, 2011, the contents of which are incorporated herein by reference in their entirety. used).

[0254] In one embodiment, the promoter is less than 1 kb. , 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 The promoter may be 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 up to 800, 600 to 700, 600 to 800, or 700 to 800.

[0255] In one embodiment, the promoters are the same or different, such as, but not limited to, CMV and CBA. It may be a combination of two or more components, regions or sequences of different promoters. , 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 or more than 800. Each component may be 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 The length may be 0 to 800, 600 to 700, 600 to 800, or 700 to 800. .

[0256] In one embodiment, the promoter comprises a CMV enhancer sequence, e.g., an immediate early CMV enhancer sequence. enhancer sequence (e.g., the 382 nucleotide CMV enhancer sequence) and chicken β-activator chin (CBA) promoter sequence (e.g., the 260 nucleotide CBA promoter sequence) It is a combination of.

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

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

[0259] In one embodiment, the AAV vector comprises the sequence: [ka] a hybrid CMV enhancer / chicken β-actin promoter comprising, for example, Includes motor.

[0260] In one embodiment, the AAV vector plasmid comprises a ubiquitous promoter. Non-limiting examples of motors include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1a, PGK, UBC, GUSB (hGBp), and UCOE (HNRPA2B1 -CBX3 promoter). In one embodiment, , Gill, Husain, Passini, Xu, Drews or Raymond Any of the promoters taught in Yu et al. may be used in the present invention. (Molecular Pain 2011, 7:63; the contents of which are incorporated herein by reference) (incorporated by reference in their entirety) used lentiviral vectors to transfect rat DRG cells and CAG, EF-1a, PGK, and UBC promoters in primary DRG cells. The expression of eGFP was evaluated, and UBC showed weaker expression than the other three promoters. However, only 10-12% glial expression was observed for either promoter. We found that...

[0261] Soderblom et al. (E. Neuro 2015; the contents of which are incorporated herein by reference) (incorporated by reference in its entirety) demonstrated that CMV and UBC promoters were expressed after injection into the motor cortex. Expression of eGFP in AAV8 containing a motor and AAV2 containing a CMV promoter Intranasal administration of plasmids containing the UBC or EF-1α promoter was evaluated. showed sustained airway expression higher than that of the V promoter (e.g., Gill et al. t al.,Gene Therapy 2001,Vol.8,1539-1546 See, e.g., Husai, et al., the contents of which are incorporated herein by reference in their entirety. n et al. (Gene Therapy 2009; the contents of which are incorporated herein by reference) (incorporated by reference in its entirety) provides an HβH construct containing the hGUSB promoter, HSV The LAT promoter and NSE promoter were evaluated in the mouse brain to assess HβH assembly. Passini and Wolf found that the expression of NSE was weaker than that of NSE. e (J. Virol. 2001, 12382-12392, the contents of which are incorporated herein by reference). (incorporated by reference in its entirety) showed that HβH vectors were expressed after intracerebroventricular injection in neonatal mice. The long-term effects of the drug were evaluated and found to be sustained for at least one year. et al. (Gene Therapy 2001,8,1323-1332; According to the description (which is incorporated herein by reference in its entirety), the NF-L and NF-H processes When using the promoter, CMV-lacZ, CMV-luc, EF, GFAP, and hE NK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8 Low expression was found in all brain regions compared with NSE (1.8kb) and NSE (1.8kb+wpre). Xu et al. found that promoter activity was enhanced by NSE (1.8 kb), EF, and NSE (0.3 kb). The results showed that the levels of GFAP, CMV, hENK, PPE, NFL, and NFH were highest in this order. NFL is a 650 nucleotide promoter, and NFH is a 920 nucleotide promoter. Both are not found in the liver, but NFH is found in proprioceptive neurons, the brain SCN8A is abundant in the spinal cord and spinal cord, while NFH is present in the heart. motor and is expressed throughout the DRG, spinal cord, and brain, particularly 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 Alternate ively Spliced ​​Sodium Channel a-subunit g enes,Journal of Biological Chemistry(200 4) 279(44) 46234-4624; the contents of each of these are incorporated herein by reference. (The entire disclosure of which is incorporated herein by reference).

[0262] In one embodiment, the AAV vector plasmid comprises a promoter that is not cell-specific. In one embodiment, the promoter is hTREM2, e.g., as described herein. Weak promoter (R) for sustained expression of antibodies or their antigen-binding fragments in neural tissue Its affinity for NA polymerase and / or sigma factors and that of other promoters In one embodiment, the promoter is a promoter including, but not limited to, To sustainably express frataxin in nervous system tissues such as neuronal and glial tissues It is a weak promoter of

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

[0264] In one embodiment, the AAV vector plasmid contains the ubiquitin c (UBC) promoter. The UBC promoter can be 300-350 nucleotides in size. As an example, the UBC promoter is 332 nucleotides.

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

[0266] In one embodiment, the AAV vector plasmid contains a neurofilament (NFL) promoter. 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 contains the 5'-promoter-CMV / globin intron- hFXN-RBG-3, wherein the AAV vector plasmid is self-complementary and the capsid may be of the DJ serotype.

[0267] In one embodiment, the AAV vector plasmid encodes a neurofilament heavy chain (NFH) promoter. The NFH promoter can be 900-950 nucleotides in size. As a non-limiting example, the NFH promoter is 920 nucleotides. For example, the AAV vector plasmid contains the 5'-promoter-CMV / globin intro hFXN-RBG-3', wherein the AAV vector plasmid is self-contained. It may be complementary and the capsid may be of the DJ serotype.

[0268] In one embodiment, the AAV vector plasmid comprises the SCN8A promoter. The 8A promoter can be 450-500 nucleotides in size. For example, the SCN8A promoter is 470 nucleotides. The vector plasmid is d'-promoter-CMV / globin intron-hFXN-R BG-3, wherein the AAV vector plasmid may be self-complementary. , the capsid may be of the DJ serotype.

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

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

[0271] In one embodiment, the AAV vector plasmid contains a chicken beta-actin (CBA) promoter. Includes the controller.

[0272] In one embodiment, the AAV vector plasmid is an immediate early cytomegalovirus (CMV) Contains a promoter.

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

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

[0275] In one embodiment, the AAV vector plasmid is a hepatic promoter or a skeletal muscle promoter. Non-limiting examples of liver promoters include hAAT and TBG. Non-limiting examples of skeletal muscle promoters include desmin, MCK, and C5-12. do.

[0276] In one embodiment, the AAV vector plasmid contains an engineered promoter, e.g., Includes promoters derived from, but not identical to, the promoters described herein. .

[0277] Augmenting Element In one embodiment, the AAV vector plasmid contains at least one enhancer and / or An enhancer or expression element can be a regulatory sequence (e.g., a promoter). In one embodiment, the AAV vector may be used in combination with a promoter. The mid contains a transgene enhancer, promoter and / or 5'UTR intron. Transgene enhancers, also referred to herein as "enhancers," include, but are not limited to: However, it is possible to use a CMV enhancer (or a fragment thereof, e.g., as described herein). The promoter may be, but is not limited to, CMV, CBA, UBC, GUSB, It may be the NSE, synapsin, MeCP2, or GFAP promoter. The R / intron may be, but is not limited to, SV40 and CBA-MVM.

[0278] In one embodiment, the AAV vector comprises an intron, which optionally comprises a promoter -element and an hTREM2 antibody or antigen-binding fragment thereof (e.g., a and a polynucleotide encoding the sequence as follows. However, including the 5' intron may result in an hTREM2 antibody or antigen-binding fragment thereof (e.g., and the steady-state level of mRNA encoding a nucleotide sequence (as described herein) is enhanced. In embodiments, the enhancer is a 5' endogenous enhancer derived from SV40. In one embodiment, the SV40 intron has the sequence: [ka] It comprises, for example consists of,

[0279] In one embodiment, the AAV vector plasmid (e.g., of the AAV vector) Although not specified, (1) CMV enhancer, CMV promoter, SV40 5'UT (2) an R intron (e.g., as described herein); (3) a CMV enhancer; CBA promoter, SV40 5'UTR intron (e.g., as described herein) (3) CMV enhancer, CBA promoter, CBA-MVM 5'U enhancers, promoters, etc., such as TR introns (e.g., as described herein); It includes combinations of transgenes and / or introns.

[0280] Enhancement of transgenes In one embodiment, the AAV vector plasmid increases target specificity and expression of the transgene. at least one transgene enhancer element capable of enhancing the , Powell et al.Viral Expression Cassette Elements to Enhance Transgene Target Spe significance and expression in Gene Therapy, 2015, the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of transgene enhancer elements that enhance target specificity and expression of transgenes Typical examples include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), Polyadenylation (polyA) signal sequence and upstream enhancer (USE), CMV enhancer These include sensors and introns.

[0281] In one embodiment, the AAV vector plasmid contains at least one CMV enhancer. In one embodiment, the AAV vector contains one transgene enhancer element. The transgene contains at least one transgene enhancer element, which is a promoter. nothing.

[0282] In one embodiment, the AAV vector plasmid contains at least one gene that is an intron. Contains transgenic enhancer elements.

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

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

[0285] In one embodiment, the AAV vector plasmid contains a polyadenylation (polyA) signal sequence. In embodiments, the transgene comprises at least one transgene enhancer element that is a sequence of A The AAV vector plasmid of the AV vector contains a growth hormone poly(A) signal. In morphology, the growth hormone poly(A) signal is similar to the bovine growth hormone (BGH) poly(A) signal. An example of a BGH poly A signal sequence is [ka] is.

[0286] In embodiments, the AAV vector plasmid is, for example, a hAAV vector as described herein. downstream of the polynucleotide encoding the TREM2 antibody or antigen-binding fragment thereof (e.g., On the 3' side, it contains a polyA signal (for example, a BGH polyA signal).

[0287] In one embodiment, the AAV vector plasmid contains a nucleotide sequence that is an upstream enhancer (USE). Contains at least one transgene enhancer element.

[0288] Tissue-specific expression In one embodiment, the vector genome comprises hTREM, e.g., as described herein. 2. Tissue-specific expression enzymes that enhance expression of antibodies or antigen-binding fragments thereof in tissues and / or cells. As a non-limiting example, the promoter may include, but is not limited to, the promoter of a human Long factor Ia-subunit (EF-1a), immediate early cytomegalovirus (CMV), Chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB) , and ubiquitin C (UBC).

[0289] In one embodiment, the vector genome may be used to direct expression to, but not limited to, neurons, astrocytes, or other tissues. Neuronal promoters that can be used to restrict Tissue-specific expression elements, such as promoters, that can be used to restrict expression to specific cell types It may include a ment.

[0290] In one embodiment, the vector genome may comprise, but is not limited to, a gene encoding a 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), metabotropic glutamate mGluR2, NFL, NFH, ηβ2, PPE, Enk, and EAAT The promoter may include tissue-specific expression elements for neurons, such as the .2 promoter.

[0291] In one embodiment, the vector genome may be a gene encoding, but not limited to, glial fibrillary acidic protein (GFAP). Tissue-specific expression for astrocytes, including the (GFAP) and EAAT2 promoters It may contain elements.

[0292] In one embodiment, the vector genome may be a gene encoding, but not limited to, myelin basic protein ( The promoter of the ribosomal protein MBP is a tissue-specific expression element for oligodendrocytes. It may include.

[0293] Introns In one embodiment, the AAV vector plasmid contains one or more introns or portions thereof. It contains at least one element that enhances transgene expression, such as

[0294] In one embodiment, the payload construct comprises a transgene, such as one or more introns or portions thereof. It contains at least one element that enhances transgene expression.

[0295] Non-limiting examples of introns include MVM (67-97 bp), FIX truncated Type intron 1 (300 bp), β-globin SD / immunoglobulin heavy chain splice accession Sceptor (250bp), adenovirus splice donor / immunoglobulin (immunoglobulin noglobin) splice acceptor (500bp), SV40 late spliced splice acceptor (19S / 16S) (180bp) and hybrid adduct Examples include the viral splice donor / IgG splice acceptor (230 bp) do.

[0296] In one embodiment, the intron or intron portion is 100 to 500 nucleotides in length. Introns may be 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 intron may be 60, 470, 480, 490 or 500 in length. 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 Or it may be 400 to 500 in length.

[0297] In one embodiment, the AAV vector comprises an intron, which optionally comprises a promoter -element and an hTREM2 antibody or antigen-binding fragment thereof (e.g., a and a polynucleotide encoding the sequence as follows. However, including the 5' intron may result in an hTREM2 antibody or antigen-binding fragment thereof (e.g., and the steady-state level of mRNA encoding a nucleotide sequence (as described herein) is enhanced. In embodiments, the enhancer is a 5' endogenous enhancer derived from SV40. In one embodiment, the SV40 intron has the sequence: [ka] It comprises, for example consists of,

[0298] In one embodiment, the AAV vector comprises an AAV vector plasmid comprising: (1) a CMV enhancer; (1) a CBA promoter (e.g., SEQ ID NO: 134); 5), (3) SV40 intron (e.g., SEQ ID NO: 137), (4) hTREM2 antibody or a polynucleotide encoding an antigen-binding fragment thereof (e.g., as described herein). and (5) a BGH polyA signal (e.g., SEQ ID NO: 138). In this configuration, elements (1) to (5) are arranged 5' to 3' on the AAV vector plasmid. In an embodiment, elements (1) to (5) are located in the ITRs (e.g., 5'ITR and In one embodiment, the I and II vectors are placed on an AAV vector plasmid further comprising the I and II ITRs. The TR is derived from the AAV2 ITR.

[0299] In one embodiment, the AAV vector plasmid is a self-complementary AAV vector plasmid. "Self-complementary" or the abbreviation "sc" refers to self-complementary. "AAV" or "scAAV" refers to a recombinant AAV in which the coding region carried by the AAV nucleic acid sequence is Refers to a construct that is designed to form an intramolecular double-stranded DNA template. These strands of scAAV are not bundled, but rather undergo cell-mediated second strand synthesis during infection. The two complementary halves join together to form a single double-stranded DNA that is ready for replication and transcription. (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 text is incorporated by reference) For self-complementary AAVs, see, e.g., U.S. Patent No. 6,449,693, which is incorporated by reference. Nos. 596,535; 7,125,717; and 7,456,68 No. 3, each of which is incorporated herein by reference in its entirety. For example, mutating the 5' ITR, for example by deleting the terminal resolution site, This can allow hairpin formation of

[0300] Capsids and Capsid Serotypes In some embodiments, the AAV particles of the invention are packaged into capsid structures. Alternatively, it may be a capsid-free A. For viral vector donor and / or acceptor sequences such as AV, see, e.g., U.S. Patent Application Publication No. 2014 / 0107186 (the entire contents of which are incorporated by reference) (The text appears to be incomplete and should be omitted.)

[0301] In some embodiments, the AAV particles produced by the present invention are Enhanced transduction into specific cell types of interest, sustained transgene expression and / or safety Hybrid serotypes may include hybrid serotypes with transgenic and transgenic profiles. Capsidation, Bispecific Antibody Capsid Surface, Mosaic Capsid, and Texture may be produced by adsorption to the capsid and / or other capsid protein modifications. .

[0302] In some embodiments, the AAV particles of the invention are capsid-specific for specific therapeutic applications. Rational mutagenesis of proteins (e.g., Pulicherla et al., Mol Ther., 2011, 19:1070-1078), peptides to the capsid peptides derived from NMDA receptor agonists to enhance retrograde transport. uptake (Xu et al., Virology, 2005, 341:203-21 4) and novel AAV barriers through directed evolution, e.g., to increase CNS transduction. The gene may be further modified by the production of a nucleotide.

[0303] In some embodiments, the AAV particles produced by the present invention include, 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 variants thereof (e.g., A AAV3A and AAV3B), either naturally occurring and / or recombinant. One or more AAV capsid proteins useful in the present invention may comprise various capsid proteins. The nucleic acid sequence encoding the protein is described in WO 2015191508 ( (the contents of which are incorporated herein by reference in their entirety).

[0304] In some embodiments, the AAV particles of the invention comprise any natural or recombinant AAV serotype. According to the present invention, the AAV particles may comprise or be derived from the following: 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 serotypes, and The present invention may utilize or be based on a serotype selected from any of these variants. .

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

[0306] In some embodiments, the AAV particles of the present invention can be derived from, but are not limited to, AAV1 (US SEQ ID NOs: 6 and 64 in the specification of Japanese Patent Application Publication No. 20030138772), AAV2 (US SEQ ID NOs: 7 and 70 in the specification of Japanese Patent Application Publication No. 20030138772), AAV3( SEQ ID NOs: 8 and 71 of U.S. Patent Application Publication No. 20030138772), AAV4 (SEQ ID NO: 63 in U.S. Patent Application Publication No. 20030138772), AAV5 ( US Patent Application Publication No. 20030138772, SEQ ID NO: 114), AAV6 (US SEQ ID NO: 65 in Patent Application Publication No. 20030138772), AAV7 (U.S. Pat. SEQ ID NOS: 1 to 3 in the specification of published application No. 20030138772), AAV8 (U.S. Pat. No. SEQ ID NOS: 4 and 95 in the specification of published application No. 20030138772), AAV9 (U.S. Pat. SEQ ID NOs: 5 and 100 in the specification of published application No. 20030138772), AAV10 (US US Patent Application Publication No. 20030138772, SEQ ID NO: 117), AAV11 (US US Patent Application Publication No. 20030138772, SEQ ID NO: 118), AAV12 (US SEQ ID NO: 119 of Japanese Patent Application Publication No. 20030138772), AAVrhlO (Amino acids 1-7 of SEQ ID NO: 81 in U.S. Patent Application Publication No. 20030138772) 38), AAV16.3 (US Patent Application Publication No. 20030138772, Sequence No. No. 10), AAV29.3 / bb. l (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 11), AAV29.4 (US Patent Application Publication No. 20030138772 No. 12), AAV29.5 / bb.2 (U.S. Patent Application Publication No. 20030 No. 138772, SEQ ID NO: 13), AAV1.3 (U.S. Patent Application Publication No. 20030 No. 138772, SEQ ID NO: 14), AAV13.3 (U.S. Patent Application Publication No. 2003 No. 0138772, SEQ ID NO: 15), AAV24.1 (U.S. Patent Application Publication No. 200 No. 30138772, SEQ ID NO: 16), AAV27.3 (U.S. Patent Application Publication No. 20 No. 030138772, SEQ ID NO: 17), AAV7.2 (U.S. Patent Application Publication No. 20 AAVC1 (US Pat. App. Pub. No. 20030138772, SEQ ID NO: 18), No. 30138772, SEQ ID NO: 19), AAVC3 (U.S. Patent Application Publication No. 2003 AAVC5 (US Patent Application Publication No. 2003 ... 138772, SEQ ID NO: 21), AAVF1 (U.S. Patent Application Publication No. 200301 38772, SEQ ID NO: 22), AAVF3 (U.S. Patent Application Publication No. 2003013 8772, SEQ ID NO: 23), AAVF5 (U.S. Patent Application Publication No. 20030138 772, SEQ ID NO: 24), AAVH6 (U.S. Patent Application Publication No. 200301387 72, SEQ ID NO: 25), AAVH2 (U.S. Patent Application Publication No. 2003013877 No. 2, SEQ ID NO: 26), AAV42-8 (U.S. Patent Application Publication No. 200301387 72, SEQ ID NO: 27), AAV42-15 (U.S. Patent Application Publication No. 2003013 8772, SEQ ID NO: 28), AAV42-5b (U.S. Patent Application Publication No. 20030 No. 138772, SEQ ID NO: 29), AAV42-lb (U.S. Patent Application Publication No. 200 No. 30138772, SEQ ID NO: 30), AAV42-13 (U.S. Patent Application Publication No. 2002 / 0022994), No. 0030138772, SEQ ID NO: 31), AAV42-3a (U.S. Patent Application Publication No. No. 20030138772, SEQ ID NO: 32), AAV42-4 (U.S. Patent Application Publication No. No. 20030138772, SEQ ID NO: 33), AAV42-5a (U.S. Pat. No. Patent Publication No. 20030138772, SEQ ID NO: 34), AAV42-10 (U.S. Patent No. Patent Application Publication No. 20030138772, SEQ ID NO: 35), AAV42-3b (US Japanese Patent Application Publication No. 20030138772 (SEQ ID NO: 36), AAV42-11 (US Patent Application Publication No. 20030138772, SEQ ID NO: 37), AAV42- 6b (US Patent Application Publication No. 20030138772, SEQ ID NO: 38), AAV4 3-1 (US Patent Application Publication No. 20030138772, SEQ ID NO: 39), AAV 43-5 (US Patent Application Publication No. 20030138772, SEQ ID NO: 40), AA V43-12 (US Patent Application Publication No. 20030138772, SEQ ID NO: 41), AAV43-20 (US Patent Application Publication No. 20030138772, SEQ ID NO: 42 ), AAV43-21 (US Patent Application Publication No. 20030138772, SEQ ID NO: 43), AAV43-23 (US Patent Application Publication No. 20030138772, sequence No. 44), AAV43-25 (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 45), AAV44.1 (US Patent Application Publication No. 20030138772 SEQ ID NO: 46), AAV44.5 (US Patent Application Publication No. 20030138772 SEQ ID NO: 47), AAV223.1 (U.S. Patent Application Publication No. 20030138772 SEQ ID NO: 48), AAV223.2 (U.S. Patent Application Publication No. 2003013877 No. 2, SEQ ID NO: 49), AAV223.4 (U.S. Patent Application Publication No. 20030138 772 SEQ ID NO: 50), AAV223.5 (U.S. Patent Application Publication No. 200301 38772, SEQ ID NO: 51), AAV223.6 (U.S. Patent Application Publication No. 2003 No. 0138772, SEQ ID NO: 52), AAV223.7 (U.S. Patent Application Publication No. 2003 / 0138772), No. 030138772, SEQ ID NO: 53), AAV A3.4 (U.S. Patent Application Publication No. No. 20030138772, SEQ ID NO: 54), AAV A3.5 (U.S. Patent Application Publication No. No. 20030138772, SEQ ID NO: 55), AAV A3.7 (U.S. Pat. No. Patent Publication No. 20030138772, SEQ ID NO: 56), AAV A3.3 (U.S. Patent Application Publication No. Patent Application Publication No. 20030138772, SEQ ID NO: 57), AAV42.12 (US Japanese Patent Application Publication No. 20030138772, SEQ ID NO: 58), AAV44.2( US Patent Application Publication No. 20030138772 (SEQ ID NO: 59), AAV42-2 (US Patent Application Publication No. 20030138772, SEQ ID NO: 9), or a version thereof Rianto et al., U.S. Patent Application Publication No. 20030138772 (the contents of which are incorporated herein by reference). or may be as described in It may comprise or be derived from any AAV serotype it may possess.

[0307] In some embodiments, the AAV particles of the present invention can be prepared using a variety of vectors, including, but not limited to, AAV2 (US SEQ ID NOs: 7 and 23 in the specification of Japanese Patent Application Publication No. 20150159173), rh20 (US SEQ ID NO: 1 in the specification of US Patent Application Publication No. 20150159173), rh32 / 33 (US SEQ ID NO: 2 in the specification of US Patent Application Publication No. 20150159173), rh39 (U.S. Patent SEQ ID NOs: 3, 20 and 36 in the specification of Patent Publication No. 20150159173), rh46( SEQ ID NOs: 4 and 22 of U.S. Patent Application Publication No. 20150159173, rh73 (SEQ ID NO: 5 in U.S. Patent Application Publication No. 20150159173), rh74 (U.S. SEQ ID NO: 6 in the specification of Patent Application Publication No. 20150159173), AAV6.1 (U.S. Patent Application Publication No. SEQ ID NO: 29 in U.S. Patent Application Publication No. 20150159173), rh.8 (U.S. Patent No. SEQ ID NO: 41 in the specification of Patent Publication No. 20150159173), rh.48.1 (U.S. Patent SEQ ID NO: 44 in U.S. Patent Publication No. 20150159173), hu.44 (U.S. Patent No. SEQ ID NO: 45 in the specification of Patent Publication No. 20150159173), hu.29 (U.S. Patent Application Publication No. SEQ ID NO: 42 in U.S. Patent Application Publication No. 20150159173), hu.48 (U.S. Patent Application Publication No. SEQ ID NO: 38 in U.S. Patent Application Publication No. 20150159173), rh54 (U.S. Patent Application Publication No. SEQ ID NO: 49 in U.S. Patent Application Publication No. 20150159173), AAV2 (U.S. Patent Application Publication No. 20150159173), SEQ ID NO: 7 of US Patent Application Publication No. 20150159173), cy.5 (US Patent Application Publication No. 20150159173), 159173), SEQ ID NOs: 8 and 24, rh.10 (U.S. Patent Application Publication No. 201 SEQ ID NOS: 9 and 25 of U.S. Pat. No. 5,015,9173), rh.13 (U.S. Pat. App. Pub. No. 2002 / 0109994), SEQ ID NOs: 10 and 26 of U.S. Pat. No. 5,917,373), AAV1 (U.S. Pat. App. Pub. No. 2001 / 0150159173), SEQ ID NOS: 11 and 27 of U.S. Patent Application Publication No. 20150159173), AAV3 (U.S. Patent Application Publication No. 20150159173), SEQ ID NOS: 12 and 28 of Publication No. 20150159173), AAV6 (U.S. Pat. SEQ ID NOs: 13 and 29 in the specification of Patent Publication No. 20150159173), AAV7 (U.S. SEQ ID NOs: 14 and 30 in the specification of Japanese Patent Application Publication No. 20150159173), AAV8( SEQ ID NOs: 15 and 31 of U.S. Patent Application Publication No. 20150159173), hu. 13 (SEQ ID NOs: 16 and 32 in U.S. Patent Application Publication No. 20150159173), hu.26 (SEQ ID NOs: 17 and 3 of U.S. Patent Application Publication No. 20150159173) 3), hu.37 (SEQ ID NO: 18 in U.S. Patent Application Publication No. 20150159173). and 34), hu.53 (sequence number of U.S. Patent Application Publication No. 20150159173) Nos. 19 and 35), rh.43 (U.S. Patent Application Publication No. 20150159173 SEQ ID NOs: 21 and 37), rh2 (US Patent Application Publication No. 20150159173 SEQ ID NO: 39), rh.37 (US Patent Application Publication No. 20150159173 SEQ ID NO: 40), rh.64 (sequence number of US Patent Application Publication No. 20150159173) Column number 43), rh.48 (array of U.S. Patent Application Publication No. 20150159173) No. 44), ch.5 (sequence number of U.S. Patent Application Publication No. 20150159173) 46), rh.67 (SEQ ID NO: 4 in U.S. Patent Application Publication No. 20150159173) 7), rh.58 (SEQ ID NO: 48 in U.S. Patent Application Publication No. 20150159173). ), or variants thereof, such as, 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 No. 20150159173, including U.S. Patent Application Publication No. 20150159173 (the contents of which are incorporated herein by reference) The sequences may be as described in the US Pat. No. 6,499,499, which is incorporated by reference in its entirety. The antigenic antigen may comprise or be derived from an AAV serotype which may have the antigenic antigen.

[0308] In some embodiments, the AAV particles of the present invention include, but are not limited to, AAV9 (US SEQ ID NOS: 1 to 3 in the specification of Japanese Patent No. 7198951), AAV2 (U.S. Patent No. 719895 No. 1, SEQ ID NO: 4), AAV1 (U.S. Pat. No. 7,198,951, SEQ ID NO: 5 ), AAV3 (SEQ ID NO: 6 in U.S. Pat. No. 7,198,951), and AAV8 (U.S. Pat. No. SEQ ID NO: 7 of Patent No. 7198951, etc., U.S. Patent No. 7198951 (the contents of which are incorporated herein by reference in their entirety) It may comprise or be derived from an AAV serotype which may be or may have.

[0309] In some embodiments, the AAV vector includes, 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. AAV9 serotypes that may be or have mutations in the AAV9 sequence as expressed In some embodiments, the AAV capsid comprises or is derived from a blood-brain type. It contains one or more sequences engineered to deliver the vector across a barrier (e.g., B. E.Deverman et al,Nature Biotech,Vol.34,N o.2, p. 204-211 (online publication February 1, 2016) and Caltech press release, A. Wetherston, www.neurolog y-cenfrd.com / 2016 / 02 / 10 / successfd / brain- See barrier; see also WO 2016 / 0492301 brochure. See also U.S. Pat. Nos. 8,734,809 and 8,734,809, the contents of each of which are incorporated herein by reference. (Incorporated by reference in its entirety).

[0310] In some embodiments, the AAV particles of the invention include, but are not limited to, AAV3B ( SEQ ID NOs: 1 and 10 of US Patent No. 6156303), AAV6 (US Patent No. 615 SEQ ID NOS: 2, 7 and 11 of U.S. Patent No. 6,156,303), AAV2 (U.S. Patent No. 6,156,303), No. 3 and 8 of the specification), AAV3A (SEQ ID NO: 3 of the specification of U.S. Patent No. 6,156,303), Nos. 4 and 9), or derivatives thereof, etc., as described in U.S. Pat. No. 6,156,303 (the contents of which are incorporated herein by reference) (which is incorporated by reference herein in its entirety) It may comprise or be derived from any AAV serotype that may or may not possess.

[0311] In some embodiments, the AAV particles of the present invention include, but are not limited to, AAV8 (US SEQ ID NO: 1 in the specification of Patent Application Publication No. 20140359799), AAVDJ (U.S. Pat. SEQ ID NOs: 2 and 3 of Patent Publication No. 20140359799, or barriers thereof See, for example, U.S. Patent Application Publication No. 20140359799, the contents of which are incorporated herein by reference. The sequences may be as described in the entirety of the patent application Ser. No. 09 / 199,925, which is incorporated by reference in its entirety. The AAV serotype may comprise or be derived from an AAV serotype which may have

[0312] In some embodiments, the AAV particles are prepared as described by Grimm et al. (Journal of Virology 82(12):5887-5911(2008), herein (which is incorporated by reference in its entirety) including, but not limited to: Serotypes such as AAVDJ or variants thereof such as AAVDJ8 (or AAV-DJ8) The amino acid sequence of AAVDJ8 may include a capsid from The mutation may include two or more mutations to remove the nucleotide sequence (SEQ ID NO: 1). ,588,772, the contents of which are incorporated herein by reference in their entirety. The AAV-DJ sequence set forth as SEQ ID NO: 1 in R587Q, in which arginine (R; Arg) is changed to glutamine (Q; Gln), and (2) The arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, the mutation may include three mutations: (1) R590T, which is present at amino acid 4 K406R, in which lysine (K; Lys) at position 6 is changed to arginine (R; Arg), 2) The amino acid 587, arginine (R; Arg), is changed to glutamine (Q; Gln) (3) R587Q, which contains arginine (R; Arg) at amino acid 590, and threonine (T ;Thr).

[0313] In some embodiments, the AAV particles of the invention include, but are not limited to, AAV4 (International Sequence numbers 1 to 20 in the pamphlet of International Publication No. 1998011244 are also included. No. 98011244, the contents of which are incorporated herein by reference in their entirety. AA which may be or have the sequence of AAV4 as described in It may comprise or be derived from the V serotype.

[0314] In some embodiments, the AAV particles of the invention are those described in WO2014144229. AAV2G as described in the pamphlet and incorporated herein by reference in its entirety. 9. It may comprise or be derived from an AAV serotype.

[0315] In some embodiments, the AAV particles of the present invention include, but are not limited to, AAV3-3( SEQ ID NO: 217 in the pamphlet of International Publication No. 2005033321), AAV1 (International Publication No. SEQ ID NOs: 219 and 202 in the pamphlet of Patent Publication No. 2005033321), AAV106 .1 / hu.37 (SEQ ID NO: 10 in WO 2005033321), Sequence of AAV114.3 / hu.40 (International Publication No. 2005033321) No. 11), AAV127.2 / hu.41 (International Publication No. 2005033321 Pamphlet SEQ ID NOs: 6 and 8 of AAV128.3 / hu.44 (WO 200503 SEQ ID NO: 81 in International Publication No. 3321), AAV130.4 / hu.48 (International Publication No. Sequence number 78 in the pamphlet of No. 2005033321), AAV145.1 / hu.53 (SEQ ID NOs: 176 and 177 in WO 2005033321), AA V145.6 / hu.56 (sequence number of International Publication No. 2005033321) 168 and 192), AAV16.12 / hu.11 (International Publication No. 2005033321 SEQ ID NOs: 153 and 57 in the International Publication No. SEQ ID NOs: 156 and 56 in the pamphlet of No. 2005033321), AAV161.10 / hu.60 (SEQ ID NO: 170 in WO 2005033321), A AV161.6 / hu.61 (International Publication No. 2005033321 Pamphlet Sequence Number No. 174), AAV1-7 / rh.48 (International Publication No. 2005033321 Brochure SEQ ID NO: 32), AAV1-8 / rh.49 (WO 2005033321 SEQ ID NOs: 103 and 25 of the pamphlet), AAV2 (WO 2005033321 SEQ ID NOs: 211 and 221 in the pamphlet), AAV2-15 / rh.62 (International Publication No. SEQ ID NOs: 33 and 114 in the pamphlet of No. 2005033321), AAV2-3 / rh .61 (SEQ ID NO: 21 in WO 2005033321), AAV2- 4 / rh.50 (SEQ ID NO: 23 and 1 in the pamphlet of International Publication No. 2005033321) 08), AAV2-5 / rh.51 (International Publication No. 2005033321 brochure) SEQ ID NOs: 104 and 22), AAV3.1 / hu.6 (International Publication No. 2005033321 SEQ ID NOs: 5 and 84 in International Publication No. 2005 SEQ ID NOs: 155 and 58 in the pamphlet of No. 033321), AAV3-l l / rh.5 3 (SEQ ID NOs: 186 and 176 in the pamphlet of International Publication No. 2005033321), A AV3-3 (SEQ ID NO: 200 in the pamphlet of International Publication No. 2005033321), AA V33.12 / hu.17 (SEQ ID NO. of International Publication No. 2005033321) 4), AAV33.4 / hu.15 (International Publication No. 2005033321 Brochure SEQ ID NO: 50), AAV33.8 / hu.16 (WO 2005033321 B1 FRET sequence number 51), AAV3-9 / rh.52 (International Publication No. 200503332 SEQ ID NOs: 96 and 18 in International Publication No. 1), AAV4-19 / rh.55 (International Publication No. SEQ ID NO: 117 in the pamphlet of International Publication No. 2005033321), AAV4-4 (International Publication No. SEQ ID NOs: 201 and 218 in the pamphlet of No. 005033321), AAV4-9 / rh .54 (SEQ ID NO: 116 in WO 2005033321), AAV5 (SEQ ID NOs: 199 and 216 in WO 2005033321), AA V52.1 / hu.20 (SEQ ID NO: 6 in the pamphlet of International Publication No. 2005033321) 3), AAV52 / hu. l9 (International Publication No. 2005033321 Brochure Distribution Row No. 133), AAV5-22 / rh.58 (International Publication No. 2005033321 Pan FRET sequence number 27), AAV5-3 / rh.57 (International Publication No. 200503332 SEQ ID NO: 105 in the pamphlet of International Publication No. 2005), AAV5-3 / rh.57 (International Publication No. SEQ ID NO: 26 in the International Publication No. 033321 pamphlet), AAV58.2 / hu.25 (International Publication No. SEQ ID NO: 49 in International Publication No. 2005033321), AAV6 (WO 200 SEQ ID NOs: 203 and 220 in International Publication No. 5033321), AAV7 (International Publication No. SEQ ID NOs: 222 and 213 in the pamphlet of No. 005033321), AAV7.3 / hu .7 (SEQ ID NO: 55 in WO 2005033321), AAV8 ( SEQ ID NOs: 223 and 214 in the pamphlet of International Publication No. 2005033321), AAVH -l / hu.1 (SEQ ID NO: 46 in WO 2005033321), A AVH-5 / hu.3 (SEQ ID NO: 44 in the pamphlet of International Publication No. 2005033321) ), AAVhu.1 (SEQ ID NO: 144 in the pamphlet of International Publication No. 2005033321) ), AAVhu.10 (SEQ ID NO: 15 in the pamphlet of International Publication No. 2005033321) 6), AAVhu.11 (SEQ ID NO: 1 in the pamphlet of International Publication No. 2005033321) 53), AAVhu.12 (International Publication No. 2005033321, SEQ ID NO: 5 9), AAVhu.13 (SEQ ID NO: 1 in the pamphlet of International Publication No. 2005033321) 29), AAVhu.14 / AAV9 (International Publication No. 2005033321 Brochure SEQ ID NOs: 123 and 3), AAVhu.15 (WO 2005033321 B1 FRET sequence number 147), AAVhu.16 (International Publication No. 2005033321 SEQ ID NO: 148 of the pamphlet), AAVhu.17 (WO 2005033321 SEQ ID NO: 83 in the pamphlet), AAVhu.18 (WO 2005033321 pamphlet), AAVhu.19 (International Publication No. 2005033321 No. 133 in the pamphlet of International Publication No. 2005033321 No. 143 in the pamphlet of the International Publication No. 200503332 SEQ ID NO: 134 in the pamphlet of International Publication No. 20050333 SEQ ID NO: 135 in the pamphlet of International Publication No. 2005033 SEQ ID NO: 138 in International Publication No. 321), AAVhu.23.2 (International Publication No. 2005 SEQ ID NO: 137 in International Publication No. 033321), AAVhu.24 (International Publication No. 200 SEQ ID NO: 136 in the pamphlet of International Publication No. 5033321), AAVhu.25 (International Publication No. 20 SEQ ID NO: 146 in the pamphlet of International Publication No. 05033321), AAVhu.27 (International Publication No. SEQ ID NO: 140 in the pamphlet of International Publication No. 005033321), AAVhu.29 (International Publication No. SEQ ID NO: 132 in the pamphlet of International Publication No. 2005033321), AAVhu.3 (International Publication No. Sequence number 145 in the pamphlet of International Publication No. 2005033321), AAVhu.31 (International Publication No. No. 2005033321 pamphlet, sequence number 121), AAVhu.32 (International Publication 2005033321 pamphlet sequence number 122), AAVhu.34 (International Publication No. 2005033321 pamphlet sequence number 125), AAVhu.35 (country SEQ ID NO: 164 in the pamphlet of International Publication No. 2005033321), AAVhu.37( SEQ ID NO: 88 in the pamphlet of International Publication No. 2005033321), AAVhu.39( Sequence number 102 in the pamphlet of International Publication No. 2005033321), AAVhu.4( Sequence number 141 in the pamphlet of International Publication No. 2005033321), AAVhu.40 (SEQ ID NO: 87 in WO 2005033321), AAVhu.41 (SEQ ID NO: 91 in WO 2005033321), AAVhu.42 (SEQ ID NO: 85 in WO 2005033321), AAVhu.43 (SEQ ID NO: 160 in WO 2005033321), AAVhu.4 4 (SEQ ID NO: 144 in WO 2005033321), AAVhu. 45 (SEQ ID NO: 127 in WO 2005033321), AAVhu .46 (SEQ ID NO: 159 in WO 2005033321), AAVh u.47 (SEQ ID NO: 128 in WO 2005033321), AAV hu.48 (SEQ ID NO: 157 in WO 2005033321), AA Vhu.49 (SEQ ID NO: 189 in WO 2005033321), A AVhu.51 (SEQ ID NO: 190 in WO 2005033321), AAVhu.52 (SEQ ID NO: 191 in WO 2005033321) , AAVhu.53 (SEQ ID NO: 186 in the pamphlet of International Publication No. 2005033321) ), AAVhu.54 (SEQ ID NO: 18 in the pamphlet of International Publication No. 2005033321) 8), AAVhu.55 (SEQ ID NO: 1 in the pamphlet of International Publication No. 2005033321) 87), AAVhu.56 (SEQ ID NO: 1 in the pamphlet of International Publication No. 2005033321) 192), AAVhu.57 (sequence number in the pamphlet of International Publication No. 2005033321) No. 193), AAVhu.58 (International Publication No. 2005033321 Brochure Sequence No. 194), AAVhu.6 (International Publication No. 2005033321 Pamphlet Sequence No. 84), AAVhu.60 (International Publication No. 2005033321 Pamphlet Sequence No. 184), AAVhu.61 (International Publication No. 2005033321 Brochure Distribution Column number 185), AAVhu.63 (International Publication No. 2005033321 pamphlet) SEQ ID NO: 195), AAVhu.64 (International Publication No. 2005033321) SEQ ID NO: 196), AAVhu.66 (International Publication No. 2005033321, SEQ ID NO: 197), AAVhu.67 (International Publication No. WO 2005033321) AAVhu.7 (International Publication No. WO 2005033321, brochure SEQ ID NO: 150 of the AAVhu.8 (International Publication No. WO 2005033321) 12), AAVhu.9 (International Publication No. WO 2005033321) SEQ ID NO: 155), AAVLG-10 / rh.40 (International Publication No. 2005033321 SEQ ID NO: 14 in the pamphlet of International Publication No. 20050 SEQ ID NO: 86 in the pamphlet of International Publication No. 33321), AAVLG-4 / rh.38 (International Publication No. Sequence number 7 in the pamphlet of No. 2005033321), AAVN721-8 / rh.43 (SEQ ID NO: 163 in the pamphlet of International Publication No. 2005033321), AAVN721 -8 / rh.43 (SEQ ID NO: 43 in WO 2005033321), AAVpi. l (International Publication No. 2005033321, SEQ ID NO: 28), A AVpi.2 (International Publication No. 2005033321, SEQ ID NO: 30), AAV pi.3 (SEQ ID NO: 29 in the pamphlet of International Publication No. 2005033321), AAVrh .38 (SEQ ID NO: 86 in WO 2005033321), AAVrh .40 (SEQ ID NO: 92 in WO 2005033321), AAVrh .43 (SEQ ID NO: 163 in WO 2005033321), AAVr h.44 (International Publication No. 2005033321, SEQ ID NO: 34), AAVrh .45 (International Publication No. 2005033321, SEQ ID NO: 41), AAVrh. 47 (International Publication No. 2005033321, SEQ ID NO: 38), AAVrh.4 8 (SEQ ID NO: 115 in WO 2005033321), AAVrh. 49 (SEQ ID NO: 103 in WO 2005033321), AAVrh .50 (SEQ ID NO: 108 in WO 2005033321), AAVr h.51 (SEQ ID NO: 104 in WO 2005033321), AAV rh.52 (SEQ ID NO: 96 in WO 2005033321), AAV r h.53 (SEQ ID NO: 97 in WO 2005033321), AAVr h.55 (International Publication No. 2005033321, SEQ ID NO: 37), AAVrh .56 (SEQ ID NO: 152 in WO 2005033321), AAVr h.57 (SEQ ID NO: 105 in WO 2005033321), AAV rh.58 (SEQ ID NO: 106 in WO 2005033321), AA Vrh.59 (International Publication No. 2005033321, SEQ ID NO: 42), AAV rh.60 (International Publication No. 2005033321, SEQ ID NO: 31), AAVr h.61 (SEQ ID NO: 107 in WO 2005033321), AAV rh.62 (SEQ ID NO: 114 in WO 2005033321), AA Vrh.64 (SEQ ID NO: 99 in WO 2005033321), AA Vrh.65 (International Publication No. 2005033321, SEQ ID NO: 35), AAV rh.68 (International Publication No. 2005033321, SEQ ID NO: 16), AAVr h.69 (International Publication No. 2005033321, SEQ ID NO: 39), AAVrh .70 (International Publication No. 2005033321, SEQ ID NO: 20), AAVrh. 72 (International Publication No. 2005033321, SEQ ID NO: 9), or variations thereof Ant, but 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 h.36, AAVrh.37, and AAVrhl4, International Publication No. 200503332 No. 1, the contents of which are incorporated herein by reference in their entirety. and / or AAV serotypes which may be or have the sequences as described in Non-limiting examples of variants include those derived from WO 2005033321. SEQ ID NO: 1 of the pamphlet, the contents of which are incorporated herein by reference in their entirety. 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 19, 224-236.

[0316] In some embodiments, the AAV particles of the invention include, but are not limited to, AAVrh8R (SEQ ID NO: 9 in WO 2015168666), AAVrh8R A 586R mutant (SEQ ID NO: 10 in WO 2015168666), A AVrh8R R533A mutant (discussion in the pamphlet of International Publication No. 2015168666) Column No. 11), or variants thereof, etc., WO 2015168666 As described in Lett., the contents of which are incorporated herein by reference in their entirety. and may comprise or be derived from an AAV serotype which may be or have the sequence of do.

[0317] In some embodiments, the AAV particles of the invention include, but are not limited to, AAVhu68 (e.g., SEQ ID NO: 2 in WO 2018160582), or its variants Rianto et al., International Publication No. 2018 / 160582 (the contents of which are incorporated herein by reference) The sequences may be as described in the US Pat. No. 6,499,499, which is incorporated by reference in its entirety. The antigenic antigen may comprise or be derived from an AAV serotype which may have the antigenic antigen.

[0318] In some embodiments, the AAV particles of the invention include, but are not limited to, AAVhE1. 1 (SEQ ID NO: 44 in U.S. Pat. No. 9,233,131), AAVhErl.5 (U.S. Pat. No. 9,233,131), No. 9233131), SEQ ID NO: 45, AAVhER1.14 (U.S. Pat. No. 9233131), SEQ ID NO: 46 of U.S. Patent No. 33131), AAVhErl.8 (U.S. Patent No. 9,233,131 No. 47), AAVhEr1.16 (U.S. Pat. No. 9,233,131 No. 48 of U.S. Pat. No. 9,233,131), AAVhEr1.18 (SEQ ID NO: 48 of U.S. Pat. No. 9,233,131), No. 49), AAVhEr1.35 (SEQ ID NO: 50 in U.S. Pat. No. 9,233,131) , AAVhEr1.7 (SEQ ID NO: 51 in U.S. Pat. No. 9,233,131), AAVh Er1.36 (SEQ ID NO: 52 in U.S. Patent No. 9,233,131), AAVhEr2. 29 (SEQ ID NO: 53 in U.S. Pat. No. 9,233,131), AAVhEr2.4 (U.S. Pat. No. 9,233,131), SEQ ID NO: 54 in the specification of Japanese Patent No. 9233131), AAVhEr2.16 (U.S. Patent No. 9233131), SEQ ID NO: 55 of U.S. Patent No. 233131), AAVhEr2.30 (U.S. Patent No. 92331 31), AAVhEr2.31 (U.S. Pat. No. 9,233,131), No. 58 in the specification), AAVhEr2.36 (sequence number 58 in the specification of U.S. Pat. No. 9,233,131), row number 57), AAVhER1.23 (SEQ ID NO: 5 in U.S. Pat. No. 9,233,131), 3), AAVhEr3.1 (SEQ ID NO: 59 in U.S. Pat. No. 9,233,131), AA V2.5T (SEQ ID NO: 42 in U.S. Pat. No. 9,233,131), or a barrier thereof No. 9,233,131, the contents of which are incorporated herein by reference in their entirety. A may be or have the sequence as described in It may comprise or be derived from an AV serotype.

[0319] In some embodiments, the AAV particles of the invention include, but are not limited to, AAV-PAE C (SEQ ID NO: 1 in U.S. Patent Application Publication No. 20150376607), AAV-LK Ol (SEQ ID NO: 2 in U.S. Patent Application Publication No. 20150376607), AAV-L K02 (SEQ ID NO: 3 in U.S. Patent Application Publication No. 20150376607), AAV- LK03 (SEQ ID NO: 4 in U.S. Patent Application Publication No. 20150376607), AAV -LK04 (SEQ ID NO: 5 in U.S. Patent Application Publication No. 20150376607), AA V-LK05 (SEQ ID NO: 6 in U.S. Patent Application Publication No. 20150376607), A AV-LK06 (SEQ ID NO: 7 of U.S. Patent Application Publication No. 20150376607); AAV-LK07 (SEQ ID NO: 8 of U.S. Patent Application Publication No. 20150376607) , AAV-LK08 (SEQ ID NO: 9 of U.S. Patent Application Publication No. 20150376607) ), AAV-LK09 (SEQ ID NO: 1 in U.S. Patent Application Publication No. 20150376607) 10), AAV-LK10 (sequence of U.S. Patent Application Publication No. 20150376607) No. 1), AAV-LK11 (sequence number of U.S. Patent Application Publication No. 20150376607) Row No. 12), AAV-LK12 (US Patent Application Publication No. 20150376607 SEQ ID NO: 13), AAV-LK13 (US Patent Application Publication No. 20150376607) SEQ ID NO: 14 of the specification), AAV-LK14 (U.S. Patent Application Publication No. 20150376607 No. 201503766), AAV-LK15 (US Patent Application Publication No. 201503766 07), AAV-LK16 (U.S. Patent Application Publication No. 2015037 SEQ ID NO: 17 of U.S. Patent Application Publication No. 20150 SEQ ID NO: 18 of U.S. Patent Application Publication No. 376607), AAV-LK18 (U.S. Patent Application Publication No. 201 SEQ ID NO: 19 of U.S. Patent Application Publication No. 50376607), AAV-LK19 (U.S. Patent Application Publication No. 2002 / 0022994), No. 0150376607), AAV-PAEC2 (U.S. Pat. App. Pub. No. 2001002434), 21 in the specification of Patent Publication No. 20150376607), AAV-PAEC4 (U.S. Patent No. SEQ ID NO: 22 in the specification of published application No. 20150376607), AAV-PAEC6 (US SEQ ID NO: 23 in the specification of Japanese Patent Application Publication No. 20150376607), AAV-PAEC 7 (SEQ ID NO: 24 in U.S. Patent Application Publication No. 20150376607), AAV-P AEC8 (SEQ ID NO: 25 in U.S. Patent Application Publication No. 20150376607), AA V-PAEC11 (SEQ ID NO: 26 in U.S. Patent Application Publication No. 20150376607) ), AAV-PAEC12 (sequence of U.S. Patent Application Publication No. 20150376607) No. 27), or variants thereof, such as U.S. Patent Application Publication No. 20150376607 No. 6,299,433, the contents of which are incorporated herein by reference in their entirety. and / or comprising or derived from an AAV serotype which may be or have a sequence similar to that of obtain.

[0320] In some embodiments, the AAV particles of the invention include, but are not limited to, AAV-2-particles. miRNA-101 (SEQ ID NO: 1, U.S. Patent No. 9,163,261), or its derivatives Rianto et al., U.S. Pat. No. 9,163,261, the contents of which are incorporated herein in their entirety. The nucleic acid sequence may be or may have the sequence as described in The antigen may comprise or be derived from any AAV serotype.

[0321] In some embodiments, the AAV particles of the present invention include, but are not limited to, AAV-8h ( SEQ ID NO: 6 in U.S. Patent Application Publication No. 20150376240), AAV-8b (U.S. US Patent Application Publication No. 20150376240, SEQ ID NO: 5), AAV-h (US Patent Application Publication No. SEQ ID NO: 2 in U.S. Patent Application Publication No. 20150376240), AAV-b (U.S. Patent No. SEQ ID NO: 1 in the specification of Patent Publication No. 20150376240), or variants thereof, etc. and U.S. Patent Application Publication No. 20150376240, the entire contents of which are incorporated herein by reference. and the sequences may be or have the sequences as described in The vector may comprise or be derived from any suitable AAV serotype.

[0322] In some embodiments, the AAV particles of the invention include, but are not limited to, AAV SM. 10-2 (SEQ ID NO: 22 in U.S. Patent Application Publication No. 20160017295), AA V Shuffle 100-1 (U.S. Patent Application Publication No. 20160017295) SEQ ID NO: 23), AAV Shuffle 100-3 (U.S. Patent Application Publication No. 2016 SEQ ID NO: 24 of the specification of US Pat. No. 0017295), AAV Shuffle 100-7 (US Pat. No. 0017295), SEQ ID NO: 25 in Patent Application Publication No. 20160017295), AAV Shuff le 10-2 (SEQ ID NO: 34 of U.S. Patent Application Publication No. 20160017295) , AAV Shuffle 10-6 (U.S. Patent Application Publication No. 20160017295) SEQ ID NO: 35 in the specification), AAV Shuffle 10-8 (U.S. Patent Application Publication No. 201 SEQ ID NO: 36 in US Pat. No. 6,001,7295), AAV Shuffle 100-2 (US Pat. No. 6,001,7295), SEQ ID NO: 37 in the specification of Japanese Patent Application Publication No. 20160017295), AAV SM 1 0-1 (SEQ ID NO: 38 in U.S. Patent Application Publication No. 20160017295), AAV SM 10-8 (SEQ ID NO: 39 in U.S. Patent Application Publication No. 20160017295) ), AAV SM 100-3 (U.S. Patent Application Publication No. 20160017295) SEQ ID NO: 40), AAV SM 100-10 (U.S. Patent Application Publication No. 201600172 95), or variants thereof, No. 160017295, the contents of which are incorporated herein by reference in their entirety. ) and AAV serotypes which may be or have sequences as described in , or may be derived therefrom.

[0323] In some embodiments, the AAV particles of the invention include, but are not limited to, BNP61 A AV (SEQ ID NO: 1 in U.S. Patent Application Publication No. 20150238550), BNP62 AAV (SEQ ID NO: 3 in U.S. Patent Application Publication No. 20150238550), BNP 63 AAV (SEQ ID NO: 4 in U.S. Patent Application Publication No. 20150238550), and These variants and others are described in U.S. Patent Application Publication No. 20150238550 (the The sequences were as described in (the contents of which are incorporated herein by reference in their entirety). It may comprise or be derived from an AAV serotype which may or may have.

[0324] In some embodiments, the AAV particles of the invention include, but are not limited to, AAVrh.5 0 (SEQ ID NO: 108 in U.S. Patent Application Publication No. 20150315612), AAVr h.43 (SEQ ID NO: 163 in U.S. Patent Application Publication No. 20150315612), A AVrh.62 (SEQ ID NO: 114 in U.S. Patent Application Publication No. 20150315612) ), AAVrh.48 (SEQ ID NO: 1 in U.S. Patent Application Publication No. 20150315612) 115), AAVhu.19 (the arrangement of the specification of U.S. Patent Application Publication No. 20150315612) Column No. 133), AAVhu.ll (U.S. Patent Application Publication No. 20150315612 SEQ ID NO: 153 of the specification), AAVhu.53 (U.S. Patent Application Publication No. 2015031561 No. 186 of the specification of U.S. Patent Application Publication No. 2014 / 010999), AAV4-8 / rh.64 (U.S. Patent Application Publication No. 2014 / 0109999), No. 5,031,561, SEQ ID NO: 15), AAVLG-9 / hu.39 (U.S. Pat. No. 5,031,561, SEQ ID NO: 16), SEQ ID NO: 24 in the specification of Patent Publication No. 20150315612), AAV54.5 / hu.2 3 (SEQ ID NO: 60 in U.S. Patent Application Publication No. 20150315612), AAV54 .2 / hu.22 (SEQ ID NO: 67 of U.S. Patent Application Publication No. 20150315612) ), AAV54.7 / hu.24 (U.S. Patent Application Publication No. 20150315612 SEQ ID NO: 66), AAV54.1 / hu.21 (U.S. Patent Application Publication No. 20150315 SEQ ID NO: 65 of U.S. Patent Application Publication No. 612), AAV54.4R / hu.27 (U.S. Patent Application Publication No. SEQ ID NO: 64 of the specification of US Patent Application Publication No. 20150315612), AAV46.2 / hu.28 (US SEQ ID NO: 68 in Patent Application Publication No. 20150315612), AAV46.6 / h u.29 (SEQ ID NO: 69 in U.S. Patent Application Publication No. 20150315612), AA V128.1 / hu.43 (sequence of U.S. Patent Application Publication No. 20150315612) No. 80), or variants thereof, such as U.S. Patent Application Publication No. 20150315612 No. 6,299,433, the contents of which are incorporated herein by reference in their entirety. and / or comprising or derived from an AAV serotype which may be or have a sequence similar to that of obtain.

[0325] In some embodiments, the AAV particles of the present invention include, but are not limited to, wild-type AAV ( ttAAV) (SEQ ID NO: 2 in WO 2015 / 121501 pamphlet), "UP enn AAV10" (SEQ ID NO: 8 in WO 2015 / 121501 pamphlet) , "Japanese AAV10" (SEQ ID NO: 9 in WO 2015 / 121501 pamphlet ), or AAV serotypes such as variants thereof, as described in WO 2015 / 121501 pamphlet (the content of which is incorporated herein by reference in its entirety), may include or have an AAV serotype that is an array as described or may have it, or be derived therefrom .

[0326] According to the present invention, the AAV particles may include an AAV capsid serotype that can be selected from or derived from various species. In one embodiment, the AAV may be avian AAV (AAAV). The AAAV serotype may include, but is not limited to, AAAV (SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14 in US Patent No. 9,238,8 00 specification), or variants thereof, etc., as described in US Patent No. 9,238,800 specification (the content of which is incorporated herein by reference in its entirety), may be an array as described or may have it, or have it .

[0327] In one embodiment, the AAV particles may include an AAV capsid serotype that can be or derived from bovine AAV (BAAV). The BAAV serotype may include, but is not limited to, BAAV (SEQ ID NOs: 1 and 6 in US Patent No. 9,193,769 specification), or variants thereof, etc., as described in US Patent No. 9193769 specification (the content of which is incorporated herein by reference ​The BA may be or have a sequence as described in the BA. AV serotypes include, but are not limited to, BAAV (sequence number 1 ... No. 7,427,396 (including U.S. Pat. Nos. 5 and 6), or variants thereof. the sequences of which are incorporated herein by reference in their entirety. It may also have.

[0328] In one embodiment, the AAV particles may be or may be derived from caprine AAV. Caprine AAV serotypes may include, but are not limited to, caprine AAV (U.S. Pat. No. 6,423,199). No. 7,427,396, or variants thereof, such as U.S. Pat. No. 27396, the contents of which are incorporated herein by reference in their entirety. It may be or have the sequence as shown.

[0329] In other embodiments, the AAV particles are hybrid AAVs from two or more parent serotypes. In one embodiment, the AAV may comprise an AAV capsid serotype that may be engineered to: AAV2G9 may also be used, which contains sequences from AAV2 and AAV9. AV serotypes are described in U.S. Patent Application Publication No. 20160017005, the contents of which are incorporated herein by reference. (the entire document is incorporated by reference), or may have it.

[0330] In one embodiment, the AAV particles are prepared as described by Pulicherla et al. lar Therapy 19(6):1070-1078(2011) (This content is a book 39 amino acids as described by Generated by an AAV9 capsid library containing mutations in 0 to 627 (VP1 numbering) The serotypes and corresponding nucleotide and The amino acid substitutions include, but are not limited to, AAV9.1 (G1594C;D532H), AAV9.2 (G1594C;D532H), AAV9.3 (G1594C;D532H), AAV9.4 (G1594C;D532H), AAV9.5 (G1594C;D532H), AAV9.6 (G1594C;D532H), 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).

[0331] In one embodiment, the AAV particles of the invention have increased tropism for the brain, as described in WO 2014 No. 160092, the contents of which are incorporated herein by reference in their entirety. It may comprise capsid proteins having the sequences of sequence numbers 1 and 3.

[0332] In one embodiment, the AAV particles of the invention target oligodendrocytes in the central nervous system. The capsid protein may comprise a capsid protein capable of producing the desired nucleotide sequence. 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 to 4 It may comprise a capsid protein.

[0333] In one embodiment, the AAV particles of the invention are those described in U.S. Pat. No. 8,927,514 ( the contents of which are incorporated herein by reference in their entirety) The capsid protein may have an increased ability to cross the blood-brain barrier. The amino acid and nucleic acid sequences of proteins include, but are not limited to, those described in U.S. Pat. No. 8,922,929. Examples of the compounds of the present invention include SEQ ID NOs: 2 to 17 and 25 to 33 in the specification of No. 7,514. can.

[0334] In some embodiments, the AAV particles of the invention comprise AAV2 capsid proteins or their equivalents. AAV particles containing AAV2 capsid proteins may deliver genes to the brain, It has been shown to effectively deliver AA to neurons in the retina and spinal cord. The V2 capsid protein is described in U.S. Pat. Nos. 6,691,948 and 8,299 ,215 (the contents of each of which are incorporated herein by reference in their entirety). As disclosed in the paper, a targeting peptide that targets AAV particles to the endothelium of cerebral blood vessels is Such AAV particles may be further modified, for example, by adding a nucleotide sequence to the capsid protein. The antibody may be a functional hTREM2 antibody or antigen-binding fragment thereof, e.g., as described herein. It may be used to deliver the fragments.

[0335] In some embodiments, the AAV particles of the invention comprise AAV5 capsid proteins or their equivalents. AAV particles containing the AAV5 capsid protein are expressed in the cortex, hippocampus ( Various regions of the CNS, including the HPC, cerebellum, substantia nigra (SN), striatum, globus pallidus, and spinal cord Neurons in the nucleus 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). In this state, AAV-5 containing the AAV-5 capsid protein increased transduction of cells in the CNS. AV particles are described in U.S. Pat. No. 7,056,502, the entire contents of which are incorporated herein by reference. The particles may be from the group consisting of cellulose, cellulose acetate, cellulose nitrite, cellulose nitrite, cellulose nitrite, cellulose acetate, cellulose acetate nitrite ...

[0336] In some embodiments, the AAV particles of the invention comprise AAV6 capsid proteins or their equivalents. Recombinant AAV6 serotypes can be administered intracerebroventricularly (ICV) into the spinal cord. It is possible to target motor neurons (Dirren E et al., Hum Gene Ther.,2014,25(2):109-120). In addition, San S According to a study by Ebastian et al., AAV6 serotype terminates from neuronal cell bodies in the rat brain. It has been shown that the endothelial cells can be retrogradely transported to the endothelium (San Sebastian et al., en Ther.,2014,20(12):1178-1183).

[0337] In some embodiments, the AAV particles of the invention comprise AAV8 capsid proteins or their equivalents. AAV particles containing AAV8 capsid proteins may be used to express AAV in the hippocampus, for example. Neurons can be transduced (Klein RL et al, Mol Th er., 2006, 13(3):517-527). In one embodiment, the AAV8 capsid The protein may be prepared as described in U.S. Pat. No. 8,318,480, the contents of which are incorporated herein in their entirety. The amino acid sequence of SEQ ID NO:2 of the nucleotide sequence of SEQ ID NO:1 of the nucleotide ...2 of the nucleotide sequence of SEQ

[0338] In some embodiments, the AAV particles of the invention comprise AAV9 capsid proteins or their equivalents. AAV9 capsid serotype-mediated gene delivery in the brain may include variants. Efficient and long-term expression of the transgene has been observed after intraparenchymal injection into the CNS (Kle in RL et al,Eur J Neurosci.,2008,27:1615 AAV9 serotypes have been shown to be effective in neonatal subjects after peripheral and systemic (e.g., intravenous) administration. This subsequently resulted in robust and widespread neuronal transduction throughout the CNS. (Foust KD et al, Nat. Biotechnol, 2009, 27 :59-65; and Duque S et al, Mol Ther., 2009, 17 Intrathecal (intracisternal) administration of AAV9 serotypes has also been widely used. In one embodiment, the AAV9 serotype can be used in a variety of ways, including the use of a recombinant AAV9 vector, as described in U.S. Pat. No. 7,198,951, the contents of which are incorporated herein by reference in their entirety. In another embodiment, the AAV capsid protein may comprise the amino acid sequence of SEQ ID NO:2. The AAV9 serotype is described in U.S. Patent Application Publication No. 20130224836, the contents of which are incorporated herein by reference. the surface in the amino acid sequence as disclosed in a sequence in which at least one of the exposed tyrosine residues is replaced by another amino acid residue; In embodiments, the VP1 capsid protein may be of sequence number 2, 4, or 6. The AAV vector containing the plasmid is administered systemically, for example, intravenously. AAV vectors containing AV9 capsids can be delivered to the brain, spine, or cerebrospinal fluid (CSF), e.g., the intrathecal cavity. It is administered internally.

[0339] In some embodiments, the AAV vector is engineered to achieve specific properties. Such methods for obtaining engineered capsids include the use of modified variant capsid proteins. For details of such a method, see, for example, International Publication No. 2011038187 (the contents of which are (The entire contents of which are incorporated herein by reference.) For the purpose of the present invention, see, for example, WO 2012112832 and WO 2012112832. Publication No. 2015054653 (the entire contents of which are incorporated herein by reference). (incorporated by reference in the entirety of this application). Such variant capsids include, for example, SEQ ID NO: 23 of WO 2015054653, or a variant thereof Further variant capsids include, for example, those described in WO 2017 / 01 Capsules described in Brochure No. 9994, the contents of which are incorporated by reference in their entirety. In embodiments, the AAV vector includes an Anc80 AAV capsid sequence. Capsid of the sequence (e.g., SEQ ID NO: 1 in WO 2017019994) , for example, Anc80L65 (see, for example, International Publication No. 2017019994 pamphlet) SEQ ID NO: 23), or, for example, Anc110 (e.g., WO 20170199 (SEQ ID NO: 42 in pamphlet No. 94).

[0340] In some embodiments, the AAV particles of the invention comprise an AAV rh10 capsid protein or AAV particles containing the AAVrh10 capsid protein may include variants thereof. Neurons in the spinal cord as well as other cells can be targeted after intrathecal (IT) administration. In one embodiment, the AAVrh10 capsid protein is the AAVrh10 capsid protein described in EP 2341068. In some embodiments, the amino acid sequence may comprise the amino acid sequence of SEQ ID NO: 81 of the specification. The AAV of the present invention comprises an AAVDJ capsid protein, an AAVDJ / 8 capsid protein, Holehonnur et al. demonstrated that the cytochrome P4500 can target neurons within the basolateral amygdala (BLA) (Hole honnur R et al.,BMC Neurosci,2014,Feb 18 :15:28). In one embodiment, the AAVDJ capsid protein and / or the AAVDJ The / 8 capsid protein is derived from a first AAV serotype (e.g., AAV2). a second region derived from a second AAV serotype (e.g., AAV8), and a third AA and a third region derived from a V serotype (e.g., AAV9). wherein the first, second, and third regions comprise any amino acid sequence disclosed herein. It can be seen.

[0341] In one embodiment, the AAV particles of the invention are capable of transducing dorsal root ganglia (DRG). It may include capsid proteins as shown or as known.

[0342] In one embodiment, the AAV particles of the invention have been shown to transduce motor neurons. The capsid protein may comprise any capsid protein that is known or has been identified.

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

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

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

[0346] In one embodiment, the serotypes of AAV particles described herein are capable of achieving the desired distribution, transduction, and This may depend on the efficiency and cell targeting required. 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; the contents of which are incorporated herein by reference in their entirety. As described by et al., which is incorporated by reference herein, AAV serotypes have different distribution, transduction, and To achieve the desired efficacy, the targeted The AAV serotype best suited not only to the target cells but also to the desired transduction efficiency and distribution is chosen. You need to choose.

[0347] In one embodiment, the AAV vector comprises an AAV9 capsid (as described herein). (1) ITRs from AAV2; (2) a CMV enhancer (e.g., SEQ ID NO: No. 134), (3) CBA promoter (e.g., SEQ ID NO: 135), (4) SV40 (5) an hTREM2 antibody or an antigen-binding fragment thereof (e.g., SEQ ID NO: 137); (6) a polynucleotide encoding B, e.g., as described herein; and an AAV vector plasmid containing a GH polyA signal (e.g., SEQ ID NO: 138). In one embodiment, elements (2) to (6) are located 5' on the AAV vector plasmid. In one embodiment, elements (2) to (6) are located 3' from the AAV vector promoter. In one embodiment, the AAV vector is placed between the 5' ITR and the 3' ITR on the smid. is an scAAV vector.

[0348] Monoclonal antibody production Monoclonal antibodies (mAbs) can be generated using conventional monoclonal antibody methodologies, e.g., Koh et al. ler and Milstein, 1975 Nature 256:495 standard These can be produced by a variety of techniques, including somatic cell hybridization techniques. Many techniques for producing monoclonal antibodies (e.g., viral or oncogenic immunization of B lymphocytes) have been developed. transformation) can be used.

[0349] The animal system for preparing hybridomas is the murine system. Doma production is a well-established procedure for the isolation of immunized splenocytes for fusion. Immunization protocols and techniques are known in the art. Mouse myeloma cells and fusion procedures are also known.

[0350] In some embodiments, the antibodies of the invention are humanized monoclonal antibodies. The chimeric or humanized antibodies and antigen-binding fragments thereof can be prepared by the use of mouse monoclonal antibodies prepared as described above. The sequences encoding the heavy and light immunoglobulin chains can be prepared based on the sequences of the monoclonal antibody. DNA can be obtained from the mouse hybridoma of interest and purified using standard molecular biology techniques. and can be engineered to include non-mouse (e.g., human) immunoglobulin sequences. To create chimeric antibodies, murine variable regions are fused to human variable regions using methods known in the art. The nucleic acid sequence can be linked to a nucleic acid sequence containing a nucleic acid sequence that is a nucleotide sequence of interest (e.g., U.S. Pat. No. 4,816,566 to Cabilly et al.). To generate a humanized antibody, the murine CDR regions are ligated to the humanized antibody using techniques known in the art. The human framework can be inserted using methods known in the art. No. 5,225,539 to Nter, and U.S. Pat. No. 6,225,539 to Queen et al. Specification No. 5,530,101; Specification No. 5,585,089; No. 5,693,762 See US Pat. Nos. 6,180,370 and 6,180,370.

[0351] In some embodiments, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies against 2 utilize parts of the human immune system rather than the mouse system. can be generated using transgenic or transchromosomic mice carrying These transgenic and transchromosomic mice are referred to herein as These mice are referred to as HuMAb mice and KM mice, respectively, and are collectively referred to herein as They are called "human Ig mice."

[0352] HuMAb Mouse® (Medarex, Inc.) is an unrearranged human Human immunoglobulin genes encoding heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences The minilocus contains targeted mutations that inactivate the endogenous μ and κ chain loci (e.g., Lonberg, et al., 1994 Nature 368(6474):8 56-859). Thus, mice with reduced expression of mouse IgM or K In response to immunization, the introduced human heavy and light chain transgenes undergo class switching. undergoing cross-linking and somatic mutation to generate a high-affinity human IgG-κ monoclonal (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 The genome modifications carried by such mice are described in 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 ure Biotechnology 14:845-851, The contents of all of which are expressly incorporated herein by reference in their entirety. U.S. Patent Nos. 5,545,806; 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 No. 5,545,807 to Surani et al.; all PCT Publication WO 92103918 to Lonberg and Kay; International Publication No. 93 / 12227 Brochure, International Publication No. 94 / 25585 Brochure International Publication No. 97113852, International Publication No. 98 / 24884 and PCT Publication No. WO 99 / 45962; and PCT Publication No. Korman et al. See WO 01 / 14424.

[0353] In some embodiments, human antibodies comprise a human heavy chain transgene and a human light chain transgene. Such mice have transgenes and human immunoglobulins on transhomosomes. These can be generated using mice carrying the Purin sequence, referred to herein as "KM mice." Such mice are described in PCT Publication WO 02 / 43478 to Ishida et al. Details will be provided in the pamphlet.

[0354] Still further, alternative transgenic animal systems expressing human immunoglobulin genes. are available in the art and are used to raise TREM2-binding antibodies and antigen-binding fragments thereof. For example, a device called Xenomouse (Abgenix, Inc.) Other transgenic lines that can be used include mice that are, for example, U.S. Patent Nos. 5,939,598 to Kucherlapati et al.; 6,075 ,181; 6,114,598; 6,150,584 and 6,1 No. 62,963.

[0355] Furthermore, alternative transchromosomal animal systems expressing human immunoglobulin genes are are available in the art and can be used to raise the TREM2-binding antibodies of the present invention. For example, a human heavy chain transchromosome and a human light chain transchromosome, called "TC mouse," are obtained. Mice carrying both transchromosomes can be used; The results are from Tomizuka et al., 2000 Proc. Natl. Acad. S ci. USA 97:722-727. Additionally, human heavy and light chain transfectants are Cattle carrying the chromosome have been described in the art (Kuroiw a et al.,2002 Nature Biotechnology 20:88 9-894), can be used to raise TREM2-binding antibodies of the invention.

[0356] Human monoclonal antibodies can also be generated by screening libraries of human immunoglobulin genes. Human antibodies can be prepared using phage display methods for isolating human antibodies. Such phage display methods for this purpose are well established in the art or are described in detail below. See, e.g., U.S. Patent No. 5,223,409 to Ladner et al. Nos. 5,403,484 and 5,571,698; Dowe U.S. Pat. No. 5,427,908 to McCafferty et al.; U.S. Pat. No. 5,427,908 to McCafferty et al. Nos. 5,580,717, 5,969,108, and 6,172,19 No. 7; and U.S. Pat. No. 5,885,793 to Griffiths et al.; Specification No. 6,521,404; Specification No. 6,544,731; No. 6,555,313 See also: 6,582,915 and 6,593,081. sea ​​bream.

[0357] The human monoclonal antibodies of the present invention can also be used in SCID mice reconstituted with human immune cells. such that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Pat. No. 5,476,996 to Wilson et al. and No. 5,698,767.

[0358] Framework or Fc manipulation The engineered antibodies and antigen-binding fragments thereof of the present invention may be modified, e.g., to improve the properties of the antibody. These include those in which modifications have been made to framework residues within VH and / or VL. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to align one or more framework residues with the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations are called "back mutations." It may contain framework residues that differ from the germline sequence from which the antibody is derived. The basis for this is to compare the antibody framework sequences to the germline sequences from which the antibody is derived. To restore framework region sequences to their germline configuration, e.g., Somatic mutations can be "reverted" to germline sequences by site-directed mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0359] Another type of framework modification is within the framework region, or even one or more Mutating one or more residues within the CDR region to remove T cell epitopes, thereby enhancing the This involves reducing the body's immunogenic potential. This approach is also known as "deimmunization." Further details are found in U.S. Patent Application Publication No. 20030153043 to Carr et al. is described in.

[0360] In addition to, or instead of, modifications made within the framework or CDR regions, the present invention The antibodies typically have a serum half-life, complement fixation, Fc receptor binding, and / or antigen dependency. The antibodies may contain modifications within the Fc region to alter one or more functional properties of the antibody, such as cytotoxicity. Furthermore, the antibodies of the present invention may again be engineered to have one or more functions of the antibody. The antibody may be chemically modified (e.g., by adding one or more chemical It may be modified to alter its glycosylation (e.g., to add a nucleotide moiety to the nucleotide sequence), or to alter its glycosylation. Each of these embodiments is described in further detail below. The numbering is from Kabat's EU index.

[0361] In one embodiment, the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. The hinge region of CH1 is modified so that it can be expressed as a nucleotide sequence similar to that of the CH1. This approach is described in detail in Bodmer et al. The hinge of CH1 is further described in U.S. Patent No. 5,677,425 to Varying the number of cysteine ​​residues in a region can affect, for example, the assembly of light and heavy chains. This may enhance or decrease the stability of the antibody.

[0362] In another embodiment, the Fc hinge region of the antibody is altered to decrease the biological half-life of the antibody. More specifically, one is located in the CH2-CH3 domain junction region of the Fc-hinge fragment. These amino acid mutations were introduced, resulting in the staphylococcal protein A (Staph Spirococcyl protein A (SpA) binding to the native Fc-hinge domain This approach was described by Ward et al. This is described in further detail in US Pat. No. 6,165,745.

[0363] In another embodiment, the antibody is modified to increase its biological half-life. For example, U.S. Patent No. 6,277,375 to Ward discloses One of the following mutations: T252L, T254S, T256F, as described in the product description. Alternatively, to increase the biological half-life, one or more Pre Nos. 5,869,046 and 6,121,022 to Sta et al. As described in the specification, the fragments are taken from two loops of the CH2 domain of the Fc region of IgG. The antibody was altered within the CH1 or CL region to include the salvage receptor binding epitope. It is possible.

[0364] In one embodiment, at least one amino acid residue is replaced with a different amino acid residue. By modifying the Fc region, the effector functions of the antibody can be altered. For example, the affinity of the antibody for the effector ligand may be altered, but the antigen binding of the parent antibody may be maintained. One or more amino acids can be replaced with different amino acid residues so that the activity is maintained. The effector ligand to which affinity is changed can be, for example, an Fc receptor or a complement receptor. This approach may be the C1 component of the body. and 5,648,260 (both by Winter et al.) further It is described in detail.

[0365] In another embodiment, the antibody has altered C1q binding and / or complement dependent cytotoxicity (CD C) One or more amino acids selected fr...

Claims

1. An anti-TREM2 antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), (i) As defined by the Kabat numbering system, HCDR1 comprises the amino acid sequence of SEQ ID NO:7; HCDR2 comprises the amino acid sequence of SEQ ID NO:5; HCDR3 comprises the amino acid sequence of SEQ ID NO:6; LCDR1 comprises the amino acid sequence of SEQ ID NO:17; LCDR2 comprises the amino acid sequence of SEQ ID NO:18; and LCDR3 comprises the amino acid sequence of SEQ ID NO:19; (ii) As defined by the Chothia numbering system, HCDR1 comprises the amino acid sequence of SEQ ID NO:8; HCDR2 comprises the amino acid sequence of SEQ ID NO:9; HCDR3 comprises the amino acid sequence of SEQ ID NO:6; LCDR1 comprises the amino acid sequence of SEQ ID NO:20; LCDR2 comprises the amino acid sequence of SEQ ID NO:21; and LCDR3 comprises the amino acid sequence of SEQ ID NO:22; (iii) As defined by the IMGT numbering system, HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; HCDR2 comprises the amino acid sequence of SEQ ID NO: 11; HCDR3 comprises the amino acid sequence of SEQ ID NO: 12; LCDR1 comprises the amino acid sequence of SEQ ID NO: 23; LCDR2 comprises the amino acid sequence of SEQ ID NO: 21; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 19; or (iv) As defined by the combined numbering system, HCDR1 comprises the amino acid sequence of SEQ ID NO:4; HCDR2 comprises the amino acid sequence of SEQ ID NO:5; HCDR3 comprises the amino acid sequence of SEQ ID NO:6; LCDR1 comprises the amino acid sequence of SEQ ID NO:17; LCDR2 comprises the amino acid sequence of SEQ ID NO:18; and LCDR3 comprises the amino acid sequence of SEQ ID NO:19; An anti-TREM2 antibody or antigen-binding fragment thereof.

2. An anti-TREM2 antibody or its antigen-binding fragment described in claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and a light chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:

24.

3. An anti-TREM2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

4. An anti-TREM2 antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region.

5. An anti-TREM2 antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region.

6. An anti-TREM2 antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc region having reduced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity compared to the parent antibody.

7. An anti-TREM2 antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a human Ig kappa light chain constant region.

8. An anti-TREM2 antibody or its antigen-binding fragment described in claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 37 and a light chain that is at least 95% identical to the amino acid sequence of SEQ ID NO:

26.

9. An anti-TREM2 antibody or its antigen-binding fragment, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 37 and a light chain having the amino acid sequence of SEQ ID NO:

26.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A pharmaceutical composition for treating a neuroinflammatory or neurodegenerative disease, comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9.

12. The disease is Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Nasu-Hakola disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), anti-NMDA receptor encephalitis, autism, cerebral lupus erythematosus (NP-SLE), chemotherapy-induced peripheral neuropathy (CIPN), postherpetic neuralgia (PSN), or neuralgia), chronic inflammatory demyelinating polyneuropathy (CIDP), epilepsy, Guillain-Barré syndrome (GBS), inclusion body myositis, lysosomal storage disease, sphingomyelin lipidosis (Niemann-Pick disease type C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, myasthenia gravis, neuro-Behcet's disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, Rasmussen's encephalitis, Rett syndrome, stroke, transverse myelitis, traumatic brain injury, spinal cord injury, viral encephalitis, or bacterial meningitis.

13. The pharmaceutical composition described in claim 12, wherein the disease is Alzheimer's disease.

14. The pharmaceutical composition described in claim 12, wherein the disease is multiple sclerosis.

15. The pharmaceutical composition described in claim 12, wherein the disease is amyotrophic lateral sclerosis (ALS).

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