EA042890B1 - ANTIBODIES TO TREM2 AND METHODS FOR THEIR APPLICATION - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 62/035336 filed August 8, 2014, U.S. Provisional Application No. 62/135110 filed March 18, 2015, and U.S. Provisional Application No. 62/135122 filed August 18 March 2015, the contents of each of which are hereby incorporated by reference in their entirety.

Representation of a list of sequences in an ASCII text file.

The content of the following representation in an ASCII text file is incorporated herein by reference in its entirety: Machine Readable Form (CRF) of the Sequence Listing (File Name: 735022000440SEQLISTING.TXT, File Date: August 7, 2015, Size: 240 kB).

Field of invention

The present invention relates to antibodies to TREM2 and to DAP12 and the therapeutic use of such antibodies.

Technical field

The trigger receptor that is expressed on myeloid cells-2 (TREM2) is an immunoglobulin-like receptor that is expressed primarily on myeloid cells such as macrophages, dendritic cells, monocytes, skin Langerhans cells, Kupffer cells, osteoclasts, and microglia; and is essential for modulation (eg, suppression) of the Toll-like receptor (TLR) signaling pathway, modulation of inflammatory cytokines, and normal osteoclast development. TREM2 has been discovered as a member of the TREM transmembrane glycoproteins, which belong to the single variable domain (IgV) immunoglobulin receptor family. The genes encoding human and mouse TREMs are mapped to human chromosome 6p21.1 and mouse chromosome 17C3, respectively. The TREM cluster includes genes encoding TREM1, TREM2, TREM4, and TREM5, as well as TREM-like genes in humans and mice. Additionally, TERM3 and plasmacytoid dendritic cell (rDC)-TREM have been identified in mice. TREM-like genes, TREML1 and TREML2 in humans and Treml1 and Treml2 in mice, encode TLT-1 and TLT-2, respectively. The two best characterized of these receptors, TREM1 and TREM2, show some sequence homology with other Ig-SF members such as NK cell activating receptors (20% identity with NKp44) and act through association with a DAP12-mediated signaling pathway.

TREM2 was originally cloned as a cDNA encoding a TREM1 homologue (Bouchon A. et al., J. Exp. Med., 2001. 194(8): p. 1111-22). This receptor is a glycoprotein of about 40 kDa, which decreases to 26 kDa after N-deglycosylation. The TREM2 gene encodes a 230 amino acid long protein that contains an extracellular domain, a transmembrane region, and a short cytoplasmic tail. The extracellular region encoded by exon 2 consists of a single type V Ig-SF domain containing three potential N-glycosylation sites. The putative transmembrane region contains a charged lysine residue. The cytoplasmic tail of TREM2 has lost signaling motifs and presumably transmits the signal through the DAP12/TRYROBP signaling adapter molecule.

The DAP12 signaling adapter molecule is expressed as a homodimer on the surface of a variety of cells involved in the innate immune response, including microglia, macrophages, granulocytes, NK cells, and dendritic cells (DCs). DAP12 is a member of the type I transmembrane adapter protein family based on homology to human T cell receptor (TCR)-associated CD3 chains and Fc receptor γ-chain (FcR) (Turnbull IR and Colonna M., Nat. Rev. Immunol. , 2007. 7(2): pp. 155-61). These proteins have many structural and functional characteristics, including one or more ITAM motifs in their cytoplasmic domain, a charged acidic residue in the transmembrane region (required for interaction with a chain partner), and the ability to recruit proteins containing the Src homologous domain-2 (SH2) after tyrosine phosphorylation. The ITAM motif mediates signal propagation through the activation of ZAP70 or Syk tyrosine kinase. Both kinases phosphorylate multiple substrates, thus facilitating the formation of a signaling complex that leads to cellular activation. Interestingly, some B cells and T cells also express DAP12 in inflammatory processes. In humans, a subset of CD4+ ^CD28 T cells, aeTCR ⁺ CD4 ⁺ T cells, and CD8+ T cells expressing this protein have been described in patients suffering from chronic inflammatory diseases in the context of autoimmune T cells (Schleinitz, N. et al., PLoS ONE, 4 (2009), p. e6264). Due to the significant level of DAP12 expression in mouse peritoneal macrophages, this protein is thought to be expressed in other macrophage-associated cells such as osteoclasts in the bone marrow, Kupffer cells in the liver, lung alveolar macrophages, Langerhans cells in the skin, and microglial cells in the brain (Takaki , R. et al., Immunol Rev., 2006. 214: pp. 118-29).

TREM2 has been identified as being expressed on the surface of monocyte-derived dendritic cells and as an mRNA transcript in the RAW264 mouse macrophage cell line.

- 1 042890 (Bouchon, A. et al., J. Exp. Med., 2001. 194(8): p. 1111-22). Human TREM2 was the first DAP12-associated receptor described on the DC surface. Studies have shown that, compared with wild-type cells, cell surface expression of TREM2 is reduced in DAP12-deficient bone marrow dendritic cells (BMDCs) and in DAP12-deficient macrophages (Ito, H. and Hamerman, J.A., Eur. J. Immunol. 42(1): pp. 176-85, Hamerman, J. A. et al., J. Immunol., 2006. 177(4): pp. 2051-5, and Hamerman, J. A. et al., Nat. Immunol., 2005 6(6): pp. 579-86). This indicates that the formation of the TREM2/DAP12 complex is necessary for maximum expression of TREM2 on the cell surface.

Recent studies have also shown expression of TREM2 on the cell surface of macrophages infiltrated from the bloodstream, as well as on macrophages activated by IL-4 or IL-13 (Turnbull, IR et al., J. Immunol., 2006. 177(6): p 3520-4). However, TREM2 expression has not always been detected in other cell populations, such as tissue resident macrophages, circulating monocytes, or corresponding progenitor cells in the bone marrow, suggesting that TREM2 expression is not induced centrally, but locally during tissue infiltration or by cytokines. mediated activation. Moreover, it has also been observed that IFN-γ and LPS reduce or otherwise stop the expression of TREM2. In addition, TREM2 has recently been reported to be highly expressed on microglial cells and infiltrated macrophages in the central nervous system during experimental autoimmune encephalomyelitis or Alzheimer's disease (Piccio, L. et al., Eur. J. Immunol., 2007. 37 (5): pp. 1290-301 and Wang, Y., Cell 2015 Mar 12; 160(6):1061-71).

TREM2 has been shown to signal through DAP12. Downstream, this leads to the activation of the Syk/Zap70 tyrosine kinase family, PICK, and other intracellular signals. For myeloid cells, TLR signals are important for activation, for example, as in the response to infection, and also play a key role in the pathological inflammatory response, for example, as in the case of macrophages and dendritic cells (Hamerman, J.A. et al., ( 2006) J. Immunol.177: 2051-2055; Ito, H. et al., Eur. J. Immunol. 42: 176-185; Neumann, H. et al., (2007) J. Neuroimmunol. 184: 92 -99; Takahashi, K. et al., (2005) J. Exp. Med. 201: 647-657; and Takahashi, K. et al., (2007) PLoS Med 4: e124). It is believed that TREM2 or DAP12 deficiency leads to increased pro-inflammatory signaling. The effect of TREM2 deficiency in vitro has been shown in the context of stimulation with typical TLR ligands such as LPS, CpG DNA and zymosan. TREM-2-deficient dendritic cells show increased release of IL-12p70, TNF, IL-6 and IL-10 in the presence but not in the absence of stimulation.

Several recent studies have examined intracellular signaling events induced by activation of the TREM2/DAP12 pathway. For example, TREM2 is believed to activate signaling pathways involved in cell survival (eg, protein kinase B-Akt), cell activation and differentiation (eg, Syk, Erk1/2, PLC-γ, etc.), and in control of the actin cytoskeleton. (e.g. Syk, Vav et al.) (Peng, Q. et al., Sci Signal. 3 (122): p. ra38; and Whittaker, G. C. et al., J. Biol. Chem. 285(5): p. 297685). Following TREM2 ligation, ITAM tyrosines in DAP12 are phosphorylated by SRC family kinases, resulting in the recruitment and activation of Syk kinase and/or ZAP70 kinase. In the mouse, Syk may be the predominant kinase involved, while in humans, both Syk and ZAP70 appear to efficiently interact with such ITAM-containing subunits by binding to them through their tandem SH2 domains.

TREM2 signaling studies have shown that, similar to TREM1, TREM2-mediated signaling via DAP12 also results in increased levels of intracellular calcium ions and ERK1/2 phosphorylation of ERK1/2 (Bouchon, A. et al., J. Exp. Med., 2001 194(8): pp. 1111-22 and Sharif, O. and Knapp, S., Immunobiology, 2008. 213(9-10): pp. 701-13). Importantly, ligation of the TREM2 receptor does not cause degradation of IkB-α and subsequent nuclear translocation of NF-kB, indicating a possible difference between TREM2 and TREM1 signaling (Bouchon, A. et al., J. Exp. Med., 2001. 194(8): pp. 1111-22). Cross-linking of the TREM2 receptor on immature dendritic cells triggers the activation of molecules involved in costimulation of T cells, such as CD86, CD40, and MHC class II, as well as activation of the CCR7 chemokine receptor (Bouchon, A. et al., J. Exp. Med. , 2001. 194(8): pp. 1111-22). TREM2 is also expressed on microglia, where cross-linking of the receptor results in increased phosphorylation of ERK1/2 and CCR7, but not increased expression of CD86 or MHC class II, suggesting possible cell-type specific differences in TREM2 signaling. Additionally, TREM2 overexpression in myeloid cells resulted in increased phagocytosis of degenerated myelin (Takahashi, K. et al., PLoS Med, 2007. 4(4): p. e124; and Neumann, H. and Takahashi, K., J. Neuroimmunol ., 2007. 184(1-2): pp. 92-9).

It has also been shown that bone marrow macrophages (BMDMs) in which TREM2 has been downregulated by shRNA show increased TNF release in response to TLR2/6 ligand zymosan and TLR9 CpG ligand compared to control BMDM cells that have been treated with non-specific shRNA, indicating that TREM2 negatively regulates cytokine synthesis in macrophages (Ito, H. and Hamerman, J.A., Eur. J. Immunol. 42(1): p. 176-85; Hamerman, J.A. et al., J. Immunol .,

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2006. 177(4): p. 2051-5; and Hamerman, JA et al., Nat. Immunol., 2005. 6(6): p. 579-86). These results were confirmed using BMDM cells from TREM2 knockout mice and additionally showed that compared to wild-type BMDM cells, TNF and IL-6 levels in response to LPS were also higher in TREM2' ^/_ BMDM cells (Turnbull, IR, et al., J. Immunol., 2006. 177(6): p.3520-4, and Turnbull, IR and Colonna, M., Nat. Rev. Immunol., 2007. 7(2): p. 155-61). Additionally, it has been demonstrated that TREM2 overexpression in microglia leads to a decrease in TNF and inducible nitric oxide synthase (iNOS) mRNA after culturing these cells with apoptotic neurons, while TREM2 knockdown results in a modest increase in TNF and iNOS mRNA levels. This indicates that, unlike TREM1, which is a positive regulator of cytokine synthesis, TREM2 is a negative regulator of cytokine synthesis. This effect of TREM2 on inflammation may be independent of macrophage type, as occurs in microglial and BMDM cells.

It has also been shown that in resident myeloid cells of the central nervous system, activation of microglia can lead to inflammation (Neumann, H. et al., (2007) J. Neuroimmunol. 184: 92-99; Takahashi, K. et al., (2005 ) J. Exp. Med. 201: 647-657; Takahashi, K. et al., (2007) PLoS Med. 4: el24; and Hsieh, C. L. et al., (2009) J. Neurochem. 109: 1144- 1156). Moreover, microglial activation has also been observed in frontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease, stroke/ischemic brain injury, and multiple sclerosis. While a decrease in TREM2 activation leads to an increase in some markers of activation and inflammation, such as NOS2 gene transcription in myeloid cells, an increase in TREM2 activation leads to a decrease in NOS2 transcription. It is believed that dying neurons express an endogenous ligand for TREM2. HSP60 has been implicated as a TREM2 ligand on neuroblastoma cells (Stefani, L. et al., (2009) Neurochem 110: 284-294). Overexpression of TREM2 also leads to increased phagocytosis of dying neurons by microglial cells and similarly increases phagocytosis by other myeloid cells.

In humans, complete absence of TREM2 has been shown to cause Nasu-Hakola disease, a rare neurodegenerative disease with late onset dementia, demyelination, and brain atrophy (Paloneva, J. et al., (2002) Am. J. Hum. Genet. 71: 656-662 and Paloneva, J. et al., (2003) J. Exp. Med. 198: 669-675). Nasu-Hakola disease can also be caused by DAP12 deficiency.

TREM2 gene expression has also been shown to be upregulated in APP23 transgenic mice, a model of Alzheimer's disease in which mice express a mutant form of an amyloid precursor protein that is associated with familial Alzheimer's disease (Melchior, B. et al., ASN Neuro 2: e00037) . Amyloid 1-42 uptake has also been shown to be increased in BV-2 microglial cell lines that overexpress TREM2.

Additionally, increased expression of TREM2 has been shown in the EAE mouse model of multiple sclerosis (Neumann, H. et al., (2007) J. Neuroimmunol. 184: 92-99; Takahashi, K. et al., (2005) J. Exp. Med 201: 647-657 and Takahashi, K. et al. (2007) PLoS Med. 4: e124). Transduction of myeloid progenitor cells from bone marrow (BM-DC) in vitro with TREM2 results in enhanced phagocytosis of degenerated myelin. In response to LPS, these cells show an increase in IL-10 and a decrease in ΚΠ-1β. Intravenous transplantation of myeloid cells overexpressing TREM2 can suppress EAE in vivo.

Additionally, exome sequencing of individuals with described frontotemporal dementia (FTD) revealed homozygous mutations in TREM2 (Guerreiro, R. J. et al., JAMA Neurol. 70: 78-84; and Guerreiro, R. J. et al., Arch Neurol: 1-7 ). Some of these mutations result in truncation and likely loss of TREM2 function. These same TREM2 mutations can also cause Naku-Hakula disease in some individuals. Image analysis in some individuals with homozygous TREM2 mutations also showed signs of demyelination.

Heterozygous mutations in TREM2, which are the same as the mutation that causes Naku-Hakula disease and FTD, also increase the risk of Alzheimer's disease (Guerreiro, R. et al., N. Engl. J. Med 368: 117-127; Jonsson, T. et al., N. Engl. J. Med 368: 107-116 and Neumann, H. et al., N. Engl. J. Med. 368: 182-184). Although these TREM2 mutations are less common than the known variants associated with the risk of Alzheimer's disease (eg, APOE4), the effect of having these mutations is just as severe; approximately 3 times the risk of developing Alzheimer's disease. Moreover, even individuals without Alzheimer's disease who carry a heterozygous TREM2 mutation show worse cognitive performance compared to individuals with two normal TREM2 alleles. Additionally, the R47H TREM2 variant (arginine to histidine amino acid substitution at position 47 in TREM2), which is the most common TREM2 mutation (up to 1 in 200 individuals), has been shown to be located in the immunoglobulin domain of TREM2 and thus can alter ligand binding. (Wang Y., Cell. 2015 Mar 12;160 (6): 1061-71).

Additionally, an integrative network approach to a rank-ordered structure of molecular networks of gene expression relevant for the development of late-onset Alzheimer's disease (LOAD) has identified TYROBP/DAP12 as a signaling molecule for TREM2 as a key regulator of immune/microglial gene modules that are associated with LOAD. . TYROBP was found to be the causal regulator of immune/microglial module with the highest

- 3 042890 quantified as ranked based on the number of other genes regulated by TREM2 and the amount of downregulation as well as differential expression in LOAD brains. TYROBP levels were significantly elevated in LOAD brains and there was a progressive change in TYROBP expression in mild cognitive impairment (MCI) that often precedes LOAD (Zhang et al., (2013) Cell 153, 707-720). Orienting such causal networks in ways that restore them to their normal state may be a way to treat disease.

Accordingly, there is a need for antibodies that specifically bind TREM2 and/or its DAP12/TRYROBP signal adapter molecule on the cell surface and that modulate (e.g., activate or inhibit) one or more TREM2 and/or DAP12 activities for the treatment of one or more diseases. , disorders and conditions associated with decreased TREM2 and/or DAP12 activity, as well as conditions associated with undesirable TREM2 and/or DAP12 activity.

Moreover, the tumor microenvironment consists of a heterogeneous immune infiltrate that includes T-lymphocytes, macrophages, and myeloid/granulocytic cells.

Therapeutic approaches that modulate certain immune cell subtypes are changing the standard of care. Checkpoint-blocking antibodies targeting immune-modulating molecules expressed on T cells (such as CTLA-4 and PD-1) have demonstrated clinical activity in a variety of tumor types (Naidoo et al., (2014) British Journal of Cancer 111, 2214-2219).

Cancer immunotherapy targeting tumor-associated macrophages (eg, M2-type macrophages) is an area of intense research. The presence of M2 macrophages in tumors is associated with poor prognosis. Accordingly, there is a need for antibodies that specifically bind TREM2 and/or DAP12 and modulate (e.g., activate or inhibit) one or more of TREM2 and/or DAP12 activities in tumor-associated immune system cells such as macrophages, dendritic cells, myeloid /granulocytic cells, T cells and monocytes.

All references cited in this document, including patent applications and publications, are incorporated herein by reference in their entirety.

The essence of the invention

The invention generally relates to methods and compositions that include antibodies, such as monoclonal antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibody fragments, and the like, that specifically bind to the TREM2 protein and/or its DAP12 signaling adapter molecule, such as TREM2 mammalian, TREM2 human, DAP12 mammalian or DAP12 human, including wild-type proteins and their variants of natural origin. Antibodies as described herein may include agonist antibodies, inert antibodies, and/or antagonist antibodies. The methods provided herein find use in the prevention, risk reduction, or treatment of an individual with dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, or multiple sclerosis; in inducing or promoting the survival of cells of the innate immune system in a person in need thereof and/or in reducing the survival of cells of the innate immune system in a person in need thereof.

Some aspects of the present description relate to different classes of antibodies to TREM2. In some embodiments, anti-TREM2 antibodies are agonistic antibodies that bind to TREM2 and activate, induce, promote, stimulate, or otherwise increase one or more TREM2 activities, survival of one or more innate immune system cells, and/or IL expression. -6. In some embodiments, anti-TREM2 agonist antibodies as described herein compete with TREM2 ligands to bind to TREM2 expressed on the cell surface. In some embodiments, anti-TREM2 agonist antibodies as described herein do not compete with TREM2 ligands for binding to TREM2 expressed on the cell surface. In some embodiments, anti-TREM2 antibodies are inert or antagonist antibodies that bind to TREM2 and reduce, inhibit, or otherwise reduce one or more TREM2 activities and/or the survival of one or more cells of the innate immune system. In some embodiments, the inert or anti-TREM2 antagonist antibodies described herein block or otherwise inhibit ligand binding to TREM2 expressed on the cell surface.

Other aspects of the present disclosure relate to an isolated agonist antibody that binds to the TREM2 protein, the DAP12 protein, or both proteins, wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both activities.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a mammalian protein or a human protein. In certain implementations

- 4 042890 of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a wild-type protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a naturally occurring variant. In certain embodiments, which may be combined with any of the preceding embodiments, the TREM2 protein, the DAP12 protein, or both, is expressed on human dendritic cells, human macrophages, human monocytes, human osteoclasts, human skin Langerhans cells, Kupffer cells. human cells and/or human microglial cells. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on the cell surface. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities include binding TREM2 to DAP12. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more DAP12 activities include binding DAP12 to TREM2. In certain embodiments, which may be combined with any of the preceding embodiments, one or more of TREM2 activities, DAP12 activities, or both, include DAP12 phosphorylation, TREM2 phosphorylation, or both. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, DAP12 phosphorylation, TREM2 phosphorylation, or both, is induced by one or more SRC families of tyrosine kinases. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more SRC-family of tyrosine kinases contains the kinase Syk. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities, include the activation of PIB. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include increased expression of one or more anti-inflammatory cytokines. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include increased expression of one or more anti-inflammatory mediators (for example, cytokines) selected from the group consisting of IL-12r70, IL-6 and IL-10. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, increased expression occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and cells microglia. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include reduced expression of one or more pro-inflammatory cytokines. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities, include reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN -b, IL-6, IL-12 p70, IL-1b, TNF, TNF-α, IL-10, IL-8, CRP, members of the TGF-beta chemokine protein families, members of the IL-20 family, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18 and CRP. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities include reduced expression of TNF-α, IL-6, or both. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, reduced expression of one or more pro-inflammatory mediators occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, Langerhans cells of the skin , Kupffer cells and microglial cells. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities, include extracellular signal regulated kinase (ERK) phosphorylation. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities include increased expression of the C-C chemokine receptor 7 (CCR7). In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more activities

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TREM2, DAP12 activities, or both activities include the induction of microglial cell chemotaxis towards cells expressing CCL19 and CCL21. In certain embodiments, which may be combined with any of the preceding embodiments, one or more of TREM2 activities, DAP12 activities, or both, include enhancing, normalizing, or both the ability of bone marrow dendritic cells to induce proliferation of antigen-specific T cells. . In certain embodiments, which may be combined with any of the preceding embodiments, one or more of TREM2 activities, DAP12 activities, or both, include inducing osteoclast production, increasing levels of osteoclastogenesis, or both. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include increasing the survival of macrophages, microglial cells, or both. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities include increasing the function of macrophages, microglial cells, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, and /or Kupffer cells. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the macrophages are M1 macrophages and/or microglia, M2 macrophages and/or microglia, or both. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the M1 macrophages and/or microglia are activated M1 macrophages and/or microglia. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities, include the induction of one or more types of clearance selected from the group consisting of clearance of apoptotic neurons, clearance of debris nervous tissue, clearance of debris from non-nervous tissue, clearance of bacteria or other foreign bodies, clearance of a pathogenic protein, clearance of a pathogenic peptide, and clearance of a pathogenic nucleic acid. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of the TREM2 activities, DAP12 activities, or both activities include the induction of phagocytosis of one or more apoptotic neurons, nervous tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, pathogenic proteins, pathogenic peptides or pathogenic nucleic acid. In certain embodiments, which may be combined with any of the preceding embodiments, the disease-causing protein is selected from the group consisting of beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein , prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin , cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and prolinarginine (PR) repeat peptides. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the pathogenic nucleic acid is a GGCCCC (G2C4) repeat expansion antisense RNA. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include the normalization of abnormal gene expression, which depends on TREM2/DAP12. In certain embodiments, which may be combined with any of the preceding embodiments, one or more TREM2 activities, DAP12 activities, or both, involve the involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of TREM2 activities, DAP12 activities, or both activities include Syk phosphorylation. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include increased expression of CD83 and/or CD86 on dendritic cells, macrophages and/or monocytes. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include a reduced release of one or more inflammatory cytokines. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more inflammatory cytokines are selected from the group consisting of TNF-α, IL-10, IL-6, MHB-1, IFN-a4, IFN -b, IL-1c, IL-8, CRP, members of the chemo protein families

- 6 042890 kines of TGF-beta, members of the IL-20, IL-33, FIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL- 18 and SRB. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include reduced expression of one or more inflammatory receptors. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more inflammatory receptors include CD86. In certain embodiments, which may be combined with any of the preceding embodiments, one or more of TREM2 activities, DAP12 activities, or both, involve increasing phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia under conditions of reduced levels of MCSF. In certain embodiments, which may be combined with any of the preceding embodiments, one or more of TREM2 activities, DAP12 activities, or both, involve the reduction of phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal levels of MCSF. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include an increase in the activity of one or more TREM2-dependent genes. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2-dependent genes include one or more nuclear factors from activated T cell transcription factors (NFAT). In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class, the IgM class, or the IgA class. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody induces one or more TREM2 activities, DAP12 activities, or both activities, regardless of binding to the Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region that contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering. In certain embodiments, which may be combined with any of the preceding embodiments, the human IgG2 constant region comprises a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG1 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG1 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB). In some embodiments of the invention, which may be combined with any of the preceding

- 7 042890 in the embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a heavy chain constant domain 1 (CH1) of the IgG2 isotype and a hinge region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the IgG2 isotype CH1 and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS

WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397) .

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region of the antibody contains the amino acid substitution S267E, the amino acid substitution L328F, or both substitutions, and/or the amino acid substitution N297A or N297Q, and the numbering of residues on IgG1 is presented according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG4 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG4 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any combination thereof, the residue numbering being EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2/4 hybrid isotype. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, and the numbering of the residues is presented in accordance with the numbering EU or Kabat. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG4 constant region. In certain embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, human DAP12 and a naturally occurring human DAP12 variant, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, DAP12 human and naturally occurring human DAP12 variant. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv.

Other aspects of the present description relate to an isolated inactive antibody that binds to the TREM2 protein. Other aspects of the present description relate to the selected antagonist

- 8 042890 static antibody that binds to the TREM2 protein.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a mammalian protein or a human protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a wild-type protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein, the DAP12 protein, or both, is a naturally occurring variant. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody inhibits one or more TREM2 activities, DAP12 activities, or both activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities, include reducing the activity of one or more TREM2-dependent genes. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2-dependent genes include one or more nuclear factors from activated T cell transcription factors (NFAT). In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more of the TREM2 activities, DAP12 activities, or both activities, include a decrease in the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, microglial cells M2, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is not able to bind the Fc-gamma receptor (FcyR). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG1 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG1 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbers given according to EU or Kabat numbering . In some embodiments, which may be combined with any of the previous embodiments, the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous options

- 9 042890 implementation of the invention, the antibody has the IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG4 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG4 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, the residue numbering being EU or Kabat numbering. In some embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12 and a naturally occurring variant of human DAP12. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv.

In some embodiments, which may be combined with any of the preceding embodiments, the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, and the numbering of residues is presented in accordance with the numbering of the EU or Kabat. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering residues are presented according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region further comprises the amino acid substitution S228P according to EU or Kabat numbering. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody competes for TREM2 binding to one or more TREM2 ligands. In some embodiments, which may be combined with any of the preceding embodiments, the one or more TREM2 ligands are selected from the group consisting of E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid beta peptide. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, a conjugated antibody, or a chimeric antibody. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In some embodiments, which may be combined with any of the preceding embodiments, the first antigen is human TREM2 or a naturally occurring variant thereof, and the second antigen is a disease-causing protein selected from the group consisting of beta-amyloid or its fragments, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medina, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DRP) , glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides); the blood-brain barrier specific protein is selected from the group consisting of: transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1 and LRP1; or ligands and/or proteins expressed on the surface of immune cells. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody

- 10 042890 is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12, and naturally occurring human DAP12 variant; and wherein the antibody is used in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of: beta-amyloid or fragments thereof, Tau, IAPP, alphasynuclein, TDP-43, FUS protein, prion protein, prion PrPsc protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin , immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, repeat peptides glycine-arginine (GR), proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides, and any combination thereof. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a monoclonal antibody.

In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody binds to a linear epitope on TREM2. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the linear epitope on TREM2 is within the extracellular domain of TREM2. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the linear epitope on TREM2 is within the extracellular immunoglobulin-like domain of the variable type (IgV) of TREM2. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody binds to the TREM2 protein, and wherein the isolated antibody binds to one or more amino acids within amino acid residues selected from the group consisting of: i. amino acid residues 29-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1; ii amino acid residues 29-41 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1; iii. amino acid residues 40-44 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 40-44 of SEQ ID NO: 1; iv. amino acid residues 47-69 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 47-69 of SEQ ID NO: 1; v. amino acid residues 67-76 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1; vi. amino acid residues 76-86 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1; vii. amino acid residues 91-100 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 91100 of SEQ ID NO: 1; viii. amino acid residues 99-115 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1; ix. amino acid residues 104-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 104-112 of SEQ ID NO: 1; their. amino acid residues 114-118 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the highlighted the antibody binds to one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 4350 of SEQ ID NO: 1. In certain embodiments of the invention, which can be combined with any from previous embodiments, the isolated antibody binds to one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certain embodiments implementation of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody binds to an epitope containing one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1. In certain embodiments of the invention, which can be combined with any of the preceding embodiments, the isolated antibody binds to an epitope containing one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments , the isolated antibody binds to an epitope containing one or more amino acid residues selected from the group consisting of: i. amino acid residues of Arg47 or Asp87 of SEQ ID NO: 1; ii. amino acid residues 40-44 of SEQ ID NO: 1; iii. amino acid residues 67-76 of SEQ ID NO: 1; and iv. amino acid residues 114-118 of SEQ ID NO: 1. In certain embodiments, which may be combined with any of the previous embodiments, an isolated antibody binds to one or more amino acids within amino acid residues 22-40 of SEQ ID NO: 2 or amino acid residues on the DAP12 protein corresponding to amino acid residues 22-40 of SEQ ID NO: 2. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody is a bispecific antibody which binds to one or more amino acids selected from the group consisting of: i. one or more amino acid residues from SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues from SEQ ID NO: 1; and ii. one or more amino acid residues from SEQ ID NO: 2, or amino acid residues on the DAP12 protein corresponding to amino acid residues from SEQ ID NO: 2.

In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2, and/or HVR -H3 monoclonal antibody At52; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2, and/or HVR-L3 of the At52 monoclonal antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H1 contains the amino acid sequence of SEQ ID NO: 398. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR -H2 contains the amino acid sequence from SEQ ID NO: 399. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H3 contains the amino acid sequence from SEQ ID NO: 400. In certain embodiments of the invention, which may be combined with any of the prior embodiments, HVR-L1 comprises the amino acid sequence from SEQ ID NO: 401. In certain embodiments that may be combined with any of the prior embodiments, HVR-L2 comprises the amino acid sequence from SEQ ID NO: 402. In certain embodiments of the invention that can be combined with any of the previous embodiments, HVR-L3 contains the amino acid sequence from SEQ ID NO: 403. In certain embodiments of the invention, which can be combined with any of the previous embodiments, the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: the level of homology with the amino acid sequence of SEQ ID NO: 398; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 399 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 399; and/or (c) an HVRH3 comprising the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 401 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 401; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 402 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 402; and; and/or (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403. In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention, the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the At21 monoclonal antibody; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H1 contains the amino acid sequence of SEQ ID NO: 404. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR -H2 contains the amino acid sequence from SEQ ID NO: 405. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H3 contains the amino acid sequence from SEQ ID NO: 406. In certain embodiments of the invention, which may be combined with any of the previous embodiments, HVR-L1 contains the amino acid sequence from SEQ ID NO: 407. In certain embodiments that can be combined with any of the previous embodiments, HVRL2 contains the amino acid sequence from SEQ ID NO: : 408. In certain variants of reality

- 12 042890 of the invention, which can be combined with any of the previous embodiments of the invention, HVR-L3 contains the amino acid sequence of SEQ ID NO: 409. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the highlighted the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404 or an amino acid sequence with at least about 95% homology to the amino acid sequence SEQ ID NO: 404; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 405 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 405; and/or (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 407 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 407; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 408 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 408; and; and/or (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 409, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 409.

Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the At52 monoclonal antibody; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At52 monoclonal antibody. In certain embodiments of the invention that can be combined with any of the previous embodiments of the invention, HVRH1 contains the amino acid sequence of SEQ ID NO: 398. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H2 contains the amino acid sequence from SEQ ID NO: 399. In certain embodiments, which may be combined with any of the previous embodiments, HVR-H3 contains the amino acid sequence from SEQ ID NO: 400. In certain embodiments, which may be combined with any of the previous embodiments of the invention, HVR-L1 contains the amino acid sequence from SEQ ID NO: 401. In certain embodiments of the invention that can be combined with any of the previous embodiments of the invention, HVR-L2 contains the amino acid sequence from SEQ ID NO: : 402. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-L3 contains the amino acid sequence from SEQ ID NO: 403. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody contains a heavy chain variable domain and a light chain variable domain, in which the heavy chain variable domain contains HVR-H1 containing the amino acid sequence from SEQ ID NO: 398, HVR-H2 containing the amino acid sequence from SEQ ID NO: 399, and HVR-H3 containing the amino acid sequence from SEQ ID NO: 400 and/or wherein the light chain variable domain comprises HVR-L1 containing the amino acid sequence from SEQ ID NO: 401, HVR-L2 containing the amino acid sequence from SEQ ID NO : 402, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 403.

Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 398 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 398; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 399 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 399; and/or (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 401 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 401; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 402 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 402; and; and/or (c) an HVR-L3 containing

- 13 042890 a common amino acid sequence from SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403.

Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the At21 monoclonal antibody; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVRH1 contains the amino acid sequence of SEQ ID NO: 404. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H2 contains the amino acid sequence from SEQ ID NO: 405. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-H3 contains the amino acid sequence from SEQ ID NO: 406. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-L1 contains the amino acid sequence from SEQ ID NO: 407. In certain embodiments of the invention that can be combined with any of the previous embodiments of the invention, HVR-L2 contains the amino acid sequence from SEQ ID NO: : 408. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, HVR-L3 contains the amino acid sequence of SEQ ID NO: 409. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody contains a heavy chain variable domain and a light chain variable domain, in which the heavy chain variable domain contains HVR-H1 containing the amino acid sequence from SEQ ID NO: 404, HVR-H2 containing the amino acid sequence from SEQ ID NO: 405, and HVR-H3 containing the amino acid sequence from SEQ ID NO: 406, and/or wherein the light chain variable domain comprises HVR-L1 containing the amino acid sequence from SEQ ID NO: 407, HVR-L2 containing the amino acid sequence from SEQ ID NO: : 408, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 409.

Other aspects of the description relate to an isolated anti-human TREM2 antibody, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 404; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 405 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 405; and/or (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 407 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 407; (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 408 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 408; and; and/or (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 409, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 409.

Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody that binds substantially the same TREM2 epitope as the Ab52 antibody. Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody that binds substantially the same TREM2 epitope as the At21 antibody.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both activities. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on the cell surface. In certain embodiments of the invention that can be combined with any of the previous embodiments of the invention, one or more TREM2 activities, DAP12 activities, or both activities are selected from the group consisting of TREM2 binding to DAP12; binding DAP12 to TREM2; TREM2 phosphorylation, DAP12 phosphorylation; physical activation; increased expression of one or more anti-inflammatory mediators (eg, cytokines),

- 14 042890 selected from the group consisting of IL-12p70, IL-6 and IL-10; reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1b, TNF, TNF-α, IL-10, IL-8, CRP , members of the TGF-beta chemokine protein families, members of the IL-20, IL-33, FIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL families -18 and SRP; reduced expression of TNF-α, IL-6, or both; phosphorylation of kinase regulated by extracellular signals (ERK); increased expression of the C-C chemokine receptor 7 (CCR7); inducing chemotaxis of microglial cells towards cells expressing CCL19 and CCL21; increasing, normalizing, or both, the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; inducing the production of osteoclasts, increasing the level of osteoclastogenesis, or both; increase the survival and/or function of one or more dendritic cells, macrophages, microglial cells, macrophages and/or M1 microglial cells, activated macrophages and/or M1 microglial cells, macrophages and/or M2 microglial cells, monocytes, osteoclasts, skin Langerhans cells and Kupffer cells; induction of one or more types of clearance selected from the group consisting of apoptotic neuron clearance, neural debris clearance, non-nervous tissue debris clearance, bacteria or other foreign body clearance, pathogenic protein clearance, pathogenic peptide clearance, and pathogenic nucleic acid clearance; inducing phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, or disease-causing nucleic acid; normalization of disturbed TREM2/DAP12 dependent gene expression; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; phosphorylation of Syk; increased expression of CD83 and/or CD86 on dendritic cells, macrophages, monocytes and/or microglia; decreased secretion of one or more inflammatory cytokines selected from the group consisting of TNF-α, IL-10, IL-6, MHB-1, IFN-a4, IFN-b, IL-1b, IL-8, CRP, family members chemokine proteins TGF-beta, family members IL-20, IL-33, LIF, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18 and SRP; decreased expression of one or more inflammatory receptors; increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of reduced levels of MCSF; decreased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia in the presence of normal levels of MCSF; increasing the activity of one or more TREM2-dependent genes; and any combination of them. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class, the IgM class, or the IgA class. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody induces one or more TREM2 activities, DAP12 activities, or both activities, regardless of binding to the Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering. In certain embodiments, which may be combined with any of the preceding embodiments, the human IgG2 constant region comprises a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG1 isotype. In some embodiments of the invention, which may be combined with any

- 15 042890 of the previous embodiments of the invention, the antibody contains a human IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG1 constant region contains an Fco region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a heavy chain constant domain 1 (CH1) of the IgG2 isotype and a hinge region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the IgG2 isotype CH1 and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS

WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397) .

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region of the antibody contains the amino acid substitution S267E, the amino acid substitution L328F, or both substitutions, and/or the amino acid substitution N297A or N297Q, and the numbering of the residues is presented in accordance with EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG4 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG4 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any combination thereof, the residue numbering being EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2/4 hybrid isotype. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, and the numbering of the residues is presented in accordance with the numbering EU or Kabat. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG4 constant region. In certain embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring variant.

- 16 042890

human TREM2, human DAP12, and a naturally occurring human DAP12 variant, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring TREM2 variant human, human DAP12, and a naturally occurring variant of human DAP12. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an inert antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody is an antagonistic antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody inhibits one or more TREM2 activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities are selected from the group consisting of reducing the activity of one or more TREM2-dependent genes; reducing the activity of one or more nuclear factors of activated T-cell transcription factors (NFAT); reduced survival of macrophages, microglial cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells; and any combination of them. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is not able to bind the Fc-gamma receptor (FcyR). In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG1 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG1 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbers given according to EU or Kabat numbering . In some embodiments, which may be combined with any of the previous embodiments, the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a mouse IgG1 constant region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG4 constant region. In some embodiments of the invention,

- 17 042890 which can be combined with any of the previous embodiments of the invention, the human IgG4 constant region contains an Fc region. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more modifications. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region contains one or more amino acid substitutions. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more amino acid substitutions in the Fc region are at a residue position selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, the residue numbering being EU or Kabat numbering. In some embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12 and a naturally occurring variant of human DAP12. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, and the numbering of residues is presented in accordance with the numbering of the EU or Kabat. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering residues are presented according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region further comprises the amino acid substitution S228P according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a monoclonal antibody.

Other aspects of the present disclosure relate to an isolated antibody that binds to the TREM2 protein, wherein the isolated antibody promotes the survival of one or more cells of the innate immune system. Other aspects of the present description relate to an isolated antibody that binds to the TREM2 protein, and the selected antibody increases the expression of IL-6. Other aspects of the present disclosure relate to an isolated antibody that binds to the TREM2 protein, wherein the isolated antibody promotes the survival of one or more cells of the innate immune system or increases the expression of IL-6. Other aspects of the present disclosure relate to an isolated antibody that binds to the TREM2 protein, wherein the isolated antibody promotes the survival of one or more cells of the innate immune system or increases the expression of IL-6. In certain embodiments of the invention, one or more cells of the innate immune system are selected from the group consisting of macrophages, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, dendritic cells, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes , osteoclasts, skin Langerhans cells, Kupffer cells, and any combination thereof. In some embodiments of the invention, one or more cells of the innate immune system are macrophages. In some embodiments of the invention, one or more cells of the innate immune system are microglial cells. In some embodiments, the one or more cells of the innate immune system are M1 microglial cells. In some embodiments of the invention, one or more cells of the innate immune system are activated M1 microglial cells. In some embodiments, the one or more cells of the innate immune system are M2 microglial cells. In some embodiments of the invention, one or more cells of the innate immune system are dendritic cells (DC). In some embodiments of the invention, one or more cells of the innate immune system are M1 macrophages. In some embodiments of the invention, one or more cells of the innate immune system are activated M1 macrophages. In some embodiments of the invention, one or more cells of the innate immune system are M2 macrophages. In some embodiments of the invention, one or

- 18 042890 more cells of the innate immune system are monocytes. In some embodiments of the invention, one or more cells of the innate immune system are osteoclasts. In some embodiments of the invention, one or more cells of the innate immune system are Langerhans cells of the skin. In some embodiments of the invention, one or more cells of the innate immune system are Kupffer cells.

Other aspects of the present disclosure relate to an isolated antibody that binds to the TREM2 protein, wherein the isolated antibody binds to one or more amino acids within amino acid residues selected from the group consisting of: i. amino acid residues 29-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1; ii. amino acid residues 29-41 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1; iii. amino acid residues 40-44 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 40-44 of SEQ ID NO: 1; iv. amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1; v. amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1; vi. amino acid residues 47-69 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 47-69 of SEQ ID NO: 1; vii. amino acid residues 67-76 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1; viii. amino acid residues 76-86 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1; ix. amino acid residues 91-100 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 91-100 of SEQ ID NO: 1; X. amino acid residues 99-115 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1; xi. amino acid residues 104-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 104-112 of SEQ ID NO: 1; xi. amino acid residues 114-118 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1; xiii. amino acid residues 130171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-171 of SEQ ID NO: 1; xiv. amino acid residues 139-146 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 139-146 of SEQ ID NO: 1; xv. amino acid residues 140-153 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1; xvi. amino acid residues 130-144 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-144 of SEQ ID NO: 1; and xviii. amino acid residues 158-171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to one or more amino acids within amino acid residues 43 -50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to one or more amino acids within amino acid residues 139-146 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to one or more amino acids within amino acid residues 140-153 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to an epitope comprising one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1. In certain embodiments, carrying out the invention, the isolated antibody binds to an epitope comprising one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1. In certain embodiments of the invention, the isolated antibody binds to an epitope comprising one or more amino acids within amino acid residues 139-146 from SEQ ID NO: 1. In certain embodiments, the isolated antibody binds to an epitope comprising one or more amino acids within amino acid residues 140-153 of SEQ ID NO: 1.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein is a mammalian protein or a human protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein is a wild-type protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the TREM2 protein is

- 19 042890 is a naturally occurring variant. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody induces or maintains TREM2 clustering on the cell surface. In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention, one or more TREM2 activities are selected from the group consisting of: i. binding TREM2 to DAP12; ii. phosphorylation of DAP12; iii. Increasing the survival of macrophages, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, skin Langerhans cells and / or Kupffer cells; iv. phosphorylation of Syk; v. increased expression of CD83 and/or CD86 on dendritic cells; vi. increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia; vii. increasing the activity of one or more TREM2-dependent genes, optionally one or more TREM2-dependent genes including one or more nuclear factors from activated T cell transcription factors (NFAT); and viii. increasing the expression of one or more mediators selected from the group consisting of IL-12p70, IL-6 and IL-10. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class, the IgM class, or the IgA class. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody induces one or more TREM2 activities, regardless of binding to the Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention: i. the isolated antibody is human IgG2 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T , T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering; ii. the isolated antibody has a human IgG2 isotype, in which the human IgG2 contains a constant region and in which the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU or Kabat numbering; iii. the isolated antibody is of human or mouse IgG1 isotype and contains one or more amino acid substitutions in the Fc region at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E , P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering; iv. the isolated antibody is of the IgG1 isotype and contains the heavy chain constant domain 1 (CH1) of the IgG2 isotype and the hinge region, optionally the CH1 of the IgG2 isotype and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS

WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS

NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397), and optionally the Fc region of the antibody contains the S267E amino acid substitution, the L328F amino acid substitution or both substitutions, and/or the N297A or N297Q amino acid substitution, the residue numbering being EU or Kabat numbering; v. the isolated antibody is human or mouse IgG4 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any their combinations, with the numbering of the residues presented in accordance with the EU or Kabat numbering; or vi. the isolated antibody has an IgG2/4 hybrid isotype, and optionally the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with the numbering remaining

- 20 042890 kov is represented by EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an inert antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody is an antagonistic antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody inhibits one or more TREM2 activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more inhibited TREM2 activities are selected from the group consisting of a decrease in the activity of one or more TREM2-dependent genes; reducing the activity of one or more nuclear factors of activated T cell transcription factors (NFAT); reduced survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells; reducing the expression of one or more mediators selected from the group consisting of IL-12p70, IL-6 and IL-10; and any combination of them. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is not able to bind the Fc-gamma receptor (FcyR). In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention: i. the isolated antibody is human or mouse IgG1 isotype and contains one or more amino acid substitutions in the Fc region at the position of a residue selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A , A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being EU or Kabat numbering, optionally the Fco region additionally containing an amino acid deletion at position corresponding to glycine 236 according to the EU or Kabat numbering; ii. the isolated antibody is human IgG2 isotype and contains one or more amino acid substitutions in the Fc region at a position of a residue selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T , T256E, and any combination thereof, the numbering of residues being given according to the EU or Kabat numbering; or iii. the isolated antibody is human or mouse IgG4 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y , S254T, T256E, N297A, N297Q, and any combination thereof, with residue numbering according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, moreover, the numbering of residues is presented in accordance with the numbering of the EU or Kabat. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fco region additionally contains the amino acid substitution S228P according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering residues are presented according to EU or Kabat numbering. In certain embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2 and a naturally occurring human TREM2 variant, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12, and naturally occurring human DAP12 variant. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an antibody fragment, and wherein the antibody fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody competes for TREM2 binding to one or more TREM2 ligands. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 ligands are selected from the group consisting of E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negative

- 21 042890 charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides and lipidated amyloid beta peptide. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In certain embodiments of the invention, which may be combined with any of the preceding embodiments, the first antigen is human TREM2 or a naturally occurring variant thereof, and the second antigen is a disease-causing protein selected from the group consisting of beta-amyloid or its fragments, Tau, IAPP, alphasynuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medina, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), peptides glycine-alanine (GA) repeat, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides); or a blood-brain barrier specific protein selected from the group consisting of: transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1 and LRP1. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2 and a naturally occurring human TREM2 variant; and moreover, the antibody is used in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of: beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein , prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2 microglobulin, gelsolin, keratoepithelin, qi statin , immunoglobulin AL light chain, SIBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine- arginine (GR), proline-alanine (PA) repeat peptides, and prolinarginine (PR) repeat peptides, and any combination thereof. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a monoclonal antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is a bispecific antibody that binds to TREM2 and DAP12.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody contains a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain contains: (a) HVR-H1 containing the amino acid sequence selected from the group consisting of SEQ ID NOs: 3-24, 398 and 404; HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-49, 399 and 405; and (c) HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-119, 400 and 406; and/or where the variable domain of the light chain contains: (a) HVR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137, 401 and 407; (b) HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 138-152, 402 and 408; and (c) an HVR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 153-236, 403 and 409.

Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: (a) HVR-H1 comprising an amino acid sequence selected from the group consisting of from SEQ ID NOs: 3-24, 398 and 404; HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-49, 399 and 405; and (c) HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-119, 400 and 406; and/or where the variable domain of the light chain contains: (a) HVR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137, 401 and 407; (b) HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 138-152, 402 and 408; and (c) HVR-L3 containing the amino acid

- 22 042890 a sequence selected from the group consisting of SEQ ID NOs: 153-236, 403 and 409. Other aspects of the present description relate to an isolated anti-human TREM2 antibody that binds essentially the same TREM2 epitope as the monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35,

At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52,

At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69,

At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87. Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody that competes with a monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13 , At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38 ,

At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52, At53, At54, At55,

At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72,

At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87 for binding to TREM2.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody induces or maintains TREM2 clustering on the cell surface. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more TREM2 activities are selected from the group consisting of TREM2 binding to DAP12; phosphorylation of DAP12; increasing the survival of macrophages, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, skin Langerhans cells and/or Kupffer cells; increased expression of IL-6; phosphorylation of Syk; increased expression of CD83 and/or CD86 on dendritic cells; increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia; and increasing the activity of one or more TREM2-dependent genes, optionally one or more TREM2-dependent genes including one or more nuclear factors from activated T cell transcription factors (NFAT); and any combination of them. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class, the IgM class, or the IgA class. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody has an IgG2 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains a human IgG2 constant region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the human IgG2 constant region contains an Fc region. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody induces one or more TREM2 activities regardless of binding to the Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody binds to an inhibitory Fc receptor. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention: i. the isolated antibody is human IgG2 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T , T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering; ii. the isolated antibody has a human IgG2 isotype, in which the human IgG2 contains a constant region and in which the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU or Kabat numbering; iii. the isolated antibody is of human or mouse IgG1 isotype and contains one or more amino acid substitutions in the Fc region at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E , P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU or Kabat numbering; iv. the isolated antibody has the IgG1 isotype and contains a constant domain

- 23 042890 (CH1) of the IgG2 isotype heavy chain and the hinge region, optionally the IgG2 isotype CH1 and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS

WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS

NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397), and optionally the Fc region of the antibody contains the S267E amino acid substitution, the L328F amino acid substitution or both substitutions, and/or the N297A or N297Q amino acid substitution, the residue numbering being EU or Kabat numbering; v. the isolated antibody is of human or mouse IgG1 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any their combinations, with the numbering of the residues presented in accordance with the EU or Kabat numbering; or vi. the isolated antibody has an IgG2/4 hybrid isotype, and optionally the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with residue numbering according to EU or Kabat numbering. In certain embodiments, which may be combined with any of the preceding embodiments, an isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2 and a naturally occurring human TREM2 variant, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12, and naturally occurring human DAP12 variant. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an antibody fragment, and wherein the antibody fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an inert antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody is an antagonistic antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody inhibits one or more TREM2 activities. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, one or more inhibited TREM2 activities are selected from the group consisting of a decrease in the activity of one or more TREM2 dependent genes; reducing the activity of one or more nuclear factors of activated T-cell transcription factors (NFAT); reduced survival of macrophages, microglial cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells; reducing the expression of one or more mediators selected from the group consisting of IL-12p70, IL-6 and IL-10; and any combination of them. In some embodiments, which may be combined with any of the preceding embodiments, the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is not able to bind the Fc-gamma receptor (FcyR). In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention: i. the isolated antibody is human or mouse IgG1 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A , A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being EU or Kabat numbering, optionally the Fc region additionally containing an amino acid deletion at the position corresponding to glycine 236 according to EU or Kabat numbering; ii. the isolated antibody is human IgG2 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E and any combination thereof, with residue numbering according to EU or Kabat numbering; or iii. The isolated antibody is human or mouse IgG4 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T , T256E, N297A, N297Q, and any combination thereof, with residue numbering according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, an isolated antibody is an antibody fragment that

- 24 042890 which binds to one or more human proteins selected from the group consisting of human TREM2 and a naturally occurring human TREM2 variant. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is an antibody fragment, and wherein the antibody fragment is a fragment of Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, and the numbering of residues is presented in accordance with the numbering of the EU or Kabat. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region further comprises the amino acid substitution S228P according to EU or Kabat numbering. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering residues are presented according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a bispecific antibody that recognizes a first antigen and a second antigen. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody is a monoclonal antibody.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody specifically binds to both human TREM2 and mouse TREM2. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody has a dissociation constant (K _D ) for human TREM2 and mouse TREM2 that ranges from less than about 5.75 nM to less than about 0.09 nM. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody has a dissociation constant (KD) for the human TREM2-Fc fusion protein that ranges from less than about 1.51 nM to less than about 0.35 nM. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody has a dissociation constant (KD) for the monomeric human TREM2 protein that ranges from less than about 5.75 nM to less than about 1, 15 nM. In certain embodiments, which may be combined with any of the prior embodiments, the isolated antibody has a dissociation constant (K _D ) for the mouse TREM2-Fc fusion protein that ranges from less than about 0.23 nM to less than about 0.09 nM. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody has a dissociation constant (K _D ) for human TREM2 and mouse TREM2 that ranges from less than about 6.70 nM to less than about 0.23 nM. In certain embodiments, which may be combined with any of the preceding embodiments, the isolated antibody has a dissociation constant (KD) for the human TREM2-Fc fusion protein that ranges from less than about 0.71 nM to less than about 0.23 nM. In certain embodiments, which may be combined with any of the prior embodiments, the isolated antibody has a dissociation constant (K _D ) for the monomeric human TREM2 protein that ranges from less than about 6.70 nM to less than about 0 .66 nM. In certain embodiments, which may be combined with any of the prior embodiments, the isolated antibody has a dissociation constant (K _D ) for the mouse TREM2-Fc fusion protein that ranges from less than about 4.90 nM to less than about 0.35 nM.

Other aspects of the present description relate to the selected nucleic acid encoding the antibody according to any of the previous embodiments of the invention. Other aspects of the present description relate to a vector containing a nucleic acid according to any of the previous embodiments of the invention. Other aspects of the present description relate to a host cell containing a vector according to any of the previous embodiments of the invention. Other aspects of the present description relate to an isolated host cell containing a vector according to any of the previous embodiments of the invention. Other aspects of the present description relate to a method for producing an antibody, comprising culturing a cell according to any of the preceding embodiments of the invention, which produces an antibody. In some variants

- 25 042890 max implementation of the invention, the method further includes the selection of antibodies produced by the cell. Other aspects of the present description relate to the selected antibody, obtained by any of the previous methods for obtaining antibodies. Other aspects of the present description relate to a pharmaceutical composition containing an antibody according to any of the preceding embodiments of the invention and a pharmaceutically acceptable carrier.

Other aspects of the present disclosure relate to a method for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, and multiple sclerosis, comprising administering to the individual therapeutically an effective amount of isolated agonist antibody according to any of the previous embodiments of the invention. Other aspects of the present disclosure relate to an isolated agonist antibody of any of the previous embodiments of the invention for use in the prevention, risk reduction, or treatment of an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease. , Nasu-Hakola disease and multiple sclerosis. Other aspects of the present disclosure relate to the use of an isolated agonist antibody of any of the previous embodiments of the invention in the manufacture of a medicament for the prevention, risk reduction, or treatment of an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease and multiple sclerosis. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the individual has a heterozygous TREM2 variant, and the variant contains one or more substitutions selected from the group consisting of: i. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1; ii. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1; iii. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1; iv. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1; v. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1; vi. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1; vii. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1. the nucleotide corresponding to the nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both of these deletions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the individual has a heterozygous DAP12 variant, and the variant contains one or more variants selected from the group consisting of: i. replacing methionine with threonine at the amino acid corresponding to the amino acid residue Met1 of SEQ ID NO: 2; ii. amino acid substitution of glycine for arginine in the amino acid corresponding to the amino acid residue Gly49 of SEQ ID NO: 2; iii. deletions within exons 1-4 of the nucleic acid sequence encoding SEQ ID NO: 2; iv. inserts of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2; and v. a nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2.

Other aspects of the present description relate to a method of inducing or promoting the survival of innate immune cells of an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated agonistic antibody according to any of the previous embodiments of the invention. Other aspects of the present description relate to the selected agonist antibody according to any of the previous embodiments of the invention, for use in inducing or promoting the survival of innate immune cells of an individual in need of it. Other aspects of the present description relate to the use of an isolated agonistic antibody according to any of the preceding embodiments of the invention, in the manufacture of a medicinal product for inducing or promoting the survival of innate immune cells of an individual in need thereof. Other aspects of the present disclosure relate to a method of inducing or promoting wound healing in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated agonist antibody that binds to the TREM2 protein. Other aspects of the present disclosure relate to an isolated agonist antibody that binds to the TREM2 protein,

- 26 042890 for use in inducing or promoting wound healing in an individual in need thereof. Other aspects of the present disclosure relate to the use of an isolated agonist antibody that binds to the TREM2 protein in the manufacture of a medicament to induce or promote wound healing in an individual in need thereof.

Other aspects of the present description relate to a method for reducing the survival of innate immune cells of an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated antagonistic antibody according to any of the previous embodiments of the invention. Other aspects of the present description relate to an isolated antagonist antibody according to any of the previous embodiments of the invention, for use in reducing the survival of innate immune cells of an individual in need thereof. Other aspects of the present disclosure relate to the use of an isolated antagonist antibody of any of the preceding embodiments of the invention in the manufacture of a medicament for reducing the survival of the innate immune cells of an individual in need thereof.

Other aspects of the present disclosure relate to a method for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakol disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, disease of aging, epileptiform seizures , spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis , a disease associated with osteoporosis, Paget's disease of bone, and cancer, comprising administering to an individual a therapeutically effective amount of an isolated antibody according to any of the previous embodiments of the invention. Other aspects of the present description relate to an isolated antibody according to any of the previous embodiments of the invention, with the aim of using to prevent, reduce the risk or treat an individual having a disease, disorder or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease , multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakol's disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory disease bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, disease of aging, epileptiform seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteoporosis related disease, Paget's disease of bone, and cancer. Other aspects of the present description relate to the use of an isolated antibody according to any of the previous embodiments of the invention, in the manufacture of a medicinal product for the prevention, risk reduction or treatment of an individual having a disease, disorder or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakol disease, stroke, acute injury, chronic injury, lupus, acute and chronic colitis, wound healing, disease Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, disease of aging, epileptiform seizures, spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteoporosis-related disease, Paget's disease of the bone, and

- 27 042890 cancer.

In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated antibody is: (a) an agonist antibody; (b) an inert antibody; or (c) an antagonistic antibody. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, (a) the antibody belongs to the IgG class, the IgM class, or the IgA class; and/or (b) the antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the antibody contains one or more amino acid substitutions in the Fc region located at the position of the residue selected from the group consisting of: (a) V234A, G237A, H268Q , V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E and any combination thereof; (b) N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof; (c) L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any combination thereof; (d) N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L , M252Y, S254T, T256E and any their combinations; (e) V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof; or (f) E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, the residue numbering being given according to EU or Kabat numbering. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody: (a) binds to one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein, corresponding to amino acid residues 43-50 of SEQ ID NO: 1; or (b) one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certain embodiments of the invention, which may be combined with any of the preceding embodiments of the invention, the isolated antibody: (a) binds substantially the same TREM2 epitope as the Ab52 antibody; (b) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibody At52; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At52 monoclonal antibody; (c) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 398, or an amino acid sequence with at least about 95% homology to the amino acid sequence from SEQ ID NO: 398, HVR-H2 containing the amino acid sequence from SEQ ID NO: 399 or an amino acid sequence with at least about 95% homology with the amino acid sequence from SEQ ID NO: 399, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400, and/or wherein the light chain variable domain comprises an HVR-L1 containing the amino acid sequence of SEQ ID NO: : 401 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 401, HVR-L2 containing the amino acid sequence of SEQ ID NO: 402 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 402, and an HVR-L3 containing the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403; (d) binds essentially the same TREM2 epitope as the At21 antibody; (e) contains a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibody At21; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody; or (f) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404 or an amino acid sequence with at least about 95% homology to the amino acid sequence from SEQ ID NO: 404, HVR-H2 containing the amino acid sequence from SEQ ID NO: 405, or an amino acid sequence with at least about 95% homology with the amino acid sequence from SEQ ID NO: 405, HVR-H3 containing the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406, and/or wherein the light chain variable domain comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: : 407, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 407, HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, or

- 28 042890 an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 408, and an HVR-L3 containing the amino acid sequence of SEQ ID NO: 409 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 409. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the selected antibody is an isolated antibody according to any of the previous embodiments of the invention. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the isolated agonist antibody is an isolated agonist antibody according to any of the previous embodiments of the invention. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the individual has a heterozygous TREM2 variant, and the variant contains one or more substitutions selected from the group consisting of: i. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1; ii. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1; iii. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1; iv. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1; v. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1; vi. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1; vii. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1. the nucleotide corresponding to the nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both of these deletions. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the individual has a heterozygous DAP12 variant, and the variant contains one or more variants selected from the group consisting of: i. replacing methionine with threonine at the amino acid corresponding to the amino acid residue Met1 of SEQ ID NO: 2; ii. amino acid substitution of glycine for arginine in the amino acid corresponding to the amino acid residue Gly49 of SEQ ID NO: 2; iii. deletions within exons 1-4 of the nucleic acid sequence encoding SEQ ID NO: 2; iv. inserts of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2; and v. a nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2.

In certain embodiments, which may be combined with any of the preceding embodiments, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer. , kidney cancer, renal cell carcinoma, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the method further comprises administering to the individual at least one antibody that specifically binds to a checkpoint inhibitory molecule and/or other standard or experimental cancer therapy. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with the isolated antibody. In certain embodiments of the invention that may be combined with any of the preceding embodiments of the invention, at least one antibody that specifically binds to a checkpoint inhibitory molecule is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA4 antibody, an antibody to PD-L2, antibodies to PD-1, antibodies to B7-H3, antibodies to B7-H4, antibodies to HVEM, antibodies to B- and T-lymphocyte attenuator (BTLA), antibodies to cytotoxicity inhibition receptor (KIR), antibodies KGAL9, TIM3 antibodies, A2AR antibodies, LAG-3 antibodies, phosphatidylserine antibodies, KCD27 antibodies, and any combination thereof. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the standard or experimental anticancer therapy is one or more therapies selected from the group consisting of radiotherapy, chemotherapy, targeted therapy, imatinib (Gleevec®), trastuse

- 29 042890 mab (Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy and cytokine therapy. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory cytokine. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an isolated antibody. In certain embodiments that may be combined with any of the preceding embodiments, at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of a KCCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody. and any combination of them. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the method further comprises administering to the individual at least one agonistic antibody that specifically binds to a checkpoint stimulating protein. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, at least one agonist antibody that specifically binds to a checkpoint stimulating protein is administered in combination with the isolated antibody. In certain embodiments, which may be combined with any of the preceding embodiments, at least one agonist antibody that specifically binds to the checkpoint stimulating protein is selected from the group consisting of an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, an ICOS agonist antibody, an anti-CD28 agonist antibody, an anti-CD137/4-1BB agonist antibody, an anti-CD27 agonist antibody, an agonist antibody for glucocorticoid-induced TNFR-related GITR protein, and any combination thereof. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, the method further comprises administering to the individual at least one stimulatory cytokine. In certain embodiments of the invention, which can be combined with any of the previous embodiments of the invention, at least one stimulatory cytokine is administered in combination with an isolated antibody. In certain embodiments of the invention that may be combined with any of the preceding embodiments of the invention, at least one stimulatory cytokine is selected from the group consisting of TNF-α, IL-10, IL-6, IL-8, CRP, members chemokine protein families TGF-beta, members of the IL-20, IL-33, FIL, OSM, CNF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α, IFN families -β, IL-2, IL-18, GM-CSF, G-CSF and any combination thereof.

Brief description of graphic materials

In FIG. 1A illustrates an amino acid sequence alignment between the human TREM2 protein (SEQ ID NO: 426) and the human NCTR2 protein (SEQ ID NO: 427) showing the homology between the two proteins. The consensus sequence is SEQ ID NO: 446. FIG. 1B illustrates structure-based sequence alignment between several TREM proteins and other family members, human TREM-1 (SEQ ID NO: 429), human TREM-2 (SEQ ID NO: 430), mouse TREM-1 (SEQ ID NO: 431). ), mouse TREM-2 (SEQ ID NO: 432), mouse TREM-3 (SEQ ID NO: 433), NKp44 (SEQ ID NO: 434), human aTCR (SEQ ID NO: 435), human bTCR (SEQ ID NO: 436), human gTCR (SEQ ID NO: 437), human dTCR (SEQ ID NO: 438), human Vd (SEQ ID NO: 439), mouse hIGG1 (SEQ ID NO: 440), mouse l!GG1 ( SEQ ID NO: 441), human CD8 (SEQ ID NO: 442), and human CTLA4 (SEQ ID NO: 443). The numbering of amino acid residues corresponds to the mature human TREM1 protein sequence. The secondary structure elements of TREM1 are illustrated as arrows for β strands and cylinders for α helices. Amino acid residues involved in the formation of homo- and heterodimers are shown on a black background. Cysteine residues that form disulfide bonds and that are conserved for Ig type V folding are indicated in bold and marked with asterisks. Spaces are marked with -. M-1 residues that disrupt the mode of formation of the antibody-like dimer are marked with closed triangles as in (eg, Radaev et al., (2003) Structure. 11 (12): 1527-1535).

In FIG. 2A illustrates an amino acid sequence alignment between the human TREM1 protein (SEQ ID NO: 428) and the human TREM2 protein (SEQ ID NO: 426) showing the homology between the two proteins. The consensus sequence is SEQ ID NO: 447. FIG. 2B illustrates the amino acid sequences of the heavy chain variable regions of the antibodies At21 and At52. The CDR sequences are underlined in each sequence. The sequence regions in between each underlined CDR sequence correspond to the frame regions. In FIG. 2C illustrates the amino acid sequences of the light chain variable regions of the antibodies At21 and At52. CDR sequences are underscore

- 30 042890 nutted in each sequence. The sequence regions in between each underlined CDR sequence correspond to the frame regions.

In FIG. 3A illustrates FACS histograms showing the binding of Ab21, At52, At16, At20, At66 and At68 TREM2 antibodies to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2. In FIG. 3B illustrates Ab21 and At52 antibodies binding to DT (Trem +/+) and TREM2 (TREM2 -/-) mouse bone marrow macrophages (BMMac). Shaded histograms show a negative population of TREM2 antibodies. The black outlined histograms show a positive TREM2 antibody population.

In FIG. 4A illustrates FACS histograms showing the binding of Ab21, At52, At43, and At60 TREM2 antibodies to a human (293) cell line expressing the recombinant human TREM2-DAP12 fusion protein. The shaded histograms show the TREM2 antibody negative population. The black outlined histograms show a positive TREM2 antibody population. In FIG. 4B illustrates Ab21, At52, At43, and At60 antibodies binding to human primary dendritic cells (hDCs). Shaded histograms show negative control of secondary antibodies only. The black outlined histograms show a positive TREM2 antibody population.

In FIG. 5A illustrates FACS dot plots demonstrating the expression of cell surface markers CD83 and CD86 on human dendritic cells (DCs) after incubation with plate-bound anti-TREM2 antibodies At21 or At52. The Ab88 antibody is a negative isotype control. Graphs were based on CD11c ⁺ HLA-DR+ LIN-DC threshold. The percentage of cells within the CD83+CD86+ threshold is displayed on each graph. In FIG. 5B illustrates FACS histograms showing expression of the cell surface marker CD86 on human dendritic cells (DCs) after incubation with anti-TREM2 cross-linked At21 or At52 antibodies. The antibodies were cross-linked with a human secondary antibody. The Ab88 antibody is a negative isotype control.

In FIG. 6A illustrates Western blot Syk phosphorylation in TREM2-deficient (Trem2 -/-) and wild-type mice (left and center panels) and human macrophages (right panel) after incubation with anti-TREM2 antibodies At21 and At52. Antibodies At89 and At92 are non-agonistic negative controls. In FIG. 6B illustrates Western blot Syk phosphorylation in primary human dendritic cells after incubation with anti-TREM2 antibodies At21 and At52.

In FIG. 7A illustrates Western blot DAP12 phosphorylation in mouse macrophages after incubation with anti-TREM2 antibody At52. In FIG. 7B illustrates DAP12 phosphorylation as determined by Western blotting in mouse wild-type macrophages and TREM2-deficient (TREM2 -/-) mouse macrophages after incubation with anti-TREM2 Ab21 antibody.

In FIG. 8 illustrates competitive binding between TREM2 antibodies and E. coli bacteria expressing the putative TREM2 ligand with mouse and human TREM2 expressing cell lines. Bacterial binding is expressed as a percentage of control. Average of two independent experiments; black bars: no difference in isotype control, red bars: significant difference from isotype control (ANOVA).

In FIG. 9A illustrates the protein levels of the inflammatory cytokines TNFa, IL-6, IL-10, and MHB-1 secreted in response to stimulation of DT and TREM2 KO macrophage macrophages by inflammatory mediators LPS or zymosan. In FIG. 9B illustrates the protein levels of the inflammatory cytokines IL-6 and TNFa secreted in response to stimulation of DT macrophages, heterozygous (Het) TREM2 and TREM2 KO macrophages by IL-4 or IFNg.

In FIG. 10A illustrates FACS data demonstrating the expression of cell surface markers CD86 and CD206 on DT macrophages, heterozygous (Het) TREM2 and TREM2 KO macrophages after stimulation with cytokines IL-4 or IFNg. In FIG. 10B illustrates expression of the cell surface marker CD86 on DT macrophages and TREM2 KO macrophages after stimulation with inflammatory mediators LPS or Zymosan.

In FIG. 11A illustrates the number of live DT macrophages heterozygous for TREM2 (TREM2 +/-) and TREM2 KO (TREM2 -/-) after culturing in growth factor MKSF for the indicated number of days. In FIG. 11B illustrates FACS plots showing staining of DT macrophages heterozygous for TREM2 (TREM2 +/-) and TREM2 KO (TREM2 -/-) after culture in

MCSF for 6 days (+ MCSF) or in MCSF for 4 days, after which MCSF was not used for 36 hours (- MCSF). The percentage of live macrophages within the CD11b+DAPI threshold is indicated on each graph. In FIG. 11C illustrates the levels of luminescence detected in the luciferase viability assay after culturing DT and TREM2 KO dendritic cells, M1 macrophages, and M2 macrophages in growth factors GM-CSF, M-CSF, or M-CSF+IL-4, respectively. In FIG. 11D shows the frequency of live TD macrophages heterozygous for TREM2 (Het) and TREM2 KO (CD11b+) after cultivation in inflammatory mediators IFNg, LPS, or zymosis.

- 31 042890 no.

In FIG. 12 illustrates the phagocytosis of apoptotic cells and E. coli macrophages derived from bone marrow (BMmacs), wild type (DT) and TREM2 KO (TREM2 -/-) cultured without the addition of MCSF.

In FIG. 13 illustrates the epitope map of the anti-TREM2 antibody At52.

In FIG. 14 illustrates Syk phosphorylation as determined by Western blotting in wild-type mouse macrophages deficient in TREM2 (Trem2 -/-) after incubation with anti-TREM2 or 78.18 antibody MAB17291 (RD), demonstrating that antibody 78.18 does not induce Syk phosphorylation or TREM2 signaling. .

In FIG. 15A illustrates a Fortebio assay demonstrating the simultaneous binding of the MAB17291 antibody and the At21 antibody to TREM2-Fc. In FIG. 15A illustrates a Fortebio assay demonstrating the simultaneous binding of the MAB17291 antibody and the At52 antibody to TREM2-Fc.

In FIG. 16 illustrates the percentage increase in survival of macrophages derived from the bone marrow of wild-type (DT) and TREM2 knockout (KO) mice cultured in the presence of plate-bound, cross-linked anti-TREM2 Fabs antibody At21 or At52 and M-CSF. The Ab88 antibody is a negative isotype control.

In FIG. 17A illustrates a luminescence viability assay of mouse bone marrow-derived macrophages cultured in the presence of soluble, non-cross-linked, anti-TREM2 Fabs antibody At21 or At52 and M-CSF. The Ab99 antibody is a negative isotype control. In FIG. 17B illustrates a luminescence viability assay of mouse bone marrow-derived macrophages cultured in the presence of soluble full-length anti-TREM2 antibody At21 or At52 and M-CSF. The Ab91 antibody is a negative isotype control. The dotted line NT indicates the average viability obtained from untreated macrophages (without the addition of antibodies). The dotted line without MCSF indicates the average viability obtained by culturing macrophages in the absence of M-CSF.

In FIG. 18A illustrates the induction of TREM2-dependent gene expression with plate-bound, full-length anti-TREM2 antibodies, At21 and At52, using the luciferase reporter gene in cell assay. In FIG. 18B illustrates the induction of TREM2-dependent gene expression with plate-bound phasphatidylserine (PS). In FIG. 18C illustrates activation of the TREM2-dependent IL-6 gene in mouse macrophages by plate-coupled At21 and At52 Fab anti-TREM2 Fab antibodies. In FIG. 18D illustrates activation of the TREM2 dependent MCB-1 gene in mouse macrophages by plate-coupled At21 and At52 Fab anti-TREM2 Fab antibodies. The data in FIG. 18C and 18D are illustrated as means±SD; n=3 mice per group. In FIG. 18C and 18D without Ab illustrates the negative control without antibody treatment, 21 denotes treatment with At21 Fab, 52 denotes treatment with At52 Fab, and kp denotes treatment with control Fab antibody.

In FIG. 19 illustrates the inhibition of TREM2-dependent gene expression by soluble, full-length anti-TREM2 antibodies, At21 and At52, using the luciferase reporter gene in a cell-based assay.

In FIG. 20A illustrates the amino acid sequences of the heavy chain variable regions of anti-TREM2 antibodies. The CDR sequences are underlined in each sequence. The sequence regions in between each underlined CDR sequence correspond to the frame regions. In FIG. 20B illustrates the amino acid sequences of the light chain variable regions of anti-TREM2 antibodies. The CDR sequences are underlined in each sequence. The sequence regions in between each underlined CDR sequence correspond to the frame regions.

In FIG. 21A illustrates FACS histograms showing the binding of the antibodies At1, At9, At14, At22, At45 and At65 TREM2 to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2. In FIG. 21B illustrates antibodies At1, At9, At14, At22, At45, and At65 binding to DT (Trem +/+) and TREM2-deficient (TREM2 -/-) mouse bone marrow macrophages (BMMac). The Ab88 antibody is a negative isotype control. Shaded histograms show a negative population of TREM2 antibodies. The black outlined histograms show a positive TREM2 antibody population.

In FIG. 22A illustrates FACS histograms showing TREM2 antibody binding of At1, At9, At14, At22, At43, At45, At60, and At65 to a human (293) cell line expressing recombinant human TREM2-DAP12 fusion protein. The shaded histograms show the TREM2 antibody negative population. The black outlined histograms show a positive TREM2 antibody population. In FIG. 22B illustrates antibodies At1, At9, At14, At22, At43, At45, At60, and At65 binding to human primary dendritic cells (hDCs). The Ab88 antibody is a negative isotype control. Shaded histograms show negative control of secondary antibodies only. The black outlined histograms show a positive TREM2 antibody population.

- 32 042890

In FIG. 23A illustrates FACS dot plots demonstrating the expression of cell surface markers CD83 and CD86 on human dendritic cells (DCs) after incubation with TREM2 plate bound antibodies At1, At9, At14, At22, At45, and At65. The Ab88 antibody is a negative isotype control. Graphs were based on CD11c ⁺ HLA-DR+ LIN threshold ^- DC. The percentage of cells within the CD83+CD86+ threshold is displayed on each graph. In FIG. 23B illustrates FACS histograms showing expression of the cell surface marker CD86 on human dendritic cells (DCs) after incubation with TREM2 cross-linked antibodies At1, At9, At14, At22, At45, and At65. The antibodies were cross-linked with a human secondary antibody. The Ab88 antibody is a negative isotype control.

In FIG. 24A illustrates Syk phosphorylation as determined by Western blotting in TREM2 deficient mouse wild-type macrophages (Trem2 -/-) after incubation with anti-TREM2 antibodies At1, At9 or At45. Antibodies At89 and At92 are negative isotype controls. In FIG. 24B illustrates Syk phosphorylation as determined by Western blotting in human macrophages after incubation with anti-TREM2 antibodies At1, At9, At14, At20, At22, At45, and At65. Antibodies At16 and At77 are non-agonistic negative controls. In FIG. 24C illustrates Syk phosphorylation as determined by Western blotting in primary human dendritic cells after incubation with At1, At5, At9, At22, At45, or At65k TREM2 antibodies.

In FIG. 25 illustrates competitive binding between anti-TREM2 antibodies At1, At9, At14, At22, At45, At65, At66 and At68 and E. coli cells expressing the putative TREM2 ligand with mouse and human TREM2 expressing cell lines. Bacterial binding is expressed as a percentage of control. Average of two independent experiments; black bars: no difference in isotype control, red bars: significant difference from isotype control (ANOVA).

In FIG. 26A illustrates DAP12 phosphorylation as determined by western blotting in mouse macrophages after incubation with anti-TREM2 antibody At45 or At65. In FIG. 26B illustrates DAP12 phosphorylation as determined by Western blotting in mouse wild-type macrophages and TREM2-deficient (TREM2 -/-) mouse macrophages after incubation with anti-TREM2 antibody At1, At9, At22, or At45.

In FIG. 27A illustrates the epitope map of the anti-TREM2 antibodies At1 and At9. In FIG. 27B illustrates an epitope map of the anti-TREM2 antibodies At45 and At65.

In FIG. 28 illustrates a Fortebio assay demonstrating the simultaneous binding of the MAB17291 antibody and the antibodies At1, At9, At14, At22, At45 and At65 to TREM2-Fc. Control antibodies At63 and At87 did not bind simultaneously to TREM2-Fc.

In FIG. 29 illustrates the percentage increase in survival of macrophages derived from the bone marrow of wild-type (DT) and TREM2 knockout (KO) mice cultured in the presence of plate-bound cross-linked anti-TREM2 Fabs At22, At45 or At65 antibody and M-CSF. The Ab88 antibody is a negative isotype control.

In FIG. 30A illustrates a luminescence viability assay of mouse bone marrow-derived macrophages cultured in the presence of Fab soluble, non-crosslinked anti-TREM2 antibody and M-CSF. The Ab99 antibody is a negative isotype control. In FIG. 30B illustrates a luminescence viability assay of mouse bone marrow-derived macrophages cultured in the presence of soluble full-length antibodies to TREM2 and M-CSF. The Ab91 antibody is a negative isotype control. The dotted line NT indicates the average viability obtained from untreated macrophages (without the addition of antibodies). The dotted line without MCSF indicates the average viability obtained by culturing macrophages in the absence of M-CSF.

In FIG. 31A illustrates the induction of TREM2-dependent gene expression with plate-bound, full-length anti-TREM2 antibodies using the luciferase reporter gene in cell assay. In FIG. 31B illustrates the induction of TREM2-dependent gene expression with plate-bound, full-length anti-TREM2 antibodies using the luciferase reporter gene in a cellular assay. In FIG. 31C illustrates the induction of TREM2 dependent gene expression with plate-bound phasphatidylserine (PS).

In FIG. 32 illustrates the inhibition of TREM2-dependent gene expression by soluble, full-length anti-TREM2 antibodies using the luciferase reporter gene in a cellular assay.

In FIG. 33 illustrates competitive binding between anti-TREM2 antibodies At22 and At45 and Phosphatidylserine (PS) or Sphingomyelin (SM) in mouse and human TREM2-expressing cell lines.

In FIG. 34A illustrates activation of the TREM2-dependent IL-6 gene in mouse macrophages by plate-coupled At22 and At65 Fab anti-TREM2 Fab antibodies. In FIG. 34B illustrated

TREM2-3aeucuMoro activation of the MHB-1 gene in mouse macrophages with plate-bound At22 and At65 Fab anti-TREM2 Fab antibodies. The data in FIG. 34A and 34B are illustrated as means±SD; n=3 mice per group. In FIG. 34A and 34B without Ab illustrates the negative control without antibody treatment, 22 denotes treatment with At22 Fab, 65 denotes treatment with At65 Fab, and kp denotes treatment with control Fab antibody.

Detailed Description of the Invention

General methods.

The methods and procedures described or referred to herein are generally well known and commonly used using conventional techniques by those skilled in the art, such as, for example, the widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

Definitions.

As used herein, the term prevention includes providing prophylaxis against the occurrence or recurrence of a particular disease, disorder, or condition in an individual. An individual may be predisposed, susceptible to, or at risk of developing such a disease, disorder, or condition, but not yet diagnosed with the disease, disorder, or condition.

As used herein, an individual at risk for developing a particular disease, disorder, or condition may or may not have a detected disease or symptoms of a disease, and may or may not have evidence of a detected disease or symptoms of a disease prior to using the methods of treatment described herein. At risk means that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or pathological condition, as is known in the art. An individual with one or more of these risk factors is more likely to develop a particular disease, disorder or condition than an individual without one or more of these risk factors.

In this context, the term treatment refers to clinical interventions aimed at changing the natural course of the disease in the individual being treated, in the process of clinical pathology. Desired effects of treatment include a reduction in the rate of progression, a decrease in intensity or a temporary amelioration of the disease state, and remission, or an improved prognosis of a particular disease, disorder, or disease state. An individual is successfully treated, for example, if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

An effective amount refers to an amount effective at such dosages and for such periods of time as are necessary to achieve the desired therapeutic or prophylactic result. An effective amount may be provided in one or more administrations.

A therapeutically effective amount is at least the minimum concentration necessary to effect a measurable improvement in a particular disease, disorder, or condition. In this context, a therapeutically effective amount may vary depending on such factors as the course of the disease, the age, sex and body weight of the patient and the ability of the anti-TREM2 antibody and/or anti-DAP12 antibody to elicit the desired response in the individual. A therapeutically effective amount is also the amount at which the therapeutically beneficial effects of an anti-TREM2 antibody and/or an anti-DAP12 antibody are superior to

- 34 042890 toxic or harmful effects.

In this context, administration in combination with another compound or composition includes simultaneous administration and/or administration at different times. Administration in combination also includes administration as both a co-formulation and administration in the form of separate compositions, including different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

Individual for the purpose of treatment, prevention or risk reduction refers to any animal belonging to the class of mammals, including humans, domestic and farm animals, as well as zoo animals, animals for sports activities or pets, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats and the like. Preferably, the subject is a human.

In this context, the term immunoglobulin (Ig) is used interchangeably with the term antibody. As used herein, the term antibody is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

The basic unit of a 4-chain antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. Combining a pair of VH and V _L leads to the formation of a single antigen-binding site. The structure and properties of various classes of antibodies are presented in, for example, Basic and Clinical Immunology, 8 Ed, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, pp 71 Vol 6.

The L chain of any vertebrate species can be assigned to one of two distinct types, called kappa (k) and lambda (λ), based on the amino acid sequences of their conserved domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains labeled alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively. The γ and α classes are further subclassified based on relatively minor differences in CH sequence and function, for example, the following subclasses are expressed in humans: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structure and three-dimensional configuration of various classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Molecular Immunology, 4 Ed. (W.B. Saunders Co., 2000).

Native antibodies are typically heterotetrameric glycoproteins with a molecular weight of approximately 150,000 Da, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has equally spaced intrachain disulfide bridges. At one end of each heavy chain is a variable domain (VH) and then a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. Specific amino acid residues are expected to form a region of separation between the light chain and heavy chain variable domains.

An isolated antibody, such as an isolated anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein, is an antibody that is identified and isolated and/or recovered from a component of its natural environment (eg, naturally or recombinantly). Preferably, the isolated polypeptide is not associated with all other contaminant components from its natural environment. Contaminant components from its natural environment, such as those derived from recombinant transfected cells, are materials that may interfere with research, diagnostic, or therapeutic applications of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solubles. substances. In preferred embodiments, the polypeptide will be: (1) greater than 95% by weight of the antibody, according to, for example, Lowry's method, and in some embodiments, greater than 99% by weight; (2) to a level sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating dish sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie staining blue or preferably silver staining. An isolated antibody includes the antibody in situ in recombinant T cells since at least one component of the antibody's natural environment will not be present. In this case, as a rule, the isolated polypeptide or

- 35 042890 antibody will be obtained by at least one purification step.

The variable region or variable domain of an antibody such as an anti-TREM2 antibody or an anti-DAP12 antibody as used herein refers to the amino-terminal domains of an antibody heavy or light chain. The heavy chain and light chain variable domains may be referred to as VH and V _L , respectively. These domains are usually the most variable parts of an antibody (compared to other antibodies of the same class) and contain antigen-binding sites.

The term "variable" refers to the fact that, among antibodies, certain segments of the variable domains differ greatly in sequence, such as anti-TREM2 and/or anti-DAP12 antibodies as described herein. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. At the same time, variability is not evenly distributed over the entire range of variable domains. Instead, it is concentrated in three segments of both the light and heavy chain variable domains called hypervariable regions (HVRs). More conserved fragments of variable domains are called framework regions (FRs). Each native light and heavy chain variable domain contains four FRs predominantly in the beta-sheet configuration and connected by three HVRs that form loops connecting, and in some cases part of, the beta-type structures. The HVRs of each strand are associated with each other in close proximity to the FR regions and, together with the HVRs of the other strand, participate in the formation of the antibody antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5 Ed, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

As used herein, the term monoclonal antibody refers to an antibody, such as an anti-TREM2 and/or DAP12 monoclonal antibody as described herein, derived from a population of substantially homogeneous antibodies, ie. from a population in which individual antibodies are identical, with the exception of possible natural mutations and/or post-translational modifications (eg, isomerization, amidation, etc.), which may be present in small quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Unlike polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against one determinant per antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier monoclonal indicates the property of the antibody as being derived from a substantially homogeneous population of antibodies and should not be interpreted as a requirement for antibody production by any particular method. For example, monoclonal antibodies contemplated for use in accordance with the present invention can be prepared by various methods, including, for example, the hybridoma method (eg, Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al. , Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 Ed. 1988) Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, for example, US Pat. No. 4,816,567), phage display methods (see, for example, Clackson et al., Nature, 352 :624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 ( 2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA 101 (34): 12467-472 (2004 and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for the production of human antibodies or human antibody analogues in animal organisms that partially or completely contain immunoglobulin loci or genes. human coding sequences for human immunoglobulins (see, for example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jacobovits et al., Proc. Nat'l Acad. sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US patents No. 5545807; 5545806; 5569825; 5625126; 5633425; and 5661016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

The terms full antibody, intact antibody, or complete antibody are used interchangeably to refer to an antibody, such as an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein, in its substantially intact form, as opposed to an antibody fragment. More specifically, full antibodies include those having heavy and light chains, including an Fc fragment. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

An antibody fragment contains a portion of an intact antibody, preferably an antigen-binding

- 36 042890 or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabody; linear antibodies (see US patent No. 5641870, example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies, such as anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, produces two identical antigen-binding fragments, termed Fab fragments, and a residual Fc fragment, indicating good crystallizability. The Fab fragment consists of the entire L chain together with the H chain variable region domain (VH) and the first constant domain of one of the heavy chains ( _CH 1 ). Each Fab fragment is monovalent with respect to antigen binding, ie. contains a single antigen binding site. Treatment of the antibody with pepsin allows the isolation of a single large F(ab')2 fragment, which approximately corresponds to two Fab fragments with different antigen-binding activity, connected by a disulfide bond, and retains the ability to cross-link the antigen. Fab' fragments differ from Fab fragments in that they carry several additional residues at the carboxy-terminus of the C _H 1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH in this document is the designation Fab', in which the cysteine residue(s) in the constant domains bears(ut) a free thiol group. Antibody fragments F(ab') ₂ originally received in the form of pairs of Fab' fragments, between which are hinged cysteine residues. In addition, other variants of the chemical connection of antibody fragments are known.

The Fc fragment contains the carboxy-terminal fragments of both H-chains connected to each other by disulfide bonds. The effector functions of antibodies are determined by the sequences of the Fc region, which is also recognized by Fc receptors (FcR) found on some cell types.

Fv is the minimum antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of the variable domains of one heavy and one light chain, tightly linked through non-covalent bonds. When these two domains are folded, six hypervariable loops protrude (3 loops each from the H- and L-chains), the amino acid regions of which are involved in binding to the antigen and give the antibody specificity for antigen binding. At the same time, even a single variable domain (or half of the Fv fragment, including only three antigen-specific HVRs) has the ability to recognize and bind an antigen, albeit with a lower affinity compared to a full-length binding site.

Single chain Fvs, also abbreviated as sFv or scFv, are antibody fragments containing VH and VL domains joined in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore red, Springer-Verlag, New York, pp 269-315 (1994).

Functional antibody fragments, such as anti-TREM2 and/or DAP12 antibodies as described herein, comprise an intact antibody portion, typically including an antigen-binding or variable region of an intact antibody, or an antibody F region that retains FcR binding capacity or has a modified FcR binding capacity. Examples of antibody fragments include a linear antibody, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term diabody refers to small antibody fragments obtained by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of the V domains, leading to the formation bivalent fragment, i.e. fragment carrying two antigen binding sites. Bispecific diabodies are heterodimers of two overlapping sFv fragments in which the VH and _VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161 and Hollinger et al., Proc. Nat'l Acad. sci. USA 90:6444-48 (1993).

As used herein, a chimeric antibody refers to an antibody (immunoglobulin), such as a chimeric anti-TREM2 and/or DAP12 antibody of the present disclosure, in which the heavy and/or light chain portion is identical or homologous to the corresponding sequences in antibodies derived from a particular species. or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US patent No. 4816567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, wherein the antigen-binding region of the antibody is formed by an antibody obtained, for example, by immunizing monkeys with an antigen of interest. In this context, a humanized antibody is used as a subtype of chimeric antibodies.

- 37 042890

Humanized forms of non-human (eg, murine) antibodies, such as the humanized forms of anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the HVR of the recipient are replaced with HVR residues from a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity and capacity. In some cases, human immunoglobulin FR residues are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient's antibody or in the donor's antibody. These modifications can be made to further improve antibody performance, such as binding affinity. Typically, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains wherein all or substantially all of the hypervariable loops correspond to hypervariable loops of a non-human immunoglobulin sequence, and all or substantially all of the regions FRs are FR regions of a human immunoglobulin sequence, although FR regions may include substitution of one or more distinct FR residues that improve antibody characteristics such as binding affinity, isomerization, immunogenicity, and the like. The number of these amino acid substitutions in FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. For more information, see, for example, Jones et al., Nature 321:522525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, All. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428433 (1994); and US Pat. Nos. 6,982,321 and 7,087,409.

A human antibody is an antibody that has an amino acid sequence corresponding to that of an anti-TREM2 antibody and/or an anti-DAP12 antibody disclosed herein, produced in a human and/or obtained using any of the methods for producing human antibodies as described herein. Specifically excluded from this definition of a human antibody is a humanized antibody containing antigen-binding residues of non-human origin. Human antibodies can be obtained using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be produced by administering an antigen to a transgenic animal that is modified to produce such antibodies in response to immunization with an antigen and whose endogenous loci are blocked, such as those immunized with xenomice (see, for example, US Pat. . See also, for example, Li et al., Proc. Nat'l Acad. sci. USA, 103:3557-3562 (2006) for human antibodies produced by human B-cell hybridoma technology.

As used herein, the term hypervariable region, HVR or HV refers to regions of the variable domain of an antibody, such as in an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, that possess hypervariable sequences and/or form structurally defined loops. Typically, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 show the most diversity of the six HVRs, and in particular, H3 is believed to play a unique role in providing high antibody specificity. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, natural camelid antibodies, consisting only of a heavy chain, are functional and stable in the absence of a light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

This document uses and covers a number of HVR definitions. HVR regions, which are Kabat Complementarity Determining Regions (CDRs), are based on sequence variability and are the most widely used (Kabat et al., supra). In contrast, Chothia pays attention to the localization of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVR regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modulation software. Contact HVRs are based on the analysis of available complex crystal structures. Remains from each of these HVRs are listed below.

- 38 042890

Loop Kabat AbM Chothia Contact

LI L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

Hl H31-H35B H26-H35B H26-H32 H30-H35B (no Kabat numbering)

Hl H31-H35 H26-H35 H26-H32 H30-H35 (no Chothia numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

HVR regions may contain extended HVRs as follows: 24-36 or 24-34 (L1), 4656 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102 94-102 or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al., supra, for each of these HVR extended definitions.

Framework residues or FRs are variable domain residues other than HVR residues as defined herein.

The expression Kabat variable domain numbering or Kabat amino acid position numbering, and variants thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains from the collection of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR variable domain. For example, a heavy chain variable domain can include a single amino acid insertion (residue 52a according to Kabat) after residue 52 H2 and inserted residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. Kabat numbering of residues can be determined for a given antibody by aligning regions of antibody sequence homology with a standard Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest. 5 Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Typically, the EU numbering system or EU suffix is used when referring to a residue in an immunoglobulin heavy chain constant region (eg, the EU suffix described in Kabat et al., supra). The Kabat EU index refers to the residue numbering of the EU human IgG1 antibody. References to residue numbers in the variable domain of antibodies mean the numbering of residues according to the Kabat numbering system. References to residue numbers in the constant domain of antibodies refer to residue numbering according to the EU numbering system (eg, see US Patent Publication No. 2010-280227).

As used herein, a human acceptor framework is a framework containing a VL or VH framework amino acid sequence derived from a human immunoglobulin framework or a human framework consensus sequence. A human acceptor framework region formed from a human immunoglobulin framework region or a human framework consensus sequence may contain the same amino acid sequence as these regions or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. If pre-existing amino acid changes are present in the VH, preferably these changes occur only at three, two or one position 71H, 73H and 78H; for example, amino acid substitutions at these positions can be 71A, 73T and/or 78A. In one embodiment, the human VL acceptor framework sequence is identical to the human VL framework sequence or the human VL framework consensus sequence.

The human framework consensus sequence is a framework that represents the most frequently occurring amino acid residues in a selection of VL or VH human immunoglobulin framework sequences. Typically, the selection of VL or VH human immunoglobulin sequences is made from a subset of the variable domain sequences. Typically, a subset of sequences is a subset of Kabat et al., Sequences of Proteins of Immunological Interest, 5 Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for VL, the subgroup may be kappa I, kappa II, kappa I, kappa IV, as in Kabat et al., supra. Additionally for VH, the subgroup may be subgroup I, subgroup II, or subgroup III, as in Kabat et al., supra.

Modification of amino acids at a specified position, for example, an antibody such as an anti-TREM2 antibody or an anti-DAP12 antibody as used herein refers to a substitution or deletion of a specified residue, or the insertion of at least one amino acid residue adjacent to a specified residue.

- 39 042890

An insert next to a specified residue means an insert within one or two residues of it. The insert may be N-terminal or C-terminal with respect to the specified residue. The preferred amino acid modification herein is substitution.

An affinity matured antibody, such as an anti-TREM2 antibody and/or an affinity matured anti-DAP12 antibody of the present disclosure, is an antibody with one or more changes in one or more of its HVRs that result in an increase in the antibody's affinity for the antigen from baseline. an antibody that does not possess the change(s). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are prepared by methods known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation via VH and VL domain shuffling. Random mutagenesis of HVR residues and/or framework region is described, for example: Barbas et al Proc Nat. Acad. sci. USA 91:3809-3813 (1994); Schier et al Gene 169:147-155 (1995); Yelton et al J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

In this context, the term specifically recognizes or specifically binds refers to measurable and reproducible interactions, such as attraction or binding between a target and an antibody, e.g. heterogeneous population of molecules, including biological molecules. For example, an antibody, such as an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein, that specifically or preferentially binds a target or epitope is an antibody that binds that target or epitope with greater affinity, avidity, more readily, and/or more longer than it binds to other targets or other target epitopes. It also becomes clear from this definition, for example, that an antibody (or fragment thereof) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, specific binding or preferred binding does not necessarily require (although it may include) exclusive binding. An antibody that specifically binds to a target may have an association constant of at least about 103 M ^-1 or 10 ⁴ M ^-1 , sometimes about 105 M ^-1 or 106 M ^-1 , in other cases about 106 M ^-1 or 10 ⁷ M ^-1 , about 108 M ^-1 to 10 ⁹ M ^-1 , or about 10 ¹⁰ M ^-1 to 10 ¹¹ M ^-1 or higher. Various immunoassays can be used to select for antibodies having specific immunoreactivity for a particular protein. For example, ELISA immunoassays, which have become commonplace, are used to select for monoclonal antibodies that have specific immunoreactivity with a protein. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of the formats and conditions of an immunoassay that can be used to determine specific immunoreactivity.

In this context, the interaction between the TREM2 protein or DAP12 protein and the second protein includes, but is not limited to, protein-protein interaction, physical interaction, chemical interaction, bonding, covalent bonding, and ionic bonding. In this context, an antibody inhibits an interaction between two proteins when the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody or fragment thereof according to the present disclosure inhibits the interaction between two proteins if the antibody or fragment thereof binds to one of the two proteins.

An agonist or activating antibody is an antibody, such as an anti-TREM2 agonist antibody or an anti-DAP12 agonist antibody of the present disclosure, that induces (eg, increases) one or more activities or functions of an antigen after the antibody binds to the antigen.

An antagonist or blocking antibody is an antibody, such as an anti-TREM2 agonist antibody or an anti-DAP12 agonist antibody of the present disclosure, that reduces or eliminates (e.g., reduces) the binding of an antigen to one or more ligands after the antibody has bound to the antigen, and /or, which reduces or eliminates (eg, reduces) one or more activities or functions of the antigen after the antibody binds to the antigen.

The effector functions of an antibody refer to the biological activity conferred by the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody and vary depending on the isotype of the antibody.

As used herein, the term Fc region is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as the region from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The C-terminal lysine residue (EU residue 447) of the Fc region can be removed, for example, during antibody production or purification, or by

- 40 042890 recombinant construction of a nucleic acid encoding the heavy chain of an antibody. Accordingly, an intact antibody composition may comprise antibody populations that have all K447 residues removed, antibody populations that do not have any K447 residue removed, and antibody populations containing a mixture of antibodies with and without a K447 residue. Suitable native sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2, IgG3 and IgG4.

The native sequence Fc region contains an amino acid sequence identical to the amino acid sequence of the native Fc region. Native sequence Fc regions include the native sequence Fc region of human IgG1 (non-A and A allotypes); Fc region of native human IgG2 sequence; Fc region of native human IgG3 sequence; and Fc region of native human IgG4 sequence, as well as naturally occurring variants thereof.

The variant Fc region contains an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by modifying at least one amino acid, preferably by one or more amino acid substitution(s).

Preferably, the Fc region variant contains at least one amino acid substitution compared to the native sequence of the Fc region or to the Fc region of the parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region as described herein is preferably at least about 80% homologous to the Fc region of the native sequence and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homologous to them, more preferably at least about 90% homologous to them. least by about 95%.

Fc receptor or FcR describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is a receptor that binds. Moreover, a preferred FcR is a receptor that binds an IgG antibody (gamma receptor) and includes receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibiting receptor), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The FcyRIIA activating receptor contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain. (See, for example, M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, (Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab Clin Med 126:330-41 (1995) The term FcR as used herein encompasses other FcRs to be identified in the future FcRs can also increase the half-life of antibodies.

The in vivo human FcRn binding assay and serum half-life of polypeptides with high binding affinity for human FcRn can be performed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with a variant Fc region. WO 2000/42072 (Presta) describes antibody variants with increased or decreased binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).

As used herein, percent (%) amino acid sequence identity and homology relative to a peptide, polypeptide, or antibody sequence means the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after alignment of the sequences and insertion of gaps, if necessary for achieving maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are known to those skilled in the art, for example using open source software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR). Those skilled in the art can determine the appropriate parameters for measuring the degree of alignment, including any algorithms known in the art, necessary to achieve maximum alignment over the entire length of the compared sequences.

An isolated nucleic acid molecule encoding an antibody, such as an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure, is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule.

- 41 042890 acid, with which it is usually associated in the environment in which it was produced. Preferably, the isolated nucleic acid is not associated with all the components to which it is associated in the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are presented in a form other than the form or conditions in which they are found in nature. Therefore, the isolated nucleic acid molecules are different from the nucleic acid molecule encoding the polypeptides and antibodies described herein that exists naturally in cells.

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a plasmid, which refers to circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is the phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thus replicate along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. In this document, such vectors are referred to as recombinant expression vectors or simply expression vectors. Typically, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present description, the terms plasmid and vector can be used interchangeably, since the plasmid is the most commonly used form of the vector.

The terms polynucleotide or nucleic acid, used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or a synthesis reaction. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, the modification of the nucleotide structure can be carried out before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may contain modification(s) carried out after synthesis, such as conjugation with a label. Other types of modification include, for example, caps, replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications, such as modifications due to chargeless bonds (eg, methylphosphonates, phosphotriesters, phosphoamidates, etc.), and beyond charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), modifications containing side chains, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.). ), modifications with intercalating substances (e.g. acridine, psoralen, etc.), modifications containing substances that form chelates (e.g. metals, radioactive metals, boron, metal oxidizing agents, etc.), modifications containing alkylating agents , linkage-modified modifications (eg, alpha-anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any of the hydroxyl groups normally found in sugars may be substituted, for example, with phosphonate groups, phosphate groups, protected with conventional protecting groups or activated to form additional bonds to other nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' OH terminal groups may be phosphorylated or may be replaced by amino groups or organic capping groups containing from 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized with conventional protecting groups. Polynucleotides may also contain analogs of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'- azidoribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and basic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments of the invention in which the phosphate group is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR ₂ (amidate), P( O)R, P(O)OR', CO or CH ₂ (formacetal), where each substituent R or R' is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) bond , aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. Not all bonds in a polynucleotide need to be identical. The above applies to all polynucleotides mentioned in this document, including RNA and DNA.

The host cell includes an individual cell or cell culture that may or has been the recipient of the vector(s) for incorporating polynucleotide inserts. Host cells include the precursor of an individual host cell, said

- 42 042890 the precursor does not have to be completely identical (morphologically or at the level of complementary genomic DNA) to the original parent cell, due to natural, accidental or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of the present invention.

In this context, carriers include pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cell or mammal being exposed to them, in the dosages and concentrations used. Often the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

In this context, the term about refers to the usual range of errors for the corresponding value, well known to a person skilled in the art. In this document, reference to about in relation to a value or parameter includes (and describes) embodiments of the invention that relate to the value or parameter per se.

In this context and in the appended claims, the singular forms include references to the plural, unless the context clearly dictates otherwise. For example, a reference to an antibody is a reference to one to more antibodies, such as molar amounts, and includes equivalents known to those skilled in the art, and so on.

It should be understood that the aspect and embodiments of the invention described herein include aspects and embodiments of the invention defined in terms of comprising, consisting of, and/or consisting essentially of.

Review.

The present invention relates to antibodies to TREM2 and/or to DAP12, which have one or more agonistic or antagonistic activities; methods for making and using such antibodies; pharmaceutical compositions containing such antibodies; nucleic acids encoding such antibodies; and host cells containing nucleic acids encoding such antibodies.

In some embodiments, and without being limited by theory, it is believed that the agonist activities of the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure are due at least in part to the ability of the antibodies to induce or maintain clustering of the TREM2 receptor on the cell surface. In some embodiments, it is believed that anti-TREM2 antibodies and/or anti-DAP12 antibodies can induce or maintain clustering of the TREM2 receptor in vivo not only by specific binding to TREM2, but also by binding to Fc receptors on neighboring cells, resulting in clustering antibody, which in turn aggregates TREM2. Preferably, certain immunoglobulin isotypes, including but not limited to IgG2 and IgM, have the natural ability to induce or maintain clustering of target antigens (eg, TREM2) without binding to Fc receptors on neighboring cells. In some embodiments, TREM2 agonist activities can be determined or tested in vitro using any of several methods described herein (see, for example, Examples 23-26, 34-37, 41-44, 52-55, and 67-68), including, but not limited to, fully diluted anti-TREM2 antibodies associated with a petri dish/plate to increase the density of antibodies exposed to TREM2, and cross-linked anti-TREM2 antibodies.

Accordingly, certain aspects of the present disclosure are based at least in part on the identification of anti-TREM2 antibodies and/or anti-DAP12 antibodies that are capable of binding both human and murine TREM2 with high affinity (see, for example, Examples 1 and 40 ); which compete with TREM2 ligand for binding to the ligand-binding site on human TREM2 and mouse TREM2 (see, for example, Examples 26 and 43); and which exhibit one or more TREM2 agonist activities, including, but not limited to, induction of CD83+CD86+ dendritic cells (see, for example, Examples 23 and 41), induction of TREM2 downstream signaling molecule Syk in macrophages and dendritic cells (see, for example, Examples 24 and 42), induction of TREM2 signaling adapter molecule DAP12 in macrophages (see, for example, Examples 25 and 44), induction of cell survival of innate immune cells such as macrophages (see, for example, Examples 34 and 52), and TREM2 activation -dependent gene expression (see, for example, Examples 36, 38, 54, 56 and 68).

Additional aspects of the present disclosure are based at least in part on the surprising discovery that the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can also elicit antagonistic activities when the antibody is produced or otherwise

- 43 042890 is formatted in such a way that it loses the ability to induce or maintain clustering of the TREM2 receptor. In some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure have one or more TREM2 antagonistic activities, including, but not limited to, inhibition of innate immune cell survival ((see, e.g., Examples 35 and 53) and inhibition of TREM2-dependent gene expression (see, for example, Examples 37, 55 and 67).

TREM2 proteins.

In one aspect, the present invention provides antibodies that bind to a TREM2 protein as described herein and modulate one or more TREM2 activities upon binding to a TREM2 protein that is expressed in a cell.

TREM2 proteins as described herein include, but are not limited to, mammalian TREM2 protein, human TREM2 protein (UniProt accession number Q9NZC2), mouse TREM2 protein (Accession number Q99NH8), rat TREM2 protein (Uniprot accession number D3ZZ89), rhesus monkey TREM2 protein ( Uniprot accession number F6QVF2), bovine TREM2 protein (Uniprot accession number Q05B59), equine TREM2 protein (Uniprot accession number F7D6L0), porcine TREM2 protein (Uniprot accession number H2EZZ3), and dog TREM2 protein (Uniprot accession number E2RP46). In this context, the TEM2 protein refers to both wild-type and naturally occurring variant sequences.

Trigger receptor that is expressed on myeloid cells-2 (TREM2) is known by names such as TREM-2, Trem2a, Trem2b, Trem2c, trigger receptor that is expressed on myeloid cells-2a and trigger receptor that is expressed on monocytes-2 . TREM2 is a 230 amino acid membrane protein. TREM2 is an immunoglobulin-like receptor that is primarily expressed in cells of myeloid origin, including, but not limited to, macrophages, dendritic cells, osteoclasts, microglia, monocytes, skin Langerhans cells, Kupffer cells. In some embodiments, TREM2 forms a receptor-signaling complex with DAP12. In some embodiments of the invention, TREM2 phosphorylates and sends signals through DAP12 (an adapter protein containing an ITAM domain). In some embodiments of the invention, TREM2 signaling results in downstream activation of FICK or other intracellular signals. Signals on myeloid cells, Toll-like receptors (TLRs), are important for the activation of TREM2 activities, for example in the context of response to infection. TLRs also play a key role in the pathological inflammatory response, for example, TLRs are expressed in macrophages and dendritic cells.

In some embodiments, an exemplary human TREM2 amino acid sequence is shown below in SEQ ID NO: 1:

1_0 2_0 30 40 50_6_0

MEPLRLLILL FVTELSGAHN TTVFQGVAGQ SLQVSCPYDS MKHWGRRKAW CRQLGEKGPC

£7 8£ 90 100 11£12£

QRWSTHNLW LLSFLRRWNG STAITDDTLG GTLTITLRNL QPHDAGLYQC QSLHGSEADT

13_0 14_0 150 160 170_18_0

LRKVLVEVLA DPLDHRDAGD LWFPGESESF EDAHVEHSIS RSLLEGEIPF PPTSILLLLA

£19 £20 £210 £22023

CIFLIKILAA SALWAAAWHG QKPGTHPPSE LDCGHDPGYQ LQTLPGLRDT

In some embodiments, human TREM2 is a precursor protein that includes a signal peptide. In some embodiments, human TREM2 is a mature protein. In some embodiments, the mature human TREM2 protein does not include a signal peptide. In some embodiments, the mature TREM2 protein is expressed in the cell. In some embodiments, TREM2 comprises a signal peptide located at amino acid residues 1-18 of human TREM2 (SEQ ID NO: 1); an extracellular variable-type immunoglobulin-like domain (IgV) located at amino acid residues 29-112 of human TREM2 (SEQ ID NO: 1); additional extracellular sequences located at amino acid residues 113-174 of human TREM2 (SEQ ID NO: 1); a transmembrane domain located at amino acid residues 175-195 of human TREM2 (SEQ ID NO: 1); and an intracellular domain located at amino acid residues 196-230 of human TREM2 (SEQ ID NO: 1).

The human TREM2 transmembrane domain contains a lysine at amino acid residue 186, which can interact with aspartic acid in DAP12, which is a key adapter protein that transduces signals from TREM2, TREM1 and other related members of the IgV family.

Human TREM2 homologues include, but are not limited to, NK-p44 natural killer (NK) cell receptor (NCTR2), polymeric immunoglobulin receptor (pIgR), CD300E, CD300A, CD30°C, and TREML1/TLT1. In some embodiments of the invention, NCTR2 has similarities with TREM2 in the IgV domain.

DAP12 proteins.

- 44 042890

In one aspect, the present invention provides antibodies that bind to a DAP12 protein as described herein and modulate one or more DAP12 activities upon binding to a DAP12 protein that is expressed in a cell.

DAP12 proteins as described herein include, but are not limited to, mammalian DAP12 protein, human DAP12 protein (Uniprot accession number O43914), mouse DAP12 protein (Uniprot accession number O54885), rat DAP12 protein (Uniprot accession number Q6X9T7), rhesus monkey DAP12 protein (Uniprot accession number Q8WNQ8), bovine DAP12 protein (Uniprot accession number Q95J80), and porcine DAP12 protein (Uniprot accession number Q9TU45). In this context, the DAP12 protein refers to both wild-type and naturally occurring variant sequences.

DNAX activating protein 12 (DAP12) is known by names such as killer-activating receptor-associated protein, KAR-associated protein (KARAP), PLOSL, PLO-SL, TYRO protein, and tyrosine kinase binding protein. DAP12 is a 113 amino acid membrane protein. In some embodiments, DAP12 functions as a transmembrane signal polypeptide that contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. It can associate with membrane glycoproteins of the inhibitory killer cell receptor (KIR) family and may act as a signal transduction activator. In other embodiments, the DAP12 protein can bind 70 kDa protein kinase (ZAP-70) associated with the zeta chain (TCR) and spleen tyrosine kinase (Syk) and play an important role in signal transduction, bone modeling, brain myelination and inflammation.

Mutations within the gene encoding DAP12 have been associated with polycystic lipomembrane osteodysplasia with sclerosing leukoencephalopathy (PLSL), also known as Nasu-Hakol's disease. Without wishing to be bound by theory, the DAP12 receptor may be considered to be TREM2, which also causes PLSL. Many alternative transcripts have been identified encoding various isoforms of DAP12. DAP12 binds non-covalently to activating receptors of the CD300 family. cross-linking of CD300-TYROBP/DAP12 complexes results in cellular activation, such as integrin-mediated activation of neutrophils. DAP12 is a homodimer; disulfide-linked protein. In some embodiments, DAP12 interacts with SIRPB1, TREM1, CLECSF5, SIGLEC14, CD300LB, CD300E, and CD300D through affinity and through the ITAM domain, and with SYK through the SH2 domain. In other embodiments, DAP12 activates Syk, which causes integrin-mediated activation of neutrophils and macrophages. In other embodiments of the invention, DAP12 interacts with KLRC2 and KIR2DS3.

In some embodiments, an example of the human DAP12 amino acid sequence is shown below in SEQ ID NO: 2:

30 40 £5 £6

MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA

7_0 8 0. 9 0. 100 110

VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

In some embodiments, human DAP12 is a precursor protein that includes a signal peptide. In some embodiments, human DAP12 is a mature protein. In some embodiments, the mature human DAP12 protein does not include a signal peptide. In some embodiments, the mature DAP12 protein is expressed in the cell. DAP12 is a type 1 single pass membrane protein. It contains an extracellular domain located at amino acid residues 22-40 of human DAP12 (SEQ ID NO: 2); a transmembrane domain located at amino acid residues 41-61 of human DAP12 (SEQ ID NO: 2); and an intracellular domain located at amino acid residues 62-113 of human DAP12 (SEQ ID NO: 2). The immunoreceptor tyrosine activating motif (ITAM) domain is located at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2).

In some embodiments, the aspartic acid residue in DAP12 interacts with the transmembrane domain of human TREM2, which contains lysine at amino acid residue 186, and transduces signals from TREM2, TREM1, and other related members of the IgV family.

Antibodies to TREM2 and antibodies to DAP12.

Some aspects of the present description relate to antibodies that specifically bind to TREM2 and/or DAP12. In some embodiments, the antibodies of the present disclosure bind to mature TREM2 protein and/or mature DAP12 protein. In some embodiments, the antibodies of the present disclosure bind to mature TREM2 protein and/or mature DAP12 protein. In some embodiments, the antibodies of the present disclosure bind to the TREM2 protein and/or the DAP12 protein that is expressed on one or more human cells selected from human dendritic cells, human macrophages, human monocytes, human osteoclasts, human skin Langerhans cells, Kupffer cells. human cells and/or

- 45 042890 human microglial cells. In some embodiments, the antibodies of the present disclosure are agonist antibodies. In some embodiments, the antibodies of the present disclosure are inert antibodies. In some embodiments, the antibodies of the present disclosure are antagonistic antibodies.

Agonistic antibodies.

The anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure typically bind to one or more TREM2 and/or DAP12 proteins expressed in a cell. One class of antibodies is agonist antibodies. For example, the TREM2 receptor is believed to require clustering on the cell surface for signal transduction. Thus, agonist antibodies may be uniquely capable of stimulating, for example, the TREM2 receptor. For example, they may have the correct antigenic determinant specificity compatible with receptor activation, as well as the ability to induce or maintain receptor clustering on the cell surface.

Antibodies can induce receptor clustering in vivo using several potential mechanisms. Some human antibody isotypes, such as IgG2, due to their unique structure, have the natural ability to cause receptor clustering or to keep receptors in a clustered configuration, thereby activating receptors such as TREM2 without binding to the Fc receptor (e.g., White et al., (2015) Cancer Cell 27, 138-148).

Other antibodies induce receptor clustering (eg TREM2) by binding to Fcg receptors on neighboring cells. Binding of the Fc constant portion of an IgG antibody to Fc receptors results in antibody clustering and the antibodies in turn aggregate the receptors to which they bind via their variable regions (Chu and et al (2008) Mol. Immunol., 45:39263933; and Wilson et al., (2011) Cancer Cell 19, 101-113). Binding to the Fcg inhibitory receptor FcgR (FcgRIIB), which does not cause cytokine secretion, oxidative response, increased phagocytosis, and enhanced antibody-dependent cellular cytotoxicity (ADCC), is often the preferred method for in vivo antibody clustering because binding to FcgRIIB does not lead to unwanted immune responses.

Other mechanisms can also be used for receptor clustering (eg TREM2). For example, antibody fragments (eg, Fab fragments) cross-linked with each other can be used to cluster receptors (eg, TREM2) in a manner similar to antibodies with Fc fragments that bind to Fcg receptors as described above. Without wishing to be bound by any theory, cross-linked antibody fragments (eg, Fab fragments) can be considered to function as agonistic antibodies if they cause receptor clustering on the cell surface and bind to the appropriate epitope on the target (eg, TREM2).

Thus, in some embodiments, antibodies that bind TREM2 protein and/or DAP12 protein may include agonistic antibodies that, due to their epitope specificity, bind TREM2 and/or DAP12 and activate one or more TREM2 and/or DAP12 activities. Without wishing to be bound by theory, it is believed that such antibodies can bind to a ligand-binding site on a target antigen (e.g., TREM2 and/or DAP12) and mimic the action of a natural ligand or stimulate the target antigen to signal transduce by binding to one or more domains that are not ligand binding sites. Such antibodies will not interfere with ligand binding and may act additively or synergistically with natural ligands.

In some embodiments, an antibody of the present disclosure is an agonist antibody that induces one or more TREM2 activities, one or more DAP12 activities, or one or more TREM2 activities and one or more DAP12 activities. In some embodiments, the antibody elicits one or more TREM2 and/or DAP12 activities upon binding to the TREM2 protein and/or DAP12 protein that is expressed in the cell. In some embodiments of the invention, one or more TREM2 activities, one or more DAP12 activities, or both activities are selected from the group consisting of: TREM2 binding to DAP12; binding DAP12 to TREM2; TREM2 phosphorylation, DAP12 phosphorylation; physical activation; increased expression of one or more anti-inflammatory cytokines; increased expression of one or more anti-inflammatory mediators (eg, cytokines) selected from IL-12p70, IL-6 and IL-10; decreased expression of one or more pro-inflammatory cytokines, decreased expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1b, TNF, TNF-α, IL -10, IL-8, CRP, TGF-beta chemokine protein family members, IL-20 family members, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GMCSF, IL-11, IL-12 , IL-17, IL-18, MHB-1 and SRB; reduced expression of TNF-α, reduced expression of IL-6; phosphorylation of kinase regulated by extracellular signals (ERK); increased expression of the C-C chemokine receptor 7 (CCR7); induction of chemotaxis of microglial cells in relation to

- 46,042,890 cells expressing CCL19 and CCL21; increasing, normalizing, or both, the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; inducing the production of osteoclasts, increasing the level of osteoclastogenesis, or both; increasing the survival and/or function of one or more macrophages, microglial cells, macrophages and/or M1 microglial cells, activated macrophages and/or M1 microglial cells, macrophages and/or M2 microglial cells, monocytes, osteoclasts, skin Langerhans cells and Kupffer cells; induction of one or more types of clearance selected from the group consisting of apoptotic neuron clearance, neural debris clearance, non-nervous tissue debris clearance, bacteria or other foreign body clearance, pathogenic protein clearance, pathogenic peptide clearance, and pathogenic nucleic acid clearance; inducing phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, or disease-causing nucleic acids (e.g., antisense GGCCCC (G2C4) RNA repeat expansion); normalization of impaired TREM2/DAP12-dependent gene expression; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; phosphorylation of Syk; increased expression of CD83 and/or CD86 on dendritic cells, macrophages, monocytes and/or microglia; decreased secretion of one or more inflammatory cytokines, decreased secretion of one or more inflammatory cytokines selected from the group consisting of TNF-α, IL-10, IL-6, MHB1, IFN-a4, IFN-b, IL-1c, IL- 8, CRP, TGF-beta chemokine protein family members, IL-20, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL- 17 and IL-18; decreased expression of one or more inflammatory receptors; increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of reduced levels of MCSF; decreased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia in the presence of normal levels of MCSF; an increase in the activity of one or more TREM2-dependent genes (eg, nuclear factors from activated T cell transcription factors (NFAT) of the transcription factor family).

An antibody that depends on binding to the FcgR receptor to activate target receptors may lose its agonist activity if designed to eliminate FcgR binding (see, for example, Wilson et al., (2011) Cancer Cell 19, 101-113; Armor et al., (2003) Immunology 40 (2003) 585-593); and White et al., (2015) Cancer Cell 27, 138-148). Thus, it is believed that an antibody of the present disclosure with the correct antigenic determinant specificity can be an agonist antibody and activate the target antigen with minimal adverse reactions if the antibody has an Fc domain from the human IgG2 isotype (CH1 and hinge region) or another type of domain. Fc that is capable of preferentially binding FcgRIIB inhibitory receptors or variants thereof.

Exemplary isotypes and Fc modifications of agonist antibodies are shown in Table 1. 2 below. In some embodiments, the agonist antibody has the Fc isotype listed in Table 1. 2 below.

- 47 042890

Table 2 Exemplary anti-TREM2 agonist Fc isotypes

Fc isotype Mutation (no EU and Kabat numbering scheme) IgGl N2 97A IgGl D265A and N297A IgGl L234A and L235A L234A and G237A L234A & L235A & G237A IgG2 V234A and G237A IgG4 L235A and G237A and E318A IgG4 S228P and L236E Hybrid IgG2/4 IgG2 a.k. 118-260 and IgG4 a.k. 261-447 IgG2 H268Q and V309L; and A330S and P331S IgGl C226S, C229S, E233P, L234V, L235A IgGl L234F & L235E & P331S IgG2 C232S or C233S IgG2 A330S and P331S IgGl S267E and L328F S267E only IgG2 S267E and L328F IgG4 S267E and L328F IgG2 TC DT with kappa LC (light chain) TC C127S with kappa LC Kappa LC C214S Kappa LC C214S and TC C233S Kappa LC C214S and TC C232S Any of the above mutations together with P330S and P331S mutations F(ab')2 fragment of IgGl DT and any of the above mutations IgGl Replacement of heavy chain 1 constant domain (CH1) and IgGl hinge region with CH1 and IgG2 hinge region ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP (SEQ ID NO: 397) on kappa LC IgGl Any of the above mutations together with A330L and/or L234F and/or L235E and/or P331S mutations IgGl, IgG2 or IgG4 Any of the above mutations together with the M252Y and/or S254T and/or T256E mutations Mouse IgGl For mouse disease models IgG4 DT

-48042890

In addition to the isotypes described in Table. 2 and without wishing to be bound by any theory, human IgG1 or IgG3 antibody isotypes and mutants thereof (e.g., Strohl (2009) Current Opinion in Biotechnology 2009, 20:685-691) that bind Fcg I activating receptors, Human IIA, IIC, IIIA, IIIB and/or mouse Fcg I, III and IV receptors may also act as agonist antibodies in vivo, but may lead to adverse reactions associated with ADCC. However, such Fcg receptors appear to be less accessible to antibody binding in vivo than the inhibitory Fcg receptor FcgRIIB (see, for example, White, et al., (2013) Cancer Immunol. Immunother. 62, 941-948 and Li et al., (2011) IScience 333(6045):1030-1034).

In some embodiments, the agonist antibody is of the IgG class, the IgM class, or the IgA class. In some embodiments, the agonist antibodies are IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the agonist antibody is of the IgG2 isotype. In some embodiments, the agonist antibody comprises a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the agonist antibody induces one or more TREM2 activities, DAP12 activities, or both, regardless of Fc receptor binding. In some embodiments, the agonist antibody binds an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fc gamma receptor IIB (FcyIIB). In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from V234A (Alegre et al., (1994) Transplantation 57:1537-1543.31; Xu et al., (2000) Cell Immunol, 200:16-26) , G237A (Cole et al (1999) Transplantation, 68:563-571), H268Q, V309L, A330S, P331S (US 2007/0148167; Armor et al (1999) Eur. J. Immunol 29: 2613-2624; Armor et al (2000) The Haematology Journal 1 (Suppl.1):27 Armor et al (2000) The Haematology Journal 1 (Suppl.1):27), C232S, and/or C233S (White et al ( 2015) Cancer Cell 27, 138-148), S267E, L328f (Chu et al., (2008) Mol Immunol, 45:3926-3933), M252Y, S254T, and/or T256E, where the amino acid position is represented by with EU or Kabat numbering system.

In some embodiments, the agonist antibody is an IgG2 isotype with a heavy chain constant domain that contains a C127S amino acid substitution, where the amino acid position is represented according to the EU or Kabat numbering system. (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO 2008079246).

In some embodiments, the agonist antibody is an IgG2 isotype with a light chain kappa constant domain that contains the C214S amino acid substitution, where the amino acid position is represented according to the EU or Kabat numbering system (White et al., (2015) Cancer Cell 27, 138 -148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO 2008079246).

In some embodiments, the agonist antibody is of the IgG1 isotype. In some embodiments, the agonist antibody comprises a mouse IgG1 constant region. In some embodiments, the agonist antibody comprises a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, the agonist antibody binds an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB). In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from N297A (Bolt S. et al., (1993) Eur. J. Immunol. 23:403-411), D265A (Shields et al (2001) R. J. Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al (1995) Proc. Natl. Acad. Sci. USA, 92:11980-11984; Alegre et al., (1994) Transplantation 57:1537- 1543. 31; Xu et al., (2000) Cell Immunol., 200:16-26), G237A (Alegre et al (1994) Transplantation 57:1537-1543. 31; Xu et al (2000) Cell Immunol., 200:16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) ) Proc. Natl. Acad. Sci. USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T, and/or T256E, where the amino acid position is represented according to the EU or Kabat numbering system.

In some embodiments, the antibody comprises an IgG2 isotype heavy chain 1(CH1) constant domain and a hinge region (White et al., (2015) Cancer Cell 27, 138-148). In some embodiments, the CH1 and hinge region of the IgG2 isotype contains the amino acid sequence

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ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397) .

In some embodiments, the Fc region of an antibody comprises an S267E amino acid sequence, an L328F amino acid sequence, or both, and/or an N297A or N297Q amino acid sequence, where the amino acid position is represented according to the EU or Kabat numbering system.

In some embodiments, the agonist antibody is of the IgG4 isotype. In some embodiments, the agonist antibody comprises a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, the agonist antibody binds an inhibitory Fc receptor. In some embodiments, the inhibitory Fc receptor is an inhibitory Fc gamma receptor IIB (FcyIIB). In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from L235A, G237A, S228P, L236E (Reddy et al., (2000) J. Immunol., 164:1925-1933), S267E, E318A, L328F, M252Y, S254T and/or T256E, where the amino acid position is represented according to the EU or Kabat numbering system.

In some embodiments, the agonist antibody is an IgG2/4 hybrid isotype. In some embodiments, the antibody comprises an amino acid sequence comprising amino acids 118 to 260, according to Kabat numbering, from human IgG2, and amino acids 261 to 447 from human IgG4, according to Kabat numbering (WO 1997/11971; WO 2007 /106585).

In some embodiments, the antibody comprises a mouse IgG4 constant region (Bartholomaeus et al. (2014). J. Immunol. 192, 2091-2098).

In some embodiments, the Fc region further comprises one or more additional amino acid substitutions selected from the group consisting of A330L, L234F, L235E, and/or P331S, where the amino acid position is represented according to the EU or Kabat numbering system.

inert antibodies.

Another class of antibodies according to the present description includes inert antibodies. In this context, inert antibodies refer to antibodies that specifically bind target antigens, but do not modulate (eg, reduce/inhibit or activate/induce) the function of the antigen. For example, in the case of TREM2, inert antibodies do not modulate TREM2 ligand binding and/or activity. Without wishing to be bound by theory, it may be considered that antibodies that do not have the ability to cluster TREM2 on the cell surface may be inert antibodies, even if they have an antigenic determinant specificity compatible with receptor activation.

In some embodiments, antibodies that bind the TREM2 protein and/or the DAP12 protein may include antibodies that bind TREM2 and/or DAP12 but due to their epitope specificity do not modulate the function of the protein. Such functionally inert antibodies can be used as a toxin delivery vehicle as described for the anti-CD33 antibody gemtuzumab ozogamicin (sold as Mylotarg), which is conjugated to a cytotoxic agent from the calicheamicin class and is used to identify and kill tumors in acute myeloid leukemia (Naito et al., (2000), Leukemia, 14, 1436-1443; Ricart (2011) Clin Cancer Res 17; 64176436; Hamann et al., (2002) Journal: Bioconjugate Chemistry, 13, 47-58; and Beitz et al., (2001) Clin Cancer Res 7;1490-6). Thus, in some embodiments, the antibodies of the present disclosure are inert antibodies that bind TREM2 and/or DAP12 but are unable to induce one or more TREM2 activities (e.g., the TREM2 activity described herein) and/or DAP12 activities ( e.g. DAP12 activity described herein).

Typical isotypes and Fc modifications of inert antibodies are given in table. 3 below. In some embodiments of the invention, the inert antibody has the Fc isotype listed in Table. 3 below.

Antagonistic antibodies.

The third class of antibodies according to the present description includes antagonistic antibodies. In some embodiments, antibodies that bind TREM2 protein and/or DAP12 protein may include antagonistic antibodies that bind TREM2 and/or DAP12 and inhibit one or more TREM2 and/or DAP12 activities, either by preventing an interaction between TREM2 and/or DAP12 and its ligand(s), or by preventing signal transduction from the extracellular domain of TREM2 and/or DAP12 into the cell cytoplasm in the presence of the ligand. In some embodiments, the antagonist antibodies of the present disclosure

- 50 042890 may have the epitope specificity of an agonist antibody of the present disclosure but have an Fc domain that is unable to bind Fcg receptors and thus unable, for example, to cluster the DAP 12 receptor and/or TREM2.

In some embodiments of the invention, the antibody according to the present description is an antagonistic antibody. In some embodiments, the antagonist antibody inhibits one or more TREM2 and/or DAP 12 activities. In some embodiments, the antagonist antibody reduces the activity of one or more TEEM2-dependent genes. In some embodiments of the invention, one or more TREM2-dependent genes include, without limitation, one or more nuclear factors from activated T cell transcription factors (NFAT). In some embodiments, the agonist antibody reduces the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, skin Langerhans cells, Kupffer cells, and/or dendritic cells. In some embodiments, the antagonist antibody inhibits the interaction between TREM2 and/or DAP 12 and one or more TREM2 and/or DAP 12 ligands. In some embodiments, the antagonist antibody inhibits TREM2 and/or DAP 12 signaling. In some embodiments, the antagonist antibody the antagonist antibody inhibits the interaction between TREM2 and/or DAP 12 and one or more TREM2 and/or DAP 12 ligands and inhibits TREM2 and/or DAP 12 signaling.

In some embodiments of the invention, cross-linking of an antibody is necessary for the agonist function of the antibody. Cross-linking of an antibody can occur by binding to a secondary antibody in vitro or by binding to Fc receptors in vivo. For example, antagonist antibodies can be converted to agonist antibodies by biotin/streptavidin crosslinking or in vitro binding of a secondary antibody (see, for example, Gravesteifi et al. (1996) J. Exp. Med. 184:675-685; Gravestein et al., (1994) International Immunol 7:551-557). Agonistic antibodies can exert their activity by mimicking the biological activity of a receptor ligand or by enhancing receptor clustering, thereby activating receptor signaling. In some embodiments of the invention, the absence of cross-linking of the antibody is necessary for antagonistic activity. Antagonistic antibodies can exert their activity by blocking receptor-ligand interactions.

Typical isotypes and modifications of Fc antagonistic antibodies are given in table. 3 below. In some embodiments, the antagonist antibody has the Fc isotype listed in Table 1. 3 below.

Isotypes of inert and antagonistic antibodies.

In some embodiments, the inert and/or antagonistic anti-TREM2 antibodies described herein comprise one or more of the Fc isotypes and modifications listed in Table 1. 3.

Table 3. Exemplary isotypes of inert and antagonistic antibodies to TREM2

Fc isotype Mutation (numbering scheme no EU and Rabat) IgGl N297A or N297Q IgGl D265A and N297A IgGl L234A and L235A IgG2 V234A and G237A IgG4 L235A and G237A and E318A E233P and/or F234V N297A or N297Q IgG4 S228P and L236E S241P S241P and L248E IgG2 H268Q and V309L and A330S and P331S IgGl C220S and C226S and C229S and P238S

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IgGl C226S, C229S, E233P, L234V, and L235A IgGl E233P and L234V and L235A and 6236 deletion R238A D265A N2 97A A327Q or A327G R329A IgGl K322A and L234A and L235A IgGl L234F and L235E and P331S IgGl or IgG4 T394D IgG2 C232S or C233S N297A or N297Q IgGl, IgG2 or IgG4 modifications delta, a, b, c, ab, ac, g IgGl Any of the above mutations together with A330L or L234F and/or L235E and/or P331S mutations IgGl, IgG2 or Any of the above mutations together with IgG4 mutations M252Y and/or S254T and/or T256E

In some embodiments, the antibody is of the IgG1 isotype. In some embodiments, the antibody comprises a mouse IgG1 constant region. In some embodiments, the antibody comprises a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from N297A, N297Q (Bolt S. et al., (1993) Eur. J. Immunol. 23:403-411), D265A, L234A, L235A (McEarchern et al. , (2007) Blood, 109:11851192), C226S, C229S (McEarchern et al., (2007) Blood, 109:1185-1192), P238S (Davis et al., (2007) J. Rheumatol., 34:2204-2210), E233P, L234V (McEarchern et al., (2007) Blood, 109:1185-1192), P238A, A327Q, A327G, P329A (Shields R.L. et al., (2001) J. Biol Chem. 276(9):6591-604), K322A, L234F, L235E (Hezareh et al., (2001) J. Virol. 75, 12161-12168; Oganesyan et al., (2008) Acta Crystallographica 64, 700-704), P331S (Oganesyan et al., (2008) Acta Crystallographica 64, 700-704), T394D (Wilkinson et al (2013) MAbs 5(3): 406-417), A330L, M252Y , S254T and/or T256E, where the amino acid position is represented by EU or Kabat numbering. In some embodiments, the Fc region further includes a deletion of the amino acid at the position corresponding to glycine 236 according to EU or Kabat numbering.

In some embodiments, the antibody is an IgG1 isotype with a heavy chain constant domain that contains the C220S amino acid substitution, according to EU or Kabat numbering.

In some embodiments, the Fc region further comprises one or more additional amino acid substitutions selected from A330L, L234F; L235E, and/or P331S according to EU or Kabat numbering.

In some embodiments, the antibody is of the IgG2 isotype. In some embodiments, the antibody comprises a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, and/or T256E, where the amino acid position is represented by EU numbering or Kabat.

- 52 042890

In some embodiments, the antibody is of the IgG4 isotype. In some embodiments, the antibody comprises a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments of the invention, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (eg, compared to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from E233P, F234V, L235A, G237A, E318A (Hutchins et al. (1995) Proc. Natl. Acad. Sci. USA, 92:11980-11984), S228P, L236E , S241P, L248E (Reddy et al., (2000) J. Immunol., 164:1925-1933; Angal et al., (1993) Mol. Immunol. 30(1):105-8; US 8614299 B2 ), T394D, M252Y, S254T, T256E, N297A, and/or N297Q, where the amino acid position is represented by EU or Kabat numbering.

In some embodiments, the Fc region further comprises one or more additional amino acid substitutions selected from M252Y, S254T, and/or T256E, where the amino acid position is represented by EU or Kabat numbering.

Additional IgG mutations.

In some embodiments, one or more of the IgG1 variants described herein may be combined with the A330L mutation (Lazar et al., (2006) Proc. Natl. Acad. Sci. USA, 103:4005-4010) or one or more L234F, L235E and/or P331S mutations (Sazinsky et al., (2008) Proc. Natl. Acad. Sci. USA, 105:20167-20172), where the amino acid position is represented by EU or Kabat numbering. In some embodiments of the invention, the IgG variants described herein can be combined with one or more mutations to increase the half-life of the antibody in human serum (for example, mutations M252Y, S254T, T256E according to EU or Kabat numbering) (Dall 'Acqua et al., (2006) J Biol Chem, 281:23514-23524; and Strohl et al., (2009) Current Opinion in Biotechnology, 20:685-691).

In some embodiments, an IgG4 variant as described herein may be combined with an S228P mutation according to EU or Kabat numbering. (1993) Mol. Immunol., 30:105-108) and/or with one or more of the mutations described in Peters et al., (2012) J. Biol. Chem. 13; 287 (29): 24525-33) to improve antibody stability.

Antibodies to TREM2.

Some aspects of the present description relate to antibodies to TREM2.

In some embodiments, anti-TREM2 antibodies as described herein are agonist antibodies that induce one or more TREM2 activities. In some embodiments, the anti-TREM2 antibodies of the present disclosure are agonist antibodies that promote the survival of one or more innate immune cells. In some embodiments, anti-TREM2 antibodies of the present disclosure promote survival of macrophages, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts , skin Langerhans cells and/or Kupffer cells. In some embodiments of the invention, promoting the survival of one or more innate immune cells includes increasing cell survival or otherwise slowing down cell death. Accordingly, in some embodiments, the anti-TREM2 antibodies of the present disclosure promote the survival of one or more innate immune cells. In some embodiments, the anti-TREM2 antibodies of the present disclosure slow down the cell death of one or more innate immune cells. In some embodiments, promoting cell survival and/or increasing cell survival is determined by measuring the cell survival of one or more innate immune cells in the presence of an anti-TREM2 antibody compared to the cell survival of the corresponding one or more innate immune cells in the absence of an anti-TREM2 antibody. In some embodiments, cell death delay is determined by measuring the cell death of one or more innate immune cells in the presence of an anti-TREM2 antibody compared to the cell death of the corresponding one or more innate immune cells in the absence of an anti-TREM2 antibody. Any suitable methods for measuring cell survival or cell death known in the art and described herein can be used (see, for example, Examples 30, 34, 35, 38, 52, 53 and 56). In some embodiments, the anti-TREM2 antibodies of the present disclosure are agonist antibodies that increase the expression of IL-6. In some embodiments, the anti-TREM2 antibodies of the present disclosure are agonist antibodies that promote the survival of one or more innate immune cells and increase the expression of IL-6. Any suitable methods for measuring IL-6 expression in a cell, known in the art and described herein, can be used (see, for example, Examples 28, 38 and 68).

In some embodiments, the anti-TREM2 antibodies of the present disclosure are inert or antagonistic antibodies that inhibit one or more active

- 53 042890 TREM2. In some embodiments, the anti-TREM2 antibodies of the present disclosure are inert or agonistic antibodies that reduce the survival of one or more innate immune cells. In some embodiments, anti-TREM2 antibodies of the present disclosure reduce the survival of macrophages, microglial cells, M1 microglial cells, activated M1 microglial cells, M2 microglial cells, dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts , skin Langerhans cells and/or Kupffer cells. In some embodiments, cell survival reduction is determined by measuring the cell survival of one or more innate immune cells in the presence of an anti-TREM2 antagonist antibody compared to the cell survival of the corresponding one or more innate immune cells in the absence of an anti-TREM2 antagonist antibody. Any suitable methods for measuring cell survival or cell death known in the art and described herein can be used (see, for example, Examples 30, 34, 35, 38, 52, 53 and 56).

In some embodiments, an isolated anti-TREM2 antibody as described herein competes for binding to TREM2 with one or more TREM2 ligands. In some embodiments, the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. Exemplary descriptions of such antibodies are provided herein. In some embodiments, the antibody is a bispecific antibody that recognizes a first antigen and a second antigen.

In some embodiments, the TREM2 protein is expressed on the cell surface. In some embodiments, the anti-TREM2 antibodies of the present disclosure modulate (eg, induce or inhibit) one or more TREM2 activities. TREM2 activities modulated (eg, induced or inhibited) by anti-TREM2 antibodies may include, but are not limited to, DAP12 phosphorylation; TREM2 phosphorylation; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; physical activation; increased expression of anti-inflammatory mediators (eg, cytokines); decreased expression of one or more pro-inflammatory mediators; phosphorylation of ERK; increased expression of CCR7, induction of chemotaxis of microglial cells in relation to cells expressing CCL19 and CCL21; increase, normalization, or both the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; induction of osteoclast production, increased levels of osteoclastogenesis, or both; increased survival and/or function of microglial and/or macrophage cells (such as M1 macrophage cells and/or M1 microglial cells, activated M1 macrophages and/or M1 microglial cells and/or M2 macrophages and/or M2 microglial cells), dendritic cells , monocytes, osteoclasts, skin Langerhans cells and/or Kupffer cells; induction of clearance of apoptotic neurons; reduced expression of TNF-α; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells, monocytes, macrophages and/or microglia; decreased secretion of one or more inflammatory cytokines (such as TNF-α, IL-10, IL-6 and/or MHB-1); decreased expression of one or more inflammatory receptors (such as CD86); increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of reduced levels of MCSF; reduced phagocytosis by macrophages, dendritic cells, monocytes and/or microglia in conditions of reduced levels of MCSF; and/or increased activity of one or more TREM2-dependent genes (eg, nuclear factors from activated T cell transcription factors (NFAT) from the family of transcription factors). The anti-TREM2 antibodies of the present disclosure may be used to prevent, reduce risk, or treat dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakola, stroke, acute injury, chronic injury, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease , Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, aging disease, epileptiform seizures, spinal cord injury, traumatic brain injury, age-related degeneration macular macula, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteoporosis-related disease, Paget's disease of bone, and cancer . Anti-TREM2 antibodies of the present disclosure may also be used in wound care. In some embodiments, the anti-TREM2 antibodies of the present disclosure are monoclonal antibodies. Anti-TREM2 antibodies of the present disclosure can be tested to induce one or more TREM2 activities (e.g., TREM2 autophosphorylation; DAP12 phosphorylation; phosphorylation

- 54 042890 Syk; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; physical activation; increased expression of cytokines; decreased expression of pro-inflammatory mediators; phosphorylation of ERK; increased expression of CCR7; inducing chemotaxis of microglial cells towards cells expressing CCL19 and CCL21; maturation of dendritic cells from the bone marrow; increase, normalization, or both the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; increased ability of dendritic cells, monocytes, microglia and/or macrophages to induce T cell proliferation; inducing the production of osteoclasts, increasing the level of osteoclastogenesis, or both; increased survival and function of dendritic cells, macrophages, monocytes, osteoclasts, skin Langerhans cells and Kupffer cells, and/or microglia; induction of one or more types of clearance; inducing phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, disease-causing nucleic acid, or cancer cells; reduced secretion of one or more inflammatory cytokines, reduced expression of one or more inflammatory receptors, increased phagocytosis by macrophages, dendritic cells, monocytes, and/or microglial cells under conditions of reduced levels of MCSF; reduced phagocytosis by macrophages, dendritic cells, monocytes and/or microglial cells in the presence of normal levels of MCSF; normalization of impaired TREM2/DAP12-dependent gene expression; and increased activity of one or more DAP12 dependent genes), using any suitable method known in the art and/or described herein. For example, anti-TREM2 antibodies can be assayed in vitro for tyrosine phosphorylation of TREM2, DAP12, Syk, and/or ERK, by Syk and/or ZAP70 recruitment assay to DAP12, by assay of PISA activation, by assay of induction of cytokine expression (e.g., IL-12p70, IL-6 and IL-10) or CCR7, or by assaying down-expression of pro-inflammatory mediators (eg IL-β and TNF) with TLR stimulation (eg LPS, CpG DNA or zymosan). Useful assays may include Western blot (eg, for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PKK kinase substrates), ELISA (eg, for secreted interleukin or cytokine secretion), FACS (eg, for anti-TREM2 antibody binding to TREM2), immunocytometry (e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PIBK kinase substrates), reporter gene assays (e.g., for TLR activation), increased survival and/or function of dendritic cells, macrophages, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or microglia, increased phagocytosis of apoptotic neurons, damaged synapses, amyloid beta or its fragments, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin , Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein , translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline repeat peptides -alanine (PA) and proline-arginine (PR) repeat peptides, nerve tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing protein, disease-causing peptide, disease-causing nucleic acid, or cancer cells by macrophages, dendritic cells, skin Langerhans cells , Kupffer cells, monocytes, osteoclasts, and/or microglial cells, increased cytoskeletal reorganization, and reduced pro-inflammatory response, or other assays known in the art.

In some embodiments, the anti-TREM2 antibodies of the present disclosure modulate (i.e., increase or decrease) the expression and/or secretion of one or more inflammatory cytokines (e.g., TNF-α, IL-10, IL-6, MHB-1, IFN-a4, IFN-b, IL-1b, IL-8, CRP, members of the TGF-beta chemokine protein families, members of the IL-20 family, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17 and IL-18). In some embodiments, the anti-TREM2 antibodies of the present disclosure increase the expression and/or secretion of one or more inflammatory cytokines. In some embodiments, the anti-TREM2 antibodies of the present disclosure reduce the expression and/or secretion of one or more inflammatory cytokines. In some embodiments, the anti-TREM2 antibodies of the present disclosure modulate (ie, increase or decrease) the expression and/or secretion of one or more inflammatory receptors (eg, CD86). In some embodiments, the anti-TREM2 antibodies of the present disclosure increase the expression and/or secretion of one or more inflammatory receptors. In some embodiments, the anti-TREM2 antibodies of the present disclosure reduce the expression and/or secretion of one or more inflammatory receptors.

In some embodiments, anti-TREM2 antibodies as described herein bind to human TREM2 or a homologue thereof, including, but not limited to, mammalian TREM2 protein, mouse TREM2 protein (UniProt accession number Q99NH8), rat TREM2 protein (Account No.

- 55 042890 Uniprot D3ZZ89), rhesus monkey TREM2 protein (Uniprot accession number F6QVF2), bovine TREM2 protein (Uniprot accession number Q05B59), equine TREM2 protein (Uniprot accession number F7D6L0), porcine TREM2 protein (Uniprot accession number H2EZZ3) and canine TREM2 protein (Uniprot accession number E2RP46). In some embodiments, anti-TREM2 antibodies as described herein specifically bind to human TREM2. In some embodiments, anti-TREM2 antibodies as described herein specifically bind to mouse TREM2. In some embodiments, anti-TREM2 antibodies as described herein specifically bind to both human TREM2 and mouse TREM2. In some embodiments, the anti-TREM2 antibodies of the present disclosure modulate (eg, induce or inhibit) at least one TREM2 activity. In some embodiments, at least one TREM2 activity is DAP12 phosphorylation, TREM2 phosphorylation; physical activation; increased expression of one or more anti-inflammatory mediators (eg, cytokines); reduced expression of one or more pro-inflammatory mediators, increased survival and/or function of microglial cells, dendritic cells, macrophages, monocytes, osteoclasts, skin Langerhans cells, and Kupffer cells; decreased expression of TNF-α, SYK phosphorylation, increased expression of CD83 and/or CD86 on dendritic cells, macrophages, monocytes and/or macrophages, decreased secretion of one or more inflammatory cytokines, decreased expression of one or more inflammatory receptors, increased phagocytosis by macrophages, dendritic cells , monocytes, and/or microglia in the setting of reduced levels of MCSF, decreased phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal levels of MCSF, and/or increased activity of one or more TREM2-dependent genes (e.g., nuclear factors from transcription factors activated T-cell (NFAT) family of transcription factors).

In some embodiments, anti-TREM2 antibodies as described herein bind to a TREM2 protein as described herein and/or naturally occurring variants. In some embodiments, anti-TREM2 antibodies bind to human TREM2.

In some embodiments, the anti-TREM2 antibodies of the present disclosure are agonist antibodies or antagonist antibodies that bind to the TREM2 protein of the present disclosure that is expressed on a cell surface and modulate (e.g., induce or inhibit) at least one TREM2 activity of the present disclosure. described after binding to the surface-expressed TREM2 protein. In some embodiments, the anti-TREM2 antibodies of the present disclosure are inert antibodies.

In some embodiments, anti-TREM2 antibodies as described herein bind to one or more amino acids within amino acid residues 29-112 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 29-41 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 47-69 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on a protein TREM2 corresponding to amino acid residues 4769 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 76-86 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 91-100 of human TREM2 (SEQ ID NO: 1 ) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 91-100 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 99-115 of human TREM2 ( SEQ ID NO: 1) or within the amino acid residues on the TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1. -112 human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 104-112 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 114-118 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind with one or more amino acids within amino acid residues

- 56 042890

130-171 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 130-171 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 139-153 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 139-153 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 139-146 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 139-146 of SEQ ID NO: 1. In some embodiments, antibodies to TREM2 as described herein bind to one or more amino acids within amino acid residues 130-144 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 130-144 of SEQ ID NO: 1. in some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 158-171 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1.

In some embodiments, anti-TREM2 antibodies as described herein bind to one or more amino acids within amino acid residues 43-50 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 49-57 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 139-146 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on a protein TREM2 corresponding to amino acid residues 139146 of SEQ ID NO:1. residues on the TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1.

The TREM2 proteins of the present disclosure include complementarity determining region 1 (CDR1) located at amino acid residues corresponding to amino acid residues 40-44 of human TREM2 (SEQ ID NO: 1); complementarity determining region 2 (CDR2) located at amino acid residues corresponding to amino acid residues 67-76 of human TREM2 (SEQ ID NO: 1); and complementarity determining region 3 (CDR3) located at amino acid residues corresponding to amino acid residues 114-118 of human TREM2 (SEQ ID NO: 1). Accordingly, in some embodiments, anti-TREM2 antibodies as described herein bind to one or more amino acids within amino acid residues 40-44 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 40-44 of SEQ ID NO: 1. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 67-76 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1. In some embodiments, anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 114-118 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1.

In other embodiments, antibodies of the present disclosure bind to an epitope that includes the amino acid residue Arg47 or Asp87 of human TREM2 (SEQ ID NO: 1). In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 40-44 of human TREM2 (SEQ ID NO: 1). In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 67-76 of human TREM2 (SEQ ID NO: 1). In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 114-118 of human TREM2 (SEQ ID NO: 1).

In some embodiments, the anti-TREM2 antibodies of the present disclosure competitively inhibit the binding of at least one antibody selected from any of the antibodies listed in Table 1. 1 and/or table. 8. In some embodiments, the anti-TREM2 antibodies of the present disclosure competitively inhibit the binding of at least one antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At 38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49,

- 57 042890

At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At 74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87. In some embodiments, the anti-TREM2 antibodies of the present disclosure competitively inhibit the binding of at least one of the following anti-TREM2 antibodies: At1, At9, At14, At22, At45, and At65. In some embodiments, anti-TREM2 antibodies as described herein bind to a human TREM2 epitope that is the same as or matches a TREM2 epitope bound by at least one antibody selected from any of the antibodies listed in Table 1. 1 and/or table. 8. In some embodiments, anti-TREM2 antibodies as described herein bind to a human TREM2 epitope that is the same as or matches a TREM2 epitope bound by at least one antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28,

At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45,

At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62,

At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79,

At80, At81, At82, At83, At84, At85, At86 and At87. In some embodiments, anti-TREM2 antibodies as described herein bind to a human TREM2 epitope that is the same as or matches a TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: At1, At9, At14, At22, At45 and At65. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind substantially the same TREM2 epitope bound by at least one antibody selected from any of the antibodies listed in Table 1. 1 and/or table. 8. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind substantially the same TREM2 epitope bound by at least one antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33,

At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50,

At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67,

At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84,

At85, At86 and At87. In some embodiments, anti-TREM2 antibodies as described herein bind substantially the same TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: At1, At9, At14, At22, At45, and At65. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) Epitope Mapping Protocols, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In a typical competition assay, immobilized TREM2 or cells expressing TREM2 on the cell surface are incubated in a solution containing a first labeled antibody that binds to TREM2 (e.g. human or non-human primate) and a second unlabeled antibody tested for ability to compete with the first. antibody for binding to TREM2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized TREM2 or cells expressing TREM2 are incubated in a solution containing the first labeled antibody but not containing the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TREM2, the excess of unbound antibody is removed and the amount of label associated with immobilized TREM2 or cells expressing TREM2 is measured. If the amount of label associated with immobilized TREM2 or cells expressing TREM2 is significantly reduced in the analyzed sample compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to TREM2. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise (a) a heavy chain variable region comprising at least one, two, or three HVRs selected from HVR-H1, HVR-H2, and HVR-H3 of any of the antibodies listed in table. 1 and/or table. 8, or selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23 , At24, At25, At26, At27, At28, At29, At30, At31, At32,

At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49,

At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66,

At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83,

At84, At85, At86 and At87; and/or (b) a light chain variable region containing at least one, two, and three HVRs selected from HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies listed in Table. 1 and/or table. 8, or selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23 , At24, At25, At26, At27, At28, At29,

At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46,

At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63,

At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80,

- 58 042890

At81, At82, At83, At84, At85, At86 and At87. In some embodiments, HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contain Kabat CDRs, Chothia CDRs, or CDR contact sequences as shown in Table 1. 1 and/or table. 8, or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21 , At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37,

At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52, At53, At54,

At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71,

At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87.

In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 containing the amino acid sequence of any of the HVR-H1 sequences listed in Table . 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At23, At24, At25, At26, At27,

At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44,

At45, At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61,

At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78,

At79, At80, At81, At82, At83, At84, At85, At86 and At87; (ii) HVR-H2 containing the amino acid sequence of any of the HVR-H2 sequences listed in table. 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At23, At24, At25, At26, At27, At28, At29, At30, At31,

At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48,

At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65,

At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82,

At83, At84, At85, At86 and At87; (iii) HVR-H3 containing the amino acid sequence of any of the HVR-H3 sequences listed in table. 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35,

At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52,

At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69,

At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87; (iv) HVR-L1 containing the amino acid sequence of any of the HVR-L1 sequences listed in table. 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39,

At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56,

At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73,

At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87; (v) HVR-L2 containing the amino acid sequence of any of the HVR-L2 sequences listed in table. 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At2 3, At24, At25, At26, At27,

At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44,

At45, At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61,

At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78,

At79, At80, At81, At82, At83, At84, At85, At86 and At87; and (vi) HVR-L3 containing the amino acid sequence of any of the HVR-L3 sequences listed in table. 1 and/or table. 8 or from an antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22 , At23, At24, At25, At26, At27, At28, At29, At30, At31,

At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48,

At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65,

At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82,

At83, At84, At85, At86 and At87.

In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises one or more of: (a) HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3-24 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-24; (b) HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25-49 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 25-49; and (c) an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-119 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from

- 59 042890 of the group consisting of SEQ ID NO: 50-119; and/or wherein the light chain variable domain comprises one or more of: (a) an HVR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-137 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137; (b) HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 25-49; and (c) an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 153-236 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NO. : 138-152.

In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise the heavy chain variable region of any of the antibodies listed in Table 1. 1 and/or table. 8, or selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23 , At24, At25, At26, At27, At28, At29, At30, At31, At32,

At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49,

At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66,

At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83,

At84, At85, At86 and At87; and/or the variable region of the light chain of any of the antibodies listed in table. 1 and/or table. 8, or selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23 , At24, At25, At26, At27, At28, At29,

At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46,

At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63,

At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80,

At81, At82, At83, At84, At85, At86 and At87.

Any of the antibodies according to the present description can be obtained using a cell line. In some embodiments, the cell line may be a yeast cell line. In some embodiments, the cell line may be a mammalian cell line. In some embodiments, the cell line may be a hybridoma cell line. Any cell line known in the art suitable for antibody production can be used to produce an antibody as described herein. Typical cell lines for the production of antibodies are described in the present description.

In some embodiments, the anti-TREM2 antibody is an anti-TREM2 monoclonal antibody selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17 , At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28,

At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45,

At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62,

At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79,

At80, At81, At82, At83, At84, At85, At86 and At87. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.

In some embodiments, the anti-TREM2 antibody is an At1 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as Ab1. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At1 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At1 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At1 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence at least about 95% homology with the amino acid sequence of SEQ ID NO: 3; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 25; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 50; (iv) HVR-L1 containing

- 60 042890 amino acid sequence from SEQ ID NO: 120, or an amino acid sequence with at least about 95% homology with the amino acid sequence from SEQ ID NO: 120; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 138; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 153.

In some embodiments, the anti-TREM2 antibody is an At9 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as does At9. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At9 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At9 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At9 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence at least about 95% homology to the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 33; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 58; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 124; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 144; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 161.

In some embodiments, the anti-TREM2 antibody is an anti-TREM2 monoclonal antibody Ab14. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as Ab14. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At14 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At14 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At14 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence at least about 95% homology to the amino acid sequence of SEQ ID NO: 13; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 36; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 63; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 122; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 146; and (vi) an HVR-L3 containing the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence of at least

- 61 042890 with about 95% homology to the amino acid sequence of SEQ ID NO: 166.

In some embodiments, the anti-TREM2 antibody is an At22 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as Ab22. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At22 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At22 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At22 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11, or the amino acid sequence at least about 95% homology to the amino acid sequence of SEQ ID NO: 11; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 34; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 60; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 123; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 141; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 173.

In some embodiments, the anti-TREM2 antibody is an anti-TREM2 Ab45 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as Ab45. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At45 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At45 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At45 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence at least about 95% homology with the amino acid sequence of SEQ ID NO: 7; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 29; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 87; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 120; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 138; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 196 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 196.

In some embodiments, the anti-TREM2 antibody is an At65 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody that substantially binds to the same TREM2 epitope as Ab65. In some embodiments, the anti-TREM2 antibody is an isolated anti

- 62 042890 body containing HVR-H1, HVR-H2 and HVR-H3 variable domains of the heavy chain of the monoclonal antibody At65. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At65 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of the At65 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence at least about 95% homology to the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 34; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 101; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 124; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 144; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 215 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 215.

In some embodiments, anti-TREM2 antibodies as described herein bind to one or more amino acids within amino acid residues 43-50 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 49-57 of human TREM2 (SEQ ID NO: 1) or within amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. In some embodiments, the anti-TREM2 antibodies of the present disclosure bind to an epitope that includes one or more amino acid residues 43-50 of human TREM2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies as described herein bind to an epitope that includes one or more amino acid residues 49-57 of human TREM2 (SEQ ID NO: 1). In some embodiments, the anti-TREM2 antibodies of the present disclosure competitively inhibit the binding of at least one of the following anti-TREM2 antibodies: At21 and At52. In some embodiments, anti-TREM2 antibodies as described herein bind to a human TREM2 epitope that is the same as or matches a TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: At21 and At52. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise (a) a heavy chain variable region comprising at least one, two, or three HVRs selected from HVR-H1, HVR-H2, and HVR-H3 of any of the Ab21 antibodies, and At52; and/or (b) a light chain variable region comprising at least one, two, and three HVRs selected from HVR-L1, HVR-L2, and HVR-L3 of any of the At21 and At52 antibodies. In some embodiments, HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 contain Kabat CDRs, Chothia CDRs, or CDR contact sequences as shown in Table 1. 1 and/or table. 8. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise the heavy chain variable region of any of the At21 and At52 antibodies; and/or the light chain variable region of any of the antibodies At21 and At52. In some embodiments, the anti-TREM2 antibody is an At52 or At21 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody that shares the same TREM2 epitope as either At52 or At21. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.

In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains of the At52 or At21 monoclonal antibody. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains of an At52 or At21 monoclonal antibody. In some embodiments, an anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 light chain variable domains.

- 63 042890 monoclonal antibody At52 or At21. In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 398, or the amino acid sequence at least about 95% homology to the amino acid sequence of SEQ ID NO: 398; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 399 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 399; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 400; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 401, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 401; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO: 402, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 402; and (vi) HVR-L3 containing the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403. In some embodiments, the anti-TREM2 antibodies described herein comprise at least one, two, three, four five or six HVRs selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 404; (ii) HVR-H2 containing the amino acid sequence of SEQ ID NO: 405 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 405; (iii) HVR-H3 containing the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 407, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 407; (v) HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 408; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 409 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 409.

In some embodiments, the anti-TREM2 antibodies of the present disclosure comprise a heavy chain variable region, and the heavy chain variable region comprises at least one, two, or three HVR sequences selected from those listed in Table 1. 1 and/or in table. 8; and/or contain a light chain variable region, and the light chain variable region contains at least one, two, and three HVR sequences selected from those listed in Table. 1 and/or in table. 8.

In some embodiments, an anti-TREM2 antibody as described herein competes for binding to TREM2 with one or more TREM2 ligands. Examples of suitable TREM2 ligands include, but are not limited to, E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides and lipidated amyloid beta peptide. Accordingly, in some embodiments, the one or more TREM2 ligands include E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides; and/or lipidated amyloid beta peptide. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.

Dissociation constants (KD) of anti-human TREM2 antibodies (e.g., human TREM2-Fc fusion proteins and monomeric human TREM2 proteins) and mouse TREM2 (e.g., mouse TREM2-Fc fusion proteins) can be less than 10 nM, less than 9.5 nM , less than 9 nM, less than 8.5 nM, less than 8 nM, less than 7.5 nM, less than 7 nM, less than 7 nM, less than 6.9 nM, less than 6.8 nM, less than less than 6.7 nM, less than 6.6 nM, less than 6.5 nM, less than 6.4 nM, less than 6.3 nM, less than 6.2 nM, less than 6.1 nM, less than 6 nM, less than 5.9 nM, less than 5.8 nM, less than 5.75 nM, less than 5.7 nM, less than 5.6 nM, less than 5.5 nM, less than 5.4 nM, less than 5.3 nM, less than 5.2 nM, less than 5.1 nM, less than 5 nM, less than 4.5 nM, less than 4 nM, less than 3.5 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, less than 0.95 nM, less than 0.9 nM, less than 0.85 nM, less than 0 .8 nM, less than 0.75 nM, less than 0.7 nM, less than 0.65 nM, less than 0.6 nM, less than 0.55 nM, less than 0.5 nM, less than 0, 45 nM, less than 0.4 nM, less than 0.35 nM, me

- 64 042890 less than 0.3 nM, less than 0.29 nM, less than 0.28 nM, less than 0.27 nM, less than 0.26 nM, less than 0.25 nM, less than 0.24 nM, less than 0.23 nM, less than 0.22 nM, less than 0.21 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM, less than 0.095 nM, less than less than 0.09 nM, less than 0.085 nM, less than 0.08 nM, less than 0.075 nM, less than 0.07 nM, less than 0.065 nM, less than 0.06 nM, less than 0.055 nM, or less than 0 .05 nM. In some embodiments, dissociation constants range from less than about 5.75 nm to less than about 0.09 nm. In some embodiments, anti-TREM2 antibody dissociation constants for human TREM2-Fc fusion proteins range from less than about 1.51 nm to less than about 0.35 nm. In some embodiments, anti-TREM2 antibody dissociation constants for monomeric TREM2 proteins range from less than about 5.75 nm to less than about 1.15 nm. In some embodiments, anti-TREM2 antibody dissociation constants for mouse TREM2-Fc fusion proteins range from less than about 0.23 nm to less than about 0.09 nm. In some embodiments, dissociation constants range from less than about 6.70 nm to less than about 0.23 nm. In some embodiments, anti-TREM2 antibody dissociation constants for human TREM2-Fc fusion proteins range from less than about 0.71 nm to less than about 0.23 nm. In some embodiments, anti-TREM2 antibody dissociation constants for monomeric TREM2 proteins range from less than about 6.70 nm to less than about 0.66 nm. In some embodiments, anti-TREM2 antibody dissociation constants for mouse TREM2-Fc fusion proteins range from less than about 4.90 nm to less than about 0.35 nm. Dissociation constants can be determined using any analytical method, including any biochemical or biophysical method such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see e.g. Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-jet analysis, and colorimetric and fluorescent protein melting assays. In some embodiments, the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.

Additional anti-TREM2 antibodies, such as antibodies that specifically bind to the TREM2 protein as described herein, can be identified, screened, or characterized for their physical/chemical properties and/or biological activity using various assays known in the art.

Antibodies to DAP12.

Some aspects of the present description relate to antibodies to DAP12.

Anti-DAP12 antibodies of the present disclosure typically bind to one or more DAP12 proteins expressed in a cell. In some embodiments, the DAP12 protein is expressed on the cell surface.

In some embodiments, anti-DAP12 antibodies of the present disclosure are agonist antibodies or antagonist antibodies that bind to a DAP12 protein of the present disclosure that is expressed on a cell surface and modulate (e.g., induce or inhibit) at least one DAP12 activity at of the present description after binding to the surface-expressed DAP12 protein. In some embodiments of the invention, antibodies to DAP12 according to the present description are inert antibodies.

In some embodiments, the DAP12 protein is expressed on the cell surface. In some embodiments, anti-DAP12 antibodies as described herein modulate (eg, induce or inhibit) one or more DAP12 activities. DAP12 activities modulated (eg, induced or inhibited) by anti-DAP12 antibodies may include, but are not limited to, TREM2 binding, DAP12 phosphorylation; TREM2 phosphorylation; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; physical activation; increased expression of anti-inflammatory mediators (eg, cytokines); decreased expression of one or more pro-inflammatory mediators; phosphorylation of ERK; increased expression of CCR7, induction of chemotaxis of microglial cells in relation to cells expressing CCL19 and CCL21; increase, normalization, or both the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; induction of osteoclast production, increased levels of osteoclastogenesis, or both; increased survival and/or function of microglial and/or macrophage cells (such as M1 macrophage cells and/or M1 microglial cells, activated M1 macrophages and/or M1 microglial cells and/or M2 macrophages and/or M2 microglial cells), dendritic cells , monocytes, osteoclasts, skin Langerhans cells and/or Kupffer cells; induction of clearance of apoptotic neurons; reduced expression of TNF-α; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells, monocytes, macrophages and/or microglia; decreased secretion of one or more inflammatory cytokines (that

- 65 042890 CI as, TNF-α, IL-10, IL-6 and/or MHB-1); decreased expression of one or more inflammatory receptors (such as CD86); increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of reduced levels of MCSF; reduced phagocytosis by macrophages, dendritic cells, monocytes and/or microglia in conditions of reduced levels of MCSF; and/or increased activity of one or more TREM2-dependent genes (eg, nuclear factors from activated T cell transcription factors (NFAT) from the family of transcription factors). The anti-TREM2 antibodies of the present disclosure may be used to prevent, reduce the risk of, or treat dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, and multiple sclerosis. In some embodiments, the anti-TREM2 antibodies of the present disclosure are monoclonal antibodies. Anti-TREM2 antibodies of the present disclosure can be tested to induce one or more TREM2 activities (e.g., phosphorylation of DAP12; involvement of Syk, ZAP70, or both in DAP12; PHYS activation; increased expression of anti-inflammatory mediators; decreased expression of pro-inflammatory mediators; phosphorylation of ERK; increased expression of CCR7, induction of microglial cell chemotaxis towards cells expressing CCL19 and CCL21; increased, normalized, or both, ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation; induction of osteoclast production, increased levels of osteoclastogenesis, or both ; induction of clearance of apoptotic neurons; decreased expression of TNF-α; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells; decreased secretion of one or more inflammatory cytokines; decreased expression of one or more inflammatory receptors; increased survival and/or function of dendritic cells , macrophages and/or microglial cells; increased phagocytosis by macrophages, dendritic cells and/or microglia in conditions of reduced levels of MCSF; reduced phagocytosis by macrophages, dendritic cells, and/or microglia in the presence of normal levels of MCSF, and increased activity of one or more TREM2-dependent genes (e.g., nuclear factors from activated T cell transcription factors (NFAT) from the family of transcription factors) using any suitable method known in the art the art and/or described in this document; For example, anti-TREM2 antibodies can be assayed in vitro for tyrosine phosphorylation of TREM2, DAP12, and/or ERK, by assaying Syk and/or Zap70 recruitment to the DAP12/TREM2 complex, by assaying activation of PISA, by assaying induction of expression of anti-inflammatory mediators (e.g., IL- 12p70, IL-6 and IL-10) or CCR7, or by assaying down-expression of pro-inflammatory mediators (eg IL-β and TNF) with TLR stimulation (eg LPS, CpG DNA or zymosan). Useful assays may include Western blotting (eg, for tyrosine-phosphorylated DAP12 or threonine/serine phosphorylated by PIKK kinase substrates), ELISA (eg, for secreted interleukin or cytokine secretion), FACS (eg, for anti-TREM2 antibody binding to TREM2), immunocytometry (e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PIBK kinase substrates), reporter gene assays (e.g., for TLR activation), increased survival and/or function of microglial cells, dendritic cells, monocytes, and/or macrophages, increased phagocytosis of apoptotic neurons, damaged synapses, A beta and/or other cellular debris by macrophages, dendritic cells, osteoclasts, and/or microglial cells, increased cytoskeletal reorganization, and reduced pro-inflammatory microglial responses, or other assays known in the art.

Certain aspects of the present disclosure provide antibodies to DAP12 that bind to human DAP12 or a homologue thereof, including, but not limited to, mammalian DAP12 protein, mouse DAP12 protein (UniProt accession number Q99NH8), rat DAP12 protein (Uniprot accession number D3ZZ89), protein Rhesus monkey DAP12 (Uniprot accession number F6QVF2), DAP12 bovine protein (Uniprot accession number Q05B59), DAP12 equine protein (Uniprot accession number F7D6L0), DAP12 porcine protein (Uniprot accession number H2EZZ3), and DAP12 dog protein (Uniprot accession number E2RP46) ; and induce at least one DAP12 activity. In some embodiments, the at least one DAP12 activity is DAP12 phosphorylation, TREM2 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, and/or decreased expression of one or more pro-inflammatory mediators.

In some embodiments, anti-DAP12 antibodies as described herein bind to a DAP12 protein as described herein and/or naturally occurring variants. In some embodiments, anti-DAP12 antibodies bind to human DAP12.

In some embodiments, anti-DAP12 antibodies of the present disclosure bind to a DAP12 protein of the present disclosure that is expressed on a cell surface and induce at least one DAP12 activity of the present disclosure upon binding to a surface-expressed DAP12 protein.

In some embodiments, anti-DAP12 antibodies as described herein bind to one or more amino acids within amino acid residues 22-40 of human DAP12.

- 66 042890 ka (SEQ ID NO: 2) or within amino acid residues on the DAP12 protein corresponding to amino acid residues 22-40 of SEQ ID NO: 2.

The dissociation constants (K _D ) of anti-DAP12 antibodies for human DAP12 and mouse DAP12 can be less than 10 pM, less than 9.5 nM, less than 9 nM, less than 8.5 nM, less than 8 nM, less than 7, 5 nM, less than 7 nM, less than 6.5 nM, less than 6 nM, less than 5.9 nM, less than 5.8 nM, less than 5.75 nM, less than 5.7 nM, less than 5.6 nM, less than 5.5 nM, less than 5.4 nM, less than 5.3 nM, less than 5.2 nM, less than 5.1 nM, less than 5 nM, less than 4.5 nM, less than 4 nM, less than 3.5 nM, less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, less than 0.95 nM, less than 0.9 nM, less than 0.85 nM, less than 0.8 nM, less than 0.75 nM, less than 0.7 nM, less than 0.65 nM, less than 0.6 nM, less than less than 0.55 nM, less than 0.5 nM, less than 0.45 nM, less than 0.4 nM, less than 0.35 nM, less than 0.3 nM, less than 0.25 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM, less than 0.095 nM, less than 0.09 nM, less than 0.085 nM, less than 0.08 nM, less than 0.075 nM, less than 0.07 nM, less than 0.065 nM, less than 0.06 nM, less than 0.055 nM, or less than 0.05 nM. Dissociation constants can be determined using any analytical method, including any biochemical or biophysical method such as ELISA, surface plasmon resonance (SPR), biolayer interferometry (see e.g. Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-jet analysis, and colorimetric and fluorescent protein melting assays.

Additional anti-DAP12 antibodies, such as antibodies that specifically bind to the DAP12 protein as described herein, can be identified, screened, or characterized for their physical/chemical properties and/or biological activity using various assays known in the art.

bispecific antibodies.

Some aspects of the present description relate to bispecific antibodies that bind to both TREM2 and DAP12 proteins of the present description. Methods for producing bispecific antibodies are well known in the art and are described herein. In some embodiments, the bispecific antibodies of the present disclosure bind to one or more amino acid residues from human TREM2 (SEQ ID NO: 1) or amino acid residues on the TREM2 protein corresponding to amino acid residues from SEQ ID NO: 1. In other embodiments, the antibodies as described herein bind to one or more amino acid residues of human DAP12 (SEQ ID NO: 2) or amino acid residues on the DAP12 protein corresponding to amino acid residues from SEQ ID NO: 2.

In some embodiments, the bispecific antibodies of the present disclosure recognize a first antigen and a second antigen. In some embodiments, the first antigen is human TREM2 or a naturally occurring variant thereof, or human DAP12 or a naturally occurring variant thereof. In some embodiments, the second antigen is a disease-causing protein selected from beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, bodies Levi, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, products translations associated with non-ATG repeats (RAN), dipeptide repeat peptides (DRP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine repeat peptides (RA) and proline-arginine repeat peptides (PR). In some embodiments, the second antigen is a blood-brain barrier specific protein selected from transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1, and LRP1; or ligands and/or proteins expressed by immune cells, wherein the ligands and/or proteins are selected from the group consisting of: CD40, 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 may be, without limitation, a protein expressed on one or more tumor cells.

fragments of antibodies.

Some aspects of the present disclosure relate to antibody fragments that bind to one or more human proteins selected from human TREM2, native human TREM2, pathological human TREM2 variant, human DAP12, and native human DAP12. In some embodiments, the antibody fragment is a Fab, Fab', Fab'-SH, F(ab')2, Fv, or scFv fragment. In some embodiments, an antibody fragment is used in combination with one or more antibodies that specifically bind to a disease-causing protein, selected from: beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein , prion protein PrPsc, huntingtin, calciton

- 67 042890 on, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, mild immunoglobulin AL chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine- arginine (GR), proline-alanine (PA) repeat peptides, proline-arginine (PR) repeat peptides, and any combination thereof, and/or one or more antibodies that specifically bind a cancer-associated protein selected from: CD40, 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, phosphatidylserine and any combination thereof.

Antibody scaffolds.

Any of the antibodies described herein further include a framework. In some embodiments, the framework is a human immunoglobulin framework. For example, in some embodiments, the antibody (eg, an anti-TREM2 antibody) comprises an HVR according to any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. Human immunoglobulin frameworks may be part of a human antibody, or a non-human antibody may be humanized by replacing one or more endogenous frameworks with human framework(s). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the best fit method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J Immunol., 151:2623 (1993)); mature (containing somatic mutations) human frameworks or human embryonic frameworks (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611- 22618 (1996)).

In some embodiments, the antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3 as described herein and one, two, three, or four heavy chain framework regions as shown in FIG. 2B and/or FIG. 20A. In some embodiments, the antibody comprises a light chain variable region comprising an HVR-L1, HVRL2 HVR-L3 as described herein, and one, two, three, or four light chain framework regions as shown in FIG. 2C and/or FIG. 20V. In some embodiments, the antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3 as described herein and one, two, three, or four heavy chain framework regions as shown in FIG. 2B and/or FIG. 20A and further comprises a light chain variable region comprising an HVR-L1, HVR-L2 HVR-L3 of the present disclosure and one, two, three, or four light chain framework regions as shown in FIG. 2C and/or FIG. 20V.

TREM2 and/or DAP12 Binding and Phosphorylation In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce TREM2 binding to DAP12 and/or DAP12 binding to TREM2. In other embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce TREM2 phosphorylation upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein. In other embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce phosphorylation of DAP12 upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein. In other embodiments, TREM2 mediated phosphorylation of TREM2 and/or DAP12 is induced by one or more SRC family tyrosine kinases. Examples of Src family tyrosine kinases include, without limitation, Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk.

DAP12 is known by names such as tyrosine kinase binding protein TYRO, TYROBP, KARAP, and PLOSL. DAP12 is a transmembrane signaling protein that contains an immunoreceptor tyrosine activating motif (ITAM) in its cytoplasmic domain. In some embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce phosphorylation of DAP12 into its ITAM motif. Any method known in the art can be used to determine protein phosphorylation, eg DAP12 phosphorylation.

In some embodiments, DAP12 is phosphorylated by SRC family kinases, resulting in the recruitment and activation of Syk kinase, ZAP70 kinase, or both, to DAP12. Thus, in some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may recruit Syk, ZAP70, or both to the DAP12/TREM2 complex.

Without being limited by theory, it is believed that anti-TREM2 antibodies and/or anti-DAP12 antibodies are truly

- 68 042890 description can be used to prevent, reduce the risk or treat pathological conditions and / or diseases associated with reduced levels of DAP12 activity, DAP12 phosphorylation or recruitment of Syk, ZAP70 or both to the DAP12 / TREM2 complex, including dementia, fronto temporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Physical activation.

In some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce activation of PBG upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

PICKs are a family of related intracellular signaling kinases capable of phosphorylation of the hydroxyl group of the inositol ring of phosphatidylinositol at position 3 (PtdIns). The FICK family is subdivided into three distinct classes (class I, class II, and class III) based on the primary structure, regulation, and specificity of the lipid substrate in vitro.

Activated PIB produces various 3-phosphorylated phosphoinositides including, but not limited to, PtdIns3P, PtdIns(3.4) P2, PtdIns(3.5)P2, and PtdIns(3,4,5)P3. These 3-phosphorylated phosphoinositides function in the mechanism by which signaling proteins are recruited into various cell membranes. These signaling proteins contain phosphoinositide binding domains, including, but not limited to, PX domains, plextrin homologous domains (PH domains), and FYVE domains. Any method known in the art can be used to determine the activation of PBIS.

Without being limited by theory, it is believed that the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure are effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced levels of FICK activity, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Increased expression of anti-inflammatory mediators.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure have anti-inflammatory activity in the brain upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein. TREM2 has been recently reported to have an anti-inflammatory function in the brain. In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure increase the expression of anti-inflammatory mediators (e.g., cytokines) and/or decrease the expression of pro-inflammatory mediators after binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

Inflammation is part of a complex biological response of vascular tissues to harmful stimuli such as pathogens, damaged cells, and irritants. The classic signs of acute inflammation are pain, fever, redness, swelling, and decreased function. Inflammation is the body's defensive attempt to remove harmful stimuli and begin the healing process. Inflammation can be classified as acute inflammation or chronic inflammation. Acute inflammation is the body's initial response to noxious stimuli and is mediated by increased influx of plasma and white blood cells (especially granulocytes) from the blood into damaged tissues. A cascade of biochemical events propagates and propagates the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Chronic inflammation is a long-term inflammation that results in a progressive shift in the types of cells present at the site of inflammation and is characterized by the simultaneous destruction and healing of tissue from the inflammatory process.

As used herein, anti-inflammatory mediators are proteins involved directly or indirectly (eg, via an anti-inflammatory signaling pathway) in a mechanism that reduces, inhibits, or inactivates the inflammatory response. Any method known in the art can be used to identify and characterize anti-inflammatory mediators. Examples of anti-inflammatory mediators include, but are not limited to, cytokines such as IL-12p70, IL-6 and IL-10.

In some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may increase the expression of anti-inflammatory mediators such as IL12p70, IL-6, and IL-10. In some embodiments of the invention, increased expression of anti-inflammatory mediators occurs in macrophages, dendritic cells and/or microglial cells. Increased expression may include, but is not limited to, an increase in gene expression, an increase in transcriptional expression, or an increase in protein expression. Any method known in the art can be used to determine the expression of a gene, transcript (eg, mRNA), and/or protein. For example, Northern blot can be used to determine the expression levels of an anti-inflammatory mediator gene, RT-PCR can be used to determine the transcription level of an anti-inflammatory mediator, and Western blot can be used

- 69 042890 to determine the levels of anti-inflammatory mediator protein.

In this context, an anti-inflammatory mediator may be overexpressed if its expression in one or more cells of a subject receiving treatment with an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure is greater than the expression of the same anti-inflammatory mediator expressed in one or more cells of the respective subject not receiving anti-TREM2 antibody and/or anti-DAP12 antibody treatment. In some embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure may increase the expression of an anti-inflammatory mediator in one or more cells of a subject by at least 10%, at least 15%, at least 20%, at least 25%, at least

30%, at least 35%, at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, at least

70%, at least 75%, at least 80%, at least 85%, at least

90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130 %, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180% , at least 190% or at least 200%, for example, compared with the expression of an anti-inflammatory mediator in one or more cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12. In other embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure increases the expression of an anti-inflammatory mediator in one or more cells of a subject by at least 1.5-fold, at least 1.6-fold, at least 1. .7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times, at least 2.4 times, at least 2, 45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times, for example, compared with the expression of an anti-inflammatory mediator in one or more cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12.

Without being limited by theory, it is believed that, in some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are useful in the prevention, risk reduction, or treatment of conditions and/or diseases associated with reduced levels of one or more anti-inflammatory mediators, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Decreased expression of pro-inflammatory mediators.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can reduce the expression of pro-inflammatory mediators upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

As used herein, pro-inflammatory mediators are proteins involved directly or indirectly (eg, via pro-inflammatory signaling pathways) in a mechanism that induces, activates, promotes, or otherwise enhances the inflammatory response. Any method known in the art can be used to identify and characterize pro-inflammatory mediators. Examples of pro-inflammatory mediators include, without limitation, cytokines such as IFN-a4, IFN-b, IL-1b, TNF-α, IL-10, IL-6, IL-12, p70, IL-8, CRP, TNF, members of the TGF-beta chemokine protein families, members of the IL-20, IL-33, PIL, IFNgamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18 and SRP.

In some embodiments, anti-TREM2 antibodies or anti-DAP12 antibodies of the present disclosure may reduce functional expression and/or secretion of pro-inflammatory mediators such as IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1b, TNF. , TNF-α, IL-10, IL-8, CRP, TGF-beta chemokine protein family members, IL-20 family members, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF , IL-11, IL-12, IL-17, IL-18 and SRP. In some embodiments, reduced expression of pro-inflammatory mediators occurs in macrophages, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells, and/or microglial cells. Reduced expression may include, but is not limited to, reduced gene expression, reduced transcriptional expression, or reduced protein expression. Any method known in the art can be used to determine the expression of a gene, transcript (eg, mRNA), and/or protein. For example, Northern blotting can be used to determine pro-inflammatory mediator gene expression levels, RT-PCR can be used to determine the level

- 70 042890 pro-inflammatory mediator transcription, and Western blotting can be used to determine pro-inflammatory mediator protein levels.

In some embodiments, the pro-inflammatory mediators include inflammatory cytokines. Accordingly, in some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may reduce the secretion of one or more inflammatory cytokines. Examples of inflammatory cytokines whose secretion can be reduced by anti-TREM2 and/or anti-DAP12 antibodies as described herein include, but are not limited to, TNF-α, IL-10, IL-6, MHB-1, IFN-a4, IFN-6, IL-1v. IL-8, CRP, TGF-beta chemokine protein family members, IL-20, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12 family members, IL-17 and IL-18.

In some embodiments, the pro-inflammatory mediators include inflammatory receptors. Accordingly, in some embodiments of the invention, antibodies to TREM2 and/or antibodies to DAP12 according to the present description can reduce the expression of one or more inflammatory receptors. Examples of inflammatory receptors whose secretion can be reduced by anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure include, but are not limited to, CD86.

In this context, a pro-inflammatory mediator may be underexpressed if its expression in one or more cells of a subject receiving treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody of the present disclosure is less than the expression of the same pro-inflammatory mediator expressed in one or more cells of an appropriate subject not receiving treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody. In some embodiments, an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody of the present disclosure may reduce the expression of a pro-inflammatory mediator in one or more cells of a subject by at least 10%, at least 15%, at least 20%. at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least by 190% or at least 200%, for example, compared with the expression of a pro-inflammatory mediator in one or more cells of the corresponding subject who did not receive treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody. In other embodiments, the anti-TREM2 agonist antibody and/or the anti-DAP12 agonist antibody can reduce the expression of a pro-inflammatory mediator in one or more cells of the subject by at least 1.5-fold, at least 1.6-fold, at least 1. .7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times, at least 2.4 times, at least 2, 45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times, for example, compared with the expression of a pro-inflammatory mediator in one or more cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12.

Without being limited by theory, it is believed that certain anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be useful in the prevention, risk reduction, or treatment of conditions and/or diseases associated with increased levels of one or more pro-inflammatory mediators, including dementia, frontal temporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Phosphorylation of ERK.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce extracellular signal-regulated kinase (ERK) phosphorylation upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

Extracellular signal-regulated kinases (ERKs) are widely expressed intracellular signaling protein kinases that are involved, for example, in the regulation of meiosis, mitosis, and post-mitotic functions in differentiated cells. Various stimuli such as growth factors, cytokines, viral infections, ligands for heterotrimeric G-coupled receptors, transforming agents, and carcinogens activate the ERK pathways. phosphorylated

- 71 042890 ERK leads to the activation of their kinase activity.

Without being limited by theory, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are believed to be effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced levels of ERK phosphorylation, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Increased expression of C-C chemokine receptor 7.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can increase expression of C-C chemokine receptor 7 (CCR7) upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein. Increased expression may include, but is not limited to, an increase in gene expression, an increase in transcriptional expression, or an increase in protein expression. Any method known in the art can be used to determine the expression of a gene, transcript (eg, mRNA), and/or protein. For example, Northern blotting can be used to determine levels of anti-inflammatory mediator gene expression, RT-PCR can be used to determine transcription levels of anti-inflammatory mediator, and Western blotting can be used to determine levels of anti-inflammatory mediator protein.

C-C chemokine receptor 7 (CCR7) is a member of the G protein-coupled receptor family. CCR7 is expressed in various lymphoid tissues and can activate B cells and T cells. In some embodiments, CCR7 can modulate T cell memory migration to secondary lymphoid organs such as lymph nodes. In other embodiments of the invention, CCR7 can stimulate the maturation of dendritic cells. CCR7 is a receptor protein that can bind the chemokine (C-C motif) ligands CCL19/ELC and CCL21.

In this context, CCR7 may be overexpressed if its expression in one or more cells of a subject receiving treatment with an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present description is greater than the expression of CCR7 expressed in one or more cells of the corresponding subject, not receiving anti-TREM2 antibody and/or anti-DAP12 antibody treatment. In some embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can increase CCR7 expression in one or more cells of a subject by at least 10%, at least 15%, at least 20%, at least at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135 %, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200%, for example, compared with the expression of CCR7 in one or more cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12. In other embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure increases CCR7 expression in one or more cells of a subject by at least 1.5-fold, at least 1.6-fold, at least 1, 7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.15 times, at least at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times, at least 2.4 times, at least 2.45 times times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times , at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times, for example, compared with the expression of CCR7 in one or more cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12.

In some embodiments of the invention, increased expression of CCR7 occurs in macrophages, dendritic cells and/or microglial cells. Increased expression of CCR7 can cause chemotaxis of microglial cells towards cells expressing the chemokines CCL19 and CCL21. Accordingly, in some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can induce microglial cell chemotaxis towards cells expressing CCL19 and CCL21.

Without being limited by theory, it is believed that in some embodiments of the invention, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure are useful in the prevention, risk reduction, or treatment of pathological conditions and/or diseases associated with reduced levels of CCR7, including dementia, frontal temporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or

- 72 042890 multiple sclerosis.

Strengthening or normalization of the ability of bone marrow dendritic cells to induce antigen-specific T-cell proliferation.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can enhance and/or normalize the ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation upon binding to a cell-expressed TREM2 protein and/or DAP12 protein.

In some embodiments, an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody of the present disclosure may enhance and/or normalize the ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation in one or more bone marrow dendritic cells of a subject at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120% at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%, for example, compared to the ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation in one or more bone marrow dendritic cells of the respective subject who has not received treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody. In other embodiments, the anti-TREM2 agonist antibody and/or the anti-DAP12 agonist antibody can enhance and/or normalize the ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation in one or more of the subject's bone marrow dendritic cells in at least 1, 5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times times, at least 2.4 times, at least 2.45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times , at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times, for example, compared to the ability of bone marrow dendritic cells to induce antigen-specific T cell proliferation in one or more bone marrow dendritic cells of the corresponding subject, who did not receive treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody.

Without being limited by theory, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are believed to be effective in preventing, reducing the risk, or treating conditions and/or diseases associated with an increased or dysregulated ability to induce antigen-specific T cell proliferation, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

production of osteoclasts.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce osteoclast production and/or increase the rate of osteoclastogenesis upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

In this context, an osteoclast is a type of bone cell that can remove bone tissue by removing the mineralized matrix and destroying organic bone (eg, bone atrophy). Osteoclasts can be formed by cell fusion of the monocyte-macrophage cell line. In some embodiments, osteoclasts may be characterized by high expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K.

In this context, the rate of osteoclastogenesis may be increased if the rate of osteoclastogenesis of a subject treated with an anti-TREM2 agonist antibody and/or a DAP12 agonist antibody of the present disclosure is greater than the rate of osteoclastogenesis of a corresponding subject not treated with an anti-TREM2 agonist antibody and/or agonist antibody to DAP12. In some embodiments, an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody of the present disclosure can increase the rate of osteoclastogenesis in a subject by at least 10%, at least 15%, at least 20%, at least 25%. %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least

- 73 042890

60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135% , at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or by at least 200%, for example, compared to the rate of osteoclastogenesis of a corresponding subject who has not received treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody. In other embodiments, an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody of the present disclosure can increase the rate of osteoclastogenesis in a subject at least 1.5-fold, at least 1.6-fold, at least 1.7-fold , at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times, at least 2.4 times, at least 2.45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4 times .5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times , for example, for example, compared with the rate of osteoclastogenesis of the corresponding subject who did not receive treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody.

In this context, the rate of osteoclastogenesis may be reduced if the rate of osteoclastogenesis of a subject treated with an anti-TREM2 antagonist antibody and/or a DAP12 antagonist antibody of the present disclosure is less than the rate of osteoclastogenesis of a corresponding subject not treated with an anti-TREM2 antagonist antibody and/or agonist antibody to DAP12. In some embodiments, an anti-TREM2 antagonist antibody and/or an anti-DAP12 antagonist antibody of the present disclosure may reduce the rate of osteoclastogenesis in a subject by at least 10%, at least 15%, at least 20%, at least 25%. %, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least at least 200%, for example, compared with the rate of osteoclastogenesis of the corresponding subject who did not receive treatment with an antagonist antibody to TREM2 and/or an antagonist antibody to DAP12. In other embodiments, an anti-TREM2 antagonist antibody and/or an anti-DAP12 antagonist antibody of the present disclosure can reduce the rate of osteoclastogenesis in a subject at least 1.5-fold, at least 1.6-fold, at least 1.7-fold , at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times, at least 2.4 times, at least 2.45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4 times .5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times, at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times , for example, for example, compared with the rate of osteoclastogenesis of the corresponding subject who did not receive treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody.

Without being limited by theory, it is believed that the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced osteoclast production and/or rate of osteoclastogenesis, including dementia, frontal temporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis; or associated with abnormal bone formation and maintenance, including osteoporosis, which is associated with an abnormal decrease in bone density, and osteoporotic diseases, which are associated with an abnormal increase in bone density.

Proliferation and survival of macrophages, microglial cells, dendritic cells, monocytes, osteoclasts, skin Langerhans cells and Kupffer cells.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies are

- 74 042890 of the present description can increase the proliferation, survival and/or function of macrophages, microglia (microglial) cells, dendritic cell monocytes, osteoclasts, skin Langerhans cells and Kupffer cells after binding to the TREM2 protein expressed in the cell and/or the DAP12 protein.

Microglial cells are a type of glial cells that are resident macrophages in the brain and spinal cord and thus act as the first and main form of active immune defense in the central nervous system (CNS). Microglial cells make up 20% of the total number of glial cells in the brain. Microglial cells continuously clear the CNS for plaque, damaged neurons, and infectious agents. The brain and spinal cord are considered immune-privileged organs because they are separated from the rest of the body by a series of endothelial cells known as the blood-brain barrier, which prevents most infections from reaching vulnerable nerve tissue. When infectious agents are injected directly into the brain or pass through the blood-brain barrier, microglial cells must respond quickly to reduce inflammation and destroy the infectious agents before they damage sensitive nerve tissue. Due to the inaccessibility of antibodies from the rest of the body (a small number of antibodies are small enough to pass through the blood-brain barrier), microglia must be able to recognize foreign bodies, engulf them, and act as antigen-presenting cells that activate T cells. Since this process must be carried out quickly to prevent potentially lethal damage, microglial cells are extremely sensitive to even small pathological changes in the CNS. They achieve this sensitivity, among other things, by having unique potassium channels that respond to even small changes in extracellular potassium concentration.

In this context, the macrophages of the present description include, but are not limited to, M1 macrophages, activated M1 macrophages, and M2 macrophages. As used herein, the macrophages of the present disclosure include, but are not limited to, M1 microglial cells, activated M1 microglial cells, and M2 microglial cells.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase the expression of CD83 and/or CD86 in dendritic cell monocytes, monocytes, and/or macrophages.

In this context, the proliferation rate, survival and/or function of microglia, monocytes and/or dendritic macrophage macrophages may include increased expression if the proliferation rate, survival and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, skin Langerhans cells and/or Kupffer cells of a subject treated with an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure is greater than the proliferation rate, survival and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, skin Langerhans cells and/or Kupffer cells of the corresponding a subject not receiving treatment with an anti-TREM2 agonist antibody and/or an anti-DAP12 agonist antibody. In some embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can increase the proliferation rate, survival, and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, skin Langerhans cells, and Kupffer cells of a subject by at least 10%. at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, at least at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%, e.g. compared to the proliferation rate, survival and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, Langerhans cells skin and Kupffer cells of the respective subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12. In other embodiments, an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure may increase the proliferation rate, survival, and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, skin Langerhans cells, and Kupffer cells of a subject by at least 1, 5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least at least 2.1 times, at least 2.15 times, at least 2.2 times, at least 2.25 times, at least 2.3 times, at least 2.35 times times, at least 2.4 times, at least 2.45 times, at least 2.5 times, at least 2.55 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 5.5 times, at least 6.0 times , at least 6.5 times, at least 7.0 times, at least 7.5 times, at least 8.0 times, at least 8.5 times, at least 9.0 times, at least 9.5 times, or at least 10 times, for example, according to

- 75 042890 comparison with the proliferation rate, survival and/or function of macrophages, microglia, dendritic cell monocytes, osteoclasts, skin Langerhans cells and Kupffer cells of the corresponding subject who did not receive treatment with an antibody to TREM2 and/or an antibody to DAP12.

Without being limited by theory, it is believed that the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced proliferation, survival, and/or function of macrophages, microglia, dendritic monocytes. cells, osteoclasts, skin Langerhans cells and Kupffer cells, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

clearance and phagocytosis.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce clearance and/or phagocytosis upon binding to a cell-expressed TREM2 protein and/or DAP12 protein of one or more apoptotic neurons, nerve tissue debris, nervous system debris non-nervous tissue of the nervous system, bacteria, other foreign bodies, pathogenic proteins, pathogenic peptides and/or pathogenic nucleic acids. In some embodiments, pathogenic proteins include, but are not limited to, amyloid beta or fragments thereof, Tau, IAPP, alpha synuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline- alanine (RA) and proline-arginine repeat peptides (PR). In some embodiments, the pathogenic nucleic acid includes, without limitation, antisense RNA expansion of GGCCCC (G2C4) repeats.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may induce one or more types of clearance, including, but not limited to, apoptotic neuron clearance, neural debris clearance, non-nervous tissue debris clearance, bacterial or other foreign bodies, pathogenic protein clearance, pathogenic peptide clearance, and pathogenic nucleic acid clearance, and cancer cell clearance.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce phagocytosis of one or more apoptotic neurons, neural debris, non-nervous tissue debris, bacteria, other foreign bodies, pathogenic proteins, pathogenic peptides, pathogenic nucleic acids and/or cancer cells.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may increase phagocytosis of macrophages, dendritic cells, monocytes, and/or microglia under conditions of reduced levels of macrophage colony stimulating factor (MCSF). Alternatively, in some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may reduce phagocytosis of macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal macrophage colony stimulating factor (MCSF) levels.

Without being limited by theory, it is believed that the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure are effective in preventing, reducing the risk, or treating pathological conditions and/or diseases associated with apoptotic neurons, neuronal tissue debris, and non-nervous tissue debris. systems, bacteria, other foreign bodies, or disease-causing proteins, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, and/or multiple sclerosis.

Phosphorylation of Syk.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can induce phosphorylation of spleen tyrosine kinase (Syk) upon binding to a cellularly expressed TREM2 protein and/or DAP12 protein.

Splenic tyrosine kinase (Syk) is an intracellular signaling molecule that functions downstream of TREM2 in the metabolic pathway by phosphorylation of several substrates, thus promoting the formation of a signaling complex that leads to cellular activation and inflammatory processes.

Without being limited by theory, it is believed that the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced levels of Syk phosphorylation, including dementia, frontotemporal dementia, disease Alzheimer's disease, Nasu-Hakola disease, Parkinso's disease

- 76 042890 on, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

TREM2-dependent gene expression.

In some embodiments, the anti-TREM2 agonist antibodies and/or anti-DAP12 agonist antibodies of the present disclosure may increase the activity and/or expression of TREM2-dependent genes, such as one or more nuclear factors from the Activated T Cell Transcription Factors (NFAT) family transcription factors. Alternatively, the anti-TREM2 antagonist antibodies and/or anti-DAP12 antagonist antibodies of the present disclosure may inhibit the activity and/or expression of TREM2-dependent genes, such as one or more transcription factors from the NFAT family of transcription factors.

Without being limited by theory, it is believed that the anti-TREM2 agonist antibodies and/or anti-DAP12 agonist antibodies of the present disclosure are effective in preventing, reducing the risk, or treating conditions and/or diseases associated with reduced levels of TREM2-dependent genes, including dementia, frontal temporal dementia, Alzheimer's disease, Nasu-Hakola's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy and/or multiple sclerosis.

Preparation of antibodies.

Anti-TREM2 and/or anti-DAP12 antibodies as described herein may include polyclonal antibodies, monoclonal antibodies, humanized and chimeric antibodies, human antibodies, antibody fragments (e.g., Fab, Fab'-SH, Fv, scFv, and F(ab' ) ₂ ), bispecific and polyspecific antibodies, multivalent antibodies, antibodies derived from a library, antibodies having modified effector functions, fusion proteins containing an antibody portion, and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site such as an epitope, having amino acid residues of the TREM2 and/or DAP12 protein as described herein, including glycosylated antibody variants, antibody amino acid sequence variants, and covalently modified antibodies. Anti-TREM2 antibodies and/or anti-DAP12 antibodies may be of human, mouse, rat or other origin (including chimeric or humanized antibodies).

(1) Polyclonal antibodies.

Polyclonal antibodies, such as anti-TREM2 polyclonal antibodies and/or anti-DAP12 polyclonal antibodies, are typically produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and adjuvant. It may be useful to conjugate an appropriate antigen (e.g., a purified or recombinant TREM2 protein and/or a purified or recombinant DAP12 protein of the present disclosure) to a protein that is immunogenic to the species being immunized, e.g. thyroglobulin or a soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as a maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ or R1N=C=NR, where R and R ¹ represent different alkyl groups. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorinomicolate).

The immunization protocol can be chosen by one of skill in the art without undue experimentation.

Animals are immunized against the desired antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg (for rabbits) or 5 μg (for mice) of the protein or conjugate with 3 volumes of complete Freund's adjuvant and injecting this solution intradermally at multiple sites. One month later, animals are re-immunized (booster immunization) with 1/5-1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood is taken from the animals and the serum is analyzed for antibody titer. Animals are boosted until a titer plateau is obtained. Conjugates can also be produced in recombinant cell culture as fusion proteins. Also, aggregating agents such as alum are useful in enhancing the immune response.

(2) Monoclonal antibodies.

Monoclonal antibodies such as anti-TREM2 monoclonal antibodies and/or anti-DAP12 monoclonal antibodies are derived from a population of substantially homogeneous antibodies, ie. the individual antibodies that make up this population are identical except for possible naturally occurring mutations and/or post-translational modifications (eg, isomerizations, amidations), which may be present in minor amounts. Thus, the definition of monoclonal indicates the nature of the antibody as not being a mixture of discrete antibodies.

For example, anti-TREM2 monoclonal antibodies and/or anti-DAP12 monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567) .

- 77 042890

In the hybridoma method, a mouse or other suitable host animal such as a hamster is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (e.g., purified or recombinant TREM2 protein and/or a purified or recombinant DAP12 protein as described herein). Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

Typically, the immunizing agent will include an antigenic protein (eg, a purified or recombinant TREM2 protein and/or a purified or recombinant DAP12 protein as described herein) or a fusion variant thereof. Typically, if the desired source is a non-human mammal, spleen cells or lymph node cells are used, or if cells of human origin are desired, peripheral blood lymphocytes (PBLs) are used. To obtain a hybridoma cell, lymphocytes are fused to an immortalized cell line using a suitable fusion agent such as polyethylene glycol. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

Typically, immortalized cell lines are transformed mammalian cells, in particular rodent, bovine or human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused original myeloma cells. For example, if the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) is absent in the original myeloma cells, then the culture medium for hybridomas should usually include hypoxanthine, aminopterin and thymidine (HAT medium), and such substances prevent the growth of HGPRT deficient cells.

Preferred myeloma cells are those that fuse efficiently, maintain a consistently high level of antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are mouse myeloma cell lines such as lines derived from mouse tumors MOPC-21 and MPC-11 (available from the Salk Institute Cell Distribution Center, San Diego, California USA), as well as SP-2 cells and their derivatives ( eg X63-Ag8-653) (available from American Type Culture Collection, Manassas, Virginia USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 ( Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells are grown is analyzed for the production of monoclonal antibodies directed against the antigen (eg, TREM2 protein and/or DAP12 protein as described herein). The binding specificity of monoclonal antibodies produced by hybridoma cells is preferably determined by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The culture medium in which hybridoma cells are grown can be assayed for the presence of monoclonal antibodies directed against the antigen (eg, TREM2 protein and/or DAP12 protein as described herein). Binding affinity and specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme immunoassay (ELISA). Such methods are known in the art. For example, binding affinity can be determined using the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

Once hybridoma cells have been identified that produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned by limiting dilution methods and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascitic fluid, or serum by conventional immunoglobulin purification methods such as, for example, protein A-sepharose chromatography, hydroxyapatite, gel electrophoresis, dialysis, affinity chromatography, and other methods described higher.

Monoclonal antibodies to TREM2 and/or monoclonal antibodies to DAP12 can also be obtained by recombinant DNA methods, such as those described in US patent No. 4816567 and as described above. DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional techniques (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of mouse antibodies). hybrido cells

- 78 042890 we serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression of bacterial DNA encoding an antibody include Skerra et al., Curr. Opin. Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Rev. 130:151-188 (1992).

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies can be isolated from antibody phage libraries generated using the methods described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nanomolar (nM) range) human antibodies using chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy. constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, such methods are acceptable alternatives to conventional monoclonal antibody-producing hybridoma methods for isolating monoclonal antibodies (eg, those that bind the TREM2 protein of the present disclosure).

DNA encoding antibodies or fragments thereof can also be modified, for example, by replacing the sequence encoding heavy and light chain constant domains instead of homologous mouse sequences (US patent No. 4816567; Morrison, et al., Proc. Natl Acad. Sci. USA , 81:6851 (1984)), or by covalent attachment to an immunoglobulin-coding sequence of all or part of a sequence encoding a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of a single antigen-binding site of an antibody to create a hybrid bivalent antibody comprising one antigen-binding site having specificity for an antigen and another antigen-binding site having specificity for another antigen.

The monoclonal antibodies described herein (eg, anti-TREM2 antibodies and/or DAP12 antibodies as described herein or fragments thereof) can be monovalent, the production of which is well known in the art. For example, one method involves recombinant expression of a light chain and a modified heavy chain of an immunoglobulin. The heavy chain is typically shortened at any point in the Fc region to prevent crosslinking of the heavy chain. Alternatively, relevant cysteine residues may be replaced with another amino acid residue or removed to prevent crosslinking. In vitro methods are also suitable for the production of monoclonal antibodies. Cleavage of antibodies to obtain their fragments, in particular Fab fragments, can be carried out using conventional methods known in the art.

Chimeric or hybrid anti-TREM2 antibodies and/or anti-DAP12 antibodies can also be made in vitro using known methods in synthetic protein chemistry, including methods using crosslinkers. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a simple thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

(3) Humanized antibodies.

The anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure or corresponding antibody fragments may further include humanized or human antibodies.

Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antibody antigen-binding subsequences) that contain the minimum sequence obtained from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, residues from the Fv reading frame region of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not present in either the recipient antibody or the imported CDR or framework sequences. Typically, a humanized antibody contains substantially all of at least one, more often two, variable domains in which all or substantially all of the CDRs correspond to those in a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin consensus sequence regions. Preferably, the humanized antibody also contains at least an immunoglobulin constant region (Fc) region, usually

- 79 042890 human immunoglobulin constant region. Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing anti-TREM2 antibodies and/or anti-DAP12 antibodies are well known in the art. Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, which are usually derived from an imported variable domain. In general, humanization can be carried out according to the method of Winter et al., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) or by replacing rodent CDR or CDR sequences with corresponding human antibody sequences. Thus, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than one intact human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar regions in rodent antibodies.

The choice of human variable domains, both light and heavy chain, for use in making humanized antibodies is very important to reduce antigenicity. According to the so-called best fit method, the variable domain sequence of a rodent antibody is screened against a complete library of known human variable domain sequences. The human sequence that most closely resembles that of the rodent is then taken as the human framework (FR) for the humanized antibody. Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies. Carter et al., Proc. Nat'l Acad. sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993).

In addition, it is important that the antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, in accordance with the preferred method, humanized antibodies are obtained by analyzing the original sequences and various conceptual humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and known to those skilled in the art. Computer programs are available that illustrate and show possible three-dimensional conformational structures of selected immunoglobulin sequences for study. The study of such images makes it possible to analyze the probable role of the residues in the functioning of the immunoglobulin sequence selected for the study, i.e. analyze residues that affect the ability of the immunoglobulin selected for the study to bind its corresponding antigen. Thus, FR residues from recipient and import sequences can be selected and combined so as to achieve desired antibody characteristics, such as increased affinity for the target antigen(s)(s) (eg, the TREM2 proteins of the present disclosure). In general, CDR residues directly and to the greatest extent affect antigen binding.

Various forms of humanized anti-TREM2 antibody and/or humanized anti-DAP12 antibody are contemplated. For example, a humanized anti-TREM2 antibody and/or a humanized anti-DAP12 antibody may be an antibody fragment, such as a Fab, that is optionally conjugated to one or more TREM2 ligands, such as HSP60. Alternatively, the humanized anti-TREM2 antibody and/or the humanized anti-DAP12 antibody may be an intact antibody, such as an intact anti-IgG1 antibody.

(4) Human antibodies.

Alternatively, human anti-TREM2 antibodies and/or human anti-DAP12 antibodies can be generated. It is now possible to obtain transgenic animals (eg mice) which are capable of producing the full repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. Homozygous deletion of the antibody heavy chain connecting region (J _H ) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a set of human germline immunoglobulin genes into such germline mutant mice should result in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al., Proc. Nat'l Acad. sci. USA 90:2551 (1993); Jakobovits et al. Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993); US patents No. 5591669 and WO 97/17852.

Alternatively, to generate human anti-TREM2 antibodies and/or human anti-DAP12 antibodies and antibody fragments in vitro, a phage display method using immunoglobulin variable (V) domain gene repertoires from unimmunized donors can be used.

- 80 042890

McCafferty et al., Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991). According to this method, antibody V domain genes are cloned in-frame with either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded copy of the DNA of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting such properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be done in many formats, reviewed in, for example, Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993). Several sources of V gene regions can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a broad spectrum of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a wide range of antigens (including self-antigens) can be isolated essentially following the method described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also US Pat. WO 2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus and Siegel (2004) J. Immunological Methods 290:69-80). In other embodiments, the yeast display method can be used to generate human anti-TREM2 antibodies and/or human anti-DAP12 antibodies and antibody fragments in vitro (e.g., Roberts and Szostak (1997) Proc. Natl. Acad. Sci. 94:12297- 12302; Schaffitzel et al. (1999) J. Immunological Methods 231:119-135; Lipovsek and Pluckthun (2004) J. Immunological Methods 290:51-67).

The methods of Cole et al., and Boerner et al., are also available to generate human monoclonal antibodies to TREM2 and/or human monoclonal antibodies to DAP12 (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 ( 1985) and Boerner et al., J. Immunol. 147(1): 86-95 (1991).Similarly, human anti-TREM2 antibodies and/or human anti-DAP12 antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, e.g., mice. in which the endogenous immunoglobulin genes are partially or completely inactivated.When stimulated, the production of human antibodies is observed, which are very similar to antibodies detected in humans in all respects, including gene rearrangement, assembly and spectrum of antibodies.This approach is described, for example, in US patents Nos. 5545807, 5545806, 5569825, 5625126, 5633425, 5661016 and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992), Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14: 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

Finally, human antibodies to TREM2 and/or DAP12 can also be generated by B cell activation (see US Pat. Nos. 5,567,610 and 5,229,275).

(5) Antibody fragments.

In some embodiments, there are advantages to using anti-TREM2 antibody fragments and/or anti-DAP12 antibody fragments rather than full anti-TREM2 antibodies and/or full anti-DAP12 antibodies. In some embodiments of the invention, the smaller size of the fragment allows for faster clearance and better penetration into the brain.

Various methods have been developed to obtain antibody fragments. Traditionally, such fragments have been prepared by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81 (1985) ). However, at present, such fragments can be directly produced by recombinant host cells, for example, using nucleic acids encoding anti-TREM2 antibodies and/or anti-DAP12 antibodies as described herein. Fab, Fv and scFv antibody fragments can all be expressed and isolated from E. coli, thereby allowing large quantities of these fragments to be directly produced. Anti-TREM2 antibody fragments and/or anti-DAP12 antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly extracted from E. coli cells and chemically linked to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In another approach, F(ab') ₂ fragments can be isolated directly from a culture of recombinant host cells. The production of Fab and F(ab') ₂ antibody fragments with increased in vivo half-lives is described in US Pat. No. 5,869,046. In other embodiments, the antibody of choice is a single chain Fv (ScFv) fragment. See WO 93/16185; US Pat. No. 5,571,894 and US Pat. No. 5,587,458. The anti-TREM2 antibody fragment and/or the anti-DAP12 antibody fragment can also be a linear antibody, such as described in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

(6) Bispecific and polyspecific antibodies.

Bispecific antibodies (BsAbs) are antibodies that have binding specificities for at least two different epitopes, including epitopes on the same

- 81 042890 or on another protein (for example, one or more TREM2 proteins according to the present description). Alternatively, one arm can be designed to bind to the target TREM2 antigen and/or target DAP12 antigen, and the other arm can be combined with the arm that binds the second protein. Such antibodies can be made from full length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies).

Methods for producing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two pairs of heavy chains/light chains of immunoglobulin, where these two pairs of chains have different specificities. Millstein et al., Nature, 305:537-539 (1983). Due to random segregation of immunoglobulin heavy and light chain genes, these hybridomas (quadromas) form a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of this correct molecule, which is usually performed by affinity chromatography steps, is quite cumbersome, yields of this product are low. Similar procedures are described in WO 93/08829 and Traunecker et al., EMBO J. 10:3655-3659 (1991).

In another approach, antibody variable domains with the desired binding specificity (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. Hybridization preferably occurs in an immunoglobulin heavy chain constant domain containing at least a portion of the hinge CH2 and CH3 regions. It is preferred to have a first heavy chain constant region (CH1) containing the site required for light chain binding present in at least one of the fusions. The DNA encoding these immunoglobulin heavy chain and, if desired, immunoglobulin light chain fusions are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments of the invention in which unequal proportions of the three polypeptide chains used in this construct provide optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in equal ratios results in high yields or when these ratios are not particularly important.

In a preferred embodiment of this approach, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains, since the presence of an immunoglobulin light chain in only one half of these bispecific molecules provides an easy separation path. This approach is described in WO 94/04690. For additional details on the formation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121: 210 (1986); and Garber, Nature Reviews Drug Discovery 13, 799-801 (2014).

In accordance with another approach, described in WO 96/27011 or US patent No. 5731168, the contact area between a pair of antibody molecules can be designed in such a way as to maximize the percentage of heterodimers that are recovered from the culture of recombinant cells. A preferred contact region contains at least a portion of the C _H 3 region of the constant domain of the antibody. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with large side chains (eg, tyrosine or tryptophan). Compensatory cavities of identical or similar size to relatively large side chains are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (eg, alanine or threonine). This provides a mechanism to increase the yield of this heterodimer relative to other undesired end products such as homodimers.

Methods for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be made using a chemical bond. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then re-converted to a Fab'-TNB derivative to generate a bispecific antibody. The obtained bispecific antibodies can be used as agents for the selective immobilization of enzymes.

Fab' fragments can be directly extracted from E. coli and chemically linked to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the formation of fully humanized F(ab')2 bispecific antibody molecules. Each Fab' fragment was separately secreted from E. coli and subjected to targeted chemical binding in vitro to obtain a bispecific antibody. The thus formed bispecific antibody was able to

- 82 042890 able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various methods have also been described for obtaining and isolating bivalent antibody fragments directly from recombinant cell culture. For example, bivalent heterodimers have been prepared using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. These antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. The diabody method described by Hollinger et al., Proc. Nat'l Acad. sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for generating bispecific/bivalent antibody fragments. These fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the V _L and V _H domains of another fragment, thereby forming two antigen-binding sites. Another strategy has also been reported to generate bispecific/bivalent antibody fragments using single chain Fv (sFv) dimers. See Gruber et al., J. Immunol., 152:5368 (1994).

Another method for producing bispecific antibodies is controlled Fab fragment exchange (cFAE), which is an easy-to-use method for generating bispecific IgG1 (bsIgG1). The protocol includes the following steps: (i) separate expression of two parental IgG1 containing unidirectional match point mutations in the CH3 domain; (ii) mixing parental IgG1s under acceptable redox conditions in vitro to allow recombination of half of the molecules; (iii) removal of the reducing agent to allow re-oxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and end product using chromatography based methods or mass spectrometry (MS) based methods. The protocol generates BsAbs with the correct IgG architecture, characteristics, and quality attributes at both laboratory scale (micrograms to milligrams) and mini-bioreactor scale (milligrams to grams), which are designed to simulate large-scale production (kilograms). Starting with good quality purified proteins, >95% exchange efficiency can be achieved within 2-3 days (including quality control). See Labrijn et al., Natur Protocols 9, 2450-2463 (2014); and Garber, Nature Reviews Drug Discovery 13, 799-801 (2014).

Antibodies having more than two valences are also contemplated. For example, trispecific antibodies can be generated. Tutt et al., J. Immunol. 147:60 (1991).

Typical bispecific antibodies can bind to two different epitopes on a given molecule (eg, TREM2 protein and/or DAP12 protein as described herein). In some embodiments, the bispecific antibodies bind to a first antigen, such as the TREM2 protein or DAP12 protein of the present disclosure, and a second antigen to facilitate transport across the blood-brain barrier. Numerous antigens known in the art that facilitate transport across the blood-brain barrier (see, for example, Gabathuler R., Approaches to transport therapeutic drugs across the blood-brain barrier to treat brain diseases, Neurobiol. Dis. 37 (2010) 48 -57). Such second antigens include, but are not limited to, transferrin receptor (TR), insulin receptor (HIR), insulin like growth factor receptor (IGFR), low density lipoprotein receptor proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, including CRM197 (non-toxic diphtheria toxin mutant), llama single domain antibodies such as TMEM 30(A) (flippase), transduction protein domains such as TAT, Syn-B or penetratin, polyarginine or commonly positively charged peptides, angiopep peptides such as ANG1005 (see, e.g., Gabathuler, 2010) and other cell surface proteins that saturate on blood-brain barrier endothelial cells (see, e.g., Daneman et al., PLoS One. 2010 Oct 29; 5 (10): el3741). In some embodiments, the second antigen for an anti-TREM2 antibody may include, without limitation, the DAP12 antigen as described herein. In other embodiments, the second antigen for an anti-DAP12 antibody may include, without limitation, the TREM2 antigen as described herein. In other embodiments, bispecific antibodies that bind to both TREM2 and DAP12 can facilitate and enhance one or more of TREM2 and/or DAP12 activities. In other embodiments, the second antigens for an anti-TREM2 antibody and/or an anti-DAP12 antibody may include, but are not limited to, anti-A beta peptide antigen, or anti-alpha-synuclein antigen, or anti-tau antigen, or anti-TDP-43 antigen. , or antigen to prion protein, or antigen to huntingtin or RAN protein, antigen to translation products, including dipiptide repeats (DPR peptides), consisting of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR) , proline-alanine (RA) or proline-arginine (PR).

(7) Multivalent antibodies.

A multivalent antibody can be internalized (and/or catabolized) faster than bi

- 83 042890 a valent antibody, a cell that expresses an antigen to which the antibodies bind. The anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, or antibody fragments, can be multivalent antibodies (which may be in a class other than the IgM class) with three or more antigen-binding sites (e.g., tetravalent antibodies) that can be readily generated by recombinant expression of a nucleic acid encoding antibody polypeptide chains. A multivalent antibody may contain a dimerization domain and three or more antigen binding sites. A preferred dimerization domain contains an Fc region or a hinge region. In this case, the antibody will contain an Fc region and three or more antigen-binding sites on the N-terminal side of the Fc region. A preferred multivalent antibody provided herein contains three to about eight, but preferably four, antigen-binding sites.

A multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), and the polypeptide chain contains two or more variable domains. For example, the polypeptide chain or polypeptide chains may contain VD1-(X1)n-VD2(X2)n-Fc, where VD1 is the first variable domain, VD2 is the second variable domain, Fc is one polypeptide chain of the Fc region, X1 and X2 is an amino acid or polypeptide, and n is 0 or 1. Similarly, the polypeptide chain or polypeptide chains may contain a V _H -CH ₁ -flexible linker-V _{H -CH} 1 -Fc chain region; or V _H -C _H 1V _H -C _H 1-Fc region of the chain.

The multivalent antibody provided herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody provided herein, for example, may comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides provided herein comprise a light chain variable domain and optionally further comprise a CL domain. Multivalent can recognize the TREM2 antigen, as well as, without limitation, additional antigens antigen to peptide A beta or antigen to alpha-synuclein protein, or antigen to tau protein, or antigen to TDP-43 protein, or antigen to prion protein, or antigen to the huntingtin protein or RAN, an antigen to translation products, including dipiptide repeats (DPR peptides) consisting of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline -arginine (PR), insulin receptor, insulin-like growth factor receptor. A receptor for transferrin or any other antigen that facilitates the transfer of antibodies across the blood-brain barrier.

(8) Effector function engineering.

In addition, it may be desirable to modify the anti-TREM2 antibody and/or the anti-DAP12 antibody of the present disclosure to alter effector function and/or to increase the serum half-life of the antibody. For example, the Fc receptor binding site on a constant region may be modified or mutated to eliminate or reduce binding affinity for certain Fc receptors such as FcyRI, FcyRII, and/or FcyRIII (for example, to reduce antibody-dependent cell-mediated cytotoxicity. B In some embodiments, effector function is impaired by eliminating N-glycosylation of the Fc region (e.g., in the IgG CH2 domain) of the antibody In some embodiments, effector function is impaired by modifying regions such as 233-236, 297, and/or 327-331 Human IgG as described in PCT WO 99/58572 and Armor et al., Molecular Immunology 40:585-593 (2003) Reddy et al., J. Immunology 164:1925-1933 (2000) In some embodiments of the invention it may also be desirable to modify an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure to modify effector function to increase selectivity for ITIM-containing FcgRIIb (CD32b) to increase antibody clustering on adjacent cells without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

To increase the half-life, a reutilization receptor binding epitope can be inserted into an antibody (especially an antibody fragment) as described, for example, in US Pat. IgG ₂ , IgG ₃ or IgG4), which is responsible for the increase in the half-life of the IgG molecule in serum in vivo.

(9) Other amino acid sequence modifications.

Modifications to the amino acid sequence of anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure or antibody fragments are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies or antibody fragments. Amino acid sequence variants of antibodies or antibody fragments are obtained by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or antibody fragment, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any

- 84 042890 the combination of deletion, insertion and replacement is carried out to obtain the final design, provided that the final design has the desired characteristics (ie, the ability to bind or physically interact with the TREM2 protein and/or DAP12 protein according to the present description). Amino acid substitutions can alter the post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of an anti-TREM2 antibody and/or an anti-DAP12 antibody that are preferred positions for mutagenesis is called alanine scan mutagenesis as described by Cunningham and Wells in Science, 244:1081-1085 (1989). This method identifies a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) and replaces them with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to achieve amino acid-antigen interaction. Such amino acid positions that are functionally sensitive to substitutions are further modified by introducing additional or other variants at the substitution sites. Thus, while the site of introduction of amino acid sequence variants is predetermined, the nature of the mutation per se may not necessarily be predetermined. For example, to analyze the occurrence of a mutation at a given site, alanine-scanning or selective mutagenesis is performed at the target codon and target region, and expressed antibody variants are screened for the desired activity.

Insertions of amino acid sequences include amino-(N) and carboxy-(C) terminal fusions ranging in length from a single residue to a polypeptide containing one hundred or more amino acid residues, as well as sections of one or more amino acid residues within the sequences. Examples of end inserts are N-terminal methionine antagonists or antagonists fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion to the N- or C-terminus of the antibody of an enzyme or polypeptide, which increases the serum half-life of the antibody.

Another type of variant is a variant based on substitution of amino acid residues. In these embodiments, at least one amino acid residue in the antibody molecule is replaced by another residue. Sites of greatest interest for displacement mutagenesis include hypervariable regions, but FR rearrangements are also considered. Conservative substitutions are given in table. And under the heading, preferred substitutions. If such substitutions change the biological activity, then they are the most significant changes and are given in table. And under the heading generic substitutions, or as further described below with respect to classes of amino acids, can be introduced and products screened.

Table A. Amino acid substitutions

original balance Typical replacements Preferred Substitutions Ala (A) val; leu; lie val Arg(R) lys; gin; asn lys Asn(N) gin; his; asp, lys; arg gin Asp(D) glu; asn glu

- 85 042890

Cys (C) ser; ala ser Gin (Q) asn; glu asn Glu (E) asp; gin asp Gly (G) ala ala His (Η) asn; gin; lys; arg arg He(I) leu; val; met; ala; phe; norleucine leu Leu(L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gin; asn arg Met(M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser(S) thr thr Thr(T) Ser ser TRP(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val(V) ile; leu; met; phe; ala; norleucine leu

Significant modifications in the biological properties of antibodies are accompanied by selective substitutions that differ significantly in their ability to maintain (a) the structure of the polypeptide backbone at the site of substitution, such as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and (c) side chain dimensions. Residues of natural origin are divided into groups depending on the general properties of the side chains:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions include substitutions of amino acid residues of one class for amino acid residues of another class.

Cysteine residues that are not involved in maintaining the proper conformation of the antibody can also be replaced primarily with serine to enhance the oxidative stability of the molecule and prevent unwanted crosslinking. In contrast, cysteine bonds can be introduced into an antibody molecule to improve its stability (especially when the antibody is an antibody fragment, such as an Fv fragment).

A particularly preferred type of substitution variant comprises substitution of one or more hypervariable region residues of the parent antibody (eg, a humanized or human anti-TREM2 antibody and/or a humanized or human anti-DAP12 antibody). Typically, the resulting variant(s) selected for further modification will have improved biological properties relative to the original antibody from which they are derived. A suitable method for obtaining such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus obtained are then detected in monovalent form on the surface of the filamentous bacteriophage particles in the form of their fusion with the M13 bacteriophage gene III product packaged in each particle. The phage display variants are then screened for their biological activity (eg, binding affinity) as described herein. In order to identify possible sites for modification of the hypervariable regions, alanine-scanning mutagenesis can be performed to identify hypervariable region residues that provide significant antigen binding. In an alternative

- 86 042890 option or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex in order to identify points of contact between antigen and antibody (for example, the TREM2 protein of the present description). Such contact residues and neighboring residues are potential targets for replacement, according to the methods presented in this document. Once such variants have been generated, they are screened as described herein, and improved antibodies, as established in one or more relevant studies, are selected for further modification.

Another type of amino acid variant of an antibody alters the original glycosylation profile of the antibody. By alteration is meant the removal of one or more carbohydrate molecules of an antibody and/or the addition of one or more glycosylation sites that are absent from the antibody.

Glycosylation of polypeptides is usually N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X and asparagine-X-threonine, where X is any amino acid other than proline, are sequences for enzymatic attachment of a carbohydrate component to the asparagine side chain. Thus, the presence of tripeptide sequences within a polypeptide creates a potential glycosylation site. O-linked glycosylation consists of the attachment of one of the sugars, which include Nacetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, mostly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

The addition of glycosylation sites to an antibody is conveniently accomplished by changing the amino acid sequence to include one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can also be made by adding, replacing one or more serine or threonine residues in the sequence of the original antibody (O-linked glycosylation sites).

Nucleic acid molecules encoding anti-IgE antibody amino acid sequence variants are prepared using a variety of methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or generation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR-assisted mutagenesis, and cassette mutagenesis of previously generated variant or non-variant versions of antibodies (eg, anti-TREM2 and/or anti-DAP12 antibodies as described herein) or antibody fragments.

(10) Other modifications of the antibody.

The anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure or corresponding antibody fragments may be further modified to contain additional non-protein fragments that are known in the art and are readily available, or contain various types of drug conjugates that are known in the art and easy to get. Preferably, derivatizable antibodies are water-soluble polymers.

Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer /maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The specified polymer may have any molecular weight and be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used to derivatize can be determined based on factors including, but not limited to, the specific properties or functions of the antibody to be improved, whether or not the antibody derivative is to be used in therapy under certain conditions. conditions, etc. Such methods and other suitable compositions are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science (2000).

Drug conjugation involves coupling a biologically active cytotoxic (antitumor) payload or drug to an antibody that specifically targets a specific tumor marker (eg, a protein that ideally should only be found on or on tumor cells). Antibodies track these proteins in the body and attach to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or engulfs the antibody along with the cytotoxin. Once the ADC is taken up, the cytotoxic drug is released and kills the cancer. Thanks to this targeting, the drug ideally has

- 87 042890 lower side effects and gives a wider therapeutic window than other chemotherapeutic agents. Methods for antibody conjugation are disclosed, known in the art (see, e.g., Jane de Lartigue, OncLive July 5, 2012; ADC Review on antibody-drug conjugates; and Ducry et al., (2010). Bioconjugate Chemistry 21 (1) : 5-13).

Binding assays and other assays.

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure (e.g., anti-TREM2 antibody activity) can be identified, screened, or characterized for their physical/chemical activities and/or biological activity using a variety of assays known in the art. or the assays described in the examples provided herein, such as, for example, radiolabeled immunoassays, optical assays, protein binding assays, biochemical screening assays, immunoassays, fluorescent assays, and/or cell survival assays.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies as described herein can be tested for antigen-binding activity, for example, by known methods such as ELISA, Western blot, and the like.

In some embodiments of the invention, competitive assays can be used to identify an antibody that competes with any of the antibodies listed in Table. 1 and/or table. 8 selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32,

At33, At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49,

At50, At51, At52, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66,

At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83,

At84, At85, At86 and At87, and/or human mAb17291 and/or humanized mAb17291 for binding to TREM2. In some embodiments, such a competing antibody binds to the same epitope (eg, a linear or conformational epitope) that is bound by any of the antibodies listed in Table 1. 1 and/or table. 8 selected from At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At21, At22, At23, At24,

At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38, At39, At40, At41,

At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At52, At53, At54, At55, At56, At57, At58,

At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75,

At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87, and/or human mAb17291 and/or humanized mAb17291. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) Epitope Mapping Protocols, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In a typical competition assay, immobilized TREM2 or cells expressing TREM2 on the cell surface are incubated in a solution containing a first labeled antibody that binds to TREM2 (e.g. human or non-human primate) and a second unlabeled antibody tested for ability to compete with the first. antibody for binding to TREM2. The second antibody may be present in the hybridoma supernatant. As a control, immobilized TREM2 or cells expressing TREM2 are incubated in a solution containing the first labeled antibody but not containing the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TREM2, the excess of unbound antibody is removed and the amount of label associated with immobilized TREM2 or cells expressing TREM2 is measured. If the amount of label associated with immobilized TREM2 or cells expressing TREM2 is significantly reduced in the analyzed sample compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to TREM2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

In some embodiments, assays are provided to identify anti-TRME2 agonist antibodies and/or anti-DAP12 agonist antibodies of the present disclosure having biological activity. Biological activity may include, for example, inducing, promoting, stimulating or otherwise increasing another TREM2 and/or DAP12 activity. Also provided are anti-TREM2 agonist antibodies and/or anti-DAP12 agonist antibodies having such biological activity in vivo and/or in vitro.

Assays known in the art and described herein (see, for example, Examples 23-27, 33-38, 41-44, 52-55 and 67-68) can be used to identify and test the biological activity of antibodies to TRME2 and/or anti-DAP12 antibodies that induce, promote, stimulate or otherwise increase another TREM2 and/or DAP12 activity. In some embodiments, assays are provided to test for one or more activities of an anti-TRME2 agonist antibody and/or an anti-DAP12 agonist antibody. An exemplary biological activity assay may include, for example, treating a population of immune cells, such as innate immune cells expressing TREM2 and/or DAP12, with an agonistic

- 88 042890 candidate anti-TRME2 antibody and/or agonistic anti-DAP12 antibody for sufficient time to allow binding and activation of the TREM2 protein and/or DAP12 protein, and measurement of cell survival (or cell death) versus cell survival (or cell death). cells) in the respective population in the absence of the candidate antibody. Methods for measuring cell survival or cell death are well known in the art and are described herein. Anti-TRME2 agonist antibodies and/or anti-DAP12 agonist antibodies can be identified by their ability to promote or prolong cell survival or delay cell death in a cell population relative to a control cell population, e.g., a cell population that has not been treated with a candidate agonist antibody. Certain aspects of the present disclosure relate to methods for identifying a candidate agonist antibody that specifically binds to TREM2 and/or DAP12. In some embodiments, a method for identifying a candidate agonist antibody that specifically binds to TREM2 and/or DAP12 comprises: a. contacting a population of innate immune cells expressing TREM2 and/or DAP12 on their surface with a candidate agonist antibody that specifically binds to TREM2 and/or DAP12 for a period of time sufficient for the candidate agonist antibody to promote cell survival in the innate immune population cells; b. comparison of the survival of a population of contacted innate immune cells with the survival of cells from a corresponding population of innate immune cells not exposed to a candidate agonist antibody that specifically binds to TREM2 and/or DAP12, with the increased survival of cells from a population of contacted innate immune cells indicating that the antibody the candidate is an agonist. Other aspects of the present disclosure relate to an isolated antibody that specifically binds to TREM2 and/or DAP12, identified by any of the disclosed methods for identifying an antibody that specifically binds to TREM2 and/or DAP12.

Nucleic acids, vectors and host cells.

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can be made using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In some embodiments, isolated nucleic acids are provided having a nucleotide sequence encoding any anti-TREM2 and/or antibodies to DAP12 according to the present description. Such nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an anti-TREM2 antibody and/or an anti-DAP12 antibody (eg, an antibody light and/or heavy chain). In some embodiments of the invention, one or more vectors (eg, expression vectors) containing such a nucleic acid are provided. In some embodiments, the invention provides a host cell containing such a nucleic acid. In some embodiments, the host cell is an isolated host cell. In this context, an isolated host cell is a cell that is identified and separated from at least one contaminating cell with which it is normally associated in the environment in which it was produced. In some embodiments of the invention, the isolated cell is not associated with all the components with which it is associated in the production environment. An isolated cell is presented in a different form than the form or conditions in which they are found in nature. Isolated cells are different from cells that naturally exist in tissues, organs, or individuals. In some embodiments, the isolated cell is a host cell as described herein. In some embodiments, the host cell contains (e.g., has been transduced with): (1) a vector containing a nucleic acid encoding an amino acid sequence containing an antibody VL and an amino acid sequence containing an antibody VH, or (2) a first vector containing a nucleic acid encoding an amino acid sequence comprising an antibody VL; and a second vector containing a nucleic acid encoding an amino acid sequence comprising an antibody VH. In some embodiments, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell). The host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

Proposed methods for obtaining antibodies to TREM2 and/or antibodies to DAP12 according to the present description. In some embodiments, the method includes culturing a host cell of the present disclosure containing a nucleic acid encoding an anti-TREM2 antibody and/or an anti-DAP12 antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or culture medium of the host cell).

For recombinant production of an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein, a nucleic acid encoding an anti-TREM2 antibody and/or an anti-DAP12 antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such a nucleic acid can be easily isolated and sequenced using conventional

- 89 042890 procedures (for example, using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

Suitable vectors containing a nucleic acid sequence encoding any of the anti-TREM2 and/or anti-DAP12 antibodies described herein, or fragments of their polypeptides (including antibodies) described herein, include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors may be constructed according to standard techniques or may be selected from a wide variety of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell to be used, suitable cloning vectors will generally be self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used. when selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (eg pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs and shuttle vectors such as pSA3 and pAT28. These and many more cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen.

Expression vectors are typically replicable polynucleotide constructs that contain a nucleic acid as described herein. Expression vectors can replicate in host cells as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components typically may include, but are not limited to, one or more of the following: a signal sequence; start of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers and terminator). For expression (ie, translation), one or more translational regulatory elements, such as ribosome binding sites, translation initiation sites, and stop codons, are usually also required.

Vectors containing the nucleic acids of interest can be introduced into the host cell by any of a number of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection; and infection (eg, when the vector is an infectious agent such as vaccinia virus). The choice of vectors or polynucleotides to be administered often depends on the characteristics of the host cell. In some embodiments of the invention, the vector contains a nucleic acid containing one or more amino acid sequences encoding antibodies to TREM2 and/or antibodies to DAP12 according to the present description.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, anti-TREM2 antibodies and/or anti-DAP12 antibodies can be produced in bacteria, in particular if glycosylation and Fc effector functions are not needed. With respect to the expression of antibody fragments and polypeptides in bacteria (e.g., US Pat. pp. 245-254, which describes the expression of antibody fragments in E. coli.). After expression, the antibody can be converted from the mass of bacterial cells into a soluble fraction, and then further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts with humanized glycosylation pathways, are also suitable hosts for cloning or expression of vectors encoding an antibody, resulting in the production of an antibody with a glycosylation profile partially or wholly characteristic for humans (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210215 (2006)).

Suitable host cells for expression of the glycosylated antibody can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells. In addition, plant cell cultures can be used as hosts (eg, US Pat.

In addition, vertebrate cells can be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be used. Other examples of suitable mammalian cell lines as host cells include SV40 (COS-7) transformed monkey kidney cell line CV1; a human embryonic kidney cell line (293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells described, for example, in Mather, Biol.

- 90 042890

reproduction. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other suitable mammalian-derived host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. An overview of some mammalian-derived host cell lines suitable for antibody production can be found in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

pharmaceutical compositions.

The anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be formulated into various dosage forms for therapeutic administration by combining the antibodies with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into solid, semi-solid, liquid, or gaseous forms. Examples of such dosage forms include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Depending on the dosage form, pharmaceutical compositions may include pharmaceutically acceptable, non-toxic carriers or diluents, which are conventional carriers for the manufacture of pharmaceutical compositions for administration to animals or humans. The diluent is chosen so as not to affect the biological activity of the combination. Examples of such diluents are, but are not limited to, distilled water, buffered water, saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical composition or dosage form of the present disclosure may further include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The compositions may also include additional substances to achieve near physiological conditions, such as buffering agents, pH and toxicity adjusting agents, wetting agents and detergents.

The pharmaceutical composition of the present disclosure may also include any of a variety of stabilizing agents such as, for example, an antioxidant. When a pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, decrease its toxicity, and increase solubility or uptake). Examples of such modifications or complexing agents include, but are not limited to, sulfate, gluconate, citrate, and phosphate. The composition polypeptides can also be combined with molecules that enhance their in vivo qualities. Such molecules include, but are not limited to, carbohydrates, polyamines, amino acids, other peptides, ions (eg, sodium, potassium, calcium, magnesium, manganese), and lipids.

Other examples of dosage forms that are suitable for various types of administration can be found in Remington Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985). For a summary of drug delivery methods, see Langer, Science 249:1527-1533 (1990).

For oral administration, the active ingredient may be administered in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. The active ingredient(s) can be encapsulated in gelatin capsules along with inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, carbonate magnesium. Examples of additional inactive ingredients that may be added to provide the desired color, taste, stability, buffering capacity, dispersion, or other known desirable properties are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Such diluents can be used to make compressed tablets. Both tablets and capsules can be formulated as sustained release products to provide continuous drug release over several hours. Compressed tablets may be sugar-coated or coated to mask any unpleasant taste and protect the tablet from weathering, or enteric-coated for selective degradation in the gastrointestinal tract. Liquid dosage forms for oral administration may contain dyes and flavors that increase the susceptibility of the patient.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that maintain the drug isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents. agents, solubilizers, thickeners, stabilizers and preservatives.

- 91 042890

Components used to prepare pharmaceutical compositions are preferably of high purity and substantially free of potentially hazardous impurities (e.g., at least food grade (NF), typically at least analytical grade, and generally at least pharmaceutical grade). In addition, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the final product is generally substantially free of any potentially toxic agents, especially any endotoxins that may be present during synthesis or purification. Compositions for parenteral administration are also sterile, substantially isotonic and manufactured under GMP conditions.

Drugs can be optimized for retention and stabilization in the brain or central nervous system. In the event that an agent is administered to the cranial region, it is desirable that the agent be retained in the region and not diffuse or otherwise cross the blood-brain barrier. Stabilization methods include cross-linking, multimerization or coupling with groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. to achieve an increase in molecular weight.

Other strategies to increase retention include placing an antibody, such as an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure, in a biodegradable or biodegradable implant. The rate of release of the therapeutically active agent is controlled by the rate of transport through the polymer matrix and biodegradation of the implant. Transport of the drug across the polymeric barrier will also depend on the solubility of the compound, the hydrophilicity of the polymer, the degree of crosslinking of the polymer, the expansion of the polymer upon absorption of water enough to make the polymeric barrier more permeable to the drug, the geometry of the implant, and the like. The implants are sized commensurate with the size and shape of the area chosen as the site of implantation. The implants can be particles, sheets, patches, plaques, fibers, microcapsules, and the like, and can be of any size or shape compatible with the chosen insertion site.

The implants may be monolithic, ie. having the active agent uniformly distributed over the polymer matrix, or encapsulated, wherein the active agent reservoir is encapsulated by the polymer matrix. The choice of polymer composition used will vary depending on the site of administration, the desired period of treatment, patient tolerance, the nature of the disease being treated, and the like. Characteristics of the polymers will include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, physiological half-life.

Biodegradable polymeric compositions that can be used include, but are not limited to, organic esters or ethers that decompose to physiologically acceptable products, including monomers. Anhydrides, amides, orthoesters, and the like, alone or in combination with other monomers, may find use. The polymers are condensation polymers. The polymers may or may not be crosslinked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids, both homo- and copolymers, and polysaccharides. Polyesters of interest include polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. The use of L-lactate or D-lactate provides a slowly biodegradable polymer, while the racemate significantly enhances degradation. Copolymers of glycolic and lactic acids are of particular interest when the rate of biodegradation is controlled by the ratio of glycolic acid to lactic acid. The most rapidly degrading copolymer has approximately equal amounts of glycolic and lactic acid, with each homopolymer being more resistant to degradation. The ratio of glycolic acid to lactic acid also affects the fragility of the implant, with a more flexible implant being suitable for larger geometries. Among the polysaccharides of interest, calcium alginate and functionalized celluloses, in particular carboxymethyl cellulose esters, are characterized in that they are insoluble in water, have a molecular weight of about 5 to 500 kDa, and so on. Biodegradable hydrogels can also be used in the implants of the present invention. As a rule, hydrogels are a copolymer material characterized by the ability to absorb liquid. Exemplary biodegradable hydrogels that can be used are described in Heller: Hydrogels in Medicine and Pharmacy, N.A. Peppes ed., Vol. III, CRC Press, Boca Raton, Fla., 1987, pp 137-149.

Dosages of drugs.

Pharmaceutical compositions of the present disclosure comprising an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure may be administered to an individual in need of treatment with an anti-TREM2 antibody and/or an anti-DAP12 antibody, preferably a human, according to known methods such as intravenous administration as a bolus or by continuous infusion over a certain period of time, intramuscular, intraperitoneal, intracerebrospinal,

- 92 042890 intracranial, intraspinal, subcutaneous, intraarticular, intrathecal, oral, or by topical or inhalation routes of administration.

The dosages and the desired concentration of drugs in the pharmaceutical compositions of the present invention may vary depending on the specific intended use. Determining the appropriate dose or route of administration is within the purview of the average person skilled in the art. Animal experiments provide reliable guidance for determining effective doses for human therapy. Interspecies scaling of effective doses can be performed according to the principles described in Mordenti, J. and Chappell, W. The Use of Interspecies Scaling in Toxicokinetics, in Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp.42-46.

For in vivo administration of any of the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure, normal dosage amounts may range from about 10 ng/kg to about 100 mg/kg of an individual's body weight per day or more, preferably from about 1 to 10 mg/kg/day, depending on the route of administration. With repeated administration for several days or longer, depending on the severity of the disease, disorder or pathological condition to be treated, treatment is maintained until the desired suppression of symptoms is achieved.

A typical application regimen may include the introduction of an initial dose of antibodies to TREM2 and/or antibodies to DAP12, from about 2 mg/kg, followed by a weekly maintenance dose from about 1 mg/kg every other week. Other regimens may be useful, depending on which pharmacokinetic decay pattern is desired by the clinician. For example, this document discusses dosing an individual from one to twenty-one times a week. In some embodiments, dosages in the range of about 3 µg/kg to about 2 mg/kg (e.g., about 3 µg/kg, about 10 µg/kg, about 30 µg/kg, about 100 µg/kg, about 300 mcg/kg, about 1 mg/kg and about 2 mg/kg). In some embodiments, the dosing frequency is three times a day, twice a day, once a day, once every two days, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks or once a month, once every two months, once every three months or longer. The course of treatment is easy to observe using conventional techniques and analyses. The dosage regimen, including the anti-TREM2 antibody and/or anti-DAP12 antibody administered, may change over time regardless of the dose used.

Doses for a particular anti-TREM2 antibody and/or anti-DAP12 antibody can be determined empirically in individuals who have received one or more administrations of an anti-TREM2 antibody and/or an anti-DAP12 antibody. Individuals receive escalating doses of anti-TREM2 antibody and/or anti-DAP12 antibody. To evaluate the efficacy of an anti-TREM2 antibody and/or an anti-DAP12 antibody, a clinical symptom of any of the diseases, disorders, or conditions described herein (e.g., dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, and multiple sclerosis) can be monitored.

Administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure may be continuous or intermittent, depending on, for example, the physiological state of the recipient, whether the intended administration is therapeutic or prophylactic, and other factors known to those skilled in the art. The administration of the anti-TREM2 antibody and/or the anti-DAP12 antibody may be substantially continuous over a preselected period of time, or may occur in a series of interval doses.

Recommendations for specific doses and routes of delivery are provided in the literature; see, for example, US Pat. Nos. 4,657,760; 5206344; and 5,225,212. Within the scope of the invention, different dosage forms will be effective for different types of treatment and different disorders, and that administration intended to treat a particular organ or tissue may require delivery in a different way than delivery to another organ or tissue. In addition, dosages may be administered via one or more separate administrations or via continuous infusion. With repeated administration for several days or longer, depending on the condition, treatment is continued until the desired suppression of the symptoms of the disease. At the same time, other regimens can be used. The course of treatment can be monitored using conventional methods and tests.

Therapeutic application.

Additional aspects of the present disclosure provide methods for modulating (eg, activating or inhibiting) TREM2, modulating (eg, activating or inhibiting) DAP12, modulating (eg, activating or inhibiting) FICK, modulating (eg, increasing or decreasing) the expression of one or more anti-inflammatory mediators. (e.g., IL-12p70, IL-6, and IL-10) or modulate (e.g., increase or decrease) the expression of one or more pro-inflammatory mediators (e.g., IL-1b and TNF) in an individual in need thereof by administering to the individual therapeutically an effective amount of an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein to modulate (e.g., induce or inhibit

- 93 042890) one or more TREM2 activities and/or DAP12 activities in an individual.

As described herein, the anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure can be used to prevent, reduce risk, or treat dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, lupus of the central nervous system, Behçet's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, disease of aging, epileptiform seizures , spinal cord injury, traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis , osteoporosis-related disease, Paget's disease of the bone, and cancers (eg, bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer). In some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies are agonistic antibodies. In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies are inert antibodies. In some embodiments, the anti-TREM2 antibodies and/or anti-DAP12 antibodies are antagonistic antibodies.

In some embodiments, the present disclosure provides methods for preventing, reducing risk, or treating an individual having dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, tauopathy , Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus, Behcet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, disease of aging, epileptiform seizures, spinal cord injury, traumatic brain injury trauma, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, osteoporosis-related disease, disease Paget's bone and cancer, by administering to an individual a therapeutically effective amount of an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein to modulate (e.g., induce or inhibit) one or more of TREM2 activities, including, but not limited to, DAP12 phosphorylation, PHYS activation, expression of one or more anti-inflammatory mediators (eg IL-12p70, IL-6 and IL-10) or expression of one or more pro-inflammatory mediators (eg IL-1b and TNF). In some embodiments, the anti-TREM2 antibody and/or the anti-DAP12 antibody is an agonist antibody. In some embodiments, the anti-TREM2 antibody and/or the anti-DAP12 antibody is an inert antibody. In some embodiments, the anti-TREM2 antibody and/or the anti-DAP12 antibody is an antagonist antibody. In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a glutamic acid stop codon substitution in the nucleic acid sequence encoding amino acid residue 14 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a glutamine-to-stop codon substitution in the nucleic acid sequence encoding amino acid residue 33 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a tryptophan-stop codon substitution in the nucleic acid sequence encoding amino acid residue 44 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having an arginine to histidine amino acid substitution at amino acid residue 47 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a tryptophan-stop codon substitution in the nucleic acid sequence encoding amino acid residue 78 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a valine to glycine amino acid substitution at the amino acid corresponding to amino acid residue 126 of the human TREM2 protein (SEQ ID

- 94 042890

NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having an amino acid substitution of aspartic acid for glycine at the amino acid corresponding to amino acid residue 134 of the human TREM2 protein (SEQ ID NO: 1). In some embodiments, the individual has a heterozygous variant of the TREM2 allele having the amino acid substitution of lysine for asparagine at the amino acid corresponding to amino acid residue 186 of the human TREM2 protein (SEQ ID NO: 1).

In some embodiments, the individual has a heterozygous variant of the TREM2 allele having a nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; amino acid substitution of threonine for methionine at the amino acid corresponding to the amino acid residue Thr66 of SEQ ID NO: 1; and/or the amino acid substitution of serine for cysteine at the amino acid corresponding to the amino acid residue Ser116 of SEQ ID NO: 1.

In some embodiments, the individual has a heterozygous variant of the DAP12 allele having a methionine to threonine amino acid substitution at the amino acid corresponding to the Met1 amino acid residue of SEQ ID NO: 2, a glycine to arginine amino acid substitution at the amino acid corresponding to the Gly49 amino acid residue of SEQ ID NO: 2 , a deletion within exons 1-4 of the nucleic acid sequence encoding SEQ ID NO: 2, an insertion of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2, and/or a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G141 nucleic acid sequence encoding SEQ ID NO: 2.

As described herein, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can also be used to induce and/or promote survival of innate immune cells. In some embodiments, the present disclosure provides methods for inducing or promoting innate immune cell survival in an individual in need thereof by administering to the individual a therapeutically effective amount of an anti-TREM2 agonist antibody and/or an anti-DAP12 antibody of the present disclosure.

As described herein, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can also be used to induce and/or promote wound healing, eg following injury. In some embodiments, the wound healing may be colonic wound healing after injury. In some embodiments, the present disclosure provides methods for inducing or promoting wound healing in an individual in need thereof by administering to the individual a therapeutically effective amount of an anti-TREM2 agonist antibody and/or an anti-DAP12 antibody of the present disclosure.

As described herein, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can also be used to inhibit the survival of innate immune cells. In some embodiments, the present disclosure provides methods for inhibiting innate immune cell survival in an individual in need thereof by administering to the individual a therapeutically effective amount of an anti-TREM2 and/or DAP12 antagonist antibody of the present disclosure.

In some embodiments, the methods of the present disclosure may include the co-administration of anti-TREM2 antibodies and/or anti-DAP12 antibodies or bispecific antibodies that bind to both TREM2 and DAP12, antagonistic TLRs, or agents that neutralize agonistic TLRs (e.g., neutralizing antibodies to cytokine or antibodies to interleukin).

In some embodiments, the methods of the present disclosure may include administering chimeric constructs including an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure in combination with a TREM2 ligand such as HSP60.

Dementia.

Dementia is a non-specific syndrome (i.e., a set of signs and symptoms) that is a severe loss of global cognitive ability in a previously healthy individual, beyond what would be expected from normal aging. Dementia can be static due to a unique global brain injury. Alternatively, dementia may be progressive, resulting in long-term deterioration in health due to damage or disease in the body. While dementia is much more common in the geriatric population, the disease can also occur before the age of 65. Cognitive areas affected by dementia include, but are not limited to, memory, concentration, language, and problem solving. Typically, symptoms must be present for at least six months before an individual is diagnosed with dementia.

Typical forms of dementia include, but are not limited to, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, semantic dementia, and dementia with Lewy bodies.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can prevent, reduce the risk of, and/or cure dementia. In some variants

- 95 042890 max implementation of the invention, the introduction of antibodies to TREM2 and/or antibodies to DAP12 can induce one or more activities of TREM2 and/or DAP12 in an individual with dementia (for example, phosphorylation of DAP12, activation of FICK, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).

Frontotemporal dementia.

Frontotemporal dementia (FTD) is a pathological condition that results in progressive destruction of the frontal lobe of the brain. Over time, degeneration can move to the temporal lobe. Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of cases of presenile dementia. Clinical features of FTD include memory impairment, behavioral abnormalities, personality changes, and language impairment (Cruts, M. & Van Broeckhoven, C., Trends Genet. 24:186-194 (2008); Neary, D., et al., Neurology 51 :1546-1554 (1998); Ratnavalli, E., Brayne, C., Dawson, K. & Hodges, J.R., Neurology 58:1615-1621 (2002)).

A significant proportion of cases of FTD are inherited in an autosomal dominant pattern, but even in the same family, symptoms can range from FTD with behavioral disturbances to primary progressive aphasia to ganglionic degeneration of the corticobasal region. FTD, like most neurodegenerative diseases, can be characterized by the abnormal presence of specific protein aggregates in the affected brain. Historically, the first descriptions of FTD identified the presence of intraneuronal accumulations of hyperphosphorylated tau protein in neurofibrillary tangles or Pick bodies. The causal role of the microtubule-associated protein has been confirmed by the identification of mutations in the gene encoding the tau protein in several families (Hutton, M., et al., Nature 393:702-705 (1998). However, most brains affected by FTD do not detect accumulation of hyperphosphorylated tau, but exhibit immunoreactivity to ubiquitin (Ub) and DNA-binding protein TAR (TDP43) (Neumann, M., et al., Arch. Neurol. 64:1388-1394 (2007)). these cases of FTD with Ub inclusions (FTD-U) carry mutations in the progranulin gene.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can prevent, reduce the risk of, and/or cure FTD. In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody may induce one or more of TREM2 and/or DAP12 activities in an individual having FTD (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or decreased expression of one or more pro-inflammatory mediators).

Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia. There is no cure for this disease, which worsens as it progresses and eventually leads to death. AD is most commonly diagnosed in people over 65 years of age. However, the less common, early-onset Alzheimer's can occur much earlier.

Common symptoms of Alzheimer's disease include behavioral symptoms such as difficulty remembering recent events; cognitive symptoms, confusion, irritability and aggression, mood swings, speech problems, and long-term memory loss. As the disease progresses, bodily functions are lost, eventually leading to death. Alzheimer's disease develops over an unknown and variable amount of time before becoming fully apparent, and it can progress undiagnosed for years.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can prevent, reduce the risk of, and/or cure Alzheimer's disease. In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce one or more of TREM2 and/or DAP12 activities in an individual having Alzheimer's disease (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).

Nasu-Hakol disease.

Nasu-Hakola disease (NHD), which may alternatively be referred to as polycystic lipomembrane osteodysplasia with sclerotic leukoencephalopathy (PLOSL), is a rare hereditary leukodystrophy characterized by progressive presenile dementia associated with recurrent bone fractures due to polycystic bone lesions of the lower and upper extremities. How the course of the disease is divided into four stages: latent, bone, early neurological and late neurological. After normal development during childhood (latent stage), BNC begins to manifest in adolescence or early adulthood (typical age of onset is 20-30 years) with pain in the hands, wrists, ankles, and feet. Patients then begin to suffer repetitive bone fractures due to polycystic bone and osteoporotic lesions in the bones of the extremities (bone phase). During the third or fourth decade of life (early neurological stage), patients experience marked personality changes (eg, euphoria, lack of concentration, loss of critical thinking, and social inhibition) characteristic of frontal lobe syndrome. Typically, patients also suffer from progressive

- 96 042890 memory disorders. Epileptic seizures are also common. Finally (late neurological stage), patients progress to profound dementia, cannot speak or move, and usually die by the age of 50.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or cure Nasu-Hakola disease. In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce one or more of TREM2 and/or DAP12 activities in an individual having Nasu-Hakola disease (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).

Parkinson's disease.

Parkinson's disease, which can be called idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or shaking palsy, is a neurodegenerative disease of the brain that affects the regulation of the motor system. The progressive death of dopamine-producing brain cells leads to the main symptoms of Parkinson's disease. Most often, Parkinson's disease is diagnosed in people over 50 years of age. Parkinson's disease is idiopathic (no known cause) in most people. However, genetic factors also play a role in the disease.

Symptoms of Parkinson's disease include, but are not limited to, tremors in the hands, arms, legs, jaw, and face, muscle rigidity in the limbs and trunk, slowness of movement (bradykinesia), postural instability, difficulty walking, neuropsychiatric problems, changes in speech or behavior, depression, anxiety, pain, psychosis, dementia, hallucinations and sleep problems.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or cure Parkinson's disease. In some embodiments, the administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce one or more of TREM2 and/or DAP12 activities in an individual having Parkinson's disease (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).

Lateral amyotrophic sclerosis.

In this context, amyotrophic lateral sclerosis (ALS) or motor neurone disease or Lou Gehrig's disease are used interchangeably and refer to a debilitating disease with different etiologies, characterized by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia) and shortness of breath (shortness of breath).

Progranulin has been shown to play a role in ALS (Schymick, J.C. et al., (2007) J. Neurol. Neurosurg Psychiatry; 78:754-6) and protect against damage caused by ALS-causing proteins such as TDP-43 ( Laird, A. S. et al., (2010) PLoS ONE 5: e13368). ProNRP has also been shown to induce p75-mediated death of oligodendrocytes and corticospinal neurons after spinal cord injury (Beatty et al., Neuron (2002),36, pp. 375-386; Giehl et al., Proc. Natl. Acad. Sci USA (2004), 101, pp 6226-30).

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or cure ALS disease. In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce one or more of TREM2 and/or DAP12 activities in an individual with ALS (e.g., DAP12 phosphorylation, FICK activation, increased expression of one or more anti-inflammatory mediators, or decreased expression of one or more pro-inflammatory mediators).

Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by an autosomal dominant mutation in the Huntingtin (HTT) gene. The spread of the cytokine-adeninguanine (CAG) triplet repeat in the Huntingtin gene results in a mutant form of the Huntingtin (Htt) protein encoded by the gene. This mutant Huntingtin (mHtt) protein is toxic and promotes neuronal death. Symptoms of Huntington's disease most often appear between 35 and 44 years of age, although they can appear at any age.

Symptoms of Huntington's disease include, but are not limited to, motor control problems, jerky, random movements (chorea), abnormal eye movements, balance problems, seizures, chewing difficulty, swallowing difficulty, cognitive problems, altered speech, memory loss, thinking difficulties, insomnia , fatigue, dementia, personality changes, depression, anxiety, and compulsive behavior.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody of the present disclosure may prevent, reduce the risk, and/or cure Huntington's disease (HD). In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody may induce one or

- 97 042890 more TREM2 and/or DAP12 activities in an individual having Huntington's disease (eg, DAP12 phosphorylation, FIBA activation, increased expression of one or more anti-inflammatory mediators, or decreased expression of one or more pro-inflammatory mediators).

Taupatia.

Taupatia disease or tupathies are a class of neurodegenerative diseases caused by the clustering of microtubule-associated tau proteins in the brain. Alzheimer's disease (AD) is the best known tauopathy and involves the accumulation of the tau protein in neurons as insoluble neurofibrillary tangles (NNCs). Other diseases and disorders of tauopathy include progressive supranuclear palsy, boxer's dementia (chromatic traumatic encephalopathy), frontotemporal dementia and parkinsonism associated with chromosome 17, lyticobodigue disease (Parkinson-Guam dementia complex), tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic granule disease (AZD), Huntington's disease, frontotemporal dementia, and frontotemporal degeneration of the frontotemporal lobe.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody of the present disclosure may prevent, reduce the risk, and/or cure tauopathy. In some embodiments, administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody can induce one or more of TREM2 and/or DAP12 activities in an individual having tauopathy (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or decreased expression of one or more pro-inflammatory mediators).

Multiple sclerosis.

Multiple sclerosis (PC) may also be referred to as multiple sclerosis or multiple sclerosis. MS is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, resulting in demyelination and scarring, as well as a wide range of signs and symptoms. PC affects the ability of nerve cells in the brain and spinal cord to communicate effectively with each other. Nerve cells communicate by sending electrical signals called action potentials along long fibers called axons that are contained in an insulating substance called myelin. In MS, the body's own immune system attacks and damages myelin. If myelin is lost, axons no longer conduct signals efficiently. The initial stage of PC usually occurs in young people and is more common in women.

The symptoms of PC include, but are not limited to, changes in perception such as loss of sensation or tingling; tingling or numbness such as hypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms; movement problems; problems with coordination and balance, such as ataxia; speech problems such as dysarthria or swallowing problems such as dysphagia; visual problems such as nystagmus, optic neuritis including phosphenes and diplopia; fatigue; acute or chronic pain; as well as problems with the bladder and intestines; cognitive impairment of varying degrees; emotional symptoms of depression or unstable mood; the Uchtoff phenomenon, which is an exacerbation of existing symptoms due to exposure to higher than usual ambient temperatures; and Lhermitte's symptom, which is a sensation of an electrical discharge going down the back when the neck is turned.

Without being limited by theory, it is believed that administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or cure multiple sclerosis. In some embodiments, the administration of an anti-TREM2 antibody and/or an anti-DAP12 antibody may induce one or more of TREM2 and/or DAP12 activities in an individual having multiple sclerosis (e.g., DAP12 phosphorylation, PICK activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).

Cancer.

Still further aspects of the present disclosure provide methods for preventing, reducing the risk of, or treating an individual having cancer, comprising administering to the individual a therapeutically effective amount of an isolated anti-TREM2 antibody and/or an anti-DAP12 antibody of the present disclosure. Any of the isolated antibodies of the present disclosure may be used in these methods. In some embodiments, the isolated antibody is an agonist antibody as described herein. In other embodiments, the isolated antibody is an antagonist antibody as described herein.

As described above, the tumor microenvironment is known to contain a heterogeneous immune infiltrate that includes T-lymphocytes, macrophages, and cells of myeloid/granulocytic origin. In particular, the presence of M2 macrophages in tumors is associated with poor prognosis. Drugs that reduce the number of these cells in tumors, such as agents that block CSF1R, show positive effects in preclinical models and in early clinical trials.

- 98 042890 It has been shown that TREM2 synergizes with CSF1 to promote macrophage survival in vitro, and that this effect is particularly pronounced for M2 macrophages compared to other phagocytic cell types. A preliminary preclinical study also demonstrated synergy between drugs that target tumor-associated macrophages (e.g., antibodies that block CSF1/CSF1R) and checkpoint-blocking antibodies that target T cells, indicating that manipulation of both types cells shows efficacy in tumor models where individual treatments are not effective enough (Zhu Y; Cancer Res. 2014 Sep 15; 74(18):5057-69). Therefore, without being limited by theory, it is believed that blocking TREM2 signaling in tumor-associated macrophages can inhibit suppression of the immune response in the tumor microenvironment, leading to a therapeutic anti-tumor immune response.

Due to the synergy between TREM2 and CSF1 and between tumor-targeting macrophages and T-cell-targeting macrophages, in some embodiments, methods for preventing, reducing risk, or treating an individual having cancer further comprise administering to the individual at least one antibody, which specifically binds to a molecule that inhibits the checkpoint. Examples of antibodies that specifically bind to a checkpoint inhibitory molecule include, but are not limited to, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, anti-PD-1 antibody, anti-B7-H3 antibody, anti-B7 antibody -H4 and anti-HVEM, anti-BTLA, anti-GAL9, anti-TIM3, anti-A2AR, anti-LAG-3, anti-phosphatidylserine, and any combination thereof. In some embodiments, at least one antibody that specifically binds to a checkpoint inhibitory molecule is administered in combination with an anti-TREM2 antagonist antibody and/or an anti-DAP12 antagonist antibody of the present invention.

In some embodiments, cancers to be prevented or treated using the methods of the present disclosure include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer, peritoneal cancer, hepatocellular cancer, gastrointestinal or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma , superficially spreading melanoma, lentigo malignant melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (eg, associated with brain tumors), Meigs syndrome, brain cancer, and head and neck cancer and associated metastases. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is selected from non-small cell lung cancer, glioblastoma, neuroblastoma, renal carcinoma, bladder cancer, ovarian cancer, melanoma, breast carcinoma, gastric cancer, and hepatocellular carcinoma. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer may be an early stage cancer or a late stage cancer. In some embodiments, the cancer may be a primary tumor. In some embodiments, the cancer may be a metastatic tumor at a secondary site from any of the above types of cancer.

In some embodiments, anti-TREM2 antibodies and/or anti-DAP12 antibodies of the present disclosure may be used to prevent, reduce risk, or treat cancer, including, but not limited to, bladder cancer, colon and rectal cancer, endometrial cancer, kidney cancer. , renal cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer.

In some embodiments, the present disclosure provides methods for preventing, reducing the risk of, or treating a subject having cancer by administering to the subject a therapeutically effective amount of an anti-TREM2 antibody and/or an anti-DAP12 antibody as described herein.

In some embodiments, the method further comprises administering to the subject at least one antibody that specifically binds to a checkpoint inhibitory molecule and/or other standard or experimental cancer therapy. In some embodiments, at least one antibody that specifically binds to a checkpoint inhibitory molecule is administered in combination with isolated anal

- 99 042890 titel. In some embodiments, at least one antibody that specifically binds to a checkpoint inhibitory molecule is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, antibodies to B7-H3, antibodies to B7-H4, antibodies to HVEM, antibodies to B- and T-lymphocyte attenuator (BTLA) antibodies to cytotoxicity suppression receptor (KIR), antibodies to GAL9, antibodies to TIM3, antibodies to A2AR, antibodies to LAG-3, antibodies to phosphatidylserine, antibodies to CD27, and any combination thereof. In some embodiments, the standard or experimental cancer therapy is one or more of radiotherapy, chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®), adaptive cell transfer (ACT), chimeric antigen receptor T -cells (CAR-T), vaccine therapy and cytokine therapy.

In some embodiments, the method further comprises administering to the subject at least one antibody that specifically binds to an inhibitory cytokine. In some embodiments of the invention, at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with an isolated antibody. In some embodiments, at least one antibody that specifically binds to an inhibitory cytokine is selected from an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.

In some embodiments, the method further comprises administering to the subject at least one antibody that specifically binds to the checkpoint stimulating protein. In some embodiments, at least one agonist antibody that specifically binds to a checkpoint stimulating protein is administered in combination with an isolated antibody. In some embodiments, the at least one agonist antibody that specifically binds to the checkpoint stimulatory protein is selected from a CD40 agonist antibody, an OX40 agonist antibody, an ICOS agonist antibody, a CD28 agonist antibody, a CD137/4- agonist antibody. 1BB, an anti-CD27 agonist antibody, an agonist antibody for glucocorticoid-induced TNFR-related GITR protein, and any combination thereof.

In some embodiments of the invention, the method further comprises administering to the individual at least one stimulatory cytokine. In some embodiments of the invention, at least one stimulatory cytokine is administered in combination with an isolated antibody. In some embodiments, at least one stimulatory cytokine is selected from TNF-α, IL-10, IL-6, IL-8, CRP, members of the TGF-beta chemokine protein families, members of the IL-20 family, IL-33, PHIL, OSM, CNF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α, IFN-β, IL-2, IL-18, GM-CSF, G- CSF and any combination thereof.

Kits/products.

The present disclosure also refers to kits containing an isolated antibody of the present disclosure (eg, an anti-TREM2 antibody or an anti-DAP12 antibody described herein) or a functional fragment thereof. Kits according to the present description may include one or more containers containing a purified antibody according to the present description. In some embodiments of the invention, the kits further include instructions for use in accordance with the methods of the present description. In some embodiments, these instructions describe the administration of an isolated antibody of the present disclosure (e.g., an anti-TREM2 antibody or an anti-DAP12 antibody described herein) to prevent, reduce risk, or treat an individual having a disease, disorder, or injury selected from dementia. , frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease and multiple sclerosis in accordance with any of the methods of this description.

In some embodiments of the invention, the instructions contain a description of how to determine TREM2 and/or DAP12, for example, in an individual in a tissue sample or in a cell. The kit may further comprise a description of selecting an individual suitable for treatment based on a determination of whether the individual has the disease and stages of the disease.

In some embodiments, kits may further comprise another antibody as described herein (e.g., at least one antibody that specifically binds to a checkpoint inhibitory molecule, at least one antibody that specifically binds to an inhibitory cytokine, and/or at least at least one agonist antibody that specifically binds to a checkpoint stimulatory protein) and/or at least one stimulatory cytokine. In some embodiments, the kits may further include instructions for using an antibody and/or a stimulatory cytokine in combination with an isolated antibody of the present disclosure (e.g., an anti-TREM2 agonist antibody described herein), instructions for using the isolated antibody of the present disclosure in combination with antibody and/or stimulating cytokine or instruction

- 100 042890 for use of an isolated antibody of the present disclosure and an antibody and/or stimulatory cytokine according to any of the methods of this disclosure.

As a rule, the instructions include information about the dosage, regimen and route of administration for the intended treatment. The containers may be in the form of unit doses, multi-dose packs (eg, multi-dose packs), or sub-unit doses. The instructions provided in the kits herein are typically written instructions on a label or package insert (for example, the paper sheet included in the kit), but machine-readable instructions (for example, instructions recorded on a magnetic or optical storage disk). ) are also acceptable.

The label or package insert indicates that the composition is used to treat, for example, a disease as described herein. Instructions may be provided for the practical application of any of the methods described herein.

Kits according to this description are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Also contemplated are packages intended for use in combination with a particular device, such as an inhaler, a nasal device (eg, a nebulizer), or an infusion device, such as a minipump. The kit may have a sterile inlet (for example, the container may be an intravenous solution bag or a vial having a stopper pierced by a hypodermic needle). The container may also have a sterile inlet (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active drug in the composition is an isolated antibody as described herein (eg, an anti-TREM2 antibody or an anti-DAP12 antibody described herein). The container may further contain a second pharmaceutically active preparation.

Containers may optionally offer additional components such as buffers and interpretive information. Typically, a kit comprises a container and a label or package insert associated with or on the container.

diagnostic application.

The isolated antibodies of the present disclosure (eg, the anti-TREM2 antibody or the anti-DAP12 antibody described herein) are also of diagnostic value. Thus, this description provides methods for using antibodies according to this description or functional fragments thereof for diagnostic purposes, such as the detection of TREM2 and/or DAP12 in an individual or in tissue samples obtained from an individual.

In some embodiments of the invention, the individual is a human. In some embodiments, the individual is a patient suffering from or at risk of developing dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, tauopathy, multiple sclerosis, or cancer. In some embodiments of the invention, diagnostic methods include the detection of TREM2 and/or DAP12 in a biological sample, such as a biopsy sample, tissue or cell. An isolated antibody of the present disclosure (eg, an anti-TREM2 antibody or an anti-DAP12 antibody described herein) is contacted with a biological sample and detects the antibody bound to the antigen. For example, a tissue sample (eg, a biopsy sample) can be stained with an anti-TREM2 antibody or an anti-DAP12 antibody described herein to detect and/or quantify disease-associated macrophages (eg, M2 type macrophages). The detection method may include quantification of an antibody bound to an antigen. Detection of antibodies in biological samples can be by any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micropositron emission tomography. In some embodiments, the antibody is radiolabelled, eg, with ¹⁸ F, and subsequently detected using micropositron emission tomography analysis. Antibody binding can also be quantified in a patient using non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single photon emission computed tomography (SPECT), computed tomography (CT), and axial computed tomography (ACT).

In other embodiments, an isolated antibody as described herein (e.g., an anti-TREM2 antibody or an anti-DAP12 antibody described herein) can be used to detect and/or quantify, for example, microglia in a brain sample taken from a preclinical disease model. (e.g., a non-human disease model). Thus, an isolated antibody of the present disclosure (e.g., an anti-TREM2 antibody or an anti-DAP12 antibody described herein) may be useful in evaluating post-treatment therapeutic response in a model for a neurological disease or injury such as

- 101 042890 as dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease or multiple sclerosis compared to control.

Numbered embodiments of the invention.

The following numbered embodiments of the invention represent some aspects of the invention.

1. An isolated agonist antibody that binds to the TREM2 protein, the DAP12 protein, or both, wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both.

2. The isolated antibody of embodiment 1, wherein the TREM2 protein, DAP12 protein, or both is a mammalian protein or a human protein.

3. The isolated antibody of embodiment 2, wherein the TREM2 protein, the DAP12 protein, or both, is a wild-type protein.

4. The isolated antibody of embodiment 2, wherein the TREM2 protein, DAP12 protein, or both, is a naturally occurring variant.

5. An isolated antibody according to any one of embodiments 1-4, characterized in that one or more TREM2 activities include TREM2 binding to DAP12.

6. An isolated antibody according to any one of embodiments 1-4, characterized in that one or more DAP12 activities include binding DAP12 to TREM2.

7. An isolated antibody according to any one of embodiments 1-6, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve phosphorylation of DAP12.

8. The isolated antibody of embodiment 7 wherein DAP12 phosphorylation is induced by one or more SRC family tyrosine kinases.

9. An isolated antibody according to any one of embodiments 1-8, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve activation of PIB.

10. An isolated antibody according to any one of the embodiments of the invention 1-9, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include increased expression of one or more anti-inflammatory mediators selected from the group consisting of IL-12p70, IL -6 and IL-10.

11. The isolated antibody of embodiment 10, wherein the overexpression of one or more anti-inflammatory mediators occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, and microglial cells.

12. An isolated antibody according to any one of the embodiments of the invention 1-11, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN -b, IL-6, IL-12 p70, IL-1r, TNF.

13. The isolated antibody of embodiment 12, wherein the reduced expression of one or more pro-inflammatory mediators occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, and microglial cells.

14. The isolated antibody of any one of embodiments 1-13, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve extracellular signal-regulated kinase (ERK) phosphorylation.

15. An isolated antibody according to any one of embodiments 1-14, wherein one or more of TREM2 activities, DAP12 activities, or both activities include increased expression of the C-C chemokine receptor 7 (CCR7).

16. An isolated antibody according to any one of embodiments 1-15, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve the induction of microglial cell chemotaxis towards cells expressing CCL19 and CCL21.

17. An isolated antibody according to any one of embodiments 1-16, wherein one or more of TREM2 activities, DAP12 activities, or both of the activities include reducing, normalizing, or both the ability of bone marrow dendritic cells to induce proliferation of antigen-specific T cells.

18. The isolated antibody of any one of embodiments 1-17, wherein one or more of TREM2 activities, DAP12 activities, or both include induction of osteoclast production, increased levels of osteoclastogenesis, or both.

19. An isolated antibody according to any one of embodiments 1-18, wherein one or more of the TREM2 activities, DAP12 activities, or both activities involve an increase in the survival of macrophages, microglial cells, or both.

20. An isolated antibody according to any one of embodiments 1-19, characterized in that one or more TREM2 activities, DAP12 activities, or both activities comprise

- 102 042890 increased function of macrophages, microglial cells, or both.

21. An isolated antibody according to any one of embodiments 1-20, wherein one or more TREM2 activities, DAP12 activities, or both activities comprise induction of one or more types of non-inflammatory clearance selected from the group consisting of non-inflammatory clearance of apoptotic neurons. inflammation, clearance of neural debris without inflammation, clearance of non-nervous debris without inflammation, clearance of bacteria or other foreign bodies without inflammation, and clearance of pathogenic protein without inflammation.

22. An isolated antibody according to any one of embodiments 1-21, characterized in that one or more of TREM2 activities, DAP12 activities, or both activities include the induction of phagocytosis without inflammation of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, or pathogenic proteins without inflammation.

23. An isolated antibody according to embodiment 21 or embodiment 22, wherein the disease-causing protein is selected from the group consisting of A beta peptide, alpha-synuclein protein, Tau protein, TDP-43 protein, prion protein, and huntingtin protein.

24. An isolated antibody according to any one of embodiments 1-23, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve normalization of abnormal gene expression that is TREM2/DAP12 dependent.

25. An isolated antibody according to any one of embodiments 1-24, wherein one or more of the TREM2 activities, DAP12 activities, or both activities involve the involvement of Syk, ZAP70, or both to DAP12.

26. An isolated antibody according to any one of embodiments 1-25, wherein the isolated agonistic antibody that binds to the TREM2 protein binds to one or more amino acids within amino acid residues selected from the group consisting of:

i. amino acid residues 29-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1;

ii. amino acid residues 29-41 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1;

iii. amino acid residues 40-44 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 40-44 of SEQ ID NO: 1;

iv. amino acid residues 47-69 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 47-69 of SEQ ID NO: 1;

v. amino acid residues 67-76 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1;

vi. amino acid residues 76-86 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1;

vii. amino acid residues 91-100 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 91-100 of SEQ ID NO: 1;

viii. amino acid residues 99-115 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1;

ix. amino acid residues 104-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 104-112 of SEQ ID NO: 1; And

X. amino acid residues 114-118 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1.

27. An isolated antibody according to any one of embodiments 1-25, wherein the isolated agonistic antibody that binds to the TREM2 protein binds to an epitope containing one or more amino acid residues selected from the group consisting of:

i. amino acid residues of Arg47 or Asp87 of SEQ ID NO: 1; ii. amino acid residues 40-44 of SEQ ID NO: 1; iii. amino acid residues 67-76 of SEQ ID NO: 1; and iv. amino acid residues 114-118 of SEQ ID NO: 1.

28. An isolated antibody according to any one of the embodiments of the invention 1-25, characterized in that the isolated agonistic antibody that binds to the DAP12 protein binds to one or more amino acids within amino acid residues 22-40 of SEQ ID NO: 2 or amino acids residues on the DAP12 protein corresponding to amino acid residues 22-40 of SEQ ID NO: 2.

29. An isolated antibody according to any one of embodiments 1-25, wherein the isolated agonist antibody is a bispecific antibody that binds to one or more amino acids selected from the group consisting of:

i. one or more amino acid residues from SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues from SEQ ID NO: 1; And

- 103 042890 ii. one or more amino acid residues from SEQ ID NO: 2, or amino acid residues on the DAP12 protein corresponding to amino acid residues from SEQ ID NO: 2.

30. An isolated antibody according to any of the preceding embodiments of the invention, wherein the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.

31. An isolated antibody according to any of the preceding embodiments of the invention, characterized in that the antibody is a monoclonal antibody.

32. An isolated antibody according to any of the preceding embodiments of the invention, characterized in that the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12 and a naturally occurring human DAP12 variant.

33. The isolated antibody of embodiment 32, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment.

34. An isolated nucleic acid encoding an antibody according to any of the preceding embodiments of the invention.

35. A vector containing a nucleic acid according to an embodiment of the invention 34.

36. Host cell containing the vector of embodiment 35.

37. A method for producing an antibody, comprising culturing a host cell according to embodiment 36, accompanied by the production of an antibody.

38. The method of Embodiment 37, further comprising isolating an antibody produced by the host cell.

39. A pharmaceutical composition comprising an antibody according to any one of embodiments 1-33 and a pharmaceutically acceptable carrier.

40. A method for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola's disease, and multiple sclerosis, comprising administering to the individual a therapeutically effective amount of the isolated antibody according to any one of the embodiments of the invention 1-33.

41. The method of embodiment 40, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains one or more substitutions selected from the group consisting of:

i. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1;

ii. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1;

iii. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1;

iv. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1

v. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1;

vi. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1;

vii. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1.

42. The method of embodiment 40, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains a nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both of these deletions.

43. The method of embodiment 40, wherein the individual has a heterozygous DAP12 variant, the variant comprising one or more variants selected from the group consisting of:

i. replacing methionine with threonine at the amino acid corresponding to the amino acid residue Met1 of SEQ ID NO: 2;

ii. amino acid substitution of glycine for arginine in the amino acid corresponding to the amino acid residue Gly49 of SEQ ID NO: 2;

iii. deletions within exons 1-4 of the nucleic acid sequence encoding SEQ ID

- 104 042890

NO: 2;

iv. inserts of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2; And

v. nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2.

An isolated agonist antibody that binds to the TREM2 protein, the DAP12 protein, or both, wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both.

The isolated antibody of embodiment 44, wherein the TREM2 protein, DAP12 protein, or both is a mammalian protein or a human protein.

The isolated antibody of embodiment 45, wherein the TREM2 protein, the DAP12 protein, or both, is a wild-type protein.

The isolated antibody of Embodiment 45, wherein the TREM2 protein, DAP12 protein, or both, is a naturally occurring variant.

The isolated antibody of any one of embodiments 44-47, wherein the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on the cell surface.

An isolated antibody according to any one of the embodiments of the invention 44-48, characterized in that one or more TREM2 activities include TREM2 binding to DAP12.

An isolated antibody according to any one of the embodiments of the invention 44-48, characterized in that one or more DAP12 activities include the binding of DAP12 to TREM2.

The isolated antibody of any one of embodiments 44-50, wherein one or more of the TREM2 activities, DAP12 activities, or both include DAP12 phosphorylation, TREM2 phosphorylation, or both.

The isolated antibody of embodiment 51 wherein DAP12 phosphorylation, TREM2 phosphorylation, or both are induced by one or more SRC family tyrosine kinases.

The isolated antibody of embodiment 52, wherein one or more SRC family tyrosine kinases contain the Syk kinase.

An isolated antibody according to any one of embodiments 44-53, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve activation of PIB.

An isolated antibody according to any one of embodiments 44-54, wherein one or more TREM2 activities, DAP12 activities, or both activities include increased expression of one or more anti-inflammatory cytokines.

An isolated antibody according to any one of embodiments 44-54, wherein one or more of TREM2 activities, DAP12 activities, or both activities include increased expression of one or more anti-inflammatory mediators selected from the group consisting of IL-12p70, IL-6 and IL-10.

The isolated antibody of Embodiment 55 or Embodiment 56, characterized in that overexpression occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells, and microglial cells .

An isolated antibody according to any one of embodiments 44-57, wherein one or more of TREM2 activities, DAP12 activities, or both activities include reduced expression of one or more pro-inflammatory cytokines.

An isolated antibody according to any one of the embodiments of the invention 44-57, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN- b, IL-6, IL-12 p70, IL-1b, TNF, TNF-α, IL-10, IL-8, CRP, TGF-beta chemokine protein family members, IL-20 family members, IL-33, PIL , IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18 and CRP.

An isolated antibody according to any one of embodiments 44-58, wherein one or more of the TREM2 activities, DAP12 activities, or both activities include reduced expression of TNF-α, IL-6, or both.

An isolated antibody according to embodiment 58 or embodiment 60, characterized in that the reduced expression of one or more pro-inflammatory mediators occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, Langerhans cells of the skin, Kupffer cells and microglial cells.

An isolated antibody according to any one of the embodiments of the invention 44-60, featuring

- 105 042890 in that one or more of TREM2 activities, DAP12 activities, or both activities involve phosphorylation of extracellular signal regulated kinase (ERK).

An isolated antibody according to any one of embodiments 44-62, wherein one or more of TREM2 activities, DAP12 activities, or both activities include increased expression of the C-C chemokine receptor 7 (CCR7).

An isolated antibody according to any one of embodiments 44-63, wherein one or more of TREM2 activities, DAP12 activities, or both activities involve induction of microglial cell chemotaxis towards cells expressing CCL19 and CCL21.

An isolated antibody according to any one of embodiments 44-64, wherein one or more of TREM2 activities, DAP12 activities, or both activities include enhancing, normalizing, or both the ability of bone marrow dendritic cells to induce proliferation of antigen-specific T cells.

The isolated antibody of any one of embodiments 44-65, wherein one or more of TREM2 activities, DAP12 activities, or both include induction of osteoclast production, increased levels of osteoclastogenesis, or both.

An isolated antibody according to any one of the embodiments of the invention 44-66, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include an increase in the survival of macrophages, microglial cells, dendritic cells, monocytes, osteoclasts, skin Langerhans cells and / or cells Kupfer

An isolated antibody according to any one of embodiments of the invention 44-67, characterized in that one or more of TREM2 activities, DAP12 activities, or both activities include an increase in the function of macrophages, microglial cells, dendritic cells, monocytes, osteoclasts, skin Langerhans cells and / or cells Kupfer

The isolated antibody of Embodiment 67 or Embodiment 68, wherein the macrophages and/or microglial cells are M1 macrophages and/or microglial cells, M2 macrophages and/or microglial cells, or both.

An isolated antibody according to an embodiment of the invention 69, characterized in that the macrophages and/or M1 microglial cells are activated macrophages and/or M1 microglial cells.

An isolated antibody according to any one of embodiments 44-68, wherein one or more of TREM2 activities, DAP12 activities, or both, include induction of one or more types of clearance selected from the group consisting of apoptotic neuron clearance, neural debris clearance tissue, clearance of debris from non-nervous tissue, clearance of bacteria or other foreign bodies, clearance of pathogenic protein, clearance of pathogenic peptide, and clearance of pathogenic nucleic acid.

An isolated antibody according to any one of the embodiments of the invention 44-71, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include the induction of phagocytosis of one or more apoptotic neurons, nervous tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease proteins, disease peptides or disease nucleic acids.

An isolated antibody according to embodiment 71 or embodiment 72, wherein the pathogenic protein is selected from the group consisting of amyloid beta, Tau, IAPP, alpha synuclein, TDP-43, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain , S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine repeat peptides (GR ), proline-alanine (PA) repeat peptides, and proline-arginine repeat (PR) peptides.

The isolated antibody of Embodiment 71 or Embodiment 72, wherein the pathogenic nucleic acid is GGCCCC repeat expansion antisense RNA (G2C4).

An isolated antibody according to any one of embodiments 44-74 wherein one or more of TREM2 activities, DAP12 activities, or both activities involve normalization of abnormal gene expression that is TREM2/DAP12 dependent.

An isolated antibody according to any one of embodiments 44-75, wherein one or more TREM2 activities, DAP12 activities, or both activities involve the involvement of Syk, ZAP70, or both, to the DAP12/TREM complex.

An isolated antibody according to any one of embodiments 44-76 wherein one or more of TREM2 activities, DAP12 activities, or both activities involve Syk phosphorylation.

- 106 042890

An isolated antibody according to any one of embodiments 44-77, wherein one or more of TREM2 activities, DAP12 activities, or both activities include increased expression of CD83 and/or CD86 on dendritic cells.

An isolated antibody according to any one of embodiments 44-78, wherein one or more of TREM2 activities, DAP12 activities, or both activities include reduced release of one or more inflammatory cytokines.

An isolated antibody according to embodiment 79, characterized in that one or more inflammatory cytokines are selected from the group consisting of TNF-α, IL-10, IL-6, MHB-1, IFNa4, IFN-b, IL-1c, IL -8, CRP, TGF-beta chemokine protein family members, IL-20, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL -17 and IL-18.

An isolated antibody according to any one of embodiments 44-79, wherein one or more TREM2 activities, DAP12 activities, or both activities include downregulation of one or more inflammatory receptors.

The isolated antibody of embodiment 81, characterized in that one or more inflammatory receptors contains CD86.

An isolated antibody according to any one of embodiments 44-82, wherein one or more of TREM2 activities, DAP12 activities, or both, include an increase in phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia under conditions of reduced levels of MCSF.

An isolated antibody according to any one of embodiments 44-82, wherein one or more of TREM2 activities, DAP12 activities, or both, include a reduction in phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal levels of MCSF.

An isolated antibody according to any one of embodiments 44-84, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include increased activity of one or more TREM2-dependent genes.

The isolated antibody of embodiment 85, characterized in that one or more TREM2-dependent genes include one or more nuclear factors from activated T cell transcription factors (NFAT).

An isolated antibody according to any one of the embodiments of the invention 44-86, characterized in that the antibody belongs to the IgG class, the IgM class, or the IgA class.

An isolated antibody according to embodiment 87, characterized in that the antibody belongs to the IgG class and has the isotype IgG1, IgG2, IgG3, or IgG4.

An isolated antibody according to embodiment 87, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 89, characterized in that the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 90, wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 89-91, wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both activities, regardless of binding to the Fc receptor.

An isolated antibody according to any one of the embodiments of the invention 89-91, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 93 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

The isolated antibody of Embodiment 93 or Embodiment 94, characterized in that the human IgG2 constant region contains an Fc region that contains one or more modifications.

The isolated antibody of embodiment 95, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 96, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 90, characterized in that the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU numbering.

An isolated antibody according to embodiment 87, characterized in that the antibody has an IgG1 isotype.

- 107 042890

An isolated antibody according to embodiment 99, characterized in that the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 100, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 99-101, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 102, wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB).

An isolated antibody according to any one of the embodiments of the invention 101-103, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 104, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 105, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 101-103, characterized in that the antibody comprises a heavy chain 1(CH1) constant domain of the IgG2 isotype and a hinge region.

An isolated antibody according to embodiment 107, characterized in that CH1 of the IgG2 isotype and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE

STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT

YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397).

An isolated antibody according to embodiment 107 or embodiment 108, characterized in that the Fc region of the antibody contains the S267E amino acid substitution, the L328F amino acid substitution, or both substitutions, and/or the N297A or N297Q amino acid substitution, and the numbering of the residues is presented in accordance with the numbering EU.

An isolated antibody according to an embodiment of invention 99, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 87, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 111, characterized in that the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 112, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 111-113, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 114 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to any one of the embodiments of the invention 113-115, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 116, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 117, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E and any combination thereof, the numbering of the residues being presented in accordance with the EU numbering.

An isolated antibody according to embodiment 87, characterized in that the antibody has an IgG2/4 hybrid isotype.

An isolated antibody according to embodiment 119, characterized in that the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with residue numbering according to EU numbering.

An isolated antibody according to embodiment 111, characterized in that the antibody contains a mouse IgG4 constant region.

The isolated antibody of any one of embodiments 1-121, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, DAP12 human, and the naturally occurring DAP12 variant

- 108 042890 loveca, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, human DAP12, and a naturally occurring variant DAP12 human.

The isolated antibody of embodiment 122, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment.

An isolated inert antibody that binds to the TREM2 protein, the DAP12 protein, or both.

An isolated antagonist antibody that binds to the TREM2 protein, the DAP12 protein, or both.

The isolated antibody of Embodiment 124 or Embodiment 125, wherein the TREM2 protein, DAP12 protein, or both is a mammalian protein or a human protein.

The isolated antibody of embodiment 126, wherein the TREM2 protein, the DAP12 protein, or both, is a wild-type protein.

The isolated antibody of embodiment 126, wherein the TREM2 protein, DAP12 protein, or both, is a naturally occurring variant.

The isolated antibody of any one of embodiments 125-128, wherein the isolated antibody inhibits one or more of TREM2 activities, DAP12 activities, or both.

The isolated antibody of embodiment 129, characterized in that one or more TREM2 activities, DAP12 activities, or both activities include reduced activity of one or more TREM2-dependent genes.

The isolated antibody of embodiment 130, characterized in that one or more TREM2-dependent genes include one or more nuclear factors from activated T cell transcription factors (NFAT).

An isolated antibody according to any one of the embodiments of the invention 129-131, characterized in that one or more of the TREM2 activities, DAP12 activities, or both activities include a decrease in the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells.

The isolated antibody of any one of embodiments 125-132, wherein the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both.

An isolated antibody according to any one of the embodiments of the invention 124-133, characterized in that the antibody is not capable of binding the Fc-gamma receptor (FcyR).

An isolated antibody according to embodiment 134, characterized in that the antibody has an IgG1 isotype.

An isolated antibody according to embodiment 135, characterized in that the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 136 wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to embodiment 137, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 138, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to an embodiment of the invention 139, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU numbering.

The isolated antibody of embodiment 140, characterized in that the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to EU numbering.

An isolated antibody according to embodiment 135, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 134, characterized in that the antibody has an IgG2 isotype.

An isolated antibody according to embodiment 143, characterized in that the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 144, wherein the human IgG2 constant region contains an Fc region.

- 109 042890

An isolated antibody according to embodiment 145, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 146, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 147, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 134, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 149, characterized in that the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 150, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to embodiment 151, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 152, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 153, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, with residue numbering following the EU numbering.

The isolated antibody of any one of embodiments 124-154, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, DAP12 human and naturally occurring human DAP12 variant.

The isolated antibody of embodiment 155, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment.

The isolated antibody of Embodiment 106, Embodiment 140, or Embodiment 141, wherein the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, the numbering of the residues being given in accordance with the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 95-157, characterized in that the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering of residues presented according to the EU numbering.

The isolated antibody of Embodiment 118 or Embodiment 154, characterized in that the Fc region further comprises the S228P amino acid substitution according to EU numbering.

The isolated antibody of any one of embodiments 44-159, wherein the isolated antibody competes for binding to TREM2 with one or more TREM2 ligands.

An isolated antibody according to embodiment 160, characterized in that one or more TREM2 ligands are selected from the group consisting of E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides and lipidated amyloid beta peptide.

An isolated antibody according to any one of embodiments 44-161, wherein the isolated antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.

The isolated antibody of any one of embodiments 44-162, wherein the isolated antibody is a bispecific antibody that recognizes a first antigen and a second antigen.

The isolated antibody of embodiment 163, wherein the first antigen is human TREM2 or a naturally occurring variant thereof, or human DAP12 or a naturally occurring variant thereof, and the second antigen is a disease-causing protein selected from the group consisting of beta -amyloid or its fragments, Tau, IAPP, alpha

- 110 042890 synuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin , transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine repeat peptides alanine (GA), glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides); or a blood-brain barrier-specific protein selected from the group consisting of transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1 and LRP1.

The isolated antibody of any one of embodiments 44-161, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, DAP12 human and naturally occurring human DAP12 variant; and moreover, the antibody is used in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of: beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein , prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin , cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides, and any combination thereof.

An isolated antibody according to any one of the embodiments of the invention 44-165, characterized in that the antibody is a monoclonal antibody.

An isolated antibody according to any one of the embodiments of the invention 44-166, characterized in that the isolated antibody binds to the TREM2 protein, and wherein the isolated antibody binds to one or more amino acids within amino acid residues selected from the group consisting of:

i. amino acid residues 29-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1;

ii. amino acid residues 29-41 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 2 9-41 of SEQ ID NO: 1;

iii. amino acid residues 40-44 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 40-44 of SEQ ID NO: 1;

iv. amino acid residues 47-69 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 47-69 of SEQ ID NO: 1;

v. amino acid residues 67-76 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1;

vi. amino acid residues 76-86 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1;

vii. amino acid residues 91-100 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 91-100 of SEQ ID NO: 1;

viii. amino acid residues 99-115 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1;

ix. amino acid residues 104-112 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 104-112 of SEQ ID NO: 1; And

X. amino acid residues 114-118 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1.

An isolated antibody according to embodiment 167, characterized in that the isolated antibody binds to one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO : 1.

An isolated antibody according to embodiment 167, characterized in that the isolated antibody binds to one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO : 1.

An isolated antibody according to any one of the embodiments of the invention 44-167, characterized in that the isolated antibody binds to an epitope containing one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1.

An isolated antibody according to any one of the embodiments of the invention 44-167, differ

- 111 042890 in that the isolated antibody binds to an epitope containing one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1.

An isolated antibody according to any one of embodiments 44-167 wherein the isolated antibody binds to an epitope containing one or more amino acid residues selected from the group consisting of:

i. amino acid residues of Arg47 or Asp87 of SEQ ID NO: 1;

ii. amino acid residues 40-44 of SEQ ID NO: 1;

iii. amino acid residues 67-76 of SEQ ID NO: 1; and iv. amino acid residues 114-118 of SEQ ID NO: 1.

An isolated antibody according to any one of the embodiments of the invention 44-167, characterized in that the isolated antibody binds to one or more amino acids within amino acid residues 22-40 of SEQ ID NO: 2 or amino acid residues on the DAP12 protein corresponding to amino acid residues 22- 40 of SEQ ID NO: 2.

An isolated antibody according to any one of embodiments 44-167, wherein the isolated antibody is a bispecific antibody that binds to one or more amino acids selected from the group consisting of:

i. one or more amino acid residues from SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues from SEQ ID NO: 1; and ii. one or more amino acid residues from SEQ ID NO: 2, or amino acid residues on the DAP12 protein corresponding to amino acid residues from SEQ ID NO: 2.

The isolated antibody of any one of embodiments 44-174, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibodies At52; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At52 monoclonal antibody.

An isolated antibody according to embodiment 175, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 398.

An isolated antibody according to embodiment 175 or embodiment 176, characterized in that HVR-H2 contains the amino acid sequence from SEQ ID NO: 399.

An isolated antibody according to any one of the embodiments of the invention 175-177, characterized in that HVR-H3 contains the amino acid sequence from SEQ ID NO: 400.

An isolated antibody according to any one of the embodiments of the invention 175-178, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 401.

An isolated antibody according to any one of the embodiments of the invention 175-179, characterized in that HVR-L2 contains the amino acid sequence from SEQ ID NO: 402.

An isolated antibody according to any one of the embodiments of the invention 175-180, characterized in that HVR-L3 contains the amino acid sequence from SEQ ID NO: 403.

The isolated antibody of any one of embodiments 44-174, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises:

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 398, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 398;

(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 399, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 399; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400; and/or wherein the light chain variable domain comprises:

(a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 401, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 401;

(b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 402, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 402; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403.

The isolated antibody of any one of embodiments 44-174, wherein the isolated antibody comprises a heavy chain variable domain and a

- 112 042890 light chain, and the variable domain of the heavy chain contains HVR-H1, HVR-H2 and/or HVR-H3 monoclonal antibody At21; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody.

An isolated antibody according to embodiment 183, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 404.

An isolated antibody according to embodiment 183 or 184, characterized in that HVR-H2 contains the amino acid sequence from SEQ ID NO: 405.

An isolated antibody according to any one of the embodiments of the invention 183-185, characterized in that HVR-H3 contains the amino acid sequence from SEQ ID NO: 406.

An isolated antibody according to any one of the embodiments of the invention 183-186, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 407.

An isolated antibody according to any one of the embodiments of the invention 183-187, characterized in that HVR-L2 contains the amino acid sequence from SEQ ID NO: 408.

An isolated antibody according to any one of the embodiments of the invention 183-188, characterized in that HVR-L3 contains the amino acid sequence from SEQ ID NO: 409.

The isolated antibody of any one of embodiments 44-174, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises:

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 404, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 404;

(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 405, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 405; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406; and/or wherein the light chain variable domain comprises:

(a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 407 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 407;

(b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 408; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 409 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 409.

An isolated antibody to human TREM2, characterized in that the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the At52 monoclonal antibody; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At52 monoclonal antibody.

An isolated antibody according to embodiment 191, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 398.

An isolated antibody according to embodiment 191 or embodiment 192, characterized in that HVR-H2 contains the amino acid sequence from SEQ ID NO: 399.

An isolated antibody according to any one of the embodiments of the invention 191-193, characterized in that HVR-H3 contains the amino acid sequence from SEQ ID NO: 400.

An isolated antibody according to any one of the embodiments of the invention 191-194, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 401.

An isolated antibody according to any one of the embodiments of the invention 191-195, characterized in that HVR-L2 contains the amino acid sequence from SEQ ID NO: 402.

An isolated antibody according to any one of the embodiments of the invention 191-196, characterized in that HVR-L3 contains the amino acid sequence from SEQ ID NO: 403.

The isolated antibody of Embodiment 191, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 398, HVR-H1, containing the amino acid sequence from SEQ ID NO: 399, and HVR-H3 containing the amino acid sequence from SEQ ID NO: 400, and/or in which the light chain variable domain contains HVR-L1 containing the amino acid sequence from SEQ ID NO: 401, HVR -L2 containing the amino acid sequence from SEQ ID NO: 402, and

- 113 042890

HVR-L3 containing the amino acid sequence from SEQ ID NO: 403.

An isolated antibody to human TREM2, characterized in that the isolated antibody contains a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain contains.

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 398, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 398;

(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 399, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 399; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400; and/or wherein the light chain variable domain comprises:

(a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 401, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 401;

(b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 402, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 402; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403.

An isolated antibody to human TREM2, characterized in that the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibody At21; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody.

The isolated antibody of embodiment 200, wherein HVR-L1 contains the amino acid sequence of SEQ ID NO: 404.

An isolated antibody according to embodiment 200 or embodiment 201, characterized in that HVR-H2 contains the amino acid sequence from SEQ ID NO: 405.

An isolated antibody according to any one of the embodiments of the invention 200-202, characterized in that HVR-H3 contains the amino acid sequence from SEQ ID NO: 406.

An isolated antibody according to any one of the embodiments of the invention 200-203, characterized in that HVR-L1 contains the amino acid sequence from SEQ ID NO: 407.

An isolated antibody according to any one of the embodiments of the invention 200-204, characterized in that HVR-L2 contains the amino acid sequence from SEQ ID NO: 408.

An isolated antibody according to any one of the embodiments of the invention 200-205, characterized in that HVR-L3 contains the amino acid sequence from SEQ ID NO: 409.

The isolated antibody of embodiment 200, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404, HVR-H1, containing the amino acid sequence from SEQ ID NO: 405, and HVR-H3 containing the amino acid sequence from SEQ ID NO: 406, and/or in which the light chain variable domain contains HVR-L1 containing the amino acid sequence from SEQ ID NO: 407, HVR -L2 containing the amino acid sequence from SEQ ID NO: 408, and HVR-L3 containing the amino acid sequence from SEQ ID NO: 409.

An isolated antibody to human TREM2, characterized in that the isolated antibody contains a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain contains.

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 404, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 404;

(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 405, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 405; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406; and/or wherein the light chain variable domain comprises:

- 114 042890 (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 407 or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 407;

(b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 408; and (c) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 409, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 409.

An isolated anti-human TREM2 antibody that binds substantially the same TREM2 epitope as the At52 antibody.

An isolated anti-human TREM2 antibody that binds substantially the same TREM2 epitope as the At21 antibody.

The isolated antibody of any one of embodiments 191-210, wherein the isolated antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both.

The isolated antibody of embodiment 211, wherein the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on the cell surface.

The isolated antibody of Embodiment 211 or Embodiment 212, wherein one or more TREM2 activities, DAP12 activities, or both are selected from the group consisting of: TREM2 binding to DAP12; binding DAP12 to TREM2; TREM2 phosphorylation, DAP12 phosphorylation; physical activation; increased expression of one or more anti-inflammatory mediators; decreased expression of one or more pro-inflammatory mediators; reduced expression of TNF-α, IL-6, or both; phosphorylation of kinase regulated by extracellular signals (ERK); increased expression of the C-C chemokine receptor 7 (CCR7); inducing chemotaxis of microglial cells towards cells expressing CCL19 and CCL21; increasing, normalizing, or both, the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; inducing the production of osteoclasts, increasing the level of osteoclastogenesis, or both; increase the survival and/or function of one or more dendritic cells, macrophages, microglial cells, macrophages and/or M1 microglial cells, activated macrophages and/or M1 microglial cells, macrophages and/or M2 microglial cells, osteoclasts, skin Langerhans cells and Kupffer cells ; induction of one or more types of clearance selected from the group consisting of apoptotic neuron clearance, neural debris clearance, non-nervous tissue debris clearance, bacteria or other foreign body clearance, pathogenic protein clearance, pathogenic peptide clearance, and pathogenic nucleic acid clearance; inducing phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, or disease-causing nucleic acids; normalization of disturbed TREM2/DAP12 dependent gene expression; involvement of Syk, ZAP70, or both in the DAP12/TREM2 complex; phosphorylation of Syk; increased expression of CD83 and/or CD86 on dendritic cells; decreased secretion of one or more inflammatory cytokines; decreased expression of one or more inflammatory receptors; increased phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of reduced levels of MCSF; decrease in phagocytosis by macrophages, dendritic cells, monocytes and/or microglia under conditions of normal levels of MCSF; increasing the activity of one or more TREM2-dependent genes; and any combination of them.

An isolated antibody according to any one of the embodiments of the invention 211-213, characterized in that the antibody belongs to the IgG class, the IgM class, or the IgA class.

An isolated antibody according to embodiment 214, characterized in that the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype.

An isolated antibody according to embodiment 215, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 216, wherein the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 217, wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 216-218, characterized in that the antibody induces one or more TREM2 activities, DAP12 activities, or both activities, regardless of binding to the Fc receptor.

An isolated antibody according to any one of the embodiments of the invention 216-218, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 220, characterized in that inhibition

- 115 042890 Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to embodiment 220 or embodiment 221, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 222, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to an embodiment of the invention 223, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 217, characterized in that the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU numbering.

An isolated antibody according to embodiment 215, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 226, wherein the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 227, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 226-228, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 229 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB).

An isolated antibody according to any one of the embodiments of the invention 228-230, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 231, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 232, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to the EU numbering.

An isolated antibody according to any one of embodiments of the invention 228-230, characterized in that the antibody comprises a heavy chain 1(CH1) constant domain of the IgG2 isotype and a hinge region.

An isolated antibody according to embodiment 234, characterized in that CH1 of the IgG2 isotype and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE

STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT

YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397) .

An isolated antibody according to embodiment 234 or embodiment 235, characterized in that the Fc region of the antibody contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and/or an N297A or N297Q amino acid substitution, wherein the numbering of the residues is presented in accordance with the numbering EU.

The isolated antibody of embodiment 226, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 215, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 238, wherein the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 239, characterized in that the human IgG4 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 238-240, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 241 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to any one of the embodiments of the invention 240-242, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 243, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 244, characterized in that one or

- 116 042890 more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E and any combination thereof, and the numbering of residues is presented in according to EU numbering.

An isolated antibody according to embodiment 214, characterized in that the antibody has an IgG2/4 hybrid isotype.

An isolated antibody according to embodiment 246, characterized in that the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with residue numbering according to EU numbering.

An isolated antibody according to embodiment 239, characterized in that the antibody contains a mouse IgG4 constant region.

The isolated antibody of any one of embodiments 211-248, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, DAP12 a human and a naturally occurring human DAP12 variant, wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12 and a naturally occurring human DAP12 variant.

The isolated antibody of embodiment 249, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

The isolated antibody of any one of the embodiments of the invention 191-210, wherein the isolated antibody is an inert antibody.

An isolated antibody according to any one of the embodiments of the invention 191-210, characterized in that the isolated antibody is an antagonistic antibody.

The isolated antibody of embodiment 252, wherein the isolated antibody inhibits one or more TREM2 activities.

An isolated antibody according to an embodiment of the invention 253, characterized in that one or more TREM2 activities are selected from the group consisting of a decrease in the activity of one or more TREM2-dependent genes; reducing the activity of one or more nuclear factors of activated T-cell transcription factors (NFAT); reduced survival of macrophages, microglial cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells; and any combination of them.

The isolated antibody of any one of embodiments 252-254, wherein the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both.

An isolated antibody according to any one of the embodiments of the invention 251-255, characterized in that the antibody is not capable of binding the Fc-gamma receptor (FcyR).

An isolated antibody according to embodiment 256, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 257, characterized in that the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 258, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to embodiment 259, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 260, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 261, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU numbering.

The isolated antibody of embodiment 262, characterized in that the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to EU numbering.

The isolated antibody of embodiment 257, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 256, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 264, wherein the antibody contains a human IgG2 constant region.

- 117 042890

The isolated antibody of embodiment 266 wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to embodiment 267, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 268, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 269, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 256, characterized in that the antibody is of the IgG4 isotype.

The isolated antibody of embodiment 271, characterized in that the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 272, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to embodiment 273, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 274, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 275, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, with residue numbering following the EU numbering.

The isolated antibody of any one of embodiments 208-276, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, DAP12 human and naturally occurring human DAP12 variant.

The isolated antibody of embodiment 277, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

The isolated antibody of Embodiment 233, Embodiment 262, or Embodiment 263, characterized in that the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, the numbering of the residues being given in accordance with the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 223-279, characterized in that the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering of residues presented according to the EU numbering.

The isolated antibody of Embodiment 245 or Embodiment 276, characterized in that the Fc region further comprises the S228P amino acid substitution according to EU numbering.

An isolated antibody according to any one of embodiments 191-281, wherein the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.

An isolated antibody according to any one of the embodiments of the invention 191-282, characterized in that the antibody is a bispecific antibody that recognizes a first antigen and a second antigen.

An isolated antibody according to any one of the embodiments of the invention 191-283, characterized in that the antibody is a monoclonal antibody.

An isolated antibody according to any of the preceding embodiments of the invention, wherein the isolated antibody specifically binds to both human TREM2 and mouse TREM2.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (KD) for human TREM2 and mouse TREM2 that ranges from less than about 5.75 nM to less than about 0.09 nM.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (KD) for the human TREM2-Fc fusion protein that ranges from less than about 1.51 nM to less than about 0.35 nM .

- 118 042890

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (K _D ) for the monomeric human TREM2 protein that ranges from less than about 5.75 nM to less than about 1.15 nM.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (K _D ) for the mouse TREM2-Fc fusion protein that ranges from less than about 0.23 nM to less than about 0.09 nM.

An isolated nucleic acid encoding an antibody according to any of the preceding embodiments of the invention.

A vector containing the nucleic acid of embodiment 290.

Host cell containing the vector of embodiment 291.

A method for producing an antibody, comprising culturing a cell according to embodiment 292, accompanied by the production of an antibody.

The method of Embodiment 293, further comprising isolating an antibody produced by the cell.

A pharmaceutical composition comprising an antibody according to any one of the embodiments of the invention 44-289 and a pharmaceutically acceptable carrier.

A method for preventing, reducing risk, or treating an individual having a disease, disorder, or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakol's disease, and multiple sclerosis, comprising administering to the individual a therapeutically effective amount of an isolated antibody that binds to the TREM2 protein, the DAP12 protein, or both.

The method of embodiment 296 wherein the isolated antibody is the isolated antibody of any one of embodiments 44-289.

The method of Embodiment 296 or Embodiment 297, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains one or more substitutions selected from the group consisting of:

i. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1;

ii. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1;

iii. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1;

iv. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1;

v. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1;

vi. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1;

vii. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1.

The method of embodiment 296, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains a nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both of these deletions.

The method of embodiment 296, wherein the individual has a heterozygous DAP12 variant, the variant comprising one or more variants selected from the group consisting of:

i. replacing methionine with threonine at the amino acid corresponding to the amino acid residue Met1 of SEQ ID NO: 2;

ii. amino acid substitution of glycine for arginine in the amino acid corresponding to the amino acid residue Gly49 of SEQ ID NO: 2;

iii. deletions within exons 1-4 of the nucleic acid sequence encoding SEQ ID NO: 2;

iv. inserts of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2; And

v. nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2.

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A method for inducing or promoting the survival of innate immune cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated agonist antibody that binds to the TREM2 protein, the DAP12 protein, or both proteins.

The method of embodiment 301, wherein the isolated agonist antibody is the isolated antibody of any one of embodiments 44123, 157-250, and 277-289.

An isolated agonist antibody that binds to the TREM2 protein, wherein the antibody induces one or more TREM2 activities.

The isolated antibody of embodiment 303, wherein the TREM2 protein is a mammalian protein or a human protein.

The isolated antibody of embodiment 304, wherein the TREM2 protein is a wild-type protein.

The isolated antibody of embodiment 304, wherein the TREM2 protein is a naturally occurring variant.

An isolated antibody according to any one of the embodiments of the invention 303-306, characterized in that the TREM2 protein is expressed on human dendritic cells, human macrophages, human monocytes, human osteoclasts, human skin Langerhans cells, human Kupffer cells and/or human microglia.

The isolated antibody of any one of embodiments 303-307, wherein the isolated antibody induces or retains TREM2 clustering on the cell surface.

An isolated antibody according to any one of the embodiments of the invention 303-308, characterized in that one or more TREM2 activities include TREM2 binding to DAP12.

An isolated antibody according to any one of the embodiments of the invention 303-309, characterized in that one or more TREM2 activities include autophosphorylation of TREM2.

An isolated antibody according to any one of the embodiments of the invention 303-310, characterized in that one or more TREM2 activities include phosphorylation of DAP12.

The isolated antibody of embodiment 310 or embodiment 311, wherein TREM2 phosphorylation, DAP12 phosphorylation, or both are induced by one or more SRC family tyrosine kinases.

The isolated antibody of embodiment 312, wherein one or more SRC family tyrosine kinases contain the Syk kinase.

An isolated antibody according to any one of the embodiments of the invention 303-313, characterized in that one or more of the activities of TREM2 include the activation of PIB.

An isolated antibody according to any one of the embodiments of the invention 303-314, characterized in that one or more TREM2 activities include increased expression of one or more anti-inflammatory cytokines.

An isolated antibody according to any one of the embodiments of the invention 303-314, characterized in that one or more TREM2 activities include increased expression of one or more cytokines selected from the group consisting of IL-12p70, IL-6 and IL-10.

The isolated antibody of Embodiment 315 or Embodiment 316, characterized in that overexpression occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells, and microglial cells .

An isolated antibody according to any one of the embodiments of the invention 303-317, characterized in that one or more TREM2 activities include reduced expression of one or more pro-inflammatory cytokines.

An isolated antibody according to any one of the embodiments of the invention 303-317, characterized in that one or more TREM2 activities include reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-1b, TNF -α, IL-10, IL-6, IL-8, CRP, TGF-beta chemokine protein family members, IL-20 family members, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM -CSF, IL-11, IL-12, IL-17, IL-18 and SRP.

An isolated antibody according to any one of the embodiments of the invention 303-318, characterized in that one or more TREM2 activities include reduced expression of TNF-α, IL-6, or both.

An isolated antibody according to embodiment 319 or embodiment 320, characterized in that the reduced expression of one or more pro-inflammatory mediators occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and microglial cells.

An isolated antibody according to any one of the embodiments of the invention 303-320, differ

- 120 042890 in that one or more of the activities of TREM2 involve phosphorylation of an extracellular signal regulated kinase (ERK).

An isolated antibody according to any one of the embodiments of the invention 303-322, characterized in that one or more TREM2 activities include increased expression of the C-C chemokine receptor 7 (CCR7).

An isolated antibody according to any one of the embodiments of the invention 303-322, characterized in that one or more TREM2 activities include the induction of chemotaxis of microglial cells in relation to cells expressing CCL19 and CCL21.

An isolated antibody according to any one of the embodiments of the invention 303-324, characterized in that one or more TREM2 activities include an increased ability of dendritic cells, monocytes, microglia and/or macrophages to induce T cell proliferation.

The isolated antibody of any one of embodiments 303-324, wherein one or more of the TREM2 activities include enhancement, normalization, or both of the ability of bone marrow dendritic cells to induce proliferation of antigen-specific T cells.

The isolated antibody of any one of embodiments 303-326, wherein one or more of TREM2's activities include induction of osteoclast production, increased levels of osteoclastogenesis, or both.

An isolated antibody according to any one of the embodiments of the invention 303-327, characterized in that one or more TREM2 activities include an increase in the survival of dendritic cells, macrophages, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or microglia.

An isolated antibody according to any one of the embodiments of the invention 303-328, characterized in that one or more TREM2 activities include an increase in the function of dendritic cells, macrophages and/or microglia.

An isolated antibody according to any one of the embodiments of the invention 303-329, characterized in that one or more TREM2 activities include an increase in phagocytosis by dendritic cells, macrophages, monocytes and/or microglia under conditions of reduced levels of MCSF.

An isolated antibody according to any one of the embodiments of the invention 303-330, characterized in that one or more TREM2 activities include a decrease in phagocytosis by dendritic cells, macrophages, monocytes and/or microglia in the presence of normal levels of MCSF.

An isolated antibody according to any embodiment of the invention 328-331, wherein the macrophages and/or microglia are M1 macrophages and/or microglia, M2 macrophages and/or microglia, or both.

The isolated antibody of embodiment 332, wherein the macrophages and/or M1 microglia are activated macrophages and/or M1 microglia.

An isolated antibody according to any one of embodiments of the invention 303-333, characterized in that one or more TREM2 activities include the induction of one or more types of clearance selected from the group consisting of apoptotic neuronal clearance, neural debris clearance, non-nervous tissue debris clearance , clearance of bacteria or other foreign bodies, clearance of a disease-causing protein, and clearance of a tumor cell.

An isolated antibody according to any one of the embodiments of the invention 303-334, characterized in that one or more TREM2 activities include the induction of phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, pathogenic nucleic acids or tumor cells.

The isolated antibody of embodiment 335, wherein the pathogenic nucleic acid is GGCCCC (G2C4) repeat expansion antisense RNA.

An isolated antibody according to embodiment 334 or embodiment 335, characterized in that the disease-causing protein is selected from the group consisting of beta-amyloid or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medina, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, peptides glycine-arginine (GR) repeat, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides.

An isolated antibody according to any one of the embodiments of the invention 303-337, characterized in that one or more TREM2 activities include the normalization of impaired gene expression, which depends on TREM2/DAP12.

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An isolated antibody according to any one of the embodiments of the invention 303-338, characterized in that one or more TREM2 activities include the involvement of Syk, ZAP70, or both to the DAP12/TREM complex.

An isolated antibody according to any one of the embodiments of the invention 303-339, characterized in that one or more TREM2 activities include Syk phosphorylation.

An isolated antibody according to any one of the embodiments of the invention 303-340, characterized in that one or more TREM2 activities include increased expression of CD83 and/or CD86 on dendritic cells, monocytes, macrophages, or both.

The isolated antibody of embodiment 341 wherein the dendritic cells are bone marrow dendritic cells.

An isolated antibody according to any one of the embodiments of the invention 303-342, characterized in that one or more TREM2 activities include reduced release of one or more inflammatory cytokines.

An isolated antibody according to an embodiment of the invention 343, characterized in that one or more inflammatory cytokines are selected from the group consisting of IFN-a4, IFN-b, IL-1b, TNF-α, IL-10, IL-6, IL-8 , CRP, members of the TGF-beta chemokine protein families, members of the IL-20, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17 families , IL-18, SRB and MHB-1.

An isolated antibody according to any one of the embodiments of the invention 303-344, characterized in that one or more TREM2 activities include reduced expression of one or more inflammatory receptors.

The isolated antibody of embodiment 345, characterized in that one or more inflammatory receptors contains CD86.

An isolated antibody according to any one of the embodiments of the invention 303-346, characterized in that one or more TREM2 activities include increased activity of one or more TREM2-dependent genes.

The isolated antibody of embodiment 347, characterized in that one or more TREM2-dependent genes include one or more nuclear factors from activated T cell transcription factors (NFAT).

An isolated antibody according to any one of the embodiments of the invention 303-348, characterized in that the antibody belongs to the IgG class, the IgM class, or the IgA class.

An isolated antibody according to embodiment 349, characterized in that the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype.

An isolated antibody according to embodiment 349, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 351, characterized in that the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 352, wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 351-353, characterized in that the antibody induces one or more TREM2 activities independent of binding to the Fc receptor.

An isolated antibody according to any one of the embodiments of the invention 351-353, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 355 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

The isolated antibody of embodiment 355 or embodiment 356, characterized in that the human IgG2 constant region contains an Fc region that contains one or more modifications.

The isolated antibody of embodiment 357, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 358, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 352, characterized in that the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU numbering.

An isolated antibody according to embodiment 349, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 361, wherein the antibody

- 122 042890 contains the human IgG1 constant region.

The isolated antibody of embodiment 362, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 361-363, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 364 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB).

An isolated antibody according to any one of the embodiments of the invention 363-365, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 366, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 367, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 363-365, characterized in that the antibody includes a heavy chain constant domain 1(CH1) of the IgG2 isotype and a hinge region.

An isolated antibody according to embodiment 369, characterized in that CH1 of the IgG2 isotype and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE

STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT

YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397) .

An isolated antibody according to embodiment 369 or embodiment 370, characterized in that the Fc region of the antibody contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and/or an N297A or N297Q amino acid substitution, wherein the numbering of the residues is presented in accordance with the numbering EU.

The isolated antibody of embodiment 361, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 349, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 373, characterized in that the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 374, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 373-375, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 376 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to any one of the embodiments of the invention 375-377, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 378, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 379, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E and any combination thereof, the numbering of the residues being presented in accordance with the EU numbering.

An isolated antibody according to embodiment 349, characterized in that the antibody has an IgG2/4 hybrid isotype.

An isolated antibody according to embodiment 381, characterized in that the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with residue numbering according to EU numbering.

The isolated antibody of embodiment 373, characterized in that the antibody contains a mouse IgG4 constant region.

An isolated antibody according to any one of embodiments 1-383, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, and variant human TREM2 in pathology, and wherein the antibody fragment is cross-linked to a second antibody fragment that binds to one or

- 123 042890 more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, and a pathological human TREM2 variant.

The isolated antibody of embodiment 384, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

An isolated inert antibody that binds to the TREM2 protein.

An isolated antagonistic antibody that binds to the TREM2 protein.

The isolated antibody of Embodiment 386 or Embodiment 387, wherein the TREM2 protein is a mammalian protein or a human protein.

The isolated antibody of embodiment 388, wherein the TREM2 protein is a wild-type protein.

The isolated antibody of embodiment 388, wherein the TREM2 protein is a naturally occurring variant.

The isolated antibody of embodiment 388, wherein the TREM2 protein is a pathological variant.

An isolated antibody according to any one of embodiments of the invention 387-391, wherein the isolated antibody inhibits one or more TREM2 activities.

The isolated antibody of embodiment 392, characterized in that one or more TREM2 activities include reduced activity of one or more TREM2-dependent genes.

The isolated antibody of embodiment 393, wherein the one or more TREM2 dependent genes comprise one or more nuclear factors from activated T cell transcription factors (NFATs).

An isolated antibody according to any one of the embodiments of the invention 392-394, characterized in that one or more of the TREM2 activities include a decrease in the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells.

The isolated antibody of any one of embodiments 387-395, wherein the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both.

An isolated antibody according to any one of the embodiments of the invention 386-396, characterized in that the antibody is not capable of binding the Fc-gamma receptor (FcyR).

An isolated antibody according to embodiment 397, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 398, wherein the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 399, wherein the human IgG1 constant region contains an Fc region.

The isolated antibody of embodiment 400, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 401, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 402, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU numbering.

The isolated antibody of embodiment 403, characterized in that the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to EU numbering.

The isolated antibody of embodiment 398, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 397, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 406, wherein the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 407, wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to embodiment 408, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 409, characterized in that the Fc region contains one or more amino acid substitutions.

- 124 042890

The isolated antibody of embodiment 410, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 397, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 412, wherein the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 413, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to embodiment 414, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 415, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 416, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, with residue numbering following the EU numbering.

An isolated antibody according to any one of embodiments 386-417, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, and variant TREM2 in pathology.

The isolated antibody of embodiment 418, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

The isolated antibody of Embodiment 368, Embodiment 403, or Embodiment 404, wherein the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, the numbering of the residues being given in accordance with the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 357-420, characterized in that the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering of residues presented according to the EU numbering.

The isolated antibody of Embodiment 380 or Embodiment 417, wherein the Fc region further comprises the S228P amino acid substitution according to EU numbering.

The isolated antibody of any one of embodiments 303-422, wherein the antibody competes for binding to TREM2 with one or more TREM2 ligands.

An isolated antibody according to embodiment 423, characterized in that one or more TREM2 ligands are selected from the group consisting of E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholine, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides and lipidated amyloid beta peptide.

The isolated antibody of any one of embodiments 303-422, wherein the antibody does not compete for TREM2 binding to a TREM2 ligand.

An isolated antibody according to any one of embodiments 303-425, wherein the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, a conjugated antibody, or a chimeric antibody.

An isolated antibody according to any one of the embodiments of the invention 303-426, characterized in that the antibody is a bispecific antibody that recognizes a first antigen and a second antigen.

The isolated antibody of embodiment 427, wherein the first antigen is human TREM2 or a naturally occurring variant thereof, and the second antigen is a disease-causing protein selected from the group consisting of: beta-amyloid or fragments thereof, Tau, IAPP , alpha-synuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin , transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products, association

- 125 042890 non-ATG repeat peptides (RAN), dipeptide repeat peptides (DPP), glycinalanine repeat peptides (GA), glycine-proline repeat peptides (GP), glycine-arginine repeat peptides (GR), proline-alanine repeat peptides ( RA) and proline-arginine repeat peptides (PR); a blood-brain barrier specific protein selected from the group consisting of: transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1 and LRP1; or ligands and/or proteins expressed by immune cells, wherein the ligands and/or proteins are selected from the group consisting of: CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD -L2, PD-1, B7H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG and phosphatidylserine.

The isolated antibody of embodiment 427 wherein the first antigen is human TREM2 or a naturally occurring variant thereof and the second antigen is a protein expressed on one or more tumor cells.

The isolated antibody of any one of embodiments 303-424, wherein the antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, human DAP12 and a naturally occurring human DAP12 variant; and wherein the antibody is used in combination with one or more antibodies that specifically bind to a disease-causing protein selected from the group consisting of: beta-amyloid, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, prion protein PrPsc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide repeat peptides (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine repeat peptides -arginine (GR), proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides, and any combination thereof, and/or one or more antibodies that specifically bind a cancer-associated protein selected from the group consisting of from: CD40, 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, phosphatidylserine and any combination thereof.

An isolated antibody according to any one of the embodiments of the invention 303-430, characterized in that the antibody is a monoclonal antibody.

An isolated antibody according to any one of the embodiments of the invention 303-431, characterized in that the antibody binds to one or more amino acids within amino acid residues selected from the group consisting of:

xi. amino acid residues 130-171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-171 of SEQ ID NO: 1;

xi. amino acid residues 140-153 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1;

xiii. amino acid residues 139-146 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 139-146 of SEQ ID NO: 1;

xiv. amino acid residues 130-144 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-144 of SEQ ID NO: 1; and xv. amino acid residues 158-171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1.

An isolated antibody according to any one of the embodiments of the invention 303-431, characterized in that the antibody binds to an epitope containing one or more amino acids within amino acid residues selected from the group consisting of:

i. amino acid residues 130-171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-171 of SEQ ID NO: 1;

ii. amino acid residues 140-153 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 139-153 of SEQ ID NO: 1;

iii. amino acid residues 139-146 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 139-146 of SEQ ID NO: 1;

iv. amino acid residues 130-144 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 130-144 of SEQ ID NO: 1; And

v. amino acid residues 158-171 of SEQ ID NO: 1, or amino acid residues on the TREM2 protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1.

An isolated antibody according to embodiment 432 or embodiment 433, characterized in that the antibody binds to an epitope additionally containing one or more amino acid residues selected from the group consisting of:

v. amino acid residues of Arg47 or Asp87 of SEQ ID NO: 1;

vi. amino acid residues 40-44 of SEQ ID NO: 1;

vii. amino acid residues 67-76 of SEQ ID NO: 1; And

- 126 042890 viii. amino acid residues 114-118 of SEQ ID NO: 1.

The isolated antibody of any one of embodiments 303-433, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain. wherein the heavy chain variable domain comprises an HVR-H1, HVR-H2 and/or HVR-H3 monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11 , At12, At13, At14, At15, At16, At17, At18, At19, At20, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37 , At38, At39, At40, At41, At42, At43,

At44, At45, At46, At47, At48, At49, At50, At51, At53, At54, At55, At56, At57, At58, At59, At60, At61,

At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77, At78,

At79, At80, At81, At82, At83, At84, At85, At86 and At87, and/or wherein the light chain variable domain comprises an HVR-L1, HVR-L2 and/or HVR-L3 monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17,

At18, At19, At20, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35,

At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50, At51, At53,

At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68, At69, At70,

At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87.

An isolated antibody according to embodiment 435, wherein HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-24.

The isolated antibody of Embodiment 435 or Embodiment 436, wherein HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-49.

An isolated antibody according to any one of the embodiments of the invention 435-437, characterized in that HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 50-119.

An isolated antibody according to any one of the embodiments of the invention 435-438, characterized in that HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137.

An isolated antibody according to any one of the embodiments of the invention 435-439, characterized in that HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152.

An isolated antibody according to any one of the embodiments of the invention 435-440, characterized in that HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 153-236.

The isolated antibody of any one of embodiments 303-433, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises:

(a) HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-24 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 324;

(b) HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25-49 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 2549; and (c) an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50-119 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NO. : 50119; and/or;

wherein the light chain variable domain contains:

(a) HVR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137;

(b) HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152; and (c) an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 153-236 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NO. : 138-152.

- 127 042890

An isolated antibody to human TREM2, characterized in that the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of a monoclonal antibody selected from the group consisting of from: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At22, At2 3, At24, At25 , At26, At27, At28, At29, At30, At31, At32, At33,

At34, At35, At36, At37, At38, At39, At40, At41, At42, At43, At44, At45, At46, At47, At48, At49, At50,

At51, At53, At54, At55, At56, At57, At58, At59, At60, At61, At62, At63, At64, At65, At66, At67, At68,

At69, At70, At71, At72, At73, At74, At75, At76, At77, At78, At79, At80, At81, At82, At83, At84, At85,

At86 and At87, and/or wherein the light chain variable domain comprises an HVR-L1, HVR-L2 and/or HVR-L3 monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12, At13, At14, At15, At16, At17, At18, At19, At20, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33 , At34, At35, At36, At37, At38, At39, At40, At41, At42,

At43, At44, At45, At46, At47, At48, At49, At50, At51, At53, At54, At55, At56, At57, At58, At59, At60,

At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77,

At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87.

An isolated antibody according to embodiment 443, wherein HVR-H1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-24.

The isolated antibody of Embodiment 443 or Embodiment 444, wherein HVR-H2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-49.

An isolated antibody according to any one of the embodiments of the invention 443-445, characterized in that HVR-H3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 50-119.

An isolated antibody according to any one of the embodiments of the invention 443-446, characterized in that HVR-L1 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137.

An isolated antibody according to any one of the embodiments of the invention 443-447, characterized in that HVR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152.

An isolated antibody according to any one of the embodiments of the invention 443-448, characterized in that HVR-L3 contains an amino acid sequence selected from the group consisting of SEQ ID NO: 153-236.

The isolated antibody of embodiment 443, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-24, an HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-49 and an HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-119, and /or in which the light chain variable domain contains HVR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 120-137, HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 138 -152, and HVR-L3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 153236.

An isolated anti-human TREM2 antibody that binds substantially the same TREM2 epitope as a monoclonal antibody selected from the group consisting of: At1, At2, At3, At4, At5, At6, At7, At8, At9, At10, At11, At12 , At13, At14, At15, At16, At17, At18, At19, At20, At22, At23, At24, At25, At26, At27, At28, At29, At30, At31, At32, At33, At34, At35, At36, At37, At38 , At39, At40, At41, At42,

At43, At44, At45, At46, At47, At48, At49, At50, At51, At53, At54, At55, At56, At57, At58, At59, At60,

At61, At62, At63, At64, At65, At66, At67, At68, At69, At70, At71, At72, At73, At74, At75, At76, At77,

At78, At79, At80, At81, At82, At83, At84, At85, At86 and At87.

An isolated antibody to human TREM2, characterized in that the isolated antibody contains a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain contains:

(a) HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3-24 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 324;

(b) HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 25-49 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 2549; and (c) an HVR-H3 containing an amino acid sequence selected from the group consisting of

- 128 042890 of SEQ ID NO: 50-119 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 50119; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120-137 or an amino acid sequence with at least about 95% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: 120-137;

(b) HVR-L2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152 or an amino acid sequence with at least about 95% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 138-152; and (c) an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 153-236 or an amino acid sequence with at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NO. : 138-152.

An isolated antibody according to any one of embodiments of the invention 443-452, wherein the isolated antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities.

The isolated antibody of embodiment 453, wherein the isolated antibody induces or retains TREM2 clustering on the cell surface.

The isolated antibody of Embodiment 453 or Embodiment 454, characterized in that one or more TREM2 activities are selected from the group consisting of: TREM2 binding to DAP12; binding DAP12 to TREM2; TREM2 phosphorylation, DAP12 phosphorylation; physical activation; increased expression of one or more cytokines selected from the group consisting of IL-12p70, IL-6 and IL-10; reduced expression of one or more pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-1b, TNF-α, IL-10, IL-6, IL-8, CRP, members of the TGF-beta chemokine protein families, family members IL-20, IL-33, FIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18 and CRP; reduced expression of TNF-α, IL-6, or both; phosphorylation of kinase regulated by extracellular signals (ERK); increased expression of the C-C chemokine receptor 7 (CCR7); inducing chemotaxis of microglial cells towards cells expressing CCL19 and CCL21; increasing the ability of dendritic cells, monocytes, microglia and/or macrophages to induce T cell proliferation; increasing, normalizing, or both, the ability of bone marrow dendritic cells to induce the proliferation of antigen-specific T cells; inducing the production of osteoclasts, increasing the level of osteoclastogenesis, or both; increasing the survival and/or function of one or more dendritic cells, macrophages, M1 macrophages, activated M1 macrophages, M2 macrophages, osteoclasts, skin Langerhans cells, Kupffer cells, microglial cells, M1 microglial cells, activated M1 microglial cells, and M2 microglial cells; inducing one or more types of clearance selected from the group consisting of cancer cell clearance, apoptotic neuron clearance, neural debris clearance, non-nervous tissue debris clearance, bacterial or other foreign body clearance, and pathogenic protein clearance; inducing phagocytosis of one or more apoptotic neurons, neural tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, pathogenic proteins, or tumor cells; normalization of impaired TREM2/DAP12-dependent gene expression; involvement of Syk, ZAP70, or both in the TREM2/DAP12 complex; phosphorylation of Syk; increased expression of CD83 and/or CD86 on dendritic cells, microglia, monocytes or macrophages; decreased secretion of one or more inflammatory cytokines selected from the group consisting of IFN-a4, IFN-b, IL-1b, TNF-α, IL-10, IL-6, IL-8, CRP, members of the TGF-chemokine protein families beta, family members IL-20, IL-33, PIL, IFN-gamma, OSM, CNF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, CRP and MCP- 1; decreased expression of one or more inflammatory receptors; increased phagocytosis by macrophages, monocytes, dendritic cells and/or microglia under conditions of reduced levels of MCSF; decreased phagocytosis by macrophages, monocytes, dendritic cells and/or microglia in the presence of normal levels of MCSF; increased activity of one or more TREM2-dependent genes; and any combination of them.

An isolated antibody according to any one of the embodiments of the invention 453-455, characterized in that the antibody belongs to the IgG class, the IgM class, or the IgA class.

An isolated antibody according to embodiment 456, characterized in that the antibody belongs to the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype.

An isolated antibody according to embodiment 457, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 458, characterized in that the antibody contains a human IgG2 constant region.

- 129 042890

The isolated antibody of embodiment 459, characterized in that the human IgG2 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 458-460, characterized in that the antibody induces one or more TREM2 activities independent of binding to the Fc receptor.

An isolated antibody according to any one of the embodiments of the invention 458-461, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 462 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to embodiment 462 or embodiment 463, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 464, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 465, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

An isolated antibody according to embodiment 459, characterized in that the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the residue numbering being EU numbering.

An isolated antibody according to embodiment 457, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 468, wherein the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 469, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 468-470, characterized in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 471 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyRIIB).

An isolated antibody according to any one of the embodiments of the invention 470-472, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 473, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 474, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, the residue numbering being given according to the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 470-472, characterized in that the antibody includes a heavy chain constant domain 1(CH1) of the IgG2 isotype and a hinge region.

An isolated antibody according to embodiment 476, characterized in that CH1 of the IgG2 isotype and the hinge region contain the amino acid sequence

ASTKGPSVFP LAPCSRSTSE

STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSWT VPSSNFGTQT

YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO:397).

An isolated antibody according to embodiment 476 or embodiment 477, characterized in that the Fc region of the antibody contains an S267E amino acid substitution, an L328F amino acid substitution, or both, and/or an N297A or N297Q amino acid substitution, wherein the residue numbering is presented in accordance with the numbering EU.

The isolated antibody of embodiment 468, wherein the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 457, characterized in that the antibody has an IgG4 isotype.

The isolated antibody of embodiment 480, wherein the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 481, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to any one of the embodiments of the invention 480-482, differ

- 130 042890 in that the antibody binds to an inhibitory Fc receptor.

The isolated antibody of embodiment 483 wherein the inhibitory Fc receptor is an inhibitory Fc receptor gamma IIB (FcyIIB).

An isolated antibody according to any one of the embodiments of the invention 482-484, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 485, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 486, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E and any combination thereof, the numbering of the residues being presented in accordance with the EU numbering.

An isolated antibody according to embodiment 456, characterized in that the antibody has an IgG2/4 hybrid isotype.

An isolated antibody according to embodiment 488, characterized in that the antibody contains an amino acid sequence containing amino acids 118 to 260 from human IgG2 and amino acids 261 to 447 from human IgG4, with residue numbering according to EU numbering.

The isolated antibody of embodiment 481, characterized in that the antibody contains a mouse IgG4 constant region.

An isolated antibody according to any one of embodiments 451-490, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, and a variant TREM2 in pathology, and wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring human TREM2 variant, and a pathological TREM2 variant.

The isolated antibody of embodiment 491, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

An isolated antibody according to any one of embodiments of the invention 443-452, wherein the isolated antibody is an inert antibody.

An isolated antibody according to any one of the embodiments of the invention 443-452, wherein the isolated antibody is an antagonistic antibody.

The isolated antibody of embodiment 494, wherein the isolated antibody inhibits one or more TREM2 activities.

An isolated antibody according to embodiment 495, characterized in that one or more TREM2 activities are selected from the group consisting of a decrease in the activity of one or more TREM2-dependent genes; reducing the activity of one or more nuclear factors of activated T-cell transcription factors (NFAT); reduced survival of macrophages, microglial cells, monocytes, osteoclasts, skin Langerhans cells, Kupffer cells and/or dendritic cells; and any combination of them.

The isolated antibody of any one of embodiments 494-496, wherein the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signaling, or both.

An isolated antibody according to any one of the embodiments of the invention 493-497, characterized in that the antibody is not capable of binding the Fc-gamma receptor (FcyR).

An isolated antibody according to embodiment 498, characterized in that the antibody has an IgG1 isotype.

The isolated antibody of embodiment 499, wherein the antibody contains a human IgG1 constant region.

The isolated antibody of embodiment 500, wherein the human IgG1 constant region contains an Fc region.

An isolated antibody according to embodiment 501, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 502, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 503, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to EU numbering.

The isolated antibody of embodiment 504, characterized in that the Fc region

- 131 042890 further includes a deletion of the amino acid at the position corresponding to glycine 236 according to EU numbering.

The isolated antibody of embodiment 499, characterized in that the antibody contains a mouse IgG1 constant region.

An isolated antibody according to embodiment 498, characterized in that the antibody has an IgG2 isotype.

The isolated antibody of embodiment 507, wherein the antibody contains a human IgG2 constant region.

The isolated antibody of embodiment 508, wherein the human IgG2 constant region contains an Fc region.

An isolated antibody according to embodiment 509, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 510, characterized in that the Fc region contains one or more amino acid substitutions.

An isolated antibody according to embodiment 511, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof, with residue numbering following EU numbering.

The isolated antibody of embodiment 498, characterized in that the antibody is of the IgG4 isotype.

The isolated antibody of embodiment 513, characterized in that the antibody contains a human IgG4 constant region.

The isolated antibody of embodiment 514, wherein the human IgG4 constant region contains an Fc region.

An isolated antibody according to embodiment 515, characterized in that the Fc region contains one or more modifications.

The isolated antibody of embodiment 516, characterized in that the Fc region contains one or more amino acid substitutions.

The isolated antibody of embodiment 517, characterized in that one or more amino acid substitutions in the Fc region are at the position of a residue selected from the group consisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combination thereof, with residue numbering following the EU numbering.

An isolated antibody according to any one of embodiments 443-518, wherein the isolated antibody is an antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, naturally occurring human TREM2 variant, and variant TREM2 in pathology.

The isolated antibody of embodiment 519, wherein the fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, or scFv fragment.

The isolated antibody of Embodiment 475, Embodiment 504, or Embodiment 505, characterized in that the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, the numbering of the residues being given in accordance with the EU numbering.

An isolated antibody according to any one of the embodiments of the invention 465-521, characterized in that the Fc region additionally contains one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E and any combination thereof, and the numbering of residues presented according to the EU numbering.

The isolated antibody of Embodiment 487 or Embodiment 518, characterized in that the Fc region further comprises a serine to proline amino acid substitution at position 228 according to EU numbering.

An isolated antibody according to any one of embodiments 443-523, wherein the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.

An isolated antibody according to any one of the embodiments of the invention 443-524, characterized in that the antibody is a bispecific antibody that recognizes the first antigen and the second antigen.

An isolated antibody according to any one of the embodiments of the invention 443-525, characterized in that the antibody is a monoclonal antibody.

An isolated antibody according to any of the preceding embodiments of the invention, characterized in that the isolated antibody specifically binds to both human TREM2 and

- 132 042890

TREM2 mice.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (K _D ) for human TREM2 and mouse TREM2 that ranges from less than about 6.70 nM to less than about 0.23 nM .

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (K _D ) for the human TREM2-Fc fusion protein that ranges from less than about 0.71 nM to less than about 0.23 nM.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (KD) for the monomeric human TREM2 protein that ranges from less than about 6.70 nM to less than about 0.66 nM.

An isolated antibody according to any of the preceding embodiments, wherein the isolated antibody has a dissociation constant (KD) for the mouse TREM2-Fc fusion protein that ranges from less than about 4.90 nM to less than about 0.35 nM .

An isolated nucleic acid encoding an antibody according to any of the preceding embodiments of the invention.

A vector containing the nucleic acid of embodiment 532.

Host cell containing the vector of embodiment 533.

A method for producing an antibody, comprising culturing a cell according to embodiment 534, accompanied by the production of an antibody.

The method of Embodiment 535, further comprising isolating an antibody produced by the cell.

A pharmaceutical composition containing an antibody according to any one of the embodiments of the invention 303-531 and a pharmaceutically acceptable carrier.

A method for preventing, reducing risk or treating an individual having a disease, disorder or injury selected from the group consisting of dementia, frontotemporal dementia, Alzheimer's disease, multi-infarct dementia, mixed dementia, Creutzfeldt-Jakob disease, normotensive hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, taupathies, Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn's disease, inflammatory bowel disease, ulcerative colitis, obesity, malaria, essential tremor, central nervous system lupus , Behçet's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Shy-Drager syndrome, Steele-Richardson-Olszewski disease, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomatosis, sarcoidosis, aging disease, epileptiform seizures, spinal cord injury , traumatic brain injury, age-related macular degeneration, glaucoma, retinitis pigmentosa, retinal degeneration, respiratory tract infection, sepsis, eye infection, systemic infection, lupus, arthritis, multiple sclerosis, low bone density, osteoporosis, osteogenesis, disease associated with osteoporosis, Paget's disease of bone and cancer, comprising administering to an individual a therapeutically effective amount of an isolated antibody that binds to the TREM2 protein.

A method for inducing or promoting innate immune cell survival or wound healing in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated agonist antibody that binds to the TREM2 protein.

The method of embodiment 538 or embodiment 539, wherein the isolated antibody is:

i. (a) an agonist antibody;

(b) an inert antibody; or (c) an antagonistic antibody.

The method of embodiment 540, wherein:

ii. (a) the antibody is of the IgG class, the IgM class, or the IgA class; and/or iii. (b) the antibody is IgG1, IgG2, IgG3 or IgG4 isotype.

The method of embodiment 541, wherein the antibody contains one or more amino acid substitutions in the Fc region located at the position of a residue selected from the group consisting of:

iv. (a) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and any combination thereof;

v. (b) N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof;

vi. (c) L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any combination thereof;

vii. (d) N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L , M252Y, S254T, T256E and any their combi

- 133 042890 nation;

viii. (e) V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and any combination thereof; or ix. (f) E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q and any combination thereof,

X. moreover, the numbering of the residues is presented in accordance with the EU numbering.

The method according to any one of the embodiments of the invention 538-542, characterized in that the selected antibody:

xi. (a) binds to one or more amino acids within amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1; or xi. (b) one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.

The method according to any one of the embodiments of the invention 538-543, characterized in that the isolated antibody:

xiii. (a) binds essentially the same TREM2 epitope as the Ab52 antibody;

xiv. (b) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibody At52; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At52 monoclonal antibody;

xv. (c) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an HVR-H1 containing the amino acid sequence of SEQ ID NO: 398 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 398, HVR-H2 containing the amino acid sequence from SEQ ID NO: 399 or an amino acid sequence with at least about 95% homology to the amino acid sequence from SEQ ID NO: 399, and HVRH3 containing the amino acid sequence from SEQ ID NO: : 400, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 400, and/or wherein the light chain variable domain comprises an HVR-L1 containing the amino acid sequence of SEQ ID NO: 401, or an amino acid a sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 401, HVR-L2 containing the amino acid sequence of SEQ ID NO: 402, or an amino acid sequence of at least about 95% homology to the amino acid sequence of SEQ ID NO: 402, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 403, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 403;

xvi. (d) binds essentially the same TREM2 epitope as the At21 antibody;

xviii. (e) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2 and/or HVR-H3 of the monoclonal antibody At21; and/or wherein the light chain variable domain comprises HVR-L1, HVR-L2 and/or HVR-L3 of the At21 monoclonal antibody; or viii. (f) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an HVR-H1 containing the amino acid sequence of SEQ ID NO: 404 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 404, HVR-H2 containing the amino acid sequence of SEQ ID NO: 405 or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 405, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 406, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO: 406, and/or wherein the light chain variable domain comprises HVR-L1 containing the amino acid sequence of SEQ ID NO: 407, or an amino acid sequence with at least about 95% homology with the amino acid sequence of SEQ ID NO: 407, HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, or an amino acid sequence with at least about 95% homology with an amino acid sequence from SEQ ID NO: 408, and an HVR-L3 containing the amino acid sequence from SEQ ID NO: 409, or an amino acid sequence with at least about 95% homology to the amino acid sequence from SEQ ID NO: 409.

The method of embodiment 538, wherein the isolated antibody is the isolated antibody of any one of embodiments 303-531.

The method of embodiment 539 wherein the isolated agonist antibody is an isolated antibody of any one of the embodiments of the invention 303-134 042890

385, 420-492 and 519-531.

The method of any one of embodiments 538 and 540-545, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains one or more substitutions selected from the group consisting of:

viii. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1;

ix. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1;

X. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1;

xi. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1;

xi. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1;

xiii. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1;

xiv. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1.

The method according to any one of embodiments of the invention 538, 540-545 and 547, characterized in that the individual has a heterozygous variant of TREM2, and the variant contains a nucleotide deletion of guanine in the nucleotide corresponding to the nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; nucleo

- 135 042890 (Herceptin®), adaptive cell transfer (ACT), chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy and cytokine therapy.

The method of any one of embodiments 538, 540-545, and 550, further comprising administering to the individual at least one antibody that specifically binds to an inhibitory cytokine.

The method of embodiment 555, wherein at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with the isolated antibody.

The method of Embodiment 555 or Embodiment 556, wherein at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of a KCCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination of them.

The method of any one of embodiments 538, 540-545, and 550, further comprising administering to the subject at least one agonist antibody that specifically binds to a checkpoint stimulating protein.

The method of embodiment 558 wherein at least one agonist antibody that specifically binds to a checkpoint stimulating protein is administered in combination with an isolated antibody.

The method of Embodiment 558 or Embodiment 559, wherein the at least one agonist antibody that specifically binds to the checkpoint stimulating protein is selected from the group consisting of an anti-CD40 agonist antibody, an anti-OX40 agonist antibody, an agonist antibody to ICOS, an agonist antibody to CD28, an agonist antibody to CD137/4-1BB, an agonist antibody to CD27, an agonist antibody to glucocorticoid-induced TNFR-related GITR protein, and any combination thereof.

The method of any one of embodiments 538, 540-545, and 550, further comprising administering to the individual at least one stimulatory cytokine.

The method of embodiment 561 wherein at least one stimulatory cytokine is administered in combination with the isolated antibody.

The method of embodiment 561 or embodiment 562 wherein at least one stimulatory cytokine is selected from the group consisting of TNF-α, IL10, IL-6, IL-8, CRP, members of the TGF-beta chemokine protein families , family members IL-20, IL-33, FIL, OSM, CNF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α, IFN-β, IL-2 , IL-18, GM-CSF, G-CSF and any combination thereof.

The present invention will be better understood with reference to the following examples. However, they should not be construed as limiting the scope of the present invention. All references throughout the specification are expressly incorporated herein by reference.

Examples

Example 1 Production, identification and characterization of anti-TREM2 agonist antibodies and anti-DAP12 agonist antibodies.

Introduction.

The amino acid sequence of the human TREM2 precursor protein is shown below in SEQ ID NO: 1. TREM2 contains a signal peptide located at amino acid residues 1-18 of SEQ ID NO: 1. residues 29-112 of SEQ ID NO: 1; additional extracellular sequences located at amino acid residues 113-174 of SEQ ID NO: 1; a transmembrane domain located at amino acid residues 175-195 of SEQ ID NO: 1; and an intracellular domain located at amino acid residues 196-230 of SEQ ID NO: 1.

TREM2 amino acid sequence (SEQ ID NO: 1):

£2 £3 40 5060

MEPLRLLILL FVTELSGAHN TTVFQGVAGQ SLQVSCPYDS MKHWGRRKAW CRQLGEKGPC

£8 £9 100 110120

QRWSTHNLW LLSFLRRWNG STAITDDTLG GTLTITLRNL QPHDAGLYQC QSLHGSEADT

130 14 £ 15 £ 160 170180

LRKVLVEVLA DPLDHRDAGD LWFPGESESF EDAHVEHSIS RSLLEGEIPF PPTSILLLLA

190 20 £ 210 220230

CIFLIKILAA SALWAAAWHG QKPGTHPPSE LDCGHDPGYQ LQTLPGLRDT

A known feature of human TREM2 is that the transmembrane domain of human TREM2 contains a lysine (a186) that can interact with aspartic acid in DAP12, a key adapter protein that transduces signals from TREM2, TREM1, and other related

- 136 042890 members of the IgV family.

Human BLAST TREM2 analysis revealed 18 related homologues. These homologues include the NK-p44 natural killer (NK) cell receptor (NCTR2), the polymeric immunoglobulin receptor (pIgR), CD300E, CD300A, CD30°C and TREML1/TLT1. The closest homologue was identified as NCTR2, which shares similarities with TREM2 in the IgV domain (FIG. 1A). The BLAST analysis also compared the TREM proteins with other IgV family proteins (FIG. 1B).

TREM2 is also closely related to TREM1. The amino acid sequence alignment of TREM1 and TREM2 was generated using a bidirectional blast (FIG. 2A). This limitation also applies to the IgV domain.

Previously, agonist antibodies to TREM1, NK-p44 and other members of this family have been described. Antibodies that bind the extracellular domain of TREM2, in particular the IgV domain (amino acid residues 29-112 of SEQ ID NO: 1) are generated using mouse hybridoma technology, phage display technology and yeast display technology. The antibodies are then screened for their ability to activate the TREM2 signaling pathway and function in cells and in the whole animal in vivo as described in Examples 2-39 below.

For example, anti-TREM2 agonist antibodies can be made that target the IgV domain (amino acid residues 29-112). IgV domains bind to targets and, through multimerization of receptors such as IgG itself or NKp44, result in activation. Thus, these domains are rational targets for agonistic antibodies. They are also extremely divergent.

Anti-TREM2 agonist antibodies can also be generated that target amino acid residues 99-115 of human TREM2. Amino acid residues 99-115 are believed to correspond to a peptide that blocks TREM2 binding to its endogenous target, as the corresponding peptide in mouse TREM1 (amino acid residues 83-99) blocks TREM1 binding to its endogenous target (Gibot et al., Infect. Immunity 2004 ). The murine TREM1 peptide is called LP17 (LQVTDSGLYRCVIYHPP (SEQ ID NO: 414)). The equivalent region in human TREM2 is located in the CD3 domain and is located at amino acid residues 99-115 of SEQ ID NO: 1 (LQPHDAGLYQCQSLHG). Antibodies that block ligand binding can activate receptors similar to the ligand itself.

Another approach to predict a relevant (e.g., agonist) site in the human TREM2 protein is to target sites that are found to have mutations in Alzheimer's disease (e.g., R47H), polycystic lipomembrane osteodysplasia with sclerotic leukoencephalopathy (PLOSL), or Nasu-Hakola disease. Also relevant is the site of major mutations associated with human disease, which are typically found in the IgV domain.

The crystal structures of TREM2-related TREM1 structures have been described (Kelker, M.S. et al., J. Mol. Biol., 2004. 344(5): p. 1175-81; Kelker, M.S. et al., J. Mol. Biol. , 2004. 342(4): pp. 1237-48 and Radaev, S. et al., Structure, 2003. 11(12): pp. 1527-35), TLT1 (Gattis, J. L. et al., J. Biol Chem., 2006. 281(19): pp. 13396-403), and NKp44, and thus structural regions/features have been identified in the IgV domain that are particularly often central to interactions with natural agonists. These studies support the belief that complementarity determining regions (CDR1, CDR2, CDR3) play a major role in ligand binding. TREM1 is reported to be monomeric (Gattis, J.L. et al., J. Biol. Chem., 2006. 281(19): p. 13396-403) or dimeric (Radaev, S. et al., Structure, 2003. 11 (12): pp. 1527-35) in vitro under cell-free conditions, but its in vivo oligomeric state remains unclear, as does TREM2.

The amino acid sequence of human DAP12 is shown below in SEQ ID NO: 2:

2_0 3_0 4 £ 5_0 b_0

MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA

0 8_0 9_0 10_0 110

VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

DAP12 is a type 1 single pass membrane protein. It contains an extracellular domain located at amino acid residues 22-40 of human DAP12 (SEQ ID NO: 2); a transmembrane domain located at amino acid residues 41-61 of human DAP12 (SEQ ID NO: 2); and an intracellular domain located at amino acid residues 62-113 of human DAP12 (SEQ ID NO: 2). The immunoreceptor tyrosine activating motif (ITAM) domain of DAP12 is located at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2). The aspartic acid residue in DAP12 interacts with the transmembrane domain of human TREM2, which contains lysine at amino acid residue 186, and transduces signals from TREM2, TREM1 and other related members of the IgV family.

Anti-DAP12 agonist antibodies can be generated that target amino acid residues 22-40 of human DAP12. It is believed that DAP12 is a disulfide-linked dimer associated with TREM2, and that dimerization of DAP12 with an antibody to the extracellular domain, encompassing

- 137 042890 amino acid residues 22-40 activates one or more TREM2 and/or DAP12 activities.

The studies discussed herein describe the production of agonist antibodies that bind TREM2. Antibodies are screened for binding to cells expressing TREM2 and their ability to activate TREM2 signaling and functionality.

Results.

Anti-TREM2 antibody production.

Antibodies that bind the extracellular domain of TREM2, in particular the IgV domain (amino acid residues 29-112 of SEQ ID NO: 1) were generated using the following procedure. As previously described, eight naïve synthetic yeast libraries of ~10 ⁹ cells each were designed, generated, and propagated (see e.g. WO 2009036379; WO 2010105256; WO 2012009568; Xu et al. Sel. 26(10):663-670). The ADIMAB yeast-based antibody detection platform used in this document allowed the identification of fully human full-length IgG1 monoclonal antibodies with broad epitopic coverage. ADIMAB yeast is designed to transport high quality, complete IgG through the secretory pathway and then to present it on the surface or release it directly into the medium.

For the first selection rounds, a magnetic bead sorting method based on the Miltenyi MACS system was used as previously described (Siegel et al., (2004) J. Immunol. Methods 286(1-2):141-53). Briefly, yeast cells (~10 ¹⁰ cells/library) were incubated with 3 ml of 200 nM biotinylated TREM2 antigen or 10 nM biotinylated TREM2-Fc fusion antigen for 15 min at room temperature in FACS wash buffer PBS with 0.1% BSA. Biotinylation was performed using the EZ-Link Sulfo-NHS biotinylation kit (Thermo Scientific, Cat #21425). After washing once with 50 ml of ice-cold wash buffer, the cell conglomerate was resuspended in 40 ml of wash buffer and 500 µl of streptavidin microspheres (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat # 130-048-101) was added to the yeast and incubated for 15 min at 4°C. The yeast was then pelleted, resuspended in 5 ml wash buffer and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat.# 130-042-401). After loading 5 ml, the column was washed 3 times with 3 ml FACS wash buffer. The column was then removed from the magnetic field and the yeast was eluted with 5 ml growth medium and then grown overnight. The next three sorting rounds were performed using flow cytometry. Approximately 1x10 ⁸ yeast cells were pelleted, washed three times with wash buffer and incubated respectively with 200, 100 or 10 nM biotinylated TREM2 for 10 minutes at room temperature. Yeast was then washed twice and stained with goat anti _- human kappa F(ab') FITC antibody diluted 1:100 (Southern Biotech, Birmingham, Alabama, Cat # 2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies , Grand Island, NY, Cat # S21375) diluted 1:500, or extravidin-phycoerythrin (Sigma-Aldrich, St Louis, Cat # E4011) diluted 1:50 with secondary reagents for 15 min at 4°C. After washing twice with ice-cold wash buffer, the cell conglomerates were resuspended in 0.4 ml wash buffer and transferred to sorting tubes with a filter. Sorting was done using a FACS ARIA sorter (BD Biosciences) and only clones for TREM2 binding were selected for sorting for two rounds and the third round was a negative sort to reduce reagent binding. After the final sorting round, the yeasts were plated and individual colonies were collected for characterization.

Yeast clones were grown to saturation and then induced for 48 h at 30°C with shaking. After induction, yeast cells were pelleted and supernatants were collected for purification. IgG was purified using a protein A column and eluted with acetic acid pH 2.0. Fab fragments were generated by papain digestion and purified with KappaSelect (GE Healthcare LifeSciences, Cat # 17-5458-01). Two antibodies (At21 and At52) were selected for further analysis.

Heavy chain and light chain variable domain sequences of antibodies At21 and At52.

Using standard methods, the amino acid sequences encoding the heavy chain (FIG. 2B) and light chain (FIG. 2C) of the variable domain of the At21 antibody and the At52 antibody were determined.

The CDR sequences of the At21 antibody and the Kabat antibody At52 are shown in Table 1. 1.

The amino acid sequence of the heavy chain variable domain of the antibody At21 is

EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSF

QGQVTISADKSIS TAYLQWS S LKAS DTAMYYCARAGHYDGGHLGMDVWGQGT TVTVS S (SEQ

ID NO:410), and the amino acid sequence of the light chain variable domain of antibody 21 is

- 138 042890

EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFS

GSGSGTDFTLTISRLEPEDFAVYYCQQDDSAPYTFGGGTKVEIK (SEQ ID NO:411).

The amino acid sequence of the heavy chain variable domain of the antibody At52 is

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGIINPSGGSTSYAQKF

QGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREADDSSGYPLGLDVWGQGTMVTVSS (SEQ

ID NO:412) and the amino acid sequence of the light chain variable domain of antibody 52 is

EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSG

SGSGTEFTLTISSLQSEDFAVYYCQQVNSLPPTFGGGTKVEIK (SEQ ID NO:413).

Table 1 A. Kabat heavy chain CDR sequences

Name of the antibody CDR H1 CDR H2 CDR H3 At21 YSFTTYWIG (SEQ ID NO:404) IIYPGDSDTRYSPSFQG (SEQ ID NO:405) ARAGHYDGGHLGMDV (SEQ ID NO:406) At 5 2 YTFTSYYIH (SEQ ID NO:398) IINPSGGSTSYAQKFQG (SEQ ID NO:399) AREADDSSGYPLGLDV (SEQ ID NQ:400)

Table 1B. Light chain CDR sequences by Kabat

Name of the antibody CDR-LI CDR L2 CDR L3 At21 RASQSVSSSYLA (SEQ ID NO:407) GAS N RAT (SEQ ID NO:408) QQDDSAPYT (SEQ ID NO:409) At 5 2 RASQSVSSNLA (SEQ ID NO:401) GAST RAT (SEQ ID NO:402) QQVNSLPPT (SEQ ID NQ:403)

Characterization of the binding of At21 and At52.

Initial characterization of TREM2 antibodies included determining their ability to bind TREM2 expressed on human or mouse dendritic and other primary immune cells. Cells were harvested, plated at 10 ⁵ /ml in a 96-well plate, washed and incubated in 100 μl PBS containing 10-50 μg/ml mAb and Fc blocking reagent for 1 hour on ice. The cells were then washed twice and incubated in 100 μl of PBS containing 5 μg/ml of secondary antibody conjugated with PE for 30 min on ice. Cells were washed twice in cold PBS and quantified using BD FACS Canto. Data analysis and calculation of MFI values were performed using FlowJo software (TreeStar) version 10.0.7.

Antibodies At21, At52, At16, At20, Atb and At68 showed binding to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2 as shown by positive staining of TREM2 antibodies detected by FACS analysis (bar graphs with black outline) (Fig. . BEHIND). Antibodies At21 and At52 showed antibody binding to mouse bone marrow-derived DT (TREM +/+) macrophages (HMMac, mMac), but not to TKEM2-deficient (TREM2 -/-) mouse macrophages (HMMac, mMac) (Fig. ZV). Antibodies At21 and At52 show binding to both a human (293) cell line expressing recombinant human TREM2 (FIG. 4A) and human primary dendritic cells (hDC) (FIG. 4B). In contrast, At43 and AtbO antibodies bound to a human cell line expressing recombinant human TREM2 (FIG. 4A), but did not bind to primary human dendritic cells (FIG. 4B).

Mean fluorescence intensity (MFI) values for cell types bound by antibodies At21 and At21 to TREM2 are shown in Table 1. 4. Binding compared to parental mouse cell line (mTREM2 parental cell line BWZ), primary human cell line (4TREM2 parental cell line (293)), primary TREM2-deficient mouse macrophages (mPac KO MFI), and primary TREM2-deficient mouse dendritic cells (mDK KO MFI).

- 139 042890

Table 4 Anti-TREM2 antibody binding to human and mouse cells

Antibody MTREM2 Cell line (BWZ parental) MFI MTREM2 Cell line (BWZ T2) MFI 4TREM2 parent cell line (293) MFI 4TREM2 cell line (293) MFI mMac KO MFI mMac DT MFI mDC KO MFI mDC DT MFI hDV % positive At52 1021 15613 89 1411 73.3 174.0 203 663 69, 1 At21 1036 13840 81 1884 64.7 111.0 187 464 77.1

The binding affinity of each antibody to TREM2 was determined by measuring their KD using ForteBio or MSD-SET. Affinity measurements with ForteBio were performed as previously described (Estep et al., (2013) MAbs 5(2):270-8). Briefly, ForteBio affinity measurements were performed by uploading IgG online to AHQ sensors. The sensors were equilibrated offline in assay buffer for 30 min and then monitored online for 60 s to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen for 5 min, then transferred to assay buffer for 5 min to measure the rate of response. The kinetics were analyzed using a 1:1 binding model.

Equilibrium affinity measurements were performed as previously described (Estep et al., (2013) MAbs 5(2):270-8). Titrations to the end point (SET) were performed in PBS + 0.1% BSA without IgG (PBSF) at a constant antigen concentration of 50 pM and incubated with 3-5-fold serial dilution of the antibody starting from 10 nM. Antibodies (20 nM in PBS) were applied to standard MSD-ECL binding plates overnight at 4°C or at room temperature for 30 min. The plates were then blocked for 30 minutes with shaking at 700 rpm followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds with shaking at 700 rpm followed by a single wash. Antigen captured on the plate was detected with 250 ng/ml sulfotag-labeled streptavidin in PBSF by incubation on the plate for 3 minutes. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 using a single Read Buffer T with surfactant. The percentage of free antigen was plotted as a function of titratable antibody in Prism and squared to give the KD value. Liquid handling robots were used in all MSD-SET experiments, including SET sample preparation, to improve throughput.

In table. 5 lists values reflecting the binding affinity (K _D ) of Ab21 and Ab52 antibodies to human TREM2 Fc fusion protein (4TREM2-Fc), monomeric His-tagged human TREM2 protein (4TREM2-HIS), and mouse TREM2 Fc fusion protein (mTREM2-Fc).

Table 5. Binding affinity of antibodies to TREM2

Antibodies O IgG K _D 4TREM2-FC (M) Avid Monovalent IgG K _D 4TREM2-HIS (M) IgG K _D mTREM2-Fc (M) Avid At52 1.51Е-09 5.75Ε-09 8.96E-11 At21 3.44Е-10 1.14Ε-09 2.27E-10

Example 2 Normalization and reduction of Toll-like receptor (TLR) responses in dendritic cells with anti-TREM2, DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies.

Bone marrow dendritic cells (BMDC) are stimulated by culturing with TLR ligands such as LPS, CpG DNA and zymosan for 16 h. , IL-12 p70 and TNF. It is believed that BMDC cells that do not have active TREM2 can secrete significantly more IL-12, p70 and TNF than BMDC cells that have activated TREM2 after stimulation. In addition, anti-TREM2 agonist antibodies are believed to reduce expression levels of IL-12, p70, and TNF. Bone marrow dendritic cells from wild-type mice and from TREM2 heterozygous mice that have partially inactive TREM2 will serve as positive controls to determine the expression levels of IL-12, p70 and TNF cytokines, as well as their modulation by anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies; and/or TREM2/DAP12 bispecific antibodies.

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Cytokine concentrations in supernatants are determined using mouse IFN-a4, IFN-b, IL-6, IL-12 p70, TNF and IL-10 ELISA kits (eBioscience) and the VeriKine Mouse IFN-b ELISA kit (PBL source of interferon ) according to the manufacturer's protocol. mRNA levels for these cytokines are also measured by quantitative RT-PCR (qRT-PCR). Total RNA was obtained using the RNeasy plus mini kit (QIAGEN), reverse transcribed with Superscript III Reverse Transcriptase (Invitrogen) using an oligo-dT primer according to the manufacturer's protocol. Quantitative PCR was performed using the Master SYRR Green PCR Master Mix (Applied Biosystems) and 7900HT (Applied Biosystems) according to the manufacturer's protocol. The primer sequences for IFN-a4, IFN-b, IL-6, IL-12 p70 and TNF are presented as described (eg Hamerman, JA, Eur. J. Immunol. 2012. 42: 176185).

Example 3 Normalization and reduction of the ability of BMDC to induce antigen-specific T cell proliferation with anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or anti-TREM2/DAP12 bispecific antibodies may reduce and normalize the ability of bone marrow-derived dendritic cells (BMDCs) to induce antigen-specific T cell proliferation.

The ovalbumin (OVA)-specific T cell response induced by BMDC can be determined using a CFSE dilution. BMDCs were isolated by MACS after 6 days of culture and seeded 1 x 10 ⁴ cells per well of a 96-well round bottom plate with OVA (2 or 0.5 mg/ml) and CpG DNA (100 or 25 nM) in the presence of GM-CSF ( 10 ng/mL) for 4 h. CD4 T cells from the spleen and lymph nodes of OT-II transgenic mice were isolated using the Dynal Mouse CD4 Negative Isolation Kit (Invitrogen) and stained with CFSE (final concentration 0.8 mM) . After 4 hours of DC culture, 1x105 CFSE-labeled CD4 OT-II T cells are added to each well and incubated for 72 hours. II T-cells. Data analysis to calculate the percentage of division index and division index is performed with the software Flowjo (Treestar) (Eur. J. Immunol. 2012. 42: 176-185).

Example 4 Normalization and reduction of Toll-like receptor (TLR) responses in macrophages with anti-TREM2, DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies.

In the absence of TREM2, responses from bone marrow-derived macrophages (BMDM) or from primary peritoneal macrophages in TLR signaling are altered (Turnbull, I.R. et al., J. Immunol. 2006; 177:3520-3524). It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies can reduce and normalize TLR responses in macrophages.

To detect primary macrophages, mice are treated with 1.5 ml of 2% thioglycolate medium by intraperitoneal injection and then the cells are isolated by peritoneal washing. To obtain BMDM, total bone marrow was cultured in DMEM enriched with 10% calf serum, 5% horse serum and 6 ng/ml recombinant human CSF-1 (R&D Systems). Cells are cultured for 5-6 days and adherent cells are separated with 1M EDTA in PBS. Cells are stained with commercially available antibodies: anti-CD11b, anti-CD40, anti-GR1 (BD Pharmingen) and F4/80 (Caltag Laboratories).

BMDM is replated and allowed to adhere for 4 hours at 37°C, and then TLR agonists such as LPS (Salmonella abortus equi), zymosan (Saccharomyces cerevisiae) and CpG 1826 DNA (purchased from, for example, Sigma-Aldrich) are added. Cell culture supernatant is harvested 24 hours post-stimulation and IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF cytokine levels are measured by ELISA or cytometric array (BD Biosciences Mice Inflammation Kit).

Example 5 Induction of the anti-inflammatory cytokine IL-10 in bone marrow-derived myeloid progenitors by anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies.

It is believed that bone marrow-derived myeloid progenitor cells may exhibit an increase in the anti-inflammatory cytokine IL-10 after treatment with anti-TREM2 agonist antibodies, DAP12, and/or TREM2/DAP12 bispecific antibodies and stimulation with 100 ng/mL LPS (Sigma) , by co-cultivation with apoptotic cells, or by a similar stimulus.

Isolation of myeloid progenitor cells derived from bone marrow is performed as follows. Bone marrow cells were isolated from adult 6-8 week old C57BL/6 female mice (Charles River, Sulzfeld, Germany) from the medullary cavities of the tibia and hind limb femur. Removal of red blood cells is carried out by lysis using a hypotonic solution. Cells were cultured in DMEM (Invitrogen) containing 10% fetal calf serum (Pan Biotech) and 10 ng/ml GM-CSF (R&D Systems) in 75 cm ² culture flasks (Greiner Bio

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one). After 24 hours, non-adherent cells are harvested and replated in new 75 cm ² culture flasks. The medium is changed after 5 days and the cells are cultured for an additional 10-11 days. The remaining cells are bone marrow-derived myeloid progenitors transduced with TREM2 virus. The transduced cells are then examined for IL-10 levels in conditioned media both in the presence and absence of anti-TREM2 and LPS agonist antibodies. The supernatant is harvested 24 hours later and the level of IL-10 released from the cells is determined by IL-10 ELISA according to the manufacturer's instructions (QuantikineM mouse IL10, R&D Systems) (JEM (2005), 201; 647-657; and PLoS Medicine (2004), 4 | Issue 4 | e124).

Example 6. Induction of phagocytosis of apoptotic neurons, nerve tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, disease-causing proteins in myeloid cells using agonistic antibodies to TREM2, DAP12 and/or bispecific antibodies to TREM2/DAP12.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or anti-TREM2/DAP12 bispecific antibodies can induce phagocytosis of one or more apoptotic neurons, neural debris, non-nervous tissue debris, bacteria, other foreign bodies, and disease-causing proteins such as A beta peptide, alpha-synuclein protein, Tau protein, TDP-43 protein, prion protein, and Huntington protein in myeloid lineage cells such as monocytes and microglia.

Monocytes are isolated from peripheral blood collected from adult C57BL/6 mice. Hypotonic lysis buffer depletes red blood cells. Cells are seeded in culture dishes in RPMI medium (Invitrogen) containing 10% fetal calf serum (Pan Biotech). Cells are cultured for several hours at 37°C in 10% CO ₂ . After trypsin treatment, adherent cells are harvested and used for phagocytosis experiments.

Microglial cells are obtained from the brains of C57BL/6 mice 3-5 days after birth (P3P5). Briefly, the meninges are removed mechanically and the cells are dissociated by trituration and cultured in minimal medium (BME; GIBCO BRL) supplemented with 10% FCS (PAN Biotech GmbH), 1% glucose (Sigma-Aldrich), 1% L-glutamine (GIBCO BRL) and 1% penicillin/streptomycin (GIBCO BRL) for 14 days to form a confluent glial monolayer. To collect microgenic cells, cultures are shaken on a rotary shaker (200 rpm) for 2 hours. Attached astrocytes are used for immunohistochemistry. The detached microglial cells are seeded in conventional culture dishes for 1 hour, and then all non-adherent cells are removed and ignored. The purity of the isolated microglial cells is about 95% as determined by flow cytometry with anti-CD11b antibody (BD Biosciences). Microglial cells are cultured in minimal medium.

Oligodendrocytes (ie, neurons) and cells enriched in neurons are obtained from the brain of C57BL/6 mouse embryos (E15-16). Briefly, brain tissue is isolated and mechanically dispersed and plated in culture dishes pre-coated with 0.01 mg/ml poly-Lornithine (Sigma-Aldrich) and 10 μg/ml laminin (Sigma-Aldrich). Cells are cultured in neuronal conditioned medium (BME; GIBCO BRL) supplemented with 2% B-27 (GIBCO BRL), 1% glucose (Sigma-Aldrich) and 1% FCS (PAN Biotech GmbH). Cells are cultured for 5-10 days to obtain morphologically mature oligodendrocytes.

To perform phagocytosis analyzes of apoptotic neurons, nervous tissue debris, non-nervous tissue debris, bacteria, other foreign bodies, pathogenic proteins, microglia are transduced with TREM2 shRNA, control shRNA, WTREM2, GFP1 control vector, mtDAP12-GFP, and GFP2 control vector. After transduction, microglia are cultured for 72 h to achieve effective knockdown of TREM2 via RNA interference. Neurons are cultured for 510 days and then okadaic acid is added to a final concentration of 30 nM for 3 hours to induce apoptosis. Neuronal cell membranes are labeled with CellTracker CM-DiI membrane dye (Molecular Probes). After incubation, apoptotic neurons or other targets of phagocytosis are washed twice and added to the transduced microglial culture at an effector/target ratio of 1:20. At 1 and 24 hours after the addition of apoptotic neurons, the amount of microglia having phagocytosed neuronal membranes was counted under a confocal fluorescent microscope (Leica). Apoptotic cells are counted in three different areas with a magnification of 60. The amount of phagocytosis is confirmed by flow cytometry. In addition, 24, 48, or 72 hours after addition of apoptotic neurons, cells are harvested and used for cytokine RT-PCR.

For microbead granule assay or bacterial phagocytosis assay, microglia are transduced with TREM2 expression vector or GFP control vector. The cells are then treated with anti-TREM2 agonist antibodies. After 24 hours, 1.00 μm red fluorescent microsphere beads (Fluoresbrite Polychromatic Red Microspheres; Polysciences Inc.) or fluorescent labeled bacteria are added over 1 hour. Phagocytosis of microsphere beads or fluorescently labeled bacteria by microglia is analyzed by fluorescence microscopy. In addition, microglia are harvested from culture plates and analyzed by flow cytometry. The percentage of microglia containing phagocytosed granules is determined. Since phagocytosis differs from one experiment to another, the relative change in phagocytosis is also determined. The data is presented as

- 142 042890 relative change in phagocytosis between microglia cultured with agonist antibodies and control antibody.

For RT-PCR to analyze transcripts of inflammatory genes, microglia are transduced using the TREM2 vector or the GFP1 control vector. The cells are then cultured on plates and treated with anti-TREM2 agonist antibodies. At 24, 48, and 72 h, RNA was isolated from microglia using the RNeasy Mini Kit (QIAGEN). RNA was also harvested from microglia that had been transduced with shRNA TREM2, shRNA control, WTREM2, GFP1 control vector, mtDAP12-GFP, and GFP1 control vector, and co-cultured with apoptotic neurons for 48 h.

The RNA is then reverse transcribed. Quantitative RT-PCR with SYBR Green was performed on an ABI Prism 5700 Sequence Detection System (PerkinElmer). GAPDH amplification is used to normalize the sample. The amplification protocol is performed using the GeneAmp 5700 Sequence Detection System software (version 1.3). To detect GAPDH, TNF-alpha, IL-1, NOS2 and TGF-beta transcripts, the following forward and reverse primers were used at final concentrations of 200 nM:

GAPDH forward primer: 5'-CTCCACTCACGGCAAATTCAA-3' (SEQ ID NO: 416) and GAPDH reverse primer: 5'-GATGACAAGCTTCCCATTCTCG-3' (SEQ ID NO: 417);

TNF-α reverse primer: 5'-CCGTCAGCCGATTTGCTATCT-3' (SEQ ID NO: 418) and TNF-α reverse primer: 5'-ACGGCAGAGAGGAGGTTGACTT-3' (SEQ ID NO: 419) ;

IL-1a reverse primer: 5'-ACAA-CAAAAAAGCCTCGTGCTG-3' (SEQ ID NO: 420) and IL-1a reverse primer: 5'-CCATTGAGGTGGAGAGCTTTCA-3' (SEQ ID NO: 421);

NOS2 reverse primer: 5'-GGCAAACCCAAGGTCTACGTTC-3' (SEQ ID NO: 422), NOS2 reverse primer: 5'-TACCTCATTGGCCAGCTGCTT-3' (SEQ ID NO: 423); And

TGF-B1 reverse primer: 5'-AGGACCTGGGTTGGAAGTGG-3' (SEQ ID NO: 424) and TGF-B1 reverse primer: 5'-AGTTGGCATGGTAGCCCTTG-3' (SEQ ID NO: 425).

For the amyloid phagocytosis assay, 20 mM HiLyteFluor™ 647 (Anaspec)-Abeta-(1-40) was resuspended in Tris/EDTA (pH 8.2) and then incubated in the dark for 3 days at 37° C. to promote clustering. Microglial cells are pretreated in low serum (0.5% PBS supplemented with insulin), LPS (50 ng/mL), IFNc (100 units/mL), and anti-TREM2 agonist antibody for 24 h prior to the addition of aggregated fluorescently labeled beta- peptide. Amyloid phagocytosis and surface expression of TREM2 are determined by flow cytometric analysis 5 hours after the addition of 100 nM aggregated HiLyteFluor™ 647-Ab-(1-40) (ASN NEURO (2010) 2(3): 157-170). Phagocytosis of other pathogenic proteins is carried out in a similar way.

Example 7 Induction of ERK Activation by TREM2, DAP12 agonist antibodies and/or TEM2/DAP12 bispecific antibodies.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies can induce ERK activation.

Microglia are transduced with TREM2 vector and 2x105 cells are exposed to TREM2 agonist antibodies, DAP12 agonist antibodies, and/or TREM2/DAP12 bispecific antibodies for 1 hour. After stimulation, cells are lysed in Western blot dilution buffer. ERK phosphorylation and total ERK are determined by immunodetection of phospho-ERK antibodies and anti-ERK antibodies, respectively (both from Cell Signaling Technology) by Western blotting (JEM (2005), 201, 647-657).

Example 8 CCR7 induction and migration towards CCL19 and CCL21 in microglia, macrophages and dendritic cells by TREM2, DAP12 agonist antibodies and/or TEM2/DAP12 bispecific antibodies.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies can induce CCR7 and migration towards CCL19 and CCL21 in microglial cells, macrophages and dendritic cells.

Microglial cells are transduced with TREM2 vector or GFP1 control vector. The transduced microglial cells are then either cultured with anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies, or with a control antibody. Cells are harvested 72 hours later, labeled with specific anti-CCR7 antibodies and analyzed by flow cytometry.

Chemotaxis analysis is performed to determine any functional consequences of increased CCR7 expression. Microglial cells are stimulated with TREM2 using anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies and placed in a dual chamber system. Microglial cells migrating to the chemokine ligands CCL19 and CCL21 are quantified (JEM (2005), 201, 647-657).

For chemotaxis analysis, microglial cells are exposed to agonistic antibodies

- 143 042890 mi to TREM2, agonistic antibodies to DAP12 and/or bispecific antibodies to TREM2/DAP12 and treated with 1 μg/ml LPS. The microglia are transferred to the upper chamber of the Transwell system (3 μm pore filter; Millipore) containing 450 μl of medium with 100 ng/ml CCL19 or CCL21 (both from PeproTech) in the lower chamber. After a 1 hour incubation period, the number of microglial cells that have migrated to the lower chamber is counted in three independent areas using microscopy (JEM (2005), 201, 647-657).

Example 9 Induction of F-actin in microglia, macrophages and dendritic cells by TREM2, DAP12 agonist antibodies and/or TEM2/DAP12 bispecific antibodies.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies can induce F-actin in microglial cells, macrophages and dendritic cells.

Microglia and other cells of interest that are transduced with TREM2 or express TREM2 are added to culture plates and then exposed to TREM2 agonist antibodies, DAP12 agonist antibodies, and/or TREM2/DAP12 bispecific antibodies for 1 h. Cells are fixed , blocked and then stained with Alexa Fluor 546-conjugated phalloidin (Molecular Probes) after 1 h, and Factin was labeled with a fluorescent dye. Images were collected using confocal laser scanning microscopy with a 40x objective (Leica). (JEM (2005), 201, 647-657).

Example 10 Induction of osteoclast production and increased rate of osteoclastogenesis by TREM2, DAP12 agonist antibodies and/or TEM2/DAP12 bispecific antibodies.

It is believed that anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies can induce osteoclast production and increase the rate of osteoclastogenesis.

RAW264.7 cells that form osteoclasts or bone marrow monocyte/macrophage (BMM) precursor cells are maintained in RPMI-1640 medium (Mediatech) or other suitable medium supplemented with 10% PBS (Atlantic Biologics, Atlanta, GA, USA) and penicillin-streptomycinglutamine (Mediatech). The TREM2B cDNA with the FLAG epitope added to the N-terminus is inserted into the pMXpie retroviral vector until IRES followed by the eGFP cDNA sequence. Cells were transfected with pMXpie-FLAG TREM2B using Fugene 6 (Roche) according to the manufacturer's protocol. Cells are harvested in puromycin (Sigma) at a concentration of 2 μg/ml. Stable puromycin resistant clones are screened for binding of anti-FLAG M2 monoclonal antibodies (Sigma) using flow cytometry and then subcloned and maintained on puromycin selection media.

RAW264.7 cells expressing TREM2B were seeded in 96-well plates at 3000 cells/well in alpha-MEM supplemented with 10% PBS, penicillin-streptomycin-glutamine, 50 ng/ml RANKL, and 20 ng/ml M-CSF. Medium is changed every 3 days, exposed to anti-TREM2 agonist antibodies, and multinucleated (at least trinuclear) TRACP+ osteoclasts are counted by light microscopy. To determine complexity and size, osteoclasts are counted by the number of nuclei (>10 or 3-10 nuclei). Surface area of osteoclasts is also measured using Image J (NIH) software. In addition, the levels of osteoclast gene expression are determined. Total RNA is extracted from osteoclastogenic cultures at various time points using the TRIzol reagent (Invitrogen). After synthesizing the first strand of cDNA using the Superscript III kit (Invitrogen), quantitative real-time PCR reactions were carried out for Nfatc1, Acp5, Ctsk, Calcr and Ccnd1. Relative quantification of target mRNA expression is calculated and normalized to cyclophilin expression and expressed as (target gene mRNA/cyclophilin mRNA) 3x106. (J. OF BONE AND MINERAL RESEARCH (2006), 21, 237-245; J Immunol 2012; 188:2612-2621).

Example 11 In vivo protection against EAE and cuprizone in an intact animal.

Adult female C57BL/6 mice aged 7-9 weeks (obtained from Charles River Laboratories) are injected into the coccyx on both sides with 200 μl of innoculate containing 100 μg of myelin oligodendrocyte glycoprotein peptide 35-55 (MEVGWYRSPFSRVVHLYRNGK amino acids (SEQ ID NO: 415); Seqlab), and 1 mg Mycobacterium tuberculosis H37 Ra (Difco) in incomplete Freund's adjuvant (Difco). Pertussis toxin (200 ng, List Biological Laboratories) is administered on day 0 and the second day after immunization. Clinical signs are scored as follows: 0, no clinical signs; 1, completely damaged tail; 2, completely damaged tail and abnormal gait; 3, paraparesis of one hind limb; 4, complete paraparesis of the hind limbs; and 5, paralysis or death of the forelimbs and hind limbs.

For experiments, only mice at the initial stage of the disease (clinical index 1 or more) on the 14th day are used. Anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or anti-TREM2/DAP12 bispecific antibodies are administered intraperitoneally or intravenously to mice suffering from EAE on the day of first clinical symptoms or at any other desired time (PLoS Med (2007) 4(4): e124 ).

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Young or mature wild type (Harlan) mice are fed a standard diet (Harlan) containing 0.2% cuprizone (CPZ) bis oxalate (cyclohexylidene hydrazide) powder (Sigma-Aldrich) for 4, 6, or 12 weeks. For histological and immunohistochemical analysis, the brain is removed after perfusion of the mouse with 4% paraformaldehyde (PFA), fixation in 4% PFA for 24 h, followed by immersion in 30% sucrose for 24-48 h. To assess the integrity and damage of myelin, as well as cell proliferation and inflammation sectors, mouse brains are stained with anti-MBP antibody (1:100; Abcam, ab7349), anti-dMBP antibody (1:2000; Millipore, ab5864), anti-APP antibody (1:100, Invitrogen, 51-2700 ), anti-SMI-31 (1:1000, Covance, smi-31R), anti-Iba1 (1:600, Wako, 019-19741), anti-BrdU (1:250, Abcam, Ab1893), anti-GFAP (1:200, Invitrogen, 13-0300), iNOS antibodies (1:100, BD Pharmingen, 610329), LPL antibodies (1:400, from Dr. G. Olivecrona) and MHC II antibodies (1:100 ; BD Pharmingen, 553549). For the behavioral effects of antibodies, mice were analyzed for locomotor activity using a transparent polystyrene fence and a computerized photosensor. General activity variables (mobility, vertical rearing) were analyzed, as well as measures of emotionality, including time spent, distance traveled, and reported data. A range of sensorimotor tests are performed to assess balance (plank and platform), strength (lattices), coordination (pole and tilt screens), and movement initiation (walk initiation). Movement coordination and balance are studied using the protocol of the method for assessing the ability to hold onto a rotating drum (rotarod) (Cantoni et al., Acta Neuropathol. (2015) 129 (3): 429-47).

Example 12 Characterization of the therapeutic use of anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies in valid animal models of traumatic brain injury.

The therapeutic use of anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or TEM2/DAP12 bispecific antibodies is being tested in valid animal models of traumatic brain injury (Tanaka, Y. et al. (2013) Neuroscience 231 49-60).

For example, a traumatic brain injury model is used that causes the activation of microglia and astrocytes. Male C57BL/6J DT mice (8 or 9 weeks old) or progranulin heterozygous mice (purchased from Charles River Laboratories or Jackson Laboratories) are used. Mice are anesthetized by intraperitoneal administration of xylazine hydrochloride (8 mg/kg) and chloral hydrate (300 mg/kg) dissolved in sterile saline and then placed in a stereotaxic machine (Narishige, Tokyo, Japan). An incision is made along the scalp and the skull is exposed. The periosteal hymen is removed from the skull, a hole is drilled over the right hemisphere of the brain with a drill, and the dura is removed with the tip of a needle. A stainless steel catheter with an outer diameter of 0.5 mm is used to inflict a longitudinal puncture wound in the right hemisphere. The cannula is placed 1.3 mm lateral to the midline and 1 mm posterior to the bregma, and inserted into the brain until the tip reaches a depth of 2 mm. The cannula is then moved 2 mm caudally (bregma 3 mm) and then moved back 2 mm to its original position. Finally, the cannula is removed from the brain and the scalp wound is sutured. Mice are then treated with anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies according to standard procedures and then analyzed histologically and by immunofluorescent staining and behavioral tests.

Example 13 Characterization of the therapeutic use of anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies in a model of neuroinflammation and neuronal loss following toxin-induced injury.

The therapeutic characterization of anti-TREM2, DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies was investigated in a model of neuroinflammation and neuronal loss following toxin-induced injury (Tanaka, Y. et al. (Martens, L.H. et al., (2012) The Journal of Clinical Investigation, 122, 3955).

Three-month-old mice are given 4 intraperitoneal injections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) per day for 2 days (4 μg/g body weight) (Sigma-Aldrich) or PBS. Mice are treated with anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or anti-TREM2/DAP12 bispecific antibodies according to standard protocol and then analyzed by stereological scoring to quantify dopamine neurons and microglia in substantia nigra compactus (SNpc) as described .

Example 14 Characterization of the therapeutic use of anti-TREM2 agonist antibodies, DAP12 and/or bispecific antibodies to TEM2/DAP12 in animal models of aging, paroxysm, spinal cord injury, retinal dystrophy, frontotemporal dementia and Alzheimer's disease.

Therapeutic use of anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or bispecific anti-TREM2/DAP12 antibodies is being tested in animal models of aging, paroxysm, spinal cord injury, retinal dystrophy, frontotemporal dementia, and Alzheimer's disease, as previously described (e.g., Beattie , M. S. et al., (2002) Neuron 36, 375-386 Volosin, M. et al., (2006) J. Neurosci 26, 7756-7766 Nykjaer, A. et al., (2005) Curr. Opin Neurobiol 15, 49-57 Jansen, P. et al.

- 145 042890 (2007) Nat. neurosci. 10, 1449-1457; Volosin, M. et al., (2008) J. Neurosci. 28, 9870-9879; Fahnestock, M. et al., (2001) Mol. Cell Neurosci. 18, 210-220; Nakamura, K. et al., (2007) Cell Death. Different. 14, 1552-1554; Yune, T. et al., (2007) Brain Res. 1183, 32-42; Wei, Y. et al., (2007) Neurosci. Lett. 429, 169-174; Provenzano, M.J. et al., (2Οθ8) Laryngoscope 118, 87-93; Nykjaer, A. et al., (2θθ4) Nature 427, 843-848; Harrington, A.W. et al., (2004) Proc. Natl. Acad. sci. U.S.A. 101, 6226-6230; Teng, H.K. et al., (2005) J. Neurosci. 25, 5455-5463; Jansen, P. et al., (2007) Nat. neurosci. 10, 1449-1457; Volosin, M. et al., (2008) J. Neurosci. 28, 9870-9879; Fan, Y.J. et al., (2008) Eur. J. Neurosci. 27, 2380-2390; Al-Shawi, R. et al., (2008) Eur. J. Neurosci. 27, 21032114; and Yano, H. et al., (2009) J. Neurosci. 29, 14790-14802).

Example 15 Characterization of the therapeutic use of anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies in models of atherosclerosis.

Therapeutic use of TREM2 agonist antibodies, DAP12 agonist antibodies, and/or TEM2/DAP12 bispecific antibodies is being tested in models of atherosclerosis as previously described (e.g., Lance, A. et al., (2011) Diabetes, 60, 2285; and Kjolby , M. et al., (2012) Cell Metabolism 12, 213-223).

Example 16 Characterization of the therapeutic use of anti-TREM2, DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies in infection models.

The therapeutic use of anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TEM2/DAP12 bispecific antibodies is being tested in infection models. For example, Listeria monocytogenes or other infection in normal mice or progranulin heterozygous mice can be used as previously described (eg, Yin, F. et al., (2009) J. Exp. Med, 207, 117-128).

Example 17 Characterization of the therapeutic use of anti-TREM2, DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies in inflammatory disease models.

The therapeutic use of anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies and/or anti-TREM2/DAP12 bispecific antibodies is being tested in inflammatory disease models. For example, rheumatoid arthritis or a valid model for another inflammatory disease (Mizoguchi (2012) Prog Mol. Biol. Transl Sci., 105:263-320; and Asquith et al., (2009) Eur. J. Immunol. 39:2040-4 ).

Example 18 Screening for KTREM2 antibodies, anti-DAP12 antibodies and/or TREM2/DAP12 bispecific antibodies that induce DAP12, ERK and AKT phosphorylation, which are indicative of activation of the FICK pathway.

Cells (J774, RAW 264.7, HMM cells or osteoclasts) are removed from PBS-EDTA tissue culture dishes, washed with PBS and counted. J774 (40x106) or RAW 264.7 cells (10x106 HMM or osteoclasts) are incubated with anti-TREM2 antibody, anti-DAP12 antibody and/or anti-TREM2/DAP12 bispecific antibody or isotype-matched control antibody at 1 μg/10 ⁶ cells for 20 minutes per ice or other conditions. Cells are lysed in ice-cold buffer for radioimmunoprecipitation assay (RIPA) [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton-100, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM 0, 25% sodium deoxycholate, aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml)] for 20 min followed by centrifugation at 16,000 g for 10 min at 4°C to remove insoluble materials. The resulting supernatant is subjected to immunoprecipitation reactions with the indicated antibodies (DAP12, ERK or AKT) and A- or G-agarose protein (Sigma). the microspheres are washed extensively with RIPA buffer and the proteins are separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The proteins are then transferred to nitrocellulose membranes by Western blotting, incubated with appropriate antibodies (antibodies that specifically recognize the phosphorylated form of DAP12, ERK, or AKT), and visualized using the Advanced Chemiluminescence (ECL) System (Pierce) as described (e.g., Peng et al. al., (2010) Sci Signal., 3(122): ra38).

Example 19 Screening for kTREM2 antibodies, anti-DAP12 antibodies and/or TREM2/DAP12 bispecific antibodies that induce calcium flux.

HMM cells are washed twice with HEPES-containing buffer [20 mM HEPES (pH 7.3), 120 mM NaCl, 1 mM CaCl, 1 mM MgCl, 5 mM KCl, glucose (1 mg/ml), bovine serum albumin (1 mg/ml) ] followed by incubation in 0.05% Pluronic F-127 (Invitrogen) and 1 μM Indo-1 AM (Invitrogen) for 20 min at 37°C. Cells were washed twice with HEPES buffer and then stimulated with anti-TREM2 antibody, anti-DAP12 antibody and/or TREM2/DAP12 bispecific antibody (16 µg/ml) or control antibody (16 µg/ml) and measured with a spectrophotometer (PTL Photon Technology International ). Indo-1 fluorescent light is converted to calcium (Ca ²⁺ ) according to the manufacturer's instructions (eg Peng et al., (2010) Sci Signal., 3(122): ra38).

Example 20 Screening for kTREM2 antibodies, anti-DAP12 antibodies and/or TREM2/DAP12 bispecific antibodies that prevent apoptosis.

Mature osteoclast cell cultures are differentiated in 24-well dishes with RANKL and M-CSF. After 4 days, complete medium is replaced with serum-free medium to induce apoptosis. Cells treated with RANKL, PBS and anti-TREM2 antibody, anti-DAP12 antibody, and/or TREM2/DAP12 bispecific antibody or isotype-matched control antibody during an overnight serum fast. Cells were fixed in 1% paraformaldehyde and stained with TUNEL Kit (Milli

- 146 042890 pore Corporation) in accordance with the manufacturer's instructions. Apoptotic nuclei are counted using a Nikon TE2000-E microscope at 20x magnification. The results are expressed as the percentage of apoptotic cells relative to the total number of cells in six randomly selected fields of two wells, as described (for example, Peng et al., (2010) Sci. Signal., 3(122): ra38). Similar analyzes are carried out with primary microglial cells.

Example 21 Screening for kTREM2 antibodies, anti-DAP12 antibodies and/or TREM2/DAP12 bispecific antibodies that induce osteoclast differentiation.

HMM cells are plated in triplicate wells and treated with RANKL, M-CSF and anti-TREM2 antibody, anti-DAP12 antibody and/or TREM2/DAP12 bispecific antibody or isotype-matched control antibody. The medium is changed every 3 days until large multinucleated cells appear. After 3-5 days of cultivation, the cells are fixed with 3.7% formaldehyde in PBS for 10 min. Then the plates are washed twice in PBS, incubated for 30 s in a solution of 50% acetone and 50% ethanol and washed with PBS. Cells are stained with tartrate-resistant acid phosphatase (TRAP) using a kit from Sigma (product 435). Multinucleated (more than two nuclei) TRAP-positive cells are then counted by light microscopy as described (eg, Peng et al., (2010) Sci Signal., 3(122): ra38).

Example 22 Screening for kTREM2 antibodies, anti-DAP12 antibodies and/or TREM2/DAP12 bispecific antibodies that normalize TREM2/TYROBP-dependent gene expression changes in the immune/microglia regulatory module.

Microglial cells derived from mouse embryonic stem cells are genetically modified with lentiviral vectors to overexpress either a full-length or truncated version of Tyrobp that lacks both intracellular immunoreceptor tyrosine activating motifs (ITAMs). Microglial cells are also derived from mouse embryonic stem cells that are heterozygous for TREM2. To assess changes in genome-wide gene expression in response to Tyrobp or TREM2 retrubation, gene expression data are obtained by RNA sequencing of mouse microglial cell lines overexpressing: (1) vehicle, (2) full-length Tyrobp, or (3) dominant-negative truncated Tyrobp; or (4) overexpressing a TREM2 knockout construct such as siRNA and cells that are heterozygous for TREM2. Approximately 2638 and 3415 differentially expressed genes have been identified for overexpression of full-length Tyrobp and truncated Tyrob, respectively (Zhang et al., (2013) Cell 153, 707-720). Approximately 99% of differentially expressed microglial genes overexpressing intact Tyrobp are downregulated compared to vehicle control. For example, 658 genes associated with vacuolation/autophagy, as well as genes involved in RNA metabolism and cell cycle mitosis, are downregulated by active Tyrobp but upregulated in cells expressing dominant negative truncated Tyrobp. Conversely, about 2865 genes for vacuoles/autophagy and for mitochondria are selectively upregulated in microglia expressing dominant-negatively truncated Tyrobp.

Anti-TREM2 agonist antibodies, anti-DAP12 agonist antibodies, and/or anti-TREM2/DAP12 bispecific antibodies are screened for their ability to elicit gene expression profiles similar to those observed in normal microglial cells and in microglial cells overexpressing intact Tyrobp in cells that dominantly express -negative truncated Tyrobp (Zhang et al., (2013) Cell 153, 707-720), in cells that express a knockout construct for TREM2 or in cells that are heterozygous for TREM2. Antibodies are selected that are capable of altering the gene expression network.

Example 23 Antibodies to TREM2 induce expression of CD83 and CD86 on human dendritic cells (DC).

To evaluate the ability of anti-TREM2 antibodies to modify CD83 and CD86 expression, both plate-bound and soluble antibodies were incubated with dendritic cells (DCs) and CD83 and CD86 expression was measured.

Antibodies were seeded overnight at 4°C in 12-well plates at a concentration of 2 or 5 μg/ml in PBS. The next day, the wells were washed 3 times with PBS. On day 5, human immature DCs were harvested and seeded at 1 million cells/well and incubated at 37° C., 5% CO ₂ in the absence of cytokine. FACS analysis of CD86, CD83, CD11c, HLA-DR and LIN (BD Biosciences) was performed on BD FACS Canto after 48 hours. Data analysis was performed using FlowJo software (TreeStar) version 10.0.7. CD83 and CD86 levels were assessed on populations of CD11c+HLA-DR+LIN cells.

Alternatively, 5-day-old immature human dendritic cells were seeded at 100,000 cells/well in a 96-well U-bottom plate, untreated with TC, in media without cytokine. Antibodies were added at a concentration of 5 μg/ml with or without a removed secondary human anti-LPS antibody (Jackson ImmunoResearch) at a concentration of 20 μg/ml. FACS analysis of CD86, CD83, CD11c, HLA-DR and LIN (BD Biosciences) was performed 48 hours after antibody addition as described above.

Plate-bound antibodies At21 and At52 to TREM2 increased the frequency of CD83+CD86+DCs by

- 147 042890 compared with the Ab88 isotype control antibody (FIG. 5A). Soluble antibodies At21 and At52, either alone or cross-linked with an anti-human secondary antibody, induced equivalent expression of CD86 on DC compared to an isotype control antibody (At88) (FIG. 5B). Based on these results, the anti-TREM2 antibodies At21 and At52 function as agonists to induce expression of the inflammatory surface markers CD83 and CD86 on human dendritic cells.

Example 24 Anti-TREM2 antibodies At21 and At52 induce Syk phosphorylation.

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule that functions downstream of TREM2 in the metabolic pathway by phosphorylation of several substrates, thus promoting the formation of a signaling complex that leads to cellular activation and inflammatory processes. The ability of anti-TREM2 agonist antibodies to induce Syk activation was determined by culturing human and mouse macrophages and human primary dendritic cells and measuring the phosphorylation state of the Syk protein in cell extracts.

Macrophages from bone marrow (BMDM), DT mouse BMDM, TREM2 knockout (KO) mouse BMDM, and primary human dendritic cells were placed on depleted medium containing 1% RPMI serum for 4 h and then removed from tissue culture dishes using PBS-EDTA, washed with PBS and counted. Cells were coated with full length anti-TREM2 Ab21 and At52 agonist antibodies or control antibodies (Ab89 or At92) for 15 min on ice. After washing with cold PBS, the cells were incubated at 37° C. for the indicated time period in the presence of goat anti-human IgG. After stimulation, cells were lysed with lysis buffer (1% v/v NP-40%, 50 ml Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl ₂ , 10% glycerol, plus protease and phosphatase inhibitors) followed by centrifugation at 16,000 g for 10 min at 4°C to remove insoluble materials. The lysates were then immunoprecipitated with an anti-Syk antibody (N-19 for BMDM or 4D10 for human DC, Santa Cruz Biotechnology). Precipitated proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-phosphotyrosine antibody (4G10, Millipore). To confirm that all substrates were adequately immunoprecipitated, immunoblots were reprobed with an anti-Syk antibody (Abcam, for BMDM) or an anti-Syk antibody (Novus Biological, for human DC). Imaging was performed using the Advanced Chemiluminescence (ECL) System (GE Healthcare) as described (eg, Peng et al., (2010) Sci Signal., 3(122): ra38).

Anti-TREM2 antibodies At21 and At51 induced Syk phosphorylation in DT mouse TREM2 BMDM cells but did not induce phosphorylation in TREM2 KO (TREM2'/') cells (Fig. 6A). Antibodies At21 and At51 also induced Syk phosphorylation in both human BMDM (FIG. 6A, right panel) and primary human dendritic cells (FIG. 6B). Control antibodies At89 and At92 did not induce Syk phosphorylation. Based on these results, the anti-TREM2 antibodies At21 and At52 function as agonists to induce Syk phosphorylation in macrophages and dendritic cells.

Example 25 Anti-TREM2 antibodies At21 and At52 induce DAP12 phosphorylation in mouse macrophages.

TREM2 signals through DAP12, resulting in downstream activation of PHYS and other intracellular signals. The ability of anti-TREM2 agonist antibodies to induce DAP12 activation was determined by culturing mouse macrophages and measuring the phosphorylation status of the DAP12 protein in cell extracts.

Before antibody stimulation, mouse macrophages (MT) from bone marrow (BMDM) and BMDM mice with knockout (KO) TREM2 were placed on depleted medium for 4 h containing 1% serum RPMI. 15x106 cells were incubated on ice for 15 min with full length agonist or control antibodies. Cells were washed and incubated at 37° C. for the indicated time period in the presence of goat anti-human IgG. After stimulation, cells were lysed with lysis buffer (1% v/v NP-40%, 50 ml Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCb, 10% glycerol, plus protease inhibitors and phosphatase) followed by centrifugation at 16,000 g for 10 min at 4°C to remove insoluble materials. The cell lysate was immunoprecipitated with a second TREM2 antibody (R&D Systems). Precipitated proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-phosphotyrosine antibody (4G10, Millipore). The membrane was detached and reprobed with an anti-DAP12 antibody (Cells Signaling, D7G1X). Each cell lysate used for TREM2 immunoprecipitation contained an equal amount of proteins, as shown by a control antibody (anti-actin antibody, Santa Cruz).

DAP12 is co-precipitated with TREM2 and phosphorylated in DT macrophages incubated with anti-TREM2 (FIG. 7A) and At21 (FIG. 7B) agonist antibodies At52. Conversely, DAP12 phosphorylation was not observed in TREM2 KO (TREM2'/') macrophages incubated with Ab21 (Fig. 7B). These results demonstrate that DAP12 is associated with the TREM2 protein and that TREM2 agonist antibodies are capable of inducing DAP12 phosphorylation in vitro.

- 148 042890

Example 26 The anti-TREM2 antibodies At21 and At52 compete with the TREM2 ligand for binding to human and mouse TREM2.

The ability of TREM2 agonist antibodies to recognize the TREM2 ligand binding site was assessed by competitive binding assay using E. coli cells expressing the putative TREM2 ligand.

E. coli were grown in 10 ml of LB O/N medium, collected by centrifugation and washed twice in 10 ml of PBS. The E. coli were then thermally inactivated by incubation in a water bath at 70° C. for 30 minutes. E. coli were labeled with CellTracker DeepRed (ThermoFisher/Invitrogen, final concentration 1 μM) and then washed three times in 10 ml PBS, and resuspended in 1 ml PBS at a concentration of 10 ⁸ /ml. For competitive binding, bacteria were added to mouse TREM2 and to a cell line (BWZ) expressing DAP12 or to a BW cell line expressing human TREM2/DAP12 fusion protein together with 10 μg/mL TREM2 full-length agonist antibodies and incubated for one hour for ice. Cells were analyzed by FACS for binding of CellTracker-labeled bacteria to cell lines.

Anti-TREM2 antibodies At21 and At52 inhibited E. coli binding to both human and mouse cells, indicating competitive antibody binding to the ligand-binding site on TREM2 (FIG. 8). Control non-agonistic anti-TREM2 antibodies (At66 and At68) inhibited binding of bacteria to human cells expressing TREM2, but did not inhibit binding to mouse TREM2.

Example 27 Summary of TREM2 antibody function studies.

In table. 6 shows the results of the function tests described in Examples 24-26 above. Antibodies At21 and At52 demonstrated the induction of Syk phosphorylation in human dendritic cells (hDC), mouse dendritic cells (mDC) and mouse macrophages (mMac). However, the At52 antibody induced higher levels of phosphorylated Syk compared to the At21 antibody in human and mouse DC. Both antibodies were able to mimic ligand binding to human and mouse TREM2, although the Ab21 antibody showed more efficient binding, as evidenced by a greater reduction in bacterial binding (see Example 26 above).

Table 6. TREM2 antibody function studies

Antite LO Phospho Syk PhDA induction Phospho Syk mDC induction Induction Phospho DAP12 mMac Mimic ligand-binding site on Trem2 human Mimic ligand-binding site on the Trem2 mouse At 5 2 +++ +++ +++ ++ +++ At21 ++ ++ +++ +++ +++ Isotype control A

Example 28 TREM2 reduces the secretion of inflammatory cytokines from mouse macrophages.

To determine the role of TREM2 in the production of inflammatory cytokines, wild-type (WT) mouse macrophages, TREM2 knockout (KO) mouse macrophages, and TREM2 heterozygous (Het) macrophages with various inflammatory mediators were cultured, and cytokine levels were measured in culture supernatants.

To obtain BMDM, total bone marrow from WT, TREM2 KO (KO), and TREM2 heterozygous (Het) mice are cultured in RPMI enriched with 10% calf serum, 5% horse serum, and 50 ng/mL mouse recombinant CSF-1 (R&D Systems). Cells were cultured for 5-6 days and adherent cells were separated with 1M EDTA in PBS. BMDM was seeded on 96-well plates at 10 ⁵ cells/well and left to adhere for 4 hours at 37°C. Cells were then stimulated with TLR agonists LPS (Salmonella abortus equi) or Zymosan (Saccharomyces cerevisiae) at concentrations of 0.01 to 100 ng/mL (LPS) or 0.01 to 100 μg/mL (Zymosan). Alternatively, macrophages isolated from DT, KO and Het mice were cultured in the presence of 10 ng/ml cytokine IL-4 or 50 ng/ml IFN-γ. Cell culture supernatant was harvested 24 or 48 h after stimulation and levels of TNFa, IL-6, IL-10 and MHB-1 cytokines were measured using the Cytometric Bead Array Mouse Inflamation (BD) cytometry kit according to the

- 149 042890 manufacturer.

Wild-type (WT) macrophages stimulated with inflammatory mediators LPS or zymosan release less inflammatory cytokines TNF-alpha, IL-6, IL-10, and MHB-1 compared to TREM2 KO (TREM2 ^- / ^- ) macrophages (Fig. 9A) . Similarly, DT and Het (TREM2 ^+/- ) macrophages treated with the mediator IFNy produced less inflammatory cytokines IL-6 and TNF-alpha compared to TREM2 KO macrophages (Fig. 9B). DT, Het, and KO macrophages cultured in the presence of the cytokine IL-4 produce similar low levels of IL-6 and TNF-alpha (Fig. 9B). Based on these results, anti-TREM2 antibodies may reduce the secretion of inflammatory cytokines from macrophages.

Example 29 TREM2 reduces the expression of inflammatory markers on the cell surface of mouse macrophages.

To determine the role of TREM2 in the expression of inflammatory markers, wild-type (DT) mouse macrophages, TREM2 knockout (KO) mouse macrophages, and TREM2 heterozygous (Het) macrophages with various inflammatory mediators were cultured, and the expression of surface markers CD86 and CD206 was measured.

Macrophages isolated from DT, KO and Het mice were plated and left to adhere for 4 h at 37°C, and TLR agonists LPS (Salmonella abortus equi) and zymosan (Saccharomyces cerevisiae) were added at concentrations from 0.01 to 100 ng /ml (LPS) or 0.01-10 µg/ml (zymosan). Alternatively, macrophages isolated from DT, KO and Het mice were cultured in the presence of the cytokines IL4 (10 ng/ml) or IFN-y (0.5-50 ng/ml). FACS analysis of CD86 and CD206 was performed on a BD FACS Canto after 48 hours. Data analysis was performed using FlowJo software (TreeStar) version 10.0.7.

Wild-type (WT) macrophages treated with the inflammatory mediators IFN-y (Fig. 10A), LPS, or zymosan (Fig. 10B) expressed lower levels of the CD86 inflammatory receptor, but not the CD206 receptor, compared to TREM2 KO macrophages ( TREM2' ^/_ ). Similarly, Het (TREM2 ^+/- ) macrophages treated with IFN-y expressed lower levels of CD86 but not CD206 compared to TREM2 KO macrophages (FIG. 10A). Based on these results, anti-TREM2 agonist antibodies can reduce the expression of inflammatory receptors on macrophages.

Example 30 TREM2 increases the survival of macrophages and dendritic cells.

To assess the role of TREM2 in cell survival, wild-type (DT) macrophages and TREM2 knockout (KO) macrophages and dendritic cells were cultured in the presence of inflammatory mediators and cell survival was measured.

Mouse bone marrow progenitors from TREM2 DT, Het, and KO mice were obtained by washing tibial and femoral bone marrow cells with cold PBS. After one PBS wash, erythrocytes were lysed with ASA Lysing Buffer (Lonza), washed twice with PBS, and suspended at 0.5x106 cells/ml in complete RPMI medium (10% FCS, Pen/Strep, Gln, neAA) with the indicated amounts 50 ng/ml M-CSF for macrophage production or 10 ng/ml GM-CSF for dendritic cell production. For M2 type macrophages, 10 ng/ml IL-4 was added to cultured cells. For M2 type macrophages, 10 ng/ml IL-4 was added to cultured cells. In some experiments, LPS or zymosan was added to the cell culture on day 5 at concentrations ranging from 1 µg/mL to 0.01 ng/mL. Recombinant cytokines were purchased from Peprotech.

For viability analysis of bone marrow-derived macrophages, cells were obtained as described above and cultured in MKSF. Cells were plated at 105/200 µl in a 96-well plate (for viability analysis using a luciferase-based assay) or 0.5x106/1 ml in a 6-well plate (for Tripan Blue exclusion counting), no processed for tissue culture. On the third day, medium containing fresh MCSF was added. At the indicated time points, cells were carefully detached from the plates with 3 mM EDTA and counted using a Berker chamber. For live cell FACS analysis, macrophages were cultured in either 50 μg/ml MCSF for 6 days (+ MCSF) or 50 μg/ml MCSF for 4 days before MCSF was removed for an additional 36 h (- MCSF). Cells were stained with anti-CD11b antibody and DAPI. For luciferase viability analysis, cell viability was measured on day 5 of culture using graduated concentrations of growth factors GMCSF (dendritic cells), MCSF (M1 macrophages) or MCSF+IL-4 (M2 macrophages). Cells were directly incubated with ToxGlo reagent (Promega) and luciferase activity (luminescence) was read using an XY reader. For FACS analysis of viable macrophages cultured in the presence of inflammatory mediators IFN-y, LPS, or zymosan, cells were harvested on day 5 and stained with anti-CD11b and DAPI.

After 7 days of culture in MCSF, significantly more viable (Tripan Blue exclusion count) TREM2 DT and Het (TREM2 ^+/- ) macrophages than TREM2 KO macrophages (TREM2' ^/_ ) were observed (FIG. 11A). FACS analysis showed that DT macrophages treated with MCSF for either 6 (+ MCSF) or 4 days (- MCSF) showed increased survival compared to Het and KO macrophages, as evidenced by a higher percentage of survivors.

- 150 042890 (CD11b+DAPI-) cells (Fig. 11B). Based on luciferase assays, DT cells cultured in the presence of growth factors GMCSF (dendritic cells), MCSF (M1 macrophages), or MCSF+IL-4 (M2 macrophages) survived better than KO cells, as demonstrated by a higher luminescence reading in the range growth factor concentrations (FIG. 11C). Wild-type macrophages cultured with inflammatory mediators (IFN-γ, LPS, or zymosan) had a higher survival rate than TREM2 Het and KO macrophages, as evidenced by a higher percentage of CD11b+ living cells (Fig. 11D). Based on these results, anti-TREM2 agonistic antibodies can increase the survival of macrophages and dendritic cells, while anti-TREM2 antagonistic antibodies can reduce cell survival.

Example 31 Phagocytosis modulated by TREM2.

TREM2 signaling is involved in phagocytic pathways including bacterial clearance from the lungs and phagocytosis of apoptotic neurons. The role of TREM2 in phagocytosis was assessed by measuring the ability of wild-type (WT) mouse macrophages and TREM2 knockout (KO) macrophages to phagocytose E. coli and apoptotic cells.

BMDM DT and TREM2 KO were placed on depleted medium containing 1% RPMI serum or kept in culture in the presence of MCSF (50 ng/ml) overnight. The next day, cells were plated at 2x105 in 96-well plates (round bottom non-tissue culture) in the presence or absence of MCSF. Target cells (CCL119) were cultured overnight with 0.5 μM staurosporine to induce apoptosis. The next day, cells were washed and labeled with 20 ng/ml pHrodo-SE (Invitrogen). E. coli apoptotic cells or bioparticles (pHrodo, Invitrogen) were incubated at 37°C for 1 h and 30 min. The analysis was stopped on ice. After washing with cold PBS, cells were stained for CD11b (pacific blue-CD11b, BD) and analyzed by FACS. In all samples, BMDMs were distinguished from apoptotic cells and granules by CD11b staining, and gating was performed based on effector cells cultured without target cells. As a negative control, effector cells were incubated throughout the assay with cytochalasin D (2 μM, SIGMA). Phagocytosis was quantified as (percent or UPC from PhRodo positive cells) - (percentage or UPC from PhRodo positive negative control cells treated with cytochalasin D).

Wild-type (WT) macrophages cultured with MCSF exhibit reduced phagocytosis of apoptotic and E. coli cells compared to TREM2 KO (TREM2'/') macrophages, as evidenced by a lower percentage of pHrodo+ cells (Fig. 12). Conversely, DT macrophages cultured without MCSF exhibit increased phagocytosis of apoptotic and E. coli cells compared to TREM2 KO macrophages, as evidenced by a higher percentage of pHrodo+ cells (Fig. 12). Based on these results, anti-TREM2 agonist antibodies can enhance phagocytosis in the absence of MCSF and decrease phagocytosis in the presence of MCSF. Conversely, it is believed that antagonistic antibodies to TREM2 can enhance phagocytosis in the presence of MCSF and reduce phagocytosis in the absence of MCSF.

Example 32 Anti-TREM2 Epitope Mapping.

TREM2 antibodies were tested for their ability to bind 15 or 25 mer peptides spanning all human and mouse TREM2.

Linear 15-mer peptides were synthesized based on the human or mouse TREM2 sequence with a 14-residue overlap. In addition, linear 25-mer peptides were synthesized based on the human or mouse TREM2 sequence with a 1-residue shift. Binding of TREM2 antibodies to each of the synthesized peptides was examined by ELISA. In this assay, peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 diluted antibody-peroxidase conjugate (SBA, cat. no. 2010-05) for one hour at 25°C. After washing, peroxidase substrate 2,2'azinobis-3-ethylbenzthiazolinesulfonate (ABTS) and 2 μl/ml 3% H ₂ O ₂ were added. Color formation was measured after one hour. Color formation was determined using a charge-coupled device (CCD) camera and an image processing system.

Antibody At52 shows reliable binding in all sets of peptides. The Ab52 antibody was shown to recognize an N-terminal peptide region between human and mouse TREM2 amino acid residues 49-57 (49AWCRQLGEK ⁵⁷ (SEQ ID NO: 444)) (FIG. 13). The epitope region recognized by At52 corresponds to amino acid residues 49-57 of SEQ ID NO: 1 and has the amino acid sequence: AWCRQLGEK (SEQ ID NO: 444).

The Ab21 antibody has been shown to recognize an N-terminal peptide region between amino acid residues 43-50 of human and mouse TREM2 (43HWGRRAW ⁵⁰ (SEQ ID NO: 445)). The epitope region recognized by At21 corresponds to amino acid residues 43-50 of SEQ ID NO: 1 and has the amino acid sequence: HWGRAW (SEQ ID NO: 445).

Example 33: Comparison of anti-TREM2 agonist antibodies At52 and At21 with reference anti-TREM2 antibodies.

TREM2 reference antibodies were compared with the agonist antibodies At21 and At52, assessing their

- 151 042890 the ability to induce Syk phosphorylation and determining their TREM2 binding site.

Cells were plated with 1 μg/10 ⁶ cells with anti-TREM2 antibody MAB17291 (R&D Systems) or rat IgG1 monoclonal antibody 78,18 (obtained from the University of California, San Francisco) and stimulated by cross-linking with a secondary antibody (goat anti-IgG 1.5 microns/10 ⁶ cells). Phosphorylation of Syk was assessed in accordance with the methods described in example 24 above.

To evaluate binding regions, human TREM2-Fc antibodies were incubated with immobilized full length anti-TREM2 agonist antibodies At21 or At52 and then anti-TREM2 antibodies MAB17291 or 78.18 were added. Epitope-specific antibody sorting was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA) using a standard binding sandwich assay (see, Estep et al., (2013) MAbs 5(2):270-8) . Target control IgG was loaded onto AHQ sensors and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human anti-IgG1 antibody. The sensors were then exposed to 100 nM target antigen followed by a second target antibody. The data were processed using ForteBio's Data Analysis Software 7.0. Additional binding by the second antibody after association with the antigen indicates an unoccupied epitope (non-competing), while no binding indicates blocking of the epitope (competing).

The reference anti-TREM2 antibody MAB17291 induces Syk phosphorylation in DT cells but not in TREM2 KO cells (TREM2'/'). However, the reference anti-TREM2 antibody 78.18 did not induce Syk phosphorylation under experimental conditions (FIG. 14). This is different from At21 and At52, which are both capable of inducing Syk phosphorylation in TREM2 BMDM of the DT mouse (Fig. 6).

The reference antibody MAB17291 was able to simultaneously bind TREM2 together with either the At21 antibody (FIG. 15A) or the At52 antibody (FIG. 15B). These results demonstrate that the anti-TREM2 agonist antibodies At21 and At52 bind to different regions of the TREM2 protein than the reference antibody MAB17291.

Example 34 Assay for the ability of anti-TREM2 antibody Fab fragments to stimulate viability of innate immune cells.

The agonistic functional properties of plate-bound, cross-linked Fab fragments of anti-TREM2 antibodies derived from antibodies At21 and At52 were evaluated on innate immune cells (eg, macrophages).

Macrophages derived from the bone marrow of wild-type (DT) and TREM2 knockout (KO) mice were cultured in the presence of M-CSF and anti-TREM2 antibody plate-bound Fab fragments, and cell viability was measured.

Macrophages isolated from the bone marrow of DT and KO mice were plated in untreated 96-well plates pre-coated with either 12.5 nM or 100 nM cross-linked At21 or At52 Fab fragments. Cells were cultured for 48 h in the presence of 10 ng/ml M-CSF. Viability analysis was performed using the Cell Titer Glo kit (Promega). Plates were read with a BioTek Synergy microplate reader using GEN5 2.04 software.

Crosslinked anti-TREM2 Fab fragments derived from At21 and At52 antibodies increased the number of viable mouse bone marrow derived macrophages compared to the isotype control Ab88 Fab as evidenced by a higher % increased survival (FIG. 16). This increase in cell viability was not observed in KO mouse macrophages. These data indicate that the biological activity of the antibodies At21 and At52 is specific for TREM2, and that plate-bound, cross-linked Fab fragments of At21 and At52 function as agonists to increase the survival of macrophages cultured in M-CSF.

Example 35 Analysis of the ability of anti-TREM2 antibodies to reduce the survival of innate immune cells.

The antagonistic functional properties of Fab fragments of both soluble, non-cross-linked anti-TREM2 antibodies derived from Ab21 and At52 antibodies and soluble full-length anti-TREM2 antibodies, At21 and At52, were evaluated on innate immune cells (eg, macrophages).

Bone marrow-derived macrophages from wild-type (WT) mice and TREM2 knockout (KO) macrophages were cultured in the presence of M-CSF and TREM2 soluble antibody Fab fragments or soluble full-length antibodies, and cell viability was measured.

Macrophages isolated from the bone marrow of DT and KO mice were seeded in non-tissue-cultured 96-well plates in the presence of 20 ng/ml M-CSF and increased amounts of the indicated Fab fragments of soluble, non-cross-linked TREM2 antibodies or soluble full-length antibodies. Each variant was seeded in triplicate. Viability analysis was performed using the Cell Titer Glo kit (Promega) after 3 days. Plates were read with a BioTek Synergy microplate reader using GEN5 2.04 software.

In FIG. 17 dotted line NT indicates the average cell viability obtained from

- 152 042890 raw macrophages (no antibody added). The dotted line without MCSF indicates the average cell viability obtained by culturing macrophages in the absence of M-CSF.

When macrophage cell viability was assessed with Fab fragments of soluble, non-cross-linked TREM2 antibodies, the results indicated that Fab fragments of soluble, non-cross-linked TREM2 antibodies derived from the At52 antibody reduced cell viability (FIG. 17A). In contrast, Fab fragments of soluble, non-cross-linked TREM2 antibodies derived from the At21 antibody did not inhibit viability and had an effect comparable to the Ab99 isotype control (FIG. 17A). The results show that a soluble non-crosslinked Fab derived from the TREM2 Ab52 antibody can function as an antagonist and inhibit macrophage survival in vitro.

When macrophage cell viability was assessed with soluble full-length antibodies, Ab52 reduced cell viability more effectively than Ab21 (FIG. 17B). Indeed, the soluble full-length Ab21 antibody had an effect on the cell that was comparable to the isotype control At91 (FIG. 17B). The results show that the soluble full-length anti-TREM2 Ab52 antibody can function as an antagonist if not cross-linked or clustered and inhibit macrophage survival.

The results of these experiments show that the anti-TREM2 Ab52 antibody, in the absence of clustering, can inhibit the survival of innate immune cells such as macrophages. In contrast, the results of these experiments show that the anti-TREM2 Ab52 antibody, in the absence of clustering, can inhibit the survival of innate immune cells such as macrophages.

Example 36 Analysis of the ability of anti-TREM2 antibodies to induce TREM2-dependent genes.

The ability of plate-bound antibodies to TREM2 At21 and At52 to activate TREM2-dependent genes was assessed using the luciferase reporter gene under the control of the NFAT promoter (nuclear factor of activated T cells).

A cell line derived from BW5147.G.1.4 mouse thymic lymphoma T-lymphocytes (ATCC® TIB48™) was infected with mouse Trem2 and Dap12 and Cignal Lenti NFAT-Luciferase virus (Qiagen). Full-length anti-TREM2 antibodies were plate bound at 10 μg/ml in DPBS on tissue culture-treated clean-bottomed white 96-well plates (100 μl/well), overnight at 4°C. The wells were washed three times with DPBS and then seeded at 100,000 cells/well in medium with 1% serum. PMA (0.05 μg/ml) and ionomycin (0.25 mM) were added as positive controls for signaling. Cells were incubated for 6 hours and luciferase activity was measured by adding OneGlo Reagent (Promega) to each well and incubating for 3 minutes at room temperature on a plate shaker. The luciferase signal was measured using a BioTek plate reader.

As illustrated in FIG. 18A, anti-TREM2 antibodies Ab 21 and Ab 52 increased luciferase activity, indicating that the antibodies are capable of inducing TREM2-dependent gene transcription.

As illustrated in FIG. 18B plate-bound phosphatidylsern (PS) induces TREM2-dependent gene expression. PS is believed to be a natural TREM2 ligand. Thus, the results illustrated in FIG. 18 show that anti-TREM2 agonist antibodies can mimic natural TREM2 ligand.

The ability of Fab-coupled anti-TREM2 antibodies At21 and At52 to activate TREM2-dependent genes was assessed using primary mouse macrophages. Bone marrow-derived macrophages (BMM) were obtained with M-CSF for 5 days. HMW (10 ⁵ /well) were sown on 96-well plates, which were pre-coated with the Fab portion of the antibodies At22 and At65 (50 nm). Ultrapure LPS (100 ng/ml Escherichia coli 0111:B4) was added after centrifuging the plates for 1 min at 1200 rpm. After 18 hours of stimulation with LPS, cytokines present in cell supernatants were measured by microsphere cytometric assay (CBA; BD Biosciences, CBA mouse inflammation kit).

As illustrated in FIG. 18C and 18D, plate-bound Ab22 and At52 TREM2 antibody Fab increased IL-6 and MCB-1 levels, indicating the ability of the antibody to activate TREM2-dependent genes in primary immune cells.

Example 37 Assay for the ability of anti-TREM2 antibodies to inhibit TREM2-dependent genes.

The ability of soluble full-length anti-TREM2 antibodies Ab21 and At52 to inhibit TREM2-dependent genes was assessed using the luciferase reporter gene under the control of the NFAT promoter (nuclear factor of activated T cells).

A cell line derived from BW5147.G.1.4 mouse thymic lymphoma T-lymphocytes (ATCC® TIB48™) was infected with mouse Trem2 and Dap12 and Cignal Lenti NFAT-Luciferase virus (Qiagen). Soluble full-length Ab 21 or At52 anti-TREM2 antibodies were added to the cells at elevated concentrations. Cells were incubated for 6 h at 37° C. and luciferase activity was measured using OneGlo Reagent (Promega).

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Cells display tonic TREM2-dependent signaling due to the presence of an endogenous ligand or spontaneous receptor clustering resulting in TREM2 signaling.

The dotted line in Fig. 19 shows TREM2 activity levels without stimulation.

As illustrated in FIG. 19, a soluble full-length anti-TREM2 antibody, At52, was able to inhibit tonic, TREM2-dependent gene expression. Soluble full-length anti-TREM2 antibody At21 was able to partially inhibit tonic, TREM2-dependent gene expression; however, gene expression levels were nearly the same as TREM2 activity levels without stimulation (FIG. 19).

Example 38 Summary of the agonist and antagonist activity of the TREM2 antibody.

In table. 7 shows the results of the function tests described in Examples 35-37 above. The antibodies At21 and At52 showed agonist activity in the activation of TREM2-dependent gene expression using the luciferase reporter gene (FIG. 18) or the beta-GAL reporter gene (Table 7). As indicated in Table. 7, At21 showed an increased level of gene induction compared to At52 when the luciferase reporter gene was used. However, At52 showed an increased level of gene induction compared to At21 when the beta-GAL reporter gene was used (Table 7). The antibodies At22 and At52 also showed agonist activity in promoting survival of innate immune cells (FIG. 16). In table. 7 further summarizes the results demonstrating the antagonistic effect of soluble, non-crosslinked Ab52 antibody on inhibition of innate immune cell survival (FIG. 17). In contrast, the soluble, non-crosslinked Ab21 antibody had minimal antagonistic activity in inhibiting cell survival (FIG. 17).

Table 7 Agonistic and antagonistic activity of anti-TREM2 antibody

Antibody Luciferase Agonistic activity of an antibody beta-gal antibody agonist activity Survive Agonistic activity of an antibody Luciferase Antagonistic activity of the antibody in solution/antagonistic Ab format At 5 2 +++ +++ +++ +++ At21 ++++ ++ +++ -/+ Isotype control

Example 39 Analysis of the effect of anti-TREM2 agonist antibodies on Alzheimer's disease.

To evaluate the ability of anti-TREM2 agonist antibodies to slow, prevent, or promote regression in the development of Alzheimer's disease (AD), 5X FAD mice were used. 5X FAD mice overexpress human (695) mutant APP along with Swedish (K670N, M671L), Floridian (I716V) and London (V717I) Alzheimer's disease (FAD) familial mutations along with PS1 carrying two FAD mutations, M146L and L286V. Both transgenes are regulated by the mouse Thy1 promoter to transfer expression to the brain and replicate the main features of AD. Mice treated with anti-TREM2 agonist antibodies or control antibodies were tested for A beta plaque loading by immunohistochemistry and tissue extract ELISA. They are further tested for the amount of microglia in the brain and for the reduction of cognitive deficits using the Morris water maze, spatial learning and memory tasks, radial water eight-arm maze, Y-maze (quantifies spontaneous alternation as a measure of spatial cognition), novelty preference in open field, operant learning for assessing learning and memory, and conditioned freezing (mousebiology.org website; Wang et al., (2015) Cell, pii: S00928674(15)00127-0).

Example 40 Production, identification and characterization of anti-TREM2 agonist antibodies.

Introduction.

Antibodies that bind the extracellular domain of TREM2, in particular the extracellular domain of IgV (amino acid residues 19-174 of SEQ ID NO: 1) are generated using mouse hybridoma technology, phage display technology and yeast display technology according to the methods described in Example 1 above . The antibodies were then screened for their ability to bind TREM2 expressing cells and for their ability to activate TREM2 signaling and function in cells and in the whole animal in vivo as described in Examples 41-67 below.

Results.

Anti-TREM2 antibody production.

Antibodies that bind the extracellular domain of TREM2, particularly within the extracellular

- 154 042890 sequences located at amino acid residues 113-174 of SEQ ID NO: 1 were obtained using the following procedure described in example 1.

87 antibodies were received. The antibodies were then screened for TREM2 binding. Antibodies that were positive for binding to primary cells were tested for agonist activity. Of the 87 antibodies, certain antibodies (eg, At1, At9, At14, At22, At45, and At65) were selected for further analysis.

Heavy chain and light chain variable domain sequences of an antibody.

Using standard methods, the amino acid sequences encoding the heavy chain (FIG. 20A) and light chain (FIG. 20B) of antibody variable domains were determined. The CDR sequences of antibodies according to Kabat are given in table. 8.

Table 8. Kabat CDR sequences

Name of the antibody CDR H1 CDR H2 CDR H3 CDR-LI CDR L2 CDR L3 At1 FTFSSYAMS VISGSGGSTYYADSVKG AKGTPTLLFQH RASQSVSSNLA GAST RAT QQLPYWPPT (SEQ ID NO:3) (SEQ ID NO:25) (SEQ ID NO:50) (SEQID NO:120) (SEQ ID NO:138) (SEQ ID NO:153) At 2 FTFSSSAMS AISGSGGSTYYADSVKG AKVPSYDYWSGYSNYYYYMDV RASQSVGSNLA GAST RAT QQYFFYPPT (SEQ ID NO:4) (SEQ ID NO:26) (SEQ ID NO:51) (SEQID NO:121) (SEQ ID NO:138) (SEQ ID NO:154) AtZ GTFSSYAIS GIIPIFGTANYAQKFQG AREQYHVGMDV QASQDISNYLN DASNLAT QQPFNFPYT (SEQ ID NO:5) (SEQ ID NO:27) (SEQ ID NO:52) (SEQID NO:122) (SEQ ID NO:139) (SEQ ID NO:155) At 4 GTFSSYAIS GIIPIFGTASYAQKFQG ARGVDSIMDY RASQSVSSNLA SASTRAT QQDHDYPFT (SEQ ID NO:5) (SEQ ID NO:28) (SEQ ID NO:53) (SEQ ID NO:120) (SEQ ID NO:140) (SEQ ID NO:156) At 5 YTFTSYYIH IINPSGGSTSYAQKFQG ARAPQESPYVFDI RASQSVSSSYLA GASS RAT QQYFSSPFT (SEQ ID NO:6) (SEQ ID NO:29) (SEQ ID NO:54) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:157) At 6 YTFTSYYMH IINPGGGSTSYAQKFQG ARGSPTYGYLYDP RASQSVSSYLA DASKRAT QQRVNLPPT (SEQ ID NO:7) (SEQ ID NO:30) (SEQ ID NO:55) (SEQ ID NO:124) (SEQ ID NO:142) (SEQ ID NO:158) At 7 YTFTSYYMH IINPSGGSTTYAQKFQG ARTSSHERDY RASQSVSSYLA DASKRAT QQRISYPIT (SEQ ID NO:7) (SEQ ID NO:31) (SEQ ID NO:56) (SEQID NO:124) (SEQ ID NO:142) (SEQ ID NO:159) At 8 GSISSSSYYWG SISYSGSTYYNPSLKS ARGPYRLLLGMDV RASQSISSYLN GASSLQS QQIDDTPIT (SEQ ID NO:8) (SEQ ID NO:32) (SEQ ID NO:57) (SEQID NO:125) (SEQ ID NO:143) (SEQ ID NO:160) At 9 YSFTSYWIG IIYPGDSDTTYSPSFQG ARLHISGEVNWFDP RASQSVSSYLA DASNRAT QQFSYWPWT (SEQ ID NO:9) (SEQ ID NO:33) (SEQ ID NO:58) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:161) ATU YSFTSNWIG IIYPGDSDTRYSPSFQG AREAGYDYGELAFDI RASQSVSSSYLA GASS RAT QQHDSSPPT (SEQID NO:10) (SEQ ID NO:34) (SEQ ID NO:59) (SEQ ID NO:123) (SEQ ID NO:141) (SEQ ID NO:162) At11 YSFTTYWIG IIYPGDSDTRYSPSFQG ARAGHYDGGHLGMDV RASQSVSSDYLA GASS RAT QQDYSYPWT (SEQ ID NO:11) (SEQ ID NO:34) (SEQ ID NO:60) (SEQ ID NO:126) (SEQ ID NO:141) (SEQ ID NO:163) At12 YSFTSYWIG IIYPGDSDTRYSPSFQG ARLGHYS GTVS SYGMDV RASQSISSYLN AASSLQS QQEYAVPYT (SEQ ID NO:9) (SEQ ID NO:34) (SEQ ID NO:61) (SEQ ID NO:125) (SEQ ID NO:145) (SEQ ID NO:164) At13 YTFTSYGIS WISAYNGNTNYAQKLQG ARGPSHYYDLA RASQSVSSYLA DASNRAT QQVSNYPIT (SEQID NO:12) (SEQ ID NO:35) (SEQ ID NO:62) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:165)

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Ατ14 GSISSGGYYWS NIYYSGSTVYNPSLKS ARGLYGYGVLDV QASQDISNYLN DASNLET QQVDNIPPT (SEQID NO:13) (SEQ ID NO:36) (SEQ ID NO:63) (SEQID NO:122) (SEQ ID NO:146) (SEQ ID NO:166) At 15 GSISSGGYYWS NIYYSGSTVYNPSLKS ARGLYGYGVLDV QASQDISNYLN DASNLET QQFDTYPT (SEQ ID NO:13) (SEQ ID NO:36) (SEQ ID NO:63) (SEQID NO:122) (SEQ ID NO:146) (SEQ ID NO:167) Ατ16 GSISSNSYYWG SIYYSGSTYYNPSLKS (SEQ ID NO:37) ARGVLGYGVFDY QASQDISNYLN DASNLET QQFLNFPT (SEQ ID NO:14) (SEQ ID NO:64) (SEQ ID NO:122) (SEQ ID NO:146) (SEQ ID NO:168) At 17 GSISSNSYYWG SIYYSGSTYYNPSLKS ARGVLGYGVFDY QASQDISNYLN DASNLET QQFFNFPT (SEQ ID NO:14) (SEQ ID NO:37) (SEQ ID NO:64) (SEQ ID NO:122) (SEQ ID NO:146) (SEQ ID NO:169) Ατ18 GSISSYYWS SIYYSGSTNYNPSLKS ARDGGGEYPSGTPFDI QASQDISNYLN DASNLET QQFIDLPFT (SEQ ID NO:15) (SEQ ID NO:38) (SEQ ID NO:65) (SEQID NO:122) (SEQ ID NO:146) (SEQ ID NO:170) Ατ19 GSISSYYWS SIYYSGSTNYNPSLKS ARDGGGEYPSGTPFDI QASQDISNYLN DASNLET QQYYDLPFT (SEQID NO:15) (SEQ ID NO:38) (SEQ ID NO:65) (SEQ ID NO:122) (SEQ ID NO:146) (SEQ ID NO:171) Ατ2 0 GSISSYYWS SIYYSGSTNYNPSLKS ARSGMASFFDY RASQSVSSDYLA GASS RAT QQFSSHPFT (SEQID NO:15) (SEQ ID NO:38) (SEQ ID NO:66) (SEQID NO:126) (SEQ ID NO:141) (SEQ ID NO:172) Ατ22 YSFTTYWIG IIYPGDSDTRYSPSFQG ARAGHYDGGHLGMDV RASQSVSSSYLA GASS RAT QQDDRSPYT (SEQID NO:11) (SEQ ID NO:34) (SEQ ID NO:60) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:173) Ατ2 3 FTFSSYAMS AISGSGGSTYYADSVKG AKLGGHSMDV KSSQSVLYSSNNKN YLA WASTRES QQAYLPPIT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:67) (SEQID NO:127) (SEQ ID NO:147) (SEQ ID NO:174) Ατ2 4 FTFSSYAMS AISGSGGSTYYADSVKG AKPLKRGRGFY RASQSISSYLN AASSLQS QQAFSPPPWT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:68) (SEQID NO:125) (SEQ ID NO:145) (SEQ ID NO:175) Ατ2 5 FTFSSYAMS VISGSGGSTYYADSVKG AKEGRTITMD RASQSVSSSYLA GASS RAT QQDDRSPT (SEQ ID NO:3) (SEQ ID NO:25) (SEQ ID NO:69) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:176) Ατ2 6 FTFSSYAMS VISGSGGSTYYADSVKG AKDQYSVLDY RASQSVSSYLA DASNRAT QQEFDLPFT (SEQ ID NO:3) (SEQ ID NO:25) (SEQ ID NO:70) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:177) Ατ27 FTFSSYAMS AISGSGGSTYYADSVKG AKKYSSRGVYFDY RASQSVSSYLA DASNRAT QQYNNFPPT

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(SEQ ID N0:3) (SEQ ID NO:26) (SEQ ID NO:71) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:178) Ατ2 8 FTFSSYAMS AISGSGGSTYYADSVKG ARLGGAVGARHVTYFDY RASQSVSSYLA DASKRAT QQRYLRPIT (SEQ ID N0:3) (SEQ ID NO:26) (SEQ ID NO:72) (SEQID NO:124) (SEQ ID NO:142) (SEQ ID NO:179) Ατ2 9 FTFSSYGMH VISYDGSNKKYYADSVKG ARGQYYGGSGWFDP RASQSVSSSYLA GASS RAT QQPGAVPT (SEQ ID NO:16) (SEQ ID NO:39) (SEQ ID NO:73) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:180) AtZO FTFSSYAMS AISGSGGSTYYADSVKG ARLGQEYAYFQH RASQSISSYLN GASSLQS QQVYITPIT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:74) (SEQID NO:125) (SEQ ID NO:143) (SEQ ID NO:181) Ατ31 FTFSSYGMH LIWYDGSNKYYADSVKG ARRRDGYYDEVFDI QASQDISNFLN DASNLET QQPVDLPFT (SEQID NO:16) (SEQ ID NO:40) (SEQ ID NO:75) (SEQ ID NO:128) (SEQ ID NO:146) (SEQ ID NO:182) Ατ32 FTFSSYAMS AISGSGGSTYYADSVKG ARVPKHYWLDY RASQSVSSYLA DASNRAT QQYSFFPPT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:76) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:183) Ατ33 FTFSSYGMH VISYDGSNKKYYADSVKG ARAGGHLFDY RASQSVSSYLA DASNRAT QQDSSFPT (SEQ ID NO:16) (SEQ ID NO:39) (SEQ ID NO:77) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:184) Ατ34 FTFSSYGMH VISYDGSNKKYYADSVKG ARDRGGEYVDFAFDI RASQSISSYLN AASSLQS QQSDFPPWT (SEQ ID NO:16) (SEQ ID NO:39) (SEQ ID NO:78) (SEQID NO:125) (SEQ ID NO:145) (SEQ ID NO:185) Ατ35 FTFSSYAMS AISGSGGSTYYADSVKG ARTRSGYGASNYFDY RASQSISSYLN AASSLQS QQGYSAPIT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:79) (SEQ ID NO:125) (SEQ ID NO:145) (SEQ ID NO:186) Ατ36 FTFSTYGMH VIWYDGSNKYYADSVKG ARGTGAAAASPAFDI RASQSVSSYLA DASNRAT QQLFDWPT (SEQ ID NO:17) (SEQ ID NO:41) (SEQ ID NO:80) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:187) Ατ37 FTFSSYAMS AISGSGGSTYYADSVKG ARVGQYMLGMDV RASQSVSSYLA DASNRAT QQRAFLFT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:81) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:188) Ατ38 FTFSTYGMH VIWYDGSNKYYADSVKG ARGAPVDYGGIEPEYFQH RASQSVSSYLA DASNRAT QQIDFLPYT (SEQID NO:17) (SEQ ID NO:41) (SEQ ID NO:82) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:189) Ατ39 FTFSSYAMS AISGSGGSTYYADSVKG AKHYHVGIAFDI RASQSISSYLN AASSLQS QQVYSPPIT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:83) (SEQ ID NO:125) (SEQ ID NO:145) (SEQ ID NO:190)

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Am 4 0 FTFSSYAMS AISGSGGSTYYADSVKG ARTRSGYGASNYFDY RASQSISSYLN AASSLQS QQGYAAPIT (SEQ ID NO:3) (SEQ ID NO:26) (SEQ ID NO:79) (SEQID NO:125) (SEQ ID NO:145) (SEQ ID NO:191) At41 FTFSTYAMS AISGSGGSTYYADSVKG ARAMARKSVAFDI RASQSVSSYLA DASNRAT QQRYALPIT (SEQ ID NO:18) (SEQ ID NO:26) (SEQ ID NO:84) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:192) At42 FTFSSSAMS AISGSGGSTYYADSVKG AKVPSYQRGTAFDP RASQSVSSSYLA GASS RAT QQYASPPIT (SEQ ID NO:4) (SEQ ID NO:26) (SEQ ID NO:85) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:193) At 4 3 FTFSSSAMS AISGSGGSTYYADSVKG AKSPAVAGIYRADY RASQSISRYLN AASSLQS QQVYSTPIT (SEQ ID NO:4) (SEQ ID NO:26) (SEQ ID NO:86) (SEQID NO:129) (SEQ ID NO:145) (SEQ ID NO:194) At44 FTFSTYGMH VIWYDGSNKYYADSVKG ARGTGAAAASPAFDI RASQSVSSYLA DSSNRAT QQLVHWPT (SEQ ID NO:17) (SEQ ID NO:41) (SEQ ID NO:80) (SEQID NO:124) (SEQ ID NO:148) (SEQ ID NO:195) At 4 5 YTFTSYYMH IINPSGGSTSYAQKFQG ARGPGYTTALDYYYMDV RASQSVSSNLA GAST RAT QQLDDWFT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:87) (SEQID NO:120) (SEQ ID NO:138) (SEQ ID NO:196) At 4 6 YTFTSYYMH IINPSGGSTSYAQKFQG ARPAKTADY RASQSVSSYLA DSSNRAT QQRSNYPIT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:88) (SEQ ID NO:124) (SEQ ID NO:148) (SEQ ID NO:197) At47 YTFTSYYMH IINPSGGSTTYAQKFQG ARPGKSMDV RASQSVSSYLA DASNRAT QQRILYPIT (SEQ ID NO:7) (SEQ ID NO:31) (SEQ ID NO:89) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:198) At4 8 YTFTSYYMH IINPSGGSTTYAQKFQG ARPGKSMDV RASQSVSSYLA DASNRAT QQRAAYPIT (SEQ ID NO:7) (SEQ ID NO:31) (SEQ ID NO:89) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:199) At 4 9 YTFTSYYMH IINPSGGSTSYAQKFQG ARPAKTADY RASQSVSSYLA DASKRAT QQRTSHPIT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:88) (SEQID NO:124) (SEQ ID NO:142) (SEQ ID NO:200) Am 5 0 YTFTSYYIH IINPSGGSTSYAQKFQG ARAPQESPYVFDI RASQSVSSSYLA GASS RAT QQYAGSPFT (SEQ ID NO:6) (SEQ ID NO:29) (SEQ ID NO:54) (SEQ ID NO:123) (SEQ ID NO:141) (SEQ ID NO:201) At51 YTFTSYYMH IINPSGGSTSYAQKFQG ARGVGGQDYYYMDV RASQSISSYLN AASSLQS QQFDDVFT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:90) (SEQ ID NO:125) (SEQ ID NO:145) (SEQ ID NO:202) At53 YTFTSYYIH IINPSGGSTSYAQKFQG ARAPQESPYVFDI RASQSVSSSYLA GASS RAT QQYVNSPFT (SEQ ID NO:6) (SEQ ID NO:29) (SEQ ID NO:54) (SEQID NO:123) (SEQ ID NO:141) (SEQ ID NO:203)

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At 5 4 YTFTSYYMH IINPSGGSTSYAQKFQG ARGPGYTTALDYYYMDV RASQSINSYLN AASSLQS QQSDDDPFT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:87) (SEQ ID NO:130) (SEQ ID NO:145) (SEQ ID NO:204) At55 YTFTGSYMH WINPNSGGTNYAQKFQG ARGPLYHPMIFDY RASQSVSSYLA DASNRAT QQLSTYPLT (SEQ ID NO:19) (SEQ ID NO:42) (SEQ ID NO:91) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:205) At56 YTFTGYYMH SINPNSGGTNYAQKFQG ARASSVDN RASQSVSSYLA DASNRAT QQRSVYPIT (SEQ ID NO:20) (SEQ ID NO:43) (SEQ ID NO:92) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:206) At 5 7 YTFTNYGIS WISAYNGNTNYAQKLQG ARGPTKAYYGSGSYWFDP RASQSVSSYLA DASKRAT QQVSLFPLT (SEQ ID NO:21) (SEQ ID NO:35) (SEQ ID NO:93) (SEQ ID NO:124) (SEQ ID NO:142) (SEQ ID NO:207) At58 YSFTSYWIG IIYPGDSDTRYSPSFQG ARLGIYSTGATAFDI RASQSISSWLA DASSLES LDYNSYSPIT (SEQ ID NO:9) (SEQ ID NO:34) (SEQ ID NO:94) (SEQ ID NO:131) (SEQ ID NO:149) (SEQ ID NO:208) At59 YTFTGSYMH WINPNSGGTNYAQKFQG ARGGVWYSLFDI QASQDISNYLN DASNLET QQHIALPFT (SEQ ID NO:19) (SEQ ID NO:42) (SEQ ID NO:95) (SEQID NO:122) (SEQ ID NO:146) (SEQ ID NO:209) At 60 YTFTGYYMH WINPNSGGTSYAQKFQG ARASKMGDD RASQSVSSYLA DASKRAT QQRASMPIT (SEQ ID NO:20) (SEQ ID NO:44) (SEQ ID NO:96) (SEQID NO:124) (SEQ ID NO:142) (SEQ ID NO:210) At61 YTFTSYGIH WISAYNGNTNYAQKLQG ARGGVPRVSYFQH RASQSVSSYLA DSSNRAT QQAFNRPPT (SEQID NO:22) (SEQ ID NO:35) (SEQ ID NO:97) (SEQ ID NO:124) (SEQ ID NO:148) (SEQ ID NO:211) At62 YSFTSYWIG IIYPGDSDTRYSPSFQG ARAGHYDDWSGLGLDV RASQSVSSYLA DASKRAT QQSSVHPYT (SEQ ID NO:9) (SEQ ID NO:34) (SEQ ID NO:98) (SEQ ID NO:124) (SEQ ID NO:142) (SEQ ID NO:212) AtbZ YTFTSYGIS WISTYNGNTNYAQKLQG ARGSGSGYDSWYD RASQGIDSWLA AASSLQS QQAYSLPPT (SEQ ID NO:12) (SEQ ID NO:45) (SEQ ID NO:99) (SEQID NO:132) (SEQ ID NO:145) (SEQ ID NO:213) At 6 4 YSFTSYWIG IIYPGDSDTRYSPSFQG ARLGRWSSGSTAFDI RASQSVSSNLA GAST RAT QQDDDGYT (SEQ ID NO:9) (SEQ ID NO:34) (SEQ ID NO:100) (SEQ ID NO:120) (SEQ ID NO:138) (SEQ ID NO:214) At65 YSFTSYWIG IIYPGDSDTRYSPSFQG ARLGRKPSGSVAFDI RASQSVSSYLA DASNRAT QQDYSWPYT (SEQ ID NO:9) (SEQ ID NO:34) (SEQ ID NO:101) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:215) At 6 6 YTFTGSYMH WINPNSGGTNYAQKFQG ARAGHKTHDY RASQSVSSYLA DASNRAT QQRSAYPIT (SEQID NO:19) (SEQ ID NO:42) (SEQ ID NO:102) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:216)

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At 6 7 YTFTSYYMH IINPSGGSTTYAQKFQG ARPGKSMDV RASQSVSSYLA DASNRAT QQRSHFPIT (SEQ ID NO:7) (SEQ ID NO:31) (SEQ ID NO:89) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:217) At 6 8 FTFSSYGMH LIWYDGSNKYYADSVKG AKPGSMTDY RASQSVSSYLA DASNRAT QQRANYPIT (SEQID NO:16) (SEQ ID NO:40) (SEQ ID NO:103) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:218) At 6 9 YTFTGSYMH WINPNSGGTNYAQKFQG ARAKSVDHDY RASQSVSSYLA DASNRAT QQRADYPIT (SEQID NO:19) (SEQ ID NO:42) (SEQ ID NO:104) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:219) At7 0 YTFTGYYMH WINPNSGGTSYAQKFQG ARASKMGDD RASQSVSSYLA DASNRAT QQRSVYPIT (SEQ ID NO:20) (SEQ ID NO:44) (SEQ ID NO:96) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:206) At71 YTFTSYYMH IINPSGGSTSYAQKFQG ARDISTHDYDLAFDI RASQSVSSSYLA GAS N RAT QQAGSHPFT (SEQ ID NO:7) (SEQ ID NO:29) (SEQ ID NO:105) (SEQ ID NO:123) (SEQ ID NO:150) (SEQ ID NO:220) At72 GSISSYYWS SIYYSGSTNYNPSLKS ARSGTETLFDY QASQDITNYLN DASNLET QQDVNYPPT (SEQ ID NO:15) (SEQ ID NO:38) (SEQ ID NO:106) (SEQ ID NO:133) (SEQ ID NO:146) (SEQ ID NO:221) At73 YSFTSYWIG IIYPGDSDTTYSPSFQG ARAKMLDDGYAFDI RASQSVSSNLA GAST RAT QQDDNYPYT (SEQ ID NO:9) (SEQ ID NO:33) (SEQ ID NO:107) (SEQID NO:120) (SEQ ID NO:138) (SEQ ID NO:222) At74 YTFTGSYMH WINPNSGGTNYAQKFQG ARAGHKTHDY RASQSVSSYLA DASNRAT QQRSTFIT (SEQ ID NO:19) (SEQ ID NO:42) (SEQ ID NO:102) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:223) At7 5 YTFTGYYMH WINPNSGGTNYAQKFQG ARDLGYSSLLALDI RASQSVSSYLA DASNRAT QQVSNYPFT (SEQ ID NO:20) (SEQ ID NO:42) (SEQ ID NO:108) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:224) At7 6 FTFSSYSMN SISSSSSYIYYADSVKG ARGGGRRGDNNWFDP KSSQSVLYSSNNKN YLA WASTRES QQYHDAPIT (SEQID NO:23) (SEQ ID NO:46) (SEQ ID NO:109) (SEQ ID NO:127) (SEQ ID NO:147) (SEQ ID NO:225) At 7 7 FTFSSYGMH VISYDGSNKKYYADSVKG ARGPPHEMDY KSSQSVLYSSNNKN YLA WASTRES QQAYWPPT (SEQ ID NO:16) (SEQ ID NO:39) (SEQ ID NO:110) (SEQID NO:127) (SEQ ID NO:147) (SEQ ID NO:226) At7 8 FTFSSYGMH VIWYDGSNKYYADSVKG ARTPYPWIYFDL RASQSVSSYLA DASNRAT QQADNWPFT (SEQID NO:16) (SEQ ID NO:41) (SEQ ID NO:111) (SEQID NO:124) (SEQ ID NO:144) (SEQ ID NO:227)

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At 7 9 FTFSSYSMN YISGSSSTIYYADSVKG ARGGRRHYGGMDV RSSQSLLHSNGYNY LD LGSHRAS MQALESPRT (SEQ ID NO:23) (SEQ ID NO:47) (SEQ ID NO:112) (SEQ ID NO:134) (SEQ ID NO:151) (SEQ ID NO:228) At80 GTFSSYAIS GIIPIFGTANYAQKFQG ARGGGTFWSGSWALY RASQSVSSYLA DASNRAT QQYVNWPFT (SEQ ID NO:5) (SEQ ID NO:27) (SEQ ID NO:113) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:229) At81 GTFSSYAIS GIIPIFGTANYAQKFQG ARDSGNYDYWSGALRY RASQSVSSYLA DASNRAT QQSSNWPWT (SEQ ID NO:5) (SEQ ID NO:27) (SEQ ID NO:114) (SEQ ID NO:124) (SEQ ID NO:144) (SEQ ID NO:230) At82 GSISSGGYYWS YIYYSGSTVYNPSLKS ARVSSSWYKA RASQGISSWLA AASSLQS QQASTFPIT (SEQID NO:13) (SEQ ID NO:48) (SEQ ID NO:115) (SEQ ID NO:135) (SEQ ID NO:145) (SEQ ID NO:231) At83 GSFSGYYWS EIDHSSGSTKYNPSLKS ARVGVWGRPGYSAFDI RASQGISSWLA AASSLQS QQRNSLPLT (SEQID NO :24) (SEQ ID NO:49) (SEQ ID NO:116) (SEQID NO:135) (SEQ ID NO:145) (SEQ ID NO:232) At 8 4 YTFTSYGIS WISTYNGNTNYAQKLQG ARGSGSGYDSWYD RASQSISSYLN AASSLQS QQSYDFPIT (SEQ ID NO:12) (SEQ ID NO:45) (SEQ ID NO:99) (SEQ ID NO:125) (SEQ ID NO:145) (SEQ ID NO:233) Am 8 5 FTFSSYGMH VIWYDGSNKYYADSVKG AKDLGGYYGGAAYGMDV RASQDISSWLA AASSLQS QQEVDYPPLT (SEQID NO:16) (SEQ ID NO:41) (SEQ ID NO:117) (SEQ ID NO:136) (SEQ ID NO:145) (SEQ ID NO:234) At 8 6 FTFSSYGMH VISYDGSNKKYYADSVKG AKDGVYYGLGNWFDP RASQSISSWLA KASSLES QQLNSYSPT (SEQID NO:16) (SEQ ID NO:39) (SEQ ID NO:118) (SEQID NO:131) (SEQ ID NO:152) (SEQ ID NO:235) At87 GSISSYYWS SIYYSGSTNYNPSLKS ARHGWDRVGWFDP RASQSVSRYLA DASNRAT QQYIFWPPT (SEQID NO:15) (SEQ ID NO:38) (SEQ ID NO:119) (SEQID NO:137) (SEQ ID NO:144) (SEQ ID NO:236)

Binding characterization of At1, At9, At14, At22, At45 and At65.

Initial characterization of TREM2 antibodies included determining their ability to bind TREM2 expressed on dendritic and other human or mouse primary immune cells. Cells were harvested, plated at 10 ⁵ /ml in a 96-well plate, washed and incubated in 100 µl PBS containing 10-50 µg/ml mAb and Fc blocking reagent for 1 hour on ice. The cells were then washed twice and incubated in 100 µl of PBS containing 5 µg/ml of secondary antibody conjugated with PE for 30 min on ice. Cells were washed twice in cold PBS and quantified using BD FACS Canto. Data analysis and calculation of MFI values were performed using FlowJo software (TreeStar) version 10.0.7.

Antibodies At1, At9, At14, At22, At45, and At65 showed binding to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2 as shown by positive staining of TREM2 antibodies detected by FACS analysis (bar graphs with black outline) (Fig. .21A). The negative isotype control (At88 antibody) showed no binding. Antibodies At1, At9, At14, At22, At45 and At65 demonstrated antibody binding to DT (Trem +/+) macrophages derived from mouse bone marrow (BMMac, mMac), but not to TREM2-deficient (TREM2 -/-) mouse macrophages (VMMac, mMac) (FIG. 21B). The antibodies At1, At9, At14, At22, At45, and At65 show binding to both a human (293) cell line expressing recombinant human TREM2 (FIG. 4A) and human primary dendritic cells (hDC) (FIG. 22B). Conversely, At43 and At60 antibodies bind to a human cell line expressing recombinant human TREM2 (FIG. 4A), but do not bind to primary human dendritic cells (FIG. 22B).

The average values of fluorescence intensity (MFI) for cell types associated with antibodies At1, At9, At14, At22, At45 and At65 TREM2 are shown in Table. 9. Binding compared to parental mouse cell line (mTREM2 parental cell line BWZ), primary human cell line (4TREM2 parental cell line (293)), primary TREM2-deficient mouse macrophages (mPac KO MFI), and primary TREM2-deficient mouse dendritic cells (mDC KO MFI). The results in the table. 9 show that At1, At9, At14, At22, At45, and At65 bind to cell lines overexpressing human and mouse TREM2 on the cell membrane, but not

- 161 042890 to control cell lines that do not express TREM2. The antibodies also bind to primary human macrophages and primary mouse macrophages, and dendritic cells. Binding to primary mouse cells is specific as it is not detectable on primary cells derived from TREM2 KO mice.

Table 9 Anti-TREM2 Antibody Binding to Human and Mouse Cells

Antibody mTget2 Cell line (BWZ parental) MFI mTget2 Cell line (BWZ T2) MFI hTget2 parental cell line (293) MFI hTget2 cell line (293) MFI mMac KO MFI mMac DT MFI mDC KO MFI mDK DT MFI NPV % positive At1 960 12626 86 1491 69.8 82.3 235 393 64.9 At 9 1044 15691 83 2126 97.2 171.0 200 538 65.8 At14 852 11550 87 1656 77.9 145.0 218 529 75.8 At22 828 12451 82 1837 67.2 110.0 191 451 76.9 At 4 5 1022 16288 141 2058 86.2 141.0 277 652 78.8 At 6 5 1354 16122 93 1734 92.5 165.0 260 642 76.9

The binding affinity of each antibody to TREM2 was determined by measuring their KD using ForteBio or MSD-SET. Affinity measurements with ForteBio were performed as previously described (Estep et al., (2013) MAbs 5(2):270-8). Briefly, ForteBio affinity measurements were performed by uploading IgG online to AHQ sensors. The sensors were equilibrated offline in assay buffer for 30 min and then monitored online for 60 s to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen for 5 min, then transferred to assay buffer for 5 min to measure the rate of response. The kinetics were analyzed using a 1:1 binding model.

Equilibrium affinity measurements were performed as previously described (Estep et al., (2013) MAbs 5(2):270-8). Titrations to the end point (SET) were performed in PBS + 0.1% BSA without IgG (PBSF) at a constant antigen concentration of 50 pM and incubated with 3-5-fold serial dilution of the antibody starting from 10 nM. Antibodies (20 nM in PBS) were applied to standard MSD-ECL binding plates overnight at 4°C or at room temperature for 30 min. The plates were then blocked for 30 minutes with shaking at 700 rpm followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds with shaking at 700 rpm followed by a single wash. Antigen captured on the plate was detected with 250 ng/ml sulfotag-labeled streptavidin in PBSF by incubation on the plate for 3 minutes. The plates were washed three times with wash buffer and then read on an MSD Sector Imager 2400 using a single Read Buffer T with surfactant. The percentage of free antigen was plotted as a function of titratable antibody in Prism and squared to give the K _D value. Liquid handling robots were used in all MSD-SET experiments, including SET sample preparation, to improve throughput.

In table. 10 lists the values reflecting the binding affinity (KD) of antibodies At1, At9, At14, At22, At45, and At65 for the human TREM2 Fc fusion protein (4TREM2-Fc), the monomeric His-tagged human TREM2 protein (4TREM2-HIS), and the TREM2 Fc fusion protein. mice (mTREM2-Fc).

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Table 10: Anti-TREM2 binding affinity

Antibody IgG K _D 4TREM2-FC (M) Avid Monovalent IgG K _D 4TREM2-HIS (M) IgG K _D mTREM2-Fc (M) Avid At1 7.05Е-10 6.67Е-09 4.86E-09 At 9 3.48E-10 6.32E-09 N.V. At14 5.51E-10 3.19E-09 6.20E-10 At22 3.06E-10 1.01Е-09 3.40E-10 At 4 5 2.29E-10 6.54Е-10 N.B. At 6 5 5.46E-10 5.01Е-09 1.56E-09

Example 41 TREM2 antibodies induce CD83 and CD86 expression on human dendritic cells (DC) and induce T cell proliferation.

To evaluate the ability of anti-TREM2 antibodies to modify the expression of CD83 and CD86, both plate bound and soluble antibodies were incubated with dendritic cells (DC) and the expression of CD83 and CD86 and phosphorylated ERK was measured. To evaluate the ability of anti-TREM2 antibodies to modulate T cell proliferation, DCs were incubated with T cells and anti-TREM2 antibodies and the level of T cell proliferation was measured.

Antibodies were seeded overnight at 4°C in 12-well plates at a concentration of 2 or 5 μg/ml in PBS. The next day, the wells were washed 3 times with PBS. On day 5, immature human DCs were harvested and plated at 1 million cells/well and incubated at 37° C., 5% CO ₂ in the absence of cytokine. FACS analysis of CD86, CD83, CD11c, HLA-DR and LIN (BD Biosciences) was performed on BD FACS Canto after 48 hours. Data analysis was performed using FlowJo software (TreeStar) version 10.0.7. CD83, CD86 and CCR7 levels were assessed on populations of CDllc+HLADR+LIN cells. For intracellular ERK phosphorylation, cells were fixed with 1% formaldehyde permeabilized with Cytofix/Cytoperm kit (BD) and intracellular Erk phosphorylation was determined by flow cytometry after staining with PE-ERK antibody (BD).

Alternatively, 5-day-old immature human dendritic cells were seeded at 100,000 cells/well in a 96-well U-bottom plate, untreated with TC, in media without cytokine. Antibodies were added at a concentration of 5 μg/ml with or without a removed secondary human anti-LPS antibody (Jackson ImmunoResearch) at a concentration of 20 μg/ml. FACS analysis of CD86, CD83, CDllc, HLA-DR and LIN (BD Biosciences) was performed 48 hours after antibody addition as described above.

In addition, 5-day-old immature dendritic cells (CD14CDllc ⁺ LIN) were plated in 12-well plates coated the previous day with 2 μg/ml antibody. Before adding T cells, the plates were washed with PBS three times. CD4 ⁺ T cells from non-autologous donors were isolated and labeled with CFSE prior to addition to DC at a ratio of 1:10, 1:50, or 1:250. CD3/CD28 Dynalbead serve as a positive control. On day 5 after co-culture, cells were analyzed by flow cytometry on a BD FACSCanto II to dilute CFSE. For each condition with FlowJo (TreeStar), the percentage of CFSE ^hl compared to CFSE ¹⁰ cells was calculated.

Plate-bound TREM2 antibodies At1, At9, At14, At22, At45, and At65 increased the frequency of CO83+CO86+DC compared to the isotype control antibody At88 (FIG. 23A). Soluble antibodies At1, At9, At14, At45 and At65, when cross-linked with a human secondary antibody, induce CD86 expression on DC. Conversely, the cross-linked At22 antibody and the cross-linked soluble antibodies did not induce CD86 expression (FIG. 23B). Based on these results, the antibodies At1, At9, At14, At22, At45 and At65 TREM2 function as agonists to induce expression of the inflammatory surface markers CD83 and CD86 on human dendritic cells.

Example 42 TREM2 antibodies At1, At9, At14, At20, At22, At45 and At65 induce Syk phosphorylation.

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule that functions downstream of TREM2 in the metabolic pathway by phosphorylation of several substrates, thus promoting the formation of a signaling complex that leads to cellular activation and inflammatory processes. The ability of TREM2 agonist antibodies to induce Syk activation was determined by culturing human and mouse macrophages and human primary dendritic cells, and measuring the phosphorylation state of the Syk protein in cell extracts.

Macrophages from bone marrow (BMDM), BMDM mouse DT, BMDM mouse with knockout (KO) TREM2 and primary human dendritic cells were placed on depleted medium for 4 h containing

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1% serum RPMI, and then removed from tissue culture dishes with PBS-EDTA, washed with PBS and counted. Cells were coated with full-length TREM2 agonist antibodies At1, At9, At14, At20, At22, At45, At65, non-agonistic antibodies (At16, At77), or control antibodies (At89 or At92) for 15 min on ice. After washing with cold PBS, the cells were incubated at 37° C. for the indicated time period in the presence of goat anti-human IgG. After stimulation, cells were lysed with lysis buffer (1% v/v NP-40%, 50 ml Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl ₂ , 10% glycerol , plus protease and phosphatase inhibitors) followed by centrifugation at 16,000 g for 10 min at 4°C to remove insoluble materials. The lysates were then immunoprecipitated with an anti-Syk antibody (N-19 for BMDM or 4D10 for human DC, Santa Cruz Biotechnology). Precipitated proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-phosphotyrosine antibody (4G10, Millipore). To confirm that all substrates were adequately immunoprecipitated, immunoblots were reprobed with an anti-Syk antibody (Abcam, for BMDM) or an anti-Syk antibody (Novus Biological, for human DC). Imaging was performed using the Advanced Chemiluminescence (ECL) System (GE Healthcare) as described (eg, Peng et al., (2010) Sci Signal., 3(122): ra38).

The TREM2 antibodies At1, At9, At14, At20, At22, At45, and At65 induce TREM2-mediated Syk phosphorylation in BMDM (FIG. 24A). Syk phosphorylation induced by At1, At9 and At45 antibodies is TREM-2 specific because Syk phosphorylation is not induced when TREM2 KO BMDM is used as a control (FIG. 24B). TREM2 antibodies At22, At45 and At65 induce Syk phosphorylation in primary human dendritic cells (FIG. 24C). Non-agonistic antibodies (At16 and At77) or control antibodies (At89 and At92) do not induce Syk phosphorylation. Based on these results, the TREM2 antibodies At1, At9, At14, At20, At22, At45, At65 function as agonists to induce Syk phosphorylation in macrophages and dendritic cells.

Example 43 The TREM2 antibodies At1, At9, At14, At22, At45 and At65 compete with the TREM2 ligand for binding to human and mouse TREM2.

The ability of TREM2 agonist antibodies to recognize the TREM2 ligand binding site was assessed by competitive binding assay using E. coli cells expressing the putative TREM2 ligand.

E. coli were grown in 10 ml of LB O/N medium, collected by centrifugation and washed twice in 10 ml of PBS. The E. coli were then thermally inactivated by incubation in a water bath at 70° C. for 30 minutes. E. coli were labeled with CellTracker DeepRed (ThermoFisher/Invitrogen, final concentration 1 μM) and then washed three times in 10 ml PBS, and resuspended in 1 ml PBS at a concentration of 10 ⁸ /ml. For competitive binding, bacteria were added to mouse TREM2 and to a cell line (BWZ) expressing DAP12 or to a BW cell line expressing human TREM2/DAP12 fusion protein together with 10 μg/mL TREM2 full-length agonist antibodies and incubated for one hour for ice. Cells were analyzed by FACS for binding of CellTracker-labeled bacteria to cell lines.

TREM2 antibodies At1, At9, At14, At22, At45 and At65 inhibited E. coli binding to both human and mouse cells, indicating competitive antibody binding to the ligand-binding site on TREM2 (FIG. 25). Control non-agonist TREM2 antibodies (At66 and At68) partially inhibited bacterial binding to human cells expressing TREM2, but did not inhibit binding to mouse TREM2, indicating that they are weaker competitors in ligand binding.

Example 44 TREM2 antibodies At1, At9, At22, At45 and At65 induce DAP12 phosphorylation in mouse macrophages.

TREM2 signals through DAP12, resulting in downstream activation of PHYS and other intracellular signals. The ability of anti-TREM2 agonist antibodies to induce DAP12 activation was determined by culturing mouse macrophages and measuring the phosphorylation state of the DAP12 protein in cell extracts.

Before mAb stimulation, mouse macrophages (MT) from bone marrow (BMDM) and BMDM mice with knockout (KO) TREM2 were placed on depleted medium containing 1% RPMI serum for 4 h. 15x106 cells were incubated on ice for 15 min with full length agonist or control antibodies. Cells were washed and incubated at 37° C. for the indicated time period in the presence of goat anti-human IgG. After stimulation, cells were lysed with lysis buffer (1% v/v NP-40%, 50 ml Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl ₂ , 10% glycerol , plus protease and phosphatase inhibitors) followed by centrifugation at 16,000 g for 10 min at 4°C to remove insoluble materials. The cell lysate was immunoprecipitated with a second TREM2 antibody (R&D Systems). Precipitated proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-phosphotyrosine antibody (4G10, Millipore). The membrane was separated and re-probed with an antibody to

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DAP12 (Cells Signaling, D7G1X). Each cell lysate used for TREM2 immunoprecipitation contained an equal amount of proteins, as shown by a control antibody (anti-actin antibody, Santa Cruz).

DAP12 is co-precipitated with TREM2 and phosphorylated in DT macrophages incubated with TREM2 agonist antibodies At1, At9, At22, At45, and At65 (FIGS. 26A and 26B). Conversely, DAP12 phosphorylation was not observed in TREM2 KO macrophages (TREM2' ^/_ ) incubated with At1, At9, At22, or At45 antibodies (FIG. 26B). These results demonstrate that the agonist antibodies At1, At9, At22, and At45 induce phosphorylation of TREM-2-associated DAP12 in a specific manner, since DAP12 phosphorylation is absent in TREM-2-deficient BMDMs.

Example 45 Epitope Mapping of TREM2 Antibodies and Comparison of TREM2 Antibodies to Reference TREM2 Antibodies.

TREM2 antibodies were tested for their ability to bind 15 or 25 mer peptides spanning all human and mouse TREM2. TREM2 antibodies were also compared to a reference TREM2 antibody by determining their TREM2 binding region.

Linear 15-mer peptides were synthesized based on the human or mouse TREM2 sequence with a 14-residue overlap. In addition, linear 25-mer peptides were synthesized based on the human or mouse TREM2 sequence with a 1-residue shift. The binding of anti-TREM2 antibodies to each of the synthesized peptides was examined by an ELISA-based method. In this assay, peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 diluted antibody-peroxidase conjugate (SBA, cat. no. 2010-05) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azinobis-3-ethylbenzthiazolinesulfonate (ABTS) and 2 μl/ml 3% H ₂ O ₂ were added. Color formation was measured after one hour. Color formation was determined using a charge-coupled device (CCD) camera and an image processing system.

To evaluate binding regions, human TREM2-Fc antibodies were incubated with immobilized TREM2 full-length agonist antibodies At1, At9, At14, At22, At45, At63, At65, or At87, and then the TREM2 antibody MAB17291 (R&D Systems) was added. Epitope-specific antibody sorting was performed on a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park, CA) using a standard binding sandwich assay (see, Estep et al., (2013) MAbs 5(2):270- 8). Target control IgG was loaded onto AHQ sensors and unoccupied Fc binding sites on the sensor were blocked with an irrelevant human anti-IgG1 antibody. The sensors were then exposed to 100 nM target antigen followed by a second target antibody. The data were processed using ForteBio's Data Analysis Software 7.0. Additional binding by the second antibody after association with the antigen indicates an unoccupied epitope (non-competing), while no binding indicates blocking of the epitope (competing).

The antibodies At1, At9, At28 and At29 showed strong and strong binding exclusively for the peptides from Set1 and Set2, which are derived from the human TREM2 sequence. All four antibodies bound peptides that contain a region between amino acid residues 139146 of human TREM2 ( ¹³⁹ GDLWFPGE ¹⁴⁶ (SEQ ID NO: 237)) (FIG. 27A). The epitope region recognized by At1, At9, At28 and At29 corresponds to amino acid residues 139-146 of SEQ ID NO: 1 and has the amino acid sequence: GDLWFPGE (SEQ ID NO: 237).

Antibodies At45 and At65 bind only to 25-mer peptides from Set2 and Set4. Both antibodies recognized a highly conserved region of TREM2 near its C-terminus between amino acid residues 140-153 of human TREM2 ( ¹⁴⁰ DLWFPGESESFEDA ¹⁵³ (SEQ ID NO: 238)) and mouse TREM2 ( ¹⁴⁰ DLWVPEESSSFEGA ¹⁵³ (SEQ ID NO: 239)) (Fig. 27B). ). The epitope region recognized by At45 and At65 corresponds to amino acid residues 140-153 of SEQ ID NO: 1 and has the amino acid sequence: DLWFPGESESFEDA (SEQ ID NO: 238).

The region of the epitope recognized by the reference antibody MAB17291 was also determined. The reference antibody MAB17291 was found to recognize a first peptide that contains a region between amino acid residues 130-144 of human TREM2 ( ¹³⁰ ADPLDHRDAGDLWFP ¹⁴⁴ (SEq ID NO: 240)) and a second peptide that contains a region between amino acid residues 158-170 of human TREM2 ( ¹⁵⁸ SISRSLLEGEIPF ¹⁷⁰ (SEQ ID NO: 241)). The epitope regions recognized by MAB17291 correspond to amino acid residues 130-144 and 158-170 of SEQ ID NO: 1 and have the amino acid sequences ADPLDHRDAGDLWFP (SEQ ID NO: 240) and SISRSLLEGEIPF (SEQ ID NO: 241).

Reference antibody MAB17291 was able to simultaneously bind TREM2 to antibody At1, At9, At14, At22, At45, or At65, but not to At63 or At87 (FIG. 28). These results demonstrate that the TREM2 agonist antibodies At1, At9, At14, At22, At45 and At65 bind to different regions of the TREM2 protein than the reference antibody MAB17291.

Example 46 Summary of TREM2 antibody function studies.

In table. 11 shows the results of the function tests described in Examples 41-45 above. Antibodies At1, At9, At14, At22, At45 and At65 demonstrated the induction of CD83 and CD86 in human

- 165 042890 dendritic cells (hDC), and higher induction was observed with antibody bound on the plate, compared with cross-linked soluble antibody (values in the table represent the percentage of CD83+CD86+ cells). The antibodies At1, At9, At14, At22, At45 and At65 induced various levels of Syk phosphorylation in human dendritic cells (hDC), mouse dendritic cells (mDC) and mouse macrophages (mMac) and were able to mimic ligand binding (by blocking bacterial binding) of human TREM2 and mice.

Table 11. TREM2 antibody function studies

Antibody Induction of CD86 and CD83 human phDa plate Induction of CD86 and CD83 human hDA solution Phospho Syk PhDK induction Phospho Syk mDC induction Phospho induction TREM2 and DAP12 mMac Blocked bacterial binding of 4TREM2 Blocked bacterial At1 87.2 3.0 -/+ ++ ++ +++ +++ At 9 79.3 5.6 -/+ +++ ++++ +++ +++ At14 78.7 1.9 + ++ n/a +++ +++ At22 74.5 1.2 ++ ++ ++ +++ +++ At 4 5 65.5 4.3 +++ +++ +++ +++ +++ At 6 5 74 1.7 ++ +++ +++ +++ +++ Isotype control - - - - - - -

Example 47 Tumor growth assay in deficient mice.

TREM2 Groups of 10 TREM2 wild-type (WT), TREM2 heterozygous (NO) and TREM2 knockout (KO) mice (littermates, matched sex and age, 8 weeks (+/- 2 weeks)) are tested by subcutaneous administration of tumor cells (for example, 1x10 ⁵ -1x10 ⁶ colon carcinoma MC38, Lewis lung carcinoma, or B16 melanoma cells) suspended in 100 μl of PBS. Animals are anesthetized with isoflurane prior to implantation. To measure tumor growth, starting on day 4, tumor growth is monitored with a caliper every two weeks. The end point of the experiment is a tumor volume of 2000 mm ³ or 60 days. Tumor growth and % survival are baseline. Decreased tumor engraftment and growth rates and reduced numbers of tumor-infiltrating immunosuppressive macrophages indicate an increased influx of effector T cells into the tumor in TREM2 KO mice.

To determine the number of infiltrating, tumor-associated immunosuppressive macrophages and T cells, groups of 6-8 littermates of the corresponding sex and age are used. At 8 weeks of age (+/- 2 weeks), DT-NET-KO littermates are injected subcutaneously with tumor cells (i.e., 1 x 10 ⁵ -1 x 10 ⁶ MC38, Lewis lung cells, or B16 cells) suspended in 200 µl of Matrigel Matrix Growth Factor Reduced; BD). Animals are anesthetized with isoflurane prior to implantation. 7 and 10 days after the tumor cell injection, the matrigel plug is excised, incubated for 1 hour at 37°C with 1 mg/ml collagenase D (Sigma), dissociated to obtain a single cell suspension, and filtered through a cell filter. To determine the number of T cells present in the tumor and the ratio between effector T cells and regulatory T cells, 5 x 10 ⁶ cells are stained with anti-CD45.2 anti-CD45.2 antibody with FITC, anti-CD3 antibody with PECY7, PECY7 anti-CD45.2 antibody. CD8, APC antibodies to CD4, PE antibodies to FoxP3 (BD) and DAPI. To determine the number of cells of the monocyte/macrophage line present in the tumor, 5x10 ⁶ cells are stained with RegcpCy5.5 to CD45.2, PECY7 to CDllb, FITC to F4/80, APC to Ly6C/G. , antibodies with PE to CD86 and DAPI. Cells were obtained on BD FACS Canto. Data analysis was performed using FlowJo software (TreeStar) version 10.0.7.

Example 48 Anticancer Effect Assay of TREM2 Antagonist Antibodies.

Groups of 10 C57B16/NTac mice at 8 weeks of age (+/- 2 weeks) are injected subcutaneously with tumor cells (eg, 1 x 10 ⁵ to 1 x 10 ⁶ MC38, Lewis lung cells, or

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B16) suspended in 100 µl of PBS. Animals are anesthetized with isoflurane prior to implantation. Starting on day 2, groups of mice are injected ip every 3 days with 4 doses of 200 μg of each of the anti-TREM2 antagonist antibodies, such as those described in examples 38 and 40. To measure tumor growth, starting on day 4 days, tumor growth is monitored with a caliper every two weeks. The end point of the experiment is a tumor volume of 2000 mm ³ or 60 days. Tumor growth and % survival are baseline. Decreased tumor engraftment and growth rate, reduced numbers of tumor-infiltrating immunosuppressive macrophages, and increased influx of effector T cells into the tumor indicate anti-cancer effects of blocking anti-TREM2 antibodies.

Example 49 Analysis of the additive antitumor effect of a combination therapy that combines antibodies to TREM2 with antibodies to checkpoint inhibitory proteins or inhibitory cytokines/chemokines and their receptors.

Groups of 15 C57B16/NTac mice aged 8 weeks (+/- 2 weeks) are subjected to subcutaneous injection of tumor cells as described in Example 35. Animals are anesthetized with isoflurane prior to implantation. Beginning on day 2, mice receive 4 doses ip every 3 days with 200 µg of antibodies to TREM2 alone or in combination with antibodies to checkpoint proteins (e.g. anti-PDL1 mAb clone 10F.9G2 and/or anti-CTLA4 mAb clone UC10 -4F10-11) on the 3rd, 6th and 9th day. Treatment groups include anti-TREM2 antibody; anti-CTLA4 antibody; anti-PDL1 antibody; antibody to TREM2 and CTLA4; antibody to TREM2 and PDL1; and isotype control. To measure tumor growth, starting on day 4, tumor growth is monitored with a caliper every two weeks. The end point of the experiment is a tumor volume of 2000 mm ³ or 60 days. Tumor growth and % survival are baseline. The reduction in tumor growth and increased % survival with combination therapy indicate that antibodies to TREM2 have an additive or synergistic therapeutic effect with antibodies to checkpoint proteins. Checkpoint antagonist antibodies include antibodies to PDL1, PDL2, PD1, CTLA4, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG-3, and phosphatidylserine. Antagonistic antibodies to inhibitory cytokines include antibodies to CCL2, CSF-1 and IL-2.

Example 50 Analysis of the additive antitumor effect of a combination therapy that combines anti-TREM2 antibodies with antibodies that activate checkpoint stimulating proteins.

Groups of 15 C57B16/NTac mice aged 8 weeks (+/- 2 weeks) are subjected to subcutaneous injection of tumor cells as described in Example 35. Animals are anesthetized with isoflurane prior to implantation. Beginning on day 2, mice receive 4 doses ip every 3 days with 200 µg of anti-TREM2 antibodies alone or in combination with agonist antibodies that activate checkpoint-stimulating proteins (eg mAb OX40 or ICOS) for 3-, 6 - and the 9th day. To measure tumor growth, starting on day 4, tumor growth is monitored with a caliper every two weeks. The end point of the experiment is a tumor volume of 2000 mm ³ or 60 days. Tumor growth and % survival are baseline. The reduction in tumor growth and increased % survival with combination therapy indicate that anti-TREM2 antibodies have an additive or synergistic therapeutic effect with checkpoint stimulating antibodies. Checkpoint stimulating antibodies include CD28, ICOS, CD137, CD27, CD40 and GITR agonist/stimulatory antibodies.

Example 51 Analysis of the additive antitumor effect of a combination therapy that combines anti-TREM2 antibodies with stimulatory cytokines.

Groups of 15 C57B16/NTac mice aged 8 weeks (+/- 2 weeks) are subjected to subcutaneous injection of tumor cells as described in Example 35. Animals are anesthetized with isoflurane prior to implantation. Beginning on day 2, mice are treated intraperitoneally every 3 days with 4 doses of 200 μg anti-TREM2 antibodies alone or in combination with stimulatory cytokines (eg, IL-12, IFN-α). To measure tumor growth, starting on day 4, tumor growth is monitored with a caliper every two weeks. The end point of the experiment is a tumor volume of 2000 mm ³ or 60 days. Tumor growth and % survival are baseline. The reduction in tumor growth and increased % survival with combination therapy indicate that anti-TREM2 antibodies have an additive or synergistic therapeutic effect with immunostimulatory cytokines. Stimulating cytokines include IFN-a/b, IL-2, IL-12, IL-18, GM-CSF and G-CSF.

Example 52 Assay for the ability of anti-TREM2 antibody Fab fragments to stimulate viability of innate immune cells.

The agonist functional properties of plate-bound cross-linked anti-TREM2 Fab fragments derived from the antibodies At22, At45 and At65 were evaluated on innate immune cells (eg, macrophages).

Macrophages derived from the bone marrow of wild-type (WT) and TREM2 knockout (KO) mice were cultured in the presence of M-CSF and anti-TREM2 antibody plate-bound Fab fragments, and

- 167 042890 cell viability was measured.

Macrophages isolated from the bone marrow of DT and KO mice were seeded in untreated 96-well plates precoated with either 12.5 nM or 100 nM cross-linked At22, At45, or At65 Fab fragments. Cells were cultured for 48 h in the presence of 10 ng/ml M-CSF. Viability analysis was performed using the Cell Titer Glo kit (Promega). Plates were read with a BioTek Synergy microplate reader using GEN5 2.04 software.

Cross-linked anti-TREM2 Fab fragments derived from the antibodies At22, At45 and At65 increased the number of viable macrophages derived from mouse bone marrow compared to the Ab88 isotype control Fabs, as evidenced by a higher % increased survival (Fig. 29). This increase in cell viability was not observed in mouse KO macrophages, with the exception of At65. These data indicate that the biological activity of the antibodies At22 and At45 is specific for TREM2, and that plate-bound, cross-linked Fab fragments of At22 and At45 function as agonists to increase the survival of macrophages cultured in M-CSF.

Example 53 Analysis of the ability of anti-TREM2 antibodies to decrease the survival of innate immune cells.

The antagonistic functional properties of Fab fragments of both soluble, non-cross-linked TREM2 antibody-derived antibodies and soluble full-length TREM2 antibodies were evaluated on innate immune cells (eg, macrophages).

Bone marrow-derived macrophages from wild-type (WT) mice and TREM2 knockout (KO) macrophages were cultured in the presence of M-CSF and TREM2 soluble antibody Fab fragments or soluble full-length antibodies, and cell viability was measured.

Macrophages isolated from the bone marrow of DT and KO mice were seeded in non-tissue-cultured 96-well plates in the presence of 20 ng/ml M-CSF and increased amounts of the indicated Fab fragments of soluble, non-cross-linked TREM2 antibodies derived from At22, At45 and At65, or soluble full length antibodies At9, At14, At22, At45 and At65. Each variant was seeded in triplicate. Viability analysis was performed using the Cell Titer Glo kit (Promega) after 3 days. Plates were read with a BioTek Synergy microplate reader using GEN5 2.04 software.

In FIG. 30A and 30B, the dotted NT line indicates the mean cell viability obtained from untreated macrophages (no antibody added). The dotted line without MCSF indicates the average cell viability obtained by culturing macrophages in the absence of M-CSF.

When macrophage cell viability was assessed with Fab fragments of soluble non-cross-linked TREM2 antibodies, the results indicated that Fab fragments of soluble non-cross-linked TREM2 antibodies derived from the At45 antibody reduced cell viability (FIG. 30A). In contrast, Fab fragments of soluble non-cross-linked TREM2 antibodies derived from the Ab22 antibody did not inhibit viability and had an effect comparable to the isotype control Ab99 (FIG. 30A). Fab fragments of soluble, non-cross-linked TREM2 antibodies derived from the At65 antibody partially inhibited cell viability (FIG. 30A). The results show that the soluble non-cross-linked Fab derived from the TREM2 Ab45 and At65 antibodies can function as antagonists and inhibit macrophage survival in vitro.

When macrophage cell viability was assessed with soluble full-length antibodies, Ab14, At45, At65, and At9 antibodies reduced cell viability (FIG. 30B).

The soluble full-length Ab22 antibody had an effect on cell viability that was nearly comparable to that of the isotype control Ab91 (FIG. 30B). The results show that soluble full length anti-TREM2 antibodies At14, At45, At65 and At9 can function as antagonists if they are not cross-linked or clustered and inhibit macrophage survival.

The results of these experiments show that antibodies to TREM2 At45, At65 and At9, in the absence of clustering, can inhibit the survival of innate immune cells such as macrophages. In contrast, the anti-TREM2 Ab22 antibody, even in the absence of clustering, does not inhibit the survival of innate immune cells such as macrophages.

Example 54 Assay for the ability of anti-TREM2 antibodies to induce TREM2-dependent genes.

The ability of full-length anti-TREM2 antibodies Ab1, At9, At10, At14, At15, At17, At18, At20, At22, At24, At25, At28, At29, At45, At54, At51, At64, At65 and At66, to activate TREM2-dependent genes was evaluated using the luciferase reporter gene under the control of the NFAT promoter (nuclear factor of activated T cells).

A cell line derived from BW5147.G.1.4 mouse thymic lymphoma T-lymphocytes (ATCC® TIB48™) was infected with mouse Trem2 and Dap12 and Cignal Lenti NFAT-Luciferase virus (Qiagen). Full-length anti-TREM2 antibodies were plate bound at 10 μg/ml in DPBS on treated tissues.

- 168 042890 new culture white 96-well clean-bottomed plates (100 µl/well), overnight at 4°C. The wells were washed three times with DPBS and then seeded at 100,000 cells/well in medium with 1% serum. PMA (0.05 μg/ml) and ionomycin (0.25 mM) were added as positive controls for signaling. Cells were incubated for 6 hours and luciferase activity was measured by adding OneGlo Reagent (Promega) to each well and incubating for 3 minutes at room temperature on a plate shaker. The luciferase signal was measured using a BioTek plate reader.

As illustrated in FIG. 31A and 31B, antibodies to TREM2 At1, At9, At10, At14, At15, At17, At20, At22, At45, At54, At64, At65, and At66 increased luciferase activity, indicating that the antibodies are capable of inducing TREM2-dependent gene transcription. . As illustrated in FIG. 31C bound on the plate phosphatidylsern (PS) induces TREM2-dependent gene expression. PS is believed to be a natural TREM2 ligand. Thus, the results illustrated in FIG. 31A-C show that anti-TREM2 agonist antibodies can mimic natural TREM2 ligand.

Example 55 Assay for the ability of anti-TREM2 antibodies to inhibit TREM2-dependent genes.

The ability of soluble full-length antibodies to TREM2 At9, At14, At22, At45 and At65 to inhibit TREM2-dependent genes was assessed using the luciferase reporter gene under the control of the NFAT promoter (nuclear factor of activated T cells).

A cell line derived from BW5147.G.1.4 mouse thymic lymphoma T-lymphocytes (ATCC® TIB48™) was infected with mouse Trem2 and Dapl2 and Cignal Lenti NFAT-Luciferase virus (Qiagen). Soluble full length anti-TREM2 antibodies were added to the cells at elevated concentrations. Cells were incubated for 6 hours at 37° C. and luciferase activity was measured using OneGlo Reagent (Promega).

Cells display tonic TREM2-dependent signaling due to the presence of an endogenous ligand or spontaneous receptor clustering resulting in TREM2 signaling.

The dotted line in Fig. 32 shows TREM2 activity levels without stimulation.

As illustrated in FIG. 32, soluble full-length anti-TREM2 antibodies At9, At14, At45 and At65 were able to inhibit tonic, TREM2-dependent gene expression. In contrast, soluble full-length anti-TREM2 antibody At22 does not appear to block TREM2 tonic signaling (FIG. 32).

Example 56 Summary of the agonist and antagonist activity of the TREM2 antibody.

In table. 12 shows the results of the function studies described in Examples 52-55 above. The antibodies At1, At9, At14, At22, At45 and At65 showed agonist activity in the activation of TREM2-dependent gene expression using the luciferase reporter gene (FIG. 31A) or the beta-GAL reporter gene (Table 12). As indicated in Table. 12, At1 and At65 showed an increased level of gene induction compared to At9, At22 and At45 when the beta-GAL reporter gene was used (Table 12). The antibodies At22, At45 and At65 also showed agonistic activity in promoting survival of innate immune cells (FIG. 29). In table. 12 further summarizes the results demonstrating the antagonistic effect of soluble, non-crosslinked Ab9, At14, At45, and At65 antibodies on inhibition of innate immune cell survival (FIG. 30). In contrast, the soluble, non-crosslinked Ab22 antibody has minimal antagonistic activity in inhibiting cell survival (FIG. 30).

Table 12. TREM2 antibody function studies

Antibody Luciferase Agonistic activity of an antibody betaGAL Agonistic activity of an antibody Survive Agonistic activity of an antibody Luciferase Antagonistic activity of an antibody in solution/antagonistic format At1 ++++ +++ nd nd At 9 ++++ + nd ++++ At14 ++++ nd nd +++ At22 ++++ + +++ - At 4 5 ++ + ++ ++++ At 6 5 +++ +++ + +++

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Control isotype

Example 57 Summary of Binding and Function Studies for Additional TREM2 Antibodies.

In table. 13 shows the results of a binding and function study for additional full-length TREM2 antibodies prepared as described in Example 40. The following results were obtained using the methods described in Examples 40 and 42.

Table 13. Binding and function studies for other TREM2 antibodies

Antibody IgG K _D 4TREM2-FC (M) Avid IgG K _D 4TREM2HIS (M) t IgG K _D mTREM2-Fc (M) Avid MFI cellular binding of mTget2 MFI cell binding hTget2 Phospho Syk ChDK Phospho Syk mdk At 2 5.92E-10 2.41E-08 N.V. 1432 1407 nd nd AtZ 4.90E-10 1.42E-08 N.V. 488 1693 nd nd At 4 9.71Е-10 P. F. N.V. 295 1343 nd nd At 5 6.97E-10 3.00E-08 N.V. 310 1336 - nd At 6 4.44Е-09 P. F. 8.51E-09 228 816 ND nd At 7 1.05Е-09 3.34E-08 1.11E-09 10934 1249 nd nd At 8 7.23E-10 1.50E-08 N.B. 523 1623 nd ND ATU 5.44Е-10 4.79E-09 P.F. 11486 1788 ++ ++ At11 9.34Е-10 7.20E-09 1.19E-09 5495 1909 nd nd At12 1.19E-09 2.68E-09 7.91E-09 1552 1930 - nd At13 1.49Е-09 6.67E-08 1.71E-09 3246 971 nd nd At 15 6.05Е-10 3.03E-09 2.80E-09 4755 1611 ND + At16 1.09Е-09 2.47E-09 1.36E-09 6382 1545 ND -

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At 17 7.92E-10 3.46E-09 1.67Е-09 10312 1604 nd + At18 5.13E-10 6.77Е-09 N.V. 5164 1924 ++ + At19 4.65E-10 4.49Е-09 N.V. 2304 1993 ++ nd At2 0 6.58E-10 3.62E-09 N.V. 11245 1556 nd + At2 3 9.67E-10 R.F. 6.83Е-09 919 2653 nd nd At2 4 4.79E-10 R.F. 9.32Е-09 2760 1882 nd + At2 5 6.84Е-10 4.51Е-09 5.98Е-09 2497 1884 nd - At2 6 4.81E-10 3.60E-09 N.V. 2551 1421 nd nd At27 1,ZZE-09 R.F. 6.39Е-09 2163 1316 nd nd At2 8 9.15E-10 R.F. 7.45Е-09 5248 1487 nd - At2 9 2.36E-09 R.F. 3.27E-09 4518 1193 nd - Am 3 0 1.32E-09 R.F. N.V. 399 1421 nd nd At31 4.51Е-10 6.82E-09 N.V. 551 1760 nd nd At32 9.95E-10 2.48Е-08 1.35Е-08 756 1202 nd nd At 33 1.42E-09 1.60E-08 N.V. 413 1352 nd nd At34 7.06Е-10 2.70E-08 N.V. 395 1770 nd nd At 35 2.21E-10 7.82E-09 6.44Е-09 931 1391 nd nd At 3 6 4.65E-10 4.01Е-09 N.V. 1258 1896 nd nd At 37 1.58Е-09 R.F. N.V. 1433 1434 nd nd At 3 8 9.60E-10 1.31Е-08 N.V. 297 1832 nd nd At 3 9 2.13E-09 R.F. 6.41Е-09 588 9029 nd nd Am 4 0 1.97E-10 7.97Е-09 6.03Е-09 577 1452 nd nd At41 2.14Е-09 R.F. 8.64Е-09 373 1017 nd nd At42 4.03Е-09 R.F. N.V. 387 1234 nd nd At43 4.37Е-09 R.F. N.V. 418 918 nd nd At 4 4 5.62E-10 9.46Е-09 N.V. 1206 1249 nd nd At 4 6 4.45E-10 1.68Е-08 2.46Е-09 4257 1528 nd nd

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At 4 7 4.31E-10 7.37Е-09 1.45Е-09 10123 1833 nd nd At4 8 3.50E-10 9.08Е-09 1.41Е-09 9770 1412 nd nd At 4 9 4.35E-10 1.74Е-08 2.08Е-09 4315 1392 ND nd Am 5 0 7.29E-10 3.64Е-08 N.V. 325 1435 nd nd At 51 2.84Е-10 1.64Е-09 N.V. 12077 1928 ++ + At 5 2 1.51Е-09 5.75Е-09 8.96E-11 15613 1411 +++ +++ At 53 6.35E-10 2.16E-08 N.V. 843 1338 nd nd At 5 4 5.00E-10 3.02E-09 N.V. 11001 1931 nd ++ At 5 5 2.22E-09 9.17Е-08 1.17E-09 6221 911 nd nd At 5 6 1.03Е-09 5.18E-08 2.71Е-09 743 1157 nd nd At 5 7 4.36E-10 1.83Е-08 5.98E-10 10226 1227 nd nd At 5 8 5.75E-10 2.73Е-09 N.V. 1870 1698 nd nd At 5 9 8.54Е-10 5.72E-09 N.V. 3901 1754 nd nd At 60 5.59Е-09 R.F. R.F. 1871 741 nd nd At 61 4.61E-09 R.F. N.V. 10517 1101 nd nd At62 7.72E-10 1.42E-08 R.F. 4746 1647 nd nd At 63 1.13E-09 4.96E-08 N.V. 1722 1347 nd nd At 6 4 9.45Е-10 6.57Е-08 R.F. 8676 1458 +++ + At 6 6 1.98Е-09 6.07Е-08 3.88E-09 4098 1161 nd + At 6 7 6.60E-10 3.45Е-08 2.92E-09 6207 1118 nd nd At 6 8 6.68Е-09 1.09Е-07 3.26E-09 2103 1135 nd nd At 6 9 3.75Е-09 5.97Е-08 3.40E-09 629 957 nd nd Am 7 0 6.26Е-09 R.F. 3.45Е-09 3265 833 nd nd At 71 2.77Е-09 6.47Е-08 N.V. 804 1556 nd nd At 7 2 8.39E-10 2.94Е-08 R.F. 3746 1500 nd nd At 7 3 4.10E-09 R.F. N.V. 1117 1307 nd nd At 7 4 9.43Е-09 R.F. 4.91Е-09 1360 645 nd nd

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At 7 5 5.88Е-09 R.F. N.V. 276 676 nd nd At 7 6 4.84Е-09 R.F. N.V. 1604 580 nd nd At 7 7 R.F. R.F. N.V. 1650 951 nd nd At 7 8 8.51Е-10 3.73E-08 N.V. 433 1435 nd nd At 7 9 1.07Е-08 R.F. N.V. 738 900 nd nd Am 8 0 5.72E-09 R.F. 8.85Е-09 593 897 nd nd At81 1.01Е-08 R.F. N.V. 400 994 nd nd At 8 2 1.10E-08 N.B. 5.82E-09 712 371 nd nd At 8 3 8.21Е-09 N.B. N.V. 1576 684 nd nd At 8 4 1.16E-09 6.04Е-08 N.V. 799 1148 nd nd Am 8 5 5.72E-09 R.F. 2.19E-09 1121 460 nd nd At 8 6 3.89Е-09 R.F. 6.55Е-09 355 840 nd nd At 8 7 9.51Е-09 N.B. 4.48Е-09 1228 414 ND nd 88 control No binding nd nd 90.7 187 - - 89 control No binding nd nd 142 185 Antibody Luciferase Agonistic activity of an antibody ATU ++++ At 15 ++++ At 17 ++++ At18 - At2 0 +++ At24 - At25 - At2 8 -

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Example 58 Analysis of the anti-stroke effect of TREM2 agonist antibodies.

Temporary occlusion of the middle cerebral artery (MCAO) is a model that is very similar to human stroke and is used to induce ischemic stroke in mice. A surgical monofilament (70SPRe, Doccol Corp, USA) is inserted into the internal carotid artery through an incision in the right common carotid artery. The middle cerebral artery is occluded for 30 minutes with a reperfusion time interval (6, 12, 24 hours, 2, 7 and 28 days). The effect of surgery is monitored using control animals after 12 hours and on day 7. Control animals undergo the same surgical procedure, but without occlusion of the middle cerebral artery. MCAO animals treated with anti-TREM2 agonist antibodies or control antibodies are tested for infarct volumetry, acute inflammatory response (12 h reperfusion), transcription of the pro-inflammatory cytokines TNFa, IL-1a and IL-1b, microglial activity (CD68, Iba1), transcription of CCL2 chemokines (MCB1), CCL3 (MIP1a and chemokine receptor CX3CR1, and CD3-positive T cell invasion (Sieber et al. (2013) PLoS ONE 8(1): e52982. doi:10.1371/journal.pone.0052982.).

Example 59 Analysis of the protective effect of TREM2 antagonistic antibodies in respiratory tract infections.

To evaluate the ability of anti-TREM2 antagonist antibodies to slow, prevent, or treat respiratory tract infections, a preclinical mouse model involving infection of C57B16 mice with Streptococcus pneumoniae was used. This model includes intranasal (intraperitoneal) administration of 105 cfu of S. pneumoniae serotype 3 (ATCC 6303) as described (see e.g. Sharif O. et al., 2014 PLoS Pathog. 2014 Jun; 10(6): e1004167; and Schabbauer G. et al., 2010 J. Immunol. 185: 468476). In this model, 90% of C57B16 DT mice die from infection within 6 days of infection.

Ten to fifteen mice/group were challenged with S.pneumoniae and simultaneously treated with anti-TREM2 antagonist antibodies every other day, starting on day 0. The first dose of anti-TREM2 antibodies was administered 3 hours prior to S.pneumoniae challenge. Mice were examined daily for 15 days to check for deaths. The % of surviving mice after infection with bacteria was determined.

In some embodiments of the invention, the amount of bacterial load and the expression of cytokines in the blood and in the lungs were also determined. 24 or 48 hours after infection, blood was collected in tubes containing EDTA and placed on agar plates to enumerate bacterial CFU in plasma. Plasma was stored at -20° C. for cytokine analysis by ELISA. Lungs were harvested, homogenized and plated on agar plates for bacterial CFU enumeration or incubated for 30 min in lysis buffer and supernatants analyzed for cytokine measurements.

In separate experiments, lungs were harvested 40 hours after bacterial infection, fixed in 10% formalin, and embedded in paraffin for pathology analysis using the H&E method.

Example 60 Analysis of the protective effect of TREM2 antagonist antibodies in sepsis.

To assess the ability of anti-TREM2 antagonist antibodies to slow, prevent, or treat sepsis, a preclinical mouse model was used in C57B16 mice with systemic LPS. This model includes intraperitoneal (ip) administration of 37 mg/ml LPS as described (see, for example, Gawish R. et al., 2014 FASEB J). In this model, >95% of C57B16 DT mice die from infection within 40 h after LPS injection.

Mice cohorts were injected with LPS and simultaneously treated with anti-TREM2 antagonistic antibodies every day starting from day 0. The first dose of anti-TREM2 antibodies was administered 3 h prior to LPS administration. Mice were examined every ~4 h during the daytime to check for deaths. The percentage of surviving mice after LPS administration was determined.

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In separate experiments, peritoneal lavage fluid (PLF) was collected. Supernatants were stored at -20°C for analysis of cytokines by ELISA; the precipitated cells were counted to quantify the inflammatory cells recruited in the peritoneal cavity. Similar studies can be performed to test the efficacy of TREM2 antibodies in other models of infection (see, for example, Sun et al., (2013) Invest. Ophthalmol. Vis. Sci. 17;54(5):3451-62).

Example 61 Analysis of the protective effect of TREM2 antagonistic antibodies in acute and chronic colitis.

Preclinical mouse models of acute or chronic colitis are used to evaluate the ability of anti-TREM2 antagonist antibodies to slow, prevent, or treat colitis. For DSS-induced colitis, mice receive 3% DSS in drinking water ad libitum for 8 days. For TNBS-induced colitis, mice were anesthetized and treated with an intrarectal injection of 3 mg TNBS in 20% ethanol (v/v) or vehicle alone as controls. For the chronic colitis model, all mice were treated with 3 cycles of 2% DSS for 5 days followed by a 10 day recovery period. For all models, weight loss, stool consistency, and presence of fecal occult blood were monitored daily and used to calculate disease activity index as described (see e.g. Correale C, 2013, Gastroenterology, February 2013, pp. 346-356.e3) .

A cohort of mice were treated with anti-TREM2 agonist antibodies every day starting on day 0 and treated with DSS or TNBS. Mice were examined daily for weight loss, fecal consistency, and fecal occult blood and used to calculate disease activity index as described (see e.g. S. Vetrano, Gastroenterology, 135 (2008), pp. 173-184) .

In separate experiments, endoscopic and histological images of mucosal lesions were collected to evaluate inflammatory cell infiltration and mucosal injury. Similar studies could be done to test the benefit of TREM2 antibodies in other autoimmune models including Crohn's disease, inflammatory bowel disease, and ulcerative colitis (see, e.g., Low et al., (2013) Drug Des Devel. Ther.; 7:1341- 1357 and Sollid et al., (2008) PLoS Med 5(9): e198).

Example 62 Analysis of the protective effect of TREM2 agonistic antibodies in wound healing.

To assess the ability of anti-TREM2 agonist antibodies to improve colonic wound healing after injury, a mouse model of biopsy injury in the colon was used. In this model, an endoscope with an outer operating sheath is inserted into the middle descending colon and the mucosa is examined prior to the anorectal junction. Then, one full-thickness area of the entire mucosa and submucosa is removed using flexible biopsy forceps with a diameter of 3 French diameters, while avoiding penetration into the muscular layer. Each mouse was biopsied at 3-5 sites along the dorsal side of the colon (see e.g. Seno H., 2008, Proc. Natl. Acad. Sci. USA. 2009 Jan. 6; 106(1): 256-261) .

A cohort of mice were treated with anti-TREM2 agonist antibodies 2 or 3 days after biopsy injury. The mice are monitored every day for 15 days to check for weight loss and wound healing by measuring the surface area of the lesions.

Example 63 Analysis of the protective effect of TREM2 antagonist antibodies in retinal degeneration.

TREM2 antagonist antibodies reduce the accumulation and/or function of inflammatory macrophages and, as a result, slow, prevent and/or treat age-related macular degeneration (AMD).

AMD is a degenerative disease of the outer retina. Inflammation, especially inflammatory cytokines and macrophages, is believed to contribute to the progression of AMD.

The presence of macrophages in the immediate vicinity of AMD lesions has been documented in drusen, Bruch's membrane, choroid, and retina. Macrophages secrete tissue factor (TF) and vascular endothelial growth factor (VEGF), which triggers the formation of new blood vessels in patients showing choroidal neovascularization.

The type of macrophages present in the macula vasculature of the eye changes with age, showing increased levels of M2 macrophages in the eyes of older people compared to the eyes of younger people. However, dilated AMD patches had higher M1 to M2 ratios compared to normal age-matched autopsy eyes (see e.g. Cao X et al. (2011), Pathol. Int. 61(9): pp.528- 35). This suggests a link between classical M1 macrophage activation in the eye during late onset AMD progression.

Retinal microglial cells are resident tissue macrophages that are also commonly present in the inner retina. In case of damage, microglia can be activated and act as an inflammatory mediator. Activated microglia have been found in AMD tissue samples and have been proposed as one of the potential participants in the inflammatory process that leads to the pathogenesis of AMD (Gupta et al., (2003) Exp. Eye Res., 76(4): 463-71). The ability of TREM2 antagonist antibodies to prevent, delay, or reverse AMD is tested in one or more models of AMD (see, for example, Pennesi et al., (2012) Mol. Aspects Med.; 33(4): 487-509).

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Common inflammatory macrophages (or M1 and/or activated microglia) have been documented as being correlated with AMD disease progression and therefore represent a therapeutic target for TREM2 antagonist antibodies. Similar therapeutic benefits may be achieved in glaucoma and genetic forms of retinal degeneration such as retinitis pigmentosa.

The ability of TREM2 antagonist antibodies to prevent, slow or reverse retinal ganglion cell degeneration in glaucoma has been tested in a glaucoma model (see, for example, ElDanaf et al., (2015). J. Neurosci. 11;35(6):2329-43). Similarly, the therapeutic benefit of TREM2 in genetically induced retinal degeneration and retinitis pigmentosa is being tested as described in Chiang et al., (2002) Vision Res.; 42(4):517-25 and in Retinal Degeneration Rat Model Resource Availability of P23H and S334ter Mutant Rhodopsin Transgenic Rats and RCS Inbred and RCS Congenic Strains of Rats, MM LaVail, June 30, 2011.

Example 64 Analysis of the protective effect of TREM2 antagonist antibodies in adipogenesis and diet-induced obesity.

To test the effect of TREM2 antagonist antibodies on adipogenesis and obesity, a high fat diet (HFD) mouse model is used (see, e.g., Park et al., (2015) Diabetes. 64(1):117-27).

Example 65 Analysis of the protective effect of TREM2 antibodies against malaria.

Expression of TREM2 in non-parenchymal liver cells is closely correlated with resistance to the stage of liver infection with the malarial agent Plasmodium berghei (Gongalves et al. (2013) Proc. Natl. Acad. Sci. 26;110(48):19531-6). Without being bound by theory, it is believed that TREM2 antibodies increase resistance to the P. berghei stage of liver infection.

The ability of TREM2 antibodies to increase resistance to malaria infection was tested as described in Gongalves et al., (2013) Proc. Natl. Acad. sci. 26;110(48):19531-6. Briefly, GFP-expressing P. berghei ANKA sporozoites were obtained by dissection of infected salivary glands of Anopheles Stephensi mosquitoes. Sporozoite suspensions in RPMI medium were injected intravenously into 100 μl of an inoculum containing 10 ⁴ sporozoites per mouse. The liver was harvested 40 hours after injection or survival, and parasitemia was carried out for 28 days. For experimental evaluation of cerebral malaria, neurological symptoms were monitored from the 5th day after injection.

Example 66 Analysis of the protective effect of TREM2 antagonistic antibodies in osteoporosis.

Bone is a dynamic organ, constantly remodeling to maintain calcium homeostasis and structural needs. The osteoclast is the cell responsible for removing both organic and inorganic bone components. The osteoclast is derived from hematopoietic progenitors in the macrophage lineage and differentiates in response to the action of cytokine receptor activators of the NFkB ligand tumor necrosis factor family. Osteoclasts, as the only cells that resorb bone tissue, play a central role in the pathogenesis of osteoporosis and osteopetrosis (Novack et al., (2008) Annual Rev Pathol., 3:457-84).

Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which can lead to an increased risk of fracture. This is mainly manifested in the first years after menopause, when bone remodeling is accelerated, with an increase in the activity of both osteoclasts and osteoblasts. However, due to an imbalance in the processes of resorption and synthesis, the overall effect is the loss of bone tissue, which is largely the trabecular substance of the bone. Thus, the most common fracture sites in osteoporosis are the wrist, femoral neck, and vertebral bodies, in which the trabecular structure is a key factor in overall bone strength. Accelerated osteoclast differentiation and increased bone resorption capacity leading to osteoporosis have been described in animal models lacking TREM2 expression (Otero et al. (2012) J. Immunol. 188, 2612-2621).

Decreased osteoclast function leads to osteopetrosis, with increased bone mass and elimination of marrow space, as observed in animal models lacking the DAP12 ITAM signaling adapter, and also results in a significant defect in osteoclast-like cell differentiation (Koga, et al. (2004) Nature 428: 758-763).

Without being limited by theory, it is believed that administration of an anti-TREM2 antagonist antibody of the present disclosure can prevent, reduce the risk, and/or cure osteoporosis. In some embodiments, administration of an anti-TREM2 agonist antibody can induce one or more TREM2 activities in an individual having osteoporosis (e.g., DAP12 phosphorylation, Syk activation, and accelerated differentiation into osteoclasts) (Peng et al. (2010). Sci. Signal. 2010 18; 3 122; and Humphrey et al., (2006) J. Bone Miner Res., 21(2):237-45).

Example 67 Ability of the At22 and At45 TREM2 antibodies to block the induction of TREM2-dependent gene expression.

Antibodies At22 and At45 were tested for their ability to block the induction of TREM2-dependent gene expression by lipid ligands using a luciferase reporter system.

Phosphatidylserine (PS, Avanti Lipids) and sphingomyelin (SM, Avanti Lipids) were dissolved in methanol and

- 176 042890 were plated on 96-well Immulon plates. The methanol was evaporated overnight at room temperature and the cells were added the next day. BWZ cells expressing mouse TREM2 and DAP12 in combination with NFAT:luciferase reporter (Qiagen) were harvested and added at 100,000 cells/well in DMEM+1% FBS. Cells were either seeded alone or in combination with 50 or 5 nM human IgG. Six hours later, luciferase activity was measured using the OneGlo reagent (Promega) and Biotek plate reader, according to the manufacturer's instructions.

At45 blocks the induction of TREM2-dependent gene expression by both PS and SM, as evidenced by a decrease in luminescence (Fig. 33).

Example 68 Assay for the ability of TREM2 antibodies to induce TREM2 dependent cytokine genes.

The ability of Fab-coupled anti-TREM2 antibodies At22 and At65 to activate TREM2-dependent genes was assessed using primary mouse macrophages.

Bone marrow-derived macrophages (BMDM) were prepared with M-CSF for 5 days. BMDM (105/well) were plated in 96-well plates which were pre-coated with the Fab portion of the Ab22 and At65 antibodies (50 nM). Ultrapure LPS (100 ng/ml Escherichia coli 0111:B4) was added after centrifuging the plates for 1 min at 1200 rpm. After 18 hours of stimulation with LPS, cytokines present in cell supernatants were measured by microsphere cytometric assay (CBA; BD Biosciences, CBA Mouse Inflammation Kit).

As illustrated in FIG. 34A and 34B, plate-coupled, Ab22 anti-TREM2 Fab increased IL-6 and MHB-1 levels, indicating the antibody's ability to activate TREM2-dependent cytokine genes in primary immune cells.

Claims (43)

1. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the antibody is derived from an epitope containing amino acid residues 43-50 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. 2. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the antibody is derived from an epitope containing amino acid residues 49-57 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1. 3. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the antibody is derived from an epitope containing amino acid residues 140-153 of SEQ ID NO: 1 or amino acid residues on the TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1. 4. An isolated antibody according to any one of claims 1 to 3, wherein said antibody promotes the survival of one or more cells of the innate immune system, selected from macrophages or microglial cells, when cross-linked or linked on a plate. 5. An isolated antibody according to any one of claims 1-4, wherein the antibody competes for binding to TREM2 with one or more TREM2 ligands. 6. The isolated antibody of claim 5, wherein the one or more TREM2 ligands are selected from the group consisting of E. coli cells, phosphatidylserine, and sphingomyelin. 7. An isolated antibody according to any one of claims 1 to 6, wherein the antibody: i. increases the expression of CD83 and CD86; and/or ii. induces Syk phosphorylation; and/or iii. increases the expression of IL-6; and/or iv. increases the expression of MHB-1. 8. An isolated antibody according to any one of claims 1 to 7, wherein the isolated antibody: i. has a dissociation constant (KD) for the human TREM2-Fc fusion protein that ranges from about 0.23 nM to about 1.51 nM; and/or ii. has a dissociation constant (K _D ) for the monomeric human TREM2 protein that ranges from about 0.65 nM to about 5.75 nM. 9. An isolated antibody according to any one of claims 1 and 4-8, wherein when the antibody is not cross-linked or linked on the plate, the antibody does not inhibit macrophage survival. 10. An isolated antibody according to any one of claims 2-8, wherein when the antibody is not cross-linked or linked on the plate, the antibody inhibits macrophage survival. 11. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 404, HVR- H2 containing the amino acid sequence of SEQ ID NO: 405, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 406, and the light chain variable domain contains HVR-L1 containing the amino acid sequence - 177 042890 validity of SEQ ID NO: 407, HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 409. 12. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the isolated antibody contains a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO: 11, HVR- H2 containing the amino acid sequence of SEQ ID NO: 34, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 60, and the light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 123, HVR-L2 containing the amino acid the sequence of SEQ ID NO: 141, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 173. 13. An isolated antibody according to any one of claims 1 and 4-9, wherein the isolated antibody competes with a reference anti-TREM2 antibody for binding to the TREM2 protein, wherein the reference anti-TREM2 antibody contains a heavy chain variable domain and a light chain variable domain, wherein: i. the heavy chain variable domain contains HVR-H1 containing the amino acid sequence of SEQ ID NO: 404, HVR-H2 containing the amino acid sequence of SEQ ID NO: 405, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 406, and a light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 407, HVR-L2 containing the amino acid sequence of SEQ ID NO: 408, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 409; or ii. the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 410, and the light chain variable domain contains the amino acid sequence of SEQ ID NO: 411. 14. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the isolated antibody contains a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR- H2 containing the amino acid sequence of SEQ ID NO: 29, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 87, and the light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 120, HVR-L2 containing the amino acid the sequence of SEQ ID NO: 138, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 196. 15. An isolated human or humanized antibody that specifically binds to the TREM2 protein, wherein the isolated antibody contains a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO: 398, HVR- H2 containing the amino acid sequence of SEQ ID NO: 399, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 400, and the light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 401, HVR-L2 containing the amino acid the sequence of SEQ ID NO: 402, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 403. 16. An isolated antibody according to any one of claims 3-8 and 10, wherein the isolated antibody competes with a reference anti-TREM2 antibody for binding to the TREM2 protein, wherein the reference anti-TREM2 antibody contains a heavy chain variable domain and a light chain variable domain, wherein: i. the heavy chain variable domain contains HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 29, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 87, and a light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 120, HVRL2 containing the amino acid sequence of SEQ ID NO: 138, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 196; or ii. the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 322, and the light chain variable domain contains the amino acid sequence of SEQ ID NO: 323. 17. An isolated antibody according to any one of claims 2, 4-8 and 10, wherein the isolated antibody competes with a reference anti-TREM2 antibody for binding to the TREM2 protein, wherein the reference anti-TREM2 antibody contains a heavy chain variable domain and a light chain variable domain, wherein: i. the heavy chain variable domain contains HVR-H1 containing the amino acid sequence of SEQ ID NO: 398, HVR-H2 containing the amino acid sequence of SEQ ID NO: 399, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 400, and a light chain variable domain contains HVR-L1 containing the amino acid sequence of SEQ ID NO: 401, HVR-L2 containing the amino acid sequence of SEQ ID NO: 402, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 403; or ii. the heavy chain variable domain contains the amino acid sequence of SEQ ID NO: 412, and the light chain variable domain contains the amino acid sequence of SEQ ID NO: 413. 18. An isolated antibody according to any one of claims 1 to 17, wherein the antibody is of the IgG class, the IgM class, or the IgA class. 19. An isolated antibody according to claim 18, wherein the antibody belongs to the IgG class and has the IgG1 isotype, - 178 042890 IgG2, IgG3 or IgG4. 20. An isolated antibody according to claim 19, wherein: i. the isolated antibody is human IgG2 isotype and includes one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T , T256E and any combination thereof, with the numbering of the residues presented in accordance with the EU numbering system; ii. the isolated antibody has a human IgG2 isotype, wherein the human IgG2 contains a constant region, and while the human IgG2 constant region contains a light chain constant region containing the C214S amino acid substitution, the numbering of the residues is presented in accordance with the EU numbering system; iii. the isolated antibody is of human or murine IgG1 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E , P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, with residue numbering according to the EU numbering system; iv. the isolated antibody has an IgG1 isotype and contains a heavy chain constant domain 1 (CH1) of the IgG2 isotype and a hinge region; v. the isolated antibody is human or mouse IgG4 isotype and contains one or more amino acid substitutions in the Fc region at a position of a residue selected from the group consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E and their any combination, and the numbering of the residues is presented in accordance with the EU numbering system; or vi. the isolated antibody has an IgG2/4 hybrid isotype. 21. The isolated antibody of claim 20, wherein the isolated antibody is of human or murine IgG1 isotype and contains one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of N297A, D265A, L234A, L235A, G237A, C226S , C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof, wherein the residue numbering is in accordance with the EU numbering system, and wherein the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of A330L, L234F, L235E, P331S, and any combination thereof, the residue numbering being in accordance with the EU numbering system. 22. An antibody fragment of an isolated antibody according to any one of claims 1-21, wherein the antibody fragment is a Fab, Fab', Fab'-SH, F(ab') ₂ , Fv or scFv fragment. 23. An isolated bispecific antibody that binds to the TREM2 protein, wherein the isolated bispecific antibody comprises an antibody or antibody fragment according to any one of claims 1-22. 24. An isolated multivalent antibody that binds to the TREM2 protein, wherein the isolated multivalent antibody comprises an antibody or antibody fragment according to any one of claims 1-22. 25. An isolated antibody, antibody fragment, bispecific antibody, or multivalent antibody according to any one of claims 1 to 24, wherein the antibody, antibody fragment, bispecific antibody, or multivalent antibody is a monoclonal antibody, a monoclonal antibody fragment, a monoclonal bispecific antibody, or a monoclonal multivalent antibody. 26. An antibody fragment of an isolated antibody according to any one of claims 1-21 or an antibody fragment according to claim 22, wherein the antibody fragment binds to one or more human proteins selected from the group consisting of wild-type human TREM2 and a naturally occurring human TREM2 variant wherein the antibody fragment is cross-linked with a second antibody fragment that binds to one or more human proteins selected from the group consisting of wild type human TREM2, naturally occurring human TREM2 variant, human DAP12, and naturally occurring human DAP12 variant. 27. An isolated bispecific antibody according to claim 23 or 25, wherein the bispecific antibody recognizes a first antigen and a second antigen, wherein the first antigen is wild type human TREM2 or a naturally occurring variant thereof and the second antigen is DAP12 or a protein selected from the group , consisting of beta-amyloid or its fragments, Tau, IAPP, alphasynuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide peptides repeat (DPP), glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeat peptides; or a blood-brain barrier specific protein selected from the group consisting of: transferrin receptor, insulin receptor, insulin-like growth factor receptor, LRP-1 and LRP1. 28. An isolated antibody or antibody fragment according to any one of claims 1 to 22, characterized in that you - 179 042890 the divided antibody or antibody fragment binds to one or more human proteins selected from the group consisting of wild-type human TREM2 and a naturally occurring variant of human TREM2. 29. An isolated antibody, bispecific antibody, multivalent antibody, or antibody fragment according to any one of claims 1-28, wherein the isolated antibody, bispecific antibody, multivalent antibody, or antibody fragment specifically binds to both human TREM2 and murine TREM2. 30. An isolated nucleic acid encoding an antibody according to any one of claims 1-21, 25, 28 and 29. 31. An isolated nucleic acid encoding a bispecific antibody according to any one of claims 23, 25, 27 and 29. 32. An isolated nucleic acid encoding a multivalent antibody according to any one of claims 24, 25 and 29. 33. An isolated nucleic acid encoding an antibody fragment according to any one of claims 22, 25, 26, 28 and 29. 34. A vector containing a nucleic acid according to any one of claims 30-33. 35. An isolated host cell containing the vector of claim 34. 36. A pharmaceutical composition containing an isolated antibody, a bispecific antibody, a multivalent antibody, or an antibody fragment according to any one of paragraphs. 1-29 and a pharmaceutically acceptable carrier. 37. A method of preventing, reducing the risk of, or treating an individual with dementia, frontotemporal dementia, Alzheimer's disease, or multiple sclerosis, comprising administering to the individual a therapeutically effective amount of an isolated antibody, bispecific antibody, multivalent antibody, or antibody fragment according to any one of claims 1- 29. 38. The method of claim 37, wherein the subject has Alzheimer's disease. 39. The method according to claim 37 or 38, wherein the individual has a heterozygous TREM2 variant, wherein the variant contains one or more substitutions selected from the group consisting of: i. replacement of glutamic acid with a stop codon in the nucleic acid sequence encoding the amino acid residue Glu14 of SEQ ID NO: 1; ii. replacing glutamine with a stop codon in the nucleic acid sequence encoding the amino acid residue Gln33 of SEQ ID NO: 1; iii. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the Trp44 amino acid residue of SEQ ID NO: 1; iv. amino acid substitution of arginine for histidine in the amino acid corresponding to the amino acid residue Arg47 of SEQ ID NO: 1; v. replacement of tryptophan with a stop codon in the nucleic acid sequence encoding the amino acid residue Trp78 of SEQ ID NO: 1; vi. amino acid substitution of valine for glycine in the amino acid corresponding to the amino acid residue Val126 of SEQ ID NO: 1; vii. amino acid substitution of aspartic acid with glycine in the amino acid corresponding to the amino acid residue Asp134 of SEQ ID NO: 1; and viii. amino acid substitution of lysine for asparagine at the amino acid corresponding to the amino acid residue Lys186 of SEQ ID NO: 1. 40. The method according to item 37 or 38, characterized in that the individual has a heterozygous variant of TREM2, and the variant contains a nucleotide deletion of guanine in the nucleotide corresponding to the nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a nucleotide deletion of guanine at the nucleotide corresponding to the nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both of these deletions. 41. The method according to claim 37 or 38, wherein the individual has a heterozygous DAP12 variant, the variant comprising one or more variants selected from the group consisting of: i. replacing methionine with threonine at the amino acid corresponding to the amino acid residue Met1 of SEQ ID NO: 2; ii. amino acid substitution of glycine for arginine in the amino acid corresponding to the amino acid residue Gly49 of SEQ ID NO: 2; iii. deletions within exons 1-4 of the nucleic acid sequence encoding SEQ ID NO: 2; iv. inserts of 14 amino acid residues in exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2; And v. nucleotide deletion of guanine at the nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2. 42. The method of any one of claims 37-41, wherein the isolated antibody, bispecific antibody, multivalent antibody, or antibody fragment is administered in combination with one or more antibodies that specifically bind a protein selected from the group consisting of beta-amyloid or - 180 042890 fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPsc prion protein, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy bodies, atrial natriuretic peptide, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta-2-microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin AL light chain, S-IBM protein, translation products associated with non-ATG repeats (RAN), dipeptide peptides repeat (DPP), glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, proline-arginine (PR) repeat peptides, and any combination of them. 43. The use of an isolated antibody, bispecific antibody, multivalent antibody, or antibody fragment according to any one of claims 1 to 29 to induce or promote survival of macrophages or microglial cells in an individual in need thereof.