US20170240631A1 - Anti-trem2 antibodies and methods of use thereof - Google Patents

Anti-trem2 antibodies and methods of use thereof Download PDF

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US20170240631A1
US20170240631A1 US15/502,766 US201515502766A US2017240631A1 US 20170240631 A1 US20170240631 A1 US 20170240631A1 US 201515502766 A US201515502766 A US 201515502766A US 2017240631 A1 US2017240631 A1 US 2017240631A1
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amino acid
antibody
trem2
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Kate MONROE
Tina SCHWABE
Francesca AVOGADRI-CONNORS
IIaria TASSI
Helen Lam
Arnon Rosenthal
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This invention relates to anti-TREM2 and anti-DAP12 antibodies and therapeutic uses of such antibodies.
  • Triggering receptor expressed on myeloid cells-2 is an immunoglobulin-like receptor that is expressed primarily on myeloid lineage cells, such as macrophages, dendritic cells, monocytes, Langerhans cells of skin, Kupffer cells, osteoclasts, and microglia; and is required for modulation (e.g., suppression) of Toll-like receptor (TLR) signaling, the modulation of inflammatory cytokines, as well as for normal osteoclast development.
  • TLR Toll-like receptor
  • TREM2 was discovered as a member of the TREM transmembrane glycoproteins, which belong to the single immunoglobulin variable (IgV) domain receptor family.
  • the genes encoding human and mouse TREMs map to human chromosome 6p21.1 and mouse chromosome 17C3, respectively.
  • the TREM cluster includes genes encoding TREM1, TREM2, TREM4, and TREM5, as well as the TREM-like genes in both human and mouse. Additionally TREM3 and plasmocytoid dendritic cell (pDC)-TREM were identified in mouse.
  • TREM1 and TREM2 display some sequence homology with other members of the Ig-SF such as activating NK cells receptors (20% identity with NKp44) and act through association with a DAP12-mediated pathway for signaling.
  • 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 is reduced to 26 kDa after N-deglycosylation.
  • the TREM2 gene encodes a 230 amino acid-length protein that includes an extracellular domain, a transmembrane region and a short cytoplasmic tail.
  • the extracellular region, encoded by exon 2 is composed 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 lacks signaling motifs and is thought to signal through the signaling adaptor molecule DAP12/TRYROBP.
  • the signaling adaptor molecule DAP12 is expressed as a homodimer at the surface of a variety of cells participating in innate immune response, including microglia, macrophages, granulocytes, NK cells, and dendritic cells (DC).
  • DAP12 is a member of the type I transmembrane adapter protein family on the basis of homology with the human T-cell receptor (TCR)-associated CD3 chains and the Fc receptor (FcR) ⁇ -chain (Turnbull, I R and Colonna, M, Nat Rev Immunol, 2007. 7(2): p. 155-61).
  • ITAM motifs in their cytoplasmic domain, charged acidic residue in transmembrane region (critical for interaction with its partner chain) and the ability to recruit Src homology domain-2 (SH2)-containing proteins following tyrosine phosphorylation.
  • the ITAM motif mediates signal propagation by activation of the ZAP70 or Syk tyrosine kinase. Both kinases phosphorylate several substrates, thereby facilitating the formation of a signaling complex leading to cellular activation.
  • B-cells and T-cells also express DAP12 under inflammatory conditions.
  • CD4 + CD28 ⁇ T-cells In humans, subsets of CD4 + CD28 ⁇ T-cells, ⁇ TCR + 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).
  • this protein is believed to be expressed in other macrophage-related cells, such as osteoclasts in the bone marrow, Kupffer cells in the liver, alveolar macrophages of the lung, Langerhans cells of skin, and microglial cells in the brain (Takaki, R et al., Immunol Rev, 2006. 214: p. 118-29).
  • TREM2 has been identified as expressed on the surface of human monocyte-derived dendritic cells and as an mRNA transcript in the mouse macrophage cell line RAW264 (Bouchon, A et al., J Exp Med, 2001. 194(8): p. 1111-22).
  • Human TREM2 was the first DAP12-associated receptor described on the surface of DCs.
  • Studies have demonstrated that TREM2 cell surface expression is reduced in DAP12-deficient bone marrow-derived dendritic cells (BMDCs) and in DAP12-deficient macrophages, as compared to wild-type cells (Ito, H and Hamerman, J A, Eur J Immunol. 42(1): p.
  • TREM2 expression was not always found in other cell populations, such as tissue-resident macrophages, circulating monocytes, or the corresponding progenitor cells in the bone marrow, suggesting that TREM2 expression is not induced centrally, but locally during tissue infiltration or by cytokine-mediated activation.
  • IFN- ⁇ and LPS reduce or otherwise abrogate TREM2 expression.
  • TREM2 is highly expressed on microglia and infiltrating macrophages in the central nervous system during experimental autoimmune encephalomyelitis or Alzheimer's disease (Piccio, L et al., Eur J Immunol, 2007. 37(5): p. 1290-301; and Wang Y, Cell. 2015 Mar. 12; 160(6):1061-71).
  • TREM2 signals through DAP12. Downstream this leads to activation of the Syk/Zap70 tyrosine kinase family, PI3K, and other intracellular signals.
  • TLR signals are important for activation, such as with infection response, but also play a key role in the pathological inflammatory response, such as with 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).
  • TREM2 or DAP12 Deficiency of either TREM2 or DAP12 is thought to lead to increased pro-inflammatory signaling.
  • 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.
  • TREM2 is thought to activate signaling pathways involved in cell survival (e.g., protein kinase B-Akt), cell activation and differentiation (e.g., Syk, Erk1/2, PLC- ⁇ , etc.), and in the control of the actin cytoskeleton (e.g., Syk, Vav, etc.) (Peng, Q et al., Sci Signal. 3(122): p. ra38; and Whittaker, G C et al., J Biol Chem. 285(5): p. 2976-85).
  • signaling pathways involved in cell survival e.g., protein kinase B-Akt
  • cell activation and differentiation e.g., Syk, Erk1/2, PLC- ⁇ , etc.
  • actin cytoskeleton e.g., Syk, Vav, etc.
  • the ITAM tyrosines in DAP12 are phosphorylated by SRC-family kinases leading to the recruitment and activation of the Syk kinase and/or ZAP70 kinase.
  • Syk may be the predominant kinase involved, whereas in humans both Syk and ZAP70 appear to couple efficiently with such ITAM-containing subunits, binding them through their tandem SH2 domains.
  • TREM2 signaling has shown that, like TREM1, TREM2-mediated signaling through DAP12 also leads to an increase in intracellular calcium ion levels and ERK1/2 phosphorylation of ERK1/2 (Bouchon, A et al., J Exp Med, 2001. 194(8): p. 1111-22; and Sharif, O and Knapp, S, Immunobiology, 2008. 213(9-10): p. 701-13).
  • TREM2 receptor ligation does not induce the degradation of IkB-a and the subsequent nuclear translocation of NF-kB, which points to a possible difference between TREM2 and TREM1 signaling (Bouchon, A et al., J Exp Med, 2001. 194(8): p.
  • TREM2 Receptor cross-linking of TREM2 on immature dendritic cells triggers the up-regulation of molecules involved in T-cell co-stimulation, such as CD86, CD40, and MHC class II, as well as the up-regulation of the chemokine receptor CCR7 (Bouchon, A et al., J Exp Med, 2001. 194(8): p. 1111-22).
  • TREM2 is also expressed on microglia, where receptor cross-linking results in an increase in ERK1/2 phosphorylation and CCR7, but not an increase in CD86 or MHC class II expression, suggesting possible cell type-specific differences in TREM2 signaling.
  • TREM2 over-expression of TREM2 in myeloid cells resulted in an increase in 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): p. 92-9).
  • BMDM bone marrow-derived macrophages
  • shRNAi bone marrow-derived macrophages
  • TREM2 overexpression in microglia has been demonstrated to lead to a decrease in TNF and inducible nitric oxide (iNOS) mRNA after culture of these cells with apoptotic neurons, whereas TREM2 knockdown resulted in a modest increase in TNF and iNOS mRNA levels.
  • 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 the type of macrophage as it occurs in both microglia and BMDM cells.
  • microglia activation has also been implicated in frontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease, stroke/ischemic brain injury, and multiple sclerosis.
  • FTD frontotemporal dementia
  • TREM2 activation leads to increases in certain activation and inflammation markers, such as NOS2 gene transcription in myeloid cells
  • increased TREM2 activation leads to reduced NOS2 transcription.
  • dying neurons express an endogenous ligand for TREM2.
  • HSP60 has been implicated as a ligand of TREM2 on neuroblastoma cells (Stefani, L et al., (2009) Neurochem 110: 284-294).
  • TREM2 over-expression also leads to increased phagocytosis of dying neurons by microglia, and similarly increases phagocytosis by other myeloid lineage cells.
  • Nasu-Hakola disease a rare neurodegenerative disease with late-onset dementia, demyelination, and cerebral 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 increased in APP23 transgenic mice, an Alzheimer's disease model in which the mice express a mutant form of the amyloid precursor protein that is associated with familial Alzheimer's disease (Melchior, B et al., ASN Neuro 2: e00037). Uptake of Amyloid 1-42 has also been shown to be increased in BV-2 microglial cell lines that overexpress TREM2.
  • TREM2 has further been shown to be upregulated 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).
  • the transduction of bone marrow-derived myeloid precursor cells (BM-DC) in vitro with TREM2 leads to increased phagocytosis of degenerated myelin. In response to LPS, these cells show increased IL-10 and decreased IL-1 ⁇ . Intravenous transplantation of myeloid cells overexpressing TREM2 can suppress EAE in vivo.
  • BM-DC bone marrow-derived myeloid precursor cells
  • exome sequencing of individuals with frontotemporal dementia (FTD) presentation has identified 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 lead to truncation and likely loss-of-function of TREM2. These same TREM2 mutations can also cause Naku-Hakula disease in some individuals. Imaging analysis in certain individuals with TREM2 homozygous mutations has also shown evidence of demyelination.
  • TREM2 Heterozygous mutations in TREM2, which are the same as the mutation that cause Naku-Hakula 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 rarer than the known risk variants of Alzheimer's disease (e.g., APOE4), the effect of carrying these mutations is just as serious; around a 3 fold increase in the risk of developing Alzheimer's disease.
  • APOE4 the known risk variants of Alzheimer's disease
  • TYROBP/DAP12 as the signaling molecule for TREM2 as a key regulator of the immune/microglia gene modules that is associated with LOAD.
  • TYROBP was found to be the causal regulator of the highest scoring immune/microglia module as rank-ordered based on the number of other genes that TREM2 regulated and the magnitude of loss of regulation, as well as differential expression in LOAD brains.
  • TYROBP was significantly upregulated in LOAD brains and there was a progression of TYROBP expression changes across mild cognitive impairment (MCI), which often precedes LOAD (Zhang et al., (2013) Cell 153, 707-720). Targeting such causal networks in ways that restore them to a normal state may be a way to treat disease.
  • MCI mild cognitive impairment
  • TREM2 and/or its signaling adapter molecule DAP12/TRYROBP on a cell surface and that modulate (e.g., activate or inhibit) one or more TREM2 and/or DAP12 activities in order to treat one or more diseases, disorders, and conditions associated with decreased TREM2 and/or DAP12 activity, as well as conditions associated with undesired TREM2 and/or DAP12 activity.
  • the tumor microenvironment is composed of a heterogeneous immune infiltrate, which include T lymphocytes, macrophages and cells of myeloid/granulocytic lineage.
  • Therapeutic approaches that modulate specific subsets of immune cells are changing the standard of care.
  • “Checkpoint blocking” antibodies targeting immune-modulatory molecules expressed on T cells have demonstrated clinical activity across a variety of tumor types (Naidoo-et al., (2014) British Journal of Cancer 111, 2214-2219).
  • tumor-associated macrophages e.g., M2-type macrophages
  • M2-macrophages e.g., M2-macrophages in tumors
  • TREM2 and/or DAP12 e.g., activate or inhibit
  • TREM2 and/or DAP12 activities in tumor-associated immune cells, such as macrophages, dendritic cells, myeloid/granulocytic cells, T cells, and monocytes.
  • the invention is generally directed to methods and compositions that include antibodies, e.g., monoclonal antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibody fragments, etc., that specifically bind a TREM2 protein and/or its signaling adaptor molecule DAP12, e.g., a mammalian TREM2, a human TREM2, a mammalian DAP12, or a human DAP12, including wild-type proteins and naturally occurring variants thereof.
  • the antibodies of the present disclosure may include agonist antibodies, inert antibodies, and/or antagonist antibodies.
  • the methods provided herein find use in preventing, reducing risk, or treating an individual having dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, or multiple sclerosis; in inducing or promoting innate immune cell survival in an individual in need thereof, and/or in decreasing innate immune cell survival in an individual in need thereof.
  • anti-TREM2 antibodies are agonist 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 cells, and/or expression of IL-6.
  • agonist anti-TREM2 antibodies of the present disclosure compete with TREM2 ligands for binding to TREM2 expressed on a cell surface.
  • agonist anti-TREM2 antibodies of the present disclosure do not compete with TREM2 ligands for binding to TREM2 expressed on a cell surface.
  • anti-TREM2 antibodies are inert or antagonist antibodies that bind to TREM2 and decrease, inhibit, or otherwise reduce one or more TREM2 activities and/or survival of one or more innate immune cells.
  • inert or antagonist anti-TREM2 antibodies of the present disclosure block or otherwise inhibit ligand binding to TREM2 expressed on a cell surface.
  • aspects of the present disclosure relate to an isolated agonist antibody that binds to a TREM2 protein, a DAP12 protein, or both, wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both.
  • the TREM2 protein, the DAP12 protein, or both is a mammalian protein or a human protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein, the DAP12 protein, or both is a wild-type protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein, the DAP12 protein, or both is a naturally occurring variant.
  • the TREM2 protein, the DAP12 protein, or both is expressed on human dendritic cells, human macrophages, human monocytes, human osteoclasts, human Langerhans cells of skin, human Kupffer cells, and/or human microglia.
  • the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on a cell surface.
  • the one or more TREM2 activities comprise TREM2 binding to DAP12.
  • the one or more DAP12 activities comprise DAP12 binding to TREM2.
  • the one or more TREM2 activities, DAP12 activities, or both comprise DAP12 phosphorylation, TREM2 phosphorylation, or both.
  • DAP12 phosphorylation, TREM2 phosphorylation, or both is induced by one or more SRC family tyrosine kinases.
  • the one or more SRC family tyrosine kinases comprise a Syk kinase.
  • the one or more TREM2 activities, DAP12 activities, or both comprise PI3K activation. In certain embodiments that may be combined with any of the preceding embodiments, the one or more TREM2 activities, DAP12 activities, or both comprise increased expression of one or more anti-inflammatory cytokines. In certain embodiments that may be combined with any of the preceding embodiments, the one or more TREM2 activities, DAP12 activities, or both comprise increased expression of one or more anti-inflammatory mediators (e.g., cytokines) selected from the group consisting of IL-12p70, IL-6, and IL-10.
  • cytokines e.g., cytokines
  • the increased expression occurs in one or more cells selected from the group consisting of macrophages, dendritic cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, and microglial cells.
  • the one or more TREM2 activities, DAP12 activities, or both comprise reduced expression of one or more pro-inflammatory cytokines.
  • the one or more TREM2 activities, DAP12 activities, or both comprise 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-1 ⁇ , TNF, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.
  • pro-inflammatory mediators selected from the group consisting of IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1 ⁇ , TNF, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, O
  • the one or more TREM2 activities, DAP12 activities, or both comprise reduced expression of TNF- ⁇ , IL-6, or both.
  • the reduced expression of the 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 skin, Kupffer cells, and microglial cells.
  • the one or more TREM2 activities, DAP12 activities, or both comprise extracellular signal-regulated kinase (ERK) phosphorylation.
  • ERK extracellular signal-regulated kinase
  • the one or more TREM2 activities, DAP12 activities, or both comprise increased expression of C-C chemokine receptor 7 (CCR7).
  • the one or more TREM2 activities, DAP12 activities, or both comprise induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells.
  • the one or more TREM2 activities, DAP12 activities, or both comprise an enhancement, normalization, or both of the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation.
  • the one or more TREM2 activities, DAP12 activities, or both comprise induction of osteoclast production, increased rate of osteoclastogenesis, or both.
  • the one or more TREM2 activities, DAP12 activities, or both comprise increasing the survival of macrophages, microglial cells, or both.
  • the one or more TREM2 activities, DAP12 activities, or both comprise increasing the function of macrophages, microglial cells, dendritic cells, monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells.
  • the macrophages are M1 macrophages and/or microglia, M2 macrophages and/or microglia, or both.
  • the M1 macrophages and/or microglia are activated M1 macrophages and/or microglia.
  • the one or more TREM2 activities, DAP12 activities, or both comprise induction of one or more types of clearance selected from the group consisting of apoptotic neuron clearance, nerve tissue debris clearance, non-nerve tissue debris clearance, bacteria or other foreign body clearance, disease-causing protein clearance, disease-causing peptide clearance, and disease-causing nucleic acid clearance.
  • the one or more TREM2 activities, DAP12 activities, or both comprise induction of phagocytosis of one or more of apoptotic neurons, nerve tissue debris, non-nerve tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, or disease-causing nucleic acid.
  • the disease-causing protein is selected from the group consisting of amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides,
  • DPR DiPeptide repeat
  • GA glycine-
  • the disease-causing nucleic acid is antisense GGCCCC (G2C4) repeat-expansion RNA.
  • the one or more TREM2 activities, DAP12 activities, or both comprise normalization of disrupted TREM2/DAP12-dependent gene expression.
  • the one or more TREM2 activities, DAP12 activities, or both comprise recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex.
  • the one or more TREM2 activities, DAP12 activities, or both comprise Syk phosphorylation. In certain embodiments that may be combined with any of the preceding embodiments, the one or more TREM2 activities, DAP12 activities, or both comprise increased expression of CD83 and/or CD86 on dendritic cells, macrophages, and/or monocytes. In certain embodiments that may be combined with any of the preceding embodiments, the one or more TREM2 activities, DAP12 activities, or both comprise reduced secretion of one or more inflammatory cytokines.
  • the one or more inflammatory cytokines are selected from the group consisting of TNF- ⁇ , IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1 ⁇ , IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.
  • the antibody induces the one or more TREM2 activities, DAP12 activities, or both independently of binding to an Fc receptor.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB).
  • the human IgG2 constant region comprises an Fc region that comprises one or more modifications.
  • the Fc region comprises one or more amino acid substitutions.
  • the antibody comprises an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region.
  • the IgG2 isotype CH1 and hinge region comprise the amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397).
  • the antibody Fc region comprises a S267E amino acid substitution, a L328F a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, wherein the numbering of the residues on IgG1 is according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG1 constant region.
  • the antibody has an IgG4 isotype.
  • the antibody comprises a human IgG4 constant region.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody has a hybrid IgG2/4 isotype.
  • the antibody comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 and amino acids 261 to 447 of human IgG4, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG4 constant region.
  • the TREM2 protein, the DAP12 protein, or both is a mammalian protein or a human protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein, the DAP12 protein, or both is a wild-type protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein, the DAP12 protein, or both is a naturally occurring variant. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody inhibits one or more TREM2 activities, DAP12 activities, or both.
  • the one or more TREM2 activities, DAP12 activities, or both comprise decreasing activity of one or more TREM2-dependent genes.
  • the one or more TREM2-dependent genes comprise one or more nuclear factor of activated T-cells (NFAT) transcription factors.
  • the one or more TREM2 activities, DAP12 activities, or both comprise decreasing the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or dendritic cells.
  • the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signal transduction, or both.
  • the antibody is incapable of binding an Fc-gamma receptor (Fc ⁇ R).
  • the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG1 constant region.
  • the antibody has an IgG2 isotype.
  • the antibody comprises a human IgG2 constant region.
  • the e human IgG2 constant region comprises an Fc region.
  • the Fc region comprises one or more modifications. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more amino acid substitutions. In certain embodiments that may be combined with any of the preceding embodiments, the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody has an IgG4 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG4 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG4 constant region comprises an Fc region. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more modifications. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more amino acid substitutions.
  • 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 variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • the Fc region further comprises a S228P amino acid substitution according to EU or Kabat numbering.
  • the isolated antibody competes for binding of TREM2 with one or more TREM2 ligands.
  • the one or more TREM2 ligands are selected from the group consisting of E.
  • the isolated antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, a conjugated antibody, or a chimeric antibody.
  • the isolated antibody is a bispecific antibody recognizing a first antigen and a second antigen.
  • the first antigen is human TREM2 or a naturally occurring variant thereof
  • the second antigen is a disease-causing protein selected from the group consisting of amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM
  • 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 variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12; and wherein the antibody is used in combination with one or more antibodies that specifically bind a disease-causing protein selected from the group consisting of: amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobul
  • a disease-causing protein selected from the group consisting of:
  • the isolated antibody binds to a linear epitope on TREM2. In certain embodiments that may be combined with any of the preceding embodiments, the linear epitope on TREM2 is located within the extracellular domain of TREM2. In certain embodiments that may be combined with any of the preceding embodiments, the linear epitope on TREM2 is located within the extracellular immunoglobulin-like variable-type (IgV) domain of TREM2. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody binds to a 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.
  • 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 a TREM2 protein corresponding to 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 one or more amino acids within amino acid residues 49-57 of SEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.
  • 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 that may be combined with any of the preceding 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 that may be combined with any of the preceding embodiments, the isolated antibody binds to an epitope comprising one or more amino acid residues selected from the group consisting of: i. amino acid residue Arg47 or Asp87 of SEQ ID NO: 1; ii. amino acid residues 40-44 of SEQ ID NO: 1; iii.
  • 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 a DAP12 protein corresponding to amino acid residues 22-40 of SEQ ID NO: 2.
  • the isolated antibody is a bispecific antibody that binds to one or more amino acids selected from the group consisting of: i.
  • amino acid residues of SEQ ID NO: 1 one or more amino acid residues of SEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding to amino acid residues of SEQ ID NO: 1; and ii. one or more amino acid residues of SEQ ID NO: 2, or amino acid residues on a DAP12 protein corresponding to amino acid residues of SEQ ID NO: 2.
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab52; and/or wherein the light chain variable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab52.
  • the HVR-H1 comprises the amino acid sequence of SEQ ID NO:398.
  • the HVR-H2 comprises the amino acid sequence of SEQ ID NO:399.
  • the HVR-H3 comprises the amino acid sequence of SEQ ID NO:400.
  • the HVR-L1 comprises the amino acid sequence of SEQ ID NO:401.
  • the HVR-L2 comprises the amino acid sequence of SEQ ID NO:402.
  • the HVR-L3 comprises the amino acid sequence of SEQ ID NO:403.
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain
  • the heavy chain variable domain comprises: (a) an 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) an 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; and; 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
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab21; and/or wherein the light chain variable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab21.
  • the HVR-H1 comprises the amino acid sequence of SEQ ID NO:404.
  • the HVR-H2 comprises the amino acid sequence of SEQ ID NO:405.
  • the HVR-H3 comprises the amino acid sequence of SEQ ID NO:406.
  • the HVR-L1 comprises the amino acid sequence of SEQ ID NO:407.
  • the HVR-L2 comprises the amino acid sequence of SEQ ID NO:408.
  • the HVR-L3 comprises the amino acid sequence of SEQ ID NO:409.
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain
  • the heavy chain variable domain comprises: (a) n 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) an HVR-H2 comprising 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/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:
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab52; and/or wherein the light chain variable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab52.
  • the HVR-H1 comprises the amino acid sequence of SEQ ID NO:398.
  • the HVR-H2 comprises the amino acid sequence of SEQ ID NO:399.
  • the HVR-H3 comprises the amino acid sequence of SEQ ID NO:400.
  • the HVR-L1 comprises the amino acid sequence of SEQ ID NO:401.
  • the HVR-L2 comprises the amino acid sequence of SEQ ID NO:402.
  • the HVR-L3 comprises the amino acid sequence of SEQ ID NO:403.
  • 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) an 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) an 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; and; 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
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab21; and/or wherein the light chain variable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab21.
  • the HVR-H1 comprises the amino acid sequence of SEQ ID NO:404.
  • the HVR-H2 comprises the amino acid sequence of SEQ ID NO:405.
  • the HVR-H3 comprises the amino acid sequence of SEQ ID NO:406.
  • the HVR-L1 comprises the amino acid sequence of SEQ ID NO:407.
  • the HVR-L2 comprises the amino acid sequence of SEQ ID NO:408.
  • the HVR-L3 comprises the amino acid sequence of SEQ ID NO:409.
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain
  • the heavy chain variable domain comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:404, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:405, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:406, and/or wherein the light chain variable domain comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:407, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:408, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:409.
  • 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) n 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) an HVR-H2 comprising 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/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 S
  • aspects of the present disclosure relate to an isolated anti-human TREM2 antibody which binds essentially the same TREM2 epitope as the antibody Ab52.
  • Other aspects of the present disclosure relate to an isolated anti-human TREM2 antibody which binds essentially the same TREM2 epitope as the antibody Ab21.
  • the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities, DAP12 activities, or both.
  • the isolated antibody induces or retains TREM2 clustering, DAP12 clustering, or both on a cell surface.
  • the one or more TREM2 activities, DAP12 activities, or both are selected from the group consisting of TREM2 binding to DAP12; DAP12 binding to TREM2; TREM2 phosphorylation, DAP12 phosphorylation; PI3K activation; increased expression of one or more anti-inflammatory mediators (e.g., 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-6, IL-12 p70, IL-1 ⁇ , TNF, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12,
  • the antibody is of the IgG class the IgM class, or the IgA class. In certain embodiments that may be combined with any of the preceding embodiments, the antibody is of the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody has an IgG2 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG2 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG2 constant region comprises an Fc region.
  • the antibody induces the one or more TREM2 activities, DAP12 activities, or both independently of binding to an Fc receptor.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB).
  • the Fc region comprises one or more modifications.
  • the Fc region comprises one or more amino acid substitutions.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the human IgG2 constant region comprises a light chain constant region comprising a C214S amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody has an IgG1 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG1 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG1 constant region comprises an Fc region. In certain embodiments that may be combined with any of the preceding embodiments, the antibody binds an inhibitory Fc receptor. In certain embodiments that may be combined with any of the preceding embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ RIIB). In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more modifications.
  • the Fc region comprises one or more amino acid substitutions.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody comprises an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region.
  • the IgG2 isotype CH1 and hinge region comprise the amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397).
  • the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG1 constant region.
  • the antibody has an IgG4 isotype.
  • the antibody comprises a human IgG4 constant region.
  • the human IgG4 constant region comprises an Fc region.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB).
  • the Fc region comprises one or more modifications.
  • the Fc region comprises one or more amino acid substitutions.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody has a hybrid IgG2/4 isotype.
  • the antibody comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 and amino acids 261 to 447 of human IgG4, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG4 constant region.
  • 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 variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12, and wherein the antibody fragment is cross-linked to a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • the isolated antibody is an inert antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody is an antagonist antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody inhibits one or more TREM2 activities.
  • the one or more TREM2 activities are selected from the group consisting of decreasing activity of one or more TREM2-dependent genes; decreasing activity of one or more nuclear factor of activated T-cells (NFAT) transcription factors; decreasing the survival of macrophages, microglial cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or dendritic cells; and any combination thereof.
  • the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signal transduction, or both.
  • the antibody is incapable of binding an Fc-gamma receptor (Fc ⁇ R).
  • Fc ⁇ R Fc-gamma receptor
  • the antibody has an IgG1 isotype.
  • the antibody comprises a human IgG1 constant region.
  • the human IgG1 constant region comprises an Fc region.
  • the Fc region comprises one or more modifications.
  • the Fc region comprises one or more amino acid substitutions.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to EU or Kabat numbering.
  • the antibody comprises a mouse IgG1 constant region.
  • the antibody has an IgG2 isotype.
  • the antibody comprises a human IgG2 constant region.
  • the human IgG2 constant region comprises an Fc region.
  • the Fc region comprises one or more modifications. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more amino acid substitutions. In certain embodiments that may be combined with any of the preceding embodiments, the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody has an IgG4 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG4 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG4 constant region comprises an Fc region. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more modifications. In certain embodiments that may be combined with any of the preceding embodiments, the Fc region comprises one or more amino acid substitutions.
  • the 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • 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 variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the Fc region further comprises a S228P amino acid substitution according to EU or Kabat numbering.
  • the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.
  • the antibody is a bispecific antibody recognizing a first antigen and a second antigen.
  • the antibody is a monoclonal antibody.
  • aspects of the present disclosure relate to an isolated antibody that binds to a TREM2 protein, wherein the isolated antibody promotes survival of one or more innate immune cells.
  • Other aspects of the present disclosure relate to an isolated antibody that binds to a TREM2 protein, wherein the isolated antibody increases expression of IL-6.
  • Other aspects of the present disclosure relate to an isolated antibody that binds to a TREM2 protein, wherein the isolated antibody promotes survival of one or more innate immune cells or increases expression of IL-6.
  • Other aspects of the present disclosure relate to an isolated antibody that binds to a TREM2 protein, wherein the isolated antibody promotes survival of one or more innate immune cells and increases expression of IL-6.
  • the one or more innate immune cells 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, Langerhans cells of skin, Kupffer cells, and any combination thereof.
  • the one or more innate immune cells are macrophages.
  • the one or more innate immune cells are microglial cells.
  • the one or more innate immune cells are M1 microglial cells.
  • the one or more innate immune cells are activated M1 microglial cells.
  • the one or more innate immune cells are M2 microglial cells. In certain embodiments, the one or more innate immune cells are dendritic cells (DCs). In certain embodiments, the one or more innate immune cells are M1 macrophages. In certain embodiments, the one or more innate immune cells are activated M1 macrophages. In certain embodiments, the one or more innate immune cells are M2 macrophages. In certain embodiments, the one or more innate immune cells are monocytes. In certain embodiments, the one or more innate immune cells are osteoclasts. In certain embodiments, the one or more innate immune cells are Langerhans cells of skin. In certain embodiments, the one or more innate immune cells are Kupffer cells.
  • DCs dendritic cells
  • aspects of the present disclosure relate to an isolated antibody that binds to a 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 a 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 a TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1; iii.
  • 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 a 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 a 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 a TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.
  • 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 a 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, 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, the isolated antibody binds to an epitope comprising one or more amino acids within amino acid residues 139-146 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 140-153 of SEQ ID NO: 1.
  • the TREM2 protein is a mammalian protein or a human protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein is a wild-type protein. In certain embodiments that may be combined with any of the preceding embodiments, the TREM2 protein is a naturally occurring variant. In certain embodiments that may be combined with any of the preceding embodiments, the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody induces or retains TREM2 clustering on a cell surface.
  • the one or more TREM2 activities are selected from the group consisting of: i. TREM2 binding to DAP12; ii. DAP12 phosphorylation; 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, Langerhans cells of skin, and/or Kupffer cells; iv. Syk phosphorylation; v. increased expression of CD83 and/or CD86 on dendritic cells; vi.
  • the antibody is of the IgG class the IgM class, or the IgA class.
  • the antibody is of the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody has an IgG2 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG2 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG2 constant region comprises an Fc region. In certain embodiments that may be combined with any of the preceding embodiments, the antibody induces the one or more TREM2 activities independently of binding to an Fc receptor.
  • the antibody binds an inhibitory Fc receptor.
  • the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB). In certain embodiments that may be combined with any of the preceding embodiments: i.
  • the isolated antibody has a human IgG2 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering; ii.
  • the isolated antibody has a human IgG2 isotype, wherein the human IgG2 comprises a constant region, and wherein the human IgG2 constant region comprises a light chain constant region comprising a C214S amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering; iii.
  • the isolated antibody has a human or mouse IgG1 isotype and comprises 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 numbering of the residues is according to EU or Kabat numbering; iv.
  • the isolated antibody has an IgG1 isotype and comprises an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region, optionally wherein the IgG2 isotype CH1 and hinge region comprise the amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397), and optionally wherein the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering; v.
  • the isolated antibody has a human or mouse IgG4 isotype and comprises 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 combination thereof, wherein the numbering of the residues is according to EU or Kabat numbering; or vi. the isolated antibody has a hybrid IgG2/4 isotype, optionally wherein the antibody comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 and amino acids 261 to 447 of human IgG4, wherein the numbering of the residues is according to EU or, Kabat numbering.
  • the isolated antibody is an inert antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody is an antagonist antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody inhibits one or more TREM2 activities.
  • the inhibited one or more TREM2 activities are selected from the group consisting of decreasing activity of one or more TREM2-dependent genes; decreasing activity of one or more nuclear factor of activated T-cells (NFAT) transcription factors; decreasing the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or dendritic cells; decreased expression of one or more mediators selected from the group consisting of IL-12p70, IL-6, and IL-10; and any combination thereof.
  • NFAT nuclear factor of activated T-cells
  • the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signal transduction, or both.
  • the antibody is incapable of binding an Fc-gamma receptor (Fc ⁇ R).
  • Fc ⁇ R Fc-gamma receptor
  • the isolated antibody has a human or mouse IgG1 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering, optionally wherein the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to EU or Kabat numbering; ii.
  • the isolated antibody has a human IgG2 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering; or iii.
  • the isolated antibody has a human or mouse IgG4 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the Fc region further comprises a S228P amino acid substitution according to EU or Kabat numbering.
  • the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the numbering of the residues is according to EU or Kabat numbering.
  • 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 variant of human TREM2, and wherein the antibody fragment is cross-linked to a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the isolated antibody is an antibody fragment, and wherein the antibody fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • the isolated antibody competes for binding of TREM2 with one or more TREM2 ligands.
  • 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, phosphatidylcholin, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid beta peptide.
  • the isolated antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody is a bispecific antibody recognizing a first antigen and a second antigen.
  • the first antigen is human TREM2 or a naturally occurring variant thereof
  • the second antigen is a disease-causing protein selected from the group consisting of amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA)
  • 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 variant of human TREM2; and wherein the antibody is used in combination with one or more antibodies that specifically bind a disease-causing protein selected from the group consisting of: amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-
  • a disease-causing protein selected from the group consisting of:
  • the antibody is a monoclonal antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody is a bispecific antibody that binds to TREM2 and DAP12.
  • the isolated antibody comprises a heavy chain variable domain and a light chain variable domain
  • 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) an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:3-24, 398, and 404; an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:25-49, 399, and 405; and (c) an HVR-H3 c comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:50-119, 400, and 406; and/or wherein the light chain variable domain comprises: (a) an HVR-L1 c comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:120-137, 401, and 407; (b) an HVR-L2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:138-152, 402, and 408; and (c
  • TREM2 antibody which binds essentially the same TREM2 epitope as a monoclonal antibody selected from the group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72
  • an isolated anti-human TREM2 antibody which competes with a monoclonal antibody selected from the group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab
  • the antibody is an agonist antibody, and wherein the antibody induces one or more TREM2 activities.
  • the isolated antibody induces or retains TREM2 clustering on a cell surface.
  • the antibody is of the IgG class the IgM class, or the IgA class. In certain embodiments that may be combined with any of the preceding embodiments, the antibody is of the IgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody has an IgG2 isotype. In certain embodiments that may be combined with any of the preceding embodiments, the antibody comprises a human IgG2 constant region. In certain embodiments that may be combined with any of the preceding embodiments, the human IgG2 constant region comprises an Fc region.
  • the antibody induces the one or more TREM2 activities independently of binding to an Fc receptor. In certain embodiments that may be combined with any of the preceding embodiments, the antibody binds an inhibitory Fc receptor. In certain embodiments that may be combined with any of the preceding embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB). In certain embodiments that may be combined with any of the preceding embodiments: i.
  • the isolated antibody has a human IgG2 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering; ii.
  • the isolated antibody has a human IgG2 isotype, wherein the human IgG2 comprises a constant region, and wherein the human IgG2 constant region comprises a light chain constant region comprising a C214S amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering; iii.
  • the isolated antibody has a human or mouse IgG1 isotype and comprises 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 numbering of the residues is according to EU or Kabat numbering; iv.
  • the isolated antibody has an IgG1 isotype and comprises an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region, optionally wherein the IgG2 isotype CH1 and hinge region comprise the amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397), and optionally wherein the antibody Fc region comprises a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, wherein the numbering of the residues is according to EU or Kabat numbering; v.
  • the isolated antibody has a human or mouse IgG1 isotype and comprises 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 combination thereof, wherein the numbering of the residues is according to EU or Kabat numbering; or vi. the isolated antibody has a hybrid IgG2/4 isotype, optionally wherein the antibody comprises an amino acid sequence comprising amino acids 118 to 260 of human IgG2 and amino acids 261 to 447 of human IgG4, wherein the numbering of the residues is according to EU or, Kabat numbering.
  • 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 variant of human TREM2, and wherein the antibody fragment is cross-linked to a second antibody fragment that binds to one or more human proteins selected from the group consisting of human TREM2, a naturally occurring variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the isolated antibody is an antibody fragment, and wherein the antibody fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • the isolated antibody is an inert antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody is an antagonist antibody. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody inhibits one or more TREM2 activities.
  • the inhibited one or more TREM2 activities are selected from the group consisting of decreasing activity of one or more TREM2-dependent genes; decreasing activity of one or more nuclear factor of activated T-cells (NFAT) transcription factors; decreasing the survival of macrophages, microglial cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or dendritic cells; decreased expression of one or more mediators selected from the group consisting of IL-12p70, IL-6, and IL-10; and any combination thereof.
  • NFAT nuclear factor of activated T-cells
  • the isolated antibody inhibits interaction between TREM2 and one or more TREM2 ligands, inhibits TREM2 signal transduction, or both.
  • the antibody is incapable of binding an Fc-gamma receptor (Fc ⁇ R).
  • Fc ⁇ R Fc-gamma receptor
  • the isolated antibody has a human or mouse IgG1 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering, optionally wherein the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to EU or Kabat numbering; ii.
  • the isolated antibody has a human IgG2 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering; or iii.
  • the isolated antibody has a human or mouse IgG4 isotype and comprises 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • 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 variant of human TREM2.
  • the isolated antibody is an antibody fragment, and wherein the antibody fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • 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, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the Fc region further comprises a S228P amino acid substitution according to EU or Kabat numbering.
  • the Fc region further comprises one or more additional amino acid substitutions at a position selected from the group consisting of M252Y, S254T, T256E, and any combination thereof, wherein the numbering of the residues is according to EU or Kabat numbering.
  • the antibody is a human antibody, a humanized antibody, a bispecific antibody, a multivalent antibody, or a chimeric antibody.
  • the antibody is a bispecific antibody recognizing a first antigen and a second antigen.
  • the antibody is a monoclonal antibody.
  • the isolated antibody binds specifically to both human TREM2 and mouse TREM2.
  • the isolated antibody has 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.
  • the isolated antibody has dissociation constant (K D ) for human TREM2-Fc fusion protein that ranges from less than about 1.51 nM to less than about 0.35 nM.
  • the isolated antibody has dissociation constant (K D ) for human monomeric TREM2 protein that ranges from less than about 5.75 nM to less than about 1.15 nm. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody has dissociation constant (K D ) for mouse TREM2-Fc fusion protein that ranges from less than about 0.23 nM to less than about 0.09 nM. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody has 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.
  • the isolated antibody has dissociation constant (K D ) for human TREM2-Fc fusion protein that ranges from less than about 0.71 nM to less than about 0.23 nM. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody has dissociation constant (K D ) for human monomeric TREM2 protein that ranges from less than about 6.70 nM to less than about 0.66 nM. In certain embodiments that may be combined with any of the preceding embodiments, the isolated antibody has dissociation constant (K D ) for mouse TREM2-Fc fusion protein that ranges from less than about 4.90 nM to less than about 0.35 nM.
  • aspects of the present disclosure relate to an isolated nucleic acid encoding the antibody of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to a vector comprising the nucleic acid of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to a host cell comprising the vector of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to an isolated host cell comprising the vector of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to a method of producing an antibody, comprising culturing the cell of any one of the preceding embodiments so that the antibody is produced. In certain embodiments, the method further comprises recovering the antibody produced by the cell.
  • Other aspects of the present disclosure relate to an isolated antibody produced by any of the preceding methods of producing an antibody.
  • Other aspects of the present disclosure relate to a pharmaceutical composition comprising the antibody of any one of the preceding embodiments and a pharmaceutically acceptable carrier.
  • aspects of the present disclosure relate to a method of 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 disease, and multiple sclerosis, comprising administering to the individual a therapeutically effective amount of an isolated agonist antibody of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to an isolated agonist antibody of any one of the preceding embodiments for use in 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 disease, and multiple sclerosis.
  • an isolated agonist antibody of any one of the preceding embodiments in the manufacture of a medicament 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 disease, and multiple sclerosis.
  • the individual has a heterozygous variant of TREM2, wherein the variant comprises one or more substitutions selected from the group consisting of: i. a glutamic acid to stop codon substitution in the nucleic acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1; ii.
  • the individual has a heterozygous variant of TREM2, wherein the variant comprises a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both.
  • the individual has a heterozygous variant of DAP12, wherein the variant comprises one or more variants selected from the group consisting of: i.
  • aspects of the present disclosure relate to a method of inducing or promoting innate immune cell survival in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated agonist antibody of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to an isolated agonist antibody of any one of the preceding embodiments for use in inducing or promoting innate immune cell survival in an individual in need thereof.
  • Other aspects of the present disclosure relate to use of an isolated agonist antibody of any one of the preceding embodiments in the manufacture of a medicament for inducing or promoting innate immune cell survival in an individual in need thereof.
  • 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 a TREM2 protein.
  • Other aspects of the present disclosure relate to an isolated agonist antibody that binds to a TREM2 protein for use in inducing or promoting wound healing in an individual in need thereof.
  • Other aspects of the present disclosure relate to use of an isolated agonist antibody that binds to a TREM2 protein in the manufacture of a medicament for inducing or promoting wound healing in an individual in need thereof.
  • aspects of the present disclosure relate to a method of decreasing innate immune cell survival in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an isolated antagonist antibody of any one of the preceding embodiments.
  • Other aspects of the present disclosure relate to an isolated antagonist antibody of any one of the preceding embodiments for use in decreasing innate immune cell survival in an individual in need thereof.
  • Other aspects of the present disclosure relate to use of an isolated antagonist antibody of any one of the preceding embodiments in the manufacture of a medicament for decreasing innate immune cell survival in an individual in need thereof.
  • aspects of the present disclosure relate to a method of 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, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, seizures, spinal cord injury
  • aspects of the present disclosure relate to an isolated antibody of any one of the preceding embodiments for use in 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, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis
  • aspects of the present disclosure relate to use of an isolated antibody of any one of the preceding embodiments in the manufacture of a medicament 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, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomart
  • the isolated antibody is: (a) an agonist antibody; (b) an inert antibody; or an (c) an antagonist antibody.
  • the antibody is of the IgG class the IgM class, or the IgA class; and/or (b) the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype.
  • the antibody comprises one or more amino acid substitutions in the Fc region are at a residue position 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; (c) L235A, G237
  • the isolated 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 a 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 a TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.
  • the isolated antibody (a) binds essentially the same TREM2 epitope as the antibody Ab52; (b) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab52; and/or wherein the light chain variable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab52; (c) comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an 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, an 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:
  • the isolated antibody is the isolated antibody of any one of the preceding embodiments.
  • the isolated agonist antibody is the isolated agonist antibody of any one of the preceding embodiments.
  • the individual has a heterozygous variant of TREM2, wherein the variant comprises one or more substitutions selected from the group consisting of: i. a glutamic acid to stop codon substitution in the nucleic acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1; ii.
  • the individual has a heterozygous variant of TREM2, wherein the variant comprises a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; or both.
  • the individual has a heterozygous variant of DAP12, wherein the variant comprises one or more variants selected from the group consisting of: i.
  • the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer.
  • the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule, and/or another standard or investigational anti-cancer therapy.
  • the at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with the isolated antibody.
  • he at least one antibody that specifically binds to an inhibitory checkpoint 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, an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEM antibody, an anti-B- and T-lymphocyte attenuator (BTLA) antibody, an anti-Killer inhibitory receptor (KIR) antibody, an anti-GALS antibody, an anti-TIM3 antibody, an anti-A2AR antibody, an anti-LAG-3 antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, and any combination thereof.
  • BTLA T-lymphocyte attenuator
  • KIR anti-Killer inhibitory receptor
  • the standard or investigational anti-cancer therapy is one or more therapies selected from the group consisting of radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy, and cytokine therapy.
  • the method further comprises administering to the individual at least one antibody that specifically binds to an inhibitory cytokine.
  • the at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with the isolated antibody.
  • the at least one antibody that specifically binds to an inhibitory cytokine is selected from the group consisting of an anti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any combination thereof.
  • the method further comprises administering to the individual at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein.
  • the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is administered in combination with the isolated antibody.
  • the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is selected from the group consisting of an agonist anti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28 antibody, an agonist anti-CD137/4-1BB antibody, an agonist anti-CD27 antibody, an agonist anti-glucocorticoid-induced TNFR-related protein GITR antibody, and any combination thereof.
  • the method further comprises administering to the individual at least one stimulatory cytokine.
  • the at least one stimulatory cytokine is administered in combination with the isolated antibody.
  • the at least one stimulatory cytokine is selected from the group consisting of TNF- ⁇ , IL-10, IL-6, IL-8, CRP, TGF-beta members of the chemokine protein families, IL20 family member, IL-33, LIF, OSM, CNTF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN- ⁇ , IFN- ⁇ , IL-2, IL-18, GM-CSF, G-CSF, and any combination thereof.
  • FIG. 1A shows an amino acid sequence alignment between the human TREM2 protein (SEQ ID NO: 426) and the human NCTR2 protein (SEQ ID NO: 427), depicting the homology between the two proteins.
  • the consensus sequence is SEQ ID NO: 446.
  • TREM-1_human SEQ ID NO: 429), TREM-2_human (SEQ ID NO: 430), TREM-1_mouse (SEQ ID NO: 431), TREM-2_mouse (SEQ ID NO: 432), TREM-3_mouse (SEQ ID NO: 433), NKp44 (SEQ ID NO: 434), aTCR_human (SEQ ID NO: 435), bTCR_human (SEQ ID NO: 436), gTCR_human (SEQ ID NO: 437), dTCR_human (SEQ ID NO: 438), Vd_human (SEQ ID NO: 439), hIGG1_mouse (SEQ ID NO: 440), lIGG1_mouse (SEQ ID NO: 441), CD8_human (SEQ ID NO: 442), and CTLA4_human (SEQ ID NO: 443).
  • the amino acid residue numbering is consistent with the mature sequence of the human TREM1 protein.
  • the secondary structure elements of TREM1 are illustrated as arrows for the ⁇ strands and cylinders for a helices. Amino acid residues involved in homo- and heterodimer formation are shown on black background. Cysteine residues that form disulfide bonds and that are conserved for the V-type Ig fold, are depicted in bold and marked with asterisks. Gaps are indicated by “-”. M-1 residues violating antibody-like dimer formation mode are marked with closed triangles as (e.g., Radaev et al., (2003) Structure. 11(12):1527-1535).
  • FIG. 2A shows an amino acid sequence alignment between the human TREM1 protein (SEQ ID NO: 428) and the human TREM2 protein (SEQ ID NO: 426), depicting the homology between the two proteins.
  • the consensus sequence is SEQ ID NO: 447.
  • FIG. 2B shows the amino acid sequences of the heavy chain variable regions of antibodies Ab21 and Ab52. The CDR sequences are underlined in each sequence. Sequence regions in-between each underlined CDR sequence correspond to the framework regions.
  • FIG. 2C shows the amino acid sequences of the light chain variable regions of antibodies Ab21 and Ab52. The CDR sequences are underlined in each sequence. Sequence regions in-between each underlined CDR sequence correspond to the framework regions.
  • FIG. 3A shows FACS histograms demonstrating binding of TREM2 antibodies Ab21, Ab52, Ab16, Ab20, Ab66, and Ab68 to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2.
  • FIG. 3B shows antibodies Ab21 and Ab52 binding to WT (Trem+/+) and TREM2 deficient (TREM2 ⁇ / ⁇ ) bone marrow derived mouse macrophages (BMMac). Shaded histograms represent the TREM2 antibody negative population. Black outlined histograms represent the TREM2 antibody positive population.
  • FIG. 4A shows FACS histograms demonstrating binding of TREM2 antibodies Ab21, Ab52, Ab43, and Ab60 to a human cell line (293) expressing recombinant human TREM2-DAP12 fusion protein. Shaded histograms represent a TREM2 antibody negative population. Black outlined histograms represent a TREM2 antibody positive population.
  • FIG. 4B shows antibodies Ab21, Ab52, Ab43, and Ab60 binding to primary human dendritic cells (hDCs). Shaded histograms represent secondary antibody alone negative control. Black outlined histograms represent the TREM2 antibody positive population.
  • FIG. 5A shows FACS dot plots demonstrating expression of cell surface markers CD83 and CD86 on human dendritic cells (DCs) after incubation with plate-bound TREM2 antibodies Ab21 or Ab52.
  • Antibody Ab88 represents the negative isotype control. Plots were gated on CD11c + HLA-DR + LIN ⁇ DCs. Percentage of cells within the CD83+CD86+ gate is displayed on each plot.
  • FIG. 5B shows FACS histograms demonstrating expressing of cell surface marker CD86 on human dendritic cells (DCs) after incubation with cross-linked TREM2 antibodies Ab21 or Ab52. Antibodies were cross-linked with anti-human secondary antibody. Antibody Ab88 represents the negative isotype control.
  • FIG. 6A shows Syk phosphorylation as determine by western blot in wild-type and TREM2 deficient (Trem2 ⁇ / ⁇ ) mouse (left and center panels) and human (right panel) macrophages after incubation with TREM2 antibodies Ab21 and Ab52.
  • Antibodies Ab89 and Ab92 are non-agonistic negative controls.
  • FIG. 6B shows Syk phosphorylation as determine by western blot in primary human dendritic cells after incubation with TREM2 antibodies Ab21 and Ab52.
  • FIG. 7A shows DAP12 phosphorylation as determine by western blot in mouse macrophages after incubation with TREM2 antibody Ab52.
  • FIG. 7B shows DAP12 phosphorylation as determine by western blot in wild-type and TREM2 deficient (Trem2 ⁇ / ⁇ ) mouse macrophages after incubation with TREM2 antibody Ab21.
  • FIG. 8 shows competitive binding between TREM2 antibodies and E. coli bacteria expressing putative TREM2 ligand with mouse and human cell lines expressing TREM2. Bacterial binding is expressed as a percentage of control. Average of two independent experiments; black bars: no difference to isotype control, red bars: significantly different from isotype controls (ANOVA).
  • FIG. 9A shows protein levels of inflammatory cytokines TNFa, IL-6, IL-10, and MCP-1 secreted in response to stimulation of WT and TREM2 KO macrophages with inflammatory mediators LPS or Zymosan.
  • FIG. 9B shows protein levels of inflammatory cytokines IL-6 and TNFa secreted in response to stimulation of WT, TREM2 heterozygous (Het), and TREM2 KO macrophages with the cytokines IL-4 or IFNg.
  • FIG. 10A shows FACS data demonstrating expression of cell surface markers CD86 and CD206 on WT, TREM2 heterozygous (Het), and TREM2 KO macrophages after stimulation with the cytokines IL-4 or IFNg.
  • FIG. 10B shows expression of cell surface marker CD86 on WT and TREM2 KO macrophages after stimulation with the inflammatory mediators LPS or Zymosan.
  • FIG. 11A shows numbers of live WT, TREM2 heterozygous (TREM2+/ ⁇ ), and TREM2 KO (TREM2 ⁇ / ⁇ ) macrophages after culture in growth factor MCSF for the indicated number of days.
  • FIG. 11B shows FACS plots demonstrating staining of WT, TREM2 heterozygous (TREM2+/ ⁇ ), and TREM2 KO (TREM2 ⁇ / ⁇ ) macrophages after culture in MCSF for 6 days (+MCSF) or in MCSF for 4 days, followed by no MCSF for 36 hours ( ⁇ MCSF). Percentage of live macrophages within the CD11b+DAPI ⁇ gate is indicated on each plot.
  • FIG. 11B shows FACS plots demonstrating staining of WT, TREM2 heterozygous (TREM2+/ ⁇ ), and TREM2 KO (TREM2 ⁇ / ⁇ ) macrophages after culture in MCSF for 6 days (+MCSF) or in MCSF for 4 days, followed by no MCSF
  • FIG. 11C shows luminescence levels detected in a luciferase viability assay after culture of WT and TREM2 KO dendritic cells, M1 macrophages, and M2 macrophages in growth factors GM-CSF, M-CSF, or M-CSF+IL-4, respectively.
  • FIG. 11D shows the frequency of live WT, TREM2 heterozygous (Het), and TREM2 KO macrophages (CD11b+) after culture in inflammatory mediators IFNg, LPS, or Zymosan.
  • FIG. 12 shows phagocytosis of apoptotic cells and E. coli by wild-type (WT) and TREM2 KO (TREM2 ⁇ / ⁇ ) bone marrow derived macrophages (BMmacs) cultured without MCSF.
  • WT wild-type
  • TREM2 KO TREM2 KO
  • BMmacs bone marrow derived macrophages
  • FIG. 13 shows an epitope map of TREM2 antibody Ab52.
  • FIG. 14 shows Syk phosphorylation as determined by Western blot in wild-type and TREM2 deficient (Trem2 ⁇ / ⁇ ) mouse macrophages after incubation with TREM2 antibodies MAB17291 (RD) or 78.18, demonstrating that antibody 78.18 does not induce Syk phosphorylation or TREM2 signaling.
  • FIG. 15A shows Fortebio analysis demonstrating simultaneous binding of antibody MAB17291 and antibody Ab21 to TREM2-Fc.
  • FIG. 15A shows Fortebio analysis demonstrating simultaneous binding of antibody MAB17291 and antibody Ab52 to TREM2-Fc.
  • FIG. 16 shows the percent increased survival of wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derived macrophages cultured in the presence of plate bound, cross-linked TREM2 antibody Ab21 or Ab52 Fabs and M-CSF.
  • Antibody Ab88 represents the negative isotype control.
  • FIG. 17A shows the luminescence viability assay of mouse bone marrow derived macrophages cultured in the presence of soluble, non-cross-linked TREM2 antibody Ab21 or Ab52 Fabs and M-CSF. Antibody Ab99 represents the negative isotype control.
  • FIG. 17B shows the luminescence viability assay of mouse bone marrow derived macrophages cultured in the presence of soluble, full-length TREM2 antibody Ab21 or Ab52 and M-CSF. Antibody Ab91 represents the negative isotype control.
  • the “NT” dotted line indicates the average viability obtained with untreated macrophages (no antibody added).
  • the “no MCSF” dotted line indicates the average viability obtained when macrophages are cultured in the absence of M-CSF.
  • FIG. 18A shows induction of TREM2-dependent gene expression by plate bound, full-length anti-TREM2 antibodies Ab21 and Ab52 using a luciferase reporter gene in a cell-based assay.
  • FIG. 18B shows induction of TREM2-dependent gene expression by plate bound phosphatidylserine (PS).
  • FIG. 18C shows activation of TREM2-dependent gene IL-6 in mouse macrophages by plate bound, Fab anti-TREM2 antibodies Ab21 and Ab52 Fabs.
  • FIG. 18 D shows activation of TREM2-dependent gene MCP-1 in mouse macrophages by plate bound, Fab anti-TREM2 antibodies Ab21 and Ab52 Fabs.
  • FIG. 19 shows inhibition of TREM2-dependent gene expression by soluble, full-length anti-TREM2 antibodies Ab21 and Ab52 using a luciferase reporter gene in a cell-based assay.
  • FIG. 20A shows the amino acid sequences of the heavy chain variable regions of TREM2 antibodies.
  • the CDR sequences are underlined in each sequence. Sequence regions in-between each underlined CDR sequence correspond to the framework regions.
  • FIG. 20B shows the amino acid sequences of the light chain variable regions of TREM2 antibodies. The CDR sequences are underlined in each sequence. Sequence regions in-between each underlined CDR sequence correspond to the framework regions.
  • FIG. 21A shows FACS histograms demonstrating binding of TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2.
  • FIG. 21B shows antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 binding to WT (Trem+/+) and TREM2 deficient (TREM2 ⁇ / ⁇ ) bone marrow derived mouse macrophages (BMMac).
  • Antibody Ab88 represents the negative isotype control. Shaded histograms represent the TREM2 antibody negative population. Black outlined histograms represent the TREM2 antibody positive population.
  • FIG. 22A shows FACS histograms demonstrating binding of TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab43, Ab45, Ab60, and Ab65 to a human cell line (293) expressing recombinant human TREM2-DAP12 fusion protein. Shaded histograms represent a TREM2 antibody negative population. Black outlined histograms represent a TREM2 antibody positive population.
  • FIG. 22B shows antibodies Ab1, Ab9, Ab14, Ab22, Ab43, Ab45, Ab60, and Ab65 binding to primary human dendritic cells (hDCs). Antibody Ab88 represents the negative isotype control. Shaded histograms represent secondary antibody alone negative control. Black outlined histograms represent the TREM2 antibody positive population.
  • FIG. 23A shows FACS dot plots demonstrating expression of cell surface markers CD83 and CD86 on human dendritic cells (DCs) after incubation with plate-bound TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65.
  • Antibody Ab88 represents the negative isotype control. Plots were gated on CD11c + HLA-DR + LIN ⁇ DCs. Percentage of cells within the CD83+CD86+ gate is displayed on each plot.
  • FIG. 23B shows FACS histograms demonstrating expression of cell surface marker CD86 on human dendritic cells (DCs) after incubation with cross-linked TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65. Antibodies were cross-linked with anti-human secondary antibody. Antibody Ab88 represents the negative isotype control.
  • FIG. 24A shows Syk phosphorylation as determined by western blot in wild-type and TREM2 deficient (Trem2 ⁇ / ⁇ ) mouse macrophages after incubation with TREM2 antibodies Ab1, Ab9, or Ab45. Antibodies Ab89 and Ab92 are negative isotype controls.
  • FIG. 24B shows Syk phosphorylation as determined by western blot in human macrophages after incubation with TREM2 antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45, and Ab65. Antibodies Ab16 and Ab77 are non-agonistic negative controls.
  • FIG. 24C shows Syk phosphorylation as determined by western blot in primary human dendritic cells after incubation with TREM2 antibodies Ab1, Ab5, Ab9, Ab22, Ab45, or Ab65.
  • FIG. 25 shows competitive binding between TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, Ab65, Ab66, and Ab68 and E. coli cells expressing putative TREM2 ligand to mouse and human cell lines expressing TREM2.
  • Bacterial binding is expressed as a percentage of control. Average of two independent experiments; black bars: no difference to isotype control, red bars: significantly different from isotype controls (ANOVA).
  • FIG. 26A shows DAP12 phosphorylation as determined by western blot in mouse macrophages after incubation with TREM2 antibody Ab45 or Ab65.
  • FIG. 26B shows DAP12 phosphorylation as determine by western blot in wild-type and TREM2 deficient (Trem2 ⁇ / ⁇ ) mouse macrophages after incubation with TREM2 antibody Ab1, Ab9, Ab22, or Ab45.
  • FIG. 27A shows an epitope map of TREM2 antibodies Ab1 and Ab9.
  • FIG. 27B shows an epitope map of TREM2 antibodies Ab45 and Ab65.
  • FIG. 28 shows Fortebio analysis demonstrating simultaneous binding of antibody MAB17291 and antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 to TREM2-Fc. Control antibodies Ab63 and Ab87 did not simultaneously bind to TREM2-Fc.
  • FIG. 29 shows the percent increased survival of wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derived macrophages cultured in the presence of plate bound, cross-linked TREM2 antibody Ab22, Ab45, or Ab65 Fabs and M-CSF.
  • Antibody Ab88 represents the negative isotype control.
  • FIG. 30A shows the luminescence viability assay of mouse bone marrow derived macrophages cultured in the presence of soluble, non-cross-linked TREM2 antibody Fabs and M-CSF. Antibody Ab99 represents the negative isotype control.
  • FIG. 30B shows the luminescence viability assay of mouse bone marrow derived macrophages cultured in the presence of soluble, full-length TREM2 antibodies and M-CSF. Antibody Ab91 represents the negative isotype control.
  • the “NT” dotted line indicates the average viability obtained with untreated macrophages (no antibody added).
  • the “no MCSF” dotted line indicates the average viability obtained when macrophages are cultured in the absence of M-CSF.
  • FIG. 31A shows induction of TREM2-dependent gene expression by plate bound, full-length anti-TREM2 antibodies using a luciferase reporter gene in a cell-based assay.
  • FIG. 31B shows induction of TREM2-dependent gene expression by plate bound, full-length anti-TREM2 antibodies using a luciferase reporter gene in a cell-based assay.
  • FIG. 31C shows induction of TREM2-dependent gene expression by plate bound phosphatidylserine (PS).
  • PS phosphatidylserine
  • FIG. 32 shows inhibition of TREM2-dependent gene expression by soluble, full-length anti-TREM2 antibodies using a luciferase reporter gene in a cell-based assay.
  • FIG. 33 shows competitive interactions between TREM2 antibodies Ab22 and Ab45 and Phosphatidylserine (PS) or Sphingomyelin (SM) in mouse and human cell lines expressing TREM2.
  • PS Phosphatidylserine
  • SM Sphingomyelin
  • FIG. 34A shows activation of TREM2-dependent gene IL-6 in mouse macrophages by plate bound, Fab anti-TREM2 antibodies Ab22 and Ab65 Fabs.
  • FIG. 34B shows activation of TREM2-dependent gene MCP-1 in mouse macrophages by plate bound, Fab anti-TREM2 antibodies Ab22 and Ab65 Fabs.
  • “no Ab” indicates negative control with no antibody treatment
  • “22” indicates treatment with Ab22 Fab
  • “65” indicates treatment with Ab65 Fab
  • ctr” indicates treatment with control antibody Fab.
  • the term “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a particular disease, disorder, or condition in an individual.
  • An individual may be predisposed to, susceptible to a particular disease, disorder, or condition, or at risk of developing such a disease, disorder, or condition, but has not yet been diagnosed with the disease, disorder, or condition.
  • an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
  • “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.
  • treatment refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition.
  • An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.
  • an “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease, disorder, or condition.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the anti-TREM2 and/or anti-DAP12 antibody to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-TREM2 and/or anti-DAP12 antibody are outweighed by the therapeutically beneficial effects.
  • administration “in conjunction” with another compound or composition includes simultaneous administration and/or administration at different times.
  • Administration in conjunction also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.
  • An “individual” for purposes of treatment, prevention, or reduction of risk refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like.
  • the individual is human.
  • immunoglobulin (Ig) is used interchangeably with “antibody” herein.
  • antibody herein is used in the broadest sense and specifically covers 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 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. The pairing of a V H and V L together forms a single antigen-binding site.
  • L light
  • H heavy
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (“ ⁇ ”), delta (“ ⁇ ”), epsilon (“ ⁇ ”), gamma (“ ⁇ ”) and mu (“ ⁇ ”), respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • subclasses immunoglobulins
  • the subunit structures and three dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al., Cellular and Molecular Immunology, 4 th ed. (W.B. Saunders Co., 2000).
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • an “isolated” antibody such as an isolated anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly).
  • the isolated polypeptide is free of association with all other contaminant components from its production environment.
  • Contaminant components from its production environment such as those resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant T-cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • variable domains of the heavy chain and light chain may be referred to as “V H ” and “V L ”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, such as anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • HVRs hypervariable regions
  • FR framework regions
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent-cellular toxicity.
  • the term “monoclonal antibody” as used herein refers to an antibody, such as a monoclonal anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations, etc.) that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., 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, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.
  • phage-display technologies see, e.g., 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.
  • full-length antibody “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody, such as an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, in its substantially intact form, as opposed to an antibody fragment.
  • whole antibodies include those with heavy and light chains including an Fc region.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • antibody fragment comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
  • antibody fragments include Fab, Fab′, F(ab′) 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, 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 produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.
  • Fab′ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the C H 1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
  • “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding.
  • Plückthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
  • “Functional fragments” of antibodies comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the F region of an antibody which retains or has modified FcR binding capability.
  • antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the V H and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the V H and V L domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described in greater detail in, for example, EP 404,097; WO 93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).
  • a “chimeric antibody” refers to an antibody (immunoglobulin), such as a chimeric anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci.
  • Chimeric antibodies of interest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • humanized antibody is used a subset of “chimeric antibodies.”
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like.
  • the number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is one that possesses an amino-acid sequence corresponding to that of an antibody, such as an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced 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).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.
  • hypervariable region when used herein refers to the regions of an antibody-variable domain, such as that of an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, that are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3).
  • H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • HVR delineations are in use and are encompassed herein.
  • the HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., supra). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software.
  • the “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
  • HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • the variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-HVR definitions.
  • Framework or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.
  • variable-domain residue-numbering as in Kabat or “amino-acid-position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. References to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system (e.g., see United States Patent Publication No. 2010-280227).
  • acceptor human framework is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework.
  • An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 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.
  • VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
  • a “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.
  • the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.
  • amino-acid modification at a specified position, e.g., of an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residue means insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.
  • the preferred amino acid modification herein is a substitution.
  • an “affinity-matured” antibody such as an affinity matured anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, is one with one or more alterations in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s).
  • an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen.
  • Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by VH- and VL-domain shuffling.
  • Random mutagenesis of HVR and/or framework residues is described by, 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).
  • the term “specifically recognizes” or “specifically binds” refers to measurable and reproducible interactions such as attraction or binding between a target and an antibody, such as between an anti-TREM2 antibody and TREM2, or an anti-DAP12 antibody and DAP12 that is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody such as an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, that specifically or preferentially binds to a target or an epitope is an antibody that binds this target or epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets or other epitopes of the target.
  • an antibody (or a moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • An antibody that specifically binds to a target may have an association constant of at least about 10 3 M ⁇ 1 or 10 4 M ⁇ 1 , sometimes about 10 5 M ⁇ 1 or 10 6 M ⁇ 1 , in other instances about 10 6 M ⁇ 1 or 10 7 M ⁇ 1 , about 10 8 M ⁇ 1 to 10 9 M ⁇ 1 , or about 10 10 M ⁇ 1 to 10 11 M ⁇ 1 or higher.
  • immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • an “interaction” between a TREM2 protein, or DAP12 protein, and a second protein encompasses, without limitation, protein-protein interaction, a physical interaction, a chemical interaction, binding, covalent binding, and ionic binding.
  • an antibody “inhibits interaction” between two proteins when the antibody disrupts, reduces, or completely eliminates an interaction between the two proteins.
  • an “agonist” antibody or an “activating” antibody is an antibody, such as an agonist anti-TREM2 antibody or an agonist anti-DAP12 antibody of the present disclosure, that induces (e.g., increases) one or more activities or functions of the antigen after the antibody binds the antigen.
  • an “antagonist” antibody or a “blocking” antibody is an antibody, such as an antagonist anti-TREM2 antibody or an antagonist anti-DAP12 antibody of the present disclosure, that reduces or eliminates (e.g., decreases) antigen binding to one or more ligand after the antibody binds the antigen, and/or that reduces or eliminates (e.g., decreases) one or more activities or functions of the antigen after the antibody binds the antigen.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2, IgG3 and IgG4.
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature.
  • Native sequence human Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors, Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (“ITAM”) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (“ITIM”) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
  • WO 2004/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).
  • percent (%) amino acid sequence identity and “homology” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the 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 achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.
  • an “isolated” nucleic acid molecule encoding an antibody is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • the isolated nucleic acid is free of association with all components associated with the production environment.
  • the isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
  • phage vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • viral vector capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label.
  • modifications include, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
  • the 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms 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′-azido-ribose, carbocyclic sugar analogs, a-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 linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • a “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • a host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • 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) polypeptide; 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 TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin,
  • an “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.
  • the present disclosure relates to anti-TREM2 antibodies and/or anti-DAP12 antibodies with one or more agonist or antagonist activities; methods of making and using such antibodies; pharmaceutical compositions containing such antibodies; nucleic acids encoding such antibodies; and host cells containing nucleic acids encoding such antibodies.
  • the agonistic activities of the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are due, at least in part, to the ability of the antibodies to induce or retain TREM2 receptor clustering on the surface of cells.
  • anti-TREM2 and/or anti-DAP12 antibodies can induce or retain TREM2 receptor clustering in vivo by not only binding specifically to TREM2, but by also binding to Fc receptors on adjacent cells, which leads to antibody aggregation that in turn aggregates TREM2.
  • certain immunoglobulin isotypes including without limitation, IgG2 and IgM, have an intrinsic ability to induce or retain clustering of target antigens (e.g., TREM2) without binding Fc receptors on adjacent cells.
  • target antigens e.g., TREM2
  • agonistic TREM2 activities can be determined or tested in vitro by any of several techniques disclosed herein (see, e.g., Examples 23-26, 34-37, 41-44, 52-55, and 67-68), including, without limitation, plate-binding full-length anti-TREM2 antibodies to increase the density of antibodies exposed to TREM2 and cross-linking anti-TREM2 antibodies.
  • certain aspects of the present disclosure are based, at least in part, on the identification of anti-TREM2 and/or anti-DAP12 antibodies that are capable of binding to both human and mouse TREM2 with high affinity (see, e.g., Examples land 40); that compete with TREM2-ligand for binding to the ligand-binding site on human and mouse TREM2 (see, e.g., Examples 26 and 43); and that exhibit one or more agonistic TREM2 activities, including, without limitation, induction of CD83 + CD86 + dendritic cells (see, e.g., Examples 23 and 41), induction of the TREM2 downstream signaling molecule Syk in macrophages and dendritic cells (see, e.g., Examples 24 and 42), induction of the TREM2 signaling adaptor molecule DAP12 in macrophages (see, e.g., Examples 25 and 44), induction of cell survival of innate immune cells, such as macrophages (see, e.g., Examples 34 and
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can also induce antagonistic activities when the antibody is produced or otherwise formatted such that it is incapable of inducing or retaining TREM2 receptor clustering.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure exhibit one or more antagonistic TREM2 activities, including, without limitation, inhibition of cell survival of innate immune cells ((see, e.g., Examples 35 and 53), and inhibition of TREM2-dependent gene expression (see, e.g., Examples 37, 55, and 67).
  • the invention provides antibodies that bind to a TREM2 protein of the present disclosure and modulate one or more TREM2 activities after binding to a TREM2 protein expressed in a cell.
  • TREM2 proteins of the present disclosure include, without limitation, a mammalian TREM2 protein, human TREM2 protein (Uniprot Accession No. Q9NZC2), mouse TREM2 protein (Uniprot Accession No. Q99NH8), rat TREM2 protein (Uniprot Accession No. D3ZZ89), Rhesus monkey TREM2 protein (Uniprot Accession No. F6QVF2), bovine TREM2 protein (Uniprot Accession No. Q05B59), equine TREM2 protein (Uniprot Accession No. F7D6L0), pig TREM2 protein (Uniprot Accession No. H2EZZ3), and dog TREM2 protein (Uniprot Accession No. E2RP46).
  • TREM2 protein refers to both wild-type sequences and naturally occurring variant sequences.
  • TREM2 Triggering receptor expressed on myeloid cells-2
  • TREM2 is variously referred to as TREM-2, Trem2a, Trem2b, Trem2c, triggering receptor expressed on myeloid cells-2a, and triggering receptor expressed on monocytes-2.
  • TREM2 is a 230 amino acid membrane protein.
  • TREM2 is an immunoglobulin-like receptor primarily expressed on myeloid lineage cells, including without limitation, macrophages, dendritic cells, osteoclasts, microglia, monocytes, Langerhans cells of skin, and Kupffer cells.
  • TREM2 forms a receptor-signaling complex with DAP12.
  • TREM2 phosphorylates and signals through DAP12 (an ITAM domain adaptor protein).
  • TREM2 signaling results in the downstream activation of PI3K or other intracellular signals.
  • Toll-like receptor (TLR) signals are important for the activation of TREM2 activities, e.g., in the context of an infection response.
  • TLRs also play a key role in the pathological inflammatory response, e.g., TLRs expressed in macrophages and dendritic cells.
  • an example of a human TREM2 amino acid sequence is set forth below as SEQ ID NO: 1:
  • the human TREM2 is a preprotein that includes a signal peptide. In some embodiments, the human TREM2 is a mature protein. In some embodiments, the mature TREM2 protein does not include a signal peptide. In some embodiments, the mature TREM2 protein is expressed on a cell.
  • TREM2 contains a signal peptide located at amino acid residues 1-18 of human TREM2 (SEQ ID NO: 1); an extracellular immunoglobulin-like variable-type (IgV) domain 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).
  • IgV immunoglobulin-like variable-type
  • the transmembrane domain of human TREM2 contains a lysine at amino acid residue 186 that can interact with an aspartic acid in DAP12, which is a key adaptor protein that transduces signaling from TREM2, TREM1, and other related IgV family members.
  • Homologues of human TREM2 include, without limitation, the natural killer (NK) cell receptor NK-p44 (NCTR2), the polymeric immunoglobulin receptor (pIgR), CD300E, CD300A, CD300C, and TREML1/TLT1.
  • NCTR2 has similarity with TREM2 within the IgV domain.
  • the invention provides antibodies that bind to a DAP12 protein of the present disclosure and modulate one or more DAP12 activities after binding to a DAP12 protein expressed in a cell.
  • DAP12 proteins of the present disclosure include, without limitation, a mammalian DAP12 protein, human DAP12 protein (Uniprot Accession No. 043914), mouse DAP12 protein (Uniprot Accession No. 054885), rat DAP12 protein (Uniprot Accession No. Q6X9T7), Rhesus monkey DAP12 protein (Uniprot Accession No. Q8WNQ8), bovine DAP12 protein (Uniprot Accession No. Q95J80), and pig DAP12 protein (Uniprot Accession No. Q9TU45).
  • DAP12 protein refers to both wild-type sequences and naturally occurring variant sequences.
  • DNAX-activation protein 12 is variously referred to 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.
  • DAP12 functions as a transmembrane signaling polypeptide, which contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. It may associate with the killer-cell inhibitory receptor (KIR) family of membrane glycoproteins and may act as an activating signal transduction element.
  • TAM immunoreceptor tyrosine-based activation motif
  • the DAP12 protein may bind zeta-chain (TCR) associated protein kinase 70 kDa (ZAP-70) and spleen tyrosine kinase (SYK), and play a role in signal transduction, bone modeling, brain myelination, and inflammation.
  • TCR zeta-chain
  • ZAP-70 zeta-chain associated protein kinase 70 kDa
  • SYK spleen tyrosine kinase
  • DAP12-encoding gene has been associated with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease.
  • PLOSL polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy
  • TREM2 sclerosing leukoencephalopathy
  • Multiple alternative transcript variants encoding distinct isoforms of DAP12 have been identified.
  • DAP12 non-covalently associates with activating receptors of the CD300 family. Cross-linking of CD300-TYROBP/DAP12 complexes results in cellular activation, such as neutrophil activation mediated by integrin.
  • DAP12 is a homodimer; disulfide-linked protein.
  • DAP12 interacts with SIRPB1, TREM1, CLECSF5, SIGLEC14, CD300LB, CD300E, and CD300D by similarity and via ITAM domain, as well as with SYK via SH2 domain.
  • DAP12 activates SYK, which mediates neutrophils and macrophages integrin-mediated activation.
  • DAP12 interacts with KLRC2 and KIR2DS3.
  • an example of a human DAP12 amino acid sequence is set forth below as SEQ ID NO: 2:
  • the human DAP12 is a preprotein that includes a signal peptide. In some embodiments, the human DAP12 is a mature protein. In some embodiments, the mature DAP12 protein does not include a signal peptide. In some embodiments, the mature DAP12 protein is expressed on a cell. DAP12 is a single-pass type I 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-based activation motif (ITAM) domain is located at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2).
  • ITAM immunoreceptor tyrosine-based activation motif
  • an aspartic acid residue in DAP12 interacts with the transmembrane domain of human TREM2 containing a lysine at amino acid residue 186, and transduces signaling from TREM2, TREM1, and other related IgV family member proteins.
  • antibodies of the present disclosure relate to antibodies that specifically bind to TREM2 and/or DAP12.
  • antibodies of the present disclosure bind a mature TREM2 protein and/or DAP12 protein.
  • antibodies of the present disclosure bind a mature TREM2 protein and/or DAP12 protein, wherein the mature TREM2 protein and/or DAP12 protein is expressed on a cell.
  • antibodies of the present disclosure bind a TREM2 protein and/or DAP12 protein expressed on one or more human cells selected from human dendritic cells, human macrophages, human monocytes, human osteoclasts, human Langerhans cells of skin, human Kupffer cells, human microglia, and any combinations thereof.
  • antibodies of the present disclosure are agonist antibodies.
  • antibodies of the present disclosure are inert antibodies.
  • antibodies of the present disclosure are antagonist antibodies.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure generally bind to one or more TREM2 proteins and/or DAP12 proteins expressed in a cell.
  • One class of antibodies is agonist antibodies.
  • the TREM2 receptor is thought to require clustering on the cell surface in order to transduce a signal.
  • agonist antibodies may have unique features to stimulate, for example, the TREM2 receptor. For example, they may have the correct epitope specificity that is compatible with receptor activation, as well as the ability to induce or retain receptor clustering on the cell surface.
  • antibodies may cluster receptors by multiple potential mechanisms.
  • Some isotypes of human antibodies such as IgG2 have, due to their unique structure, an intrinsic ability to cluster receptors, or retain receptors in a clustered configuration, thereby activating receptors such as TREM2 without binding to an Fc receptor (e.g., White et al., (2015) Cancer Cell 27, 138-148).
  • Other antibodies cluster receptors (e.g., TREM2) by binding to Fcg receptors on adjacent cells. Binding of the constant IgG Fc part of the antibody to Fcg receptors leads to aggregation of the antibodies, and the antibodies in turn aggregate the receptors to which they bind through their variable region (Chu et al (2008) Mol Immunol, 45:3926-3933; and Wilson et al., (2011) Cancer Cell 19, 101-113).
  • Binding to the inhibitory Fcg receptor FcgR (FcgRIIB) that does not elicit cytokine secretion, oxidative burst, increased phagocytosis, and enhanced antibody-dependent, cell-mediated cytotoxicity (ADCC) is often a preferred way to cluster antibodies in vivo, since binding to FcgRIIB is not associated with immune adverse effects.
  • cluster receptors e.g., TREM2
  • antibody fragments e.g., Fab fragments
  • TREM2 cluster receptors
  • cross-linked antibody fragments e.g., Fab fragments
  • TREM2 may function as agonist antibodies if they induce receptor clustering on the cell surface and bind an appropriate epitope on the target (e.g., TREM2).
  • antibodies that bind a TREM2 protein and/or a DAP12 protein may include agonist antibodies that due to their epitope specificity bind TREM2 and/or DAP12 and activate one or more TREM2 and/or DAP12 activities.
  • such antibodies may bind to the ligand-binding site on the target antigen (e.g., TREM2 and/or DAP12) and mimic the action of a natural ligand, or stimulate the target antigen to transduce signal by binding to one or more domains that are not the ligand-binding sites.
  • TREM2 and/or DAP12 target antigen
  • Such antibodies would not interfere with ligand binding and may act additively or synergistically with the natural ligands.
  • 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 induces one or more activities of TREM2 and/or DAP12 after binding to a TREM2 and/or DAP12 protein that is expressed in a cell.
  • the one or more TREM2 activities, the one or more DAP12 activities, or both are selected from TREM2 binding to DAP12; DAP12 binding to TREM2; DAP12 phosphorylation; TREM2 phosphorylation; PI3K activation; increased expression of one or more anti-inflammatory cytokines, increased expression of one or more anti-inflammatory mediators (e.g., cytokines) selected from IL-12p70, IL-6, and IL-10; reduced expression of one or more pro-inflammatory cytokines; 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-1 ⁇ , TNG, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF
  • TREM2-dependent genes e.g., transcription factors of the nuclear factor of activated T-cells (NFAT) family of transcription factors.
  • an antibody dependent on binding to FcgR receptor to activate targeted receptors may lose its agonist activity if engineered to eliminate FcgR binding (see, e.g., Wilson et al., (2011) Cancer Cell 19, 101-113; Armour at al., (2003) Immunology 40 (2003) 585-593); and White et al., (2015) Cancer Cell 27, 138-148).
  • an antibody of the present disclosure with the correct epitope specificity can be an agonist antibody and activate the target antigen, with minimal adverse effects, when the antibody has an Fc domain from a human IgG2 isotype (CH1 and hinge region) or another type of Fc domain that is capable of preferentially binding the inhibitory FcgRIIB r receptors, or a variation thereof.
  • agonist antibody Fc isotypes and modifications are provided in Table 2 below.
  • the agonist antibody has an Fc isotype listed in Table 2 below.
  • Fc Isotype Mutation (EU or Kabat numbering scheme) IgG1 N297A IgG1 D265A and N297A IgG1 L234A and L235A L234A and G237A L234A and L235A and G237A IgG2 V234A and G237A IgG4 L235A and G237A and E318A IgG4 S228P and L236E IgG2/4 hybrid IgG2 aa 118-260 and IgG4 aa 261 to 447 IgG2 H268Q and V309L; and A330S and P33 1S IgG1 C226S and C229S and E233P and L234V and L235A IgG1 L234F and L235E and P33 1S IgG2 C232S or C233S IgG2 A330S and P33 1S IgG2 C232S or C233S IgG2 A330S and P
  • antibodies with human IgG1 or IgG3 isotypes and mutants thereof e.g. Strohl (2009) Current Opinion in Biotechnology 2009, 20:685-691
  • that bind the activating Fcg Receptors I, IIA, IIC, IIIA, IIIB in human and/or Fcg Receptors I, III and IV in mouse may also act as agonist antibodies in vivo but may be associated with adverse effects related to ADCC.
  • Fcg receptors appear to be less available for antibody binding in vivo, as compared to the Inhibitory Fcg receptor FcgRIIB (see, e.g., White, et al., (2013) Cancer Immunol. Immunother. 62, 941-948; and Li et al., (2011) IScience 333(6045):1030-1034.).
  • the agonist antibody is of the IgG class, the IgM class, or the IgA class. In some embodiments, the agonist antibody has an IgG1, IgG2, IgG3, or IgG4 isotype.
  • the agonist antibody has an IgG2 isotype. In some embodiments, the agonist antibody contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the agonist antibody induces the one or more TREM2 activities, the DAP12 activities, or both independently of binding to an Fc receptor. In some embodiments, the agonist antibody binds an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB). In some embodiments, the Fc region contains one or more modifications.
  • the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the 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; Armour et al.
  • the agonist antibody has an IgG2 isotype with a heavy chain constant domain that contains a C127S amino acid substitution, where the amino acid position is according to the EU or Kabat numbering convention (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO2008079246).
  • the agonist antibody has an IgG2 isotype with a Kappa light chain constant domain that contains a C214S amino acid substitution, where the amino acid position is according to the EU or Kabat numbering convention (White et al., (2015) Cancer Cell 27, 138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; and WO2008079246).
  • the agonist antibody has an IgG1 isotype. In some embodiments, the agonist antibody contains a mouse IgG1 constant region. In some embodiments, the agonist antibody contains 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 certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB). In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the 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.
  • the antibody includes an IgG2 isotype heavy chain constant domain 1(CH1) and hinge region (White et al., (2015) Cancer Cell 27, 138-148).
  • the IgG2 isotype CH1 and hinge region contain the amino acid sequence of ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397).
  • the antibody Fc region contains a S267E amino acid substitution, a L328F amino acid substitution, or both, and/or a N297A or N297Q amino acid substitution, where the amino acid position is according to the EU or Kabat numbering convention.
  • the agonist antibody has an IgG4 isotype. In some embodiments, the agonist antibody contains 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 certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (Fc ⁇ IIB). In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the 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 according to the EU or Kabat numbering convention.
  • the agonist antibody has a hybrid IgG2/4 isotype.
  • the agonist antibody includes an amino acid sequence containing amino acids 118 to 260 according to Kabat numbering convention of human IgG2 and amino acids 261 to 447 according to EU or Kabat numbering convention of human IgG4 (WO 1997/11971; WO 2007/106585).
  • the antibody contains a mouse IgG4 constant region (Bartholomaeus, et al. (2014). J. Immunol. 192, 2091-2098).
  • the Fc region further contains 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 according to the EU or Kabat numbering convention.
  • inert antibodies refer to antibodies that specifically bind their target antigen but do not modulate (e.g., decrease/inhibit or activate/induce) antigen function.
  • inert antibodies do not modulate ligand binding and/or TREM2 activities.
  • antibodies that do not have the ability to cluster TREM2 on the cell surface may be inert antibodies even if they have an epitope specificity that is compatible with receptor activation.
  • antibodies that bind a TREM2 protein and/or a DAP12 protein may include antibodies that bind TREM2 and/or DAP12 but, due to their epitope specificity, do not modulate protein function.
  • Such functionally inert antibodies can be used as cargo to transport toxins as described for the CD33 antibody Gemtuzumab zogamicin, (marketed as Mylotarg) which is conjugated to the cytotoxic agent from the class of calicheamicins and is used to target and kill acute myelogenous leukemia tumors (Naito et al., (2000), Leukemia, 14, 1436-1443; Jamaicart (2011) Clin Cancer Res 17; 6417-6436; Hamann et al., (2002) Journal: Bioconjugate Chemistry, 13, 47-58; and Beitz et al., (2001) Clin Cancer Res 7; 1490-6.).
  • antibodies of the present disclosure are inert antibodies that bind TREM2 and/or DAP12 but are incapable of inducing one or more TREM2 activities (e.g., a TREM2 activity described herein) and/or DAP12 activities (e.g., a DAP12 activity described herein).
  • inert antibody Fc isotypes and modifications are provided in Table 3 below.
  • the inert antibody has an Fc isotype listed in Table 3 below.
  • a third class of antibodies of the present disclosure includes antagonist antibodies.
  • antibodies that bind a TREM2 protein and/or a DAP12 protein may include antagonist antibodies that bind TREM2 and/or DAP12 and inhibit one or more TREM2 activities and/or DAP12 activities, either by preventing interaction between TREM2 and/or DAP12 and its ligand(s), or by preventing the transduction of signal from the extracellular domain of TREM2 and/or DAP12 into the cell cytoplasm in the presence of ligand.
  • antagonist antibodies of the present disclosure may have the epitope specificity of an agonist antibody of the present disclosure, but have an Fc domain that is not capable of binding Fcg receptors and thus is unable to, for example, cluster DAP12 and/or the TREM2 receptor.
  • an antibody of the present disclosure is an antagonist antibody.
  • the antagonist antibody inhibits one or more TREM2 and/or DAP12 activities.
  • the antagonist antibody decreases activity of one or more TREM2-dependent genes.
  • the one or more TREM2-dependent genes include, without limitation, one or more nuclear factor of activated T-cells (NFAT) transcription factors.
  • the antagonist antibody decreases the survival of macrophages, microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2 microglial cells, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or dendritic cell.
  • the antagonist antibody inhibits interaction between TREM2 and/or DAP12 and one or more TREM2 and/or DAP12 ligands. In some embodiments, the antagonist antibody inhibits TREM2 and/or DAP12 signal transduction. In some embodiments, the antagonist antibody inhibits interaction between TREM2 and/or DAP12 and one or more TREM2 and/or DAP12 ligands and inhibits TREM2 and/or DAP12 signal transduction.
  • antibody cross-linking is required for agonist antibody function.
  • Antibody cross-linking can occur through binding to a secondary antibody in vitro or through binding to Fc receptors in vivo.
  • antagonistic antibodies can be converted to agonistic antibodies via biotin/streptavidin cross-linking or secondary antibody binding in vitro (see for example Gravestein et al., (1996) J. Exp. Med. 184:675-685; Gravestein et al., (1994) International Immunol. 7:551-557).
  • Agonistic antibodies may exert their activity by mimicking the biological activity of the receptor ligand or by enhancing receptor aggregation, thereby activating receptor signaling.
  • the absence of antibody cross-linking is required for antagonistic activity.
  • Antagonistic antibodies may exert their activity by blocking receptor-ligand interactions.
  • antagonist antibody Fc isotypes and modifications are provided in Table 3 below.
  • the antagonist antibody has an Fc isotype listed in Table 3 below.
  • inert and/or antagonist anti-TREM antibodies of the present disclosure include one or more of the Fc isotypes and modifications listed in Table 3.
  • Fc Isotype Mutation (EU or Kabat numbering scheme) IgG1 N297A or N297Q IgG1 D265A and N297A IgG1 L234A and L235A IgG2 V234A and G237A IgG4 L235A and G237A and E318A E233P and/or F234V N297Aor N297Q IgG4 S228P and L236E S241P S241P and L248E IgG2 H268Q and V309L and A330S and P331S IgG1 C220S and C226S and C229S and P238S IgG1 C226S and C229S and E233P and L234V, and L235A IgG1 E233P and L234V and L235A and G236-deleted P238A D265
  • the antibody has an IgG1 isotype. In some embodiments, the antibody contains a mouse IgG1 constant region. In some embodiments, the antibody contains a human IgG1 constant region. In some embodiments, the human IgG1 constant region includes an Fc region. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from N297A, N297Q (Bolt S et al.
  • the Fc region further includes an amino acid deletion at a position corresponding to glycine 236 according to the EU or Kabat numbering convention.
  • the antibody has an IgG1 isotype with a heavy chain constant region that contains a C220S amino acid substitution according to the EU or Kabat numbering convention.
  • the Fc region further contains one or more additional amino acid substitutions selected from A330L, L234F; L235E, and/or P331S according to EU or Kabat numbering convention.
  • the antibody has an IgG2 isotype.
  • the antibody contains a human IgG2 constant region.
  • the human IgG2 constant region includes an Fc region.
  • the Fc region contains one or more modifications.
  • the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the 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 according to the EU or Kabat numbering convention.
  • the antibody has an IgG4 isotype.
  • the antibody contains a human IgG4 constant region.
  • the human IgG4 constant region includes an Fc region.
  • the Fc region contains one or more modifications.
  • the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype).
  • the one or more amino acid substitutions are selected from E233P, F234V, L235A, G237A, E318A (Hutchins et al.
  • the Fc region further contains one or more additional amino acid substitutions selected from a M252Y, S254T, and/or T256E, where the amino acid position is according to the EU or Kabat numbering convention.
  • one or more of the IgG1 variants described herein may be combined with an A330L mutation (Lazar et al., (2006) Proc Natl Acad Sci USA, 103:4005-4010), or one or more of L234F, L235E, and/or P331S mutations (Sazinsky et al., (2008) Proc Natl Acad Sci USA, 105:20167-20172), where the amino acid position is according to the EU or Kabat numbering convention, to eliminate complement activation.
  • the IgG variants described herein may be combined with one or more mutations to enhance the antibody half-life in human serum (e.g.
  • an IgG4 variant of the present disclosure may be combined with an S228P mutation according to the EU or Kabat numbering convention (Angal et al., (1993) Mol Immunol, 30:105-108) and/or with one or more mutations described in Peters et al., (2012) J Biol Chem. 13; 287(29):24525-33) to enhance antibody stabilization.
  • Certain aspects of the present disclosure related to anti-TREM2 antibodies.
  • anti-TREM2 antibodies of the present disclosure are agonist antibodies that induce one or more TREM2 activities. In some embodiments, anti-TREM2 antibodies of the present disclosure are agonist antibodies that promote 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, M1 macrophages, activated M1 macrophages, M2 macrophages, monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells.
  • promoting survival of one or more innate immune cells encompasses prolonging cell survival or otherwise delaying cell death. Accordingly, in some embodiments, anti-TREM2 antibodies of the present disclosure prolong cell survival of one or more innate immune cells. In some embodiments, anti-TREM2 antibodies of the present disclosure delay cell death of one or more innate immune cells. In some embodiments, promoting cell survival and/or prolonging cell survival is determined by measuring cell survival of one or more innate immune cells in the presence of the anti-TREM2 antibody as compared to cell survival of corresponding one or more innate immune cells in the absence of the anti-TRME2 antibody.
  • delay in cell death is determined by measuring cell death of one or more innate immune cells in the presence of the anti-TREM2 antibody as compared to cell death of corresponding one or more innate immune cells in the absence of the anti-TRME2 antibody. Any suitable methods of measuring cell survival or cell death known in the art and disclosed herein may be used (see, e.g., Examples 30, 34, 35, 38, 52, 53, and 56).
  • anti-TREM2 antibodies of the present disclosure are agonist antibodies that increase IL-6 expression.
  • anti-TREM2 antibodies of the present disclosure are agonist antibodies that promote survival of one or more innate immune cells and increase expression of IL-6. Any suitable meathods known in the art and disclosed herein for measuring IL-6 expression in a cell may be used (see, e.g., Examples 28, 38, and 68).
  • anti-TREM2 antibodies of the present disclosure are inert or antagonist antibodies that inhibit one or more TREM2 activities. In some embodiments, anti-TREM2 antibodies of the present disclosure are inert or antagonist antibodies that decrease survival of one or more innate immune cells. In some embodiments, anti-TREM2 antibodies of the present disclosure decrease survival 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, Langerhans cells of skin, and/or Kupffer cells.
  • decreasing cell survival is determined by measuring cell survival of one or more innate immune cells in the presence of the antagonist anti-TREM2 antibody as compared to cell survival of corresponding one or more innate immune cells in the absence of the antagonist anti-TRME2 antibody. Any suitable methods of measuring cell survival or cell death known in the art and disclosed herein may be used (see, e.g., Examples 30, 34, 35, 38, 52, 53, and 56).
  • an isolated anti-TREM2 antibody of the present disclosure competes for binding of TREM2 with one or more TREM2 ligands.
  • 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 found throughout the present disclosure.
  • the antibody is a bispecific antibody recognizing a first antigen and a second antigen.
  • the TREM2 protein is expressed on a cell surface.
  • anti-TREM2 antibodies of the present disclosure modulate (e.g., induce or inhibit) one or more TREM2 activities.
  • the TREM2 activities modulated (e.g., induced or inhibited) by the anti-TREM2 antibodies may include, without limitation, DAP12 phosphorylation; TREM2 phosphorylation; recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex; PI3K activation; increased expression of anti-inflammatory mediators (e.g.
  • cytokines reduced expression of pro-inflammatory mediators; ERK phosphorylation; increased expression of CCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells; enhancement, normalization, or both of the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation; induction of osteoclast production, increased rate of osteoclastogenesis, or both; increased survival and function of microglial cells and/or macrophages (such as M1 macrophages and/or microglial cells, activated M1 macrophages and/or microglial cells, and/or M2 macrophages and/or microglial cells), dendritic cells, monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells; induction of apoptotic neuron clearance; reduced expression of TNF- ⁇ ; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells, monocytes, macrophage
  • the anti-TREM2 antibodies of the present disclosure can be used to prevent, reduce risk of, or treat dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, seizures, spinal cord injury, traumatic brain injury, age related macular degeneration, gla
  • the anti-TREM2 antibodies of the present disclosure may also be used in advanced wound care.
  • the anti-TREM2 antibodies of the present disclosure are monoclonal antibodies.
  • Anti-TREM2 antibodies of the present disclosure may be tested for inducing one or more TREM2 activities (e.g., TREM2 autophosphorylation; DAP12 phosphorylation; Syk phosphorylation; recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex; PI3K activation; increased expression of cytokines; reduced expression of pro-inflammatory mediators; ERK phosphorylation; increased expression of CCR7; induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells; maturation of bone marrow-derived dendritic cells; enhancement or normalization of the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation; increased ability of dendritic cells, monocytes, microglia, and/or macrophages to induce T-
  • the anti-TREM2 antibodies can be assayed in vitro for tyrosine phosphorylation of, TREM2, DAP12, Syk and/or ERK, by assaying for recruitment of Syk and/or ZAP70 to DAP12, by assaying for PI3K activation, by assaying for induction of expression of cytokines (e.g., IL-12p70, IL-6, and IL-10) or CCR7, or by assaying for reduced expression of pro-inflammatory mediators (e.g., IL1- ⁇ and TNF) with TLR stimulation (e.g., LPS, CpG DNA, or Zymosan).
  • cytokines e.g., IL-12p70, IL-6, and IL-10
  • CCR7 CCR7
  • pro-inflammatory mediators e.g., IL1- ⁇ and TNF
  • TLR stimulation e.g., LPS, CpG DNA, or Zymosan
  • Useful assays may include western blots (e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PI3K-kinase substrates), ELISA (e.g., for secreted interleukin or cytokine secretion), FACS (e.g., for anti-TREM2 binding to TREM2), immunocytochemistry (e.g., for e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PI3K-kinase substrates), reporter-gene assays (e.g., for TLR activation), increased survival and/or function of dendritic cells, macrophages, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or microglia, increased phagocytosis of apoptotic neurons, damaged synapses, amyloid beta or fragments thereof,
  • 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- ⁇ , IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1 ⁇ , IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18).
  • inflammatory cytokines e.g., TNF- ⁇ , IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1 ⁇ , IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CN
  • anti-TREM2 antibodies of the present disclosure increase the expression and/or secretion of one or more inflammatory cytokines. In some embodiments, anti-TREM2 antibodies of the present disclosure decrease the expression and/or secretion of one or more inflammatory cytokines. In some embodiments, anti-TREM2 antibodies of the present disclosure modulate (i.e., increase or decrease) the expression and/or secretion of one or more inflammatory receptors (e.g., CD86). In some embodiments, anti-TREM2 antibodies of the present disclosure increase the expression and/or secretion of one or more inflammatory receptors. In some embodiments, anti-TREM2 antibodies of the present disclosure decrease the expression and/or secretion of one or more inflammatory receptors.
  • inflammatory receptors e.g., CD86
  • anti-TREM2 antibodies of the present disclosure bind to a human TREM2, or a homolog thereof, including without limitation a mammalian TREM2 protein, mouse TREM2 protein (Uniprot Accession No. Q99NH8), rat TREM2 protein (Uniprot Accession No. D3ZZ89), Rhesus monkey TREM2 protein (Uniprot Accession No. F6QVF2), bovine TREM2 protein (Uniprot Accession No. Q05B59), equine TREM2 protein (Uniprot Accession No. F7D6L0), pig TREM2 protein (Uniprot Accession No. H2EZZ3), and dog TREM2 protein (Uniprot Accession No.
  • anti-TREM2 antibodies of the present disclosure specifically bind to human TREM2. In some embodiments, anti-TREM2 antibodies of the present disclosure specifically bind to mouse TREM2. In some embodiments, anti-TREM2 antibodies of the present disclosure specifically bind to both human TREM2 and mouse TREM2. In some embodiments, anti-TREM2 antibodies of the present disclosure modulate (e.g., induce or inhibit) at least one TREM2 activity.
  • the at least one TREM2 activity is DAP12 phosphorylation, TREM2 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators (e.g., cytokines), reduced expression of one or more pro-inflammatory mediators, increased survival and/or function of microglial cells, dendritic cells, macrophages, monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells, reduced expression of TNF- ⁇ , SYK phosphorylation, increased expression of CD83 and/or CD86 on dendritic cells, macrophages, monocytes, and/or macrophages, 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 microglia under conditions of reduced levels of MCSF, reduced phagocytosis by macrophages, dendritic cells, monocytes, and/
  • anti-TREM2 antibodies of the present disclosure bind to a TREM2 protein of the present disclosure and/or naturally occurring variants. In certain preferred embodiments, the anti-TREM2 antibodies bind to human TREM2.
  • anti-TREM2 antibodies of the present disclosure are agonist antibodies, or antagonist antibodies that bind to a TREM2 protein of the present disclosure expressed on the surface of a cell and modulate (e.g., induce or inhibit) at least one TREM2 activity of the present disclosure after binding to the surface-expressed TREM2 protein.
  • anti-TREM2 antibodies of the present disclosure are inert antibodies.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 29-112 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a 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 TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 29-41 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 47-69 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 47-69 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 TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 76-86 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 91-100 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a 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 TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 99-115 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 104-112 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a 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 TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 130-171 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a 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 TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 139-153 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 139-146 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 139-146 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 130-144 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 130-144 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 158-171 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 43-50 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 43-50 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 49-57 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 139-146 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 139-146 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 140-153 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 140-153 of SEQ ID NO: 1.
  • TREM2 proteins of the present disclosure include a complementary determining region 1 (CDR1) located at amino acid residues corresponding to amino acid residues 40-44 of human TREM2 (SEQ ID NO: 1); a complementary determining region 2 (CDR2) located at amino acid residues corresponding to amino acid residues 67-76 of human TREM2 (SEQ ID NO: 1); and a complementary determining region 3 (CDR3) located at amino acid residues corresponding to amino acid residues 114-118 of human TREM2 (SEQ ID NO: 1).
  • CDR1 complementary determining region 1
  • CDR2 complementary determining region 2
  • CDR3 complementary determining region 3
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 40-44 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 40-44 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 67-76 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 114-118 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 114-118 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residue Arg47 or Asp87 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 40-44 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 67-76 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies of the present disclosure bind to an epitope that includes amino acid residues 114-118 of human TREM 2 (SEQ ID NO: 1).
  • anti-TREM2 antibodies of the present disclosure competitively inhibit binding of at least one antibody selected from any of the antibodies listed in Table 1 and/or Table 8. In some embodiments, anti-TREM2 antibodies of the present disclosure competitively inhibit binding of at least one antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66
  • anti-TREM2 antibodies of the present disclosure competitively inhibit binding of at least one of the following anti-TREM2 antibodies: Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65.
  • anti-TREM2 antibodies of the present disclosure bind to an epitope of human TREM2 that is the same as or overlaps with the TREM2 epitope bound by at least one antibody selected from any of the antibodies listed in Table 1 and/or Table 8.
  • anti-TREM2 antibodies of the present disclosure bind to an epitope of human TREM2 that is the same as or overlaps with the TREM2 epitope bound by at least one antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab
  • anti-TREM2 antibodies of the present disclosure bind to an epitope of human TREM2 that is the same as or overlaps with the TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65. In some embodiments, anti-TREM2 antibodies of the present disclosure bind essentially the same TREM2 epitope bound by at least one antibody selected from any of the antibodies listed in Table 1 and/or Table 8.
  • anti-TREM2 antibodies of the present disclosure bind essentially the same TREM2 epitope bound by at least one antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76
  • anti-TREM2 antibodies of the present disclosure bind essentially the same TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65.
  • 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, N.J.).
  • immobilized TREM2 or cells expressing TEM2 on the cell surface are incubated in a solution comprising a first labeled antibody that binds to TREM2 (e.g., human or non-human primate) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to TREM2.
  • the second antibody may be present in a hybridoma supernatant.
  • immobilized TREM2 or cells expressing TREM2 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody.
  • 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 one of the antibodies listed in Table 1 and/or Table 8 or selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63
  • the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprise Kabat CDR, Chothia CDR, or Contact CDR sequences as shown in Table 1 and/or Table 8 or from an antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64
  • anti-TREM2 antibodies of the present disclosure comprise at least one, two, three, four, five, or six HVRs selected from (i) HVR-H1 comprising 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 Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab
  • 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) an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: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) an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:25-49, 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: 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 the group consisting of SEQ ID NOs: 50-119; and/or wherein the light chain variable domain comprises one or
  • anti-TREM2 antibodies of the present disclosure comprise a heavy chain variable region of any one of the antibodies listed in Table 1 and/or Table 8 or selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74,
  • any of the antibodies of the present disclosure may be produced by a cell line.
  • the cell line may be a yeast cell line.
  • the cell line may be a mammalian cell line.
  • the cell line may be a hybridoma cell line. Any cell line known in the art suitable for antibody production may be used to produce an antibody of the present disclosure. Exemplary cell lines for antibody production are described throughout the present disclosure.
  • the anti-TREM2 antibody is an anti-TREM2 monoclonal antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab1. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially 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 of the heavy chain variable domains of monoclonal antibody Ab1. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab1.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab1.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:3, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO:3; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO:25; (iii) HVR-H3 comprising 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 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
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab9. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially the same TREM2 epitope as Ab9. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains of monoclonal antibody Ab9. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab9.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab9.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:9, or an amino acid sequence with 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 comprising 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
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab14. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially 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 of the heavy chain variable domains of monoclonal antibody Ab14. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab14.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab14.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:13, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO:13; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence with at least about 95% homology to the amino acid sequence of SEQ ID NO:36; (iii) HVR-H3 comprising 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
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab22. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially 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 of the heavy chain variable domains of monoclonal antibody Ab22. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab22.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab22.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:11, or an amino acid sequence with 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 comprising 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 comprising the amino acid sequence of SEQ ID NO:141 or an amino acid
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab45. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially 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 of the heavy chain variable domains of monoclonal antibody Ab45. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab45.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab45.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:7, or an amino acid sequence with at least about 95% homology to 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 comprising 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
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab65. In some embodiments, the anti-TREM2 antibody is an isolated antibody which binds essentially the same TREM2 epitope as Ab65. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains of monoclonal antibody Ab65. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab65.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab65.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • anti-TREM2 antibodies of the present disclosure 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:9, or an amino acid sequence with 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 comprising the amino acid sequence of SEQ ID NO:101, or an amino acid sequence with at least about 95% homology to 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
  • anti-TREM2 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 43-50 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 43-50 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 49-57 of human TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 protein corresponding to amino acid residues 49-57 of SEQ ID NO: 1.
  • anti-TREM2 antibodies of the present disclosure bind to an epitope that includes one or more amino acid residues within amino acid residues 43-50 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies of the present disclosure bind to an epitope that includes one or more amino acid residues within amino acid residues 49-57 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodies of the present disclosure competitively inhibit binding of at least one of the following anti-TREM2 antibodies: Ab21 and Ab52.
  • anti-TREM2 antibodies of the present disclosure bind to an epitope of human TREM2 that is the same as or overlaps with the TREM2 epitope bound by at least one of the following anti-TREM2 antibodies: Ab21 and Ab52.
  • 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 one of antibodies Ab21 and Ab52; and/or (b) a light chain variable region comprising at least one, two, or three HVRs selected from HVR-L1, HVR-L2, and HVR-L3 of any one of antibodies Ab21 and Ab52.
  • the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 comprise Kabat CDR, Chothia CDR, or Contact CDR sequences as shown in Table 1 and/or Table 8.
  • anti-TREM2 antibodies of the present disclosure comprise a heavy chain variable region of any one of antibodies Ab21 and Ab52; and/or a light chain variable region of any one of antibodies Ab21 and Ab52.
  • the anti-TREM2 antibody is anti-TREM2 monoclonal antibody Ab52 or Ab21.
  • the anti-TREM2 antibody is an isolated antibody which binds essentially the same TREM2 epitope as Ab52 or Ab21.
  • the anti-TREM2 antibody is an agonist antibody.
  • the anti-TREM2 antibody is an antagonist antibody.
  • the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains of monoclonal antibody Ab52 or Ab21. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab52 or Ab21. In some embodiments, the anti-TREM2 antibody is an isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variable domains of monoclonal antibody Ab52 or Ab21.
  • anti-TREM2 antibodies of the present disclosure 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:398, or an amino acid sequence with 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 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; (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 comprising the amino acid sequence of SEQ ID NO:402
  • anti-TREM2 antibodies of the present disclosure 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 comprising 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; (iii) 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; (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 comprising the amino acid sequence of SEQ ID NO:
  • anti-TREM2 antibodies of the present disclosure comprise a heavy chain variable region, and the heavy chain variable region comprises at least one, at least two, or three HVR sequences selected from those listed in Table 1 and/or Table 8; and/or comprise a light chain variable region, and the light chain variable region comprises at least one, at least two, or three HVR sequences selected from those listed in Table 1 and/or Table 8.
  • anti-TREM2 antibodies of the present disclosure compete for binding of TREM2 with one or more TREM2 ligands.
  • suitable TREM2 ligands include, without limitation, TREM2 ligands expressed by E. coli cells, apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids, negatively charged phospholipids, phosphatidylserine, sulfatides, phosphatidylcholin, sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids, lipidated peptides, and lipidated amyloid beta peptide.
  • the one or more TREM2 ligands comprise E.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • the dissociation constants (K D ) of anti-TREM2 antibodies for human TREM2 (e.g., human TREM2-Fc fusion proteins and human monomeric TREM2 proteins) and mouse TREM2 (e.g., mouse TREM2-Fc fusion proteins) may 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 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 n
  • dissociation constants range from less than about 5.75 nM to less than about 0.09 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for human TREM2-Fc fusion proteins range from less than about 1.51 nM to less than about 0.35 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for human monomeric TREM2 proteins range from less than about 5.75 nM to less than about 1.15 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for mouse TREM2-Fc fusion proteins range from less than about 0.23 nM to less than about 0.09 nM.
  • dissociation constants range from less than about 6.70 nM to less than about 0.23 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for human TREM2-Fc fusion proteins range from less than about 0.71 nM to less than about 0.23 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for human monomeric TREM2 proteins range from less than about 6.70 nM to less than about 0.66 nM. In some embodiments, dissociation constants of anti-TREM2 antibodies for mouse TREM2-Fc fusion proteins range from less than about 4.90 nM to less than about 0.35 nM.
  • Dissociation constants may be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry (see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses.
  • the anti-TREM2 antibody is an agonist antibody. In other embodiments, the anti-TREM2 antibody is an antagonist antibody.
  • Additional anti-TREM2 antibodies e.g., antibodies that specifically bind to a TREM2 protein of the present disclosure, may be identified, screened, and/or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
  • Anti-DAP12 antibodies of the present disclosure generally bind to one or more DAP12 proteins expressed in a cell.
  • the DAP12 protein is expressed on a cell surface.
  • anti-DAP12 antibodies of the present disclosure are agonist antibodies or antagonist antibodies that bind to a DAP12 protein of the present disclosure expressed on the surface of a cell and modulate (e.g., induce or inhibit) at least one DAP12 activity of the present disclosure after binding to the surface-expressed DAP12 protein.
  • anti-DAP12 antibodies of the present disclosure are inert antibodies.
  • the DAP12 protein is expressed on a cell surface.
  • anti-DAP12 antibodies of the present disclosure modulate (e.g., induce or inhibit) one or more DAP12 activities.
  • the DAP12 activities modulated (e.g., induced or inhibited) by the anti-DAP12 antibodies may include, without limitation, binding to TREM2; DAP12 phosphorylation; TREM2 phosphorylation; recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex; PI3K activation; increased expression of anti-inflammatory mediators (e.g.
  • cytokines reduced expression of pro-inflammatory mediators; ERK phosphorylation; increased expression of CCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells; enhancement, normalization, or both of the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation; induction of osteoclast production, increased rate of osteoclastogenesis, or both; increased survival and function of microglial cells and/or macrophages (such as M1 macrophages and/or microglial cells, activated M1 macrophages and/or microglial cells, and/or M2 macrophages and/or microglial cells), dendritic cells, monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells; induction of apoptotic neuron clearance; reduced expression of TNF- ⁇ ; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells, monocytes, macrophage
  • the anti-TREM2 antibodies of the present disclosure can be used to prevent, reduce risk of, or treat dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis.
  • the anti-TREM2 antibodies of the present disclosure are monoclonal antibodies.
  • Anti-TREM2 antibodies of the present disclosure may be tested for inducing one or more TREM2 activities (e.g., DAP12 phosphorylation; recruitment of Syk, ZAP70, or both to DAP12; PI3K activation; increased expression of anti-inflammatory mediators; reduced expression of pro-inflammatory mediators; ERK phosphorylation; increased expression of CCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21 expressing cells; enhancement, normalization, or both of the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation; induction of osteoclast production, increased rate of osteoclastogenesis, or both; induction of apoptotic neuron clearance; reduced expression of TNF- ⁇ ; SYK phosphorylation; increased expression of CD83 and/or CD86 on dendritic cells; reduced secretion of one or more inflammatory cytokines; reduced expression of one or more inflammatory receptors; increased survival and/or function of dendritic cells
  • the anti-TREM2 antibodies can be assayed in vitro for tyrosine phosphorylation of TREM2, DAP12 and/or ERK, by assaying for recruitment of Syk and/or ZAP70 to a Dap12/TREM2 complex, by assaying for PI3K activation, by assaying for induction of expression of anti-inflammatory mediators (e.g., IL-12p70, IL-6, and IL-10) or CCR7, or by assaying for reduced expression of pro-inflammatory mediators (e.g., IL1- ⁇ and TNF) with TLR stimulation (e.g., LPS, CpG DNA, or Zymosan).
  • anti-inflammatory mediators e.g., IL-12p70, IL-6, and IL-10
  • CCR7 e.g., CCR7
  • pro-inflammatory mediators e.g., IL1- ⁇ and TNF
  • TLR stimulation e.g., LPS, CpG DNA,
  • Useful assays may include western blots (e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PI3K-kinase substrates), ELISA (e.g., for secreted interleukin or cytokine secretion), FACS (e.g., for anti-TREM2 binding to TREM2), immunocytochemistry (e.g., for e.g., for tyrosine-phosphorylated DAP12 or threonine/serine-phosphorylated PI3K-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
  • anti-DAP12 antibodies that bind to a human DAP12, or a homolog thereof, including without limitation a mammalian DAP12 protein, mouse DAP12 protein (Uniprot Accession No. Q99NH8), rat DAP12 protein (Uniprot Accession No. D3ZZ89), Rhesus monkey DAP12 protein (Uniprot Accession No. F6QVF2), bovine DAP12 protein (Uniprot Accession No. Q05B59), equine DAP12 protein (Uniprot Accession No. F7D6L0), pig DAP12 protein (Uniprot Accession No. H2EZZ3), and dog DAP12 protein (Uniprot Accession No.
  • the at least one DAP12 activity is DAP12 phosphorylation, TREM2 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, and/or reduced expression of one or more pro-inflammatory mediators.
  • anti-DAP12 antibodies of the present disclosure bind to a DAP12 protein of the present disclosure and/or naturally occurring variants. In certain preferred embodiments, the anti-DAP12 antibodies bind to human DAP12.
  • anti-DAP12 antibodies of the present disclosure bind to a DAP12 protein of the present disclosure expressed on the surface of a cell and induce at least one DAP12 activity of the present disclosure after binding to the surface expressed DAP12 protein.
  • anti-DAP12 antibodies of the present disclosure bind to one or more amino acids within amino acid residues 22-40 of human DAP12 (SEQ ID NO: 2), or within amino acid residues on a 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 may 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 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
  • Dissociation constants may be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry (see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses.
  • biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry (see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses.
  • Additional anti-DAP12 antibodies e.g., antibodies that specifically bind to a DAP12 protein of the present disclosure, may be identified, screened, and/or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
  • bispecific antibodies that bind to both TREM2 and DAP12 proteins of the present disclosure.
  • Methods of generating bispecific antibodies are well known in the art and described herein.
  • bispecific antibodies of the present disclosure bind to one or more amino acid residues of human TREM2 (SEQ ID NO: 1), or amino acid residues on a TREM2 protein corresponding to amino acid residues of SEQ ID NO: 1.
  • bispecific antibodies of the present disclosure also bind to one or more amino acid residues of human DAP12 (SEQ ID NO: 2), or amino acid residues on a DAP12 protein corresponding to amino acid residues of SEQ ID NO: 2.
  • bispecific antibodies of the present disclosure recognize a first antigen and a second antigen.
  • the first antigen is human TREM2 or a naturally occurring variant thereof, or human DAP12 or a naturally occurring variant thereof.
  • the second antigen is a disease-causing protein selected from amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products,
  • the second antigen is a blood brain barrier targeting protein selected from trasnferin receptor, insulin receptor, insulin like growth factor receptor, LRP-1, and LRP1; or ligands and/or proteins expressed on immune cells, wherein the ligands and/or proteins 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.
  • the second antigen may be, without limitation, a protein expressed on one or more tumor cells.
  • Certain aspects of the present disclosure relate to antibody fragments that bind to one or more human proteins selected from human TREM2, a naturally occurring variant of human TREM2, a disease variant of human TREM2, human DAP12, and naturally occurring variant of human DAP12.
  • the antibody fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.
  • the antibody fragment is used in combination with one or more antibodies that specifically bind a disease-causing protein selected from: amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides,
  • any of the antibodies described herein further include a framework.
  • the framework is a human immunoglobulin framework.
  • an antibody e.g., an anti-TREM2 antibody
  • Human immunoglobulin frameworks may be part of the human antibody, or a non-human antibody may be humanized by replacing one or more endogenous frameworks with human framework region(s).
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol.
  • 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)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).
  • an antibody comprises a heavy chain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3 of the present disclosure and one, two, three or four of the heavy chain framework regions as shown in FIG. 2B and/or FIG. 20A .
  • an antibody comprises a light chain variable region comprising an HVR-L1, an HVR-L2, and an HVR-L3 of the present disclosure and one, two, three or four of the light chain framework regions as shown in FIG. 2C and/or FIG. 20B .
  • an antibody comprises a heavy chain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3 of the present disclosure and one, two, three or four of the 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, an HVR-L2, and an HVR-L3 of the present disclosure and one, two, three or four of the light chain framework regions as shown in FIG. 2C and/or FIG. 20B .
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce binding of TREM2 to DAP12 and/or DAP12 binding to TREM2.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce TREM2 phosphorylation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce DAP12 phosphorylation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • TREM2-mediated TREM2 and/or DAP12 phosphorylation is induced by one or more SRC family tyrosine kinases.
  • Src family tyrosine kinases include, without limitation, Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn, and Frk.
  • DAP12 is variously referred to as TYRO protein tyrosine kinase-binding protein, TYROBP, KARAP, and PLOSL.
  • DAP12 is a transmembrane signaling protein that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the anti-TREM2 and/or anti-DAP12 antibody may induce DAP12 phosphorylation in its ITAM motif. Any method known in the art for determining protein phosphorylation, such as DAP12 phosphorylation, may be used.
  • DAP12 is phosphorylated by SRC family kinases, resulting in the recruitment and activation of the Syk kinase, ZAP70 kinase, or both, to DAP12.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may recruit Syk, ZAP70, or both to a DAP12/TREM2 complex.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are useful for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of DAP12 activity, DAP12 phosphorylation, or recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce PI3K activation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • PI3Ks are a family of related intracellular signal transducer kinases capable of phosphorylating the 3-position hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns).
  • the PI3K family is divided into three different classes (Class I, Class II, and Class III) based on primary structure, regulation, and in vitro lipid substrate specificity.
  • Activated PI3K produces various 3-phosphorylated phosphoinositides, including without limitation, PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3.
  • These 3-phosphorylated phosphoinositides function in a mechanism by which signaling proteins are recruited to various cellular membranes.
  • These signaling proteins contain phosphoinositide-binding domains, including without limitation, PX domains, pleckstrin homology domains (PH domains), and FYVE domains. Any method known in the art for determining PI3K activation may be used.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of PI3K activity, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure have an anti-inflammatory activity in the brain after binding to a TREM2 and/or DAP12 protein expressed on a cell surface. It has recently been reported that TREM2 has an anti-inflammatory role in the brain.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure increase the expression of anti-inflammatory mediators (e.g., cytokines) and/or reduce the expression of pro-inflammatory mediators after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • anti-inflammatory mediators e.g., cytokines
  • Inflammation is part of a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, and irritants.
  • harmful stimuli such as pathogens, damaged cells, and irritants.
  • the classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function.
  • Inflammation is a protective attempt by an organism to remove the injurious stimuli and to initiate the healing process.
  • Inflammation can be classified as either acute inflammation or chronic inflammation.
  • Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues.
  • a cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue.
  • Chronic inflammation is prolonged inflammation that leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
  • anti-inflammatory mediators are proteins involved either directly or indirectly (e.g., by way of an anti-inflammatory signaling pathway) in a mechanism that reduces, inhibits, or inactivates an inflammatory response. Any method known in the art for identifying and characterizing anti-inflammatory mediators may be used. Examples of anti-inflammatory mediators include, without limitation, cytokines, such as IL-12p70, IL-6, and IL-10.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase expression of anti-inflammatory mediators, such as IL-12p70, IL-6, and IL-10.
  • increased expression of the anti-inflammatory mediators occurs in macrophages, dendritic cells, and/or microglial cells.
  • Increased expression may include, without limitation, in increase in gene expression, an increase in transcriptional expression, or an increase in protein expression. Any method known in the art for determining gene, transcript (e.g., mRNA), and/or protein expression may be used. For example, Northern blot analysis may be used to determine anti-inflammatory mediator gene expression levels, RT-PCR may be used to determine the level of anti-inflammatory mediator transcription, and Western blot analysis may be used to determine anti-inflammatory mediator protein levels.
  • an anti-inflammatory mediator may have increased expression if its expression in one or more cells of a subject treated with an anti-TREM2 and/or 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 a corresponding subject that is not treated with the anti-TREM2 and/or anti-DAP12 antibody.
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may increase anti-inflammatory mediator expression 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, as compared to anti-inflammatory mediator expression in one or more cells of a corresponding subject that is not treated with the anti-TREM2 and/or anti-DAP12 antibody.
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure increases anti-inflammatory mediator expression in one or more cells of a subject by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example, as compared to anti-inflammatory mediator expression in one or more cells of a corresponding subject that is not treated with the anti-TRE
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are useful for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of one or more anti-inflammatory mediators, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may decrease the expression of pro-inflammatory mediators after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • pro-inflammatory mediators are proteins involved either directly or indirectly (e.g., by way of pro-inflammatory signaling pathways) in a mechanism that induces, activates, promotes, or otherwise increases an inflammatory response. Any method known in the art for identifying and characterizing pro-inflammatory mediators may be used.
  • pro-inflammatory mediators include, without limitation, cytokines, such as IIFN-a4, IFN-b, IL-1 ⁇ , TNF- ⁇ , IL-10, IL-6, IL-12 p70, IL-8, CRP, TNF, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.
  • cytokines such as IIFN-a4, IFN-b, IL-1 ⁇ , TNF- ⁇ , IL-10, IL-6, IL-12 p70, IL-8, CRP, TNF, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11,
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may decrease functional expression and/or secretion of pro-inflammatory mediators, such as IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1 ⁇ and TNF, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.
  • pro-inflammatory mediators such as IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1 ⁇ and TNF, TNF- ⁇ , IL-10, IL-8, CRP, TGF-beta members of the chemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM,
  • decreased expression of the pro-inflammatory mediators occurs in macrophages, dendritic cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells, and/or microglial cells.
  • Decreased expression may include, without limitation, a decrease in gene expression, a decrease in transcriptional expression, or a decrease in protein expression. Any method known in the art for determining gene, transcript (e.g., mRNA), and/or protein expression may be used. For example, Northern blot analysis may be used to determine pro-inflammatory mediator gene expression levels, RT-PCR may be used to determine the level of pro-inflammatory mediator transcription, and Western blot analysis may be used to determine pro-inflammatory mediator protein levels.
  • pro-inflammatory mediators include inflammatory cytokines.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may reduce secretion of one or more inflammatory cytokines.
  • inflammatory cytokines whose secretion may be reduced by the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure include, without limitation, TNF- ⁇ , IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1 ⁇ .
  • IL-8 CRP
  • TGF-beta members of the chemokine protein families IL-20 family members
  • IL-33 LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18.
  • pro-inflammatory mediators include inflammatory receptors. Accordingly, in certain embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may reduce expression of one or more inflammatory receptors. Examples of inflammatory receptors whose expression may be reduced by the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure include, without limitation, CD86.
  • a pro-inflammatory mediator may have decreased expression if its expression in one or more cells of a subject treated with an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure is less than the expression of the same pro-inflammatory mediator expressed in one or more cells of a corresponding subject that is not treated with the agonist anti-TREM2 and/or anti-DAP12 antibody.
  • the agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may decrease pro-inflammatory mediator expression 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, as compared to pro-inflammatory mediator expression in one or more cells of a corresponding subject that is not treated with the agonist anti-TREM2 and/or anti-DAP12 antibody.
  • the agonist anti-TREM2 and/or anti-DAP12 antibody may decrease pro-inflammatory mediator expression in one or more cells of a subject by at least at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example, as compared to pro-inflammatory mediator expression in one or more cells of a corresponding subject that is not treated with the anti-
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be useful for preventing, lowering the risk of, or treating conditions and/or diseases associated with increased levels of one or more pro-inflammatory mediators, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • pro-inflammatory mediators including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce extracellular signal-regulated kinase (ERK) phosphorylation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • ERK extracellular signal-regulated kinase
  • Extracellular-signal-regulated kinases are widely expressed protein kinase intracellular signaling kinases that are involved in, for example, the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells.
  • Various stimuli such as growth factors, cytokines, virus infection, ligands for heterotrimeric G protein-coupled receptors, transforming agents, and carcinogens, activate ERK pathways. Phosphorylation of ERKs leads to the activation of their kinase activity.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of ERK phosphorylation, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase expression of C-C chemokine receptor 7 (CCR7) after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • Increased expression may include, without limitation, in increase in gene expression, an increase in transcriptional expression, or an increase in protein expression. Any method known in the art for determining gene, transcript (e.g., mRNA), and/or protein expression may be used. For example, Northern blot analysis may be used to determine anti-inflammatory mediator gene expression levels, RT-PCR may be used to determine the level of anti-inflammatory mediator transcription, and Western blot analysis may be used to determine anti-inflammatory mediator protein levels.
  • C-C chemokine receptor 7 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 may modulate the migration of memory T-cells to secondary lymphoid organs, such as lymph nodes. In other embodiments, CCR7 may stimulate dendritic cell maturation. CCR7 is a receptor protein that can bind the chemokine (C-C motif) ligands CCL19/ELC and CCL21.
  • CCR7 may have increased expression if its expression in one or more cells of a subject treated with an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure is greater than the expression of CCR7 expressed in one or more cells of a corresponding subject that is not treated with the anti-TREM2 and/or anti-DAP12 antibody.
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may increase CCR7 expression 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, as compared to CCR7 expression in one or more cells of a corresponding subject that is not treated with the anti-TREM2 and/or anti-DAP12 antibody.
  • an anti-TREM2 and/or 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 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example, as compared to CCR7 expression in one or more cells of a corresponding subject that is not treated with the anti-TREM2
  • increased expression of CCR7 occurs in macrophages, dendritic cells, and/or microglial cells. Increased expression of CCR7 may induce microglial cell chemotaxis toward cells expressing the chemokines CCL19 and CCL21. Accordingly, in certain embodiments, anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce microglial cell chemotaxis toward CCL19 and CCL21 expressing cells.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are useful for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of CCR7, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may enhance and/or normalize the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • agonist anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may enhance and/or normalize the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation in one or more bone marrow-derived dendritic 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, as compared to the ability of bone marrow-derived dendritic cells to induce antigen
  • the agonist anti-TREM2 and/or anti-DAP12 antibody may enhance and/or normalize the ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation in one or more bone marrow-derived dendritic cells of a subject by at least at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with an increased or disregulated ability of bone marrow-derived dendritic cells to induce antigen-specific T-cell proliferation, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce osteoclast production and/or increase the rate of osteoclastogenesis after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • an osteoclast is a type of bone cell that can remove bone tissue by removing its mineralized matrix and breaking up the organic bone (e.g., bone resorption). Osteoclasts can be formed by the fusion of cells 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.
  • TRIP tartrate resistant acid phosphatase
  • cathepsin K cathepsin K.
  • the rate of osteoclastogenesis may be increased if the rate of osteoclastogenesis in a subject treated with an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure is greater than the rate of osteoclastogenesis in a corresponding subject that is not treated with the agonist anti-TREM2 and/or anti-DAP12 antibody.
  • an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may 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 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, as compared to rate of osteoclastogenesis in a corresponding subject that is not treated with the agonist anti-TREM2 and/or anti-DAP12 antibody.
  • an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may increase the rate of osteoclastogenesis in a subject by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example, as compared to rate of osteoclastogenesis in a corresponding subject that is not treated with the agonist anti-TRE
  • the rate of osteoclastogenesis may be decreased if the rate of osteoclastogenesis in a subject treated with an antagonist anti-TREM2 antibody and/or anti-DAP12 antibody of the present disclosure is smaller than the rate of osteoclastogenesis in a corresponding subject that is not treated with the antagonist anti-TREM2 antibody and/or anti-DAP12 antibody.
  • an antagonist anti-TREM2 antibody and/or anti-DAP12 antibody of the present disclosure may decrease 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 200% for example, as compared to rate of osteoclastogenesis in a corresponding subject that is not treated with the antagonist anti-TREM2 antibody and/or anti-DAP12 antibody.
  • an antagonist anti-TREM2 antibody and/or anti-DAP12 antibody of the present disclosure may decrease the rate of osteoclastogenesis in a subject by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example, as compared to rate of osteoclastogenesis in a corresponding subject that is not treated with the antagonist anti-TREM
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with a reduction in osteoclast production and/or the rate of osteoclastogenesis, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis; or associated with abnormal bone formation and maintenance including osteoporosis, which is associated with pathological decrease in bone density and osteoporotic diseases which are associated with pathological increase in bone density.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase the proliferation, survival, and/or function of macrophages, microglial cells (microglia), dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and Kupffer cells after binding to TREM2 and/or DAP12 protein expressed in a cell.
  • macrophages microglial cells (microglia), dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and Kupffer cells after binding to TREM2 and/or DAP12 protein expressed in a cell.
  • Microglial cells are a type of glial cell that are the resident macrophages of 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 constitute 20% of the total glial cell population within the brain.
  • Microglial cells are constantly scavenging the CNS for plaques, damaged neurons and infectious agents.
  • the brain and spinal cord are considered “immune privileged” organs in that 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 the vulnerable nervous tissue.
  • microglial cells In the case where infectious agents are directly introduced to the brain or cross the blood-brain barrier, microglial cells must react quickly to decrease inflammation and destroy the infectious agents before they damage the sensitive neural tissue. Due to the unavailability of antibodies from the rest of the body (few antibodies are small enough to cross the blood brain barrier), microglia must be able to recognize foreign bodies, swallow them, and act as antigen-presenting cells activating T-cells. Since this process must be done quickly to prevent potentially fatal damage, microglial cells are extremely sensitive to even small pathological changes in the CNS. They achieve this sensitivity in part by having unique potassium channels that respond to even small changes in extracellular potassium.
  • macrophages of the present disclosure include, without limitation, M1 macrophages, activated M1 macrophages, and M2 macrophages.
  • microglial cells of the present disclosure include, without limitation, M1 microglial cells, activated M1 microglial cells, and M2 microglial 1 cells.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase the expression of CD83 and/or CD86 on dendritic cells monocytes, and/or macrophages.
  • the rate of proliferation, survival, and/or function of macrophages microglia, monocytes, and/or dendritic cells may include increased expression if the rate of proliferation, survival, and/or function of macrophages, microglia, dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells in a subject treated with an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure is greater than the rate of proliferation, survival, and/or function of macrophages, microglia, dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells in a corresponding subject that is not treated with the anti-TREM2 and/or anti-DAP12 antibody.
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may increase the rate of proliferation, survival, and/or function of macrophages, microglia, dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells 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 200% for example, as compared to the rate of proliferation, survival, and/or function of macrophages,
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may increase the rate of proliferation, survival, and/or function of macrophages, microglia, dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells in a subject by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 5.0 fold, at
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with a reduction in proliferation, survival, and/or function of macrophages, microglia, dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce clearance and/or phagocytosis after binding to a TREM2 and/or DAP12 protein expressed in a cell of one or more of apoptotic neurons, nerve tissue debris of the nervous system, non-nerve tissue debris of the nervous system, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, and/or disease-causing nucleic acids.
  • disease-causing protein include, without limitation, amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides,
  • DPR
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce of one or more types of clearance, including without limitation, apoptotic neuron clearance, nerve tissue debris clearance, non-nerve tissue debris clearance, bacteria or other foreign body clearance, disease-causing protein clearance, disease-causing peptide clearance, disease-causing nucleic acid clearance, and tumor cell clearance.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce phagocytosis of one or more of apoptotic neurons, nerve tissue debris, non-nerve tissue debris, bacteria, other foreign bodies, disease-causing proteins, disease-causing peptides, disease-causing nucleic acid, and/or tumor cells.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia under conditions of reduced levels of macrophage colony-stimulating factor (MCSF).
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may decrease phagocytosis by macrophages, dendritic cells, monocytes, and/or microglia in the presence of normal levels of macrophage colony-stimulating factor (MCSF).
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with apoptotic neurons, nerve tissue debris of the nervous system, non-nerve tissue debris of the nervous system, bacteria, other foreign bodies, or disease-causing proteins, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may induce spleen tyrosine kinase (Syk) phosphorylation after binding to a TREM2 and/or DAP12 protein expressed in a cell.
  • Syk spleen tyrosine kinase
  • Spleen tyrosine kinase is an intracellular signaling molecule that functions downstream of TREM2 by phosphorylating several substrates, thereby facilitating the formation of a signaling complex leading to cellular activation and inflammatory processes.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of Syk phosphorylation, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • agonist anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may increase the activity and/or expression of TREM2-dependent genes, such as one or more transcription factors of the nuclear factor of activated T-cells (NFAT) family of transcription factors.
  • antagonistic anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may inhibit the activity and/or expression of TREM2-dependent genes, such as one or more transcription factors of the NFAT family of transcription factors.
  • agonist anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are beneficial for preventing, lowering the risk of, or treating conditions and/or diseases associated with decreased levels of TREM2-dependent genes, including dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, and/or multiple sclerosis.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can encompass 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, library derived antibodies, antibodies having modified effector functions, fusion proteins containing an antibody portion, and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site, such as an epitope having amino acid residues of a TREM2 and/or DAP12 protein of the present disclosure, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • the anti-TREM2 and/or anti-DAP12 antibodies may be human, murine, rat, or of any other origin (including chimeric or humanized antibodies).
  • Polyclonal antibodies such as anti-TREM2 and/or anti-DAP12 polyclonal antibodies, are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (e.g., a purified or recombinant TREM2 and/or DAP12 protein of the present disclosure) to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N
  • adjuvants examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the animals are immunized against the desired antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g (for rabbits) or 5 ⁇ g (for mice) of the protein or conjugate with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1 ⁇ 5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • Conjugates also can be made in recombinant-cell culture as protein fusions. Also, aggregating agents such as alum are suitable to enhance the immune response.
  • Monoclonal antibodies such as anti-TREM2 and/or anti-DAP12 monoclonal antibodies, are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
  • the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the anti-TREM2 and/or anti-DAP12 monoclonal antibodies may be made using the hybridoma method first described by Köhler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization (e.g., a purified or recombinant TREM2 and/or DAP12 protein of the present disclosure).
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)).
  • the immunizing agent will typically include the antigenic protein (e.g., a purified or recombinant TREM2 and/or DAP12 protein of the present disclosure) or a fusion variant thereof.
  • the antigenic protein e.g., a purified or recombinant TREM2 and/or DAP12 protein of the present disclosure
  • a fusion variant thereof e.g., peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, while spleen or lymph node cells are used if non-human mammalian sources are desired.
  • PBLs peripheral blood lymphocytes
  • the lymphoctyes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice , Academic Press (1986), pp. 59-103.
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine or human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient-cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred are murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA), as well as SP-2 cells and derivatives thereof (e.g., X63-Ag8-653) (available from the American Type Culture Collection, Manassas, Va. 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)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen (e.g., a TREM2 and/or DAP12 protein of the present disclosure).
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen (e.g., a TREM2 and/or DAP12 protein of the present disclosure).
  • the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked assay
  • binding affinity may be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as tumors in a mammal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, and other methods as described above.
  • Anti-TREM2 and/or anti-DAP12 monoclonal antibodies may also be made by recombinant DNA methods, such as those disclosed in U.S. Pat. No. 4,816,567, and as described above.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host-cells such as E.
  • anti-TREM2 and/or anti-DAP12 antibodies can be isolated from antibody phage libraries generated using the techniques 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) described the isolation of murine and human antibodies, respectively, from phage libraries.
  • DNA encoding antibodies or fragments thereof may also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • the monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art.
  • one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues may be substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.
  • Chimeric or hybrid anti-TREM2 and/or anti-DAP12 antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure or antibody fragments thereof 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 Fab, Fab′-SH, Fv, scFv, F(ab′) 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementarity determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. 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).
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers, 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 through substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody.
  • FR human framework
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may 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).
  • humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen or antigens (e.g., TREM2 proteins of the present disclosure), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • the humanized anti-TREM2 and/or anti-DAP12 antibody may be an antibody fragment, such as an Fab, which is optionally conjugated with one or more TREM2 ligand, such as HSP60.
  • the humanized anti-TREM2 and/or anti-DAP12 antibody may be an intact antibody, such as an intact IgG1 antibody.
  • human anti-TREM2 and/or anti-DAP12 antibodies can be generated.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy-chain joining region (J H ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Nat'l Acad. Sci.
  • phage display technology can be used to produce human anti-TREM2 and/or anti-DAP12 antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. McCafferty et al., Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991).
  • antibody V domain genes are cloned in-frame into either a 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.
  • the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B-cell.
  • Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993).
  • V-gene segments can be used for phage display.
  • yeast display technology can be used to produce human anti-TREM2 and/or anti-DAP12 antibodies and antibody fragments in vitro (e.g., WO 2009/036379; WO 2010/105256; WO 2012/009568; US 2009/0181855; US 2010/0056386; and Feldhaus and Siegel (2004) J Immunological Methods 290:69-80).
  • ribosome display technology can be used to produce human anti-TREM2 and/or 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. Immunolical Methods 231:119-135; Lipovsek and Plückthun (2004) J Immunological Methods 290:51-67).
  • human anti-TREM2 and/or anti-DAP12 monoclonal antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • human anti-TREM2 and/or anti-DAP12 antibodies may also be generated in vitro by activated B-cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • anti-TREM2 and/or anti-DAP12 antibody fragments there are advantages to using anti-TREM2 and/or anti-DAP12 antibody fragments, rather than whole anti-TREM2 and/or anti-DAP12 antibodies.
  • smaller fragment sizes allow for rapid clearance and better brain penetration.
  • Anti-TREM2 and/or anti-DAP12 antibody fragments can also be isolated from the antibody phage libraries as discussed above.
  • Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
  • F(ab′) 2 fragments can be isolated directly from recombinant host-cell culture. Production of Fab and F(ab′) 2 antibody fragments with increased in vivo half-lives are described in U.S. Pat. No. 5,869,046.
  • the antibody of choice is a single chain Fv fragment (scFv).
  • the anti-TREM2 and/or anti-DAP12 antibody fragment may also be a “linear antibody,” e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes, including those on the same or another protein (e.g., one or more TREM2 proteins of the present disclosure). Alternatively, one part of a BsAb can be armed to bind to the target TREM2 and/or DAP12 antigen, and another can be combined with an arm that binds to a second protein. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab′) 2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-chain/light chain pairs, where the two chains have different specificities. Millstein et al., Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, C H 2, and C H 3 regions. It is preferred to have the first heavy-chain constant region (C H 1) containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only half of the bispecific molecules provides for an easy way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121: 210 (1986); and Garber, Nature Reviews Drug Discovery 13, 799-801 (2014).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant-cell culture.
  • the preferred interface comprises at least a part of the C H 3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chains(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytic ally cleaved to generate F(ab′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab′-TNB derivatives is then reconverted to the Fab′-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab′ fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F(ab′) 2 molecules.
  • Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T-cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bivalent heterodimers have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • the “diabody” technology described by Hollinger et al., Proc. Nat'l Acad. Sci.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • bispecific antibodies Another method to generate bispecific antibodies is controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1).
  • the protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods.
  • MS mass spectrometry
  • the protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ⁇ 95% can be obtained 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 with more than two valencies are also contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
  • Exemplary bispecific antibodies may bind to two different epitopes on a given molecule (e.g., a TREM2 and/or DAP12 protein of the present disclosure).
  • a bispecific antibody binds to a first antigen, such as a TREM2 or DAP12 protein of the present disclosure, and a second antigen facilitating transport across the blood-brain barrier.
  • a first antigen such as a TREM2 or DAP12 protein of the present disclosure
  • a second antigen facilitating transport across the blood-brain barrier.
  • Numerous antigens are known in the art that facilitate transport across the blood-brain barrier (see, e.g., 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, without limitation, transferrin receptor (TR), insulin receptor (HIR), Insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, including CRM197 (a non-toxic mutant of diphtheria toxin), llama single domain antibodies such as TMEM 30(A) (Flippase), protein transduction domains such as TAT, Syn-B, or penetratin, poly-arginine or generally positively charged peptides, Angiopep peptides such as ANG1005 (see, e.g., Gabathuler, 2010), and other cell surface proteins that are enriched on blood-brain barrier endothelial cells (see, e.g., Daneman et al., PLoS One.
  • TR transferrin receptor
  • HIR insulin receptor
  • IGFR Insulin-like growth factor receptor
  • LPR-1 and 2 low-density lipoprotein
  • second antigens for an anti-TREM2 antibody may include, without limitation, a DAP12 antigen of the present disclosure.
  • second antigens for an anti-DAP12 antibody may include, without limitation, a TREM2 antigen of the present disclosure.
  • bispecific antibodies that bind to both TREM2 and DAP12 may facilitate and enhance one or more TREM2 and/or DAP12 activities.
  • second antigens for an TREM2 and/or DAP12 antibody may include, without limitation, A beta peptide, antigen or an alpha synuclain protein antigene or, Tau protein antigene or, TDP-43 protein antigene or, prion protein antigene or, huntingtin protein antigene, or RAN, translation Products antigene, including the DiPeptide Repeats, (DPRs peptides) composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR).
  • DPRs peptides composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR).
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure or antibody fragments thereof can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • the preferred dimerization domain comprises an Fc region or a hinge region.
  • the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • the preferred multivalent antibody herein contains three to about eight, but preferably four, antigen binding sites.
  • the multivalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain or chains comprise two or more variable domains.
  • the polypeptide chain or chains may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain or chains may comprise V H -C H 1-flexible linker-V H -C H 1-Fc region chain; or V H -C H 1-V H -C H 1-Fc region chain.
  • the multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides.
  • the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • the multiivalent antibodies may recognize a TREM2 angigen as well as without limitation additional antigens
  • DPRs peptides composed of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (
  • an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may also be desirable to modify effector function and/or to increase serum half-life of the antibody.
  • the Fc receptor binding site on the constant region may be modified or mutated to remove or reduce binding affinity to certain Fc receptors, such as Fc ⁇ RI, Fc ⁇ RII, and/or Fc ⁇ RIII (e.g., to reduce antibody-dependent cell-mediated cytotoxicity.
  • the effector function is impaired by removing N-glycosylation of the Fc region (e.g., in the CH 2 domain of IgG) of the antibody.
  • the effector function is impaired by modifying regions such as 233-236, 297, and/or 327-331 of human IgG as described in PCT WO 99/58572 and Armour et al., Molecular Immunology 40: 585-593 (2003); Reddy et al., J. Immunology 164:1925-1933 (2000).
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • Amino acid sequence modifications of anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, or antibody fragments thereof, are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibodies or antibody fragments.
  • Amino acid sequence variants of the antibodies or antibody fragments are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibodies or antibody fragments, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody.
  • deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics (i.e., the ability to bind or physically interact with a TREM2 and/or DAP12 protein of the present disclosure).
  • the amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the anti-TREM2 and/or anti-DAP12 antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells in Science, 244:1081-1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the target antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.
  • Amino acid sequence insertions include amino-(“N”) and/or carboxy-(“C”) terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in the Table A below under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table A, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions entail exchanging a member of one of these classes for another class.
  • cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).
  • a particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human anti-TREM2 and/or anti-DAP12 antibody).
  • a parent antibody e.g. a humanized or human anti-TREM2 and/or anti-DAP12 antibody.
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle.
  • the phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • Nucleic acid molecules encoding amino acid sequence variants of the anti-IgE antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibodies (e.g., anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure) or antibody fragments.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, or antibody fragments thereof, can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available, or to contain different types of drug conjugates that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • Such techniques and other suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science (2000).
  • Drug conjugtation involves coupling of a biological active cytotoxic (anticancer) payload or drug to an antibody that specifically targets a certain tumor marker (e.g. a protein that, ideally, is only to be found in or on tumor cells).
  • a tumor marker e.g. a protein that, ideally, is only to be found in or on tumor cells.
  • Antibodies track these proteins down in the body and attach themselves 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 internalizes the antibody together with the cytotoxin.
  • the cytotoxic drug is released and kills the cancer. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be identified, screened for, and/or characterized for their physical/chemical properties and/or biological activities by various assays known in the art or assays described in the Examples herein, such as, for example, radiolabeled immunoassays, optical assays, protein binding assays, biochemical screening assays, immunoassays, fluorescence assays, and/or cell survival assays.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be tested for antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.
  • competition assays may be used to identify an antibody that competes with any of the antibodies listed in Table 1 and/or Table 8, selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78
  • such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by any of the antibodies listed in Table 1 and/or Table 8, selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab
  • immobilized TREM2 or cells expressing TREM2 on cell surface are incubated in a solution comprising a first labeled antibody that binds to TREM2 (e.g., human or non-human primate) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to TREM2.
  • the second antibody may be present in a hybridoma supernatant.
  • immobilized TREM2 or cells expressing TREM2 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody.
  • assays are provided for identifying agonist anti-TRME2 and/or anti-DAP12 antibodies of the present disclosure having biological activity.
  • Biological activity may include, e.g., inducing, promoting, stimulating, or otherwise increasing one more activities of TREM2 and/or DAP12.
  • Agonist anti-TRME2 and/or anti-DAP12 antibodies having such biological activity in vivo and/or in vitro are also provided.
  • Assays known in the art and described herein can be used for identifying and testing biological activities of anti-TRME2 and/or anti-DAP12 antibodies that induce, promote, stimulate, or otherwise increase one more activities of TREM2 and/or DAP12.
  • assays for testing one or more activities of an agonist anti-TRME2 and/or anti-DAP12 antibody are provided.
  • An exemplary test for biological activity may include, e.g., treating a population of immune cells, such as an innate immune cells expressing TREM2 and/or DAP12 with an agonist anti-TRME2 and/or anti-DAP12 antibody candidate for sufficient time to allow binding to, and activation of, the TREM2 and/or DAP12 protein, and measuring cell survival (or cell death) as compared to cell survival (or cell death) in a corresponding population in the absence of the antibody candidate. Methods for measuring cell survival or cell death are well known in the art and described herein.
  • Agonist anti-TRME2 and/or anti-DAP12 antibodies may 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 was not treated with the agonist antibody candidate. Certain aspects of the present disclosure provide methods for identifying a candidate agonist antibody that specifically binds to TREM2 and/or DAP12. In certain embodiments, the method for identifying a candidate agonist antibody that specifically binds to TREM2 and/or DAP12 comprises a.
  • Other aspects of the present disclosure provide 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.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
  • isolated nucleic acids having a nucleotide sequence encoding any of the anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure are provided.
  • Such nucleic acids may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of the anti-TREM2 and/or anti-DAP12 antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors containing such nucleic acids are provided.
  • a host cell containing such nucleic acid is also provided.
  • the host cell is an isolated host cell.
  • an “isolated cell” is a cell that is identified and separated from at least one contaminant cell with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated cell is free of association with all components associated with the production environment. The isolated cell is in a form other than in the form or setting in which it is found in nature. Isolated cells are distinguished from cells existing naturally in tissues, organs, or individuals. In some embodiments, the isolated cell is a host cell of the present disclosure.
  • the host cell contains (e.g., has been transduced with): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of the antibody.
  • the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • Host cells of the present disclosure also include, without limitation, isolated cells, in vitro cultured cells, and ex vivo cultured cells.
  • the method includes culturing a host cell of the present disclosure containing a nucleic acid encoding the anti-TREM2 and/or anti-DAP12 antibody, under conditions suitable for expression of the antibody.
  • the antibody is subsequently recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the anti-TREM2 and/or anti-DAP12 antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to 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 of the present disclosure, or fragments thereof polypeptides (including antibodies) described herein include, without limitation, cloning vectors and expression vectors.
  • Suitable cloning vectors can be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • Bluescript e.g., pBS SK+
  • mpl8 mpl9 mpl9
  • pBR322 pMB9
  • ColE1 pCR1
  • RP4 phage DNAs
  • shuttle vectors such as pSA3 and pAT28.
  • Expression vectors generally are replicable polynucleotide constructs that contain a nucleic acid of the present disclosure.
  • the expression vector may replicable in the host cells either as episomes or as an integral part of the 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 may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator).
  • suitable transcriptional controlling elements such as promoters, enhancers and terminator
  • the vectors containing the nucleic acids of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
  • electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
  • microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
  • infection e.g., where the vector is an infectious agent such as vaccinia virus.
  • the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli .).
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast
  • suitable cloning or expression hosts for antibody-encoding vectors including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006)).
  • Suitable host cells for the expression of glycosylated antibody can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, describing PLANTIBODIESTM technology for producing antibodies in transgenic plants.).
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can be incorporated into a variety of formulations for therapeutic administration by combining the antibodies with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms.
  • suitable pharmaceutically acceptable carriers or diluents include, without limitation, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Pharmaceutical compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination.
  • examples of such diluents include, without limitation, distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
  • a pharmaceutical composition or formulation of the present disclosure can further include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
  • a pharmaceutical composition of the present disclosure can also include any of a variety of stabilizing agents, such as an antioxidant for example.
  • the 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, reduce its toxicity, and enhance solubility or uptake).
  • modifications or complexing agents include, without limitation, sulfate, gluconate, citrate and phosphate.
  • the polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, without limitation, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
  • the active ingredient can 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 component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.
  • additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink.
  • Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process.
  • compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • Formulations may be optimized for retention and stabilization in the brain or central nervous system.
  • Stabilization techniques include cross-linking, multimerizing, or linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc. in order to achieve an increase in molecular weight.
  • Other strategies for increasing retention include the entrapment of the antibody, such as an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure, in a biodegradable or bioerodible implant.
  • the rate of release of the therapeutically active agent is controlled by the rate of transport through the polymeric matrix, and the biodegradation of the implant.
  • the transport of drug through the polymer barrier will also be affected by compound solubility, polymer hydrophilicity, extent of polymer cross-linking, expansion of the polymer upon water absorption so as to make the polymer barrier more permeable to the drug, geometry of the implant, and the like.
  • the implants are of dimensions commensurate with the size and shape of the region selected as the site of implantation. Implants may be particles, sheets, patches, plaques, fibers, microcapsules and the like and may be of any size or shape compatible with the selected site of insertion.
  • the implants may be monolithic, i.e. having the active agent homogenously distributed through the polymeric matrix, or encapsulated, where a reservoir of active agent is encapsulated by the polymeric matrix.
  • the selection of the polymeric composition to be employed will vary with the site of administration, the desired period of treatment, patient tolerance, the nature of the disease to be 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, a half-life in the physiological environment.
  • Biodegradable polymeric compositions that may be employed include, without limitation, organic esters or ethers, which when degraded result in physiologically acceptable degradation products, including the monomers. Anhydrides, amides, orthoesters or the like, by themselves or in combination with other monomers, may find use.
  • the polymers will be condensation polymers.
  • the polymers may be cross-linked or non-cross-linked.
  • polymers of hydroxyaliphatic carboxylic acids either homo- or copolymers, and polysaccharides. Included among the polyesters of interest are polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof.
  • a slowly biodegrading polymer is achieved, while degradation is substantially enhanced with the racemate.
  • Copolymers of glycolic and lactic acid are of particular interest, where the rate of biodegradation is controlled by the ratio of glycolic to lactic acid.
  • the most rapidly degraded copolymer has roughly equal amounts of glycolic and lactic acid, where either homopolymer is more resistant to degradation.
  • the ratio of glycolic acid to lactic acid will also affect the brittleness of in the implant, where a more flexible implant is desirable for larger geometries.
  • polysaccharides of interest are calcium alginate, and functionalized celluloses, particularly carboxymethylcellulose esters characterized by being water insoluble, a molecular weight of about 5 kD to 500 kD, etc.
  • Biodegradable hydrogels may also be employed in the implants of the subject invention. Hydrogels are typically a copolymer material, characterized by the ability to imbibe a liquid. Exemplary biodegradable hydrogels which may be employed are described in Heller in: Hydrogels in Medicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press, Boca Raton, Fla., 1987, pp 137-149.
  • compositions of the present disclosure containing an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure may be administered to an individual in need of treatment with the anti-TREM2 and/or anti-DAP12 antibody, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, intracranial, intraspinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following 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.
  • normal dosage amounts may vary from about 10 ng/kg up to about 100 mg/kg of an individual's body weight or more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, depending upon the route of administration.
  • the treatment is sustained until a desired suppression of symptoms is achieved.
  • An exemplary dosing regimen may include administering an initial dose of an anti-TREM2 and/or anti-DAP12 antibody, of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg every other week.
  • Other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is contemplated herein. In certain embodiments, dosing ranging from about 3 ⁇ g/kg to about 2 mg/kg (such as about 3 ⁇ g/kg, about 10 ⁇ g/kg, about 30 ⁇ g/kg, about 100 ⁇ g/kg, about 300 ⁇ g/kg, about 1 mg/kg, and about 2/mg/kg) may be used.
  • dosing frequency is three times per day, twice per day, once per day, once every other day, once weekly, 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 monthly, once every two months, once every three months, or longer. Progress of the therapy is easily monitored by conventional techniques and assays.
  • the dosing regimen including the anti-TREM2 and/or anti-DAP12 antibody administered, can vary over time independently of the dose used.
  • Dosages for a particular anti-TREM2 and/or anti-DAP12 antibody may be determined empirically in individuals who have been given one or more administrations of the anti-TREM2 and/or anti-DAP12 antibody. Individuals are given incremental doses of an anti-TREM2 and/or anti-DAP12 antibody.
  • a clinical symptom of any of the diseases, disorders, or conditions of the present disclosure e.g., dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis
  • dementia e.g., frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis
  • Administration of an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of an anti-TREM2 and/or anti-DAP12 antibody may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
  • dosages and methods of delivery are provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is within the scope of the invention that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • Further aspects of the present disclosure provide methods for modulating (e.g., activating or inhibiting) TREM2, modulating (e.g., activating or inhibiting) DAP12, modulating (e.g., activating or inhibiting) PI3K, modulating (e.g., increasing or reducing) expression of one or more anti-inflammatory mediators (e.g., IL-12p70, IL-6, and IL-10), or modulating (e.g., increasing or reducing) expression of one or more pro-inflammatory mediators (e.g., L-13 and TNF) in an individual in need thereof, by administering to the individual a therapeutically effective amount of an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure to modulate (e.g., induce or inhibit) one or more TREM2 and/or DAP12 activities in the individual.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be used for preventing, reducing risk, or treating dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, seizures, spinal cord injury, traumatic
  • the anti-TREM2 and/or anti-DAP12 antibodies are agonist antibodies. In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies are inert antibodies. In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies are antagonist antibodies.
  • the present disclosure provides methods of preventing, reducing risk, or treating an individual having dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus, amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, 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, dementia with Lewy bodies, multiple system atrophy, Shy-Drager syndrome, progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, granulomartous disorders, Sarcoidosis, diseases of aging, seizures, spinal cord injury, traumatic brain injury, age related macular degeneration, glau
  • the anti-TREM2 and/or anti-DAP12 antibody is an agonist antibody. In some embodiments, the anti-TREM2 and/or anti-DAP12 antibody is an inert antibody. In some embodiments, the anti-TREM2 and/or anti-DAP12 antibody is an antagonist antibody. In certain embodiments, the individual has a heterozygous TREM2 variant allele having an glutamic acid to stop codon substitution in the nucleic acid sequence encoding amino acid residue 14 of the human TREM2 protein (SEQ ID NO: 1).
  • the individual has a heterozygous TREM2 variant 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 certain embodiments, the individual has a heterozygous TREM2 variant allele having a tryptophan to stop codon substitution in the nucleic acid sequence encoding amino acid residue 44 of the human TREM2 protein (SEQ ID NO: 1). In certain embodiments, the individual has a heterozygous TREM2 variant allele having an arginine to histidine amino acid substitution at amino acid residue 47 of the human TREM2 protein (SEQ ID NO: 1).
  • the individual has a heterozygous TREM2 variant allele having a tryptophan to stop codon substitution in the nucleic acid sequence encoding amino acid residue 78 of the human TREM2 protein (SEQ ID NO: 1). In certain embodiments, the individual has a heterozygous TREM2 variant allele having a valine to glycine amino acid substitution at an amino acid corresponding to amino acid residue 126 of the human TREM2 protein (SEQ ID NO: 1). In certain embodiments, the individual has a heterozygous TREM2 variant allele having an aspartic acid to glycine amino acid substitution at an amino acid corresponding to amino acid residue 134 of the human TREM2 protein (SEQ ID NO: 1). In certain embodiments, the individual has a heterozygous TREM2 variant allele having a lysine to asparagine amino acid substitution at an amino acid corresponding to amino acid residue 186 of the human TREM2 protein (SEQ ID NO: 1).
  • the individual has a heterozygous TREM2 variant allele having a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1; a threonine to methionine amino acid substitution at an amino acid corresponding to amino acid residue Thr66 of SEQ ID NO: 1; and/or a serine to cysteine amino acid substitution at an amino acid corresponding to amino acid residue Ser116 of SEQ ID NO: 1.
  • the individual has a heterozygous DAP12 variant allele having a methionine to threonine substitution at an amino acid corresponding to amino acid residue Med of SEQ ID NO: 2, a glycine to arginine amino acid substitution at an amino acid corresponding to amino acid residue Gly49 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 at exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2, and/or a guanine nucleotide deletion at a nucleotide corresponding to nucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO: 2.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may also be used for inducing and/or promoting innate immune cell survival.
  • the present disclosure provides methods of inducing or promoting innate immune cell survival in an individual in need thereof, by administering to the individual a therapeutically effective amount of an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may also be used for inducing and/or promoting wound healing, such as after injury.
  • the wound healing may be colonic wound repair following injury.
  • the present disclosure provides methods of inducing or promoting wound healing in an individual in need thereof, by administering to the individual a therapeutically effective amount of an agonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may also be used for inhibiting innate immune cell survival.
  • the present disclosure provides methods of i inhibiting innate immune cell survival in an individual in need thereof, by administering to the individual a therapeutically effective amount of an antagonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • the methods of the present disclosure may involve the coadministration of anti-TREM2 and/or anti-DAP12 antibodies, or bispecific antibodies that bind to both TREM2 and DAP12, with TLR antagonists or with agents neutralizing TLR agonist (e.g., neutralizing cytokine or interleukin antibodies).
  • the methods of the present disclosure may involve the administration of chimeric constructs, including an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure in conjunction with a TREM2 ligand, such as HSP60.
  • Dementia is a non-specific syndrome (i.e., a set of signs and symptoms) that presents as a serious loss of global cognitive ability in a previously unimpaired person, beyond what might be expected from normal ageing.
  • Dementia may be static as the result of a unique global brain injury.
  • dementia may be progressive, resulting in long-term decline due to damage or disease in the body. While dementia is much more common in the geriatric population, it can also occur before the age of 65.
  • Cognitive areas affected by dementia include, without limitation, memory, attention span, language, and problem solving. Generally, symptoms must be present for at least six months to before an individual is diagnosed with dementia.
  • Exemplary forms of dementia include, without limitation, frontotemporal dementia, Alzheimer's disease, vascular dementia, semantic dementia, and dementia with Lewy bodies.
  • administering an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or treat dementia.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having dementia (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • Frontotemporal dementia is a condition resulting from the progressive deterioration of the frontal lobe of the brain. Over time, the degeneration may advance to the temporal lobe. Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for 20% of pre-senile dementia cases.
  • the clinical features of FTD include memory deficits, behavioral abnormalities, personality changes, and language impairments (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)).
  • FTD FTD
  • a causal role for the microtubule associated protein Tau was supported by the identification of mutations in the gene encoding the Tau protein in several families (Hutton, M., et al., Nature 393:702-705 (1998).
  • administering an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or treat FTD.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having FTD (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • AD Alzheimer's disease
  • Alzheimer's disease Common symptoms of Alzheimer's disease include, behavioral symptoms, such as difficulty in remembering recent events; cognitive symptoms, confusion, irritability and aggression, mood swings, trouble with language, and long-term memory loss. As the disease progresses bodily functions are lost, ultimately leading to death Alzheimer's disease develops for an unknown and variable amount of time before becoming fully apparent, and it can progress undiagnosed for years.
  • administering an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or treat Alzheimer's disease.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having Alzheimer's disease (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • Nasu-Hakola disease which may alternatively be referred to as polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), is a rare inherited leukodystrophy characterized by progressive presenile dementia associated with recurrent bone fractures due to polycystic osseous lesions of the lower and upper extremities.
  • NHD disease course is generally divided into four stages: latent, osseous, early neurologic, and late neurologic. After a normal development during childhood (latent stage), NHD starts manifesting during adolescence or young adulthood (typical age of onset 20-30 years) with pain in the hands, wrists, ankles, and feet.
  • administering an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or treat Nasu-Hakola disease (NHD).
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having NHD (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • Parkinson's disease which may be referred to as idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans, is a neurodegenerative brain disorder that affects motor system control.
  • HRS hypokinetic rigid syndrome
  • Parkinson's disease is diagnosed in people over 50 years of age. Parkinson's disease is idiopathic (having no known cause) in most people. However, genetic factors also play a role in the disease.
  • Symptoms of Parkinson's disease include, without limitation, tremors of 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.
  • administering an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or treat Parkinson's disease.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having Parkinson's disease (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • amyotrophic lateral sclerosis or, motor neuron disease or, Lou Gehrig's disease are used interchangeably and refer to a debilitating disease with varied etiology characterized by rapidly progressive weakness, muscle atrophy and fasciculations, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea).
  • progranulin play a role in ALS (Schymick, J C et al., (2007) J Neurol Neurosurg Psychiatry; 78:754-6) and protects again the damage caused by ALS causing proteins such as TDP-43 (Laird, A S et al., (2010). PLoS ONE 5: e13368). It was also demonstrated that pro-NGF induces p75 mediated death of oligodendrocytes and corticospinal neurons following 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).
  • administering an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or treat ALS.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having ALS (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • Huntington's disease is an inherited neurodegenerative disease caused by an autosomal dominant mutation in the Huntingtin gene (HTT). Expansion of a cytokine-adenine-guanine (CAG) triplet repeat within the Huntingtin gene results in production of a mutant form of the Huntingtin protein (Htt) encoded by the gene. This mutant Huntingtin protein (mHtt) is toxic and contributes to neuronal death. Symptoms of Huntington's disease most commonly appear between the ages of 35 and 44, although they can appear at any age.
  • Symptoms of Huntington's disease include, without limitation, motor control problems, jerky, random movements (chorea), abnormal eye movements, impaired balance, seizures, difficulty chewing, difficulty swallowing, cognitive problems, altered speech, memory deficits, thinking difficulties, insomnia, fatigue, dementia, changes in personality, depression, anxiety, and compulsive behavior.
  • administering an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or treat Huntington's disease (HD).
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having HD (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • Taupathy diseases are a class of neurodegenerative disease caused by aggregation of the microtubule-associated protein tau within the brain Alzheimer's disease (AD) is the most well-known Taupathy disease, and involves an accumulation of tau protein within neurons in the form of insoluble neurofibrillary tangles (NFTs).
  • AD Alzheimer's disease
  • NFTs insoluble neurofibrillary tangles
  • Taupathy diseases and disorders include progressive supranuclear palsy, dementia pugilistica (chromic traumatic encephalopathy), Frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease (Parkinson-dementia complex of Guam), Tangle-predominant dementia, Ganglioglioma and gangliocytoma, Meningioangiomatosis, Subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, Argyrophilic grain disease (AGD), Huntington's disease, frontotemporal dementia, and frontotemporal lobar degeneration.
  • ATD Argyrophilic grain disease
  • administering an anti-TREM2 antibody of the present disclosure can prevent, reduce the risk, and/or treat Taupathy disease.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having Taupathy disease (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, or reduced expression of one or more pro-inflammatory mediators).
  • MS Multiple sclerosis
  • MS can also be referred to as disseminated sclerosis or encephalomyelitis disseminata.
  • MS is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms.
  • MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other effectively. Nerve cells communicate by sending electrical signals called action potentials down long fibers called axons, which are contained within an insulating substance called myelin.
  • myelin an insulating substance
  • Symptoms of MS include, without limitation, changes in sensation, such as loss of sensitivity or tingling; pricking or numbness, such as hypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms; difficulty in moving; difficulties with coordination and balance, such as ataxia; problems in speech, such as dysarthria, or in swallowing, such as dysphagia; visual problems, such as nystagmus, optic neuritis including phosphenes, and diplopia; fatigue; acute or chronic pain; and bladder and bowel difficulties; cognitive impairment of varying degrees; emotional symptoms of depression or unstable mood; Uhthoffs phenomenon, which is an exacerbation of extant symptoms due to an exposure to higher than usual ambient temperatures; and Lhermitte's sign, which is an electrical sensation that runs down the back when bending the neck.
  • administering an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure can prevent, reduce the risk, and/or treat multiple sclerosis.
  • administering an anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12 activities in an individual having multiple sclerosis (e.g., DAP12 phosphorylation, PI3K activation, increased expression of one or more anti-inflammatory mediators, and reduced expression of one or more pro-inflammatory mediators).
  • Yet further aspects of the present disclosure provide methods for preventing, reducing risk, or treating an individual having cancer, comprising administering to the individual a therapeutically effective amount of an isolated anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • Any of the isolated antibodies of the present disclosure may be used in these methods.
  • the isolated antibody is an agonist antibody of the present disclosure.
  • the isolated antibody is an antagonist antibody of the present disclosure.
  • the tumor microenvironment is known to contain a heterogeneous immune infiltrate, which includes T lymphocytes, macrophages and cells of myeloid/granulocytic lineage.
  • a heterogeneous immune infiltrate which includes T lymphocytes, macrophages and cells of myeloid/granulocytic lineage.
  • M2-macrophages in tumors is associated with poor prognosis.
  • therapies that reduce the number of these cells in the tumor such as CSF1R blocking agents, are showing beneficial effects in preclinical models and early stage clinical studies. It has been shown that TREM2 synergizes with CSF1 to promote survival of macrophages in vitro, and that this effect is particularly prominent in M2-type macrophages, compared to other types of phagocytic cells.
  • the methods for preventing, reducing risk, or treating an individual having cancer further include administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule.
  • antibodies that specifically bind to an inhibitory checkpoint molecule include, without limitation, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEM antibody, an anti-BTLA antibody, an anti-GALS antibody, an anti-TIM3 antibody, an anti-A2AR antibody, an anti-LAG-3 antibody, an anti-phosphatidylserine antibody, and any combination thereof.
  • the at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with an antagonist anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • a cancer to be prevented or treated by the methods of the present disclosure includes, but is not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral
  • lung cancer
  • the cancer is colorectal cancer.
  • the cancer is selected from non-small cell lung cancer, glioblastoma, neuroblastoma, renal cell carcinoma, bladder cancer, ovarian cancer, melanoma, breast carcinoma, gastric cancer, and hepatocellular carcinoma.
  • the cancer is triple-negative breast carcinoma.
  • the cancer may be an early stage cancer or a late stage cancer.
  • the cancer may be a primary tumor.
  • the cancer may be a metastatic tumor at a second site derived from any of the above types of cancer.
  • anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may be used for preventing, reducing risk, or treating cancer, including, without limitation, bladder cancer breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer.
  • cancer including, without limitation, bladder cancer breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, and thyroid cancer.
  • the present disclosure provides methods of preventing, reducing risk, or treating an individual having cancer, by administering to the individual a therapeutically effective amount of an anti-TREM2 and/or anti-DAP12 antibody of the present disclosure.
  • the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory checkpoint molecule, and/or another standard or investigational anti-cancer therapy.
  • the at least one antibody that specifically binds to an inhibitory checkpoint molecule is administered in combination with the isolated antibody.
  • the at least one antibody that specifically binds to an inhibitory checkpoint molecule is selected from an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEM antibody, an anti-B- and T-lymphocyte attenuator (BTLA) antibody, an anti-Killer inhibitory receptor (KIR) antibody, an anti-GALS antibody, an anti-TIM3 antibody, an anti-A2AR antibody, an anti-LAG-3 antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, and any combination thereof.
  • BTLA T-lymphocyte attenuator
  • KIR anti-Killer inhibitory receptor
  • the standard or investigational anti-cancer therapy is one or more therapies selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy, and cytokine therapy.
  • therapies selected from radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®), adoptive cell transfer (ACT), chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy, and cytokine therapy.
  • the method further includes administering to the individual at least one antibody that specifically binds to an inhibitory cytokine.
  • the at least one antibody that specifically binds to an inhibitory cytokine is administered in combination with the isolated antibody.
  • the 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.
  • the method further includes administering to the individual at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein.
  • the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is administered in combination with the isolated antibody.
  • the at least one agonistic antibody that specifically binds to a stimulatory checkpoint protein is selected from an agonist anti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28 antibody, an agonist anti-CD137/4-1BB antibody, an agonist anti-CD27 antibody, an agonist anti-glucocorticoid-induced TNFR-related protein GITR antibody, and any combination thereof.
  • the method further includes administering to the individual at least one stimulatory cytokine.
  • the at least one stimulatory cytokine is administered in combination with the isolated antibody.
  • the at least one stimulatory cytokine is selected from TNF- ⁇ , IL-10, IL-6, IL-8, CRP, TGF-beta members of the chemokine protein families, IL20 family member, IL-33, LIF, OSM, CNTF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN- ⁇ , IFN- ⁇ , IL-2, IL-18, GM-CSF, G-CSF, and any combination thereof.
  • kits containing an isolated antibody of the present disclosure may include one or more containers comprising a purified antibody of the present disclosure.
  • the kits further include instructions for use in accordance with the methods of this disclosure.
  • these instructions comprise a description of administration of the isolated antibody of the present disclosure (e.g., an anti-TREM2 or 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 disease, and multiple sclerosis, according to any methods of this disclosure.
  • the instructions comprise a description of how to detect 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 identifying whether that individual has the disease and the stage of the disease.
  • kits may further include another antibody of the present disclosure (e.g., at least one antibody that specifically binds to an inhibitory checkpoint molecule, at least one antibody that specifically binds to an inhibitory cytokine, and/or at least one agonistic antibody that specifically binds to a stimulatory check point protein) and/or at least one stimulatory cytokine.
  • another antibody of the present disclosure e.g., at least one antibody that specifically binds to an inhibitory checkpoint molecule, at least one antibody that specifically binds to an inhibitory cytokine, and/or at least one agonistic antibody that specifically binds to a stimulatory check point protein
  • kits may further include instructions for using the antibody and/or stimulatory cytokine in combination with an isolated antibody of the present disclosure (e.g., an anti-TREM2 antagonist antibody described herein), instructions for using the isolated antibody of the present disclosure in combination with an antibody and/or stimulatory cytokine, or instructions for using an isolated antibody of the present disclosure and an antibody and/or stimulatory cytokine, according to any methods of this disclosure.
  • an isolated antibody of the present disclosure e.g., an anti-TREM2 antagonist antibody described herein
  • the instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating, e.g., a disease of the present disclosure. Instructions may be provided for practicing any of the methods described herein.
  • kits of this disclosure are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an isolated antibody of the present disclosure (e.g., an anti-TREM2 or anti-DAP12 antibody described herein).
  • the container may further comprise a second pharmaceutically active agent.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the isolated antibodies of the present disclosure also have diagnostic utility.
  • This disclosure therefore provides for methods of using the antibodies of this disclosure, or functional fragments thereof, for diagnostic purposes, such as the detection of TREM2 and/or DAP12 in an individual or in tissue samples derived from an individual.
  • the individual is a human. In some embodiments, the individual is a human patient suffering from, or at risk for developing dementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease, multiple sclerosis, or cancer.
  • the diagnostic methods involve detecting TREM2 and/or DAP12 in a biological sample, such as a biopsy specimen, a tissue, or a cell.
  • An isolated antibody of the present disclosure e.g., an anti-TREM2 or anti-DAP12 antibody described herein
  • a tissue sample e.g., a biopsy specimen
  • an anti-TREM2 or anti-DAP12 antibody described herein in order to detect and/or quantify disease-associated macrophages (e.g., M2-type macrophages).
  • the detection method may involve quantification of the antigen-bound antibody.
  • Antibody detection in biological samples may occur with any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography.
  • the antibody is radiolabeled, for example with 18 F and subsequently detected utilizing micro-positron emission tomography analysis.
  • Antibody-binding may also be quantified in a patient by non-invasive techniques such as positron emission tomography (PET), X-ray computed tomography, single-photon emission computed tomography (SPECT), computed tomography (CT), and computed axial tomography (CAT).
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • CT computed tomography
  • CAT computed axial tomography
  • an isolated antibody of the present disclosure may be used to detect and/or quantify, for example, microglia in a brain specimen taken from a preclinical disease model (e.g., a non-human disease model).
  • a preclinical disease model e.g., a non-human disease model
  • an isolated antibody of the present disclosure e.g., an anti-TREM2 or anti-DAP12 antibody described herein
  • 4. The isolated antibody of embodiment 2, wherein the TREM2 protein, the DAP12 protein, or both is a naturally occurring variant. 5.
  • the isolated antibody of any one of embodiments 1-4, wherein the one or more TREM2 activities comprise TREM2 binding to DAP12. 6.
  • DAP12 phosphorylation is induced by one or more SRC family tyrosine kinases.
  • the increased expression of the 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.
  • ERK extracellular signal-regulated kinase
  • CCR7 C-C chemokine receptor 7
  • 26. The isolated antibody of any one of embodiments 1-25, wherein the isolated agonist antibody that binds to a TREM2 protein binds to one or more amino acids within amino acid residues selected from the group consisting of:
  • Human TREM2 contains a signal peptide located at amino residues 1-18 of SEQ ID NO: 1.
  • Human TREM2 contains an extracellular immunoglobulin-like variable-type (IgV) domain located at amino residues 29-112 of SEQ ID NO: 1; additional extracellular sequences located at amino residues 113-174 of SEQ ID NO: 1; a transmembrane domain located at amino residues 175-195 of SEQ ID NO: 1; and an intracellular domain located at amino residues 196-230 of SEQ ID NO: 1.
  • IgV immunoglobulin-like variable-type
  • TREM2 A known feature of human TREM2 is that the transmembrane domain contains a lysine (aa186) that can interact with an aspartic acid in DAP12, a key adaptor protein that transduces signaling from TREM2, TREM1, and other related IgV family members.
  • aa186 a lysine that can interact with an aspartic acid in DAP12
  • a key adaptor protein that transduces signaling from TREM2, TREM1, and other related IgV family members.
  • a BLAST analysis of human TREM2 identified 18 related homologues. These homologues included the Natural Killer (NK) cell receptor NK-p44 (NCTR2), the polymeric immunoglobulin receptor (pIgR), CD300E, CD300A, CD300C, and TREML1/TLT1. The closest homologue was identified as NCTR2, having similarity with TREM2 within the IgV domain ( FIG. 1A ).
  • a BLAST analysis also compared TREM proteins with other IgV family proteins ( FIG. 1B ).
  • TREM2 is also closely related to TREM1.
  • An alignment of the amino acid sequences of TREM1 and TREM2 was generated by 2-way blast ( FIG. 2A ). This is limited to the IgV domain as well.
  • TREM1, NK-p44, and other members of this family have been previously described.
  • Antibodies that bind the extracellular domain of TREM2, particularly 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. Antibodies are then screened for their ability to activate TREM2 signaling and functions in cells and in a whole animal in vivo as described in Examples 2-39 below.
  • agonist anti-TREM2 antibodies can be produced 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, lead to activation.
  • receptors such as IgG itself or NKp44
  • Agonist anti-TREM2 antibodies can also be produced that target amino acid residues 99-115 of human TREM2. It is believed that amino acid residues 99-115 correspond to a peptide that block binding of TREM2 to its endogenous target, as the corresponding peptide in mouse TREM1 (amino acid residues 83-99) block binding of TREM1 to its endogenous target (Gibot et al., Infect. Immunity 2004).
  • the mouse TREM1 peptide is called LP17 (LQVTDSGLYRCVIYHPP (SEQ ID NO: 414)).
  • the equivalent region in human TREM2 is located within the CD3 domain and is located at amino acid residues 99-115 of SEQ ID NO: 1 (LQPHDAGLYQCQSLHG). Antibodies that block ligand binding could activate the receptors similar to the ligand itself.
  • Another approach for predicting a relevant (e.g., agonistic) site within the human TREM2 protein is by targeting the sites where the mutations are found in Alzheimer's disease (e.g., R47H), Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), or Nasu-Hakola disease. Also relevant is the site of the major mutations associated with human disease, which are generally found within the IgV domain.
  • TREM2-related structures of TREM1 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): p. 1237-48; and Radaev, S et al., Structure, 2003. 11(12): p. 1527-35), TLT1 (Gattis, J L et al., J Biol Chem, 2006. 281(19): p. 13396-403), and NKp44 have been described, and thus structural regions/features are identified within the IgV domain that are particularly likely to play a central role in interacting with natural agonists.
  • TREM1 has been reported to be either 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): p. 1527-35) in vitro under cell-free conditions, but its oligomeric state in vivo remains unclear, as well as that of TREM2.
  • amino acid sequence of human DAP12 is set forth below as SEQ ID NO: 2:
  • DAP12 is a single-pass type I 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-based activation motif (ITAM) domain of DAP12 is located at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2).
  • An aspartic acid in DAP12 interacts with the transmembrane domain of human TREM2 containing a lysine at amino acid residue 186, and transduces signaling from TREM2, TREM1, and other related IgV family member proteins.
  • Agonist anti-DAP12 antibodies can be produced that target amino acid residues 22-40 of the human DAP12. It is believed that DAP12 is a disulfide-bonded dimer, associating with TREM2, and that dimerizing DAP12 with an antibody against the extra cellular domain encompassing amino acid residues 22-40 will activate one or more TREM2 and/or DAP12 activities.
  • Antibodies that bind the extracellular domain of TREM2, particularly the IgV domain were generated using the following procedure. Eight na ⁇ ve human synthetic yeast libraries each of ⁇ 10 9 diversity were designed, generated, and propagated as described previously (see, e.g., WO2009036379; WO2010105256; WO2012009568; Xu et al., (2013) Protein. Eng. Des. Sel. 26(10):663-670).
  • the ADIMAB yeast-based antibody discovery platform used herein allowed for the identification of fully human, full-length, monoclonal IgG1 antibodies with broad epitopic coverage.
  • the ADIMAB yeast is engineered to transport high quality, whole IgGs through the secretory pathway, and then present them on the surface or secrete them directly into the medium.
  • yeast cells ⁇ 10 10 cells/library
  • yeast cells 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.
  • Biotinylations were performed using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific, Cat #21425).
  • the cell pellet was resuspended in 40 mL wash buffer, and 500 ⁇ l Streptavidin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat #130-048-101) were added to the yeast and incubated for 15 min at 4° C. Next, the yeast were pelleted, resuspended in 5 mL wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany. Cat. #130-042-401). After the 5 mL was loaded, the column was washed 3 times with 3 ml FACS wash buffer.
  • the column was then removed from the magnetic field, and the yeast were eluted with 5 mL of growth media and then grown overnight. The following three rounds of sorting were performed using flow cytometry. Approximately 1 ⁇ 10 8 yeast were pelleted, washed three times with wash buffer, and incubated with 200 nM, 100, or 10 biotinylated TREM2 for 10 min at room temperature respectively.
  • Yeast were then washed twice and stained with goat anti-human F(ab′) 2 kappa-FITC diluted 1:100 (Southern Biotech, Birmingham, Ala., Cat#2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, N.Y., Cat # S21375) diluted 1:500 or Extravidin-phycoerthyrin (Sigma-Aldrich, St Louis, Cat # E4011) diluted 1:50 secondary reagents for 15 min at 4° C. After washing twice with ice-cold wash buffer, the cell pellets were resuspended in 0.4 mL wash buffer and transferred to strainer-capped sort tubes.
  • Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sort gates were determined to select only TREM2 binding clones for two rounds and the third round was a negative sort to decrease reagent binders. After the final round of sorting, yeast were plated and individual colonies were picked 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 the supernatants were harvested for purification. IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified over KappaSelect (GE Healthcare LifeSciences, Cat #17-5458-01). Two antibodies (Ab21 and Ab52) were selected for further analysis.
  • the Kabat CDR sequences of antibody Ab21 and antibody Ab52 are set forth in Table 1.
  • the amino acid sequence of the heavy chain variable domain of antibody Ab21 is: EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARAGHYDGGHLGMDVWGQGT TVTVSS (SEQ ID NO:410), and the amino acid sequence of the light chain variable domain of antibody 21 is: EIVMTQSPGTLSLSPGERATLSCRASQS VSSSYLAWYQQKPGQAPRLLIYGASNRATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQDDSAPYTFGGGTKVEIK (SEQ ID NO:411).
  • the amino acid sequence of the heavy chain variable domain of antibody Ab52 is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGIINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREADDSSGYPLGLDVWGQG TMVTVSS (SEQ ID NO:412), and the amino acid sequence of the light chain variable domain of antibody 52 is: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQVNSLPPTFGGGTKVEIK (SEQ ID NO:413).
  • TREM2 antibodies Initial characterization of TREM2 antibodies involved determining their ability to bind TREM2 expressed on dendritic and other primary human or mouse immune cells.
  • Cells were harvested, plated at 10 5 /ml in a 96 well plate, washed, and incubated in 100 ul PBS containing 10-50 ug/ml Mab and Fc blocking reagent for 1 hour in ice. Cells were then washed twice and incubated in 100 ul PBS containing 5 ug/ml PE-conjugated secondary antibody for 30 minutes in ice. Cells were washed twice in cold PBS and acquired on a BD FACS Canto. Data analysis and calculation of MFI values was performed with FlowJo (TreeStar) software version 10.0.7.
  • Antibodies Ab21, Ab52, Ab16, Ab20, Ab66, and Ab68 demonstrated binding to a mouse cell line (BWZ T2) expressing recombinant mouse TREM2, as indicated by positive TREM2 antibody staining detected via FACS analysis (black outlined histograms) ( FIG. 3A ).
  • Antibodies Ab21 and Ab52 demonstrated antibody binding to WT (Trem+/+) bone marrow derived mouse macrophages (BMMac, mMac), but not to TREM2 deficient (TREM2 ⁇ / ⁇ ) mouse macrophages (BMMac, mMacs) ( FIG. 3B ).
  • Antibodies Ab21 and Ab52 demonstrated binding to both a human cell line (293) expressing recombinant Human TREM2 ( FIG.
  • MFI Mean fluorescent intensities
  • the binding affinity of each anti-TREM2 antibody was determined by measuring their K D by ForteBio or MSD-SET. ForteBio affinity measurements were performed as previously described (Estep et al, (2013) MAbs 5(2):270-8). Briefly, ForteBio affinity measurements were performed by loading IgGs on-line onto AHQ sensors. Sensors were equilibrated off-line in assay buffer for 30 min and then monitored on-line for 60 seconds for baseline establishment. Sensors with loaded IgGs were exposed to 100 nM antigen for 5 minutes, then transferred to assay buffer for 5 min for off-rate measurement. Kinetics were analyzed using the 1:1 binding model.
  • Antigen captured on a plate was detected with 250 ng/mL sulfotag-labeled streptavidin in PBSF by incubation on the plate for 3 min. The plates were washed three times with wash buffer and then read on the MSD Sector Imager 2400 instrument using 1 ⁇ Read Buffer T with surfactant. The percent free antigen was plotted as a function of titrated antibody in Prism and fit to a quadratic equation to extract the K D . To improve throughput, liquid handling robots were used throughout MSD-SET experiments, including SET sample preparation.
  • Table 5 lists values representing the binding affinity (K D ) of antibodies Ab21 and Ab52 to a human TREM2 Fc fusion protein (hTREM2-Fc), a human monomeric His tagged TREM2 protein (hTREM2-HIS), and a mouse TREM2 Fc fusion protein (mTREM2-Fc).
  • TLR Toll-Like Receptor
  • Bone marrow-derived dendritic cells are stimulated by culturing with TLR ligands, such as LPS, CpG DNA, and zymosan, for 16 h.
  • TLR ligands such as LPS, CpG DNA, and zymosan
  • Conditioned media is collected and ELISA assays are performed in order to evaluate secretion of the cytokines IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF.
  • TREM2 cytokines
  • BMDC cells that do not have active TREM2 may secrete significantly more IL-12, p70, and TNF than BMDC cells that have activated TREM2 after stimulation.
  • anti-TREM2 agonistic antibodies will reduce the expression levels of IL-12, p70, and TNF.
  • Bone marrow-derived dendritic cells from wild-type and from TREM2-hetrozyous mice, which would have partially inactive TREM2, will serve as positive controls for determining expression levels of the cytokines IL-12, p70, and TNF, as well as their modulation by agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies.
  • Cytokine concentrations in the culture supernatants are determined using mouse IFN-a4, IFN-b, IL-6, IL-12 p70, TNF, and IL-10 ELISA kits (eBioscience) and VeriKine Mouse IFN-b ELISA kit (PBL interferon source) according to manufacturer's protocol. Levels of mRNA for these cytokines are also measured by Quantitative RT-PCR (qRT-PCR). Total RNA is prepared by using RNeasy plus mini kit (QIAGEN) is reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen) using oligo dT primer according to manufacturer's protocol.
  • Quantitative PCR is performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and 7900HT (Applied Biosystems) according to manufacturer's protocol.
  • the sequences of IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF primers are as described. (e.g., Hamerman, J A, Eur. J. Immunol. 2012. 42: 176-185).
  • Example 3 Normalization and Reduction of the Ability of BMDCs to Induce Antigen-Specific T-Cell Proliferation by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may reduce and normalize the ability of bone marrow-derived dendritic cells (BMDC) to induce antigen-specific T-cell proliferation.
  • BMDC bone marrow-derived dendritic cells
  • Ovalbumin (OVA)-specific T-cell response induced by BMDCs can be determined by CFSE dilution.
  • BMDCs are isolated by MACS after 6 days of culture and plated at 1 ⁇ 10 4 cells per well of a round bottom 96 well 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 are isolated by using Dynal Mouse CD4 Negative Isolation Kit (Invitrogen) and stained with CFSE (final 0.8 mM).
  • TLR Toll-Like Receptor
  • BMDM Bone marrow-derived macrophages
  • TREM2 TREM2
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may reduce and normalize TLR responses in macrophages.
  • mice are treated with 1.5 ml of 2% thioglycollate medium by intraperitoneal injection, and cells are then isolated by peritoneal lavage.
  • BMDM total bone marrow is cultured in DMEM supplemented with 10% bovine 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 detached with 1 m MEDTA in PBS. Cells are stained with commercially available antibodies: anti-CD11b, anti-CD40, anti-GR1 (BD Pharmingen), and F4/80 (Caltag Laboratories).
  • BMDM are re-plated and allowed to adhere for 4 h at 37° C., and then TLR agonists, such as LPS ( Salmonella abortus equi), zymosan ( Saccharomyces cerevisiae ), and CpG 1826 DNA (purchased from e.g., Sigma-Aldrich) are added.
  • TLR agonists such as LPS ( Salmonella abortus equi), zymosan ( Saccharomyces cerevisiae ), and CpG 1826 DNA (purchased from e.g., Sigma-Aldrich) are added.
  • Cell culture supernatant is collected 24 h after stimulation and the levels of IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF cytokines are measured by ELISA or by cytometric bead array (BD Biosciences mouse inflammation kit).
  • Example 5 Induction of the Anti-Inflammatory Cytokine IL-10 in Bone Marrow-Derived Myeloid Precursor Cells by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • bone marrow-derived myeloid precursor cells may show an increase in the anti-inflammatory cytokine IL-10 following treatment with agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies and stimulation with 100 ng/ml LPS (Sigma), by co-culturing with apoptotic cells, or by a similar stimulus.
  • Bone marrow cells are isolated from adult 6-8 week-old female C57BL/6 mice (Charles River, Sulzfeld, Germany) from the medullary cavities of the tibia and femur of the hind limbs. Removal of erythrocytes is performed by lysis with hypotonic solution. Cells are cultured in DMEM medium (Invitrogen) containing 10% fetal calf serum (Pan Biotech) and 10 ng/ml of GM-CSF (R&D Systems) in 75 cm 2 culture flasks (Greiner Bio-One).
  • non-adherent cells are collected and re-seeded in fresh 75 cm 2 culture flasks. Medium is changed after 5 d and cells are cultured for an additional 10-11 d. The remaining cells are bone marrow-derived myeloid precursor cells, and are transduced with TREM2 virus. The transduced cells are then examined for the level of IL-10 in conditioned media in both the presence and absence of anti-TREM2 agonistic antibodies and LPS.
  • Example 6 Induction of Phagocytosis of Apoptotic Neurons, Nerve Tissue Debris, Non-Nerve Tissue Debris, Bacteria, Other Foreign Bodies, and Disease-Causing Proteins in Cells from the Myeloid Lineage by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may induce phagocytosis of apoptotic neurons, nerve tissue debris, non-nerve tissue debris, bacteria, other foreign bodies, and disease-causing proteins, such as A beta peptide, alpha synuclain protein, Tau protein, TDP-43 protein, prion protein, and huntingtin protein, in cells from the myeloid lineage, such as monocytes and microglia.
  • Monocytes are isolated from peripheral blood that is collected from adult C57BL/6 mice. Hypotonic lysis buffer depletes erythrocytes. Cells are plated on 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 trypsinization, adherent cells are collected and used for phagocytosis experiments.
  • Microglial cells are prepared from the brains of post-natal day 3 to 5 (P3 to P5) C57BL/6 mice. In brief, meninges are removed mechanically, and the cells are dissociated by trituration and cultured in basal 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 d to form a confluent glial monolayer. To collect microglial cells, the cultures are shaken on a rotary shaker (200 rpm) for 2 h. The attached astrocytes are used for immunohistochemistry.
  • microglial cells are seeded in normal culture dishes for 1 h, and then all non-adherent cells are removed and discarded. Purity of the isolated microglial cells is about 95% as determined by flow cytometry analysis with antibody directed against CD11b (BD Biosciences). Microglial cells are cultured in basal medium.
  • Oligodendrocytes i.e., neurons
  • neuron-enriched cells are prepared from the brain of C57BL/6 mouse embryos (E15-16).
  • brain tissue is isolated and mechanically dispersed and seeded in culture dishes pre-coated with 0.01 mg/ml poly-L-ornithin (Sigma-Aldrich) and 10 ⁇ g/ml laminin (Sigma-Aldrich).
  • Cells are cultured in neuronal condition medium (BME; GIBCO BRL) supplemented with 2% B-27 supplement (GIBCO BRL), 1% glucose (Sigma-Aldrich), and 1% FCS (PAN Biotech GmbH). Cells are cultured for 5-10 d to obtain morphologically mature oligodendrocytes.
  • microglia are transduced with sh-TREM2 RNA, sh-control RNA, wTREM2, GFP1 control, mtDAP12-GFP, and GFP2 control vector. After transduction, microglia are cultured for 72 h to achieve effective knockdown of TREM2 by RNA interference. Neurons are cultured for 5-10 d, and okadaic acid is then added at the final concentration of 30 nM for 3 h to induce apoptosis. Neuronal cell membranes are labeled with CellTracker CM-DiI membrane dye (Molecular Probes).
  • apoptotic neurons or other targets of phagocytosis are washed two times and added to the transduced microglial culture at an effector/target ratio of 1:20.
  • the number of microglia having phagocytosed neuronal cell membranes is counted under a confocal fluorescence microscope (Leica). Apoptotic cells are counted in three different areas at a magnification of 60. The amount of phagocytosis is confirmed by flow cytometry.
  • 24, 48, or 72 h after the addition of apoptotic neurons cells are collected and used for RT-PCR of cytokines.
  • microglia are transduced with a TREM2 expression vector or a GFP control vector. Cells are then treated with anti-TREM2 agonistic antibodies. After 24 h, 1.00 ⁇ m of red fluorescent microsphere beads (Fluoresbrite Polychromatic Red Mi-crospheres; Polysciences Inc.) or, fluorescent labeled bacteria are added for 1 h. Phagocytosis of microsphere beads or, fluorescent labeled bacteria, by microglia is analyzed by fluorescence microscopy. Furthermore, microglia are collected from the culture plates and analyzed by flow cytometry. The percentage of microglia having phagocytosed beads is determined. Because phagocytosis varies from one experiment to the other, the relative change in phagocytosis is also determined. Data are shown as the relative change in phagocytosis between microglia cultured with agonistic antibodies and control antibody.
  • microglia are transduced with a TREM2 vector or a GFP1 control vector. Cells are then cultured on dishes and treated with anti-TREM2 agonistic antibodies. After 24, 48, and 72 h, RNA is isolated from microglia using an RNeasy Mini Kit (QIAGEN). RNA is also collected from microglia that have been transduced with sh-TREM2 RNA, sh-control RNA, wTREM2, GFP2, mtDAP12-GFP, and GFP1 vector and co-cultured with apoptotic neurons for 48 h.
  • RNA is also collected from microglia that have been transduced with sh-TREM2 RNA, sh-control RNA, wTREM2, GFP2, mtDAP12-GFP, and GFP1 vector and co-cultured with apoptotic neurons for 48 h.
  • RNA Reverse transcription of RNA is then performed. Quantitative RT-PCR by SYBR Green is performed on an ABI Prism 5700 Sequence Detection System (PerkinElmer). Amplification of GAPDH is used for sample normalization. The amplification protocol followed the GeneAmp 5700 Sequence Detection System Software (version 1.3). For detection of 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 (SEQ ID NO: 416) 5′-CTCCACTCACGGCAAATTCAA-3′, and GAPDH reverse primer: (SEQ ID NO: 417) 5′-GATGACAAGCTTCCCATTCTCG-3; TNF- ⁇ forward primer: (SEQ ID NO: 418) 5′-CCGTCAGCCGATTTGCTATCT-3′, and TNF- ⁇ reverse primer: (SEQ ID NO: 419) 5′-ACGGCAGAGAGGAGGTTGACTT-3′; IL-1 ⁇ forward primer: (SEQ ID NO: 420) 5′-ACAA-CAAAAAAGCCTCGTGCTG-3′, and IL-1 ⁇ reverse primer: (SEQ ID NO: 421) 5′-CCATTGAGGTGGAGAGCTTTCA-3′; NOS2 forward primer: (SEQ ID NO: 422) 5′-GGCAAACCCAAGGTCTACGTTC-3′, NOS2 reverse primer: (SEQ ID NO: 423) 5′-TACCTCATTGGCCAGCTGCTT-3′; and TGF
  • HiLyteFluorTM 647 (Anaspec)-Abeta-(1-40) was resuspended in Tris/EDTA (pH 8.2) at 20 mM and then incubated in the dark for 3 d at 37° C. to promote aggregation.
  • Microglial cells are pretreated in low serum (0.5% FBS supplemented with insulin), LPS (50 ng/ml), IFNc (100 units/ml), and anti-TREM2 agonistic antibodies for 24 h prior to the addition of aggregated fluorescently labeled a beta peptide.
  • Amyloid phagocytosis and surface expression of TREM2 are determined by flow cytometric analysis 5 h post-addition of 100 nM aggregated HiLyteFluorTM 647-Ab-(1-40) (ASN NEURO (2010) 2(3): 157-170). Phagocytosis of other disease-causing proteins is conducted in a similar manner.
  • Example 7 Induction of ERK Activation by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may induce ERK activation.
  • Microglia are transduced with a TREM2 vector, and 2 ⁇ 105 cells are exposed to agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies for 1 h. After stimulation, cells are lysed in reducing sample buffer for Western blot analysis. Phosphorylation of ERK and total amount of ERK are determined by immuno-detection with anti-phospho-ERK and anti-ERK antibodies, respectively (both from Cell Signaling Technology) by Western blot analysis (JEM (2005), 201, 647-657).
  • Example 8 Induction of CCR7 and Migration Toward CCL19 and CCL21 in Microglia, Macrophages, and Dendritic Cells by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may induce CCR7 and migration toward CCL19 and CCL21 in microglial cells, macrophages, and dendritic cells.
  • Microglial cells are transduced with a TREM2 vector or a GFP1 control vector.
  • the transduced microglial cells are then either cultured with agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies, or with a control antibody.
  • Cells are collected after 72 h, immuno-labeled with CCR7 specific anti-bodies, and analyzed by flow cytometry.
  • Microglial cells are stimulated via TREM2 with the agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies and placed in a two-chamber system.
  • the number of microglial cells migrating toward the chemokine ligands CCL19 and CCL21 is quantified (JEM (2005), 201, 647-657).
  • microglial cells are exposed to the agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies and treated with 1 ⁇ g/ml LPS.
  • Microglia are transferred into the upper chamber of a transwell system (3 ⁇ m pore filter; Millipore) containing 450 ⁇ l medium with 100 ng/ml CCL19 or CCL21 (both from PeproTech) in the lower chamber. After a 1 h incubation period, the number of microglial cells that have migrated to the lower chamber is counted in three independent areas by microscopy (JEM (2005), 201, 647-657).
  • Example 9 Induction of F-Actin in Microglia, Macrophages, and Dendritic Cells by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may induce F-actin in microglial cells, macrophages, and dendritic cells.
  • Microglia and other cells of interest that are transduced with TREM2 or that express TREM2 are added to culture plates and then exposed to agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies, or a control antibody.
  • Cells are fixed, blocked, and then stained with Alexa Fluor 546-conjugated phalloidin (Molecular Probes) after 1 h and F-actin is labeled with a fluorescence dye. Images are collected by confocal laser scanning microscopy with a 40 ⁇ objective lens (Leica). (JEM (2005), 201, 647-657).
  • Example 10 Induction of Osteoclast Production and Increased Rate of Osteoclastogenesis by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies may induce osteoclast production and increase the rate of osteoclastogenesis.
  • RAW264.7 cells that make osteoclasts or bone marrow-derived monocyte/macrophage (BMM) precursor cells are maintained in RPMI-1640 medium (Mediatech), or another appropriate medium, supplemented with 10% FBS (Atlantic Biologics, Atlanta, Ga., USA) and penicillin-streptomycin-glutamine (Mediatech).
  • TREM2B cDNA with a FLAG epitope added to the N terminus is inserted into the retroviral vector pMXpie upstream of an IRES, followed by an eGFP cDNA sequence.
  • Cells are transfected with pMXpie-FLAG TREM2B, using Fugene 6 (Roche) according to manufacturer's protocol.
  • Cells are selected in puromycin (Sigma) at 2 ⁇ g/ml. Stable puromycin-resistant clones are screened for anti-FLAG M2 monoclonal antibody (Sigma) binding by using flow cytometry, and then subcloned and maintained on puromycin selection media.
  • RAW264.7 cells expressing TREM2B are seeded in 96-well plates with 3000 cells/well in alpha-MEM medium supplemented with 10% FBS, penicillin-streptomycin-glutamine, 50 ng/ml RANKL, and 20 ng/ml M-CSF.
  • the medium is changed every 3 days, exposed to anti-TREM2 agonistic antibodies and the number of multinucleated (at least three nuclei) TRACP + osteoclasts are counted and scored by light microscopy. To determine complexity and size, osteoclasts are counted by number of nuclei (>10 or 3-10 nuclei). The surface area of osteoclasts is also measured by using Image J software (NIH).
  • RNA is extracted from osteoclastogenic cultures at different time points using TRIzol reagent (Invitrogen). After first-strand cDNA synthesis using a SuperScript III kit (Invitrogen), real-time quantitative PCR reactions are performed for Nfatc1, Acp5, Ctsk, Calcr, and Ccnd1. Relative quantification of target mRNA expression is calculated and normalized to the expression of cyclophilin and expressed as (mRNA of the target gene/mRNA of cyclophilin) 3 ⁇ 10 6 . (J. OF BONE AND MINERAL RESEARCH (2006), 21, 237-245 ; J Immunol 2012; 188:2612-2621).
  • mice obtained from Charles River Laboratories
  • mice are injected in the tail base bilaterally with 200 ⁇ l of an innoculum containing 100 ⁇ g of myelin oligodendrocyte glycoprotein peptide 35-55 (amino acids MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 415); Seqlab) and 1 mg of Mycobacterium tuberculosis H37 Ra (Difco) in incomplete Freund adjuvant (Difco).
  • Pertussis toxin 200 ng; List Bio-logical Laboratories
  • Clinical signs are scored as follows: 0, no clinical signs; 1, complete limp tail; 2, complete limp tail and abnormal gait; 3, one hind-limb paraparesis; 4, complete hindlimb paraparesis; and 5, fore- and hind-limb paralysis or moribund.
  • mice having disease onset (clinical score of 1 or more) at day 14 are used for experiments.
  • Agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies are injected intraperitoneally or intravenously in EAE-diseased mice at the day of the first clinical symptoms or at any other desired time (PLoS Med (2007) 4(4): e124).
  • mice Young or aged wild-type (WT) mice are fed a standard diet (Harlan) containing 0.2% cuprizone (CPZ) powdered oxalic bis(cyclohexylidenehydrazide) (Sigma-Aldrich) for 4, 6 or 12 weeks.
  • CPZ cuprizone
  • brains are removed after mouse perfusion with 4% paraformaldehyde (PFA), fixed in 4% PFA for 24 h, followed by immersion in 30% sucrose for 24-48 h.
  • PFA paraformaldehyde
  • mice are analyzed for locomotor activity using transparent polystyrene enclosures and computerized photobeam instrumentation.
  • General activity variables total ambulations, vertical rearings, along with indices of emotionality including time spent, distance traveled and entries, are analyzed.
  • a battery of sensorimotor tests is performed to assess balance (ledge and platform), strength (inverted screen), coordination (pole and inclined screens) and initiation of movement (walking initiation). Motor coordination and balance are studied using a rotarod protocol (Cantoni et al., Acta Neuropathol (2015)129(3):429-47).
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is tested in established animal models of traumatic brain injury (Tanaka, Y et al. (2013) Neuroscience 231 49-60).
  • a model of traumatic brain injury that induces the activation of microglia and astrocytes is used.
  • Eight or nine week-old male C57BL/6J WT mice or progranulin heterozygous mice are used (purchased from Charles River Laboratories or Jackson Laboratories).
  • Mice are anesthetized by intraperitoneal administration of xylazine hydrochloride (8 mg/kg) and chloral hydrate (300 mg/kg) dissolved in sterile saline, and subsequently placed in a stereotaxic apparatus (Narishige, Tokyo, Japan). An incision is made in the scalp and the cranium is exposed.
  • the periosteum is cleaned from the skull, a hole is drilled over the right cerebral hemisphere with a dental drill, and the duramater is removed with a needle tip.
  • a stainless steel cannula with a 0.5 mm outer diameter, is used to make a longitudinal stab wound in the right hemisphere.
  • the cannula is positioned at 1.3 mm lateral to the midline, and 1 mm posterior to bregma, and introduced into the brain until the tip reaches a depth of 2 mm.
  • the cannula is then shifted 2 mm caudally (bregma 3 mm), and then shifts back 2 mm rostrally to its initial position.
  • mice are then treated with agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies according to standard procedures and then analyzed by histology and immunofluorescence staining and behavioral tests.
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is tested in a model of neuro-inflammation and neuron loss following toxin-induced injury (Martens, L H et al., (2012) The Journal of Clinical Investigation, 122, 3955).
  • mice Three-month-old mice are treated with 4 intraperitoneal injections of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) per day for 2 days (4 ⁇ g/g body weight) (Sigma-Aldrich) or PBS. Mice are treated with agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies according to standard protocols and then analyzed using Stereological counting to quantify dopamine neurons and microglia in the substantia nigra pars compacta (SNpc), as described.
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is tested in animal models for aging, seizures, 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., (2007) Nat. Neurosci.
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is 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).
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is tested in a model of infection.
  • Listeria monocytogenes or other infection in normal mice or progranulin heterozygous mice can be used, as previously described (e.g., Yin, F et al., (2009) J. Exp. Med, 207, 117-128).
  • agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies is tested in a model of inflammatory diseases. For example rheumatoid arthritis or in an established model of 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 Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12 Bispecific Antibodies that Induce Phosphorylation of DAP12, ERK, and AKT which Indicate Activation of the PI3K Pathway
  • J774, RAW 264.7, BMM cells, or osteoclasts are removed from tissue culture dishes with PBS-EDTA, washed with PBS, and counted. J774 (40 ⁇ 10 6 ) or RAW 264.7 cells (10 ⁇ 10 6 BMM or osteoclasts) are incubated with an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibody or with an isotype-matched control antibody at 1 ⁇ g/10 6 cells for 20 min on ice or under other conditions.
  • Radioimmunoprecipitation assay [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 Na VO, 0.25% sodium deoxycholate, aprotinin (1 ⁇ g/ml), leupeptin (1 ⁇ g/ml), pepstatin (1 ⁇ g/ml)] for 20 mM followed by centrifugation at 16,000 g for 10 min at 4° C. to remove insoluble materials.
  • RIPA radioimmunoprecipitation assay
  • the resulting supernatant is subjected to immunoprecipitation reactions with the indicated antibodies (DAP12, ERK, or AKT) and protein A- or protein G-agarose (Sigma).
  • the beads are extensively washed with RIPA buffer and the proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the proteins are then transferred to nitrocellulose membranes by Western blotting, incubated with the appropriate antibodies (antibodies that specifically recognize the phosphorylated form of DAP12, ERK, or AKT) and visualized with the enhanced chemiluminescence (ECL) system (Pierce), as described (e.g., Peng et al., (2010) Sci Signal., 3(122): ra38).
  • ECL enhanced chemiluminescence
  • Example 19 Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12 Bispecific Antibodies that Induce Calcium Flux
  • BMM 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 ⁇ M Indo-1 AM (Invitrogen) for 20 min at 37° C.
  • 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)
  • Pluronic F-127 Invitrogen
  • 1 ⁇ M Indo-1 AM Invitrogen
  • Cells are washed twice with HEPES buffer and are then stimulated with an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibody (16 ⁇ g/ml) or with a control antibody (16 ⁇ g/ml) and monitored by spectrophotometer (PTL Photon Technology International).
  • the Indo-1 fluorescence emission is converted to calcium (Ca 2+ ) according to manufacturer's instructions (e.g., Peng et al., (2010) Sci Signal., 3(122): ra38).
  • Example 20 Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/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 substituted with serum-free medium to induce apoptosis.
  • Cells are treated with RANKL, PBS, and an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibody, or an isotype-matched control antibody, during the overnight serum starvation.
  • Cells are fixed in 1% paraformaldehyde and stained with a TUNEL-based kit (Millipore Corporation) according to manufacturer's instructions. Apoptotic nuclei are counted with a Nikon TE2000-E microscope with 20 ⁇ magnification.
  • Results are expressed as the percentage of apoptotic cells relative to the total number of cells in six randomly selected fields of the two wells, as described (e.g., Peng et al., (2010) Sci Signal., 3(122): ra38). Similar assays are performed with primary microglial cells.
  • Example 21 Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12 Bispecific Antibodies that Induce Osteoclast Differentiation
  • BMM cells are seeded onto the plates in triplicate wells and treated with RANKL, M-CSF, and with an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibody, or an isotype-matched control monoclonal antibody.
  • the medium is changed every 3 days until large multinucleated cells are visible.
  • cells are fixed with 3.7% formaldehyde in PBS for 10 min. Plates are then washed twice in PBS, incubated for 30 s in a solution of 50% acetone and 50% ethanol, and washed with PBS.
  • Cells are stained for tartrate-resistant acid phosphatase (TRAP) with a kit from Sigma (product 435). Multinucleated (more than two nuclei), TRAP-positive cells are then counted by light microscopy, as described (e.g., Peng et al., (2010) Sci Signal., 3(122): ra38).
  • TRAP tartrate-resistant acid
  • Example 22 Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12 Bispecific Antibodies that Normalize TREM2/TYROBP-Dependent Changes in Gene Expression within the Immune/Microglia Regulatory Module
  • Microglial cells derived from mouse embryonic stem cells are genetically modified by lentiviral vectors to overexpress either full-length or a truncated version of Tyrobp that lacks both intracellular immunoreceptor tyrosine-based activation motif (ITAM) motifs.
  • Microglia cells are also derived from mouse embryonic stem cells that are heterozygous for TREM2.
  • gene-expression data is derived from the RNA sequencing of mouse microglial cell lines overexpressing: (1) vehicle, (2) full-length Tyrobp, or (3) dominant-negative truncated Tyrobp; or (4) overexpressing a knockdown construct for TREM2, such as SiRNA and cells which are heterozygous for TREM2.
  • a knockdown construct for TREM2 such as SiRNA and cells which are heterozygous for TREM2.
  • Approximately 99% of the differentially expressed genes from the microglia overexpressing intact Tyrobp are downregulated compared to the control vehicle.
  • 658 genes related to the vacuole/autophagy, as well as genes involved with RNA metabolism and cell-cycle mitosis are downregulated by active Tyrobp, but upregulated in cells expressing dominant-negative truncated Tyrobp.
  • some 2,856 genes for the vacuole/autophagy pathway and for mitochondrion are selectively upregulated in microglia expressing the dominant-negative truncated Tyrobp.
  • Agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies are screened for their ability to elicit gene expression profiles similar to that observed in normal microglial cells and in microglial cells overexpressing intact Tyrobp in cells that express dominant-negative truncated Tyrobp (Zhang et al., (2013) Cell 153, 707-720), in cells that express the knockdown construct for TREM2, or in cells that are heterozygous for TREM2.
  • Antibodies that are capable of changing the gene expression network are selected.
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